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MECHANISMS OF R-TYPE CALCIUM CHANNEL REGULATION IN THE
ENTERIC NERVOUS SYSTEM .

By

Vinogran Naidoo

A DISSERTATION
Submitted to
Michigan State University
in partial fulfillment of the requirements
for the degree of
DOCTOR OF PHILOSOPHY

Neuroscience

2009

ABSTRACT

MECHANISMS OF R-TYPE CALCIUM CHANNEL REGULATION IN THE
ENTERIC NERVOUS SYSTEM

By

Vinogran Naidoo

The enteric nervous system (ENS) regulates gastrointestinal function, and
calcium channels are crucial to ENS function. There are distinct classes of
calcium currents. L-type currents require a strong depolarization for activation,
are long lasting, and are the main calcium currents recorded in muscle and
endocrine cells. P/Q-type, N-type, and R-type currents, also require strong
depolarizations for activation. They are expressed primarily in neurons, where
they initiate neurotransmission at most fast synapses. T-type calcium currents
are activated by weak depolarizations and found in neurons and cardiac
myocytes. R-type calcium currents were previously recorded from guinea-pig
myenteric neurons suggesting that R-type calcium channels may be important in
the ENS. The aim of my dissertation was to determine their functional
significance in the ENS. I investigated the role of R-type channels in the
myenteric plexus at four different levels: 1) the cellular level by mapping 01E-
subunit immunoreactivity in various regions of the GI tract; 2) intracellular
recordings from myenteric neurons to determine whether R-type channels
mediate slow synaptic excitation in intrinsic primary afferent (AH-type) neurons,
and/or whether calcium entry via non-R-type channels mediate fast synaptic

excitation in interneurons and motorneurons (S-type); 3) functional studies of R-

type channel-mediated neuromuscular transmission because excitatory and
inhibitory motor express a1E immunoreactivity; and 4) physiologically integrative
level by analyzing the effects of R-type calcium channel antagonists on
peristalsis. My studies revealed novel mechanisms of R-type calcium regulation
in the ENS. 1) R-type channel immunoreactivity was found in intrinsic primary
afferent nerve endings, in nerve terminals of ascending interneurons, and on
excitatory and inhibitory motorneurons that supply the muscle layers. 2. The R-
type calcium channel antagonist NiCl2 inhibited the calcium shoulder and the
amplitude of the late afterhyperpolarization in AH neurons. NiCI2 also acted at
nerve terminals of fully cholinergic ascending interneurons to inhibit fast
ganglionic neurotransmission. 3) The R-type channel couples to nitric oxide
synthesis and release from nerve terminals of inhibitory motorneurons. 4) NiClz
applied to isolated ileal segments in vitro impaired peristalsis. Taken together,
these results suggest an important role for R-type channels as mediators of
neurotransmitter release or as transducers of other synaptic events in myenteric
neurons. Success with a selective activator of chloride channels that relieves
chronic idiopathic constipation, suggests that treatments with ion channel
agonists or antagonists may help alleviate motility disorders. The R-type calcium

channel may therefore be a potential drug target in combating motility disorders.

ACKNOWLEDGEMENTS

I wish to first offer my sincerest thanks to Dr. Jim Galligan. Dr. Galligan
has been the best mentor I could have ever hoped for. Thank you Dr. Galligan for
your immense support, kindness, patience, guidance, advice, dedication, and for
keeping faith in my abilities. Thank you also for accepting me into your
laboratory, and for supporting me financially during the past 3 years. I would
have never got even close to this point in my career without your help, and I am
proud to have trained under you.

I wish to also thank my guidance committee for the time they took out of
their busy schedules to serve on my committee. Thank you Drs. David Kreulen,
Stephen Schneider, Ke Dong and Hongbing Wang for your helpful discussions
and advice. I truly appreciate your efforts. I also need to thank Dr. Stephen
Schneider for the synaptophysin antibody.

Thank you also to the International Institute of Education (HE) and the
Fulbright Scholarship Board for giving me this unique opportunity to study for my
doctorate in the United States. You are a testament that dreams do come true.
Thank you also to the Nelson Mandela Fellowship Fund and Michigan State
University for additional financial support. To the Graduate School at MSU, and
the Neuroscience Program, thank you as well for funding my research.

I certainly cannot forget my friends and colleagues in both Galligan labs.
Thank you Xiaochun, Hui Wang, Dima, Hong, Hua, and Emmy for your

friendship, support and laughter, through the best and the toughest of times. It’s

iv

been a tremendous five years with you all, and I will never forget you. Eileen
ROdriguez contributed to the Neuromuscular transmission studies presented in
my dissertation, and I thank her for that great collaboration. To my fellow
students in the Neuroscience Program, thank you for your collegiality and
friendship.

Last but not least, to my wonderful family back in South Africa. It’s been a
long 5 years since I left home. You’ve been the sources of my inspiration and my
guiding light, even though you all are thousands of miles away. Thank you for
your support, love and understanding. I will be home soon. To my brother

Seelendran, this dissertation is for you.

TABLE OF CONTENTS

LIST OF TABLES .............................................................................................. viii
LIST OF FIGURES ............................................................................................. ix
CHAPTER 1 General Introduction ..................................................................... 1
The Nervous System ........................................................................................ 2
Structure and function of a neuron ................................................................... 3
Neurotransmitters, neuropeptides and synaptic transmission .......................... 4
Classical neurotransmitters ........................................................................... 5
Neuropeptides ............................................................................................... 6
The Autonomic Nervous System ...................................................................... 7
Innervation of the gastrointestinal tract ............................................................. 8
Extrinsic innervation of the ENS .................................................................... 8
lntrinisic innervation: the ENS ..................................................................... 11
Electrophysiological characteristics of myenteric neurons .............................. 18
Neurochemical coding and synaptic transmission in the ENS ........................ 20
Slow synaptic transmission in the ENS ....................................................... 20
Fast synaptic transmission in the ENS ........................................................ 23
Neuromuscular transmission in the ENS ........................................................ 24
Small intestinal motility patterns ..................................................................... 28
Peristalsis .................................................................................................... 28

GI motility disorders ........................................................................................ 30
Voltage-gated calcium channels in the nervous system ................................. 33
Overview ..................................................................................................... 33
Calcium channels in the ENS ...................................................................... 41
R-type calcium channels in the ENS ........................................................... 42

Ion channels as targets for disease treatment in the ENS .............................. 43
CHAPTER 2 Hypothesis and specific aims ................................................... 45

CHAPTER 3 lmmunohistochemical localization of R-type calcium channels

in the guinea pig enteric nervous system ...................................................... 49
Abstract .......................................................................................................... 50
Introduction ..................................................................................................... 52
Materials and methods ................................................................................... 56
Results ........................................................................................................... 65
Discussion ...................................................................................................... 73
Summary and conclusions ............................................................................. 84

vi

CHAPTER 4 Contribution of R-type calcium channels to the function of

intrinsic primary afferent neurons and ascending interneurons in the

guinea pig ileum myenteric plexus ............................................................... 108
Abstract ........................................................................................................ 109
Introduction ................................................................................................... 1 10
Materials and methods ................................................................................. 113
Results ......................................................................................................... 1 17
Discussion .................................................................................................... 123
Summary and conclusions ........................................................................... 132

CHAPTER 5 R-type calcium channels couple to nitric oxide-mediated

inhibitory neurotransmission in the guinea pig ileum ................................ 147
Abstract ........................................................................................................ 148
Introduction ................................................................................................... 1 50
Materials and methods ................................................................................. 154
Results ......................................................................................................... 158
Discussion .................................................................................................... 163
Summary and conclusions ........................................................................... 169

CHAPTER 6 Inhibition of R-type calcium channels impairs peristalsis in the

guinea pig ileum ............................................................................................. 177
Abstract ........................................................................................................ 178
Introduction ................................................................................................... 179
Materials and methods ................................................................................. 181
Results ......................................................................................................... 185
Discussion .................................................................................................... 192
Summary and conclusions ........................................................................... 200

CHAPTER 7 General discussion and conclusions ..................................... 215

REFERENCES ................................................................................................. 235

vii

LIST OF TABLES

Chapter 1.

Table 1.1. Descriptions of enteric neuronal morphologies as described
by Dogiel ....................................................................................... 12

Chapter 3.

Table 3.1. Primary antibodies used to study the distribution of R-type
calcium channels ............................................................................. 85

viii

LIST OF FIGURES

Chapter 1.
Figure. 1.1. The enteric nervous system (ENS) ......................................... 11
Figure. 1.2. Morphological characteristics of enteric neurons ........................ 13

Figure 1.3. Model depicting the membrane structure and transmembrane

topology of a voltage-gated calcium channel ............................................. 35
Chapter 3.
Figure 3.1. Western blot analyses of (11 E subunit expression ...................... 87

Figure 3.2. The a1E-subunit is not expressed in the submucosal plexus in the
ileum and colon .................................................................................. 88

Figure 3.3. Co-localization of a1E- and SP-ir in nerve fibers in the LMMP in the
colon, stomach and ileum ...................................................................... 90

Figure 3.4. Localization of a1E-ir with calbindin-ir and calretinin-ir in the LMMP in
the colon and duodenum ....................................................................... 92

Figure 3.5. Localization of a1E and 5-HT-ir in the LMMP in the small intestine.93

Figure 3.6. Co-localization of a1E-ir (red fluorescence) and VlP-ir (green
fluorescence) in the LMMP in the ileum and stomach .................................. 95

Figure 3.7. Confocal photomicrographs showing co-Iocalization of a1E-ir (red
fluorescence) and NOS-ir (green) in the myenteric plexus ........................... 97

Figure 3.8. Mean number of neurons per ganglion .................................... 99

Figure 3.9. Co-Iocalization of a1E-ir (red fluorescence) with SP-, VIP- and N03-
ir (green fluorescence) in nerve fibers supplying the circular muscle layer ...... 100

Figure 3.10. Co—localization of a1E-ir (red fluorescence) with TH-ir (green) in the

myenteric plexus ................................................................................ 101

Figure 3.11. The density of neuronal cell bodies co-Iocalizing (11 E and TH....103

ix

Figure 3.12. Distribution of a1E (red fluorescence) with calbindin, SP, calretinin,
and VIP (green fluorescence) in dissociated myenteric neurons in the guinea pig
ileum ............................................................................................... 104

Figure 3.13. Co-localization of a1E-ir with NOS, synaptophysin, SP with
synaptophysin, and a1E-ir with pan-cadherin in the guinea pig ileal whole mount

(WM) LMMP and myenteric neurons in culture ........................................ 106
Chapter 4.

Figure 4.1. CdClz inhibits action potentials in AH neurons ........................ 133
Figure 4.2. CdCIz does not affect action potentials in S-type neurons ......... 134

Figure 4.3. NiCIz inhibits the action potential in AH neurons ..................... 135

Figure 4.4. Action potentials in S-type neurons are not sensitive to NiClz....136

Figure 4.5. NiClz, SNX-482 and w—CTX inhibit the action potential in

AH neurons .................................................................................... 137
Figure 4.6. The late afterhyperpolarization (late AHP) in AH neurons is inhibited
by NiCIz ......................................................................................... 138
Figure 4.7. Effect of CCICIz on sEPSPs in AH neurons ............................ 139
Figure 4.8. NiClz (50 pM) did not inhibit sEPSPs ................................... 140
Figure 4.9. Sensitivity of sEPSPs to w-conotoxin (w-CTX) (100 pM) ......... 141

Figure 4.10. Amplitude of fEPSPs after treatment with CdCI2 and during time
controls ........................................................................................ 142

Figure 4.11. NICIz does not inhibit fEPSPs in cholinergic/purinergic S-type
neurons ....................................................................................... 143

Figure 4.12. SNX-482 does not inhibit fEPSPs in cholinergic/purinergic S-type
neurons ....................................................................................... 144

Figure 4.13. NiClz and w-CTX inhibit fEPSPs in purely cholinergic S-type
neurons ....................................................................................... 145

Fig 4.14. Effect of SNX-482 and w-CTX on purely cholinergic fEPSPs ..... 146

Chapter 5.

Figure 5.1. Representative traces showing NANC responses after transmural

electrical field stimulation of the guinea pig LMMP in vitro ........................ 170
Figure 5.2. Concentration-response curves for the area under the curve (AUC)
of relaxation responses caused by EF S .............................................. 171
Figure 5.3. LMMP contractions in the absence of histamine .................... 172

Figure 5.4. Effects of NiCIz, NLA, and apamin on LMMP contractions ....... 173

Figure 5.5. Effect of NiClz, and NiClz + NLA on inhibitory junction potentials

(IJPs) evoked in guinea pig smooth muscle cells .................................. 176
Chapter 6.

Fig 6.1. Peristalsis video imaging ....................................................... 202
Figure 6.2. EP50 pressure-response curve and representative traces ......... 203
Figure 6.3. Time controls showing stable peristaltic motor patterns ........... 205

Figure 6.4. Effect of w—conotoxin (w-CTX), MCI; and CdCIz on the AUC
expressed as percent change ............................................................ 206

Figure 6.5. Effect of MCI; and NLA on (A) frequency, (3) AUC (C) fluid volume
expelled .............................................................................................. 207

Figure 6.6. Effect of NLA and NiCl2 on (A) frequency, (B) AUC and (C) fluid
volume expelled ............................................................................. 209

Figure 6.7. The peristaltic motor pattern changes with NiCl2, and NiCI2

+ w-CTX treatment ......................................................................... 211
Figure 6.8. Effect of w-CTX alone, and w-CTX with NiClz, on peristalsis
parameters ................................................................................... 21 3
Chapter 7.

Figure 7.1. Proposed Model for Inhibitory Neuromuscular transmission...230

Figure 7.2. Schematic of the overall effect of NiClz on the peristaltic
Reflex ......................................................................................... 232

xi

LIST OF ABBREVIATIONS

B-nicotinomide adenine dinucleotide phosphate

Acetylcholine

After hyperpolarization

Area under the curve

Autonomic nervous system
Cadmium Chloride

Calbindin

Calretinin

Central nervous system

Chloride

Choline acetyltransferase
Endoplasmic reticulum

Enteric nervous system

Excitatory junction potential

Fast excitatory post synaptic potential
Functional Gastrointestinal disorder
Gamma-aminobutyric acid
Gastrointestinal

Immunoreactivity

Inhibitory junction potential

Interstitial cells of Cajal

 

xii

B-NAD
ACh
AHP
AUC
ANS
CdClz
Calb
Calret
CNS
Cl‘
ChAT
ER
ENS
EJP
fEPSP
FGID
GABA

GI

IJP

ICC

lntrinisic primary afferent neurons
Irritable bowel syndrome

Longitudinal muscle myenteric plexus
Migrating myoelectric complex
Neurokinin

Neuropeptide Y

Neurotransmitter

Nickel Chloride

Nicotinic acetylcholine receptors
Nitric oxide synthase

Nitric oxide

Nitro-L-arginine

Non adrenergic non cholinergic
Norepinephrine

Omega-agatoxin

Omega-conotoxin

Phosphate buffered saline

Potassium
pyridoxal-phosphate-6—azophenyl-2',4'-disulfonate
Serotoninl5-Hydroxytryptamine

Slow excitatory post synaptic potential
Sodium

Substance P

xiii

IPANs
IBS
LMMP
MMC
NK
NPY
NT
NiCl2
nAChRs
NOS
NO
NLA
NANC
NE
w-ATX
w-CTX
PBS
K“
(PPADS)
5-HT
sEPSP
Na+

SP

Tertiary plexus

Tetrodotoxin

Tyrosine hydroxylase
Vasoactive intestinal peptide

Water

xiv

tp
TI'X
TH
VIP

H20

CHAPTER 1 GENERAL INTRODUCTION

THE NERVOUS SYSTEM

The nervous system is composed of the central nervous system (CNS)
which includes the brain and spinal cord, and the peripheral nervous system
which comprises peripheral nerves, receptors, effectors and ganglia that exist
between the CNS and the periphery. In response to changes in the external
environment or the internal milieu, the brain can detect, interpret and respond to
these changes. This is achieved by the transduction of sensory information in the
form of electrical impulses from the periphery to the CNS that are then
transmitted via neurotransmitters to muscles, organs and glands. It is this
capability of nerve cells to wire themselves into a network and create emergent
outputs that are not simply predicted by the inputs, that is testament to the
exquisite complexity of the nervous system. The British physiologist Sir Charles
Sherrington referred to this elegance of neural control when he famously
described the integrative function of the nervous system. That is, the ability of the
nervous system to effect appropriate responses upon receiving different types of
information is due to its functional interconnectivity and sensitivity. Towards this
end, the manifestation of plasticity within neural circuits as a consequence of
sensory inputs or motor outputs is an inherent property of our continually
changing nervous system that allows us to adapt our behavior, acquire new
tasks, remember past events and recognize objects. In this regard, the concept
of how synaptic transmission shapes the connectivity and functions of neural
circuits is crucial to understanding fundamental mechanisms of nervous system

function.

STRUCTURE AND FUNCTION OF A NEURON

The great neuroanatomist Santiago Ramon y Cajal is most famous for
being the primary proponent and developer of the Neuron Doctrine which states
that “a neuron is an anatomically and functionally distinct cellular unit that arises
through differentiation of a precursor neuroblast cell” (Guillery, 2005; Glickstein,
2006). The nerve cell, or neuron, is the structural and functional unit of the
nervous system. Although several anatomical types of neurons exist, for
example, pyramidal, granule, Purkinje and myenteric neurons, they all have
several common features. Each neuron has a soma or cell body that contains a
single nucleus which contains the genetic information for protein synthesis. The
cell body is responsible for integration of incoming information. Many cellular
organelles such as mitochondria, ribosomes, endoplasmic reticulum, the Golgi
complex, Iysosomes, and cytoskeletal proteins, reside within neurons (Shepherd,
1994). Connected to the cell body are dendrites which collect afferent information
and serve to increase the surface area via which neurons can communicate. The
axon begins at the soma and extends to surrounding and distant nerve cells.
Their function is neurotransmission through action potentials that encode
signaling information. Neurons can be differentiated according to their
configuration (multipolar or unipolar) and function (sensory, interneurons and
motorneurons). Sensory neurons have typically unipolar processes, while
interneurons and motorneurons have multipolar processes. The classical
definition of a sensory neuron is a neuron that transmits information into the

CNS, and therefore has an afferent modality. Efferent (motor) neurons transmit

information to effector cells, such as muscle cells within the gastrointestinal (GI)
tract. The function of interneurons is integration of information in the
communication pathway between sensory and motorneurons. Neurons therefore
are functionally distinct entities that have specific roles that depend on their

intrinsic properties (Jessell & Kandel, 1993).

NEUROTRANSMITTERS, NEUROPEPTIDES AND SYNAPTIC
TRANSMISSION

There has been an increasing and active interest in the various
chemicals utilized by neurons. Advances in this area have led to an
understanding of their structure, biochemical and pharmacological
characteristics, functions, and how they serve as diffusible chemical messengers
between neurons. Several benchmarks need to be met before a chemical is
considered to be a neurotransmitter: 1) it must be synthesized and released from
neurons, 2) it must act at the postsynaptic cell to produce specific changes in that
cell, such as changes in input resistance, 3) its action should be blocked by
known antagonists, and 4) there must be a mechanism through which its action
is terminated (Deutch & Roth, 1999). Neurotransmitters are subdivided into the

classical transmitters and neuropeptides.

Classical neurotransmitters

The classical neurotransmitters are small, low molecular weight
chemicals that are produced in nerve terminals. Specific enzymes catalyze the
biosynthesis of neurotransmitters before they are packaged into synaptic vesicles
by a vesicular transporter. Acetylcholine, the biogenic amines (norepinephrine,
epinephrine, dopamine, serotonin, histamine), some amino acids (glutamate and
aspartate) and the ubiquitous CNS neurotransmitter gamma-aminobutyric acid
(GABA) are all classical neurotransmitters (Amara & Arriza, 1993; Amara &
Kuhar, 1993). Although neurotransmitter distribution is heterogeneous, specific
neurotransmitters modulate activity in different regions within the CNS and
peripheral nervous system. The basic stages in classical chemical
neurotransmission are: a) synaptic vesicle (~ 50 nm in diameter) synthesis and
docking of vesicle loaded with neurotransmitters at the active zone; b) action
potential initiation at the axon hillock and invasion of the synaptic terminal by the
arrival of the depolarizing action potential; 0) calcium entry though voltage-gated
calcium channels; d) fusion of vesicles with the plasma membrane and release of
neurotransmitter into the synaptic cleft; e) binding of neurotransmitters to specific
receptors; f) transduction of the signal resulting in a postsynaptic response; 9)
termination of neurotransmission by active reuptake of the transmitter at
presynaptic nerve terminals or metabolic inactivation by degradative enzymes in

the synaptic cleft (Jessell & Kandel, 1993; Kelly, 1993).

Neuropeptides

Neuropeptide synthesis is energetically more demanding than
small-molecule transmitter synthesis. Whereas the small molecule
neurotransmitters are synthesized in neuronal nerve terminals, peptide
transmitters are synthesized in the neuron cell body. The messenger RNA
(mRNA) coding for the neuropeptide is translated into a peptide precursor on
ribosomes in the rough endoplasmic reticulum (ER) within the cell body. The pre-
propeptide is then transported to the Golgi complex where it is packaged into
large dense-core vesicles (~ 75-150 nm in diameter). These vesicles are then
transported by fast axonal transport from the cell body to the nerve terminal.
lmportantly, neuropeptides can undergo post-translational modifications including
glycosylation, methylation and sulfation. This has implications for the stability of
the mature peptide. Further, large-dense core vesicles do not always undergo
exocytosis at the active zones, but can release neuropeptides at extra-synaptic
regions. Nonetheless, calcium influx into the nerve terminal is a critical
determinant of neuropeptide release. To this end, the synaptic machinery
necessary for peptide transmitter release is highly sensitive to calcium, more
than that of the small molecule transmitters. Peptide transmitters therefore do not
require large increases in intracellular calcium concentrations for exocytosis

(Jessell & Kandel, 1993; Kelly, 1993; Goda & Davis, 2003).

THE AUTONOMIC NERVOUS SYSTEM

The autonomic nervous system (ANS) is the division of the
peripheral nervous system that is responsible for involuntary sensory and motor
control of visceral organs. The function of autonomic nerves is regulated by
networks of nuclei that are located in the brainstem, hypothalamus and forebrain
(cerebral cortex, basal ganglia, Iimbic system, thalamus and olfactory bulbs and
tracts). These brain regions generally do not impart conscious sensation from
visceral reflexes (Langley, 1922). The ANS is responsible for the co-ordination of
key survival functions, such as breathing, heart-rate, digestion, and urogenital
regulation. The ANS has three divisions: sympathetic, parasympathetic, and
enteric (Richerson, 2003). Communication between the CNS and the
sympathetic and parasympathetic pathways occurs through preganglionic
neurons that are located in nuclei in the brainstem and spinal cord, and
postganglionic neurons found in peripheral ganglia that receive synaptic input
from preganglionic neurons. The cell bodies of sympathetic preganglionic
neurons are found in the interrnediolateral cell column at levels T1 through L3 in
the thoracic and lumbar regions respectively of the spinal cord. Axons from these
preganglionic cell bodies exit the spinal cord through the ventral root and enter
the white myelinated rami. They then synapse within a sympathetic chain
(paravertebral) ganglion, or may exit that paravertebral ganglion to synapse on
postganglionic sympathetic neurons in prevertebral ganglia (celiac, superior
mesenteric, and inferior mesenteric). Parasympathetic preganglionic neuronal

cell bodies are found in the craniosacral division of the autonomic nervous

system. Preganglionic axons travel in the vagus, oculomotor, facial and
glossopharyngeal cranial nerves and synapse with postganglionic
parasympathetic neurons located mostly within target organs. Both sympathetic
and parasympathetic preganglionic neurons use acetylcholine (ACh) as the
transmitter. While postganglionic parasympathetic neurons also release ACh on
to final target cells, most postganglionic sympathetic neurons release primarily
norepinephrine (NE) onto the viscera. The sympathetic and parasympathetic

divisions of the ANS produce opposite effects on the viscera (Richerson, 2003)

INNERVATION OF THE GASTROINTESTINAL TRACT
Extrinsic innervation of the ENS

Most enteric neurons are not directly innervated by the CNS (Galligan,
2002b). This salient feature of the GI tract implies that the gut must manifest this
independence from CNS input via intact intrinsic neural networks. The small
intestine and the proximal region of the large intestine are less dependent on
sympathetic and parasympathetic innervation than the esophagus, stomach and
distal colon (Goyal & Hirano, 1996). The ENS nonetheless does receive
sympathetic, parasympathetic, extrinsic vagal and spinal neuronal input (Grundy
et al., 2006). Parasympathetic innervation of the gut occurs via the vagus and
pelvic nerves. The myenteric plexus receives the majority of vagal efferent input,
and primary afferent neurons with cell bodies in nodose ganglia branch
throughout the plexus forming synapses with enteric neurons (Kirchgessner &

Gershon, 1989; Holst et al., 1997). Cell bodies of spinal afferent neurons reside

in dorsal root ganglia and their nerve endings mainly innervate the large
intestine. Spinal afferents relay nociceptive information about the GI tract to the
CNS (Cervero, 1994). These extrinsic sensory nerves, taken together, are
important determinants of the physiological status of the gut, and do so on a
moment to moment basis. Parasympathetic innervation of specific regions in the
GI tract could mean that activation of nicotinic acetylcholine receptors (nAChRs)
by nicotine needs to be strong in order for the reflex pattern of emesis and
defecation to occur. Sympathetic innervation of the gut wall occurs though
postganglionic neurons located with prevertebral ganglia. Tyrosine hydroxylase
(TH) is the rate-limiting enzyme for the synthesis of catecholamines including NE
(Grima et al., 1985). Based on the presence of TH in sympathetic nerve endings,
three subtypes of branching, varicose sympathetic axons exist that supply the
ENS: 1) those that target blood vessels contain TH and neuropeptide Y (NPY);
2) those that provide input to the submucosa contain TH and somatostatin; and,
3) those supplying myenteric neurons contain only TH (Macrae et al., 1986). In
fact, within the myenteric plexus, NE acting at az-adrenoreceptors blocks
neurotransmitter release from ACh-containing myenteric neurons, leading to an
inhibition of motility (Nishi & North, 1973b; Hirst & McKirdy, 1974; Frigo et al.,
1984). It has been suggested that the greater innervation of specific regions of
the ENS may be because of an override system that the CNS has put into place

to be able to control local gut motility.

Intrinsic innervation: the ENS

The mammalian small intestine contains approximately 100 million
neurons, about as many neurons as that found in the spinal cord (Gershon,
1997). The ENS (Fig. 1.1) consists of two ganglionated plexuses: the myenteric
plexus which is found in between the longitudinal and circular smooth muscle,
and which innervates the GI smooth muscle, and the submucosal plexus located
betWeen the circular muscle and the mucosa, and which regulates neuroimmune
interactions, local blood flow, secretion and absorption across the GI epithelium
(Kunze & Furness, 1999). The myenteric and submucosal plexuses are
connected by numerous fiber bundles that facilitate secretomotor and motility
reflexes (Furness et al., 1990). Within each plexus, the ganglia are connected via
interganglionic fiber tracts, and these two nerve plexuses therefore provide the
basis for all neural reflex activity in the GI tract. These reflexes are mediated by
three classes of enteric neurons: intrinsic primary afferent neurons (lPANs)
lsensory neurons, interneurons and motor neurons.

Between 1895 and 1899, the Russian histologist AS Dogiel conducted
extensive anatomical experiments in which he traced and characterized the
morphologies of human and guinea pig enteric neurons (Table 1). He found three
distinct subtypes, now known as Dogiel types I, II and III. Dogiel type I neurons
(Fig. 1.2) are flat, stellate cells with lamellar dendrites, are unipolar, and with a
cell body diameter between 13 and 35 pm in length and 9 and 22 pm in width.
Dogiel type II neurons are larger in size (from 22-47 pM) and have round/oval cell

bodies (Brehmer et al., 1999). A prominent feature of IPANs is that they are

10

Myenteric lnterganglionic
ganglion fiber tract

  
  
 

Submucosal

Circular ganglion

muscle

     
 

Longitudinal
muscle
Submucosal
_///- artery
200 pm

Figure. 1.1. The enteric nervous system (ENS). The mucosa with villi
protrudes towards the intestinal lumen. There are two ganglionated plexuses: the
myenteric plexus located between the longitudinal and circular muscle layers that
controls motility, and the submucosal plexus located between the mucosa and
the circular muscle. The submucosal plexus controls blood flow of arterioles
across the epithelial cell layer, as well as secretion and absorption (Furness,

2006a)

11

Table 1.1. Descriptions of enteric neuronal morphologies as described by

Dogiel (after Brehmer et al., 1999)

 

Types and numbers of
processes

Dendrites

Axonal course

 

I 4-20 dendrites, 1 axon

Branch and end within the
ganglion of origin. Short, thick,

flat, wih varicosities, lamellar.

Through neighboring ganglia,
with collaterals sporadically to

the muculature

 

ll 1-16 dendrites, 1 axon

Leave ganglion of origin.
Structural resemblance to
axons. Ramify into long, thin,

smoothly contoured branches.

To other ganglia

 

 

lll 2-10 dendrites, 1 axon

 

Ramify and end within

ganglion of origin.

 

Through other ganglia

 

12

 

Dogiel type I

 

Dogiel type II

Figure. 1.2. Morphological characteristics of enteric neurons. (A) Dogiel
type I neurons are flat with short lamellar dendrites and a single long axon.
These neurons are the interneurons and motorneurons. (B) Dogiel type II
neurons have smooth surfaces with several dendrites and multipolar axonal

processes. These neurons are the IPANs (Wood, 2006a).

multipolar. In the guinea pig small intestine, IPANs constitute 26% of the total
number of myenteric neurons and 11% of submucosal neurons (Furness et al.,
2004). Dogiel type III neurons are filamentous neurons with long branched

processes (Lomax et al., 1999; Furness, 2006a).

lPANs

IPANS have Dogiel ll morphology (Kirchgessner & Gershon, 1988;
Furness et al., 1998; Pan & Gershon, 2000). This has generated some
controversy since lPANs do not actually convey any conscious sensations from
the intestine and are therefore not really sensory afferent neurons (Wood,
2006a). However, within the context of the intestine, the definition of IPANs are:
sensory neurons in the wall of the gut that transduce mechanical or chemical
stimuli into electrical activity within .a local reflex arc (Furness ef al., 2004;
Brookes & Costa, 2006). The term IPANs will be used during the course of my
dissertation. Most IPANs synapse with other IPANs to form positive feedfonrvard
loops. This means that excitation within these loops or networks can build rapidly
so that simultaneous activation of entire neural networks around the intestinal
circumference is elicited. These bursts of excitation then drive the entire reflex
circuitry towards threshold leading to the subsequent activation of interneurons
and motorneurons that supply GI smooth muscle. The result is ascending
contraction or descending relaxation. The axons of IPANS project from the
myenteric and submucosal plexuses to the mucosal layer (Furness et al., 2004).

The mucosa also contains enterochromaffin (EC) cells that, when activated via

14

chemical or mechanical (Kunze et al., 1998) stimulation of the mucosa, release
5-hydroxytryptamine (5-HT) (Kirchgessner et al., 1992; Mawe et al., 2006) and
the other sensory mediators such as ATP (Bertrand, 2003). This indirect
activation of IPANs occurs through 5-HT acting at 5-HT3 (Bertrand et al., 2000)
and 5-HT1p (Kirchgessner et al., 1992) receptors on myenteric and submucosal
AH nerve terminals respectively, and ATP acting at P2X receptors (Furness et
al., 2004) elicit antidromic action potentials that propagate into the soma of the
IPAN. The IPAN then provides synaptic input to interneurons, which in turn
activate motor neurons to produce an excitation of GI smooth muscle oral to and
an inhibition anal to the stimulus site, along distinctly polarized neural pathways
(Galligan, 2002b). However, it should be remembered that the mucosal
processes of lPANs are also activated directly, by chemical stimuli applied to the
mucosa (Kunze et al., 1995; Brookes & Costa, 2006). According to Brookes and
Costa (2006), a number of subsets of IPANs exist: 1) submucosal IPANs that
respond to mechanical stimulation of the mucosa; 2) myenteric IPANs that
respond to chemical stimulation of the mucosa; and 3) myenteric lPANs that are
sensitive to tension along the gut wall as they posses mechanosensitive ion
channels (Kunze et al., 1998; Kunze et al., 2000). Both myenteric and
submucosal lPANs are neurochemically coded by the calcium-binding protein,
calbindin, and this is the best marker known for lPANs (Pompolo & Furness,

198 8).

15

Myenteric interneurons

The plasticity of the enteric neural circuitry is reflected by the
presence of interneurons that are organized in a chain-like fashion along
polarized pathways. All interneurons have Dogiel type I morphology (Furness et
al., 1993). In the guinea pig ileum, there are four classes of interneurons: a single
class of ascending interneurons and three classes of descending interneurons
(Brookes, 2001). lnterneurons communicate via fast synaptic transmission
(Galligan, 2002b). Ascending interneurons comprise 5 % of all myenteric neurons
and stain positive for choline acetyltransferase (ChAT)/Calretinin/Substance P
(SP)/enkephalin (Brookes et al., 1997). Their primary neurotransmitter is
acetylcholine (ACh). These neurons provide synaptic input to other ascending
interneurons and to excitatory circular and longitudinal muscle motorneurons
(Pompolo & Furness, 1993, 1995). Their functional importance is to allow rapid
spread of excitation and for the effective co-ordination of motor responses of the
gut smooth muscle.

In the guinea pig ileum, the first type of descending interneuron is
immunoreactive for ChAT and the peptide hormone somatostatin. This class (4
% of myenteric neurons) is believed to be involved in the migrating myoelectric
complex (MMC) (Furness, 2000). The second class of descending interneurons
(2%) includes those that participate in the secretomotor and motility reflex. These
neurons are immunoreactive for ChAT and 5-HT (at 5-HT3 receptors) and use
both ACh and 5-HT as their primary neurotransmitters. Because at least 90% of

the body’s 5-HT is localized within EC cells, this may represent a significant

16

source of the 5-HT that cause fast synaptic excitation within this interneuronal
pathway. The third group of descending interneurons (5%) are those involved in
the local reflex pathway. These neurons are neurochemically coded with
ChAT/nitric oxide synthase (NOS)/vasoactiVe intestinal peptide (VlP)/gastrin-
releasing peptide (GRP)/NPY, and their principal transmitters are ACh and ATP
(Furness, 2000; Brookes, 2001). Evidence indicates that ACh and ATP may be
the important transmitters released from nerve terminals of these neurons
(LePard et al., 1997). The heterogeneity of three classes of descending
interneurons may be to allow diverse communication patterns to take place, from
the spread of inhibition along long (~ 40 mm) distances of the gut, to co-
ordinating the MMC as well as being crucial mediators of relaxation (Brookes &

Costa, 2006).

Myenteric motorneurons

All enteric motorneurons have Dogiel Type I morphology. Excitatory
and inhibitory circular and longitudinal muscle motorneurons produce GI smooth
muscle. contraction and relaxation. In the guinea pig ileum, excitatory circular and
longitudinal muscle motorneurons constitute 12 % and 25 % of the total number
of myenteric neurons respectively. Excitatory circular muscle motorneurons have
the neurochemical phenotype ChAT/SP/enkephalin, while excitatory longitudinal
muscle motorneurons immunostain positive for ChAT/calretinin/SP (Brookes &
Costa, 1990). ACh and SP are the main neurotransmitters released from these

neurons. Inhibitory circular and longitudinal muscle motorneurons make up 16 %

l7

and 2 % respectively of the neurons in the myenteric plexus. These neurons
have the chemical phenotype NOSNIP/Pituitary adenylate cyclase-activating
peptide (PACAP)/gamma-aminobutyric acid (GABA) (Costa et al., 1986a;
Furness et al., 1987; Brookes & Costa, 1990). Circular muscle motorneurons are
important components that are crucial to peristalsis. Longitudinal muscle
motorneurons may be responsible for fine-tuning motility in a graded manner
(Brookes & Costa, 2006). Substantial evidence suggests that motorneurons in
each of the muscle layers are activated synchronously (Hennig et al., 1999;

Spencer et al., 2002).

ELECTROPHYSIOLOGICAL CHARACTERISTICS OF MYENTERIC
NEURONS

lPANs are electrophysiogically characterized as AH neurons (Nishi 8.
North, 19733; Hirst et al., 1974). The defining features of AH neurons are: a
resting membrane potential of approximately -70 mV, a prominent calcium
shoulder on the repolarizing phase of the action potential, the long and late
afterhyperpolarization (late AHP), and an absence of anodal-break excitation at
the offset of a hyperpolarizing pulse (Furness et al., 2004; Wood, 2006a). The
major component of the upstroke during the AH somal action potential
(approximately 75-110 mV in amplitude) is a tetrodotoxin (Tl‘X)-sensitive sodium
current, and a TTX-insensitive calcium current is most prominent on the falling
phase of the action potential (North, 1973; Hirst et al., 1985; Rugiero et al.,

2003). The longer-lasting calcium current is carried by N-type (a1B) calcium

18

channels and generates a calcium-shoulder during the falling phase of the action
potential (Rugiero et al., 2002). R-type calcium channels may also contribute to
this calcium conductance (Rugiero ef al., 2002). Calcium entry induces further
calcium release by activating ryanodine receptors on the endoplasmic reticulum
(Hillsley et al., 2000; Vogalis et al., 2001). The action potential is followed by a
characteristic long-lasting (2-30 3) afterhyperpolarization (AHP) mediated by the
amplified level of intracellular calcium that activates an intermediate-conductance
(lK) calcium-dependent potassium channel found only within the cell bodies
(Nishi & North, 1973a; Morita et al., 1982; Furness et al., 2003). Since the AHP
limits the action-potential firing rate within the cell bodies of AH neurons, these
neurons cannot fire trains of action potentials and therefore control the frequency
of sensory information output to interneurons and motorneurons (Hirst et al.,
1974). Furthermore, the hyperpolarization produced by the AHP triggers the
activation of a non-selective cationic current (In) that increases the excitability of
AH neurons (Galligan et al., 1990). If IPANs/AH-type neurons are sensory
neurons, then S-type neurons are the interneurons and motorneurons. In S-type
neurons, the AHP following the action potential is short, lasting 20-100 ms (Hirst
et al., 1974). Action potentials in these neurons are generated by the voltage-
dependent Na‘ and K‘ conductances in line with the classic Hodgkin and Huxley
model. The resting membrane potentials of S neurons are modulated by an

inwardly-rectifying potassium current (Kir) (Rugiero et al., 2002).

19

NEUROCHEMICAL CODING AND SYNAPTIC TRANSMISSION IN THE ENS
Functional classification of enteric neurons according to the
neurochemical markers they express and their axonal projections is well
established in the guinea pig (Brookes, 2001). As mentioned previously, ACh,
SP, NO, VIP, 5-HT, the calcium-binding proteins calbindin-D28K (calbindin) and
calretinin are all markers for specific subsets of enteric neurons (Costa et al.,
2000; Brookes, 2001; Quinson et al., 2001). Moreover, there are two types of
chemically-mediated synaptic mechanisms used by enteric neurons: fast and
slow neurotransmission (Bornstein et al., 1994; Costa et al., 1996; Furness,

2000)

Slow synaptic transmission in the ENS

Long-lasting changes in neuronal excitability results from
transmitter binding to a family of cell surface receptors called G-protein-coupled
receptors. These are heterotrimeric seven-transmembrane domain receptors with
on, B and y subunits. G-proteins employ two main systems to mediate their effects:
the cyclic AMP (CAMP), and phosphoinositol systems. A third system, the
arachidonic system, is also used by G-proteins, but for the purpose of my
dissertation, the arachidonic system will not be discussed here. The cAMP
system is activated when a ligand (eg. norepinephrine) binds to a receptor (eg. [3
adrenergic receptor) that is in turn linked to a Gs transducing protein. G0,s then
stimulates adenylate cyclase which then catalyzes the conversion of ATP into the

second messenger CAMP. CAMP then activates protein kinase A. The transducer

20

of the phosphoinositol pathway is G... Go,q activates the primary effector
phospholipase C which in turn cleaves phosphoinositol phosphate (PIPz) to yield
two second messengers, diacylglycerol (DAG) and inositol 1,4,5-triphosphate
(lP3). IP3 acts at ligand-gated IP3 receptors on the endoplasmic reticulum (ER) to
cause the release of calcium from these intracellular membrane stores. DAG
remains in the membrane where it activates protein kinase C (PKC) (Siegelbaum
et al., 2000). Slow synaptic excitation thus serves to modulate channel and
intracellular enzymes to produce a remarkable array of responses, from changing
the neural circuitry to altering synaptic transmission pathways. For example, ion
channels such as K+ channels can be phosphorylated, leading to a change in
their probability of opening. Once these channels are phosphorylated, they close,
causing a long-lasting increase in excitability that can be experimentally detected
as an increase in input resistance (Galligan, 1998; Galligan et al., 2000). This
particular mechanism is a hallmark of slow synaptic excitation in AH neurons.
Trains of high frequency electrical stimuli (5—20 Hz) elicit sEPSPs in AH neurons
(Morita & North, 1985). Slow synaptic excitation occurs via G-protein-coupled
receptors due to the long latency (> 50 ms) of the sEPSP (Galligan, 2002b).
sEPSPs recorded from AH neurons are mediated by ACh and SP that are co-
released from the same nerve endings (Galligan, 1999). ACh acting at M
muscarinic receptors, however, is not the major contributor to the sEPSP in AH
neurons because sEPSPs evoked in the majority of these neurons in the
presence of muscarinic receptor antagonists are unaffected (North et al., 1985).

SP acting at neurokinin-3 (NK3) receptors coupled to inositol phospholipid

21

hydrolysis and PKC is believed to be the principal mediator of slow excitatory
neurotransmission in the ENS (Guard et al., 1988; Bertrand & Galligan, 1995).
Forskolin, a direct activator of adenylate cyclase coupled-PKA, also mimics the
sEPSP implying that the PKA pathway may be also be involved in mediating the
sEPSP in IPANs (Bertrand & Galligan, 1995). NK3 receptors are located on
IPANs and on interneurons that receive synaptic input from lPANs (Holzer &
Holzer-Petsche, 2001). lPANs also receive slow synaptic input from other IPANs
and therefore form self-reinforcing networks (Kunze & Furness, 1999). SP also
acts at postsynaptic PKC-coupled NK1 receptors on excitatory and inhibitory
enteric motor neurons (Holzer & Holzer-Petsche, 2001). lPANs can therefore
directly mediate slow synaptic excitation of motor neurons (Holzer & Holzer-
Petsche, 2001; Galligan, 2002b). The sEPSP is due to: a) suppression of the
leak potassium conductance (Galligan, 1998), b) phosphorylation of calcium-
dependent potassium channels causing them to close (Bertrand & Galligan,
1995), and 0) increase in chloride conductance in about 40 % of myenteric AH
neurons (Bertrand & Galligan, 1994; Wood & Kirchgessner, 2004). Changes in
input resistance assume a biphasic mode, where the first phase of the increase
in input resistance is the closure of calcium-dependent lK channels, while
increased CI' conductance is responsible for the second phase of decreased
input resistance (Wood, 2006a). In order for the AH neuron to generate an action
potential, the sEPSP has to reach threshold. Once this occurs, calcium then
enters through N-type channels to trigger calcium-induced-calcium-release from

the ER (Rugiero et al., 2002). Calcium then activates the IK channel, increasing

22

its probability of opening. At the same time, protein phosphatase 28 (calcineurin)
is also activated by the released calcium and dephosphorylates the IK channel,
thus permitting it to re—open (Vogalis et al., 2004). The IK channel is then free to
mediate the late AHP that is the characteristic feature of AH neurons. What then
is the functional importance of sEPSPs in the ENS? Firstly, during a sEPSP,
excitatory inputs to that AH neuron will be potentiated. Second, intestinal motility
requires a sustained neural response to cause prolonged excitation or inhibition
in the effector systems (smooth muscle, secretory glands, blood vessels)
(Bertrand & Galligan, 1995; Wood & Kirchgessner, 2004), and the electrical
properties of AH neurons appear to adequately fit this description. Based on their
electrophysiological properties, IPANS/sensory neurons are classified as “AH”

neurons, and interneurons and motor neurons are defined as “S” neurons

Fast synaptic transmission in the ENS

A single focal stimulus applied to interganglionic connectives produces
fEPSPs in S neurons (Clerc et al., 1999; Alex et al., 2002; Galligan, 2002b).
fEPSPs recorded from S neurons are mediated by ACh acting at ligand-gated
pentameric nAChRs and ATP acting at trimeric P2X receptors (Galligan et al.,
2000). ACh is the principal excitatory neurotransmitter in the ENS (Nishi & North,
1973a; Zhou et al., 2002). nAChRs and P2X receptors are non-specific cation
channels. Enteric nAChRs are also calcium-permeable (Trouslard et al., 1993).
In 27% of S-type myenteric neurons with oral polarity, hexamethonium, a nAChR

antagonist completely inhibited the fEPSP (LePard et al., 1997; LePard &

23

Galligan, 1999). This indicated that ACh was the only transmitter in the
ascending pathway (Spencer et al., 2000). ATP is a co-transmitter with ACh in
approximately 67% of S neurons in selective descending pathways (LePard &
Galligan, 1999; Galligan et al., 2000). Further analysis revealed that 11% of 8-
type neurons with aboral polarity were blocked by hexamethonium and 5-HT3,
indicating that this subset of S-neuron uses both ACh and 5-HT for fast synaptic
communication. In fact, in the small intestine, 5-HT3 receptors are found in the
soma of AH and S-type neurons, where they mediate fastly desensitizing inward
currents (Zhou & Galligan, 1999; Galligan, 2002a). Taken together, these data
indicate that most myenteric neurons mediate fast excitatory synaptic
transmission predominantly by ACh and ATP. Interestingly, nAChRs have been
localized in the somatodendritic region (Torocsik et al., 1991) and nerve
terminals (McGehee et al., 1995) of myenteric interneurons and motorneurons
(Galligan, 1999, 2002a). According to Galligan (2002a), the paucity of recorded
5-HT3-mediated fEPSPs may be due to the small percentage of 5-HT-containing

S-type interneurons.

NEUROMUSCULAR TRANSMISSION IN THE ENS

Autonomic nerves release neurotransmitters en passage from
varicosities that occur at 5-15 pm intervals along axons (Blakeley & Cunnane,
1979; Blakeley et al., 1982). ACh, released from excitatory longitudinal and
circular muscle motor neurons, acts at presynaptic M2 muscarinic and

postjunctional M3 cholinergic receptors (Matsui et al., 2000; Eglen, 2001) and is

24

the major excitatory neurotransmitter responsible for contraction of GI smooth
muscle (Paton, 1.955; Paton & Zar, 1968; Bogeski et al., 2005). Cholinergic
excitatory junction potentials (EJPs) have also been recorded from guinea pig GI
smooth muscle (Burnstock & Holman, 1963). SP, acting at NK1 receptors located
on interstitial cells of Cajal (ICC) and on the smooth muscle (Sternini et al., 1995;
Southwell & Furness, 2001), and at NKZ receptors on the muscle (Maggi, 2000)
is an excitatory co-transmitter involved in these contractions (Holzer & Holzer-
Petsche, 2001; Lecci et al., 2002). NK1 receptors are localized to the soma of
NOS-containing inhibitory motor neurons (Portbury et al., 1996). Pharmacological
evidence later showed that tachykinins excite NK1 receptors on inhibitory motor
pathways within the ENS of the guinea-pig ileum, causing those inhibitory
neurons to release NO, thus depressing motor activity (Lecci et al., 1999; Saban
et al., 1999; Bian et al., 2000). nAChRs, however, are also present on the
somatodendritic and nerve terminal regions (Wonnacott, 1997) of longitudinal
and circular muscle motor neurons (Galligan, 1999; MacDermott et al., 1999;
Schneider et al., 2000). Since excitatory longitudinal and circular muscle motor
neurons contain both choline acetyltransferase (ChAT) and SP (Brookes, 2001),
ACh and SP may be released from the same nerve endings (Galligan, 1999).
Presynaptic nAChRs may therefore function as autoreceptors to facilitate SP
release, causing non-cholinergic GI smooth muscle contractions (Galligan, 1999;
MacDermott et al., 1999).

There appears to be an oral-aboral increase in inhibitory innervation to

GI smooth muscle. Burnstock and colleagues used sucrose-gap recordings with

25

transmural field stimulation to show that in the presence of the muscarinic
receptor antagonist atropine, and guanethidine which blocks adrenergic
neurotransmission, that 1 Hz stimuli pulses evoked transient hyperpolarizations
or inhibitory junction potentials (IJPs) from smooth muscle from guinea pig taenia
coli (Burnstock et al., 1963; Burnstock & Holman, 1963; Burnstock et al., 1964).
TTX completely blocked the UPS. In guinea pig, murine and human, increasing
the electrical stimuli of nerve fibers supplying the muscle layer causes UPS to
become biphasic, consisting of an initial large-amplitude response, followed by a
smaller-amplitude and slower second response (Wang et al., 2007). Relaxation
of GI smooth muscle is mediated by non-adrenergic non-cholinergic (NANC)
inhibitory enteric neurons (Furness, 2000). NO is the primary mediator of this
relaxation (Li & Rand, 1990; Sanders 8 Ward, 1992) but ATP and VIP also
contribute to inhibitory NANC responses (Makhlouf & Grider, 1993; Furness,
2000). The NO-synthesizing enzyme NOS, ATP and VIP co-Iocalize within
inhibitory circular and longitudinal muscle motor neurons and may therefore act
in concert, in parallel or in series to exert muscle relaxation (Costa et al., 1992;
Grider & Jin, 1993; Brookes, 2001). NO is synthesized on demand, diffuses into
smooth muscle cells and binds to soluble guanylate cyclase. Guanylate cyclase
catalyzes the conversion of guanosine-5'-triphosphate (GTP) to cyclic guanosine-
3',5'-monophosphate (cGMP) (Lincoln et al., 2001). If intracellular calcium levels
increase via calcium entry through voltage-gated L-type calcium channels or from
intracellular calcium stores via activation of inositol triphosphate (IP3) or

ryanodine receptors to cause excitation (contraction) of smooth muscle cells

26

(Sanders & Koh, 2006), then a decrease in intracellular calcium should produce
inhibition (relaxation) of the muscle. Elevated cytosolic cGMP lowers intracellular
calcium levels by activating a cGMP-dependent protein kinase (PKG) (Hofmann
et al., 2006). PKG phosphorylates large-conductance calcium-dependent
potassium (BK) channels (Robertson et al., 1993; Alioua et al., 1998), thus
hyperpolarizing the cell, reducing calcium influx via L-type calcium channels and
relaxing GI smooth muscle (Sausbier et al., 2000; Geiselhoringer et al., 2004).
ATP, acts at P2Y1 receptors on GI smooth muscle to produce fast IJPs. In guinea
pig circular muscle, the fast lJPs are completely inhibited by the selective P2Y1
receptor antagonist MR82179 (Boyer et al., 1998). A recent study in mouse
colonic circular muscle showed that the purinergic co-enzyme B-nicotinamide
adenine dinucleotide (B-NAD) may also be a non-adrenergic non-cholinergic
(NANC) inhibitory neurotransmitter (Mutafova-Yambolieva et al., 2007).
Hyperpolarization responses after picospritzing B-NAD onto muscle cells
revealed that in the presence of B-NAD, MR32179 drastically inhibited those
responses. Those data were confirmed with calcium-imaging studies performed
in human embryonic kidney (HEK)-293 cells where calcium transients to B-NAD
were completely blocked by MRS2179. Therefore B-NAD mediates it effects on
GI smooth muscle via P2Y1 receptors (Mutafova-Yambolieva et al., 2007).
Moreover, electrical field stimulation of murine colonic strips maintained in vitro
showed that ATP and B-NAD are co-released, and that their release may be
mediated predominantly by N-type calcium channels (Mutafova-Yambolieva et

al., 2007; Smyth ef al., 2009).

27

VIP acts at Gs-coupled vasoactive—pituitary adenylate cyclase-
activating peptide (VPAC) receptors on lCCs and GI smooth muscle cells to
produce relaxation (F urness, 2000; Kurjak et al., 2001). VIP, via cyclic AMP, may
activate a calcium-dependent potassium channel to elicit this response

(Kawasaki et al., 1997; Bayguinov et al., 1999).

SMALL INTESTINAL MOTILITY PATTERNS
Peristalsis

Peristalsis is a polarized motor reflex that is responsible for moving
intestinal contents in an oral to anal direction (Trendelenburg, 2006; Gwynne &
Bornstein, 2007). Phasic changes in intraluminal pressure are due to
contractions and relaxations of GI smooth muscle (Huizinga et al., 2006). The GI
tract produces three important motor patterns. The first is non-propulsive
segmental contractions that mix and churn food; this enhances absorption of
essential dietary nutrients. The second type of motor patterns are those that are
fully propulsive contractions that propel food anally (Trendelenburg, 2006). This
latter form of peristalsis was initially described by Bayliss and Starling (1899) and
is referred to as the peristaltic reflex or “the law of the intestine”. Using small
intestinal segments in vitro and in vivo, Bayliss and Starling proved that
ascending excitation and descending inhibition occurs above and below a
physiological stimulus presented within the lumen of the gut, one that mimicked a
bolus of food (Bayliss & Starling, 1899). Those authors further showed that the

circular and longitudinal muscle layers contract and relax synchronously to propel

28

food anally. The muscle layers also exhibits regular and rhythmic contractions
called slow waves. Slow waves are depolarizing potentials that produce
oscillations of the membrane potential of the smooth muscle, and these
oscillations are close to threshold for calcium influx via L-type calcium channels
(Sanders, 2008; Huizinga & Lammers, 2009). Slow waves are generated by the
non-neuronal non-smooth muscle interstitial cells of Cajal, and are conducted to
the adjacent smooth muscle cells via gap-junctions (Farrugia, 1999, 2008).
Contraction and inhibition of the muscle occur through neurotransmitters
released from enteric nerves that innervate the smooth muscle. The peristaltic
reflex is mediated by both chemical stimulation of the mucosa (Bertrand et al.,
2000) or distention (Hennig et al., 1999). Intestinal segments isolated from the
guinea pig show consistent fully propagative peristaltic reflex patterns. This
neuraIIy-mediated pattern is completely abolished in the presence of the voltage-
gated sodium channel blocker tetrodotoxin (TTX), but myogenic rhythmic
patterns persist (Huizinga et al., 1998). Experiments done with intestinal
segments show that fluid-induced distention of the rat and mouse intestine in
vitro does not evoke the classic Trendelenburg peristaltic reflex (Seerden et al.,
2005). Instead, oscillatory contractions of GI smooth muscle cells in those
animals have been frequently demonstrated and appear to be under low neural
excitation, similar to the TTX-resistant contractions and relaxations observed in
the guinea pig intestine (Seerden et al., 2005). Therefore, peristaltic motor

patterns differ between species, and in rats and mice they are mainly due to the

29

high frequency of slow waves that manifest as the predominant motor patterns in
those animals (Huizinga et al., 1998).

The third electrical and motor pattern in the GI tract is the MMC that
occurs during the interdigestive (fasting) state in mammals (Galligan et al., 1989).
MMCs begin in the gastroduodenal region of the GI tract and slowly propagate
towards the terminal ileum as a slowly-moving wave complex (Szurszewski,
1969). This wave is created by rapid and intense contractions that sweep along
the gut. Humans and guinea pigs have approximately the same number of MMC
cycles that occur every 70-90 minutes (Furness, 2006a). There are four known
stages of the MMC: 1) a prolonged quiescent phase; 2) an irregular phase
consisting of increasing action potential frequency coinciding with an increase in
motilin synthesis and release from the duodenal mucosa; 3) a peak phase where
the MMC propagates; and 4) a brief period of declining and irregular activity
(Carlson et al., 1972). The functional significance of the MMC is the removal of
undigested food, bacteria, and residual intestinal secretions (Huizinga &

Lammers, 2009).

GI MOTILITY DISORDERS

The GI tract is a specialized hollow tube that consists of the oral
cavity, pharynx, esophagus, stomach, small and large intestine, and anus. The
function of those organs is to allow the sequential degradation of food by
enzymes into smaller molecules during the transit of food. In this way, the body

has access to vital minerals, nutrients, and water before they are expelled. This

30

goal is accomplished by the enteric nervous system (ENS), a collection of
neurons embedded in the gut wall, that can function independently of input from
the CNS (Gershon, 1981). Dysregulation of the ENS causes changes in motility,
secretion and absorption, and leads to motility disorders (Sharkey & Kroese,
2001; Furness, 2006b). Gl motility disorders are important areas for the health of
the United States and the rest of the world, and comprise about 40% of GI
problems for which patients seek health care (Camilleri et al., 2005). They
impose a heavy burden of illness, decreased quality of life, and decreased work
productivity on those who are affected. Disorders of the ENS produce motor,
secretory, and inflammatory and immunological disturbances of GI function.
These disorders are believed to be associated with the degeneration of
inflammatory cells or altered signaling of enteric neurons, and these changes are
manifested as disturbances in GI transit or functional obstruction (Goyal &
Hirano, 1996). Patients with FGle exhibit a greater altered motility response to
psychological or physiological stressors when compared to control subjects
(Drossman et al., 2002; Parkman et al., 2004). A major impact of GI disorders is
its global social and economic burden. Irritable bowel syndrome (IBS), for
example, has a prevalence rate of over 10% in known IBS cases and costs the
United States economy at least $25 billion a year (Camilleri, 2001). IBS is
probably the best known GI motility disorder. IBS is pathophysiologically
complex, and a hallmark of this disorder is an alteration of the sensory
innervation to the GI tract, causing visceral hypersensitivity in IBS patients.

Hypersensitivity of IPANs has been implicated in patients with IBS, such that

31

their reduced threshold of activation would heighten motility reflexes (Galligan,
2004). There is no current cure for this condition. Patients with IBS have either
increased or reduced gut motility, or alternating constipation and diarrhea in the
same patient, and painful cramps (Drossman, 1999; Thompson et al., 1999).
Used as an index of hypersensitivity, balloon bowel-distention experiments show
that IBS patients possess visceral hypersensitivity (visceral hyperalgesia),
suggesting that the neural sensory system in IBS patients may be dysregulated
(Ritchie, 1973; Mertz et al., 1995; Bernstein et al., 1996). IBS is believed to have
an immune component (Furness, 2006a). In small intestinal biopsies taken from
IBS patients with elevated mast cell counts, degranulating mast cells were
localized close to nerve terminals that supply the mucosa (Barbara et al., 2004).
The resulting inflammatory insult to the GI tract increases excitability of enteric
neurons (Sharkey & Kroese, 2001; Nurgali et al., 2007). The manifestation of
plasticity within neural circuits as a consequence of sensory inputs or motor acts
is therefore an inherent property of our continually changing central and
peripheral nervous systems. Moreover, most HIV/AIDS patients (50-93%) report
GI problems including dysphagia, diarrhea, nausea, vomiting, weight loss,
abdominal pain, anorectal disease, GI bleeding and tumors during the course of
their illness (Gazzard, 1988). . There is a positive correlation between a
progressively compromised immune system and an increased prevalence of
these GI symptoms (May et al., 1993) because the small intestine is a crucial site

in immune defense due to its large surface area. Understanding mechanisms of

32

GI disorders is therefore imperative due to their pervasiveness across many

disease-types.

VOLTAGE-GATED CALCIUM CHANNELS IN THE NERVOUS SYSTEM
Overview

Voltage-gated calcium channels (Fig. 1.3) are found in virtually every
excitable cell. Initial studies in the barnacle demonstrated properties of calcium
channels: 1) intracellular calcium chelators favor excitability, 2) permeant divalent
ions compete for entry into these channels, and 3) calcium channels are blocked
by divalent metal ions including CdClz, NiCI2 and Co” (Hagiwara & Byerly,
1981b, 19813; Hille, 2001). They are physiologically crucial because they
regulate a myriad of calcium-dependent intracellular events. Moreover, they
possess the unique ability of translating electrical signals into chemical signals
critical for neurotransmitter and hormone release, muscle contraction, gene
expression, neurite outgrowth, synaptogenesis, synaptic plasticity and cell death
(Berridge, 1998; Lee et al., 2002; Catterall et al., 2005). Structural,
pharmacological and biophysical (voltage-dependence and kinetics) studies have
described several distinct classes of neuronal voltage-gated calcium channels: a
T-type referred to as a low-threshold current whose channel opens at more
negative voltages (~ -64 mV; maximum activation ~-30 mV) with rapid
inactivation (< 100 ms) (Perez-Reyes, 2003; Talavera & Nilius, 2006), and the
high-threshold currents L-, N-, HQ and R-types whose channels open at more

depolarized voltages (~ -30 mV; maximum activation ~ 0 mV) (Bean, 1989; Tsien

33

et al., 1991; Hoffman ef al., 1994) and generally inactivate slower (> 100 ms)
(Yamakage & Namiki, 2002) (Table 2). Voltage-gated calcium channels are
pentameric with six transmembrane or helices. These channels have five
subunits: a1, 8, (126 and y. The pore-forming (between 85 and S6) subunit is a1,
and is between 190-250 kDa in size with approximately 2000 amino acid
residues. The a1 subunit also contains the voltage sensor (S4), channel gates,
and drug/toxin binding sites (Isom et al., 1994; Hofmann et al., 1999; Ertel et al.,
2000). Positive charges residing in the voltage sensor serves to activate the
channel complex. The short intracellular loop between domains I and II has a
GM and PKC phosphorylation site. Interaction of a1 with GM subunits reduces
the rate of activation of the channel by reducing the open channel probability
(Dolphin, 2003b). Phosphorylation of a1, however, increases the probability of
calcium channel opening. A calcium-calmodulin site is positioned on the C-
terminal tail. The large intracellular loop connecting domains II and III has the
SNARE-binding region referred to as a synprint site. SNARES constitute the
synaptic vesicle release machinery in neurons (Catterall, 2000). The synaptic
membrane proteins syntaxin, synaptosome—associated protein of 25 kDa
(SNAP25), and vesicle-associated membrane protein of 25 kDa (synaptobrevin)
form the SNARE complex (Hofmann et al., 1999). The [3 subunit is hydrophilic
and located intracellularly. It is mainly responsible for the proper insertion of the

0:1 subunit into the membrane. (3 subunits dramatically enhance the cell surface

34

 

 

 

 

 

 

 

 

 

Figure. 1.3. Model depicting the membrane structure and transmembrane
topology of a voltage-gated calcium channel. Arrangement of the channel in
the plasma membrane. The pore forming subunit is a1. The (125, B, and y
subunits are the accessory subunits. Sites for channel glycosylation are indicated
by “g”. Voltage-gated calcium channels are tetramers, each subunit contains six
transmembrane (TM) helices. Note the conduction pore-loop (green) between

TM 5 and TM 6, and the voltage-sensor S4 helices (Catterall & Few, 2008).

35

expression of a1 subunits and affect the kinetics and voltage-gating properties of
the channel (Dolphin, 2003a). The a26 subunits are conjoined; the a2 subunit is
found extracellularly and the y subunit is a transmembrane protein. 0:25 subunits
also increase cell membrane expression of a1, but has less pronounced effects
on channel kinetics (Davies et al., 2007). The y subunit has thus far not been
intensively studied. These subunits, however, appear to have differential effects
on a1 membrane expression, that is, y subunits may either have no effect on a1
expression, or may even reduce the level of expression (Catterall, 2000). LVA
channel inactivation is similar to the voltage-dependent process of Na+ channels.
However, HVA calcium channels show either slow (voltage-dependent) or fast
(calcium-ion dependent) inactivation. Both serve to prevent the overloading the
neuron with calcium as excessive calcium concentrations become cytotoxic.
Calcium sensitive inactivation of HVA calcium channels appears to be
calmodulin-mediated. HVA channels have calmodulin-binding domains on the
intracellular C-terminal tail that bind calmodulin with extremely high affinity
(Hofmann et al., 1999). Neurons therefore use these negative feedback
mechanisms for survival.

Synaptic transmission requires a dramatic elevation in intracellular
calcium ions entering through voltage-gated calcium channels at concentrations
of 50-100 uM (Matthews, 1996). These channels are believed to be the N- and
P/Q-type calcium channels and densely associate with calcium microdomains in
the nerve terminal (Hofmann et al., 1999). The calcium ions then interact with

the synaptic machinery. While synaptotagmin is the putative calcium sensor,

36

there have been reports where calcium-mediated exocytosis still occurs in

synaptotagmin-knockout mice (Shao et al., 1996; Sudhof & Rizo, 1996).

T-type calcium channels

T-type calcium channels produce a transient current with a tiny
conductance with rapid activation and inactivation. These channels are strongly
expressed in kidney and heart but have low expression in neural tissue (Perez-
Reyes, 2003). T-type channels are very sensitive to high concentrations of MCI;
(Vassort & Alvarez, 1994; Vassort et al., 2006). a1G, H, and l are the molecular

counterparts of T-type calcium channels.

L-type calcium channels

L-type calcium channels have a large single-channel conductance
and give rise to a long-lasting current. L-type channels are the principal channels
in smooth, skeletal and cardiac muscle, where they mediate calcium entry into
cells for excitation-contraction coupling in response to long or consistent
depolarizations (Hille, 2001). L-type channels are highly sensitive to
dihydropyridines (eg. nifedipine) and this is a distinctive property of these
channels (Bean, 1989). 0:18, C, D and F are the molecular counterparts of L-type

calcium channels (Bean, 1989).

37

N-type calcium channels

N-type calcium channels were first localized in chick sensory
neurons (Fox et al., 1987). Those authors found a calcium conductance that was
dihydropyridine-insensitive and intermediate to that of T- and L-type currents. N-
type (N for neuronal) channels are blocked by the peptide toxin w-conotoxin
GVIA (w—CTX). w-CTX is a toxin isolated from the Pacific cone-shelled snail
Conus geographus (McCleskey et al., 1987; Olivera et al., 1991). N-type calcium
channels are important mediators of neurotransmitter release at central and
peripheral synapses (Smith & Cunnane, 1996, 1997). a1B is the molecular

counterpart of N-type channels (Bean, 1989).

PIQ-type calcium channels

Around the time that N-type calcium channels were being
characterized with w-CTX, Rodolfo Llinas and colleagues (1991) were working
on Purkinje cell function in the cerebellum. One of the tools at their disposal was
a peptide toxin, w-agatoxin IVA (w-ATX) from the American funnel web spider
Agelenopsis aperta. They observed a very slowly inactivating calcium current
that was especially sensitive to w-ATX. They called this current the P (for
Purkinje)-type calcium current (Hillman et al., 1991). However, four years later, a
second w-ATX-sensitive calcium current was found in cerebellar granule cells
that displayed rapid inactivation kinetics (Randall & Tsien, 1995). This was

named the Q-type calcium current. The P/Q-currents are now combined because

38

they were shown to be homologous spliced variants (Bourinet et al., 1999). a1A

is the molecular equivalent of P/Q-type channels (Bean, 1989).

R-type calcium channels

Soong and others (1993) initially classified the R-type (R for resistant,
residual or remaining) calcium current as a LVA T-type channel. However, the R-
type calcium current is really a HVA current (Schneider et al., 1994) that may
activate at somewhat more negative voltages (~ -40 mV) and often inactivates
more rapidly than the other high-threshold currents (Soong et al., 1993;
Yamakage & Namiki, 2002). Unlike the other calcium currents, a complete
understanding of the molecular properties of the R-type calcium channel remains
elusive. The principal candidate for the ion-conducting subunit of R-type calcium
channels is the a1E subunit, encoded by the Cat/2.3 gene (Zhang ef al., 1993).
The a1E subunit was initially identified in the rabbit hippocampus, cerebral cortex
and corpus striatum (Niidome et al., 1992). Using different splice-variants of the
Cav2.3 gene, a1E was expressed in Xenopus oocytes and localized in adult rat
brain (Soong et al., 1993). Recombinant a1E was subsequently expressed in
HEK cells and Xenopus oocytes (Schneider et al., 1994). Soong et al. (1993) and
Schneider et al. (1994) both confirmed a1E sensitivity to blockade by NiClz. It
has further been shown that the toxin SNX-482 (Newcomb et al., 1998) and low
concentrations of NiCl2 selectively block R-type channels (Sochivko et al., 2002).
In situ hybridization and the reverse transcriptase-polymerase chain reaction

both revealed extensive a1E mRNA expression in the adult mouse brainstem,

39

forebrain and cerebellum (Williams et al., 1994). lmmunohistochemical
localization of a1E has been demonstrated in neurons within the rat caudate-
putamen, thalamus, hypothalamus, amygdala, somata and axons of Purkinje
cells (Yokoyama et al., 1995; Grabsch et al., 1999), cell bodies of CA1 pyramidal
cells, mitral cells of the olfactory bulb (Yokoyama et al., 1995), human Purkinje
and cerebellar granule cells (Yokoyama et al., 1995; Volsen et al., 1997), rat
cardiac myocytes and human distal tubules (Weiergraber et al., 2000). R-type
calcium channels comprise a significant portion of the calcium current recorded
from murine dentate granule cells, hippocampal CA1 and cortical neurons
(Sochivko et al., 2002). The study by Sochivko and others (2002) was conducted
in transgenic mice where the a1 E gene was knocked out. Currents recorded from
those CNS neurons showed that the R-type current was not eliminated but
reduced. R-type calcium channel knockout-mice also show deficits in fear
behavior (Lee et al., 2002), spatial memory as measured by the Morris water-
maze (Kubota et al., 2001), and pain perception (Saegusa et al., 2000). The a1E-
knockout mice in the study by Saegusa and others (2000) were not susceptible
to inflammatory pain. R-channels may mediate synaptic communication by those
neurons in descending anti-nociceptive pathways. Moreover, another study
showed that a1E mutant mice are hyperglycemic (Matsuda et al., 2001). These
data together highlight the physiological importance of the R-type channel in
various organ systems. Thus Tottene and others (2000) used antisense
oligonucleotides against the Cav2.3 gene. Those authors observed both sensitive

and insensitive R-type channel conductances in response to SNX-482 in

40

cerebellar granular neurons maintained in culture. In studies with P/Q-type (01A)
channel knockout mice, neurotransmitter release at the somatic neuromuscular
junction occurred via R-type channels, suggesting that these channels are
positioned close to presynaptic calcium sensors (Urbano et al., 2003). An
interaction between R-type calcium channels and the synaptic vesicle protein
synaptotagmin has been demonstrated previously (Wiser et al., 2002), further
strengthening the importance of this channel in short-term plasticity and synaptic

transmission.

Calcium channels in the ENS

Electrophysiological and immunohistochemical studies demonstrate
the presence of all calcium channels, with the exception of T-type channels, in
the ENS (Hirning et al., 1990; Kirchgessner & Liu, 1999; Smith et al., 2003). L-
type calcium channels, however, are not major contributors to the synaptic
excitation of enteric neurons (Reis et al., 2000; Reis et al., 2002). Further, the
inhibition of L-type channels on GI smooth muscle reduces or abolishes
spontaneous or evoked muscle movement (Horowitz et al., 1996; Farrugia,
1999). FIG type channels are expressed by myenteric neurons and contribute to
neurotransmitter release from enteric nerves (Reis et al., 2000; Smith et al.,
2003). However, weak immunoreactivity has often been associated with P/Q-type
calcium channel (Kirchgessner & Liu, 1999). Nonetheless, data from guinea pig
caecal submucosal (Cunningham ef al., 1998) and ileal myenteric neurons

indicate that P/Q-type channels do contribute to neurotransmitter release (Reis et

41

al., 2000; Reis et al., 2002). N-type calcium channels are ubiquitously expressed
in the guinea pig plexuses (Kirchgessner & Liu, 1999). In myenteric AH neurons,
N-type channels were shown to be the main contributor to the calcium shoulder
on the repolarization phase of the action potential (Furness et al., 1998; Vogalis
et al., 2002). R-type calcium currents have also been recorded in guinea pig
myenteric neuron maintained in culture (Bian et al., 2004).
R-type calcium channels in the ENS

R-type calcium channels are the principal subtype in the
somatodendritic region of myenteric neurons maintained in primary culture
(Rugiero et al., 2002; Bian et al., 2004), suggesting that R-type calcium channels
may contribute to synaptic transmission and regulation of membrane excitability
of myenteric neurons. Only two studies thus far have indicated functional roles
for R-type calcium channels in the ENS (Bian et al., 2004; Bian & Galligan,
2007). Bian and others (2004) used whole-cell voltage clamp recordings to show
that at least 50% of the calcium current present in myenteric neurons maintained
in primary culture is the R-type current. After using a strong depolarization (0 mV)
pulse to activate the R-type current, they applied N-, PlQ-, and L—type calcium
channel antagonists sequentially to block the calcium currents. However, 50% of
the remaining calcium current remained. This residual current was only blocked
after application of MCI; (50 uM). This observation was confirmed when NiCl2
and SNX-482 (0.1 pM) mutually occluded the calcium current (Bian et al., 2004).
That study suggested a somatodendritic role for R-type calcium channels in the

ENS. A further role for R-type channels has been postulated based on a

42

selective coupling with az-adrenoreceptors (Bian & Galligan, 2007).
Norepinephrine released from sympathetic postganglionic neurons inhibits GI
motility (Paton & Vizi, 1969; Stebbing et al., 2001). Presynaptically-located a2-
adrenoreceptors are activated by norepinephrine and inhibit neurotransmitter
release by inhibiting calcium influx through voltage-gated calcium channels
(Paton 8. Vizi, 1969; Stebbing et al., 2001). R-type calcium channels couple to
inhibition of az-adrenoreceptors via a pertussis toxin-sensitive G-protein (Bian &
Galligan, 2007). R-type channels may therefore be a target for norepinephrine

released from sympathetic nerve terminals (Bian & Galligan, 2007).

ION CHANNELS AS TARGETS FOR DISEASE TREATMENT IN THE ENS

The concept of how alterations in synaptic transmission shape the
connectivity and functions of enteric neural circuits has a key bearing on the
consideration of normal physiological versus pathophysiological changes in the
ENS. Voltage-gated calcium channels are critical mediators of synaptic
transmission. Excessive calcium entry, however, is also cytotoxic. Calcium influx
via calcium channels must therefore be precisely regulated because alterations
in calcium channel function would produce widespread cellular changes.
Perturbations in ion channel function affect diverse organ systems. In the
neuromuscular system, ion channel diseases results from gain-of—function
mutations to cause, amongst others, epilepsy, ataxia, myotonia and cardiac
arrhythmias (Cooper & Jan, 1999). In other systems, cystic fibrosis and Bartter

syndrome, for example, result from defects in chloride (CI‘) and potassium (K*)

43

channels that alter the transepithelial transport gradients of salt and water
(Cooper & Jan, 1999). In the ENS, the type 2 Cl' channel (ClC-2) is found in
enterocytes in the small intestine and colon (Lipecka et al., 2002). ClC-2
channels regulate intracellular volume and pH levels and activation of these
channels on the enterocytes increase intestinal fluid secretion (Cuppoletti ef al.,
2004). The highly selective ClC-2 agonist lubiprostone is now being used to
successfully treat patients with chronic constipation (Cuppoletti et al., 2004). A
recent electrophysiological study using whole-cell patch clamp recordings
demonstrated that R-type calcium channels are the predominant calcium channel
subtype expressed by myenteric neurons (Bian et al., 2004). This raised the
questions of whether a) R-type calcium channels are localized selectively within
subsets of myenteric neurons, b) the mechanisms by which these calcium
channels contribute to synaptic transmission at neuro-neuronal synapses and
neuromuscular junctions in the myenteric plexus and GI smooth muscle,
respectively, and c) the physiological significance of the large R-type currents
found by Bian et al. (2003). Voltage gated calcium channels are key
pharmacological targets for treating pain, stroke, ‘epilepsy, migraine and
hypertension (French & Zamponi, 2005). These observations support the
concept that ion disturbances in ion channel function can contribute to the
pathophysiology of some diseases, and that ion channels can also be the targets
for therapeutically useful drugs. These studies will provide new insights into the

contribution of R-type calcium channels to GI motility.

44

CHAPTER 2 HYPOTHESIS AND SPECIFIC AIMS

45

OVERALL HYPOTHESIS

Gastrointestinal (GI) disorders decrease the quality of life for affected
patients (Drossman et al., 1993; Stewart et al., 1999). These disorders are
classified as either structural (organic) or functional, but patients may
simultaneously suffer from both types. Inflammatory bowel disease (IBD) is an
organic GI disorder with clearly defined injury and inflammation that affect motility
(Stewart et al., 1999). When no pathological (anatomical, physiological or
biochemical) etiology is apparent, the patient is diagnosed as having a functional
GI disorder (FGID) (Longstreth ef al., 2006). There has been a surge in the
diagnosis of FGle including IBS in recent years. The pathophysiological basis
for FGIDs is unknown because FGID is due to an interaction of psychosocial
factors and altered gut physiology via the brain-gut axis. This is pertinent as the
GI tract has its own (enteric) nervous system (ENS) that allows the gut to
function quasi-autonomously (Furness, 2000). Synaptic transmission requires
voltage—gated calcium channels as mediators for a host of important biological
events including neurotransmitter release and muscle contraction (Cooper & Jan,
1999; Lorenzon & Beam, 2000). R-type calcium currents can be recorded from
guinea-pig myenteric neurons suggesting that pore-forming 01E calcium channel
subunits are expressed in the ENS (Rugiero et al., 2002; Bian et al., 2004). At
least 50% of the current in myenteric neurons is the R-type calcium current. This
suggests an important role for R-type channels as mediators of neurotransmitter
release or as transducers of other synaptic events in myenteric neurons, and

implicates this channel as a potential drug target in combating motility disorders.

46

I therefore tested the overall hypothesis that R-type calcium channels are
therefore functionally significant in controlling GI motility. This function was

explored further through the following specific aims.

Specific Aim 1. Myenteric neurons express R-type calcium currents (Bian et al.,
2004), but the specific subset of myenteric neuron that expresses the R-type
calcium channel is not known. These studies tested the hypothesis that R-type

calcium channels are localized to AH neurons and/or S-type neurons.

Specific Aim 2. Fast and slow synaptic neurotransmission may be selectively
modulated by calcium channel subtypes. These studies tested the hypothesis
that R-type or non-R-type calcium channels are respectively responsible for
substance P (SP) release as the mediator of the slow excitatory postsynaptic
potential (sEPSP) in AH neurons, and acetylcholine (ACh) release as the

mediator of the fast EPSP in S neurons.

Specific Aim 3. lmmunohistochemical data show that a1E is present in nerve
endings of excitatory motor neurons [SP-immunoreactivity (ir)], and descending
interneurons and inhibitory motor neurons [vasoactive intestinal peptide
(VlP)/nitric oxide synthase (NOS)-ir]. These studies tested the hypothesis that R-
type calcium channels mediate nerve-to-muscle transmission in the guinea pig

isolated ileum.

47

Specific Aim 4. My goal was to use an integrative approach to examine the
functional role of R-type channels in mediating the peristaltic reflex in the guinea
pig ileum. I used a modified method of Trendelenburg (Billbring et al., 1958;
Huizinga et al., 1998; Bian et al., 2003) to test the hypothesis that R-type calcium

channels are necessary or are sufficient for propulsive peristalsis in vitro.

48

CHAPTER 3 IMMUNOHISTOCHEMICAL LOCALIZATION OF R-TYPE

CALCIUM CHANNELS IN THE GUINEA PIG ENTERIC NERVOUS SYSTEM

49

ABSTRACT
R-type calcium currents can be recorded from guinea pig myenteric neurons
suggesting that a1E calcium channel subunits are expressed in the enteric
nervous system. However, the neuronal subset(s) expressing a1E subunits are
unknown. We used whole mounts from the stomach, intestine and colon of adult
guinea pigs, and ileal myenteric neurons maintained in primary culture to map
a1E-subunit expression. I also used antibodies against specific markers to
determine if a1E was expressed selectively in neuronal subsets. a1E-
immunoreactivity (ir) was found in colonic and gastric, but not intestinal myenteric
cell bodies in the longitudinal muscle-myenteric plexus (LMMP). a1E-ir never co-
localized with serotonin (a marker for descending interneurons). a1E-ir co—
localized with substance P (SP) in myenteric nerve fibers and in the muscle
layers throughout the gut. a1E-ir co-Iocalized with calretinin in cell bodies and
nerve fibers in primary cultured neurons, in some varicosities in the LMMP, and
with calbindin in cell bodies only in dissociated neurons. a1E-ir co-Iocalized with
vasoactive intestinal peptide (VIP) and nitric oxide synthase (NOS) in myenteric
nerve fibers and in muscle layers; only the colon contained a1E-VlP-ir neurons.
a1E-NOS-ir neurons were found in the gastric myenteric plexus. a1E-ir co-
localized with tyrosine hydroxylase in myenteric nerve fibers. a1E-ir was not
detected in the submucosal plexus. I concluded that a1E-ir is expressed by cell
bodies of myenteric intrinsic primary afferent neurons (calbindin-containing) and
in ascending interneurons (calretinin-containing), inhibitory motorneurons

(VIP/NOS-ir) and nerve endings of excitatory motorneurons (SP-ir). a1E-ir is

50

contained in sympathetic nerves supplying the myenteric but not the submucous
plexus. R-type calcium channels could participate in synaptic transmission in the
myenteric but not submucosal plexus. R-type calcium channels may also

participate in neuromuscular transmission.

51

INTRODUCTION

The enteric nervous system (ENS), a collection of neurons embedded in the gut
wall, can function independently of input from the central nervous system
(Gershon, 1981). The ENS consists of two ganglionated plexuses: the myenteric
plexus which provides innervation to the gastrointestinal smooth muscle, and the
submucosal plexus which regulates neuroimmune interactions, local blood flow,
secretion and absorption across the gastrointestinal epithelium (Kunze &
Furness, 1999). Changes in enteric neuronal function can therefore cause
gastrointestinal motility disorders.

Pharmacological and biophysical studies have described several distinct
classes of neuronal voltage-activated calcium channels: the high-threshold
currents L-, Ni, P/Q- and R-types, and a T-type referred to as a low-threshold
current (Hoffman et al., 1994). The function of R-type calcium channels in the
nervous system has not been firmly established. However, In studies using P/Q-
type channel knockout mice, neurotransmitter release at the somatic
neuromuscular junction occurred via R-type channels, suggesting that these
channels are positioned close to sites of synaptic transmission (Urbano et al.,
2003). In addition, an interaction between R-type calcium channels and the
synaptic vesicle protein synaptotagmin has been demonstrated previously (Wiser
ef al., 2002), further strengthening the importance of this channel in synaptic
transmission.

Neuronal calcium channels are composed of a pore-forming and voltage-

sensing ail-subunit in association with accessory subunits which serve to

52

modulate the properties of the channel complex (Isom et al., 1994; Ertel et al.,
2000). The pore forming subunit of R-type calcium channels is the a1E-subunit,
encoded by the Cav2.3 gene. The a1E subunit was initially identified in the rabbit
hippocampus, cerebral cortex and corpus striatum (Niidome et al., 1992).
Recombinant a1E-subunits were expressed in human embryonic kidney (HEK)
cells and Xenopus oocytes (Schneider et al., 1994) and these investigators
established that R-type calcium currents could be blocked selectively by low
concentrations (5 50 pM) of nickel chloride. It has further been shown that the
spider toxin SNX-482 (0.1 pM) can also selectively block R-type calcium currents
(Vajna et al., 1998; Tottene et al., 2000; Sochivko et al., 2002).

In situ hybridization, reverse transcriptase-polymerase chain reaction and
immunohistochemical studies have revealed that al E-subunits are expressed
throughout the central nervous system of mice (Williams et al., 1994), rats
(Yokoyama et al., 1995; Grabsch ef al., 1999) and humans (Yokoyama et al.,
1995; Volsen et al., 1997). a1E-subunits have also been localized to non-
neuronal tissues such as cardiac myocytes from rats and human distal tubules
from the kidney (Weiergraber et al., 2000). a1E-subunits and R-type calcium
channels are also expressed in sympathetic neurons (Murakami et al., 2007),
sensory neurons (Fang et al., 2007) and somatic motor neurons (Urbano et al.,
2003; Pardo et al., 2006) but localization of a1 E-subunits has not been studied in
other parts of the peripheral nervous system. Electrophysiological and
immunohistochemical studies demonstrate the presence of all calcium channels,

with the exception of T-type channels, in the ENS (Hirning et al., 1990;

53

Kirchgessner & Liu, 1999; Smith et al., 2003). L-type calcium channels, however,
are not major contributors to the synaptic excitation of enteric neurons (Reis et
al., 2000; Reis et al., 2002). Furthermore, the inhibition of L-type channels on GI
smooth muscle reduces or abolishes spontaneous or evoked muscle movement
(Horowitz et al., 1996; Farrugia, 1999). PM type channels are expressed by
I myenteric neurons and contribute to neurotransmitter release from enteric nerves
(Reis et al., 2000; Smith et al., 2003). R-type calcium channels may also
contribute to somatodendritic action potentials or neurotransmitter release from
myenteric nerve terminals (Rugiero et al., 2002; Bian et al., 2004). However,
those studies did not identify specific functional classes of myenteric neurons:
intrinsic primary afferent neurons (IPANs) or interneurons and motorneurons that
express the R-type channel.

Electrophysiological studies have established that R-type calcium
channels are the principal subtype in the somatodendritic region of guinea pig
ileal myenteric neurons maintained in primary culture (Bian et al., 2004). These
data suggest that R-type calcium channels may contribute to synaptic
transmission and regulation of membrane excitability of myenteric neurons.
Functional classification of enteric neurons according to the neurochemical
markers they express and their axonal projections is well established in the
guinea pig ‘gut (Brookes, 2001; Hansen, 2003). Substance P (SP), nitric oxide
(NO), vasoactive intestinal polypeptide (VIP), 5-hydroxytryptamine (5-HT), the
calcium-binding proteins calbindin and calretinin, and tyrosine hydroxylase (TH)

are all markers for subsets of enteric neurons.

54

The predominance of R-type calcium currents in myenteric neurons
suggests an important role for these calcium channels in enteric
neurotransmission (Bian et al., 2004). However, since the specific classes of
myenteric neurons have not been identified, it remains to be determined as to
whether R-type channels are localized to subsets of these neurons.
Furthermore, there have been no studies of the cellular distribution of 01E
calcium channel subunits in specific regions of the gut. The aim of the present
investigation was to therefore localize a1E subunits with neurochemical markers

for functional classes of neurons in the ENS of the guinea pig.

55

MATERIALS AND METHODS
Tissue collection
All animal use procedures were approved by the Institutional Animal Care
and Use Committee at Michigan State University. Adult male Hartley strain
guinea pigs (n = 4; 300-400 g, Bioport, Lansing, MI) were deeply anesthetized
with isoflurane (Abbott Laboratories, Chicago, IL), stunned by a blow to the back
of the head, and then killed by severing the carotid arteries. Segments of the
ileum, duodenum, gastric corpus, proximal and distal colon were dissected out

and placed in 0.01 M phosphate-buffered saline (PBS; pH 7.2).

Protein isolation and Western blotting

The specificity and sensitivity of the a1E antibody were verified by
Western blot. Longitudinal muscle-myenteric plexus (LMMP), submucosal and
circular muscle preparations from the guinea pig ileum were isolated on ice and
placed directly into liquid nitrogen. The rat brain and heart, and guinea pig heart
served as positive controls for (11E expression. Tissues were ground to a
powder in liquid nitrogen, and ice-cold homogenization buffer (125 mmol/L Tris
(pH 6.8), 4% SDS, 20% glycerol, 0.5 mmol/L phenylmethylsulfonyl fluoride, 1
mmol/L orthovanadate, 10 pg/ml aprotinin, 10 pg/ml leupeptin] added.
Homogenates were vortexed, sonicated briefly and transferred to a plastic
centrifuge tube and spun at 4 °C to pellet debris. The supernatant was separated
from the pellet and analyzed for protein concentration (BCA protein kit, Sigma-

Aldrich, St. Louis, MO). Total protein (50 pg) was boiled for 5 minutes with

56

standard 4:1 sample buffer. Proteins were separated on 1 mm-thick, 10% SDS
polyacrylamide gels using a Mini Bio-Rad® lll apparatus and all samples were
loaded into the gel in parallel. After transfer, the nitrocellulose membranes were
blocked at 4 °C overnight in 5% milk with 0.025% NaN3 and incubated with the
01E primary antibody (Table 1). Membranes were then rinsed three times in tris-
buffered saline (TBS) with Tween (0.1%) with a final rinse in TBS and incubated
with the appropriate horseradish peroxidase-linked secondary antibody (1:2000;
Cell Signaling Technology, Beverly, MA, USA) for 1 hour at 4 °C with continuous
shaking. ECL® reagents (Amersham Life Sciences, Arlington Heights, IL, USA)
were used to visualize bands. Finally, gels were stained with Gel Code Blue

(Pierce, Rockford, IL, USA) to verify protein loading.

Whole-mount preparations
Tissues were flushed of content, cut open along the mesenteric border,
stretched out and pinned tautly onto a silicone elastomer-Iined (Sylgard, Dow
Corning, Midland, MI) petri-dish, and fixed in 10% neutral-buffered formalin
(Sigma-Aldrich) overnight at 4 0C. Forrnalin-fixed tissues were cleared using 3
washes with dimethyl sulfoxide (J T Baker, Phillipsburg, NJ) at 10 minute

intervals. Tissues were then washed in 0.01 M PBS (3 X 10 min intervals) at

room temperature and stored in 0.01 M PBS at 4 0C.
Using fine scissors and forceps, the submucosal plexus, circular muscle

and LMMP layers were sequentially peeled away and trimmed into ~8 mm2

57

pieces. Tissues were placed into separate wells of a 24-well tissue culture plate

(Corning Glass Works, Corning, NY). Each well contained 0.5 ml of 0.01 M PBS.

Primary cultures of myenteric neurons
Newborn guinea pigs (~36 h in age, ~70 g in weight) were sacrificed by severing
the major neck blood vessels after deep isoflurane anesthesia. The ileum was
removed and placed in cold oxygenated (95% Oz; 5% 002) sterile Krebs’ solution
of the following composition (mM): NaCI, 117; KCI, 4.7; CaClz, 2.5; MgClz, 1.2;
NaHCOa 25; NaHPO4, 1.2; glucose, 11. The LMMP was removed from the entire
length of the ileum and cut into 5 mm segments. The dissected LMMP was
divided into four aliquots and placed into 1 ml Krebs’ solution containing 1600 U
of trypsin (Sigma-Aldrich) for 30 min at 37 °C. The tissues were then triturated 30
times and centrifuged at 3000 g for 5 min. The supernatant was discarded and
the pellet resuspended in 1 ml Krebs’ solution containing 2000 U of crab
hepatopancreas collagenase (Calbiochem, San Diego, CA) for 30 min at 37 °C.
The suspension was again triturated and centrifuged for 5 min at 3000 g. The
pellet was then resuspended in Eagle’s minimum essential medium containing
10% fetal calf serum, gentamicin (10 ug/ml), penicillin (100 U/ml), and
streptomycin (50 pg/ml) (Sigma-Aldrich). Cells were plated on coated poly-L-
lysine glass coverslips and grown in an incubator in a 5% CO; atmosphere for up
to 3 weeks. After 2 days in culture, cytosine arabinoside (10%) was added to the

culture medium to inhibit smooth muscle and fibroblast proliferation.

58

lmmunohistochemical procedures

All primary antibodies (see Table 1) were diluted in 0.01 M PBS/1% Triton
X-100. For myenteric plexus preparations, the a1E primary antibody was
incubated in combination with anti-calbindin, anti-calretinin, anti-5-HT, anti-SP,
anti-VIP, anti-NOS and anti-TH primary antibodies. With the exception of anti-5-
HT and anti-TH, the above antibodies were also applied to circular muscle
preparations from each of the five regions. Primary antibody incubations were
performed at 4 °C overnight in a humidified chamber. The tissues were then
washed in 0.01 M PBS (3 X 10 min), and incubated with the appropriate
secondary antibodies coupled to indocarbocyanine (CY3) or fluorescein
isothiocyanate (FITC) for 90 min at room temperature. For the purposes of
consistency during co-localization experiments, the ME primary antibody was
always localized with a CY3-conjugated secondary antibody while all other
primary antibodies were localized with a secondary antibody conjugated to F ITC.
Tissue sections were subsequently washed in 0.01 M PBS (3 X 10 min) and
mounted in buffered glycerol for confocal or conventional fluorescence
microscopy. All photomicrographs are confocal images unless otherwise

indicated.

Sequential dual-immunolocalization of a1E and nNOS. Since the a1E
and NOS antibodies were raised in rabbit, an established protocol was used to
study co-localization of these antigens (Franzusoff et al., 1991; Negoescu et al.,

1994). Tissue sections were first pretreated with 10% normal goat serum (Sigma-

59

Aldrich) (diluted in 0.01 M PBS/ 1% Triton X-100) for 1 h at room temperature to
block non-specific binding sites. The sections were then incubated with the NOS
primary antibody diluted 1:200 in 0.01 M PBS/ 1% Triton X-100, in a humidified
chamber at 4 °C overnight. Sections were then washed with 0.01 PBS (3 X 10
min). To prevent non-specific binding of the secondary antibody, the sections
were then incubated with 0.01 M PBS/ 1% Triton X-100 containing 10% normal
goat serum for 1 h at room temperature. Incubation with a goat anti-rabbit
secondary antibody conjugated to FITC was performed for 2 h at room
temperature. The tissues were then washed with 0.01 PBS (3 X 10 min). Non-
immune rabbit serum (Pierce Biotechnology, Rockford, IL) was then applied to
the tissue sections for 1 h at room temperature to saturate binding sites on the
first secondary antibody (goat anti-rabbit-FITC conjugate), thus preventing the
second primary antibody (a1 E) binding. Monovalent goat anti-rabbit
unconjugated fragment antigen binding (Fab) antibodies (Jackson
lmmunoresearch Laboratories, West Grove, PA) (1:50 diluted in 1% Triton X-
100) were then added to the tissue sections and incubated in a humidified
chamber at 4 °C overnight. These Fab antibody fragments bind to free antigen-
binding regions on the first primary antibody and to sites on the non-immune
rabbit-IgG molecules, thus obstructing the binding of the second secondary
antibody. The sections were then washed in 0.01 PBS (3 X 10 min) and blocked
with 0.01 M PBS/ 1% Triton X-100/10% normal goat serum for 1 h at room
temperature. Tissue sections were then incubated with the second primary

antibody (alE) diluted 1:400 in 0.01 M PBS/1% Triton X-100 in a humidified

60

chamber at 4 °C overnight, and subsequently washed in 0.01 PBS (3 X 10 min).
The sections were again blocked with 0.01 M PBS/1% Triton X-100/ 10% normal
goat serum for 1 h at room temperature. A goat anti-rabbit-CY3 conjugate was
then applied to the tissue sections (2 h at room temperature). The tissues were
rinsed in 0.01 PBS (3 X 10 min) and mounted with buffered glycerol for confocal
and epifluorescence microscopy.

To eliminate potential sources of signal cross-talk (Brelje et al., 1993;
Schneider & Lopez, 2002) and to validate the specificity of immunostaining, three
concurrent method control experiments were performed. These method controls
included the omission of either one or the other primary antibody, and
subsequent incubation with the corresponding secondary antibodies. The first
control involved the omission of the first primary antibody, the exclusion of the
second primary antibody served as the second control, and in the third control
both primary antibodies were included. Antibody specificity for anti-aIE was
determined using preadsorption tests on whole mount sections of the guinea pig

stomach and intestine, and no a1E-ir was observed (see Fig. 3.30 inset).

Confocal and epifluorescence microscopy. Tissue sections were
examined using a Leica TCS-SLr laser scanning confocal system (Leica
Microsystems Heidelberg, Mannheim, Germany) attached to a Leica Instruments
DMLFSA microscope. Multicolor immunofluorescence analyses were then
performed using a single line argon ion laser, (488 nm), which was used to excite

FITC while a helium-neon laser (543 nm) excited CY3. Images were then

61

obtained using a 63 x 1.32 HCX PL APO oil immersion objective (Leica). The
methods of Majlof and Forsgren (1993) and Schneider and Lopez (2002) were
followed to facilitate accurate confocal imaging (Majlof & Forsgren, 1993;
Schneider & Lopez, 2002). Sequential scan settings were used to obtain images
at each wavelength individually. A xyz series range for a particular observer-
detennined area was defined, and a z-series of optical sections through the area
of interest and orthogonal to the optical axis was obtained. The number of
sections taken was calculated automatically by doubling the optimized step size.
Paired red and green images from the different focal planes were then acquired
with Leica confocal software (version 2.61) and merged to produce either an
image montage or a single composite image. In the photomicrographs shown in
this study, a1E-ir is always presented as red staining, with the alternate
neurochemical marker presented as green staining. Yellow immunolabeling in a
merged image represents antigen co-Iocalization.

Conventional epifluorescence microscopy was used to indicate the extent of
immunolabeling and to provide a histological orientation of labeled structures
within each of the layers examined. Utilizing rhodamine and FITC filter sets,
images corresponding to higher-resolution confocal images were viewed with a
40x numerical aperture objective coupled to a Nikon Eclipse TE2000-U inverted
microscope (Nikon, Kanagawa, Japan). Images were then captured using

MetaMorph 6.2r2 (Universal Imaging Corp, Downingtown, PA).

62

Colchicine treatment in vitro. To enhance a1E staining within small
intestinal myenteric cell bodies, LMMP sections were dissected and pinned tautly
onto a silicone elastomer-lined petri dish. The preparations were then incubated
in sterile cell culture media comprising Eagle’s minimum essential medium
supplemented with fetal bovine serum (10%), gentamicin (10 jig/ml), penicillin
(100 U/ml) and streptomycin (50 pg/ml) and colchicine (80 pM) (Sigma-Aldrich)
for 24 h at 37 °C (Furness et al., 1989). The tissues were then washed with 0.01
M PBS (3 X 10 min intervals) and then fixed in formalin overnight at 4 °C in

preparation for immunohistochemistry.

Data analysis
Cell counts of co-localized labeling in neurons. Co-Iocalized
a1E/nNOS-ir cell bodies in one-hundred myenteric ganglia were counted in each
of nineteen plexus preparations from four guinea pigs. These data were then
exported to GraphPad Prism (GraphPad, San Diego, USA) for statistical
analysis. A paired t-test, or a one-way ANOVA followed by a Tukey-Kramer post-
hoc test was used to determine statistical significance. P < 0.05 was considered

significant. The data are presented as mean :I: S.E.M.

Quantification of co-Iocalization. Quantification of co-localization in
merged images was assessed by a two-tiered approach: 1) line profiles,
indicating pixel intensities, were drawn between structures deemed to have

signal overlap (Zeiss Pascal LSM 5, Carl Zeiss lnc., Thornwood, NY); 2) object-

63

based analyses involving segmentation and thresholding techniques that
separate and then classify varicosities as mathematically-defined individual
objects (Silver & Stryker, 2000) were performed using Image Pro-Plus 4.5.1
(Media Cybernetics, Silver Spring, MD). A 7X7 pixel matrix high-gauss spatial
filter (single-pass, strength 1) was applied to facilitate optimum signal-to-noise
ratios. Figures were then imported into Adobe Photoshop CS 8.0 (Adobe
Systems, Mountain View, CA) for further processing. Care was taken to ensure
that ‘when the overall sharpness, brightness and contrast of images were
adjusted, that such manipulations did not alter the information content of the

original images. Images in this thesis/dissertation are presented in color.

64

RESULTS
Western blot studies
Western blot analysis (Figure. 3.1) demonstrated the presence of the 01E
subunit (~170 kDa) in the myenteric plexus of the guinea pig ileum. The guinea
pig heart and myenteric plexus showed multiple low molecular weight bands. The
strong expression of the 170 kDa a1E band in the circular muscle and its
absence from the submucosa is supported by the immunohistochemical studies

described below.

Summary of a1E-ir in the whole mount LMMP throughout the GI tract
a1E—ir was observed in cell bodies and axons of gastric neurons within myenteric
ganglia and interconnecting nerve bundles. or1 E-positive nerve fibers were
abundant and diffusely distributed throughout the ganglia and interconnective
fibers. This distribution pattern of a1E-ir was similar in all GI regions. In the small
intestine (ileum and duodenum), no a1E-ir was detected in myenteric cell bodies.
In the colon (proximal and distal), however, a1E-ir was present in cell bodies and
axonal processes of some myenteric neurons. In all GI regions, strong a1E-ir
was observed in nerve fibers supplying the circular smooth muscle. Longitudinal
muscle nerve fibers in the tertiary plexus also exhibited a1E-ir, but this labeling

was not as intense as that visualized in the circular muscle.

65

Summary of a1E-ir in ileal myenteric neurons maintained in primary culture
Since R-type calcium channel currents were recorded from dissociated ileal
myenteric neurons in primary culture, and because no a1E-ir was observed in
myenteric cell bodies from the whole-mount LMMP in the guinea pig ileum, I
investigated a1 E-ir in myenteric neurons in primary culture. While some neurons
showed labeling throughout their cell bodies and axons, a1 E-ir within the cell

bodies was predominantly intracellular.

Co-Iabeling studies
a1E is not found in the submucosal plexus

aIE-ir was completely absent in the submucosal plexus in all regions of
the gut. For example, I attempted to localize a1E-ir in submucosal ganglia of the
ileum and proximal colon (Figure. 3.2) and no labeling was detected in any of
these ganglia (Figure. 2A1 and C). Submucosal ganglia were revealed using an
antibody raised against SP and numerous SP-ir nerve fibers were found in
submucosal ganglia (Figure. 3.2A2). SP-ir but not a1E-ir was found in extrinsic
sensory nerves supplying submucosal arterioles (Figure. 3.28). TH is the rate
limiting enzyme in norepinephrine synthesis and TH-ir is a marker for
sympathetic nerves supplying the gastrointestinal tract. TH-ir revealed that
sympathetic nerves supply submucosal ganglia and arterioles but these nerve

fibers did not express a1E-ir (Figure. 3.20, D, E).

66

Co-Iocalization of a1E-ir and SP-ir in the myenteric plexus

SP-ir and a1E-ir were co-Iocalized in varicose nerve fibers in myenteric
ganglia throughout the gut, but this was not a complete overlap. This result is
illustrated with data obtained in the proximal colon (Figure. 3.3A, B) and gastric
corpus (Figure. 3.3C1, CZ). Nerve cell bodies containing a1E-ir were also
detected in myenteric ganglia of the proximal colon but SP-ir was not detected in
these neurons (Figure. 3.38). a1E-ir and SP-ir were also co-localized in nerve
fibers in myenteric ganglia (Figure. 3.3C1, CZ and D) and in varicose nerve fibers
supplying the longitudinal muscle layer (Figure. 3.3E) and circular muscle layers
(not shown). In the duodenum, co-Iocalization of a1E-ir and SP-ir occurred in
nerve fibers in ganglia, in internodal strands and in the tertiary plexus supplying
the longitudinal muscle layer. The intensity of alE-ir labeling in the duodenal
myenteric plexus was greater than that observed in the ileum, particularly in the
interganglionic fiber tracts. I also identified some a1E-SP-ir in myenteric neurons

in culture (Figure. 3.12C2, D2, and C3, D3).

a1E-ir with calbindin and calretinin
The calcium binding protein, calbindin, is the strongest marker for IPANs
in the myenteric plexus (Song et al., 1991; Costa et al., 1996), while a second
calcium binding protein, calretinin, is a marker for orally projecting interneurons
and longitudinal muscle excitatory motorneurons in the myenteric plexus
(Brookes, 2001). In whole mount LMMP sections, calbindin-ir was detected in

myenteric nerve cell bodies in the stomach, ileum, duodenum and in the colon.

67

Representative images of calbindin-ir in nerve cell bodies are provided for the
proximal colon (Figure. 3.4A, B). Although a1E-ir was readily detected in nerve
fibers and nerve cell bodies in the stomach and colon, I never detected co-
localization of a1E-ir and calbindin-ir in nerve cell bodies in the whole mount
LMMP. These data are illustrated by results obtained in the proximal colon
(Figure. 3.4A, B). a1E-calbindin co-localization was, however, present in
myenteric neurons in culture (Figure. 3.1201, D1). Calretinin-ir was detected in
nerve fibers in myenteric ganglia throughout the gut and co—Iocalized with a1E is
some varicosities. These data are illustrated by results obtained in duodenal
myenteric ganglia (Figure. 3.4C1, C2). Calretinin-ir was also detected in
myenteric neurons of the distal colon but a1E-ir was not co-localized in calretinin-
ir neurons (Figure. 3.4D1, DZ). In culture, myenteric neurons showed some co-
expression of ME and calretinin in cell bodies (Figure. 3.12C4, D4) but a1E-

calretinin-ir labeling was intense in axons (Figure. 3.12C5, D5).

a1E-ir and 5-HT-ir are not co-expressed in neurons
5-HT is contained in a subset of descending interneurons in the myenteric
plexus and I used 5-HT as a marker for this class of neuron (Costa & Furness,
1982; Costa et al., 1982). a1E and 5-HT did not co-Iocalize in the myenteric
plexus in any gut tissue examined or in dissociated neurons. a1E-ir was
detected in varicose nerve fibers which formed distinct ring-shaped structures
surrounding non-a1E-ir cell bodies in the ileum (Figure. 3.5A1). Varicose nerve

fibers in internodal strands also showed intense and extensive a1E-ir. There

68

were relatively few 5—HT-ir nerve fibers but 5-HT-ir in these individual nerve fibers
was intense (Figure. 3.5A2). Close analysis revealed intense a1E-ir and 5-HT-ir
labeling of varicose nerve fibers within myenteric ganglia in the duodenum and 5-
HT labeling was more prominent in the duodenum than in the ileum. It is
possible that myenteric neurons in the duodenum contain more 5-HT than other
regions (Meyer & Brinck, 1999). However, 5-HT-ir and a1E-ir were contained in
separate populations of nerve fibers (Figure. 3.581, 82). In the corpus, strong
a1E-ir was present in nerve fibers in myenteric ganglia and in interganglionic
connectives. In those areas, nerve fibers displaying 5-HT-ir were more abundant
than in the small intestine as reported previously (Mawe et al., 1989). a1E-ir was
detected in cell bodies and axonal processes in myenteric ganglia and internodal
strands of the proximal and distal colon but a1E-5—HT co-localization was never

observed (not shown).

Co-Iocalization of a1E- and VlP-ir
VlP-ir is localized to cell bodies and nerve fibers in myenteric ganglia, to
nerve fibers in the tertiary plexus which supplies the longitudinal muscle and to
nerve fibers supplying the circular muscle layer (Costa & F urness, 1983; Brookes
et al., 1991b; Brookes et al., 1992). I found VIP-ir nerve fibers in ileal myenteric
ganglia and in the tertiary plexus and that arrangement of a1E-ir was very similar
to that of VIP-ir in these regions (Figure. 3.6A1, A2). Closer examination of ileal

myenteric ganglia revealed evidence of some co-localization of VlP-ir and a1E-ir

in varicose nerve fibers (Figure. 3.681, 82). Similar overlap of VlP-ir and a1E-ir

69

in varicose nerve fibers was also detected in the tertiary plexus supplying ileal
longitudinal muscle (Figure. 3.6C, D). Varicose nerve fibers in myenteric ganglia
in the gastric corpus also displayed overlapping (11 E- and VlP-ir (Figure. 3.6E,

F).

Co-Iocalization of a1E-ir with NOS-ir

l detected NOS-ir in myenteric neurons in the stomach, ileum and
proximal colon (Figure. 3.7A1, 81, C and E1). However, a1E-ir labeling in
neuronal cell bodies was only detected in the stomach (Figure. 3.7A2, 82) and
colon (Figure. 3.7E2). In the gastric myenteric plexus I found 3.9 :I: 0.60 a1E-
NOS-ir neurons per ganglion, based on counts done in 100 ganglia each from
gastric myenteric plexus from four guinea pigs. There was an anal-oral gradient
in the numbers of a1E-NOS-ir neurons in the colon as there were 5.7 :l: 0.5
neurons per ganglion in the proximal colon while there were 4.7 i: 0.6 a1-NOS-ir
neurons in the distal colon (P < 0.05) (Figure. 3.8).

NOS-ir neurons were prominent in ileal myenteric ganglia, but a1E-ir
neurons were never detected in these ganglia (Figure. 3.7C). I detected co-
localization of a1 E-ir and NOS-ir in nerve fibers in the ileal myenteric plexus
(7D1, 702). However, there was not complete overlap of a1E and NOS labeling
as l detected nerve fibers which were labeled by only one or the other antibody.
Therefore, a1 E is a marker for a subpopulation of NOS-containing nerve fibers.

I did not detect a1E-ir in myenteric nerve cell bodies in the ileum although

I did detect varicose nerve fibers that contained a1E-ir and NOS-ir. It is possible

70

that a1E-ir does not accumulate in myenteric nerve cell bodies in this region of
the gut. I used a colchicine-incubation protocol in an effort to increase a1 E-ir in
ileal myenteric neurons. Colchicine disrupts axonal transport and should cause
an accumulation of proteins in nerve cell bodies (Costa et al., 1996). However,
a1E-ir was not detected in myenteric neurons even in small intestinal
preparations that had been incubated with colchicine (not shown). Intense co-
immunolabeling for a1 E and NOS was, however, observed in the cell bodies and

axons of dissociated ileal neurons (Figure. 3.13A1-A3).

Identification of excitatory and inhibitory muscle motorneurons

l next asked whether a1E-ir is co-expressed with SP-ir, VlP-ir and NOS-ir
in nerve fibers supplying the muscle layers. In all regions a1E- and SP-ir co-
localized in many nerve fibers supplying the longitudinal and circular muscle
layers. This result is illustrated by data from nerve fibers supplying ileal circular
muscle (Figure. 3.9A1, A2). SP-ir in nerve fibers in the muscle layers is a marker
for excitatory motor innervation (Brookes et al., 1991b; Brookes et al., 1992;
Brookes et al., 1998). I therefore reasoned that the a1E—ir that did not contain
SP-ir was present in inhibitory nerve fibers. VIP-ir and NOS-ir were used as
markers for inhibitory nerve fibers (Costa and Furness, 1983; Brookes, 1993) and
it was found that a1E-VlP-ir and a1E-NOS-ir were co-localized in nerve fibers
supplying the muscle layers throughout the gut. This result is illustrated with data
obtained in the circular muscle of the stomach (Figure. 3.98) and proximal colon

(Figure. 3.9C).

71

Co-Iocalization of a1E-ir and TH-ir

As discussed above TH-ir is a marker for sympathetic nerve fibers
supplying the gut. Sympathetic nerves inhibit gut motility by inhibiting myenteric
neuronal activity and myenteric ganglia are innervated by sympathetic nerve
fibers. I next determined if the sympathetic nerve supply of the myenteric plexus
expressed a1E-ir. I found that nerve fibers in the myenteric plexus that
contained TH-ir also contained a1E-ir (Figure. 3.10A, B). In the myenteric plexus
brightly fluorescent rings of a1E-TH-ir varicosities circled non-a1 E-ir cell bodies.
lntensely labeled ME and TH varicosities were also found in ganglionic fibers in
the myenteric plexus and internodal strands of the duodenum (Figure. 3.10C).
The network of ganglia and interconnecting nerve fibers in the duodenum
consistently demonstrated intense a1E-ir and TH-ir. In the stomach, a1E- and
TH-ir were localized to varicose nerve fibers in the myenteric ganglia and
interganglionic fiber tracts. In the gastric myenteric plexus, nerve fibers co-
expressing a1E-ir and TH-ir were less dense than in the corresponding areas of
the other gut regions. In the colon (Figure. 3.100), a1 E-ir and TH-ir were co-
Iocalized in nerve cell bodies and nerve fibers in myenteric ganglia. The same
pattern of immunohistochemical labeling applied to the distal colon, where both
the a1E and TH antibodies labeled nerve cell bodies and varicose nerve fibers.
There were more a1E-TH-ir neurons per myenteric ganglion in the proximal
colon (2.1 1 0.06) compared to the distal colon (1.4 1 0.02, P < 0.05) based on

counts in 100 ganglia in preparations from four guinea pigs (Figure. 3.11).

72

Furthermore, an interesting observation was that although not all a1E-cell bodies

were TH-ir, all TH-ir neurons were a1E-ir.

Co-Iocalization of a1E-synaptophysin-ir and SP-synaptophysin-ir
Synaptophysin is an integral synaptic vesicle membrane protein believed to be
involved in neurotransmitter release after phosphorylation by tyrosine kinases
(Evans & Cousin, 2005). Interestingly, in the guinea pig ileum, a1E and
synaptophysin did not co-localize in nerve fibers in myenteric ganglia or in the
tertiary plexus (Figure. 3.13A2-C2; A3-C3). Very rare co-expression of these two
proteins, however, was found in some varicosities. Prominent SP-synaptophysin-
ir was observed in varicosities in myenteric ganglia and longitudinal muscle in the

tertiary plexus (Figure. 3.13 A4-C4).

DISCUSSION
Identification of the chemical coding of functional classes of enteric
neurons has been of great help in understanding how the ENS functions. The
results reported in this study identified subtypes of neurons that express R-type
calcium channels. This information will assist in identifying the contribution of R-

type calcium channels to the control of gastrointestinal function.

a1E is not expressed by submucosal neurons
Submucosal neurons control water and electrolyte secretion by the

gastrointestinal epithelium and they also control local blood flow by providing

73

vasodilator input to submucosal arterioles. Submucosal arteriolar diameter is
also controlled by sympathetic nerves that supply these blood vessels and by
extrinsic SP-calcitonin—gene related peptide (CGRP) containing sensory nerves
(Vanner & Macnaughton, 2004). SP and CGRP are both vasodilator substances
(Uddman et al., 1986). Neither the extrinsic nerve supply of submucosal ganglia
nor arterioles (sympathetic and sensory) nor intrinsic submucosal neurons
expressed a1E-ir. Although I was unableto localize or1E-ir in the submucosal
plexus, l was able to confirm, using TH immunohistochemistry, the presence of
periarteriolar sympathetic nerve fibers and sympathetic nerve fibers terminating
in submucosal ganglia. Similarly, I was able to demonstrate the presence of SP-ir
nerve fibers in submucosal ganglia and around submucosal arterioles. These
data suggest that drug treatments which either block or facilitate the function of
R-type calcium channels would have little effect on secretomotor activity or blood

flow in the submucosa.

a1E-ir is localized to cell bodies of myenteric plexus lPANs in the small
intestine
Intestinal IPANS contain calbindin and SP and they are the first neurons
activated in reflex pathways responding to chemical and mechanical stimuli of
the mucosa (Furness et al., 1999; Furness et al., 2004). In the small intestine,
IPANS constitute approximately 26% of myenteric neurons and these same
neurons have axons that project to other myenteric ganglia (Brookes et al.,

1995). In the small intestine, I could not detect a1E-ir nerve cell bodies, even in

74

tissues that had been incubated with colchicine in an effort to enhance cell body
content of a1 E-ir. Our calbindin antibody did not label nerve fibers although I was
able to detect prominent a1E-ir in nerve fibers in the myenteric plexus. I could,
however, assess the overlap of a1E with calbindin in small intestinal myenteric
neurons maintained in culture. In these neurons, alE-calbindin-ir was co-
Iocalized mainly within the cell soma. Very little co-staining was observed in
axonal processes. This, in agreement with the study by Rugiero et al. (2002),
suggests that R-type calcium channels may contribute to the tetrodotoxin-
insensitive calcium current during the somal action potential in lPANs.

SP-ir is contained in IPANS which send processes to myenteric ganglia
and SP-ir is also contained in ascending interneurons (Brookes, 2001).
Ascending interneurons in the guinea pig myenteric plexus constitute
approximately 5% of the total number of neurons present (Brookes, 2001).
Motorneurons will not have processes in myenteric ganglia so they will not
contribute to SP-ir in the plexus (Costa et al., 2000). I found substantial co-
localization of a1E- and SP-ir in myenteric ganglia. Calretinin, however, is also a
marker for ascending interneurons in the myenteric plexus (Brookes et al.,
1991a; Brookes, 2001) and I used an antibody against this protein to localize
nerve fibers in the myenteric plexus. Within the 5% of ascending interneurons,
both SP and calretinin share the chemical coding in this type of neuron. In
doubled-stained preparations, 1 detected a1 E-ir and calretinin-ir nerve fibers, and

found that a small population of myenteric nerve fibers expressed both a1E-ir

and calretinin. Therefore, a1E-ir may be present in the nerve endings of this

75

subset of calretinin-containing ascending interneurons. Based on these data, I
conclude that the a1E-ir nerve fibers in the small intestinal myenteric plexus arise
from SP-containing and calretinin-containing excitatory interneurons.

l was able to detect a1E-ir and calbindin-ir neurons in the gastric
myenteric plexus but I never detected co-localization of these two antigens in the
same neuron. I did not determine their co-expression in gastric or colonic
myenteric neurons in culture. Similar to my data in the small intestine, I found
that, in the gastric corpus, a1E-ir and SP-ir were co-Iocalized in nerve fibers in
myenteric ganglia and in the muscle layers. SP-ir and calbindin are localized to a
subset of gastric myenteric neurons but there are also SP-ir neurons which do
not contain calbindin or calretinin (Furness et al., 1988; Reiche et al., 1999;
Reiche & Schemann, 1999; Schemann et al., 2001). The SP-ir but
calbindin/calretinin negative neurons are either a separate population of
ascending interneurons or circular muscle excitatory motorneurons (Michel et al.,
2000; Schemann et al., 2001). My data in the ileum show that while a1 E-ir co-
localized with calbindin mainly in cell bodies in culture, a1E-ir was also present in
calretinin-positive nerve fibers in the whole mount LMMP and in myenteric
neurons in culture. It would therefore seem likely that aIE-ir nerve fibers in
gastric myenteric ganglia are probably from ascending interneurons. I also show
that alE-ir and SP-ir co-Iocalized in nerve fibers supplying the muscle layers in
the stomach and these must arise from excitatory motorneurons. Therefore, R-
type calcium channels may contribute to synaptic transmission in myenteric

ganglia and to excitatory neuromuscular transmission in the gastric corpus.

76

In the colon, IPANS contain calbindin and SP-ir (Messenger & Furness,
1990; Lomax et al., 1999; Lomax & Furness, 2000) and I found numerous nerve
fibers containing both a1E-ir and SP-ir suggesting that nerve fibers might arise
from colonic IPANS. However, unlike in the small intestine I was able to detect
both a1E-ir and calbindin-ir neuronal cell bodies in the colon myenteric plexus but
these proteins were expressed in separate neurons. Therefore, the nerve fibers
containing a1E-ir and SP-ir are unlikely to come from calbindin-containing IPANS
in the colon. There is a second set of SP-ir neurons that contain choline
acetyltransferase but not calbindin in the myenteric plexus of guinea pig colon;
these are ascending excitatory motorneurons supplying the circular muscle
(Lomax and Furness, 2000). The SP-orl E-ir nerve fibers l detected are likely to
arise from this subgroup of neurons. These data suggest that R-type calcium
channels would contribute to excitatory neurotransmission to the circular muscle

layer in guinea pig colon.

a1E does not co-Iocalize with 5-HT in the myenteric plexus
5-HT is contained in some myenteric neurons and by enterochromaffin
(EC) cells (Blilbring & Gershon, 1967). The importance of 5-HT in mucosal
sensory transduction and as a transmitter in the regulation of secretory and
motor functions in the gastrointestinal tract is well documented. In this regard, 5-
HT released by EC cells may activate extrinsic and intrinsic sensory, motor and

secretomotor neurons, and effector cells such as enterocytes and smooth muscle

77

cells (Tonini, 2005). 5-HT is also a mediator of slow synaptic excitation in some
myenteric neurons (Monro et al., 2005).

In the small intestine and stomach, there are ChAT-5-HT-containing
descending interneurons (Clerc et al., 1998; Meedeniya et al., 1998; Lomax &
Furness, 2000; Reiche et al., 2000). In the stomach 5-HT-ir neurons also contain
calbindin while 5-HT-ir descending interneurons in the distal colon are similar to
the small intestine in that they contain calbindin (Lomax & Furness, 2000). I
found that a1E-ir and 5-HT-ir were present in separate populations of nerve
fibers in myenteric ganglia and interganglionic strands in all regions of the gut
examined. Therefore, I conclude that R-type calcium channels are unlikely to

contribute to 5-HT release from nerve terminals in the myenteric plexus.

a1E is expressed by enteric inhibitory neurons

NO and VIP are released from inhibitory motor neurons supplying
the muscle layers in the gut (Murthy et al., 1995; Xue et al., 2000). I found that
alE-ir co-Iocalized with VlP-ir and NOS-ir in cell bodies in myenteric ganglia,
internodal connecting fibers and in nerve fibers supplying the longitudinal and
circular muscle layers. a1 E-ir and NOS-ir were co-localized in myenteric cell
bodies and varicosities in the stomach and colon, and to varicose nerve fibers in
myenteric ganglia, internodal strands and the muscle layers. In the ileum, VIP
and NOS are present in descending interneurons, and inhibitory motor neurons.
VIP but not NOS is also co-expressed with ChAT in some descending

interneurons (Brookes, 2001). In the duodenum, however, NOS-ir was not

78

detected in VIP-containing descending interneurons, but motorneurons supplying
the muscle layers in the intestine contain these neurotransmitters (Clerc et al.,
1998). Taken together, the localization of a1E with VIP and NOS in myenteric
cell bodies and nerve fibers, and in dissociated myenteric neurons, has a number
of implications: 1) a1E-VIP/NOS-ir varicose nerve fibers in the myenteric plexus
of the small intestine and stomach arise from descending interneurons; 2) a1E-
VIP-ir cell bodies in the colon and a1E-NOS ir neurons in the stomach and colon,
are either descending interneurons or inhibitory motor neurons projecting to the
circular and longitudinal muscle layers; 3) a1E-VIP-NOS labeling in nerve fibers
supplying the muscle layers suggests that a1 E is present in the nerve endings of
inhibitory motorneurons. These results therefore suggest that R-type channels
are likely involved in inhibitory neurotransmission to the muscle layers and in

synaptic transmission in descending pathways in the myenteric plexus.

a1E-ir is expressed by sympathetic nerves supplying the myenteric plexus

TH is a marker for sympathetic nerve fibers and most TH-ir in the
gut is extrinsic in origin. The exception is the proximal colon where amine-
containing myenteric neurons have been observed (Costa et al., 1971; Furness &
Costa, 1971). In the present study I found that: 1) TH-ir was not present in
myenteric neurons in the small intestine or stomach, but TH-ir co-localized with
a1E-ir in nerve fibers in myenteric ganglia and interganglionic connectives; 2) the
proximal colon exhibited a substantially greater number of TH-ir neurons than the

distal colon, 3) both regions of the colon displayed a1E-ir/TH-ir nerve terminals.

79

Three separate subgroups of sympathetic neurons project to the gut.
These are TH/NPY neurons that supply the intestinal blood vessels (Furness et
al., 1983), TH/somatostatin neurons that target the submucosal ganglia and
mucosa (Costa & Furness, 1984) and TH-containing nerve fibers that contain
another neurochemical marker and supply the myenteric plexus. My data
suggest that a1E is a marker of sympathetic neurons that target the myenteric
plexus and these sympathetic neurons have the neurochemical code of TH/a1E.
A recent study showed that a2-adrenoreceptors couple to and inhibit the R-type
calcium channel by mediating presynaptic inhibition of slow and fast synaptic
transmission in the ENS (Bian & Galligan, 2007). Therefore, R-type calcium
channels may be important for regulation of sympathetic neural control of gut

motility.

a1 E-ir does not co-Iocalize with synaptophysin

The absence of co-localization of a1E and synaptophysin in myenteric
ganglia suggests a number of possibilities: 1) since synaptophysin constitutes
only 7% of the total synaptic vesicle membrane protein (Evans & Cousin, 2005),
alE may only be present on synaptophysin-negative varicosities; 2) the SNARE
proteins syntaxin and SNAP-25, but not synaptobrevin, interact with N-type
calcium channels by binding to the intracellular loop between domains II and III
on the (11 subunit (Sheng et al., 1994; Sheng et al., 1996; Catterall & Few, 2008).
Furthermore, N- and PIQ type calcium channels co-localize with syntaxin-1

(Catterall & Few, 2008). Putative a1E-ir varicosities may therefore contain a

80

neuronal vesicle population comprising of other synaptic vesicle membrane
proteins such as synapsin, synaptotagmin (Wiser et al., 2002), or perhaps even
an as yet unidentified vesicle membrane protein; 3) synaptophysin may not be
responsible for regulating neurotransmitter release from R-type calcium channel-
containing nerve terminals. This is supported by evidence in CA1 hippocampal
pyramidal neurons where the amplitude of excitatory postsynaptic currents,
probability of vesicle release and overall synaptic transmission were unimpeded
in mice that were synaptophysin-deficient (McMahon et al., 1996). Those authors
suggest that the function of synaptophysin may be a redundant one or that it may
have a more subtle function not necessary for regulating exocytosis. This may
indeed explain the surprising observation in my study that 0:1E and
synaptophysin do not co-localize. It is unlikely that the intense and diffuse 0:1E-ir
fibers in the myenteric plexus represent non-functional synapses or are
completely non-varicose fibers, since I had observed definitive SP-synaptophysin
punctate co-Iabeling in the LMMP (Figure. 3.13C4, D4). It would therefore be
feasible in a future study to investigate the co-Iocalization of 0:1E-ir with other
synaptic vesicle membrane proteins, or presynaptic plasma membrane—anchored

proteins such as syntaxin or neurexin.

0:1 E-ir does not co-localize with pan-cadherin
I did not observe any co-Iocalization of 0:1 E with the cell membrane marker pan-
cadherin in neurons in primary culture or in the whole mount LMMP (Figure.

3.13A5-CS, A6-C6). Four auxiliary calcium channel subunits, B, 0:28, and y are

81

essential for the function of the 0:1 protein (Hoffman et al., 1994) and all are
expressed in neural tissue (Castellano et al., 1993; Stephens et al., 1997). The [3
subunit is located intracellularly and binds to the interaction domain on the 0:1
subunit, while the 0:2 subunit is an extracellular protein. The 0:2 subunit is
anchored to 0:1 via the integral membrane protein 8. The y subunit is also an
integral membrane protein, but anchored directly to 0:1 (Catterall & Few, 2008).
The 8 subunit enhances expression of the 0:1 protein and peak calcium current
density at the cell membrane, presumably by trafficking a large number of
functional calcium channels to the surface (Brice ef al., 1997; Hofmann et al.,
1999; Bichet et al., 2000; Altier et al., 2002). Interestingly, the expression of
specific isoforrns of the [3 subunit varies during postnatal development (Vance at
al., 1998). In that study, 84 protein expression increased ten-fold, concomitant
with rat postnatal (PO-adult) development. 0:28 subunits contribute moderately to
cell membrane expression of 0:1 subunits, but y subunits have no effect on 0:1
membrane expression (Catterall & Few, 2008). In fact, 0:28 subunits strongly
facilitate 0:1E expression and function by increasing the rate of voltage-
dependent R-type calcium channel activation and inactivation (Qin et al., 1998).
Together, co-expression of 0:28 and 8 calcium channel subunits with 0:1

increases surface expression of 0:1 to approximately 5 and 35% respectively
(Williams et al., 1992; Klugbauer et al., 2003; Bernstein & Jones, 2007).

Evidence suggests that the 8 subunit unlocks a signal motif on the HI

82

intracellular loop on the 0:1 subunit, thus preventing retention of the 0:1 protein in
the endoplasmic reticulum (ER) (Bichet et al., 2000).

The absence of co—localization of 0:1E with pan-cadherin therefore
suggests that: 1) since the 0:1E subunit has a number of glycosylation and
phosphorylation sites (eg. by protein kinase C on the I-ll intracellular loop), 0:1E
may have been post-translationally modified, preventing it’s folding in the rough
ER and subsequent association with the accessory subunits for targeting to the
cell membrane; 2) the 8 subunit may not have access to the putative ER-
retention signal and thus cannot direct the movement of the 0:1E subunit from
this intracellular compartment to the plasma membrane; 3) the cytoplasmic 0:1E
staining observed could represent immature forms of the pore-forming subunit.
The 8 subunit would then function to restrict 0:1E trafficking to the membrane to
prevent cytotoxic insults such as uncontrolled calcium entry; 4) alternatively, 0:1E
may be a mature protein, but the 8 subunit has been phosphorylated by protein
kinases A and C, or undergone palmitoylation (Dolphin, 2003a). Palmitoylation
regulates neurotransmission pre and postsynaptically (eI-Husseini Ael & Bredt,
2002). This post-translational processing of the B (or even 0:28) protein would
again mean ineffective trafficking of 0:1E to the plasma membrane. 0:1E would
thus be localized mainly intracellularly; and 5) the cytoplasmic 0:1E staining in
myenteric cell bodies in culture suggest that this may be a purely developmental
occurrence. Incremental [34 expression occurs from P0 to adult in the rat brain

(Vance at al., 1998), and it is possible that in newborn guinea pigs that 8 subunits

83

may not be in their mature conformations, and therefore unable to facilitate

efficient membrane targeting of 0:1 E.

SUMMARY AND CONCLUSIONS

The 0:1E subunit of the R-type calcium channel is expressed by nerve
fibers throughout the myenteric but not submucosal plexus in the guinea pig
gastrointestinal tract. Based on an established neurochemical code for
subclasses of myenteric neurons and myenteric nerve fibers in the guinea pig, I
conclude that 0:1E-ir is found in the nerve terminals of sympathetic nerves
supplying the myenteric plexus, ascending interneurons, inhibitory motorneurons
supplying the muscle layers and the cell bodies of myenteric IPANS in the small
intestine (Table 2). These observations suggest that it may be possible to
selectively inhibit or facilitate neurotransmitter release from specific subsets of
nerve endings using drugs which act to alter the activity of R-type calcium

channels.

84

Table 3.1. Primary antibodies used to study the distribution of R-type

calcium channels

 

 

. Dilutio “PSI . . .
Antibody n Lot No specres and Supplier’s Characterization References
Source
Western blot of membrane
Rabbit extracts from rat brain
(ACC-006 ideflgies péotggig :Bfl 99 Sagggga H,
, ’ a an ~ a. ;
01E 1'300 AN'OZ £2232: Absence of staining after Fisher TE,
l s r a el) ' preincubation with control 2000
' antigen in rat cerebellar
tissue.
Mouse,
8 .- D ”3353’ 'T-Z‘”"°”"’lli"i K. .
a in in - , lneyepi eiawoece aseos
28K 1200 113K486” (fig: extracts labels a single CD, 1994)
Aldrich St protein of 28 kDa.
Louis, MO)
Goat Western blot shows a single Jacobowitz
50700582 (A31550 P grate? Of 319 ki’a'n l viiM Emil.
. . . . . ’ rea sorp ion 0 ra co ica ins y ,
ca"et'"'" 120° 8 8333?; tissue with antibody and 1991;
CA) ’ control antigen shows Winsky et al,
absence of immunolabeling. 1989
Mouse Absence of staining after
(A816007, incubation with antibody and
5-HT 1:200 522888 Abcam, antigen in cryopreserved izaafibggfi)?
Cambridge, ventral mesencephalic '
MA) neurons-
Rat, clone
(84150103505 Complete crossreactivity for
Substance P 1,200 240907 AbD ' Substance P inbrat brain Cue1llg7gt al,
issues y
gigs: radioimmunoassay
NC)
Vasoactive Sheep Pre-incubation with VIP Zhu et al
intestinal 1:2000 25030911 (A81581, antibody and antigen 1997 ’
peptide (VIP) Millipore) abolishes immunostaining

85

Table 3.1 (cont’d)

Neuronal
nitric oxide
synthase
(nNOS)

t200

Tyrosine
hydroxylase
(TH)

1:200

Pan-cadherin 1:200

Synaptophys 1,200
in '

LV144962
9

21071256

598458

32 K4862

Sheep
(A81 529,
Invitrogen);
Rabbit (Dr.
8 Mayer, U
of Graz,
Austria)

Mouse,
clone
TOHA1 .1
(MAB358,
Millipore)

Mouse,
clone CH-
19
(A86528,
Abcam)
Mouse,
clone SVP-
38 ($5768,
Sigma)

86

Preadsorption of NOS
antibody with antigen
abolishes staining in rat
frontoparietal cortex

Recognizes a~60 kDa TH
protein by Western blot in
rat cerebral cortex.

Recognizes a protein ~135
kDa by Western blot in
mouse hippocampal Iysates.

lmmunoblot shows a 38 kDa
synaptophysin-positive band
in rat brain extracts.

Cauli B et al,

2004

Rolletschek

A et al,
2001;

Semenenko,

F. et al.
1986

Bae GU et
al, 2008;
Grimm M0
at al, 2008

Eshkind and
Leube,1995

250K0-
160KB — ‘4
105K0—

 

Brain Heart Heart LMMP CM SM
k J k J
Y W
Rat Guinea Pig

 

 

Figure 3.1. Western blot analyses of 0:1E subunit expression in rat brain and
heart, guinea pig heart, longitudinal muscle-myenteric plexus (LMMP), small
intestinal circular muscle layer (CM) and submucosa (SM). Note the multiple
spliced variants detected in. the myenteric plexus, and a prominent band near
170 kDa, the same size as that present in the rat brain and heart and guinea pig

head.

87

A1 Ileum S.PlexusP 0:1E A2 Ileum S. Plexus SP 8 Ileum S Plexus 0:1E + SP

 

C Colon S.PlexusP 0:1E+TH D Colon S. Plexus 0:1E+TH E Colon S Plexus 0:1E+TH

 

Figure. 3.2. The a1E-subunit is not expressed in the submucosal plexus in

the ileum and colon. A1. SP-ir (green fluorescence) and absence of a1E-ir in a
submucosal ganglion (g) in the ileum. A2. SP-ir (arrow) is present in submucosal
ganglia. B. A merged image showing that arterioles (bv) in the submucosa were
SP-ir (arrowhead) but not a1E-ir. C. A merged image showing TH-ir nerve fibers
in a colonic submucosal ganglion do not contain 0:1E-ir. D. A merged image
shown TH-ir (arrowhead) in periarterial nerve fibers. E. A merged image showing
a submucosal ganglion adjacent to an arteriole; periarterial and intraganglionic
nerve fibers contain TH-ir but not 0:1E-ir (arrow and arrowhead, respectively).

Scale bars = 200 am (A1-A2); 50 pm (BC); 20 pm (D) and 30 um (E).

88

Figure. 3.3. Co-Iocalization of a1E- and SP-ir in nerve fibers in the LMMP in
the colon, stomach and ileum. A. Merged (yellow labeling) low-magnification
epifluorescence photomicrograph illustrating o1E-ir (red fluorescence) and SP-ir
(green) co-Iocalization in varicose nerve fibers (arrow) with no co-labeling in
nerve cell bodies (ch) in the proximal colon. The arrowhead indicates a1E-SP co-
localization in nerve fibers supplying the longitudinal muscle. Inset show a1E-ir
in a myenteric ganglion and in the tertiary plexus (tp) supplying the longitudinal
muscle. B. Confocal image showing a1E-ir and SP-ir co-localization (yellow) in
varicose nerve fibers in the proximal colon (arrow). 0:1E-IR nerve cell bodies are
also visible (arrowhead). C1. Co—Iocalization of 0:1 E and SP-ir in varicose nerve
fibers in the myenteric plexus in the stomach. Arrowheads denote examples of
varicosities containing both antigens. The dotted line A-B corresponds to the line
profile analysis shown in C2. D. Confocal image showing a1E-SP-ir co-
localization in nerve fibers in an ileal myenteric ganglion. Arrowhead indicates a
varicosity containing both antigens but fibers containing either 0:1E-fr or SP-ir are
also visible. The inset is a preadsorption control where preincubation of the 0:1E
antibody with the antigenic peptide (1pg antibody/1 pg peptide) abolished
immunolabeling in the ileum LMMP. E. Nerve fibers of the tertiary plexus
supplying the longitudinal muscle. Some nerve fibers (arrowhead in D) contained
0:1E- but not SP-ir. Scale bars = 200 pm (A); 100 pm (8); 18 pm (C1); 40 pm

(D); 20 um (E)-

89

ALLERQXI L COLON a1F + SP 8 PROXIMAL COLON

\

    
 
 
 
  

C1 STOMACH C2 STOMACH

. ' 300' _ 0:1E
250- _ SP

N

O

O
l

Intensity
8

 

‘ ‘
(34%ka . é . .8ng88.

Distance (:lM)

  

E ILEAL TP

90

Figure. 3.4. Localization of a1E-ir with calbindin-ir and calretinin-ir in the
LMMP in the colon and duodenum. A. 0:1E-ir (red fluorescence) is contained in
nerve fibers and nerve cell bodies (arrowhead) while calbindin (Calb; green
fluorescence) is contained only in nerve cell bodies (arrows) in the proximal
colon. B. Higher magnification image showing that 0:1E-ir (arrowhead) and
calbindin-ir (arrow) are contained in different neurons in the myenteric plexus of
the proximal colon. C1, C2. Images showing that a1E-ir (red) and calretinin
(Calr; green) co-Iocalize in some varicosities in the duodenal myenteric plexus
(CZ arrowhead). D1, DZ. 01E and calretinin also co-Iocalize in some varicosities
(arrowhead in DZ) but not in cell bodies in the LMMP (arrow in DZ) in the distal

colon. Scale bars = 100 pm (A); 50 pm (8); 50 pm (CD).

91

 

A Colon 0:1E +Calb. B Colon 0:1E + Calb.

C1 Duodenum 0:1E B Duodenum 0:1E+Calr..

Colon 0:1E+Calr..

 

92

Figure. 3.5. Localization of 01E and 5-HT-ir in the LMMP in the small
intestine. A1, A2. Low-magnification epifluorescencephotomicrographs showing
a1E-ir (red fluorescence) and 5-HT-ir (green) (arrowheads) are not co-localized
in nerve fibers in myenteric ganglia (g) and internodal strands (is) in the ileum.
Note the varicose clusters of a1E-ir that surround the neuron in (A). BI. Confocal
image showing 0:1E-ir (arrowhead) and 5-HT-ir (arrow) in separate nerve fibers in
the duodenum. The 5-HT-ir nerve fibers can be seen coursing through the
myenteric ganglia independent of a1E-ir fibers. Similar results were obtained in
the stomach ileum and colon. 82. The dotted line A-B indicates the pixel profile

analysis from 81. This confirms that 0:1E-ir and 5-HT-ir are localized in different

nerve fibers in the myenteric plexus. Scale bars = 100 pm (A-B); 14 pm (C).

93

 

  

    
 

    

   

Bl

  

Duodenum 0:1E+5-HT

94

Figure. 3.6. Co-Iocalization of a1E-ir (red fluorescence) and VIP-ir (green
fluorescence) in the LMMP in the ileum and stomach. A1, A2. Low
magnification epifluorescence images demonstrating 01E and VIP-ir in
varicosities (arrows) in myenteric ganglia and internodal strands in the guinea pig
ileum. Note the prominent a1E-VIP positive nerve fibers in the tertiary plexus
supplying the longitudinal muscle layer (arrowheads). B1. Confocal image of co-
Iocalized (yellow labeling) o1E-VIP-ir varicosities in the ileum. 82. Dotted line A-
B is the line along which the pixel intensity was determined. Analysis of pixel
intensities shows co-localization of 0:1E-ir and VlP-ir in this nerve fiber. C, D.
Low-magnification epifluorescence photomicrograph illustrating a1E-VIP-ir in
nerve fibers supplying the longitudinal muscle in the tertiary plexus (arrowheads).
E, F. Epifluorescence (E) and confocal (F) images showing a1E-ir and VlP-ir co-
localization (arrowheads) in varicose nerve fibers in a myenteric ganglion in the

stomach. Scale bars = 200 pm (A1,AZ); 55 pm (8); 20 um (D-F).

95

A1 ILEUM 01E A2 ILEUM

B1 ILEUM 01E+V|P

C ILEALTP a1E+V|P 'D ILEALTP

E STOMACH 01E + VIP F STOMACH 01 E + VIP

 

96

Figure. 3.7. Confocal photomicrographs showing co-localization of 0:1E-ir
(red fluorescence) and NOS-ir (green) in the myenteric plexus. A1. NOS-ir in
neurons in the gastric corpus. A2. the same ganglion contains 0:1E-ir neurons.
A3. Merged (yellow) image of A1 and AZ showing overlap of 0:1E-ir and NOS-ir in
gastric myenteric neurons. 81. High magnification images showing NOS-ir
(arrowhead) localizes to myenteric neuronal cell bodies in the gastric corpus. BZ.
0:1E-ir (arrowhead) is present in the same neurons. B3. Merged image from
panels 81 and 82 shows co-localization of 0:1E-ir and NOS-ir in nerve cell bodies
(arrowhead). Varicosities containing 0:1E-ir without NOS-ir are also visible in
these images (arrow). C. Neurons (arrowheads) containing NOS-ir in an ileal
myenteric ganglion. D1. 0:1E-ir and NOS-ir are co-localized in pericellular
varicose nerve fibers (arrows). DZ. Pixel intensity analysis through varicosities in
D1. E1-E3: Co-localization of 0:1E-ir and NOS-ir in neurons in the myenteric
plexus of the proximal colon. The inset in D3 illustrates a string of 0:1E-NOS-ir
varicosities (arrowhead) in a myenteric ganglion from the proximal colon. Scale

bars = 200 pm (AI-A3); 50 pm (8-E3); inset in D3 = 10 pm).

97

A1

81

C

E1

STOMACH

NOS A2

STOMACH NOS B2

ILEUM

P. COLON

NOS + 01E D1
«

E2

STOMACH 01 E STOMACH NOS + O1E

STOMACH 01E STOMACH NOS + (11 E

ILEUM NOS + 01E

P. COLON (11E E3 P. COLON
NOS + G1E

 

98

0:1E-NOS-ir neurons/ganglion

 

 

Stomach Proximal colon Distal colon

Figure. 3.8. Mean number of neurons per ganglion obtained from 100 myenteric
ganglia, 19 plexus tissue sections and 4 guinea-pigs. The proximal colon
displayed the highest number of 0:1E-nNOS-ir neurons (5.7 1: 0.5), followed by
the distal colon (4.7 :l: 0.6) and then the corpus (3.9 :t 0.6). There was a
significant difference in the number of 0:1E-nNOS-ir neurons between the

stomach and proximal colon.

99

 

A1 Ileum 0:1E+SP

 

 

B Stomach 0:1E+VIP C Ileum 0:1E+NOS

 

Figure. 3.9. Co-localization of 0:1E-ir (red fluorescence) with SP-, VIP- and
NOS-ir (green fluorescence) in nerve fibers supplying the circular muscle
layer. A1. 0:1E-ir co-localized (yellow) extensively with SP-ir in the circular
muscle layer of the ileum. Inset shows a low magnification image of a1E-ir in the
nerve plexus in the circular muscle layer. A2. Line A-B indicates a line profile
analysis through the varicosities indicated by the arrowhead. B. 0:1E-ir nerve
fibers in the circular muscle co-expressed VlP-ir in the stomach. C. 0:1E-ir and
NOS-ir are co-localized in nerve fibers in the circular muscle layer in the proximal

. colon. Scale bars = 20 pm.

100

Figure. 3.10. Co-Iocalization of a1E-ir (red fluorescence) with TH-ir (green)
in the myenteric plexus. A. Merged (yellow) epifluorescence image showing
co-localization of 0:1E-ir and TH-ir in varicose nerve fibers (arrowheads) in a
myenteric ganglion in the ileum. B. Merged confocal image showing co-
localization of 0:1E-ir and TH-ir in varicose nerve fibers in a myenteric ganglion in
the ileum. Insets show individual images use to produce the main picture. C.
0:1E-ir and TH-ir co-localized in nerve fibers in ganglia (arrows) in the duodenum.
Insets show individual images of 0:1E-ir and TH-ir. D. An 0:1E-TH-ir nerve cell
body (arrowhead) and axon (arrow) in a ganglion in the proximal colon. Insets
show un-merged images of 0:1E-ir and TH-ir. Scale bars = 200 pm (A); 20 pm

(8); 16 pm (C) and 20 pm (D).

101

 

A Ileum 0:1E+TH B Ileum 0:1E+TH 0:1E

u '

 

I"

C Duodenum 0:1E+TH

i
_,Q t
\

102

0:1E-TH-ir neurons/ganglion

 

 

Proximal colon Distal colon

Figure. 3.11. The density of neuronal cell bodies co-localizing 0:1E and TH was
higher in the proximal (4.2 :I: 0.6) than in the distal colon (2.8 :t 0.5), and this

difference was significant at P <0.05.

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Figure. 3.12. Distribution of 0:1E (red fluorescence) with calbindin, SP,
calretinin, and VIP (green fluorescence) in dissociated myenteric neurons
in the guinea pig ileum. A1. 0:1E-ir (red) in neuronal cell bodies was
cytoplasmic in all myenteric neurons observed. B1. Calbindin-ir was observed
only in some neuronal cell bodies. C1, D1. Merged image (A1, 81) showing co-
Iocalization (yellow) of 0:1E- and calbindin in a neuronal cell body. AZ. 0:1E-ir
strongly localized within the cell soma and to lesser extent in axons. BZ. Intense
SP-ir was only detected around the periphery of myenteric cell bodies, and in
axonal processes. C2, D2. This cell appears to have some co-localization
(yellow, C2) at the periphery of the cell body. A3-D3. A cluster of three myenteric
neurons, two of which are 0:1E-ir. The third unlabeled neuron is positive for
neither 0:1E-ir nor SP. SP-ir was only present in neuronal processes. A4.
Myenteric neurons immunoreactive for 0:1E. 84. Two calretinin-containing
neurons show intense immunoreactivity in cell bodies. C4, D4. Calretinin-ir
almost masks the effect of 0:1E-ir labeling in this image, but these two
neurochemical markers do co-Iocalize within the cell bodies. A5-05. Prominent
0:1E-calretinin-ir in myenteric nerve fibers was observed, as evidenced by pixel
intensity analysis (05). A6. Cytoplasm and nerve fiber immunostained for 0:1E-
ir. 86. VIP-ir was only seen in the nerve fibers of myenteric neurons in culture.
No VIP immunostaining within the cell soma was visualized. C6-DS. Although
some co-localization for 0:1 E and VIP was present at the periphery of this 0:1E-ir
cell, there are two distinct neurons present, one 0:1E-ir, the other VIP-ir. No or

very little co-Iocalization is seen along the length of the axons.

104

is: Calbindin

wCZ Merge

 

 

‘B4 Calretinin

Merge (tp) C4 Merge
A

 

(11 E fibers B5 Calretinin fibers

 

 

 

Figure. 3.13. Co-Iocalization of o1E-ir with NOS, synaptophysin, SP with
synaptophysin, and 0:1E-ir with pan-cadherin in the guinea pig ileal whole
mount (WM) LMMP and myenteric neurons in culture. A1-D1. Confocal
images and line pixel intensity analysis demonstrating 01E and NOS-ir co-
localization (yellow) in cell bodies and axons in myenteric neurons in culture. AZ-
DZ. Confocal image showing an absence of co-localization between 01E and
synaptophysin-ir. Insets in AZ and 82 are low-magnification epifluorescence
photomicrographs of the LMMP showing intense but restricted ganglionic (g)
synaptophysin-ir; and a1E-ir throughout the LMMP respectively. A3-D3. (11 E and
synaptophysin do not co-localize in the tertiary plexus, but rare co-expression
(arrow in C3) was sometimes observed. A4-D4. Synaptophysin and SP strongly
co-Iocalized to varicosities in the myenteric plexus. A5-D5. In myenteric neurons
maintained in culture, 01E did not co-Iocalize with the membrane marker pan-
cadherin. A6-D6. In the LMMP, and with the rare exception of a few oIE-pan-
cadherin-ir varicosities, co-Iabeling for 01E and pan-cadherin was not

consistently detectable.

106

C1 Merge f

0 4

Q.

A3 or1E (tp)

C3 Merge (tp)

A5 Pan-cadherin

A2 Synaptophysin

9

B3 Synaptophysin (tp) A4 Synaptophysin

9

r 6 Pan-cadherin

107

82 01E

B6 01E

 

CHAPTER 4 CONTRIBUTION OF R-TYPE CALCIUM CHANNELS TO THE
FUNCTION OF INTRINSIC PRIMARY AFFERENT NEURONS AND
ASCENDING INTERNEURONS IN THE GUINEA PIG ILEUM MYENTERIC

PLEXUS

108

ABSTRACT
R-type calcium channels are expressed by subsets of myenteric neurons in the
guinea pig ileum. In the present study, I used intracellular electrophysiological
techniques preparation to determine the function of R—channels in myenteric
neurons in the acutely isolated longitudinal muscle-myenteric plexus. I used the
non-selective calcium channel antagonist, CdCIz (100 uM), the R-type calcium
channel blockers NiCI2 (50 pM) and SNX-482 (0.1 0M), and the N-type calcium
channel blocker w-conotoxin GVIA (CTX 0.1 uM). I studied the effects of these
drugs on action potentials and fast and slow excitatory postsynaptic potentials
(fEPSPs, sEPSPs) in S and AH neurons. CTX, but not NiClz, blocked sEPSPs in
AH neurons. NICI2 (n = 14) and SNX-48Z (n = 6) inhibited purely cholinergic, but
not mixed cholinergic purinergic, fEPSPs in S neurons. MCI; and SNX-482
reduced the duration and amplitude of action potentials in AH but not S neurons.
NiCI2 also reduced the amplitude of the late afterhyperpolarization (n = 4). These
data show that R-type calcium channels do not contribute to calcium entry into
nerve terminals releasing mediators of sEPSPs recorded from AH neurons but
they do contribute to release of acetylcholine as the mediator of fEPSPs in a
subset of S-type neurons. These data indicate that R-type calcium channels may
be target for drugs that could selectively modulate activity of AH neurons or could

alter fast synaptic excitation is subsets of pathways in the myenteric plexus.

109

INTRODUCTION

The enteric nervous system (ENS) is a division of the autonomic nervous
system dedicated to local control of gastrointestinal (GI) function. The ENS
exerts this control via intrinsic primary afferent neurons (IPANS), interneurons
and motor neurons that reside in enteric ganglia within the gut wall. The
myenteric plexus is located between the circular and longitudinal smooth muscle
layers and controls GI motility. The submucosal plexus, found between the
mucosa and circular muscle, facilitates absorptive and secretory responses
(Costa et al., 2000; Brookes, 2001). Together, these neural networks produce a
myriad of coordinated GI responses (Wood, 2006a). Efficient functioning of the
GI tract therefore relies on the integrative behavior of the enteric neurocircuitry.

Two types of myenteric neurons have been distinguished according to
their electrophysiological properties: AH- and S-type neurons (Hirst el‘ al., 1974).
AH-type neurons are the IPANs, and S-type neurons are interneurons and
motorneurons (Furness et al., 1998). AH neurons have a prominent shoulder on
the repolarizing phase of the action potential due to inward calcium currents
through one or more voltage-gated calcium channels. Calcium influx generates a
long-lasting (1-20 s) late afterhyperpolarization mediated by activation of
intermediate conductance calcium dependent potassium (lK) channels (Furness
et al., 2004). Trains of electrical stimuli applied to interganglionic nerve fibers
evoke slow excitatory postsynaptic potentials (sEPSPs) that last seconds to
minutes in AH neurons. A prominent mediator of the sEPSP is substance P (SP)

acting at G-protein-coupled neurokinin-3 receptors (Galligan et al., 2000; Furness

110

et al., 2004; Wood 8: Kirchgessner, 2004). The action potential in S-type neurons
is blocked by the voltage-activated sodium channel antagonist tetrodotoxin ('I'I'X)
(Hirst et al., 1974). Single stimuli applied to interganglionic nerve fibers produce a
fast EPSP (fEPSP) in S-type neurons. ACh, acting at nicotinic cholinergic
receptors (nAChR), is the major excitatory neurotransmitter in S neurons.
However, non-cholinergic fEPSPs have also been observed and these fast
synaptic responses are mediated by ATP acting at P2X purinergic receptors
(LePard et al., 1997; LePard 8: Galligan, 1999; Galligan, 2002a).

Myenteric neurons express all high-voltage-activated calcium channels (L,
N, PIQ, and R-type), but not low-voltage-activated T-type calcium channels
(North 8: Tokimasa, 1987; Kirchgessner & Liu, 1999; Reis et al., 2000).
Neurotransmitter release, contraction and secretory functions in the ENS are
thus regulated by high-voltage-activated calcium channels (Smith et al., 2003).
The dihydropyridine-sensitive L-type calcium channel is found mainly on GI
smooth muscle cells, and is not an active participant in synaptic excitation within
the plexus (Marino et al., 1993). L—, N—, P/Q- and R-type channels are present
mainly on neurons and open at more depolarized voltages (~ -30 mV; maximum
activation ~ 0 mV) and generally inactivate slower (> 100 ms) (Bean, 1989;
Hoffman et al., 1994; Yamakage 8. Namiki, 2002). The R (resistant) current may
activate at somewhat more negative voltages (~ -40 mV) and often inactivates
more rapidly than the other high-threshold currents (Fox et al., 1987; Lorenzon &
Foehring, 1995; Foehring et al., 2000). HVA channels subtypes are blocked by

different drugs. N- and PIQ-type calcium channels are blocked by w-conotoxin

111

GVIA (w-CTX) (Barajas-Lopez et al., 1996; Chen & Kirchgessner, 2002) and 00-
agatoxin IVA (w-ATX) (Tsien et al., 1988; Catterall, 2000; Bian et al., 2004)
respectively. Low concentrations of NiClz (30-50 uM) and the spider toxin SNX-
482 (0.1-1 uM) block R-type calcium channels (Randall 8 Tsien, 1995; Newcomb
et al., 1998; Wang et al., 1999; Tottene et al., 2000).

R-type calcium channels have been implicated in neurotransmitter release
at rat hippocampal mossy and associative-commissural fiber synapses
(Gasparini et al., 2001), oxytocin release from rat neurohypophysial terminals
(Wang et al., 1999), and excitation-contraction coupling (Wu et al., 1998; Albillos
et al., 2000). While a role for R-type calcium channels in the central nervous
system (CNS) has been established, the function of the R-type calcium channel
in the ENS is not known. Bian et al. (2004) showed that 50 % of the calcium
current in guinea-pig ileal myenteric neurons maintained in primary culture is
carried by R-type channels. The R-type channel was also shown to be coupled
to the az-adrenoreceptor in guinea-pig myenteric neurons, implying that R-type
channels may be targets for sympathetic nerve-derived norepinephrine to
regulate synaptic transmission (Bian & Galligan, 2007). The co-Iocalization of the
R-type channel with SP, calretinin, vasoactive intestine peptide (VIP) and nitric
oxide synthase (NOS) in varicose nerve fibers and cell bodies of calbindin-
positive myenteric neurons in the guinea-pig ileum (Naidoo et al., 2006) strongly
suggests that R-type calcium channels may mediate calcium entry into specific

subsets of myenteric neurons.

112

The objective of my study was to determine the contribution of R—type
calcium channels to synaptic transmission in the acutely-isolated longitudinal
muscle-myenteric plexus (LMMP) preparations of the adult guinea pig ileum. The
native synaptic connections present in the guinea pig intestine in vivo are
preserved in this in vitro preparation. I therefore investigated whether calcium
entry through R-type calcium channels contributes to synaptic transmission or

action potentials in guinea pig ileum myenteric neurons.

MATERIALS AND METHODS
Tissue collection and preparation
All animal use protocols were approved by the Institutional Animal Care
and Use Committee at Michigan State University. Adult male Hartley guinea pigs
(Z50-300 g, Bioport, Lansing, MI) were anesthetized with isoflurane (Abbott
Laboratories, Chicago, IL), stunned by a blow to the back of the head and
exsanguinated. A segment of the ileum approximately 10 cm proximal to the ileo-
cecal junction was quickly removed and placed in pre-warmed (37 °C)
oxygenated (95% 02; 5% C02) Krebs’ solution of the following composition (in
mM): 117 NaCl, 4.7 KCI, 2.5 CaClz, 1.2 MgClz, 1.2 NaHzPO4, 25 NaHCOg, and
11 glucose. The Krebs’ solution contained scopolamine (1 pM) and nifedipine (1
pM) to block muscarinic receptors and L-type calcium channels, respectively. A 5
mm2 section of the ileum was cut open along the mesenteric border and pinned
flat (Fine Science Tools, Foster City, CA) in a silicone elastomer-Iined (Sylgard,

Dow Corning, Midland, MI) petri dish containing Krebs’ solution. The submucosa

113

and circular muscle were peeled away and the longitudinal muscle-myenteric
plexus (LMMP) was transferred to a smaller silicone elastomer-lined perfusion
chamber. The chamber was mounted on a stage of an Olympus microscope
(Tokyo, Japan). The LMMP was pinned tautly and superfused continuously with
warm (37 °C) Krebs’ solution at a flow rate of 4 ml/min. The preparation was

equilibrated for 30 min before commencing intracellular recordings.

Intracellular electrophysiological recordings

Myenteric neurons in the acutely isolated LMMP were impaled with a 2 M
KCI-containing glass microelectrode (Frederick-Haer, Brunswick, ME) with a tip
resistance of 80-120 Mn. Membrane potential was recorded using an Axoclamp
2A amplifier (Axon Instruments, Foster City, USA) in bridge mode. Synaptic
potentials were evoked focally with a monopolar glass electrode (tip diameter 60
pM) filled with Krebs’ solution (World Precision Instruments, Sarasota, Florida) by
stimulating interganglionic nerve fiber tracts. The resting membrane potential
(RMP) of the impaled neuron was allowed to stabilize for 10 minutes without
applying intracellular holding current. Myenteric (AH and S-type) neurons were
then classified electrophysiologically according to published criteria (Hirst et al.,
1974; Morita & North, 1985; Wood, 1996). Short high frequency trains of
stimulation (20 Hz, 500 ms), provided by a pulse generator (Master 8, A.M.P.I.,
Jerusalem, Israel) and a constant current stimulation unit (Grass Technologies,
West Warwick, RI) were used to elicit slow excitatory postsynaptic potentials

(sEPSPs). Single stimuli (0.5 ms duration, 0.1 Hz stimulus rate, 4-9 mA, 10 s

114

intervals) were used to evoke fast excitatory postsynaptic potentials (fEPSPs).
When recording fEPSPs, the membrane potential was hyperpolarized to -90 mV
to prevent fEPSPs from reaching action potential threshold. Signals were
recorded using an analog to digital converter (Digidata 1322A, Molecular Devices
Corp., Sunnyvale, CA) and Axoscope and Clampfit 10 software (Molecular
Devices), and a desktop computer. Amplified signals were sampled at 2 kHz,
filtered at 1 kHz and a digital average of 6 fEPSP sweeps was then obtained.
The amplitudes of synaptic responses were measured under control conditions
and after various drug treatments.

Action potentials (APs) and late afterhypolarizations (late AHPs) from AH
neurons, and APs from S-type neurons, were elicited by injection of brief
intracellular depolarizing current pulses (2 ms duration, 0.1 Hz, 0.02-1 nA, 6V, at
10 s intervals) through the recording electrode (De Laet et al., 2002; Nurgali et
al., 2007). AH neurons were identified by the prominent calcium shoulder on the
repolarization phase of the action potential (Hirst et al., 1985). Myenteric AH and
S-type APs and late AHPs were recorded at the resting membrane potentials of
those neurons. I measured the amplitude, the half-maximum width, and the area
under the curve (AUC) of the evoked APs and late AHPs. Half-maximum width
was defined as the time at which the postsynaptic response is at half of its
maximum value (Schneider, 2008). Neurons with RMPs less than -40 mV were

excluded from the study.

115

Drug application
Local drug application was accomplished using a quartz micropipette (30
-— 40 pm tip diameter) placed within 50 to 150 pm of the impaled neuron. Drugs
were superfused near the impaled neuron and solution flow was gated using a
VC-8 Valve Controller application system (Warner Instruments, Hamden, CT).
The impaled neuron was exposed to drug treatments for at least 6 minutes

before responses were recorded.

Drugs
Nifedipine, scopolamine, pyridoxaI-phosphate-6-azophenyl-2',4'-
disulfonate (PPADS), NICIz, and mecamylamine hydrochloride were obtained
from Sigma-Aldrich. w-conotoxin GVIA (CTX), SNX-482 and tetrodotoxin (TTX)
were purchased from Alomone labs (Jerusalem, Israel). All drugs were diluted in

deionized water except for nifedipine which was dissolved in 95% ethanol.

Statistical analyses
Data are presented as mean values :I: S.E.M for n neurons. Concentration-
response data were fitted using non-linear regression and the Hill equation
(GraphPad Prism 4, GraphPad, San Diego). Statistical differences between
groups were analyzed with a t-test or one-way ANOVA with Tukey-Kramer post—
hoc test comparisons. P < 0.05 was the criterion for determining statistical

significance.

116

RESULTS
CdCIz inhibits action potentials in AH but not S neurons

To establish whether blockade of calcium channels would inhibit the
calcium shoulder on the descending phase of the AH action potential, I applied
the non-selective calcium channel antagonist CdCIz (100 pM) (Jun et al., 2004) to
the perfusion bath. In all 5 neurons tested, the calcium shoulder was blocked by
CdCl2 and this shortened the action potential half-width (Figure. 4.1A,B); CdCIz
did not change action potential amplitude in AH neurons (Figure. 4.1A,C). After
washout of CdClz, the voltage-gated sodium channel antagonist TTX (0.3 pM)
was applied. TTX reduced action potential amplitude but not half-width (Figure.
4.1A—C). In all 5 neurons examined, CdClz did not affect the amplitude or half-
maximum width of the action potential (Figure. 4.2). However, the action potential

in S neurons was blocked by TTX (Figure. 4.2).

R-type calcium channel blockers inhibit action potentials AH- but not S-
neurons

l next determined the effect of the selective R-type calcium channel
blocker NlClz (50 pM) (Tottene et al., 2000; Gasparini et al., 2001) on the AH and
S action potential. NiClz reduced the AH neuron action potential half width by
44% and the peak amplitude by 9% (n=12 P < 0.05; Figure. 4.3). However, NiCl2
(50 pM) did not change the action potential in S neurons (n=1Z, Figure. 4.4).
These data provide pharmacological evidence that R-type calcium channels

contribute to the action potential in AH but not S-type neurons.

117

Previous studies indicate that the calcium hump on the descending phase
of the myenteric AH action potential is due to calcium entry through primarily N-
type calcium channels (Franklin et al., 1992; Furness et al., 1998; Kang et al.,
2003). Strong immunohistochemical evidence points to the presence of N-type
channels in calbindin-containing myenteric IPANS (Kirchgessner & Liu, 1999).
On this basis, a significant portion of the calcium current is likely to be the N-type
current. Analysis of the AH action potential in 3 neurons demonstrated that NiCl2
(50 pM) and w-CTX (0.1 pM) did not affect the amplitude of the action potential
(Fig. 4.5A,B). However, NICIz and w-CTX produced additive reductions in the
action potential half width in AH neurons.

At low concentrations, SNX-48Z (0.1 pM) is a specific antagonist of R-type
calcium channels (Newcomb et al., 1998; Gasparini et al., 2001; Bian et al.,
2004). l next determined whether SNX-482 inhibited action potentials in AH
neurons. SNX-482 did not affect the mean amplitude of the AH action potential
but it did reduce the action potential half-width (Figure. 4.SD,E). The subsequent
addition of w-CTX, produced a further inhibition of action potential half width
(Figure. 4.5D) and also reduced the peak action potential amplitude. These data
indicate that : 1) NiClz and SNX-48Z produce an almost identical effect on the
action potential in AH neurons; and 2) N- and R-type calcium channels are the
predominant contributors to the action potential in myenteric AH neurons. Since
CdClz and NiClz did not alter the action potential of S-neurons, I therefore did not

test the effects of SNX-48Z or w-CTX on the action potential of those neurons.

118

NiCI2 inhibits the slow AHP in AH neurons

As NiClz reduced the calcium shoulder on the action potential in AH
neurons then the amplitude of the slow AHP should correspondingly decrease as
the slow AHP is mediated by a calcium activated potassium channel. As
anticipated, NICIz (50 pM) reduced the peak AHP amplitude by 42 % (Figure.
4.6A,B). NiClz also shortened the time course of the slow AHP as reflected by
the reduction in the area under the curve (Figure. 4.6A,C) which was reduced by

46%.

R-type calcium channels do not contribute to slow synaptic excitation of
AH neurons.

If R-type calcium channels contribute to the somal AH action potential, the
next question I asked was whether R-type calcium channels contribute to release
of the mediators of slow excitatory synaptic potentials (sEPSPs) in AH neurons.
Enteric neurons use two means of communication, fast and slow chemical
neurotransmission (Galligan, 2002b). Because neurotransmitter release from
myenteric nerve terminals is regulated by voltage-gated calcium channels (Smith
et al., 2003), I first ensured that blockade of all calcium channels was feasible.
The non-selective calcium channel blocker, CdClz (100 uM), was used to block
sEPSPs evoked by short trains (20 Hz 500 ms) of focal electrical stimulation
applied to of interganglionic nerves strands, In all 5 AH neurons tested, CdClz
(100 uM) largely blocked the sEPSP (Figure. 4.7A). The mean amplitude of the

control sEPSP was 21.13 :I: 0.94 mV, while the mean amplitude in the presence

119

of CdClz was 1.2 :I: 0.3 mV. A complete concentration-response curve revealed a
concentration—dependent calcium channel inhibition by CdCl2 (1 — 100 uM) (n =
5; i050 ¥—- 12 pM) (Figure. 4.73). The decline in sEPSP amplitude caused by
CdClz was not related to a time-dependent decline in synaptic transmission
because time controls revealed that sEPSP amplitude was stable overthe time
course of the pharmacology experiments (Figure. 4.70).

The effect of MCI; (50 pM) on sEPSP was studied in 16 AH neurons and it
was found that NiCIz did not change sEPSP amplitude in any of these neurons
(Figure. 4.8A,B). R-type calcium channels therefore do not contribute to slow
synaptic excitation in AH myenteric neurons. To determine the voltage-gated
channel subtype responsible for the sEPSP, I tested w—CTX (0.1 uM). In all 4
neurons tested with NiCIz resistant sEPSPs, w-CTX largely blocked the sEPSP
(Figure. 4.9A,8). These results suggest that predominantly N-type calcium

channels mediate sEPSPs in myenteric IPANS.

R-type calcium channels do not contribute to cholinergic/purinergic
fEPSPs in S-type neurons

CdCI2 (1—100 pM) blocked fEPSPs recorded from S neurons in a
concentration-dependent manner (Figure. 4.10A,B; n=5; IC50=10 pM). To
demonstrate that this inhibition was due to calcium channel inhibition and not to a
time dependent decline in synaptic transmission, time-control experiments were

done where fEPSPs were evoked in drug-free conditions in 5 min intervals for 30

120

min. The amplitude of fEPSPs (n = 7; Figure. 4.10C) did not significantly change
during this time period.

Previous studies revealed the presence of three phannacologically distinct
subsets of myenteric S-type neurons based on the mediators of fEPSPs in these
cells. There are cholinergic fEPSPs (25% ‘of neurons), mixed
cholinergic/purinergic fEPSPs (67 % of neurons), and cholinergic/serotonergic
fEPSPs (11 % of neurons) (Galligan & Bertrand, 1994; LePard 8: Galligan, 1999;
Zhou & Galligan, 1999). I therefore next asked whether R-type calcium channels
contribute to fast synaptic transmission in one of more of these classes of S-type
neurons. To answer these questions, I used NiCl2 (50 pM), the nAChR
antagonist mecamylamine (10 pM), and the P2X receptor antagonist PPADS (10
pM) (Galligan, 20023; Ren et al., 2003) in an effort to block fEPSPs.
Mecamylamine is a more potent nAChR antagonist than hexamethonium in
myenteric neurons (Zhou et al., 2002). In a subset of 15 neurons tested, NiC|2 did
not affect fEPSP amplitude (Figure. 4.11 A,B). Subsequent application of
mecamylamine significantly reduced the fEPSP amplitude by 28 % (Figure.
4.11A,B; P < 0.05). Subsequent addition of PPADS further reduced the fEPSP
amplitude by 77 % (Figure. 4.11A,B; P<0.001) compared to control. I then
attempted to mimic the effect of NiCl2 (50 uM) with SNX-48Z (0.1 uM) on the
fEPSP in 3 S-type neurons with mixed cholinergic/purinergic fEPSPs. SNX-48Z
did not change fEPSP amplitude in these neurons but subsequent application of
w-CTX (0.1 uM) reduced the fEPSP amplitude by 76 % (Figure. 4.12 A,8;

P<0.05). These results indicate that calcium entry via primarily N-type, but not R-

121

type calcium channels mediate fEPSPs in cholinergic/purinergic S-type myenteric

neurons.

R-type calcium channels contribute to cholinergic fEPSPs in S-type
neurons

l next tested the hypothesis that the R-type channel is present at nerve
endings using only acetylcholine as the fast synaptic transmitter. In 14 S-type
neurons NiClz (50 0M) reduced the fEPSP by 23 % (Figure. 4.13A,B; P < 0.05).
NICI2 was washed out and in these cells mecamylamine (10 uM) inhibited the
fEPSP by 95 % (Figure. 4.13A,8; P <0.01). In the next experiments (n = 6), | first
ensured that mecamylamine completely blocked the fEPSP. Then NiClz (50 pM)
reduced the fEPSP by 45 % (Figure. 4.13C, P < 0.001) followed by w-CTX (0.1
pM) which inhibited the fEPSP amplitude by 42 % compared to NiClz alone (P <
0.05).

I finally determined whether SNX—482 (0.1 pM) would again mimic the
effect of MCI; (50 pM). In 4 neurons examined, mecamylamine completely
inhibited the fEPSP (not shown). After washout of mecamylamine SNX 482 was
applied and it reduced the fEPSP by 40 % (Figure. 4.14A,B; P<0.01). Addition of

w-CTX (0.1 uM), then further reduced the fEPSP amplitude by 69 % (Figure.

4.14A,B; P < 0.01).

122

DISCUSSION

The ability of the ENS to effect appropriate responses upon receiving
different types of information is due to its functional interconnectivity and
sensitivity (Furness & Costa, 1987). The manifestation of plasticity within enteric
neural circuits as a consequence of distention or chemical stimulation of the
mucosa, is an inherent property of the adaptive and integrative behavior of the
ENS. The neurochemistry and synaptic connections of enteric neurons shape the
function of the enteric neural circuitry. The synaptic connections focus attention
on voltage-gated gated calcium channels in synaptic transmission in the ENS.
Voltage-gated calcium channels regulate the functions of IPANS, interneurons
and motorneurons, and tailor reflex circuits to motility and secretory patterns
(Holzer et al., 1997). Electrophysiological studies demonstrated that ~50 % of the
calcium current in ileal guinea pig myenteric neurons maintained in primary
culture is the R-type calcium current (Bian et al.,2004). That study, however, did
not identify the specific subsets of myenteric neurons from which the R—currents
were recorded. lmmunohistochemical data later established the presence of R-
type calcium channels in neurons and varicosities in the myenteric plexus in the
adult guinea pig ileum (Naidoo et al., 2006). The present study provides the first
evidence that R-type calcium channels contribute to the action potential in AH-
type neurons, and to synaptic transmission in a subset of S-type neurons in the

guinea pig LMMP.

123

The AH neuron action potential is N- and R-type calcium channel-
dependent

To reconcile the above descriptions of R-type calcium channels in the
myenteric plexus, I first looked at the kinetics of action potentials from AH and S-
type neurons. NiCl2 (50 pM) and SNX-482 (0.1 pM) did not affect the action
potential amplitude or half-maximum width in S-type neurons. However, in AH
neurons, both MCI; and SNX-482 significantly attenuated the mean peak
amplitude and the half-maximum width in comparison with the control action
potential. w-CTX (0.1 pM) largely inhibited the remainder of the calcium shoulder
on the repolarization phase of the AH action potential. The calcium shoulder is
due in part to activation of a TTX-insensitive N-type channel activated calcium
conductance in myenteric AH neurons (Vogalis et al., 2001; Rugiero et al., 2002).
Moreover, strong N-type, but weak PIQ-type calcium channel immunoreactivity
was found in IPANs (Kirchgessner & Liu, 1999). PIQ-type calcium channels
(Rugiero et al., 2002) and L-type channels do not appear to contribute to the
calcium shoulder in guinea pig myenteric neurons (North & Tokimasa, 1987;
Kunze et al., 1994). Interestingly, Rugiero et al. (2002) found a residual calcium
current after blockade of N-, L- and PIQ-type channels, and postulated a
contribution by the R-type channel to the somal action potential in AH neurons.
My results demonstrate that when the R-type and N-type calcium channels are
blocked in tandem, the duration of the AH action potential is reduced by
approximately 95 %. F urtherrnore, when I examined the effect of R-type channels

on the late AHP, I found that NiCIz inhibited the late AHP amplitude by 42 %.

124

Taken together, my results confirm that primarily N-type calcium channels (~ 54
%) regulate calcium influx during action potential in AH-neurons. However, I
show for the first time that R-type calcium channels (~ 40 %) also contribute
significantly to calcium entry during action potential firing in the somatodendritic
region of lPANs.

R-type calcium channels have been localized in neurons throughout the
CNS (Gasparini et al., 2001; Lee et al., 2002; Dietrich et al., 2003; Weiergraber
et al., 2008). In mouse hippocampal CA1 pyramidal neurons (Kavalali et al.,
1997; Isomura et al., 2002; Yasuda et al., 2003) and amygdala neurons (Lee et
al., 2002), R-type calcium channels participate in dendritic action potential
generation. Inhibition of calcium channels in amygdala neurons heightened
excitability and reduced the AHP amplitude in those neurons (Schiess et al.,
1993; Schiess et al., 1999). My data indicate that in the ENS, R-type calcium
channels may contribute to the AH neuron action potential by regulating somatic
and dendritic excitability in lPANs. This is supported by previous evidence that R-
type channels require strong depolarizations and that R-type currents have fast
activation kinetics in rat cortical (Lorenzon & Foehring, 1995; Foehring et al.,
2000) and guinea pig myenteric neurons (Bian et al., 2004). The late AHP
maintains the resting conductance of AH neurons via voltage-insensitive IK
channels that limit the firing rate of AH neurons to one or two action potentials in
response to a depolarizing intracellular current pulse (Galligan et al., 2000;
Furness et al., 2004). When R-type calcium channels on the somatodendritic

region of lPANs were inhibited, the late AHP was correspondingly attenuated. R-

125

type calcium channels are therefore important contributors to the regulation of

myenteric AH neuron excitability.

R-type calcium channels do not contribute to slow synaptic excitation in
AH-type neurons

Slow synaptic excitation is mediated by G-protein coupled receptors that
produce long-lasting slow-synaptic responses with a long latency. SP acting at
neurokinin-3 (NK3) and ACh acting at M muscarinic receptors, are the principal
mediators of slow excitatory neurotransmission in the ENS (Guard et al., 1988;
Costa et al., 2000; Galligan, 2002b). A novel finding from the present study
points to N-type rather than R-type calcium channels as the chief mediators of
sEPSPs in AH myenteric neurons. This conclusion is based on the observation of
an 89 % inhibition of the evoked sEPSP amplitude by w-CTX. N-type calcium
channels predominate on autonomic nerve terminals (Mochida et al., 1996) while
PIQ-channels are more prevalent on nerve terminals in the CNS (Takahashi &
Momiyama, 1993). Those channel subtypes contribute to synaptic transmission
in those regions (Hirning et al., 1988; Miller et al., 1988; Dolphin, 2003b). My data
suggest that in the ENS, trains of stimulation could cause calcium entry through
N-type channels to release SP. SP would then act at the postsynaptic NK3
receptor through protein kinases A (Morita & North, 1985; Bertrand 8: Galligan,
1995) or C (Guard et al., 1988; Bertrand & Galligan, 1995) to produce the

sEPSP.

126

R-type calcium channels contribute to fast synaptic excitation in a subset
of S-type neurons

Since R-type calcium channels are not involved in slow synaptic
excitation, l next investigated their role in fast synaptic transmission. In the ENS,
fast synaptic transmission in the ENS is accomplished via ligand-gated ion
channels where acetylcholine (ACh) acting at nAChRs, ATP at P2X receptors
and 5-HT at 5-HT3 receptors are the predominant mechanisms of fast excitatory
neurotransmission (Galligan, 2002a). My results demonstrate that nerve
terminals releasing only acetylcholine as a fast synaptic transmitter in S-type
neurons are sensitive to NiClz and SNX-482. Further, R-type channels are not
present on nerve terminals releasing both acetylcholine and ATP as fast synaptic
transmitters as amplitude of the fEPSP does not change in the presence of either
NiClz or SNX-482. In both groups, w-CTX inhibited the fEPSP amplitude,
suggesting that N- and R-type channels are intimately involved in modulation of
neurotransmitter release from nerve terminals of fully cholinergic myenteric S-
neurons.

I believe that R-type calcium channels are localized to nerve terminals of
fully cholinergic S-type ascending interneurons. Ascending interneurons use ACh
acting at nAChRs and constitute 5 % of the total number of myenteric neurons in
the guinea pig LMMP (Brookes, 2001). Descending interneurons use ACh acting
at nAChRs, and ATP acting at P2X and PZY receptors. I concluded that R-type
calcium channels are present on the nerve endings of ascending interneurons

because those neurons were completely sensitive to mecamylamine (Galligan &

127

Bertrand, 1994; LePard et al., 1997; LePard & Galligan, 1999). In a second
subset of S-type interneurons, PPADS (10 pM) blocked the ATP-mediated
component of the mixed cholinergic/purinergic fEPSP (LePard et al., 1997). In
fact, the fully cholinergic NiClz—sensitive S-neurons observed in my study may be
calretinin-coded (Naidoo et al. to be submitted). LePard and Galligan (1999)
surgically interrupted all nerve fibers to study the ascending and descending
projections of myenteric interneurons. They found that ascending interneurons
use only ACh for neurotransmission, whereas descending interneurons use both
ACh and ATP.

The involvement of R-type calcium channels in fast synaptic excitation in
the CNS is conflicting (Gasparini ef al., 2001; Dietrich et al., 2003). Dietrich et al.
(2003) showed that presynaptic R-type calcium channels do not participate in
fast neurotransmitter release at the mossy fiber-CA1 synapse in adult mice
hippocampal neurons. However, separate evidence from neonatal rat
hippocampal neurons showed that R-type channels contribute to fast synaptic
transmission, and may be important for regulating neurotransmitter release and
modulation of synaptic plasticity (Gasparini et al., 2001). These contrasting
observations may be due to species and/or developmental differences.
Nonetheless, my data corroborate those of Gasparini and others (2001). At the
mossy and associative-commissural fiber fast synapses, low concentrations of
NICIz (50 uM) and SNX-482 (0.3-1 uM) inhibited the amplitude of excitatory
postsynaptic currents (EPSCs) equally (Gasparini et al., 2001). The addition of

SNX-482 followed by NiClz did not further inhibit the EPSC amplitude. However,

128

an antagonist of N- and PIQ-type calcium channels (to-CTX-MVIIC) significantly
reduced the EPSC amplitude, indicating a role for R-type calcium channels in
fast synaptic transmission. MCI; and SNX-482 are also mutually occlusive in
myenteric neurons maintained in culture (Bian et al., 2004). In the present study,
NiCIz (50 uM) significantly inhibited the fEPSP by 23 % (n = 14) and by 45 % (n =
6). This discrepancy could be because of low R-type channel expression in some
of those (23 %-containing) S-neurons from which fEPSPs were evoked. SNX-482
attenuated the mean fEPSP amplitude by 40 %. Taken together, these results
suggest that MCI,» and SNX-482 act specifically on the same pathway to inhibit
fast synaptic transmission in S-type neurons. Whether NiCIz and SNX-482 are
acting pre- or postsynaptically to inhibit the fEPSP remains to be determined as it
is possible that R-type calcium channels also actively participate in synaptic
integration.

A synaptic interaction protein site (synprint) site important for interaction
with the vesicular release machinery (synaptobrevin, syntaxin and SNAP-25; the
SNARE complex) was believed to be unique to N- and PIQ-type but not R-type
channels (Maximov et al., 1999). N- and PIQ-type channels physically and tightly
couple to syntaxin 1A and SNAP-25 located on the nerve terminal membrane
(Jarvis & Zamponi, 2001). R-type channels, however, may functionally interact
with the SNARE complex at a putative unidentified interaction site (Bergsman &
Tsien, 2000; Wiser et al., 2002; Cohen et al., 2003; Cohen & Atlas, 2004). This
Dre-association of the R-type channel with synaptic proteins suggests these

Calcium channels may be responsible for rapid synaptic vesicle exocytosis. My

129

data suggest that R- and N-type calcium channels spatially co-operate to
facilitate efficient neurotransmission in fully cholinergic myenteric S neurons. In
fact, this co-operativity between R- and N-type channels has been observed at
the mouse endplate (Urbano et al., 2003). R-type channels may be also located
closer to the calcium sensor than N-type channels (Urbano et al., 2003), possibly
to synaptotagmin for fast synchronous neurotransmitter release (Catterall & Few,
2008). Therefore, in myenteric S-neurons, a possible mechanism for synaptic
transmission via R-type channels could be: 1) a train of action potentials
depolarizes the (NiCIz-sensitive) S-neuron nerve terminal allowing calcium to
enter though R-type channels, 2) calcium activates calcium/calmodulin-
dependent (CAM) protein kinase II that then phosphorylates the cytoskeletal
tethering protein synapsin on the SP-containing vesicle, 3) the vesicle is targeted
to the docking site, 4) the low affinity calcium sensor synaptotagmin binds to
syntaxin once calcium enters the cell, and exocytosis occurs. Taken together, R-
type calcium channels may therefore contribute to fast synaptic excitation in the
myenteric plexus of the guinea pig ileum.

The question then emerges, why are R-type calcium channel localized to
ascending interneurons and specifically to a fully cholinergic subset of S-type
neurons in the ENS? Does the ENS, by utilizing several calcium channel
subtypes, employ a compensatory mechanism to allow efficient synaptic
transmission? First, the topographical organization of N- and R-type calcium
channels on nerve terminals may be important to short- and long-term synaptic

plasticity for the fine-tuning of information encoding. Calcium channels are

130

modulated by G87 subunits released from Gi/Go proteins (Dolphin, 2003b). R-
type calcium channels may be coupled to the inhibitory az-adrenoreceptor (Bian
& Galligan, 2007). Furthermore, the SNARE complex enhances efficient
exocytosis if calcium channels are close to the active zone. These properties
allow synaptic transmission to be precisely regulated (Catterall & Few, 2008).
The presence of both N- and R-calcium channel subtypes may serve to enhance
synaptic transmission for the effective coordination of gut effector system (GI
smooth muscle, secretory glands and blood vessels) responses.

Second, the guinea pig ileum contains only a single class of ascending
interneurons, and these neurons provide synaptic input to other ascending
interneurons and to excitatory circular and longitudinal muscle motorneurons
(Brookes, 2001). It remains possible that the R-channel is present in ascending
interneurons to ensure that transmission proceeds unimpeded. R-type calcium
channels, located on purely cholinergic S-neurons could, by co-operating with N-
type calcium channels, serve as “booster stations” for ACh release as ACh is the
predominant mediator of fast synaptic excitation in the ENS. This is supported by
data that R-type channels are more closely associated with the synaptic release
machinery than N-type channels (Urbano el‘ al., 2003). R-type channels may not
mediate fEPSPs through mixed cholinergic/purinergic S-neurons, presumably
because the release of two transmitters, ACh and ATP, could increase the
overall probability of successful neurotransmission. These neurons would rely on
calcium entry via only N-type calcium channels for exocytosis. The functional

significance of R-type channels in S-neurons may thus be one of redundancy.

131

SUMMARY AND CONCLUSIONS
R-type calcium channels in the guinea pig ileum make a substantial contribution
to the action potential in AH neurons. The calcium inflection on the downstroke of
the AH action potential is mainly mediated by both N- and R-type calcium
channels. R-type channel blockers significantly inhibited the fEPSP in ascending
interneurons that release only ACh. N-type channels also contribute to fast
synaptic excitation in ascending pathways. I did not detect R-type channel
mediation of cholinergic/purinergic S-neurons. However, N-type channels were
the primary contributors to the fEPSPs in those neurons. R-type calcium
channels may therefore be important to control of cholinergic synaptic excitation.
This could have profound implications for the overall function of the ENS as ACh
predominates fast synaptic transmission and is one of the most important

neurotransmitters in ENS neurons.

132

AH neurons

Control CdCIZ I190 PM) TTX (0-3 PM)
i
I
J ’ AA
/ IL.” /
fl b- *_T —\..-_ W, ..-.._ __ _-..-..- ‘—-\

 

 

Figure. 4.1. CdCIZ inhibits action potentials in AH neurons. (A) A 2 ms
intracellular depolarizing current pulse was used to evoke an action potential.
The calcium shoulder (arrow in Control trace) on the repolarizing phase of the
action potential was inhibited by CdCIZ. Tetrodotoxin (TTX, 0.3 pM) blocked the
Na+ sensitive component during the depolarizing phase of the action potential to
produce a smaller amplitude and slower calcium action potential. (8) Mean data
1: S.E.M (n = 5) showing that CdCIZ, but not TTX, reduced action potential half
width (***, P< 0.01 vs Control and 'I'I'X). (C) TTX but not CdCIZ reduced action

potential amplitude C“, P < 0.05 vs Control and CdCIZ).

133

S neurons

Control CdCIZ (100 Ml) TTX (0.3 MA)
I) .
W l M: K
L’M__ i \ 2...— —/
_'/—‘\ _f—'L fl

 

 

 

Figure. 4.2. CdCIZ does not affect action potentials in S-type neurons. (A)
CdClZ (100 pM) did not affect the action potential in S-type neurons while 'I‘I’X
(0.3 uM), completely blocked the action potential in the same neurons. (8) Mean

data :I: S.E.M (n = 5) from experiments similar to A.

134

AH neurons

Control NiCIZ (50 pM) Washout

.0.
AN W” L... /J\L_,_

N... .fla—v-m

-2.“ Mfl
.._..

Figure. 4.3. NiC12 inhibits the action potential in AH neurons.
Representative traces showing attenuation of the calcium shoulder (arrow in
Control trace) on the repolarizing phase of the action potential in the presence of
NiClZ. Mean data showing that NiCIZ shortened the action potential half-width (n
= 12; *** P < 0.001) in a reversible manner. NiClZ also reversibly reduced the
action potential amplitude * P < 0.05). Data are mean :I: S.E.M. n is the number of

neurons from which AH action potentials were elicited.

135

S neurons

Control NiCIZ (50 uM) Washout
WI WI N
k...___.._... ‘ L22... ' LOW“.

 

 

.f L _, a”

Figure. 4.4. Action potentials in S-type neurons are not sensitive to NiCIZ.
Representative traces showing that the amplitude and half-maximum width of the
S-type action potential are unchanged by NiCIZ. Mean data showing that NiClZ
(50 pM) did not change action potential half-width or peak amplitude. Data are

mean :I: S.E.M. n = 8 neurons.

136

AH neurons

Control NiCIZ (50 till) crx (0.1 pill)

fl) 3 /ll _ \
LL 1%..

A

— ——.~.-_—-Aa

 

 

Figure. 4.5. NiCIZ, SNX-482 and w-CTX inhibit the action potential in AH
neurons. Representative traces showing that NiCIZ (50 uM) reduced the
calcium shoulder. The residual shoulder was then completely blocked by w-CTX .
(0.1 uM). Mean data showing that the action potential half-width after NiCIZ-
treatment was smaller compared to control (n = 3; ** = P < 0.01). w-CTX further
reduced the half-width (*** = P < 0.001 vs. control). NiCIZ and w-CTX did not
affect the peak AH action potential amplitude (n = 3; P > 0.05). SNX-482 inhibited
the action potential width at half-amplitude compared to control (n = 3; * = P <
0.05). w-CTX then further attenuated the half-maximal width (n = 3; ** = P < 0.01
vs. control). The peak amplitude was inhibited by w-CTX but not SNX-482. Data

are mean :I: S.E.M. n is the number of neurons.

137

AH neurons

Control NiCIZ (50 pM) Washout

 

 

 

 

 

 

 

Figure. 4.6. The late afterhyperpolarization (late AHP) in AH neurons is
inhibited by NiCIZ. Representative traces showing that the amplitude and half-
maximum width of the late AHP are inhibited after application of NiClZ (50 uM).
Mean data showing that the amplitude of the late AHP is reduced after addition of
MCI; (50 uM) treatment compared to control (n = 4; * = P < 0.05). The late AHP
width at half-amplitude was unchanged in the presence of NiClZ (n = 4; P >
0.05). NiCIZ significantly decreased the area under the curve (AUC) of the late
AHP compared to the AUC of the control late AHP (n = 4; * = P < 0.05). Data are

mean t S.E.M. n represents the number of neurons.

138

sEPSPs in AH neurons

Control CdCI2 (100 M)

   

Figure. 4.7. Effect of CdCIZ on sEPSPs in AH neurons. A train of electrical
pulses (20 Hz, 500 ms) applied to an interganglionic connective produces a
sEPSP in an AH neuron (NS = nerve stimulation). The sEPSP is inhibited by
CdCIZ (100 pM). Concentration-response curve for sEPSP inhibition by CdCI2.
Curves are non-linear fits of the Hill equation. The peak amplitude of the sEPSP
was stable over time (P > 0.05; one-way ANOVA). Data points are mean :I:

S.E.M. n = 5 neurons.

139

sEPSPs in AH neurons

Control

Figure. 4.8. NiCIZ (50 pM) did not inhibit sEPSPs. a sEPSP in an AH neuron

is not inhibited by NiCl2.

140

sEPSPs in AH neurons

Control
%-
NS
NiCIZ (50 0M)
Hm
/I - - - — - - - 0
NS
I CTX(0.1 ltM)
4!!
NS

Figure. 4.9. Sensitivity of sEPSPs to w-conotoxin (w-CTX) (100 uM).
sEPSPs in AH neurons are not inhibited by NiCIZ, but are blocked by w-CTX.
Data are mean 1: S.E.M similar to that shown in A. * = P < 0.05 (NiCIZ versus 00-

CTX); ** = P < 0.01 (Control versus w-CTX). n = 4 neurons.

141

fEPSPs in S neurons

Control CdCI2 (100 pM) Wash

Figure. 4.10. Amplitude of fEPSPs after treatment with CdCI2 and during

 

 

 

time controls. CdCI2 (100 pM) completely inhibited the fEPSP. Concentration-
response curve for inhibition of the fEPSP by CdCI2 (n = 5). The peak amplitude
of the fEPSP was stable over time (n = 7; P > 0.05). Data are mean :I: S.E.M. n is

the number of neurons from which fEPSPs were recorded.

142

Cholinergic/purinergic fEPSPs in S neurons

Control

 

 

 

Figure. 4.11. NiCl2 does not inhibit fEPSPs in cholinergic/purinergic S-type
neurons. Representative traces showing that in a subpopulation of S-type
neurons, NiCl2 (50 pM) did not inhibit the fEPSP. Mecamylamine (10 pM)
reduced the fEPSP. Subsequent application of mecamylamine and PPADS (10
pM) then largely inhibited the fEPSP. Mean data indicating that the amplitude of
the fEPSP under control conditions is not different from the fEPSP amplitude
after NiCl2 treatment (n = 15; P > 0.05). Application of mecamylamine (10 pM)
reduced the fEPSP amplitude (n = 15; * = P < 0.05). Mecamylamine and PPADS
(10 pM) then inhibited the fEPSP (*** = P < 0.001). Data are mean :l: S.E.M. n =

15 neurons.

143

Cholinergic/purinergic fEPSPs in S neurons

Control

 

 

\-——--—--

 

Figure. 4.12. SNX-482 does not inhibit fEPSPs in cholinergic/purinergic 8-
type neurons. Representative traces showing that SNX-482 (0.1 uM) has no
effect on the fEPSP amplitude. Application of w-CTX (0.1 uM) blocked the
fEPSP. This population of S-type neurons is sensitive to inhibition by a
combination of mecamylamine (10 pM) and PPADS (10 uM). Mecamylamine (10
pM) reduced the fEPSP amplitude (data not shown). Mecamylamine (10 pM) and
PPADS (10 pM) then largely inhibited the fEPSP. Application of w-CTX (0.1 pM)

blocked the fEPSP (n = 3; * = P < 0.05). Data are mean :I: S.E.M. n = 3 neurons.

144

Cholinergic fEPSPs in S neurons

‘ Control

 

Mecamylamine (10 pl“)

~o- WMMO'-W n.“ ~-

Wash

 

Figure. 4.13. NiCl2 and w-CTX inhibit fEPSPs in purely cholinergic S-type
neurons. Representative traces showing that in a sub-population of S-type
neurons, NiC|2 (50 uM) reduced the fEPSP. Mecamylamine (10 pM) then
completely blocked the fEPSP. Mean data indicating that the amplitude of the
fEPSP after NiCl2 treatment is significantly smaller compared to control (* = P <
0.05). Mecamylamine 10 uM) reduced the fEPSP amplitude (*** = P < 0.001),
and this was smaller compared to NiC|2 treatment (*** = P < 0.001). n = 14

neurons. (C) w-CTX (0.1 pM) further inhibited the fEPSP (trace not shown).

145

 

:\ Control

|I \

‘l \~

----.,.' .......................

'CTX (0.1 pM)

Figure. 4.14. Effect of SNX-482 and w-CTX on purely cholinergic fEPSPs.
Representative traces showing that SNX-482 (0.1 pM) reduces the fEPSP
amplitude in purely cholinergic S—type neurons. w-CTX (0.1 pM) subsequently
inhibited the fEPSP. Mean data showing that SNX-482 inhibited the amplitude of
the fEPSP compared to control (n = 3; *** = P < 0.001). Mecamylamine (10 pM)
then blocked the fEPSP (data not shown). After washout of mecamylamine, w-
CTX (0.1 pM) blocked the fEPSP (n = 3; *** = P < 0.001). Data are mean 1:

S.E.M. n = 4 neurons.

146

 

CHAPTER 5 R-TYPE CALCIUM CHANNELS COUPLE TO NITRIC OXIDE-

MEDIATED INHIBITORY NEUROTRANSMISSION IN THE GUINEA PIG ILEUM

147

 

ABSTRACT
Previous immunohistochemical studies showed that R-type calcium channels
and nitric oxide synthase (NOS) co-localize in nerve terminals supplying the
muscle layers in the guinea pig ileum. It is not known if calcium entry through R-
type channels couples to NOS activation and/or ATP release. The present study
determined the contribution of R-type calcium channel activation to release of NO
and ATP from myenteric motorneurons supplying the muscle layers of the guinea
pig ileum. I measured the effects of the NOS inhibitor, nitro L-arginine (NLA) and
the R-type calcium channel blocker NiClz on neurogenic relaxations and
contractions of longitudinal muscle-myenteric plexus muscle strips maintained in
vitro. Neurogenic relaxations under histamine tone and contractions in non-
histamine-contracted tissues were evoked by trains (20 Hz, 0.3 ms) of transmural
electrical stimulation. I also used intracellular recordings from smooth muscle
cells to investigate the contribution of R-type channels to release of ATP/B-NAD
as the mediators of the inhibitory junction potential (IJP). Tetrodotoxin (TTX), and
the non-selective calcium channel antagonist CdCI2 blocked electrically-evoked
relaxations confirming that they were neurogenic. NLA inhibited neurogenic
relaxations in a concentration-dependent (10-100 pM) manner; the maximum
inhibition was 60%. NiClz produced a similar inhibition of neurogenic relaxations
and contractions. Co-application of NiCIz and NLA did not produce a greater
inhibition of neurogenic relaxations or contractions than either blocker alone. lJPs
were blocked by TTX, CdCI2 and apamin. NiClz or NLA applied alone or together

did not alter IJP amplitude or time course. R-type calcium channels may couple

148

 

to NOS activation and NO-mediated muscle relaxation in the guinea pig ileum. R-
channels do not couple to release of the mediator of apamin-sensitive IJPs. ATP
and NO released from the same inhibitory nerves may be regulated differently, or
NO and other inhibitory neurotransmitters are released from separate

populations of nerve fibers.

149

INTRODUCTION

The enteric nervous system (ENS) controls gastrointestinal (GI) function
(Furness et al., 1998). The ENS is composed of two nerve plexuses: the
submucosal plexus which controls the absorption of nutrients, secretion, and
local blood flow across the epithelium, and the myenteric plexus which has a
pivotal role in the control of GI motility (GI) (Bornstein et al., 1994; Lomax &
Furness, 2000). Non-adrenergic non-cholinergic (NANC) nerves mediate the
majority of inhibitory responses in the ENS (Sanders & Ward, 1992). Stimulation
of NANC inhibitory enteric neurons relaxes GI smooth muscle (Gao et al., 2006;
Burnstock, 2008) and causes inhibitory junction potentials (lJPs) (Burnstock et
al., 1964). Pharmacological and electrophysiological studies revealed that
inhibitory neurotransmitters released from NANC nerves use varying
mechanisms to cause smooth muscle relaxation (He & Goyal, 1993; Smits &
Lefebvre, 1996; De Man et al., 2003). The most prominent inhibitory
neurotransmitters in the ENS are ATP (Burnstock et al., 1997), vasoactive
intestinal peptide (VIP) (Fahrenkrug et al., 1979; Rekik et al., 1996), carbon
monoxide (Kwon et al., 2001), nitric oxide (NO) (Crist et al., 1992; Sanders &
Ward, 1992; Mashimo et al., 1996) and possibly B-nicotinamide dinucleotide ([3-
NAD) (Mutafova-Yambolieva et al., 2007). B-NAD/ATP, VIP and NO may all be
released from the same motor nerve varicosity (Burnstock, 2008).

ATP and or B-NAD, released from enteric motorneurons, is an
important inhibitory enteric neurotransmitter that acts at Gq-protein-coupled P2Y1

receptors on Gl circular smooth muscle (Wood, 2006b). P2Y1 receptors mediate

150

 

the fast IJP (Gallego et al., 2006). MRSZ179 is a P2Y1 receptor antagonist
(Boyer et al., 1998) that blocks the fast IJP in myenteric neurons (Gao et al.,
2006; Wang et al., 2007). Apamin (0.1 pM) is a blocker of small-conductance
calcium—dependent K‘ channels (SK) and selectively inhibits the fast IJP
(Mackenzie & Burnstock, 1980).

NO is an important signaling molecule in the central nervous system
(CNS) and the ENS. NO can function as a retrograde (Holscher, 1997)
transmitter, or as a conventional anterograde transmitter where it is dependent
on the calcium-dependent activation of the neuronal isoforrn of nitric oxide
synthase (nNOS) (Ohashi et al., 2007). In the ENS, nNOS is the dominant
isoforrn of NOS (Ward et al., 1992) and NO is an important inhibitory
neurotransmitter (Sanders & Ward, 1992). NO relaxes GI smooth muscle
(Katsoulis et al., 1996; Mashimo et al., 1996; Kim et al., 1999). The slow IJP is
NO-mediated (He & Goyal, 1993; Stark et al., 1993). Regulation of nitrergic
neurotransmission however, is still not well understood. The identification of the
NO regulatory mechanism is complicated by the fact that NO is a highly diffusible
gas that is neither pre-formed nor stored in secretory vesicles (Bredt et al., 1990).
Therefore its mechanism of regulation must be different from that which is used
to control the release of vesicle-stored neurotransmitters. Regulation of readily
releasable vesicles is consequently an important index of neurotransmitter
release after each stimulation event (Rao et al., 2008). NO production and

release may be regulated by a catalytically active pool of nNOS via PDZ binding

151

domains that allow nNOS to anchor to the plasma membrane at the active zone
(Rao et al., 2008).

Neurotransmitter release requires calcium influx through voltage-gated
calcium channels (Catterall, 2000). R-type calcium channels are high-threshold
channels and require strong depolarization for activation (Foehring et al., 2000)
and ENS (Bian et al., 2004; Bian & Galligan, 2007). R-type voltage-gated calcium
channels are expressed in myenteric neurons and together with the N-, PlQ-, and
L-type channels, account for the total calcium current recorded from those
neurons (Bian et al., 2004). The functions of R-type channels in myenteric
neurons are not yet clear. In myenteric neurons maintained in primary culture,
the R-type calcium channels may contribute to the somatodendritic action
potential or to action potential-induced calcium entry required for
neurotransmitter release from myenteric nerve terminals (Bian et al., 2004). Low
concentrations of MCI; (50 (M) and SNX-482 (0.1 pM) block R-type calcium
channels (Randall & Tsien, 1995; Newcomb et al., 1998; Wang et al., 1999;
Tottene et al., 2000). w—conotoxin GVIA (w-CTX) (0.1 uM) and w-agatoxin IVA
(0.1 pM) block N-type (Barajas-Lopez et al., 1996; Chen & Kirchgessner, 2002)
and PIQ-type channels (Tsien et al., 1988; Catterall, 2000; Bian et al., 2004)
respectively.

Previous data has shown that the R-type calcium channel and nNOS co-
localize in myenteric neurons, and in nerve fibers supplying the smooth muscle in
the guinea pig ileum (Naidoo et al., 2006). The present study investigated

whether myenteric neurons mediate descending inhibition via R-type channels,

152

 

and whether R-type calcium channels and nNOS are spatially coupled on nerve

endings of enteric motorneurons to cause GI smooth muscle relaxation.

153

 

MATERIALS AND METHODS
Ethical approval
All animal use protocols were reviewed and approved by the Institutional

Animal Care and Use Committee at Michigan State University.

Longitudinal muscle-myenteric plexus (LMMP) preparation

Adult male Hartley-strain guinea pigs weighing 250 g (Bioport, Lansing,
Michigan; animals) were anesthetized under isofiurane inhalation (Abbott
Laboratories, Chicago, IL), stunned and exsanguinated. The ileum was removed
and placed in pre-warmed oxygenated (95% 002, 5% C02) Krebs’ solution of the
following composition (mM): 117 NaCI, 4.7 KCI, 2.5 CaCIz, 1.2 M9021, 1.2
NaHzPO4, 25 NaHCOa, and 11 glucose. The Krebs’ solution contained
scopolamine (1 pM) to block muscarinic cholinergic receptors. A glass cannula
was inserted into a 2 cm segment of the ileum and the longitudinal muscle
attached with the myenteric plexus (LMMP) was then teased off with a cotton
swab soaked in Krebs’ solution. The LMMP preparation was mounted with silk
Iigatures between a platinum foil electrode and a stationary hook connected to an
isometric force transducer (Grass Instruments, FT03C, Quincy, MA, USA) and
placed in a 20 ml jacketed organ bath containing Krebs’ solution at 37 °C. A
resting tension of 1 g was applied to each of four 2 cm segments of LMMP
preparations. Tissues were allowed to equilibrate for 60 minutes, during which

the Krebs’ solution was replaced in 15 minute intervals.

154

Transmural electrical field stimulation (EFS)

To study inhibitory mechanisms in the LMMP, each tissue was first pre-
contracted with histamine (1 pM) to induce a sustained baseline resting tension
(Osthaus and Galligan, 1991). Histamine acts directly at H1 receptors on the
longitudinal muscle in the guinea pig ileum to cause an increase in muscle tone
(lzzo et al., 1998). Non-adrenergic non-cholinergic (NANC)-mediated relaxations
were induced by brief trains of transmural electrical stimuli (20 Hz, 0.3 ms pulse
duration, 0.25 Hz train rate, 50 V) (Osthaus & Galligan, 1992). To determine the
action of the antagonist on the inhibitory responses of the ileal longitudinal
muscle, non-cumulative concentration response curves were created with a 15
minute interval between successive doses. I also investigated EFS-evoked
frequency-dependent contractions in the non pre-contracted LMMP (20 Hz, 0.3
ms, 50 V). To determine the action of the antagonist on the excitatory response
of the ileal longitudinal muscle, non-cumulative experiments were conducted and
the effects of co-application of antagonists on the excitatory responses were
studied. Transmural electrical stimuli in both set of experiments were provided
by a Grass S48 stimulator and mechanical activity of the LMMP was recorded

with Labscribe (iWorx, Dover, NH, USA) and a personal computer.

Tissue preparation for intracellular electrophysiological recordings
A 1.5 cm section of the ileum was cut open along the mesenteric border
and pinned flat (Fine Science Tools, Foster City, CA) in a silicone elastomer-Iined

(Sylgard, Dow Corning, Midland, MI) petri-dish containing Krebs’ solution. The

155

Krebs’ solution contained scopolamine (1 pM) and nifedipine (1 uM) to block
muscarinic receptors and L-type calcium channels on smooth muscle cells,
respectively. The submucosa was removed to expose the circular muscle-
longitudinal muscle-myenteric plexus (CM-LMMP). A 5 mm2 section of the CM-
LMMP was then transferred to a smaller silicone elastomer-lined recording
chamber (2 ml) with the smooth muscle layer facing up. The chamber was
mounted on a stage of a microscope (Olympus, Tokyo, Japan). The CM-LMMP
was pinned tautly and superfused with oxygenated Krebs’ solution at a flow rate
of 4 mllmin at 37 °C. The preparation was equilibrated for 40 min before

commencing intracellular recordings.

Intracellular electrophysiological recordings

Single smooth muscle cells in the CM-LMMP were impaled with glass
microelectrodes filled with 2 M KCI (tip resistance 80-120 Mn). Membrane
potential was recorded using an Axoclamp 2A amplifier (Axon Instruments,
Foster City, USA) in bridge mode. IJPs were evoked focally with a monopolar
glass electrode (tip diameter 60 pM) (World Precision Instruments, Sarasota,
Florida) filled with Krebs’ solution by electrically stimulating myenteric nerve
fibers supplying the smooth muscle. Single pulses (1 Hz, 4 ms). Short frequency
trains of stimulation (5 Hz and 10 Hz, 1 s, 50 V), provided by a pulse generator
(Master 8, A.M.P.I., Jerusalem, Israel) and a constant current stimulation unit
(Grass Technologies, West Warwick, RI) were used to evoke lJPs. The resting

membrane potential (RMP) of the smooth muscle cell was allowed to stabilize for

156

10 minutes without applying intracellular holding current. Cells with RMPs less
than -40 mV were not included in the analysis. Signals were recorded using an
A/D converter (Digidata 1322A, Molecular Devices Corp., Sunnyvale, CA),
Axoscope and Clampfit 10 software (Molecular Devices), and a desktop
computer. Amplified signals were sampled at 2 kHz and filtered at 1 kHz. I
measured the area under the curve (AUC) as this is an integration of both IJP
amplitude and duration. This allowed us to study the changes in responses
induced by drugs. AUCs were measured under control conditions and after

various drug treatments.

Drugs

All chemicals were obtained from Sigma-Aldrich (St Louis, MO), except for
w-CTX, w-ATX, and tetrodotoxin (TTX) (Alomone Labs, Jerusalem, Israel).
Drugs were dissolved in deionized water with the exception of nitro-L-arginine
(NLA), which was dissolved in hydrochloric acid (0.1 N) and nifedipine which was

dissolved in 95% ethanol.

Data Analysis

Data are presented as the mean 1: S.E.M. For the EFS study, n refers to
the number of animals from which tissues were obtained. For the intracellular IJP
recordings, n is the number of smooth muscle cells. Concentration-response data
were fitted using non-linear regression and the Hill equation (GraphPad Prism,

San Diego). A one-way ANOVA, two-way ANOVA or Student’s t-test for paired or

157

unpaired data were used when applicable. P < 0.05 was considered statistically

significant.

RESULTS

Electrically induced relaxations of the histamine-contracted LMMP

Transmural stimulation of histamine (1 pM)-contracted LMMP preparations
(n = 4) produced a triphasic response. This response was composed of a fast
contraction (Figure. 5.1Aa, B1), followed by a relaxation (Fig. 1Ab, B1), and a
rebound contraction (Figure. 5.1Ac, B1). I first tested whether these responses
were neurogenic. The voltage-dependent sodium channel blocker TTX (0.3 pM)
completely inhibited the relaxation and rebound contraction of the longitudinal
smooth muscle but not the initial fast contraction (Figure. 5.1B2). The CdCI2
blocked EFS-induced contractions and relaxations (Figure. 5183, 2A). Apamin
(0.1 uM) inhibited the EFS-evoked relaxation (Figure. 5.1B4) and revealing a

large-amplitude slow contraction.

R-type calcium channel blockers inhibit neurogenic relaxation of the LMMP

Previous data showed that R-type calcium channels and NOS co-localize
in the LMMP (Naidoo et al., 2006). The R-type calcium channel blocker, NiClz
(0.1-100 uM), caused a concentration-dependent inhibition of EFS—evoked
relaxation with a maximum 41 % reduction in the control response (P < 0.0001)
(Figure. 5.2B). NLA (0.1-100 uM) also reduced the peak relaxation amplitude in

a concentration—dependent manner with a maximum inhibition of 53 % (P<

158

0.0001). Co—application of NiClz with NLA (0.1-100 pM) inhibited the relaxation
amplitude by 48 "/0. Comparison of these dose response curves revealed that
NiCI2 did not significantly affect the dose response curve for NLA, and the
converse was also true (P > 0.05).

During these initial sets of experiments, I observed that NiClz and NLA
inhibited the EFS-evoked relaxation and also produced a concentration
dependent increase in the amplitude of the rebound contraction. I next studied
the effect of MCI,» and NLA on the amplitude of this NANC contraction in the
absence of histamine induced tone. Transmural stimulation (20 Hz, 0.3 ms) of
the non-pre-contracted LMMP produced a biphasic contraction response. The
biphasic contraction response at the beginning of the stimulation contained an
early fast contraction followed by a late slow contraction (n=10, Figure. 5.3A1,
B1). There was no evidence of a time-related change in the LMMP slow
contraction (Figure. 5.3A2). I then tested the effect of MCI; (50 pM), NLA (100
uM) and apamin (0.1 pM) on this slow contraction. In these experiments I only
measured the AUC (g.s) of the slow contraction because drug treatment had a
pronounced effect on the amplitude and duration of the response. NiCI2
increased the AUC of the contraction by 147 % (Figure. 5.3B1, 382, 4A; P <
0.05). NLA in the presence of NiCIz increased the AUC by a further 13 % and this
was not statistically different from NiCIz alone (Figure. 5.383; Figure. 5.4A P >
0.05). Subsequent addition of apamin with NiCl2 and NLA and increased the AUC
by 53 % and this was statistically greater from the effect of NiCl2 by itself (Figure.

5.334; P < 0.01), and from the effect of MCI; in the presence of NLA (P < 0.05

159

Figure. 5.4A). TTX (0.3 pM) blocked the slow but not fast contraction while CdCI2
(100 pM) blocked both responses was applied to all LMMP preparations, and
subsequently blocked the slow contraction (Figure. 5.3B5 and Figure. 5.6).

In second set of experiments preparation where NLA was applied first,
NLA increased the AUC of the contraction by 118 "/0 (Figure. 5.43, P < 0.01).
NLA in the presence of NICIz did not produce any additional effect on the
contraction (P > 0.05) when compared to NLA by itself. Co-application of NLA
with NICI2 and apamin increased the AUC of the contraction by 37 % which was
greater than the effect of MCI; and NLA together (P < 0.05). In the third set of
experiments, apamin increased the AUC by 95 % (Figure. 5.40, P < 0.01). NLA
in the presence of apamin increased the AUC by an additional 42 % (P < 0.01)
while subsequent addition of MCI; did not produce any additional increase over

that caused by apamin and NLA (Figure. 5.40).

lJPs are NiCl2 insensitive

l next investigated IJPs caused by focal stimulation of myenteric plexus
interganglionic fiber tracts. Resting membrane potentials (RMPs) of impaled
smooth muscle cells under control conditions was -51 1: 1 mV and did not change
in the presence of drugs. Figures 5.5A1-A3 show control IJPs evoked in
response to 1, 5 and 10 stimuli. I chose these frequencies because increasing
electrical stimulus frequency causes the differential and additional release of
enteric neurotransmitters from the same nerve endings (Bywater & Taylor, 1986;

Crist et al., 1991; Undi et al., 2006; Benko et al., 2007). The amplitude and

160

 

duration of the IJP increased with increasing number of stimuli and NiClz (50 pM)
did not affect lJPs (Figure. 5.5B1-B3). In the presence of NiCIZ, NLA (100 uM)
also did not change the properties of the UPS (Figure. 5.5C1-03, H). In these
cells, and regardless of the order of application, neither NLA nor NiClz by
themselves affected lJPs (Figure. 5.5I, n = 4; 5 Hz and 10 Hz frequencies not
shown).

The selective purinergic P2Y1 receptor antagonist MRS2179 (10 pM)
blocked the UPS at 1 Hz, 5 Hz and 10 Hz stimulation (Figure. 5.501-D3). TTX
(0.3 uM) (Figure. 5.5 E) also blocked IJPs. CdCI2 (1-100 pM), produced a
leftward-shift in the stimulus-response curve (Figure. 5.5F, I) and caused a
concentration-dependent inhibition of the UPS (n = 4) at all stimulus frequencies.
I therefore used 100 uM CdCI2 in subsequent experiments. While MCI; and NLA
had no effect on the UPS they were blocked by CdCI2 (Figure. 5.50, H). Finally,
application of apamin (0.1 pM) (Figure. 5.5G) also inhibited the IJP responses.
Together, these data suggest that R-type calcium channels couple to NO

synthesis and release but not ATP/B-NAD release as mediator(s) of the IJP.

NiClz-insensitive lJPs are mediated by N- and PIQ-type calcium channels.

In order to provide support for selective coupling of R-type calcium
channels to NO release, and because CdCI2 inhibited the UPS, I used, together
with NiCIz, w-CTX and w-ATX to inhibit N- and PIQ-type channels respectively.
NiCl2 did not affect the UP (Figure. 5.6B1-B3, E) compared to control (Figure.

5.6A1-A3). Additive application of w-CTX (0.1 uM) in the presence of NiClz

161

reduced IJPs evoked at all frequencies (Figure. 5.601-03). Addition of w-ATX
(0.1 pM) then further reduced the AUC (Figure. 5.601-D3). The single stimuli IJP
was almost completely blocked, while at 5 and 10 Hz a residual IJP persisted.
Apamin (0.1 pM) blocked the residual lJPs. Concentration—response curves with
w—CTX (0.001-0.1 pM) and w-ATX (0.001-0.1 pM) revealed almost complete
inhibition of UPS (Figure. 5.5I). These data indicate that N- and PIQ-type calcium

channels mediate release of ATP/B-NAD as the mediators of the IJP.

162

 

DISCUSSION

This study investigated the contribution of R-type calcium channels to
inhibitory neurotransmission in the ENS of the guinea pig ileum. Enteric inhibitory
nerves release NO and ATP/B-NAD to relax GI smooth muscle. ATP/B-NAD act
at P2Y1 receptors on smooth muscle to cause apamin-sensitive inhibitory
junction potentials (UPS) and relaxation. NO increases intracellular cGMP to
cause muscle relaxation. I used transmural EFS in the LMMP and conventional
intracellular recordings from smooth muscle cells in the ileum to show that R-type
calcium channels couple to NO to mediate relaxation of GI smooth muscle. l
have further demonstrated that N- and PIQ-type calcium channels control ATP/B-

NAD release.

R-type calcium channels couple to NOS release in the guinea pig LMMP

I first investigated nerve-mediated relaxations of the guinea pig LMMP
using transmural EFS in the presence of scopolamine. My first experiments used
histamine which caused a sustained contraction of the longitudinal muscle. EFS
then evoked consistent nerve-mediated relaxations. Three responses were
observed after EFS: a fast contraction, a relaxation and then a rebound
contraction. The relaxation response and the rebound contraction were nerve
mediated as they were blocked by TTX. The initial fast contraction, however, was
TTX-resistant. There are a couple of reasons for the TTX-resistant contraction.
First, calcium entry through voltage-gated calcium channels probably does not

cause this contraction since blockade of those channels still produces a TTX-

163

resistant contraction (Kristufek et al., 1999). Nicotinic ACh receptors (nAChRs)
may be localized to nerve terminals of excitatory longitudinal muscle myenteric
motorneurons (Galligan, 1999). nAChRs are non-specific ligand-gated cation
channels that have a greater permeability to calcium than sodium and potassium
ions (Vemino et al., 1992). In the presence of TTX, EFS would cause a
depolarization of the nerve terminal and activation of presynaptic nAChRs to
allow calcium entry via the nAChR, leading to neurotransmitter release (Sacaan
et al., 1996; Lena & Changeux, 1997; Schneider et al., 2000). Second, it is
possible that high-frequency (5 Hz, and 10 Hz) nerve stimulation in myenteric
neurons still causes additional calcium entry through activation of voltage-gated
calcium channels, different to that observed in sympathetic neurons (Kristufek et
al., 1999). Calcium influx through voltage-gated calcium channels could directly
cause neurotransmitter release, or indirectly by inducing calcium release from
intracellular calcium stores within the smooth endoplasmic reticulum (Smith et al.,
2003). The TTX-resistant contraction may therefore be a calcium-mediated
contraction.

Concentration-response curves with NiCI2 and NLA showed that whether
these drugs were co-applied, or whether they were applied alone, they produced
an equivalent effect on neurogenic relaxation. Osthaus and Galligan (1992)
observed that LMMP tissues exhibiting biphasic nerve-mediated response of the
histamine-contracted LMMP showed: a fast relaxation followed by a rebound
non-cholinergic contraction, or a fast relaxation followed by a slower and smaller

amplitude relaxation. My data, however, show a fast contraction, followed by a

164

slower and large amplitude relaxation, and a slow rebound contraction of large
amplitude. These varying responses suggest that the interplay between
excitatory and inhibitory nerves is complex and that neurotransmitters released
from enteric nerves can produce several motor responses. I therefore next
determined the contribution of R-type calcium channels to facilitation of LMMP
contractions as a measure of simultaneous release of inhibitory
neurotransmitters. MCI; and NLA, applied alone, increased the contraction and
co-application of NICIz and NLA did not produce an additive effect. The addition
of apamin however, significantly increased the contraction. Apamin applied alone
increased the contraction and subsequent addition of NLA or NiClz further
increased the contraction. These data suggest that R-type calcium channels
couple to NO synthesis and release, but R-type channels are not coupled to

ATP/B—NAD release.

R-channels do not couple to ATP release as a mediator of apamin-sensitive
lJPs

I next characterized the relationship between R-type calcium channels and
inhibitory neurotransmission by recording lJPs evoked from smooth muscle cells.
NICIz and NLA added independently, and together, did not affect the UPS.
However, apamin completely blocked IJPs evoked at all frequencies.
Furthermore, MRSZ179 also completely inhibited the UPS. These responses
were neurogenic as they were blocked by 'ITX. ATP, NO and VIP are released

from the same enteric nerve endings on to GI smooth muscle, and UPS with

165

 

different kinetics have been observed (Mackenzie & Burnstock, 1980; Benko ‘et
al., 2007; Burnstock, 2008). ATP produces a fast IJP, NO produces a slower IJP
(Hirst et al., 2004), and VIP elicits very slow and tonic relaxations in the guinea
pig ileal circular muscle (He & Goyal, 1993; Burnstock, 2008). In guinea pig
jejunum and ileum, purinergic lJPs are either purely purinergic (26 %), partially
purinergic (70 %) or non-purinergic (4 %) (Wang et al., 2007). In that study,
apamin (0.5 uM) and MRSZ179 (2 uM) completely blocked the IJP. Wang and
others (2007) used a single pulse (2 ms duration) whereas I used three different
stimuli at 1 Hz, 5 Hz and at 10 Hz. In all cells tested, apamin and MRS 2179
completely blocked the IJP. This suggests that all UPS I had evoked were
ATP/BNAD responses that are not mediated by R-type calcium channels. These
NiClz-insensitive IJPs were largely blocked with w-CTX and w-ATX indicating
that the IJP is ATP-mediated. At 10 Hz, a biphasic IJP was observed. This
cannot be VIP or NO-mediated since apamin completely inhibited all evoked
lJPs. ATP acting at P2Y1 receptors on smooth muscle causes the fast IJP (Crist
et al., 1992; Gallego et al., 2006), thus corroborating my data. However, my
results further indicate that the slower and broader duration IJPs (at 5 Hz and 10

Hz) are also be due to ATP/BNAD acting at P2Y1 receptors.

Functional significance of R-type calcium channels in neuromuscular
transmission
Taken together, it is possible that R-type calcium channels may couple to

NO release to mediate inhibition of GI smooth muscle. R-type channels and NOS

166

 

co-localize in nerve fibers that supply the smooth muscle layer. ATP/B-NAD
released from the same nerve fibers would act at P2Y1 via N- and PIQ-type
calcium channels. There is presently no marker for ATP localization. Therefore,
whether R-type channels co-localize with ATP and NOS on the same or different
myenteric motor nerve terminals remains to be determined.

R-type channels may therefore be present on separate nerve fibers to
regulate NO release from motor nerve varicosities, or they may be present on the
same NOS-ATP containing fibers but couple to NOS for the differential regulation
of NO release on to GI smooth muscle. Accumulating evidence indicates that all
three major myenteric inhibitory neurotransmitters (NO, ATP and VIP) are
released from the same motor nerve terminals (Costa et al., 1986b; Crist et al.,
1992; Furness et al., 1995). It is therefore likely that R-type calcium channels are
present with NO and ATP on the same nerve endings. Recent data from the
mouse gut suggest a model in which enteric motor nerve terminals contain
catalytically active forms of NOS (nNOSa) that are anchored via PDZ-binding
domains (Lai & Lp, 2003; Kim & Sheng, 2004) to the plasma membrane (Rao et
al., 2008). My data therefore suggest that NOS may be docked at the nerve
terminal, close to R-type calcium channel release sites. A proposed mechanism
would be as follows: when the nerve terminal is depolarized by invading action
potentials, calcium, entering through R-type calcium channels, binds to
calmodulin to activate NOS that converts L-arginine into NO. NO then diffuses

across the neuroeffector junction, binds to soluble guanylate cyclase, activates

167

protein kinase G (PKG) which then phosphorylates myosin light chain kinase to
cause relaxation of the muscle (Lecci et al., 2002)..

R-type calcium channels play important roles throughout the nervous
system. For example, at hippocampal CA3-CA1 synapses, R-type channels
contribute to presynaptic calcium entry (Wu & Saggau, 1995). R-type channels
are also involved in excitation-contraction coupling in the brainstem (Wu et al.,
1998) and in chromaffin cells (Albillos et al., 2000). R-type calcium channels also
contribute to fast synaptic transmission at hippocampal mossy fiber and
associative-commissural synapses (Gasparini et al., 2001). In the ENS, R-type
channels may be important to control of descending inhibition between myenteric
motorneurons and effector tissues. NO and ATP are the most prominent
inhibitory neurotransmitters in the ENS (Sanders & Ward, 1992; Lecci et al.,
2002; Furness, 20063). It is possible that enteric motorneurons, by using multiple
calcium channel subtypes, which in turn are coupled to important inhibitory
neurotransmitters, ensure that transmission to the smooth muscle is achieved.
The functional significance of multiple inhibitory neurotransmitters could therefore
be a compensatory one, that is, deficiencies in either NOS/ATPNIP and/or a
calcium channel subtype would be compensated, at least in part, by another
inhibitory neurotransmitter or calcium channel (Mashimo et al., 1996; Mashimo et
al., 2000). The same reasoning that multiple transmitters serve as protective
mechanisms to ensure continual gut function can also be ascribed to the

excitatory transmitters ACh and SP. This is due to the paucity of CNS innervation

168

 

to the ENS (Galligan, 2002b), and implies that the GI tract needs to be able to

function in the absence of CNS input.

SUMMARY AND CONCLUSIONS

The present study demonstrated the involvement of R-type calcium
channels in neurotransmission to the GI muscle layer. My data support a model
in which R-type calcium channels couple to NOS to mediate NO release from
myenteric motor axon varicosities to the smooth muscle. This mediation occurs
via an apamin-insensitive mechanism. Therefore R-type channels do not couple
to ATP/BNAD release, but rather to NO as a mediator of relaxation responses,
demonstrated by EFS of LMMP preparations, and recordings of UPS from
smooth muscle cells. All IJPs observed in this study were completely inhibited by
apamin but not by MCI; and NLA, thus providing indirect evidence of a spatial
relationship and coupling between R-type calcium channels and NO release. The
cross-talk between R-type channels and NOS would need to be further evaluated
as to whether there is a physical coupling between the channel and the enzyme,
or whether R-type channels are linked to NOS through intracellular signaling
mechanisms. This study helps us understand how the inhibitory signaling
pathway is involved in neuromuscular control of motility via R-type channels. This
may be important as the drugs that target R-type calcium channels would help to
facilitate or inhibit neurotransmitter release form nerve endings of myenteric

motor neurons.

169

 

Resting tension
(Histamine, 1 pM)

B1 Control relaxations B2 TI’X (0.3 pM)

B3 CdCI2 (100 uM) B4 Apamin(0.1uM)

W M

Fig. 5.1. Representative traces showing NANC responses after transmural
electrical field stimulation of the guinea pig LMMP in vitro. Relaxations were
induced in the presence of histamine (1 pM) and scopolamine (1 pM). After nerve
stimulation (NS), a fast contraction (a) was followed by a relaxation (b), and then
a rebound contraction (c) (A, B1). While LMMP relaxations were abolished by
TTX (0.3 (M), a fast contraction persisted. The entire response was, however,
completely sensitive to CdCI2, a non-selective antagonist of voltage—gated

calcium channels. Apamin (0.1 pM) blocked the relaxation.

170

 

 

 

 

 

A B
d)
.E
5 6°' n =10 - NiCI2
% 125‘ n =4 % so« I NLA
g 100. .5 40.1 A NIC12 + NLA
a .s
g 75‘ %304
é 5* g zo-
92 25- «I
g a 10‘
g o ' I E, c I I I I I
OS 45‘ '5 '5 " °\o -8 -7 -a -5 .4 .3
[CdCI21(M) [Antagonist] (M)

Figure. 5.2. Concentration-response curves for the area under the curve
(AUC) of relaxation responses caused by EFS. CdCI2 caused a successive
inhibition of the AUC (A). (B) When NLA was added first, it produced a
concentration-dependent inhibition expressed as a percent of the relaxation
response against the histamine contraction (P < 0.0001). The action of NLA was
not statistically different in the presence of NiCI2 (P > 0.05). NiCI2, added first,
also caused a concentration-dependent inhibition relaxation response (P <
0.0001). NiCI2 + NLA together had no further effect on the relaxation (P > 0.05).
Data are mean 1: S.E.M. n is the number of tissues from which responses were

obtained.

171

 

A. **

 

20- ":10 **
***
15- T

 

0.5-

 

AUC (gs) slow contraction
3
l

 

 

 

 

0.0-
- Control
- NiC|2 (50 pM)
- NiCI2 + NLA(1OO prl)
I: NiC|2 + NLA+ Apamin (0.1 pM)

 

 

 

 

 

 

 

*
*

B c 1.5- n = 10 l—‘——l
.9 ***
E T
*-‘ ** ***

c -

8 1.0

E:

(I)

71? 0.5-

92

O

D

< 0.0-
- Control
- NLA(100pM)

I NLA+ NiC|2 (50 pM)
I::I NLA+ NiC|2 + Apamin (0.1 pM)

Figure. 5.3. Effects of NiCI2, NLA, and apamin on LMMP contractions. Co-
application of apamin + NLA + NiC|2 did not cause a significant increase

compared to apamin + NLA (P > 0.05).

172

Figure. 5.4. Effect of NiCI2, and NiC|2 + NLA on inhibitory junction
potentials (IJPs) evoked in guinea pig smooth muscle cells. NiCI2 (B1-B3,
H), and NiCI2 + NLA (C1-C3) did not have any effect on evoked lJPs at all
frequency levels of nerve stimulation (NS) compared to control (A1-A3). MRS
2179 (D1-D3), TTX (E) and apamin (G) completely inhibited the contractions.
CdCI2 (F) reduced the LMMP contraction but did not block it. Concentration-
response curves (I) for NiCI2 showed no effect on the UPS, whereas CdCI2
largely inhibited contraction responses. Data are mean :t S.E.M. from n muscle

cells.

173

A1 Control (1 Hz)

 

NiC|2 (1 HZ)

”If“

C1 NiC|2+NLA (1 Hz)

”'1

A2 Control (5 Hz)

 

82 NiC|2 (5 Hz)

I‘M

M

 

 

C2 NiC|2+NLA (5 Hz)

 

174

B3

 

 

A3 Control (10 Hz)

'1'

'l-II
.
\

C3

a.
I

l' 1"

I/
b.311-
Innull

III
I;

1ll

 

 

NiC|2 (10 Hz)

 

)

Figure. 5.4 (cont’d)

D1 MRSZ179 (1 Hz) DZ MR82179 (5 Hz) DB MRSZ179 (10 Hz)

 

E TTX (10 Hz) F CdCIZ (10 Hz) G ADamII) (10 Hz)

_‘ __ II M
"W w ”'Y' "' 17““

MW

 

 

H 1 Hz, 5 Hz and 10 Hz nerve stimdation I 1 HZ nerve stimulation
I Control (n = 5) I NiCI2 (n = 4) - w-c‘rx) (n = 4)
I NiC|2 (n = 5) v co-ATX) (n = 4 :1 CdC|2 (n = 4)
30000 I NiCI2 + NLA (n = 5) 125 >
g 100
E. g 75
5 < 50
3 33
< 25

 

I I

J—-9-'8-7-'6-5-‘4-3

 

1 5 10 -25
Number Of stimuli (HZ) [Calcium Antagonist] (M)

175

1 Hz, 5 Hz and 10 Hz nerve stimulation
- Control (n = 6)

ll NiC|2 (n = 6)
250001 CI NiCI2 + CTX (n = 5) m
- NiCI2 +CTX+ATX(n=5) r-——'I
zoooo- .... **
E
>' 15000-1
E.
0 10000-
D
<

E

 

 

 

 

 

O
k

 

Number of stimuli (Hz)

Figure. 5.5. Effect of NiCI2, and NiCI2 + w-CTX and NiC|2 + w-CTX + w-ATX
on lJPs. NiCI2 did not have any effect on evoked lJPs at all frequency levels of
stimulation compared to control. Additive application of w-CTX reduced the AUC.
Subsequent application of w-ATX largely blocked LMMP contractions at the 1 Hz
frequency, and significantly reduced but did not block the contractions at the
higher 5H2 and 10 Hz frequencies. This combined effect of NiC|2, w-CTX + w-
ATX was similar to the result obtained after CdCI2 application. Data are mean :t

S.E.M. from n muscle cells.

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CHAPTER 6 INHIBITION OF R-TYPE CALCIUM CHANNELS IMPAIRS

PERISTALSIS IN THE GUINEA PIG ILEUM

177

 

ABSTRACT
R-type calcium channels are expressed by nitric oxide synthase (NOS)
containing inhibitory motorneurons and some interneurons in the guinea ileum
myenteric plexus. The goals of this study were to determine if inhibition of R-type
calcium channels alters fluid induced peristalsis in the guinea pig ileum in vitro.
Tetrodotoxin (0.3 pM), and the non-specific calcium channel blocker cadmium
chloride (CdCI2, 1-100 pM) blocked peristalsis confirming that the reflex was
neurogenic. NiCI2 (50 (M, which blocks selectively R-type calcium channels),
reduced peristaltic contractions and efficiency (measured as fluid expelled).
Additional treatment with the NOS inhibitor, NLA (100 (M) further inhibited
peristalsis. NLA applied alone increased contraction amplitude, frequency and
efficiency and additional application of NiCI2 inhibited peristalsis. w-conotoxin
GVIA (0.1 “M, a concentration which selectively inhibits N-type calcium
channels), partially inhibited peristalsis, subsequent addition of MCI; produced a
further inhibitory effect. These data indicate that R-type calcium channels couple
to NOS to regulate neurotransmission at neuroeffector junctions in the peristaltic
reflex pathway. Selective inhibition of NO release from inhibitory motorneurons
by NLA enhances peristalsis. While NiCI2 also inhibits NO release from motor
neurons, it has additional sites of action which cause inhibition of peristalsis.
NiCI2 and w-conotoxin GVIA produce additive inhibition of peristalsis indicating
that R-and N-type calcium channels are localized to different components of the

reflex circuitry mediating peristalsis.

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INTRODUCTION

Control of gastrointestinal (GI) motility is accomplished in part by interactions
between the central nervous system (CNS), the enteric nervous system (ENS),
myogenic and hormonal mechanisms (Grundy et al., 2006). The myenteric
plexus is the division of the ENS that is primarily responsible for controlling GI
motility. The peristaltic reflex is a fundamental motility reflex that propels content
along the length of the gut (Gwynne & Bornstein, 2007). Peristalsis is a reflex
that is initiated by distention of the intestine and it relies on the sequential
activation of intrinsic primary afferent neurons (IPANs), interneurons and
motorneurons in the myenteric plexus to produce oral muscle contraction and
aboral muscle relaxation relative to the stimulus (Hennig et al., 1999; Huizinga et
al., 2006). Acetylcholine and Substance P, released from excitatory longitudinal
and circular muscle motorneurons, are the major excitatory neurotransmitters
responsible for contraction of GI smooth muscle and the emptying phase of
peristalsis (Paton & Zar, 1968; Bogeski et al., 2005). Relaxation of GI smooth
muscle is mediated by non-adrenergic non-cholinergic (NANC) inhibitory enteric
neurons and NO is a mediator of this relaxation (Sanders & Ward, 1992;
Furness, 2000).

Contraction of GI smooth muscle is dependent in part on activation of L-
type calcium channels (Horowitz et al., 1996; Farrugia, 1999; Sanders, 2008).
Although L-type calcium channels are also expressed in the ENS, they do not
contribute to synaptic transmission (North & Tokimasa, 1987; Franklin & Willard,

1993; Reis et al., 2000). N, P/Q and R-type calcium channels are also expressed

179

in the ENS (Hirning et al., 1990; Kirchgessner & Liu, 1999; Smith et al., 2003).
N-type channels contribute to the calcium shoulder during the repolarizing phase
on the AH action potential and to synaptic transmission by (Franklin & Willard,
1993; Rugiero et al., 2002). N-type calcium channels are blocked by w-
conotoxin GVIA (McCleskey et al., 1987). PIQ-type channels also contribute to
neurotransmitter release in the ENS (Reis et al., 2000). w-Agatoxin IVA is a
specific antagonist of PIQ channels (Mintz et al., 1992).

R-type calcium currents have been recorded from guinea pig myenteric
neurons maintained in primary cell culture (Bian et al., 2004). R-type calcium
channels also contribute to calcium currents in duodenal myenteric neurons in
the intact myenteric plexus (Rugiero et al., 2002; Furness, 2006a).
Immunoreactivity (ir) to the pore-forming subunit (a1E) of the R-type calcium
channel has been localized to subsets of myenteric neurons (Naidoo et al.,
2006). a1E-ir was co-localized with nitric oxide synthase (NOS) in nerve fibers
supplying the muscle layers and NO contributes to non-cholinergic non-
adrenergic GI smooth muscle relaxation (Lecci et al., 2002). NO-mediated
neurotransmission in particularly important for the peristaltic reflex (Ciccocioppo
et al., 1994; Waterman & Costa, 1994; Holzer et al., 1997). However, the
relationship between R-type calcium channel function, NO-mediated
neuromuscular transmission and the peristaltic has not been studied. | used a
modified Trendelenburg system (Billbring et al., 1958; Huizinga et al., 1998; Bian
et al., 2003) to determine whether antagonism of R-type calcium channels affects

fluid-induced peristalsis in the adult guinea pig ileum in vitro.

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MATERIALS AND METHODS

Tissue collection and preparation. All animal use protocols were
approved by the Institutional Animal Care and Use Committee at Michigan State
University. Adult male Hartley guinea pigs (250-300 g, Bioport, Lansing, MI) were
anesthetized via isoflurane (Abbott Laboratories, Chicago, IL) inhalation, stunned
by a blow to the back of the head and killed by severing the carotid arteries. A
segment of the ileum approximately 10 cm proximal to the ileo-cecal junction was
quickly removed and placed in pre-warmed (37 °C) oxygenated (95 % 02; 5 %
C02) Krebs’ solution of the following composition (in mM): 117 NaCl, 4.7 KCI, 2.5

CaClg, 1.2 MgClz, 1.2 NaHzPO4, 25 NaHCOa, and 11 glucose.

Assessment of peristalsis in vitro. Assessment of peristalsis in vitro
was accomplished using a modified Trendelenburg preparation. A segment (6.5
cm) of the ileum was transferred to and pinned (Fine Science Tools, Foster City,
CA) in a silicone elastomer-lined (Sylgard, Dow Corning, Midland, MI) petri dish
containing warmed Krebs’ solution. Fire-polished ends of two glass cannulas
(Radnoti Glass Technology, Monrovia, CA) were inserted into the oral and aboral
ends of the ileum respectively, and secured firmly with 4-0 silk suture (Harvard
Apparatus, Holliston, MA). The ileal segment was mounted horizontally in a
plexiglass organ bath (volume 40 ml) containing Krebs’ solution and equilibrated
at 37 °C for 45 min. The Krebs’ solution was replaced in 15 minute intervals
during the equilibration period. Fluid-induced distention was achieved by raising

the level of a Krebs’-containing reservoir connected by a plastic filling tube to the

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glass tube inserted into the oral end of the ileum. The filling tube was connected
to a pressure transducer (TRN 050; Kent Scientific Corp., Torrington, CT) via a
T-connector, allowing intraluminal pressure increases to be monitored as the
reservoir height was increased. Signals from the pressure transducer were
digitized and acquired using an analog to digital converter (Minidigi 1A, Molecular
Devices Inc., Foster City, CA), a DC strain gage amplifier (Grass Technologies,
West Warwick, RI), and Axoscope v10 software (Molecular Devices Inc). The
glass tube at the aboral end of the ileum was fitted with a one-way value to
prevent backflow of Krebs’ solution. This tube drained into a graduate cylinder for
measurement of the volume of fluid expelled during each episode (see below).
Experimental protocol. After the 45 minute equilibration period,
intraluminal pressure was first increased from a basal to a threshold (T) level. T
was defined as the minimum pressure (mmHg) required for the initiation of
peristalsis. Peristalsis was defined as a contraction that propagated from the oral
to the anal end of the segment. Typically, these contractions occurred in regular
intervals throughout the distention period. The pressure was elevated for 1
minute for each episode and then returned to baseline where the tissue was left
to recover for 5 minutes. A range of increasing intraluminal pressures, in 0.5
mmHg intervals, from T to T+5 mmHg was used. This allowed us to calculate the
half-maximal effective intraluminal pressure (EPso) for time control and antagonist
concentration-response studies. Time control experiments were performed over
a period of 50 min, and the Krebs’ solution was replaced every 15 min. I

measured the amplitude of the peak contraction (mmHg). frequency of

182

 

contractions (waves/ 3), and the area under the curve (AUC; mmHg.ms)
(Clampfit; Molecular Devices) during a 60 s recording period. The volume of fluid
expelled (ml) at the end of each distention period was also measured.

Antagonist concentration-response curves. Concentration-response
curves were obtained by using the T+EP50 (mmHg) value at increasing
antagonist concentrations for each segment of ileum. The non-selective calcium
channel antagonist CdCI2 (1-100 pM), and the R— and N-type calcium channel
antagonists NiCI2 (1—50 pM) and w-conotoxin GVIA (cu-CTX) (0.001-1 (M),
respectively, were added to the bath. The tissue was incubated in successively
higher antagonist concentrations for 10 min prior to testing its effect on
peristalsis.

Pressure-response curves. Pressure-response curves were obtained
under control drug-free conditions and at specific antagonist concentrations using
increasing intraluminal pressure values from T to T+5. The aim of the pressure-
response curve experiments was to investigated the effects of NiC|2 (50 (M), the
nitric oxide synthase inhibitor nitro-L-arginine (NLA; 100 pM), w-CTX, the sodium
channel blocker tetrodotoxin (TTX; 0.3 pM), the small-conductance potassium
channel antagonist apamin (0.1 pM) on the peristaltic reflex. All chemicals were
purchased from Sigma-Aldrich (St Louis, MO), except for w-CTX and TTX
(Alomone Labs., Jerusalem, Israel).

Video imaging and spatio-temporal mapping. The construction
of spatio-temporal maps was performed as previously described (de Backer et

al., 2008). Briefly, a camera (Logitech, Fremont, CA), placed directly above the

183

 

organ bath, was used to capture peristaltic motor movements of the ileum over a
60 s recording period (25 frames/s, 640 x 480 pixel resolution). The video file
was imported into lmageJ using a plugin for QuickTime v7 (www.gpple.com)
(Figure 6.1A). A threshold procedure was applied to all video frames and edge-
detection was used to define the gut periphery. Images were converted into
binary format (Figure. 6.1B) and a region of interest was selected. Next, two
further lmageJ plugins were used: Amplitude Profiler, which determines the
change in intestinal diameter (mm); and Spatio-temporal Mapping software,
which facilitates 3-dimensional (diameter distance and time) rendering of
intestinal motility. I measured the minimum and maximum absolute diameters
(mm) and the overall mean diameter (mm) of the ileum for a 60 s duration (de
Backer et al., 2008).

Statistics. Data are presented as mean values 1 S.E.M for n animals
from which ileal segments were obtained. Concentration-response data were
fitted using non-linear regression and the Hill equation (GraphPad Prism 4,
GraphPad, San Diego). Statistical analysis was performed using either Student's
t test for paired data, or a one-way ANOVA with Tukey—Kramer multiple
comparison as a post-hoc test when more than two groups were compared. The
effect of drugs on measured parameter responses at various intraluminal
pressures between groups was assessed using a two-way repeated-measures
ANOVA; post hoc multiple comparisons were corrected using Bonferroni's

procedure. P < 0.05 was the criterion for determining statistical significance.

184

 

 

RESULTS

Determining the EPso. To determine the EPso, I used, from a basal level,
0.5 mmHg step intervals in intraluminal pressure, ranging from T to T+5. I then
calculated the EP50 by measuring the active area of the AUC which will
incorporate frequency and amplitude of peristaltic contraction during each
episode (Figure. 6.2A, B, C) during this range. The EP50 was 1.9 :I: 0.03 mmHg (n
= 8). Using the EPso, l verified the stability of the reflex over a period of 50
minutes with peristaltic episodes at 5 minute intervals. There was no decline in

the AUC or frequency of contraction during this period (Figure. 6.3A, B).

Effects of calcium channel blockers on peristalsis. I also used the
EPso to investigate the effect of CdCI2 on peristalsis. CdCI2 (1-100 pM) inhibited
the peak amplitude of peristaltic contractions and their frequency. These changes
are indicated by the concentration-dependent decrease in the AUC (Figure. 6.4)
caused by CdCI2. Previous studies suggest that the N-type calcium channel may
be the predominant calcium channel in the ENS (Takahashi et al., 1992; Smith et
al., 1999). I therefore first investigated whether w-CTX could inhibit the peristaltic
motor pattern. w-CTX (1—100 nM) produced a concentration-dependent inhibition
of the peak contraction amplitude of 39.31 % (Figure. 6.4). Low concentrations of
NiC|2 (50 uM) selectively blocks R-type calcium channels in the nervous system
(Randall & Tsien, 1995; Tottene et al., 2000). NiCI2 (1-50 pM) also caused a
concentration-dependent reduction in the peak amplitude, reducing it by 19.95 %

(Figure. 6.4). The peristaltic reflex was impaired but not completely blocked by

185

 

 

either of NiC|2 or w-CTX. TTX (0.3 uM) completely inhibited the peristaltic reflex,
thus confirming that this observed pattern of small intestinal motility was

neurogenic through activation of myenteric neurons (data not shown).

NiCI2 and NLA inhibit the peristaltic reflex

Previous immunohistochemical data show that the pore-forming subunit of
the R-type calcium channel, a1 E, strongly co-localizes with NOS in cell bodies
and varicose nerve fibers in the myenteric plexus, and in nerve fibers innervating
the circular muscle (Naidoo et al., 2006). I therefore hypothesized that R-type
calcium channels may couple to NOS activation and NO-mediated muscle
relaxation in the guinea pig ileum. To explore the role of a possible interaction
between the R-type calcium channel and NO, I performed two sets of the
following experiments where either NiCI2 (50 nM) or NLA (100 uM) was applied
to the bath in combination with apamin (0.1 pM): 1) NiCI2 alone; NiCI2 + NLA;

NiC|2 + NLA + apamin; and 2) NLA alone; NLA + NiCI2; NLA + NiC|2 + apamin.

Peristaltic frequency. In experiments in which NiCI2 was added first, I
detected a significant decline in the peristaltic frequency compared to the control
response (Figure. 6.5, 6.6A). The frequency of contractions after co-application
of NiCI2 and NLA compared to NiC|2-treatment alone was unchanged. However,
when NLA was applied first, I detected a significant increase in the number of

propagating peristaltic contractions at intraluminal pressures 1.9 to 2.9 mmHg

186

(Figure. 6.7, 6.8A). After co-application of NLA and NiCI2, the peristaltic

frequency decreased below control values at 1.9 to 2.9 mmHg.

AUC. l detected a significant decrease in the AUC at 1.9 to 2.9 mmHg
when NiC|2 was added first (Figure. 6.5, 6.63). Subsequent application of NiCI2
and NLA did not change the AUC. In the presence of NLA first, however, the
AUC increased. When NLA and NiCI2 were subsequently applied together, the

AUC was reduced at 1.9 to 2.9 mmHg (Figure. 6.7, 6.8B).

Volume expelled. The ileum emptied less when NiC|2 was added first,
deviating from the control response at 1.9 to 2.9 mmHg (Figure. 6.6C). The
addition of MCI; and NLA did not further decrease the volume of expelled fluid.
When the ileum was treated with NLA first, the volume expelled increased over
the control response at 2.4 and 2.9 mmHg. Subsequent application of NiC|2 with
NLA significantly reduced the overall volume of fluid expelled at 1.4 to 2.9 mmHg

(Figure. 6.8C).

Mean Diameter. In the presence of NiCI2 and then NiCI2 with NLA, the

mean diameter increased significantly at intraluminal pressures 1.9 to 2.9 mmHg

(Figure. 6.6D).

Spatio-temporal maps. Throughout this study, peristalsis, when evoked

under drug-free conditions, was consistently robust with fully propagating motor

187

 

 

patterns (Figures. 6.5A1, A2; 7A1, A2; 9A1, A2, 11A1, A2). My interpretation of
these motor patterns relied on the concurrent analysis of simultaneously-
recorded ejection pressure waves (Figure. 6.5A1). In Figure. 6.5A1, the duration
of the emptying phase of peristalsis was approximately 2.5 s. Figure. 6.5A2 is a
spatio-temporal map depicting the motor patterns as cycles by characterizing the
propagation of peristaltic waves in time and space. Examination of a control
peristaltic reflex cycle (from the midpoint of one trough to the midpoint of the next
trough; Figure. 6.5A2 line a-b ) on the map shows that the time taken for one
cycle is approximately 5-6 s. This 5-6 s duration represents the emptying phase
duration (see Figure 6.5A1). The spatio-temporal map therefore indicates that
two fully propagating motor reflexes, each taking 2.5-3 s, traversed the ileal
segment within a total time period of 5-6 s. It should therefore be noted that one
cycle, as represented on my map, is a superimposition of multiple propagating
motor patterns that summate to produce that cycle. When NiC|2 was applied first,
the time taken for one cycle to occur steadily increased (Figure. 6.5B1, B2) until
the propulsive motor reflex was replaced by regular small-amplitude oscillatory
contractions. NiC|2 and NLA together, largely suppressed, but did not completely

inhibit the reflex (Figure. 6.5C1, C2).

NiCI2 and w-CTX inhibit the peristaltic reflex
Because NiCI2 did not completely inhibit the reflex (see above), I
investigated the contribution of other calcium channel subtypes to peristalsis.

This would help us determine the extent of R-type calcium channel involvement

188

 

in modulation of the peristaltic reflex. I chose to study the interaction between
NiCI2 (50 pM) and w-CTX (100 nM). I used an identical protocol to my NLA
experiments to monitor the peristaltic motor pattern, that is: 1) NiCI2 first; then

NiC|2 + w-CTX; and 2) w-CTX first; then w-CTX + NiC|2.

Peristaltic frequency. When the ileum was treated initially with NiCI2
alone, I observed a decrease in the frequency of peristaltic waves, and threshold
pressure was unaffected. The wave frequency only decreased significantly when
NiCI2 and w-CTX were co-applied (Figure. 6.9, 6.10A). However, when w-CTX
was applied first, the peristaltic reflex was impaired. In all tissues examined,
peristalsis did not commence until at least 15 s after the desired level of pressure
was attained; the initiation of the reflex appeared to be particularly affected. Co-
application of w-CTX and NiCI2 then mostly suppressed the propulsive wave at

all levels of intraluminal pressures tested (Figure. 6.11, 6.12A).

AUC. Examination of the AUC revealed a similar sensitivity of the ileum to
treatment with NiCI2 and w-CTX. Initial application of NiC|2 did not produce a
significant decrease in the AUC, and the AUC was only reduced after incubation
with both NiCI2 and w-CTX (1.4 — 2.9 mmHg) (Figure. 6.9, 6.10B). I further found
that when w-CTX was added to the bath first, and that when the ileal segment
was further incubated with both calcium channel antagonists, that this had a
similar diminishing effect on the AUC (Figure. 6.11, 6.12B, also compare Figure.

6.90 with Figure. 6.110).

189

 

 

Volume expelled. In the presence of NiCI2, the volume of fluid expelled
was not different from control values (Figure. 6.10C). When the ileum was
incubated with both NiC|2 and w—CTX, the volume emptied declined significantly
(0.9 — 2.9 mmHg). Reversibly, the application of w-CTX first, significantly
reduced the fluid volume expelled (1.9 — 2.9 mmHg). Co-application of MCI; and
w-CTX in both sets of experiments then caused a steep decline in fluid expulsion

volume at 0.9 — 2.9 mmHg (Figure. 6.12C).

Mean diameter. The presence of both NiCI2 + w-CTX (not shown), and
w—CTX + NiC|2 caused a significant increase in the mean diameter of the ileum in
both experiments (Figure. 6.120). This increase was particularly greater at higher

intraluminal pressures (1.9 - 2.9 mmHg).

Spatio-temporal maps. Analysis of spatio-temporal maps showed
consistent regular peristaltic reflexes under drug-free conditions (Figure. 6.9A2).
NiC|2 caused a progressive reduction in the strength of contraction and amplitude
of each peristaltic wave. NiCI2 also doubled the time taken for each motor pattern
to fully propagate from 2.5 s to approximately 4.5 s (Figure. 6.9B2), and this
duration increased throughout the recording session. NiC|2 together with w-CTX
completely abolished the motor pattern (Figure. 6.902). Peristaltic activity,

however, visualized as propagating oscillations uniform in amplitude, was still

190

 

 

observed. Reverse peristalsis was also found, indicating that in the presence of
NiC|2 and w-CTX, motility is not always aborally-directed.

When I applied w-CTX first, threshold pressure was not altered.
Propagating oscillations were again present, but only for approximately 30 s from
the onset of fluid-distention (Figure. 6.11B1, B2). Moreover, these oscillatory
contractions were not uniform in size and/or time. The regular motor pattern then
commenced and appeared to have, at least in part, recovered from the initial w-
CTX insult. Incubation of the ileum with both w—CTX and NiCI2 completely

inhibited facilitation of the distension-induced motor reflex (Figure. 6.11C1, C2).

191

 

 

DISCUSSION

The mammalian small intestine contains approximately 100 million
neurons, about as many neurons as that found in the spinal cord (Gershon,
1997). The functions of these neurons are controlled primarily by the ENS. The
ENS is therefore a critical regulator of GI function by coordinating motility
(peristalsis), absorption and secretory responses, thus indicating that altered
enteric neuronal signaling can therefore cause GI motility disorders. In this study
I therefore sought to understand the physiological role of the voltage-gated R-
type calcium channels in regulating peristalsis. The major findings of this study
are that the R-type calcium channels are coupled to NO release, and that this
channel, together with the N-type calcium channel are the major contributors to
fluid distention-induced peristalsis in vitro. Specifically, the evoked peristalsis
reflex is impaired by the association of the R-channel and NO, and by the R- and
N-type calcium channel subtypes. These findings have important implications for
furthering our understanding of peristalsis as they provide a mechanism by which
ion-channels can be targeted to control GI motility.

Voltage-gated calcium channels mediate neurotransmitter release and are
therefore important regulators of synaptic transmission. I first tested for a role of
calcium channels in peristalsis in the guinea pig ileum in the presence of CdCI2.
CdCI2 inhibited the peristaltic reflex by blocking calcium channels at all synapses,
consequently increasing the intraluminal threshold pressure and the amplitude of
the peristaltic wave. Peristaltic motility however, was not abolished, but still

present as small amplitude, shallow, and regular oscillatory contractions. These

192

 

 

oscillations could be due to the intrinsic myogenic mechanisms of GI smooth
muscle cells via adjacent interstitial cells of Cajal (ICC) (Huizinga et al., 2006;
Sanders, 2008). ICCs generate the slow wave that passively transmits to the
smooth muscle cell that ultimately, in conjunction with neural input, determine the
frequency and strength of contractions (Farrugia, 2008). I therefore concluded
that calcium channels are necessary for the release of neurotransmitters from
enteric motor axons to generate peristalsis. I observed identical results when
'l'l'X was applied to the ileal segment; the reflex was inhibited, confirming neural

mediation.

Inhibition of the peristaltic reflex by NiCI2 and NLA.

R-type calcium channels represent a viable control point for
neurotransmission if the ENS (Bian et al., 2004; Bian & Galligan, 2007), and a1E
subunits are localized in cell bodies of IPANS and in nerve endings of excitatory
(SP-containing) and inhibitory (NOS and VIP-containing) motorneurons (Naidoo
et al., 2006). These channels may therefore mediate synaptic excitation in the
ascending or descending reflex pathway. In my first set of experiments, I
therefore investigated whether R-type calcium channels contribute to the
peristaltic reflex. I used NLA to further investigate a selective coupling of the R-
channel to NO inhibitory neurotransmission. NiCI2 inhibited, but did not block
peristalsis. Subsequent application of NLA with NiCI2 did not produce any
additional changes in peristalsis suggesting that NLA and NiC|2 act on the same

mechanism. Interestingly, when NLA was applied first the peak amplitude,

193

 

l'll‘fl A: I. 1- -— - -

 

frequency, AUC and volume expelled all increased, particularly at higher
threshold pressures. Subsequent application of NiCI2 with NLA inhibited
peristalsis. Using the peristaltic frequency as a point of reference, this then
suggests at least two possible scenarios:

1) When NLA was applied first, the frequency of peristaltic contractions
significantly increased. NLA inhibits NOS to increase the propagating peristaltic
contraction frequency (Ciccocioppo et al., 1994). However, when NiCI2 was
subsequently applied in the presence of NLA, the contraction frequency
decreased. Since NiCI2 (50 (M) inhibits calcium entry though the R-type channel,
if the R-channel and NOS are selectively coupled, then calcium would not be
able to activate NOS. However, it also appeared that only when the R-channel
was blocked, that the ability of NLA to inhibit NOS was impaired because the
resultant frequency declined. The source of this decay points to a physical
interaction between the R-channel and NOS, and suggests that when the R-type
channel is blocked, NOS cannot be activated and therefore NOS cannot be
blocked by NLA. The fact that many peristaltic waves are still propagating
suggests that NiCI2 may be inducing a “braking” system on NLA-induced NOS
inhibition that would othenrvise produce a hyperexcitable ileum. This effect may
require synaptic release of multiple inhibitory enteric neurotransmitters from
motor axons on to the smooth muscle. Recent evidence indicates that enteric
motor nerve terminals in the mouse may contain catalytically active. forms of NOS
(nNIOSa) that are anchored via PDZ binding domains to the plasma membrane

(Stricker et al., 1997; Rao et al., 2008). NOS may therefore be docked at the

194

 

nerve terminal, close to R-type calcium channel release sites. My data suggest
however that NO is probably not the transmitter being released because a
physical association of the R-type channel to NOS requires that calcium enters
through the R-type channel to facilitate NO release. This implies that the lower
peristaltic frequency may be due to mediation by another inhibitory
neurotransmitter via another calcium channel subtype. After NiC|2 and NLA
treatment, apamin completely inhibited the peristaltic reflex. A likely candidate for
this neurotransmitter is therefore ATP/BNAD as a mediator of apamin-sensitive
inhibitory neurotransmission. ATP and NO are released from the same nerve
terminals (Costa et al., 1986b; Crist et al., 1992) and constitute the major
contributors to inhibitory neurotransmission in the ENS (Burnstock, 2008).
Moreover, NO and ATP are reported to exert independent effects on GI motility in
the rat ileum and porcine esophagus (Benko et al., 2006; Farre et al., 2006).
When NLA and apamin were co-applied to the rabbit distal colon (Ciccocioppo et
al., 1994) and guinea pig small intestine (Waterman & Costa, 1994; Holzer et al.,
1997), the gut became hyperexcitable but with non-propulsive segmental motor
patterns and very little or no emptying. However, in my study, I had added
apamin after NiCI2 + NLNNLA + NiCI2 and in both cases, apamin did not affect
the peristaltic reflex. The resulting motor pattern was similar to the effect by TTX
on the peristaltic reflex. An important test would have been to add apamin first
before either NiCI2 or NLA. The emptying phase of peristalsis involves the
activation of excitatory and inhibitory motor neurons (Waterman & Costa, 1994).

In order for efficient propagative peristalsis to occur, there must therefore be

195

 

functional synchrony between activation of these motorneuronal inputs to the
smooth muscle.

2) To confirm this coupling, I did the reverse experiment. When NiCI2 was
added first, the frequency of contractions was reduced, suggesting the possible
release of NO. Interestingly, when NiC|2 with NLA were co-applied, the frequency
remained the same. In the presence of NiCI2, NLA was unable to inhibit NOS and
could not produce an increase in the contraction frequency. This verified my
conclusion from the previous experiment that entry via the R-type calcium
channel may be coupled to the activation of NOS. Inhibiting the R-type channel
possibly prevents inhibition of NOS. If the R-channel is inhibited, and selectively
coupled to NOS, then the low peristaltic frequency observed is probably
mediated by another inhibitory neurotransmitter. I observed that apamin again
completely blocked peristalsis. Blocking the R-channel may alternatively also
prevent the release of excitatory neurotransmitters. My data however argue that
ATP release from inhibitory motor axons may be responsible for the reduced
peristaltic frequency rather than low excitatory neurotransmission.

Norepinephrine, released from sympathetic nerve endings, act at
az-adrenoreceptors to inhibit intestinal motility (Surprenant & North, 1988;
Stebbing et al., 2001). A selective coupling between the R-type calcium channel
and az-adrenoreceptors on myenteric neuronal cell bodies and nerve terminals
has recently been proposed (Bian & Galligan, 2007). az-adrenoreceptors have
been detected on cell bodies of myenteric neurons but the exact phenotype of

myenteric neuron expressing these receptors is not yet known (Bian & Galligan,

196

 

 

2007). The R-type channel may be present on the cell bodies of lPANs where it
modulates the slow excitatory postsynaptic potential and somal action potential.
That study showed that agonist-activated az-adrenoreceptors inhibit calcium
entry through R-type channels, because in the presence of NiCI2, inhibitory
control by the az-adrenoreceptor over the R-type channel was removed. In my
study, when NLA was applied first, it is possible that the R-type channel was
unable to prevent inhibition of NOS during the peristaltic reflex due to the release
of norepinephrine from sympathetic nerve endings. Norepinephrine, acting at
pre-junctional az-adrenoreceptors on myenteric motor axons, would then inhibit
calcium influx by decreasing the probability of opening of the R-type channel, or
activating presynaptic K” channels (Hein, 2006). This principle would also hold
when NiC|2 was applied first. NiCI2-block of the R-type channel presumably
removes inhibition by the az-adrenoreceptor to cause either a conformational
change in the calcium channel protein, or a change in the R-channel-NOS

signaling cascade that renders NOS inaccessible to inhibition by NLA.

Inhibition of the peristaltic reflex by NiC|2 and w-CTX

In the present study, I demonstrate that the peristaltic reflex in the guinea
pig ileum is mostly mediated by R- and N-type calcium channels. This conclusion
is based on the observation that co-application of MCI; and the N-type calcium
channel antagonist w-CTX completed inhibited the peristaltic reflex. My
concentration-response curve for NiCI2 and w-CTX shows that the additive

inhibitory effect of these two calcium channel antagonists is approximately 60 %.

197

 

When NiCI2, was added first, the number of propagating peristaltic waves, peak
peristaltic amplitude, area under the curve, and volume of fluid expelled all
significantly decreased compared to control responses. These parameters
appeared to be particularly affected at higher threshold pressures. The reflex was
impaired but was not blocked. Application of w—CTX then completely inhibited the
peristaltic reflex. When w—CTX was applied first, non-propulsive segmentation
patterns were observed, and propagating contractions only began after at least
25 5. By itself, w-CTX did not change the threshold pressure for initiation of
peristalsis. This suggests that N-type calcium channels mediate
neurotransmission between interneurons and motorneurons, and between
motorneurons and the smooth muscle. However, in the presence of w-CTX and
NiC|2, the reflex was abolished, signifying inhibition of neurotransmission at all
synapses. I still observed intrinsic myogenic activity, indicating that peristalsis still
exists, albeit with a low level of neural excitation. To date, only two studies have
reported on the functional significance of R-type calcium channels in the GI tract
(Bian et al., 2004; Bian & Galligan, 2007). Most studies on R-type channel
function have been done in the CNS and the R-channel has been localized to
nerve endings at many excitatory synapses (Wu et al., 1998; Tottene et al., 2000;
Gasparini et al., 2001; Dietrich et al., 2003). In wild-type mice, PIQ-type calcium
channels are the major calcium channel subtype found on nerve terminals in the
calyx of Held (Wu et al., 1999), cerebellum (Matsushita et al., 2002) and
hippocampus (Qian & Noebels, 2000). Furthermore, PIQ-type channels primarily

mediate acetylcholine release at mammalian endplates (Urbano et al., 2003;

198

 

Pagani et al., 2004). Urbano et al. (2003), using PIQ-type calcium channel
knockout mice showed that: 1) R- and N-type calcium channels are upregulated,
and 2) R-type channels were less numerous than N-type channels, but are
located closer to presynaptic calcium sensors at the active zone. In the ENS,
PIQ-type calcium channels display weak immunoreactivity weak in the soma and
nerve terminals of myenteric'neurons, whereas N-type calcium channel-ir was
diffuse and intense (Kirchgessner & Liu, 1999). Further, PIQ-type calcium
channels contribute only 20 % to the total calcium channel current (Bian et al.,
2004). In the present study, my data suggest that the major contributors to the
peristaltic reflex are the N-and R-type calcium channels. Previous
immunohistochemical and electrophysiological data indicate that a1E co-
localizes with NOS, VIP and SP in enteric motor neurons, and that R-type
channels are present in a specific population of purely-cholinergic calretinin-
containing ascending interneurons (Naidoo and Galligan, unpublished data). The
present data indicate that NiCI2, in the presence of fluid-induced radial distention,
clearly induces a shift in motor activity of the ileum, from neurally-mediated fully
propagating reflex patterns to segmental contractions associated with non-
propulsive churning movements of the fluid content. | speculate that this shift
may be due to a block of neurotransmission at specific synapses along the
peristaltic reflex pathway. At these synapses, the concentration of R-type
channels may be high. Ascending interneurons constitute 5 % of the total number
of myenteric neurons in the ileum (Furness, 2006a) and 25 % of these are fully

cholinergic (Galligan et al., 2000). At purely-cholinergic ascending excitatory

199

 

 

synapses, R-type channels would mediate all cholinergic neurotransmission from
those calretinin-containing interneurons. In fact, Gasparini et al. (2001) showed
that 50 (M NiC|2 reduced fast glutamatergic transmission in the CNS by at least
51-59 %. Because R-channels are also present on SP- and NOSNIP-containing
enteric motorneurons, they may control excitatory and inhibitory
neurotransmission to the muscle. By inhibiting calcium-entry with NiCI2 at all
these synapses, the reflex circuitry is dysregulated. R-type channels make an
important contribution to the peristaltic reflex, but in its absence, this reflex can
still continue, as I have observed under conditions of fluid-evoked distention.
Inhibiting its function, however, disrupts motility. The data suggest that at least
two calcium channels are necessary for the peristaltic reflex. Perhaps for the
peristaltic reflex to occur, two calcium channels, from a combination of N-type
with the R-type calcium channel are necessary for this facilitation. This may be
part of a fail-safe mechanism that has built into the reflex circuitry to ensure the

efficacy of the propagating peristaltic motor pattern in the guinea pig.

SUMMARY AND CONCLUSIONS
Inhibition of R-type calcium channels in an in vitro model of fluid-induced
peristalsis suppresses but does not completely inhibit the peristaltic reflex. The
R-type channel is coupled to NOS and when both are inhibited, the peristaltic
reflex is abolished. The R-type and N-type calcium channels function in tandem
to facilitate the peristaltic reflex. Further understanding of the interaction between

the various voltage-gated channel subtypes holds promise for improving our

200

 

 

understanding of GI motility, and the development of therapeutic strategies

against motility diseases.

 

201

 

Figure 6.1. Peristalsis video imaging. A. A single frame from a peristalsis
movie recording of the ileum imported into Image J. B. All movie frames were
then converted into binary images. Image thresholding and edge detection
delineated the ileum periphery. By selecting regions of the ileum and using
intestinal motility software, I was able to detect changes in gut diameter. These
changes were pooled and built into 3- dimensional spatio-temporal maps with an

Image J-plugin linked to the graph-plotting program Gnuplot.

202

Figure 6.2. EP5o pressure-response curve and representative traces. (A)
Pressure-response curve in drug-free Krebs’ solution for calculating the
magnitude of the area under the curve (AUC) against successive increases in
intraluminal pressure. The half maximal effective pressure (EP50) was 1.9
mmHg. Data points are mean :I: S.E.M. n represents the number of animals.
Curves are non-linear fits of the Hill equation. (B) The reservoir was raised from
a baseline pressure where no peristalsis was observed, to a threshold pressure
(T, arrow) that produced a peristaltic reflex. (C) The area under the curve (AUC)
was calculated for every pressure interval from T to T+5. These intervals were in
0.5 mmHg increments after the T value was known. Note that only the active
area of the AUC was calculated. The arrow indicates the desired pressure level,
and the dotted red line (at the arrow point) is the baseline for calculation of the
AUC. The amplitude and frequency of peristaltic waves, in addition to the volume

expelled, were also calculated.

203

 

 

A 125-

Control (n = 8)

 

 

t 3
‘1 i 5 i

45‘ Intraluninal pressure (““01

 

3 Control Pressure Trace (Threshold, T)

Baseline
pressure

 

 

 

 

C Control Pressure trace: T+5
I Frequency %
T+5 Amplitude
l
1' .......AU. #

1 L

Baseline 10 5
re ................................................................................................................

204

A 1000001 Control (n = 5)
95000(

90000J
850004

800001

AUC (mmHg.ms)

 

 

75000

0 10 20 30 40 50 60
Minutes after equilbration

0'19' Control (n = 5)

0.18“

0.17- M

0.16‘

 

 

o. 1 5 T U I V I I
0 10 20 30 40 50 60

Minutes after equilbration

Peristaltic frequency (waves! 8)

Figure. 6.3. Time controls showing stable peristaltic motor patterns after
measuring (A) the AUC and (B) the peak peristaltic frequency in 5 min
resting intervals for a period of 50 min after tissue equilibration. Peristalsis
was evoked and recorded for 60 s. The ileal segment showed no evidence of

fatigue during the 50 min experimental period.

205

 

 

 

 

 

1501 I OJ‘CTX (n = 4)
- NiC|2 (n = 8)
CI CdCIZ (n = 5)
d; 100‘
,\
U
D
< 50‘
G T I fl I
-10 -8 -6 -4 -2

[Calcium antagonist] (M)

Figure. 6.4. Effect of w-conotoxin (cu-CTX), NiC|2 and CdClz on the AUC
expressed as percent change. w-CTX (1-100 nM) and NiC|2 (1-50 pM)
decreased the AUC by 39% and 20% respectively. CdCI2 (1-100 uM) caused a
concentration-dependent inhibition of the AUC, and blocked the AUC completely

at 100 pM. Data are mean :I: S.E.M. n indicates the number of animals,

206

 

Figure. 6.5. Effect of NiCI2 and NLA on (A) frequency, (B) AUC, and (C)
fluid volume expelled. These four parameters all decreased in response to
NiCI2-treatment alone. Subsequent application of NLA did not produce a shift in
any of the parameters. This was particularly evident at higher intraluminal
pressures: frequency and AUC 1.9 — 2.9 mmHg; volume expelled 1.4 — 2.9
mmHg, and mean diameter 1.9 - 2.9 mmHg, compared to control (two-way

ANOVA; P < 0.05). Data are mean :I: S.E.M. *P < 0.05; **P < 0.01; *** P < 0.001.

207

 

 

Peristaltic frequency (waves! 3)

40°00 - Control AUC

AUC (mmHg.ms)

.§§

Volume expelled (mi)

30000 - NiCI2 + NLA
5

0.25 - Control Frequency

 

0.4 0.9 1.4 1 .9 2.4 2.9
Intraluminal Pressure (mmHg)

  
   

- NICI2

  

0.4 0.9 1.4 1.9 2.4 2.9
lntraluminal Pressure (mmHg)

Volume expelled (ml) (per 60 seconds)

- NiC|2+ NLA
n=5

 

0.4 0.9 1.4 1.9 2.4 2.9
lntraluminal Pressure (mmHg)

208

Figure 6.6. Effect of NLA and NiCI2 on (A) frequency, (B) AUC and (C) fluid
volume expelled (refer to Figure. 6.7). Addition of NLA first, increased all the
above parameters of peristalsis. Subsequent application of NiCI2 reduced these
parameters. A. The peak NLA contraction was significantly different from NLA +
NiCI2 but not from control. B. The frequency of peristalsis after NLA treatment
was higher compared to NLA + NiC|2 treatment, particularly at intraluminal
pressures 1.4 — 2.9 mmHg. NLA, and NLA + NiC|2, were significantly different
from control at pressures 1.9 - 2.9 mmHg. C. The AUC after NiCI2 and NLA
treatment was greater than either control or NiC|2 alone, at 1.9 — 2.9 mmHg.

Data are mean :I: S.E.M.

209

 

 

AUC (mmHg.ms)

i?
To
a:
i
E.
>
o
r:
o
=
a'
o
It
Io
E
8
.2
h
a:
0.

50000 - Control

0.10

0.05

0.00

 

      
 
  

Frequency '2'
0.20 - Control “M **
I NLA H
- NLA+ NiC|2
0.15 ":4

0.4 0.9 1.4 1 .9 2.4 2.9
lntraluminal Pressure (mmHg)

AUC

 

 

Volume expelled (ml)

0.4 0.9 1 .4 1.9 2.4 2.9
lntraluminal Pressure (mmHg)

Volume expelled (ml) (per 60 seconds)

  
   
 

      
  

8 - Control **
I NLA m If"
6 I NLA+NICI2
n=4
4
2
0

0.4 0.9 1.4 1.9 2.4 2.9
lntraluminal Pressure (mmHg)

210

Figure 6.7. The peristaltic motor pattern changes with NiCI2, and NiC|2 +
w-CTX treatment (refer to Figure. 6.9). A. The frequency after NiC|2 application
was reduced (1.4 - 2.9 mmHg vs. control). Addition of w-CTX completely
inhibited the reflex wave frequency (0.9 - 2.9 mmHg vs. control; (1.9 - 2.9 mmHg
vs. control). B. The AUC was inhibited after NiCI2 treatment (1.9 -- 2.9 mmHg vs.
control). Subsequent application of w-CTX then completely blocked the AUC (0.9
— 2.9 mmHg vs. control; 2.4 - 2.9 mmHg vs. NiCI2). C. Volume of fluid expelled
decreased significantly compared to control at intraluminal pressures 2.4 and 2.9
mmHg. Following NiC|2 application, the volume ejected was significantly reduced
(0.9 - 2.9 mmHg vs. control; 2.9 mmHg vs. NiCI2). D. Mean intestinal diameter
and. Under control conditions, the ileum demonstrated a small mean diameter.
After w-CTX treatment, the diameter increased. Following w-CTX and NiC|2
treatment, the diameter significantly increased (1.9 - 2.9 mmHg vs. control; 1.9 -—

2.9 mmHg vs. w-CTX). Data are mean :I: S.E.M.

211

 

 

 

Peristalfic frequency (waves! s)

AUC (mmHg.ms)

 

 

 
 
   

Frequency
0-20‘ - Control
- N'CIZ
(“5‘ .- NICI2+ CTX . * A
n - 5 H a
i it
0.10' *
0.05- ** *** *** *** I **
0.00d
0.4 0.9 1.4 1.9 2.4 2.9
lntraluminal Pressure (mmHg)
AUC
500001 I Control
I NiC|2
.I
W I NiCI2 + w-crx
sooood n = 4 . **
* H :7
200W *
rooom * ** *** *** m
0.1
0.4 0.9 1.4 1.9 2.4 2.9
lntraluminal Pressure (mmHg)
Volume expelled (ml) (per 60 seconds)
a 12' - Control
5 10- I NiCI2
3 8. I NiCI2 + m-crx **
3 n = 4
ft 64
o .l
0 4
S l
3 2
>

 

 

0.4 0.9 1.4 1.9 2.4 2.9
lntraluminal Pressure (mmHg)

212

 

Figure. 6.8. Effect of w-CTX alone, and w-CTX with NiC|2, on peristalsis
parameters. A. After w-CTX treatment, the frequency significantly decreased
compared to control (0.9 - 2.9 mmHg). Addition of NiC|2 then inhibited the
frequency (0.4 - 2.9 mmHg vs. control; 1.4 - 2.9 mmHg vs. w-CTX + NiC|2). w-
CTX together with NiC|2 blocked propagating reflex contractions, but not small
oscillating contractions B. The AUC decreased significantly after w-CTX
treatment compared to control at intraluminal pressures 1.4 and 2.9 mmHg. The
AUC was then significantly reduced after application of NiC|2 (0.9 - 2.9 mmHg vs.
control; 1.9 - 2.9 mmHg vs. w-CTX). C. Volume of fluid expelled declined
significantly in the presence of w-CTX (1.9 - 2.9 mmHg vs. control) and after w-
CTX + NiC|2 treatment (1.9 - 2.9 mmHg vs. w-CTX). D. Under control
conditions, the ileum demonstrated a small mean diameter. After w-CTX
treatment, the diameter increased. Following w-CTX and NiCI2 treatment, the
diameter significantly increased (1.9 - 2.9 mmHg vs. control; 1.9 - 2.9 mmHg vs.

w-CTX). Data are mean :I: S.E.M for n tissues.

213

 

 

AUC (mmHg.ms)

Peristaltic frequency (waved s)

 
   
 

Frequency
I Control
CI w-crx
I (o-CTX + NiCI2
n = 4

 

 

 

0.4 0.9 1.4 1.9 2.4 2.9
lntraluminal Pressure (mmHg)

 
   

 

AUC

I Control
80°00] :3 max

I -CTX + NiCI2 m ***
soooot 0’ ,2, H "'

n = 4 it
40000“ **
20000‘ W ** ***

| .
0‘ '
0.4 0 9 1 4 1 9 2.4 2.9
lntraluminal Pressure (mmHg)
Volume expelled (ml) (per 60 seconds)

i 12' I Control
" 10' C] (D'CTX
3 8) I (o-CTX + NiCI2 ***
3 .4
o
E 4‘
2 2'
o
> 0-

 

0.4 0.9 1.4 1.9 2.4 2.9
lntraluminal Pressure (mmHg)

214

 

 

 

CHAPTER 7 GENERAL DISCUSSION AND CONCLUSIONS

215

 

Gastrointestinal motility disorders pose a severe reduction on the
quality of life for affected individuals. Despite a plethora of studies aimed at
understanding the pathophysiology of GI disorders, there are still no cures. The
reason probably has to do not only with the complexities of the disorders
themselves, but also with the overall impact on the ENS. For example, in patients
with IBS and Crohn’s disease, the ENS is a specific target of inflammatory cells
including lymphocytes and mast cells that alter the enteric neural circuitry. After
tissue inflammation, signals generated by inflammatory cells induce changes in
extrinsic sensory nerve endings, lPANs, interneurons, and motorneuronal
signaling to the smooth muscle (Kirkup et al., 2001). The effect of impaired or
altered synaptic communication means that the ENS has to attempt to overcome
inflammatory insults by deconvoluting new sensory signals and combining them
within the enteric circuitry to produce an adaptive response. However, in IBD and
Crohn’s disease patients, drastic therapeutic strategies such as corticosteroid
administration, or as a last resort, surgical intervention, are used to help patients
with moderate to severe forms of those diseases. A reason for the lack of cures
for GI disorders also probably has to do with the high sensitivity and semi-
autonomous nature of the intrinsic reflex circuitry of the ENS. By high sensitivity I
mean, for example, that even a minor alteration in the signaling pathway of the
ascending excitatory pathway may have overall consequences on muscle
contraction. Similarly, dysregulation of the descending inhibitory pathway would
produce an altered result on muscle relaxation. IPANs are connected in feed-

forward circuits, are the first neurons activated by distention or mucosal

216

 

stimulation, and act to gate excitation in the myenteric and submucosal circuitry.
The integrity of the nerve endings of lPANs is therefore important for continual
gut function. Furthermore, IPANs, interneurons and motorneurons are connected
at multiple points along the GI tract, implying that a response to a given stimulus
can occur at any given point in the gut wall. The semi-autonomous nature of the
GI tract is advantageous and pragmatic in terms of having its own “little brain”, in
that transmission over long distances (from the CNS to the ENS) and the
packaging of 100 million enteric neurons into the CNS has been avoided through
evolution. However, this may also be to the detriment of the ENS. That is,
because of the paucity of connections between the CNS and the ENS, the ENS
is only able to respond to dysregulatory mechanisms mostly by itself. Therefore,
when the GI reflex circuitry is affected by an inflammatory “soup”, the outcome is
severe and can be debilitating. Psycho-social factors are also believed to be
involved in GI motility disorders, but conclusive evidence has yet to be
presented. The search for therapeutic strategies to combat motility disorders,
although challenging, is ongoing, and the studies presented in my dissertation
contribute to that search. Because of the immense complexity that GI disorders
present, I believe that a good strategy to target ENS dysfunction is to study the
mechanisms of the ENS reflex circuitry. After all, that is the region where many
GI disorders and diseases produce their overall effects.

Calcium channels are the critical conduits of synaptic transmission.
They transduce electrical signals into chemical ones, and therefore regulate

communication between excitable cells. They are also involved in contraction of

217

 

smooth and skeletal muscle, secretion, and gene expression. Calcium channels
have also been established as key components in the ENS. Seminal experiments
by Bian and Galligan (2004) demonstrated that at least 50% of the calcium
current in myenteric neurons maintained in primary culture is R-type calcium
channel-mediated. In light of that evidence, my dissertation addressed the
functional importance of the R-type channel in the ENS. The guinea pig has been
an invaluable and major contributor in furthering our understanding of ENS
function. Other species, however, such as rats and mice have also been used to
study the ENS. Much of our present understanding of the ENS is based on
structural, morphological, neurochemical and physiological studies done in
guinea pigs. The ileum is also the most studied and best characterized GI region.
Study of local ENS control can also best be studied in this region because ileal
myenteric neurons are sparsely innervated by vagal (parasympathetic)
preganglionic cholinergic neurons and sympathetic postganglionic noradrenergic
neurons located in celiac ganglia (Goyal & Hirano, 1996). CNS contribution to
motility control in the ileum is therefore limited, implying that motility is
predominantly locally regulated. The guinea pig was therefore my model system

of choice for these studies.

A major finding in my study was that a1E-ir is not present in the
submucosal plexus, and therefore does not contribute to absorptive or secretory
functions in the guinea pig GI tract. However, extensive a1E-ir in varicose nerve

fibers is present in the myenteric plexus in all regions of the GI tract. The

218

 

predominance of R-type calcium currents in myenteric neurons suggests an
important role for this class of calcium channels in enteric neurotransmission and
motility. Interestingly, there was no expression of a1E within cell bodies in the
guinea pig small intestine. Cell bodies of myenteric neurons in the stomach and
colon, however, do express R-type channel immunoreactivity, with the colon
demonstrating a greater expression of a1E-ir. Overall, a1E staining in the colon
was greater compared to the stomach and small intestine. The reasons for the
intense a1E labeling in the colon LMMP could be related to the function of the
colon. The colon absorbs water and electrolytes from the intestinal content and
converts it into fecal matter. Furthermore, migrating myoelectric complex (MMC)
patterns are absent in the colon, but slow waves are present. The MMC is an
electromechanical pattern that occurs in the fasting state. It is responsible for the
propulsion of residual undigested food and sloughed enterocytes from the
stomach through the small intestine. It therefore serves a “housekeeping”
function. The musculature in the proximal colon exhibits two basic motor
patterns: segmental (haustral) contractions, and neurogenically—mediated mass
peristalsis, while the distal colon only displays the former (Furness, 2006a). The
function of the R-type channel, present on cell bodies and on nerve terminals,
could therefore be to augment peristalsis in the colon. This could occur by
mediating calcium entry during somatodendritic action potentials, or
neurotransmitter release. I did not find a1E-calbindin-ir colocalization in the colon

LMMP. However, I did observe strong co-localization of a1E and SP, a1E and

calretinin (nerve fibers) and a1E with NOS and VIP. This suggested that a1E

219

was present in: 1) excitatory and inhibitory circular and longitudinal muscle
motorneurons, 2) ascending interneurons, and, 3) lPANs. The prevailing dogma
is that calbindin is the best known marker for myenteric lPANs. However,
according to Brookes and Costa (2006), at least two types of myenteric lPANs
exist: 1) an IPAN majority that respond to chemical stimulation of the mucosa;
and 2) an IPAN minority that are sensitive to tension along the gut wall (Kunze et
al., 1998; Kunze et al., 2000). In fact, a third subset of IPANs may also exist,
those that are sensitive to both tension and chemical stimuli. Those data suggest
that calbindin may not necessarily fit the gold-standard for confirming IPANs, and
my data support this theory. SP is a marker for both IPANs and excitatory
longitudinal muscle neurons. SP may therefore be present in IPAN populations
that are calbindin-negative. The SP antibody I had used never labeled cell
bodies, while calbindin never labeled nerve terminals. However, because SP and
a1E co-localize in the myenteric LMMP, the R-type channel may therefore be
present in nerve terminals of colonic IPANs that are calbindin-negative. They
may also be present on nerve endings of excitatory motorneurons. There are two
ways to answer these possibilities. One is to record action potentials and
sEPSPs from AH neurons in LMMP preparations in the colon, in the presence of
NiCI2 (50 nM) or SNX-482 (0.1 nM). The second method would be to perform
neuron cell culture to determine whether a1E is expressed in colonic myenteric
neurons. R-type channel staining in the small intestine deviated from the

stomach and the colon. The abundance of axonal varicosities in the intestinal

220

 

whole mount LMMP suggests that the function of the R-type channel may be
more towards that of synaptic transmission.

Intracellular recordings of action potentials from single AH showed
that NiCI2 did not reduce the amplitude of the AH action potential, but did inhibit
the half-width by almost 50 %. Addition of w-CTX then completely blocked the
calcium shoulder. The late AHP was also significantly reduced in the presence of
NiCI2. Rugiero and others (2002) showed a major contribution of N-type channels
to the action potential, and also implicated the R-type channel as a contributor to
the total inward calcium current. In my study, I have shown the N- and R-type
calcium channels contribute equally to the somatodendritic action potential in AH
neurons. NiCI2 had no effect of on action potentials in S-type neurons. The effect
of NiC|2 on fEPSPs was interesting. I initially observed that some fEPSPs were
significantly inhibited by NiC|2, whereas others were completely unaffected.
There is a single group of ascending interneurons but 3 groups of descending
interneurons in the guinea pig ileum (Brookes, 2001). The ascending
interneurons comprise just 5 % of the total number of myenteric neurons in the
myenteric plexus and are immunoreactive for ChAT and calretinin. Those
neurons use ACh as their neurotransmitter. It should be noted that some
excitatory longitudinal muscle motorneurons (25 %) are also calretinin-ir. In the
guinea pig LMMP, the axons of excitatory longitudinal muscle motorneurons are
confined to the tertiary plexus (Brookes & Costa, 2006), are short, and project in
this plexus close to their cell bodies (Lomax et al., 1999; F urness, 2000; Brookes

& Costa, 2006). In the LMMP preparation, there is therefore a greater chance of

221

 

recording from interneurons (ascending or descending) rather than longitudinal
(excitatory or inhibitory) muscle motorneurons as l was stimulating
interganglionic fiber tracts. Furthermore, motorneurons do not synapse with other
enteric neurons (LePard & Galligan, 1999). Together, descending interneurons
account for 11% of the total number of myenteric neurons in the guinea pig
LMMP and use either ACh and ATP, or ACh and 5-HT for neurotransmission
(LePard et al., 1997; LePard & Galligan, 1999; Galligan, 2002b). A third group of
descending interneurons is associated with the MMC and has Dogiel type III
(filamentous) morphology. The majority of the interneurons (67%) belongs to the
mixed cholinergic/purinergic group, while 25% are completely purinergic
(Galligan, 2002b). Components of the fEPSP can therefore be isolated using
mecamylamine (nAChR antagonist), PPADS (P2X receptor antagonist) or
ondansetron (5-HT3-receptor antagonist). My data shows that NiCI2 and SNX—482
selectively act at the nerve endings of purely cholinergic nerve fibers to inhibit R-
type calcium channels as the fEPSP amplitude was reduced. Neurons that use
both ACh and ATP for fast transmission were unaffected by NiCI2 and SNX-482
treatment. This result means that R-type calcium channels are localized to
calretinin interneurons, and this is supported by my findings that a1E and
calretinin co-Iocalize in some varicosities in the LMMP. The paucity of co-
localization between a1E and calretinin supports this conclusion as the R-type
channel is present in just 5 "/0 of purely cholinergic ascending interneurons. In
both subtypes (cholinergic/purinergic), w-CTX largely inhibited the fEPSP

following R-channel blockade. Therefore, mainly R- and N-type channels

222

 

 

contribute to fast synaptic in purely cholinergic S-type interneurons, whereas only
N-type calcium mostly mediate fast transmission neurons at mixed
cholinergic/purinergic nerve endings. The ideal way to determine whether NiCI2 is
acting pre-or postsynaptically to inhibit calcium entry through R-type calcium
channels would be to “puff” onto impaled neurons an agonist of the mediator of
the sEPSP or fEPSP in the presence of NiC|2. Previous data (DeVries M,
unpublished) showed that NiCI2 acts presynapticallly to inhibit synaptic
trasmission. My present data further indicate that CdCI2 and NiC|2 do act
presynaptically. This conclusion is based on: 1) CdCI2 did not affect the neuronal
membrane potential; 2) NiCI2 did not affect the sEPSP; 3) NiC|2 acts at nerve
terminals in fully cholinergic S-type ascending interneurons; 4) NiC|2 does not
affect fEPSPs in descending interneurons. Taken together, these data rule out a
general non-specific effect on the postsynaptic cell. Perhaps the reason why the
R-type channel is present on nerve endings of purely cholinergic ascending
interneurons is based on redundancy. That is, the ENS has included a
mechanism using two calcium channels that act as fail-safes in instances of, for
example, a toxin—attack that acts to block chemical neurotransmission. Having
two calcium channels at a single synapse would therefore help the ENS
circumvent noxious insults. Because the ENS has a single population of S-type
interneurons, the R-type channel may consequently be very important in this
regard. ACh is an important enteric neurotransmitter, and the importance of

purely cholinergic interneurons may be to spread excitation to other cell types,

223

 

 

including excitatory motorneurons, to allow the coordination of motor responses
over long distances in the GI tract.

R-type calcium channels also mediate inhibitory neurotransmission to the
muscle layers, suggesting a functional coupling between the R-type calcium
channel and NOS synthesis and NO release (Figure. 7.1). The R-channel does
not appear to be linked to ATP release from nerve terminals. It is possible that
the R-type channel, localized to myenteric nerve endings is coupled to NO
synthesis and release, physically or via an intracellular signaling mechanism, to
cause relaxation of GI smooth muscle through an ATP (and possibly B-NAD)-
insensitive mechanism. This mechanism would be as follows: intracellular
nNOSa would be anchored at the nerve terminal membrane, and activated by
the influx of calcium. NO would be synthesized by intracellular NOS, and NO
would then passively diffuse to affect the postsynaptic cell. These data therefore
have important implications for the spatial organization of calcium-channel-

mediated exocytosis.

The effect of NiC|2 on R-type calcium channel function in the whole ileum
is important for the understanding of possible therapeutic strategies against GI
motility disorders, particularly in light of ion channels as potential drug targets
(Lipecka et al., 2002; Cuppoletti et al., 2004). For example, the peptide
Ziconotide is a synthetic analogue of w-CTX used to treat chronic pain (Miljanich,
2004). Similarly, the spider toxin peptide inhibitor guangxitoxin-1 targets the

delayed rectifying potassium channel Kv2.1 and enhances glucose-dependent

224

 

 

insulin secretion. This has important implications for the treatment of type II
diabetes (Herrington et al., 2006; Kaczorowski et al., 2008). However, the
challenges in translating ion-channel drugs from bench to bedside is difficult due
to, amongst others, species differences, and just how efficacious such drugs
would be in humans (Kaczorowski et al., 2008).

NiC|2 by itself impairs the peristalsis reflex. Peristalsis can still occur,
but mixing and churning movements of the intestine were observed 30 seconds
after fluid-induced peristalsis. Where exactly is NiC|2 acting? NiC|2: 1) reduces
the calcium shoulder and the late AHP amplitude of the AH action potential; 2)
acts at nerve terminals of purely cholinergic interneurons; 3) reduces inhibitory
neurotransmission by coupling to NO to the muscle layers; 4) may also act at
other unknown sites. By disrupting synaptic communication in a number of
pathways, such as the ascending excitatory pathway, and at inhibitory
neuromuscular junctions, the function of the reflex circuitry is compromised. N-
and PIQ-type calcium channels appear to be localized near R-type channels,
such that calcium channel co-operativity between those major calcium channel
subtypes may be important for facilitation of enteric reflexes (Pietrobon, 2002,
2005). My data indicate the presence of R-type calcium channels within specific
populations of myenteric neurons. R-type channels may therefore have an
important function as a relay point for information signaling within those neurons.
lmpairing its function alters the peristaltic reflex.

A schematic of a potential coupling between voltage-gated R-type

calcium channels (VGCC) and NOS on myenteric inhibitory motor nerve

225

 

terminals is shown in Figure. 7.2. In Figure. 7.2A, under drug-free conditions,
fluid-distention induces a propagative peristaltic reflex. At an inhibitory
neuroeffector synapse, NOS is anchored within the active zone to the plasma
membrane. This synapse is also under inhibitory control by sympathetic nerves
that release norepinephrine (NE). NE, acting at presynaptic GilGo-coupled a2-
adrenoreceptors (azAR) will inhibit release of neurotransmitters from the
myenteric inhibitory motor nerve fiber. If the azAR and R-type channel are
coupled perhaps by diffusible second messengers, then the probability of the R-
channel being open will be reduced, and calcium influx will be blocked. If the R-
channel and NOS are coupled, then NO cannot be released if the R-channel is
under the control of the azAR. If the R-channel and NOS are not coupled, then
NO can be released via calcium entry through another calcium channel, possibly
the N-type calcium channel. NO can then diffuse across the neuroeffector
junction into the smooth muscle cell, bind to guanylate cyclase, to cause muscle
relaxation.

When NLA is added first (Figure. 7.28), and distention evoked, the
frequency of contractions increases. NLA inhibits NOS leading to increased
smooth muscle contraction. NE will again act at the azAR and because it is
coupled to the R-channel, calcium entry through this channel will be then blocked
by azAR activation. If the R-channel and NOS are coupled, then in the presence
of NLA alone, NOS cannot be under the control of the R-channel, because the
channel is still inhibited by the azAR. NLA is now free to exert its inhibitory

actions on NOS, leading to increased contractions.

226

 

 

When NiC|2 is added in the presence of NLA (Figure. 7.2C), and
peristalsis induced, the frequency of contractions decreased. NE is still released
from the sympathetic nerve terminal and activates the azAR. The azAR, however,
cannot inhibit the R-type channel because NiCI2 (50 pM) removes this inhibition
(Bian & Galligan, 2007). Calcium entry therefore cannot occur. Because the
contraction frequency is reduced (by NLA and NiCI2), this suggests that NLA
cannot be inhibiting NOS. Speculatively, this may be because of an intracellular
coupling pathway or a physical interaction between the R-type channel and NOS.
Reduced calcium entry via the R-type channel would lead to less calmodulin
binding, resulting in reduced NOS activation. NO therefore cannot be
synthesized via the R-channel at this synapse. The inhibitory response must be
due to release of another neurotransmitter via another calcium channel. Apamin
completely inhibits the peristaltic reflex, and my data also show that the R- and
N-type channels act in tandem to block the reflex. Therefore, N-type (or even
PlQ-) channel-mediated ATP neurotransmission must account for the low
frequency of the propagating peristaltic reflex. This would also help explain the
sustained inhibition in the reverse experiment when NiCI2 was added first,

followed by NICI2 and NLA.

Tissue damage associated with inflammation, a cellular response to injury,
evokes the production of a myriad of chemical mediators from specific cell types
such as mast cells and lymphocytes at the site of injury (Levine 8. Reichling,

1999). The initial events in tissue inflammation, provoked by chemical, thermal,

227

 

or mechanical stimuli, involve the synthesis and release of such pro-inflammatory
mediators that comprise an “inflammatory soup”. These compounds include,
amongst others, protons, histamine, peptides (bradykinin, BK), lipids
(prostaglandins), neurotransmitters (ATP and serotonin) and nerve growth factor
(NGF) (Samad et al., 2002). Specifically, these mediators, via the activation of
intracellular signal transduction pathways alter the sensitivity of voltage-gated ion
channels to produce peripheral sensitization. Sensitization may also be triggered
by inflammatory mediators from nociceptive neurons themselves (neurogenic
inflammation) (Julius & Basbaum, 2001). Chronic inflammatory GI diseases
include ulcerative colitis and Crohn’s disease and can be debilitating to affected
patients. In Crohn’s disease, inflammatory mediators produce long-term changes
that affect the mucosal and submucosal layers, and also neuromuscular
transmission. In ulcerative colitis, the mucosal and submucosal are affected.
Both those conditions produce rapid intestinal transit accompanied by abdominal
cramping (Koch et al., 1988). Animals models using the nematode worm T.
spiralis or the chemical trinitro benzene sulfonate have successfully reproduced
inflammation in the GI tract. Inflammation depolarizes the resting membrane
potentials of GI smooth muscle cells and increases their excitability. The
excitability of enteric neurons is also increased with increased input resistance
(Linden et al., 2003). Under conditions of inflammation, calcium influx through L-
type calcium channels is suppressed, which in turn inhibits the calcium-
calmodulin-dependent phosphorylation of the myosin laight chains, thus

preventing contraction. The function of L-type calcium channels is also affected

228

 

by nuclear factor KB, a ubiquitous transcription factor responsible for immune and
inflammatory reactions. Interestingly, the release of calcium from intracellular
calcium stores are unaffected (Shi and Sarna, 2000; Shi and Sarna, 2006).
According to Shi and Sama (2006), both neurotransmitter storage in, and release
from enteric nerve terminals, are altered. Taken together, those results indicate
that voltage-gated calcium channels are affected during inflammation, and that it
is therefore possible to target them pharmacologically as viable control points. In
my study, the R-type channel is clearly involved in intestinal motility, and it would
be useful to study the function of those channels in the inflamed intestine.
Inflammation increases GI transit, and I would therefore expect NiCI2 to inhibit the
peristaltic response. The studies reported in this dissertation show that R-type
channels are integral components of the ENS. The design of therapeutic
strategies using NiCI2 is challenging due to its toxic nature. However, success
has been achieved using toxins that block N-type calcium channels. Perhaps
using SNX-482 may represent a feasible way of targeting these channels.
Overall, the motility of the ileum in the presence of NiC|2 is impaired. This
suggests that R-type channel antagonists may be useful as anti-diarrheal drugs,
so that the lives of patients who suffer with GI diseases and disorders can be

improved.

229

 

 

Figure. 7.1. Proposed Model for Inhibitory Neuromuscular transmission.
(A1) R-type calcium channels may be present on the same nerve terminals as N-
and PIQ-type calcium channels. R—channels couple to NO release, whereas N-
and PIQ-type channels couple to ATP and/or B-NAD release. Both mechanisms
produce relaxation of the smooth muscle.

(A2) R-type calcium channels may be present on different nerve terminals

compared to N- and PIQ-type calcium channels.

230

 

A1.

RWCa"

Charm
N and \
_, PIQ
channel

      

 
  
 

R",.,,,;’,,’,,, SMOOTH MUSCLE

N 8. P/Q‘
channel
R-type
I calcium -
channel NOS

 

231

 

 

Figure. 7.2. Schematic of the overall effect of NiCI2 on the peristaltic reflex.
Fluid induced distention in the presence of NiCI2 impairs peristalsis but its effects
may extend to other regions of the GI as well. The response of the ileum under
control conditions (A) after NLA (applied first) (B), and NLA + NiCI2 (C)

treatments are shown.

232

 

    
   
 

\

Myenteric inhibitory
motor nerve varicosity

A1 . Control

   
 
   
  

...........,<_3o/Gi

NE , .

NOS

NOS

l

 

 

 

' N-type VDCC
@ R-type VGCC NO
NO
Sympathetic I
nerve terminal + cGMP
guanylate cyclase I

PKG
Relaxation ‘/

GI smooth muscle

 

 

A2. NLA first

   
 
  

   
  

Myenteric inhibitory
motor nerve varicosity

 

..........,C_5i/Go NLA
a2AR NOS
I NOS NOS
Gig X N-type VDCC
® R-type vocc NO
NO
' GI smooth muscle
Sympathetic I
nerve terminal GTP » cGMP
guanylate cyclase I
PKG
Relaxation ‘/

233

 

 

 

Figure. 7.2 cont’d

A3. NLA + NiC|2 Myenteric inhibitory motor
nerve varicosity

 

NLA

 

 

x X /
0LMR........§_5i/Go

NE ’ ’,// \ N08 N08 nos
I I

I
I
I
I
I
I
I
I
I
I
I
I
I
I
I
I
I
J

 

 

 

  
  
   
   
 

 

 

/ X N-type vocc 5
@ R-type VDCC NO .................. .
NiC|2
P2Y1 receptor
Sympathetic NO
nerve terminal I
+ cGMP
guanylate cyclase I
PKG
Relaxation /

 

GI smooth muscle

234

 

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