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dissertation entitled

FUNCTIONAL DIFFERENCES IN SYMPATHETIC NEURAL
CONTROL MECHANISM OF ARTERIES AND VEINS AS
PROBED BY CONTINUOUS AMPEROMETRY AND VIDEO
MICROSCOPY

presented by

Jinwoo Park

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FUNCTIONAL DIFFERENCES IN SYMPATHETIC NEURAL
CONTROL MECHANISM OF ARTERIES AND VEINS AS PROBED
BY CONTINUOUS AMPEROMETRY AND VIDEO MICROSCOPY

By

J inwoo Park

A DISSERTATION
Submitted to
Michigan State University

In partial fulfillment of the requirements
for the degree of

DOCTOR OF PHILOSOPHY

Department of Chemistry

2006

ABSTRACT

FUNCTIONAL DIFFERENCES IN SYMPATHETIC NEURAL
CONTROL MECHANISM OF ARTERIES AND VEINS AS PROBED
BY CONTINUOUS AMPEROMETRY AND VIDEO MICROSCOPY

By
J inwoo Park

Numerous studies have reported that norepinephrine (NE) levels in the plasma of
hypertensive humans and animals are elevated due to increased sympathetic nerve
activity. Little is known, however, about how the kinetics and mechanism of NE release
from perivascular sympathetic nerves as well as the postjunctional response is altered in
hypertension. The first goal of my research was to design and implement continuous
amperometric monitoring method and video imaging to record real time NE overflow
from sympathetic nerves innervating arteries and veins and the evoked contractile
response of isolated mesenteric veins (MV) and arteries (MA). The research included
fabrication and characterization of a new microelectrode material, boron-doped diamond.
The second goal was to use these two techniques to probe the functional differences in
sympathetic neural control of MA and MV, and how the control mechanisms are altered
in salt-sensitive hypertension. The deoxycorticosterone acetate (DOCA)-salt animal
model for salt sensitive hypertension was used.

The results showed that the diamond microelectrode provided superior response
performance as compared to a carbon fiber in terms of response precision and stability in
tissue. Diamond exhibited resistance to fouling, least in part, because of the non-polar,
low-oxygen, sp3-bonded carbon surface on which weak adsorption of polar biomolecules

and other contaminants occurs. The results also confirmed that first there are fundamental

differences in sympathetic neural control of MA and MV. These differences are: (i) the
density and arrangement of sympathetic nerves in MA and MV, with MA having a higher
density than MV, (ii) electrical stimulation-evoked NE overflow was exceeded in MV
than MA in sham rats because NE release and clearance in MA were more strongly
regulated by prejunctional aZ-adrenergic autoreceptor and norepinephrine transporter
(NET), respectively compared with MV, iii) frequency-response vasoconstriction in MV
was more sensitive than MA, iv) in small MA, ATP was the dominant neurotransmitter,
whereas in small MV, NE was the transmitter mediating constriction. The neural control
mechanism of MA and MV were altered in the DOCA-salt animal model for
hypertension as compared to sham control. This was evidenced by these findings: (i) NE
overflow increased in DOCA-salt rat MA compared to sham MA due to, at least in part,
an altered function of the prejunctional a2-adrenergic autoreceptor. The elevated NE
overflow caused an increase in NE uptake in DOCA-salt MA. (ii) ATP mediated the
neurogenic constriction of MA from sham rats while NE mediated most neurogenic
constriction in DOCA—salt MA. This may be due to an increased adrenergic component
of neurogenic constriction in DOCA-salt MA. (iii) the maximum contractile response was
decreased in DOCA-salt MV, though NE overflow was not different between sham and
DOCA-salt MV. It may be due to the desensitization of Oil-adrenergic receptors or/and
due to the altered vasculature in DOCA-salt MV.

Taken together, the differences in neuroeffector transmission may contribute to the
different hemodynamic function of arteries and veins. Furthermore, the altered
neuroeffector transmission in hypertension may contribute to the elevation in blood

pressure.

Cepyright by
.IINWOO PARK

2006

Dedicated to my family for their everlasting support and encouragement.
Thank you for being the wind beneath my wings.

ACKNOWLEDGEMENTS

First of all, I would appreciate and thanks sincerely to my advisor Dr. Greg M.
Swain and co-advisor Dr. James J. Galligan for their patience, great support. I respect and
appreciate their quest for excellence, vast knowledge in science. This dissertation is the
final state of five years work on my part. However, without your advice and support, I
would have not completed my study. It has been great honor working with you. I

anticipate having a life-long professional relationship with you.

I would also like to acknowledge my guidance committee Dr. Gregory D. Fink and
Dr. McGuffin L. Victoria. I appreciate their time and willingness to serve on my graduate
committee for the last five years. Thank you for all the comments concerning my
research, and also for your kindness. In addition, I would like to thank my former MS.
degree advisor Dr. Sang-Hak Lee at Kyungpook National University in Korea for his
support and encouragement. Special thank to Dr. Show for his advices on growing

diamond-films.

I owe many thanks to my colleagues in Dr. Swain lab (Dr. Jian Wang, Dr.
Malgorzata Witek, Dr. Mateuz Hupert, Dr. Shannon Haymond, Dr. Josef Cvacka, Suzana
Cvackova, Dr. Prema Sonthalia, Dr. Jason Stottor, Dr. Grace Muna, Dr. Veronika
Mocko, Dr. Karolina Peckova, Dr. Martin Novotny, Dr. Bhavik Patel, Dr. Aihua Liu, Dr.
lsao Sasaki, Dr. Jason Bennett, Dr. Ann Ficher, Audrey Martin, Dough Knigge, Yang
Song, Shihua Wang, Elizabeth McGaw, Hua Dong, Yingrui Dai, Veron Matt, Ayten Ay,

Michael Lowe, Jason Thornton, Luther Schaeffer) and Dr. Galligan’s lab (Dr. Xiaochun

vi

Bian, Dr. Xiaoling Dai, Dr. Alex Perez-Rivera, Dr. Jim Rain, Dr. Hui Xu, Hui Wang,
Sandra Hlavacova, Vino Naidoo, Dima Alkawwas, Stacie Demel). Tank you all for your

great support.

Finally, but the most important in my life, I deeply thank my parents, sisters and
brothers in laws for their endless love, sacrifices and support. I am grateful for their
encouragement to do the best work possible through out my life. So many things
happened to my life past five years while at MSU but meeting my wife, Jiwon Son was
greatest thing that happened to me. I sincerely appreciate her sacrifices and
encouragement to overcome all the problems. Thank you very much for being my soul
mate, my confidant, my lover and my best friend. I dedicate this dissertation to my

family.

vii

Table of Contents

List of Figures x

List of Tables xvr

Chapter 1 1

Introduction 1

1.1 Overall Specific Aims ............................................................................................... 1
1.2 Hypertension ............................................................................................................. 3
1.3 Blood Vessel ............................................................................................................. 5
1.4 Sympathetic Nervous System (SN S) ........................................................................ 8
1.5 Sympathetic Neuroeffector Transmission in Arteries and Veins ............................ 12
1.6 Experimental Hypertension Animal Models ........................................................... 18
1.7 Hyperactivity of the SNS in Hypertension .............................................................. 20
1.8 Increased NE Release in Hypertension ................................................................... 21
1.9 Assessment of SN S Activity ................................................................................... 24
1.10 Microelectrode ...................................................................................................... 28
1.11 Carbon Fiber Microelectrode ................................................................................ 30
1.12 Properties of Diamond and Its Application ........................................................... 33
1.13 Boron-Doped Diamond Electrodes ....................................................................... 35
1.14 Outline ................................................................................................................... 36
Chapter 2 ...... 39

Experimental Section 39

2.1 Boron-Doped Diamond Film Growth ..................................................................... 39
2.2 Diamond Film Characterization .............................................................................. 41
2.3 Microelectrode Preparation ..................................................................................... 4-4
2.4 Electrochemical Measurement ................................................................................ 46
2.5 Animals ................................................................................................................... 52
2.6 In Vitro Electrochemical and Diameter Measurement System ............................... 53
2.7 Fluorescence Histochemistry .................................................................................. 57
2.8 Chemical and Drug Application .............................................................................. 57
2.9 Data Analysis .......................................................................................................... 58

Chapter 359

Comparison of Electrochemical Properties of Diamond and Carbon
Fiber Microelectrodes During Exposure to Biological Environments...59

viii

3.1 Introduction ............................................................................................................. 59

3.2 Results and Discussion ............................................................................................ 61
3.3 Conclusion ............................................................................................................... 81
Chapter 4 ...................................................................................................... 84

Endogenous Norepinephrine and Its Effect on the Contractile Response
of a Rat Mesenteric Artery Using Continuous Amperometry and Video

Microscopy ................................................................................................... 84
4.1 Introduction ............................................................................................................. 84
4.2 Results and Discussion ............................................................................................ 87
4.3 Conclusion ............................................................................................................. 107

Chapter 5 .................................................................................................... 109

Differences in Sympathetic Neuroeffector Transmission to Rat
Mesenteric Arteries and Veins as Probed by In Vitro Continuous

Amperometry and Video Imaging .. ............. ............. ......... ...109
5.1 Introduction ........................................................................................................... 109
5.2 Results ................................................................................................................... 110
5.3 Discussion ............................................................................................................. 128
5.4 Conclusion ............................................................................................................. 135

Chapter 6 .................................................................................................... 136

Alterations in Sympathetic Neuroeffector Transmission to Mesenteric

Arteries and Veins in DOCA-salt Hypertensive Rats ............................ 136
6.1 Introduction ........................................................................................................... 136
6.2 Results ................................................................................................................... 138
6.3 Discussion ............................................................................................................. 154
6.4 Conclusion ............................................................................................................. 160

Chapter 7 .................................................................................................... 161

Conclusions ................................................................................................ 161

References .................................................................................................. 166

ix

List of Figures

Figure 1.1. A part of cross section vessel of blood vessel ................................................. 5
Figure 1.2. Sympathetic divisions of the autonomic nervous system ................................. 9
Figure 1.3. Sympathetic nerves on a blood ....................................................................... 10

Figure 1.4. Sympathetic neuroeffector transmission to blood vessels. P: purinergic

receptor, or: adrenergic receptor, ATP: adenosine 5’-triphosphate, and NE: norepinephrine
........................................................................................................................................... 12

Figure 1.5. Major steps in norepinephrine synthesis ......................................................... 15

Figure 2.1. SEM images of (A) an electrochemically sharpened Pt wire (top) and 3 Pt
wire coated with a polycrystalline boron-doped diamond film (bottom). (B) An expanded
view of the end of the diamond-coated Pt wire ................................................................. 41

Figure 2.2. Raman spectra for diamond thin film deposited on Pt wire with different
methane-to-hydrogen ratios ............................................................................................... 43

Figure 2.3. (A) Diagram of the conically shaped diamond microelectrode insulated with
polypropylene. SEM images of the polypropylene-insulated diamond microelectrode at
(B) lower and (C) higher magnification. Top-view SEM images of the diamond film
morphology without (D) and with (E) the polypropylene insulation layer ....................... 45

Figure 2.4. Background cyclic voltammetric i—E curves in (A) 1.0 M HC104 for a
diamond (—), carbon fiber (— —) and Pt-exposed diamond microelectrode (----). Cyclic
voltammetric i —E curves for a diamond and carbon fiber microelectrode in (B) 1.0 mM
Fe(CN) 6'3“ in 1 M KCl. Scan rate=100 mV/s ................................................................ 50

Figure 2.5. Block diagram of the experimental set-up ...................................................... 54

Figure 3.1. Background cyclic voltammetric i—E curves for a (A) diamond and (B) carbon
fiber microelectrode in 0.1 M phosphate buffer solutions of different pH ranging from 3
to 7.2. Scan rate = 100 mV/s. ............................................................................................ 62

Figure 3.2. Cyclic voltammetric i—E curves for a diamond and carbon fiber
microelectrode in (A) 10 M NE and (B) 10 M EE, both in 0.1 M phosphate buffer, pH
7.2. Scan rate = 100 mV/s. ................................................................................................ 65

Figure 3.3. Cyclic voltammetric i-E curves for 1 mM Fe(CN)6’3"4 in 0.1 M KCl at (A)
diamond and (B) carbon fiber microelectrode before and after laboratory atmosphere
exposure. Scan rate = 100 mV/s. ....................................................................................... 68

Figure 3.4. Cyclic voltammetric i—E curves for 1 mM Fe(CN)(,’3/'4 in 0.1 M KC1 at a (A)
diamond and (B) carbon fiber microelectrode before and after exposure to biological
tissue. Scan rate = 100 mV/s. ............................................................................................ 73

Figure 3.5. Continuous amperometric i—t responses for a (A) diamond and (B) carbon
fiber microelectrode during multiple injections of 0.1 uM NE in Krebs’ buffer, pH 7.4
before and afier exposure to tissue. The measurements were made in the flow bath.
Injection volume = 3 mL. Flow rate = 1.6 mL/min. Detection potential = +0.8 V
(diamond) and +0.4 V (carbon fiber) vs. Ag/AgCI. (C) Plot of the nominal current
response as a function of time for the diamond and carbon microelectrodes. .................. 75

Figure 3.6. In Vitro continuous amperometric i—t responses for a diamond microelectrode
during measurement of NE release from the mesenteric artery of a laboratory rat. The
electrode response at (A) 0 V after electrical stimulation of the artery and (B) +0.8 V
after electrical stimulation of the artery. The electrode response at +0.8 V after multiple
electrical stimulation events with a time interval between each of (C) 20 s and (d) 10 min.
The blood vessels were stimulated with the same pulse number and frequency (60 pulses
at 20 Hz). Flow rate = 1.6 mL/min. ................................................................................ 778

Figure 3.7. In vitro continuous amperometric i—t curves for a (A) diamond and (B) carbon
fiber microelectrode during NE release from a mesenteric artery as a function of time.
The NE-release was evoked by electrical stimulation consisting of 60 pulses at 20 Hz. (C)
Plots of the nominal current response as a function of time for the diamond and carbon
fiber microelectrodes. Flow rate = 1.6 mL/min. Detection potential = +0.8 V (diamond)
and +0.4 V (carbon fiber) vs. Ag/AgCl. ........................................................................... 80

Figure 3.8. Dose—response curves for NE at (A, C) diamond and (B, D) carbon fiber
microelectrodes. Measurements with 0.01—1.0 uM NE were made. Flow rate = 1.6
mL/min. Detection potential = +0.8 V (diamond) and +0.4 V (carbon fiber) vs. Ag/AgCl.
........................................................................................................................................... 82

Figure 4.1. Video micrographs showing a mesenteric artery (A) before and (B) after a 20
Hz electrical stimulation (60 pulses with a 0.3 ms pulse width). The bipolar stimulator
electrode and the diamond microelectrode, both positioned at the artery surface, are
evident in (A). The NE oxidation reaction mechanism is shown in (C). Characteristic
contractile (top) and NE oxidation current (bottom) response transients in response to a
20 Hz stimulation are presented in (D). Detection potential = 800 mV. Flow rate = 1.6
mL/min. Several numerical parameters are obtained from these curves including: the
maximum oxidation current, 1...“; the current rise time, T,; the time required for current
decay to 50% of the maximum, T50; the time required for full current decay, To; the
percent constriction, Co/,; the time required to reach full constriction, Tc; the time required
for full vessel relaxation, TR; and the latency period prior to the onset of constriction, TL.

........................................................................................................................................... 88

Figure 4.2. (A) An in vitro hydrodynamic voltammetric i-E curve recorded for NE
released from the sympathetic nerves innervating a mesenteric artery. Contractile (top)

xi

and NE oxidation current (bottom) response transients for a mesenteric artery in response
to a 20 Hz stimulation (60 pulses and a 0.3 ms pulse width) at detection potentials of (B)
0 and (C) 800 mV. The oxidation current was measured with a diamond microelectrode.
Flow rate =1.6 mL/min of Krebs' buffer. ......................................................................... 92

Figure 4.3. (A) Typical contractile and NE oxidation current response transients for a
mesenteric artery in response to a 20 Hz stimulation. Contractile and NE oxidation
current response transients (B) in the presence of 'ITX (0.3 uM) and (C) after washing
out the drug. The dotted circles indicate the time the electrical stimulation was applied.
The oxidation current was measured with a diamond microelectrode poised at 800 mV.
Flow rate = 1.6 mL/min of Krebs’ buffer. ....................................................................... 94

Figure 4.4. Effect of yohimbine (1.0 uM) on the contractile (top) and NE oxidation
current (bottom) response transients recorded at a mesenteric artery. The transients were
elicited by electrical stimulation at 20 and 3 Hz (60 pulses and a 0.3 ms pulse width) (A)
without and (B) with added yohimbine. The dotted circles indicate the time the 3 Hz
electrical stimulation was applied. Plots of the oxidation current for NE, with and
without (i.e., control) added yohimbine, versus the stimulation frequency are shown in
(C). Plots of the contractile response, with and without added yohimbine, versus the
stimulation frequency are shown in (D). The data are presented as mean values with the
bars reflecting the standard error of the mean. The oxidation current was measured with a
diamond microelectrode poised at 800 mV. Flow rate = 1.6 mL/min of Krebs’ buffer 96

Figure 4.5. Effect of cocaine (10 uM) on the NE oxidation current (lefi) and contractile
(right) response transients recorded at a mesenteric artery. The transients were elicited by
electrical stimulation at (A) 20 and (B) 3 Hz (60 pulse and a 0.3 ms pulse width). Plots
of the oxidation current for NE, with and without (i.e., control) added cocaine, versus the
stimulation frequency are shown in (C). Plots of the contractile response, with and
without added cocaine, versus the stimulation frequency are shown in (D). The data are
presented as mean values with the bars reflecting the standard error of the mean. The
oxidation current was measured with a diamond microelectrode poised at 800 mV. Flow
rate = 1.6 mL/min of Krebs' buffer. ............................................................................... 100

Figure 4.6. Effect of pulse frequency (3, 5, 7, 10 and 20 Hz, all at 60 pulses with a 0.3
msec pulse width) on the contractile (top) and NE oxidation current (bottom) response
transients recorded at a mesenteric artery (A). Plots of the NE oxidation current and
contractile response versus the stimulation frequency are shown in (B). The data are
presented as mean values with the bars reflecting the standard error of the mean. The
oxidation current was measured with a diamond microelectrode poised at 800 mV. Flow
rate = 1.6 mL/min of Krebs' buffer. ............................................................................... 103

Figure 4.7. Effect of pulse number (12, 30, 60, 90 and 120, all at 20 Hz) on the contractile
(top) and NE oxidation current (bottom) response transients recorded at a mesenteric
artery (A). Plots of the NE oxidation current and contractile response versus the
stimulation frequency are shown in (B). The data are presented as mean values with the
bars reflecting the standard error of the mean. The oxidation current was measured with a

xii

diamond microelectrode poised at 800 mV. Flow rate = 1.6 mL/min of Krebs' buffer. ..
........................................................................................................................................ .105

Figure 5.1. Glyoxylic acid-induced fluorescence of catecholamines in peri-vascular
sympathetic nerves. A and B show low magnification (100 X) images of the sympathetic
nerve plexus while C and D show high magnification images (400 X). The nerve plexus
in arteries has a mesh-like arrangement and nerve fibers are oriented in both the
longitudinal and circular axis of the blood vessel (C). The plexus in veins has a selective
circular arrangement with very few fibers oriented in the long-axis of the blood vessel
(D). ................................................................................................................................. 111

Figure 5.2. Contractile (top) and NE oxidation current (bottom) response transients for
(A) MA and (B) MV elicited by a 3 and 20 Hz stimulation. Plots of the (C) NE overflow
current and (D) constriction for MA and MV as a function of the stimulation frequency.
*Significantly different from MA and MV (P < 0.05). Data are mean i S.E.M. .......... .114

Figure 5.3. Calibration of a carbon fiber electrode for quantitative recording of NE
overflow. (A) Oxidation currents produced by injection of standard NE solution. (B)
Calibration plot showing how the NE oxidation current changes with concentration. Five
different electrodes were used to record the data set. Data are mean 3: SEM. ............. 116

Figure 5.4. Contribution of al-adrenergic and P2 receptors to neurogenic constrictions of
MA and MV. (A) Effects of PPADS (10 uM) on neurogenic constriction (top) and NE
oxidation current caused by a 20 Hz stimulus train in a MA. PPADS blocked the
constriction but not the NE oxidation current. (B) PPADS did not alter the neurogenic
constriction or NE oxidation current in a MV. (C) Concentration-constriction curve for
neurally-released NE in MA in the absence and presence of PPADS and prazosin (0.1
uM). Peak NE levels were measured during stimulus trains (1, 3, 7, 10 and 20 Hz) by
converting oxidation currents to NE levels using the calibration curve shown in Figure
SB. (D) Experiments similar to those shown in C except these studies were done in MV
during stimulus trains (0.5, l, 3, 7, 10 and 20 Hz). ........................................................ 117

Figure 5.5. Contribution of a2-adrenergic receptor-mediated components to NE overflow
and neurogenic constrictions of sham mesenteric arteries (MA) and mesenteric veins
(MV). F requency-response for NE oxidation current before (control) and after application
of yohimbine (1.0 uM) and UK 14,304 (1.0 uM) in MA (A) and MV (B). Frequency-
response curves for contractile response before (control) and after application of
yohimbine and UK 14,304 in MA (C) and MV (D). *Indicates significantly different
from NE oxidation current in control MA (MV) and in yohimbine MA (MV). #Indicates
significantly different from NE oxidation current in control MA (MV) and in UK 14,304
MA (MV) (P<0.05). Data are mean :t S.E.M. ............................................................... 119

Figure 5.6. Contribution of the NE reuptake to NE clearance and neurogenic constriction
of mesenteric arteries (MA) and mesenteric veins (MV). Frequency-response for NE
oxidation current before (control) and after application of cocaine (10 uM) and cocaine +
yohimbine in MA (A) and MV (B). Frequency-response curves for contractile response

xiii

before (control) and after application of cocaine (10 M) and cocaine + yohimbine in
MA (C) and MV (D). *Indicates significantly different from NE oxidation current in
control MA (MV) and in cocaine treated MA (MV) (P<0.05). “Indicates significantly
different from NE oxidation current in control MA (MV) and in cocaine + yohimbine
treated MA (MV) (P<0.05). Data are mean i S.E.M. .................................................... 124

Figure 6.1. Contractile (top) and NE oxidation current (bottom) response transients for
(A) MA and (B) MV from sham and DOCA-salt rats in response to a 3 Hz stimulation.
Comparison of the frequency response of (C) the NE oxidation current and (D)
vasoconstriction in sham and DOCA-salt MA and MV. *Significant difference between
sham and DOCA-salt MA (P < 0.05). &Significant difference between sham MA and

sham MV (P < 0.05). Data are presented as mean :1: SEM. .......................................... 140

Figure 6.2. Contribution of aZ-adrenergic receptor-mediated components to NE overflow
and neurogenic constrictions of sham and DOCA-salt mesenteric arteries (MA) and
mesenteric veins (MV). Frequency-response for NE oxidation current before (control)
and after application of yohimbine (1.0 uM) in sham and DOCA-salt MA (A) and MV
(B). F requency-response curves for contractile response before (control) and after
application of yohimbine in sham and DOCA-salt MA (C) and MV (D). *indicates
significantly different from NE oxidation current in control sham MA(MV) (P<0.05).
l”indicates significantly different from NE oxidation current in control DOCA-salt MA
(MV) (P<0.05). Data are presented as mean i S.E.M. ................................................... 142

Figure 6.3. Contribution of oLZ-adrenergic receptor-mediated components to NE overflow
and neurogenic constrictions of sham and DOCA-salt mesenteric arteries (MA) and
mesenteric veins (MV). Frequency-response for NE oxidation current before (control)
and after application of UK 14,304 (1.0 uM) in sham and DOCA-salt MA (A) and MV
(B). Frequency-response curves for contractile response before (control) and afier
application of UK 14,304 in sham and DOCA-salt MA (C) and MV (D). *Indicates
significantly different from NE oxidation current in control sham MA (MV) (P<0.05).
#Indicates significantly different from NE oxidation current in control DOCA-salt MA
(MV) (P<0.05). &Indicates significantly different from NE oxidation current in the drug
treated sham MA (P<0.05). Data are mean 1 SEM. .................................................... 145

Figure 6.4. Contribution of reuptake transporter-mediated components to NE clearance
and neurogenic constrictions of sham and DOCA-salt mesenteric arteries (MA) and
mesenteric veins (MV). Frequency-response for NE oxidation current before (control)
and after application of cocaine (10 uM) in sham and DOCA-salt MA (A) and MV (B).
F requency-response curves for the contractile response before (control) and after
application of cocaine in sham and DOCA-salt MA (C) and MV (D). *Indicates
significantly different from NE oxidation current in control sham MA (P<0.05).
”Indicates significantly different fi'om NE oxidation current in control DOCA-salt MA
(P<0.05). &Indicates significantly different from NE oxidation current in the drug treated
sham MA (P<0.05) Data are mean i S.E.M. ................................................................. 147

xiv

Figure 6.5. Contribution of uptake transporter/aZ-adrenergic receptor-mediated
components to NE clearance/release and neurogenic constrictions of sham and DOCA-
salt mesenteric arteries (MA) and mesenteric veins (MV). Frequency-response for NE
oxidation current before (control) and afier application of combined drug (cocaine (10
uM) + yohimbine (1.0 uM)) in sham and DOCA-salt MA (A) and MV (B). Frequency-
response curves for contractile response before (control) and afier application of the
combined drug in sham and DOCA-salt MA (C) and MV (D). *Indicates significantly
different from NE oxidation current in control sham MA (MV) (P<0.05). ”Indicates
significantly different from NE oxidation current in control DOCA-salt MA(MV)
(P<0.05). Data are mean i S.E.M. ................................................................................. 150

Figure 6.6. Contribution of al-adrenergic receptor and P2X—mediated components to NE
release and neurogenic constrictions of sham and DOCA-salt mesenteric arteries (MA)
and mesenteric veins (MV). Frequency-response for NE oxidation current before
(control) and after application of prazosin (0.1 uM) or/and PPADS (10 uM) in sham and
DOCA-salt arteries (A) and veins (B). Frequency-response curves for contractile
response before (control) and after application of the drug in sham and DOCA-salt MA
(C) and MV (D). Data are mean i S.E.M. ...................................................................... 152

XV

List of Tables

Table 3.1. Summary of cyclic voltammetric Em data for diamond and carbon
microelectrodes. ................................................................................................................ 66

Table 4.1. Numerical parameters measured from the oxidation current and contractile
response transients recorded in the presence and absence of yohimbine (1.0 uM). ......... 98

Table 4.2. Numerical parameters measured from the oxidation current and contractile
response transients recorded in the presence and absence of cocaine (10 uM). ............. 101

Table 5.1. Numerical parameters obtained from the NE overflow oxidation current and
contractile response transinents recorded in the presence and absence (control) of
yohimbine (1.0 uM). ...................................................................................................... 121

Table 5.2. Numerical parameters obtained from the NE overflow oxidation current and
contractile response recorded in the absence (control) and presence of UK 14,304 (1.0
uM) treatment. ............................................................................................................... 123

Table 5.3. Numerical parameters obtained from the NE overflow oxidation current and
contractile response transients recorded in the presence and absence (control) of cocaine

(10 uM). ......................................................................................................................... 126

Table 5.4. Numerical parameters obtained from the NE overflow oxidation current and
contractile response transients recorded in the presence and absence (control) of cocaine
+ yohimbine (Co +Yo). .................................................................................................. 127

Table 6.1. Maximum constriction (Em) and half maximum stimulation frequency. (S 50)
for MA and MV from sham and DOCA-salt rats. All data are expressed as the mean i
SEM and “11” value refers to the number of animals from which the data were obtained.
......................................................................................................................................... 141

Table 6.2. Maximum constriction (Em) and half maximum stimulation frequency (S50)
for MA and MV from sham and DOCA-salt rats in the absence (control) and presence of
yohimbine (Yo, 1.0 pM) and UK 14,304 (UK, 1.0 uM). All data are expressed as the
mean i SEM and “n” values refer to the number of animals from which the data were
obtained. .......................................................................................................................... 144

Table 6.3. Maximum constriction (Em) and half maximum stimulation frequency (S50)
for MA and MV from sham and DOCA-salt rats in the absence (control) and presence of
cocaine (Co, 10 uM) and the combined drug (cocaine + yohimbine). All data are
expressed as the mean i SEM and “n” values refer to the number of animals from which
the data were obtained ..................................................................................................... 148

Table 6.4. Maximum constriction (Em) and half maximum stimulation frequency (S50) in
MA and MV from sham and DOCA-salt rat in the absence (control) and presence of

xvi

prazosin (0.1 uM) and PPADS (10 uM) treatment. All data are expressed as the mean i
SEM and “n” values refer to the number of animals from which the data were obtained.
......................................................................................................................................... 153

xvii

Chapter 1

Introduction

1.1 Overall Specific Aims

Hypertension is a major risk factor for stroke and heart and kidney failure.
According to recent estimates, nearly one in three US. adults has hypertension, but
because there are no symptoms, many people are unaware that they have high blood
pressure. Numerous studies have reported that elevated levels of norepinephrine (N E), a
vasoconstrictor neurotransmitter in the sympathetic nervous system, and altered
postjunctional reactivity of the smooth muscle cells (contractile response) exist in human
hypertension and animal hypertensive models. These alterations are at least partly
associated with dysfunction of the sympathetic nervous system (SNS). The SNS plays a
key role in regulating blood pressure by controlling vascular tone. Previous studies
suggested that SNS dysfunction is due to functional changes of the presynaptic a2-
adrenergic autoreceptors and NE uptake transporter (NET). However, these results are
still controversial and the dysfimction differs from tissue-to-tissue and animal-to-animal.
Moreover, little is known about the mechanism of local NE release from perivascular
sympathetic nerves and the evoked vasoconstriction of arteries and veins in hypertension.
Techniques that enable local and real time monitoring of endogenous NE release from
sympathetic nerve endings and vasoconstriction simultaneously are needed to provide
more insight into neural control processes in hypertension. The research described herein

was conducted with three specific aims:

Specific aim 1 : Design and implement instrumentation for directly monitoring NE
overflow and vasoconstriction using continuous amperometry and a diamond
microelectrode along with video imaging.

This phase of the work involved fabricating and characterizing diamond microelectrodes,
evaluating the basic electrochemical properties of the material and demonstrating the
beneficial properties for in vitro electrochemical measurements. A test station with these
two tools was constructed to examine sympathetic nerve activity at arteries and veins

from the mesentery of laboratory test animals.

Specific aim 2 : Identify fundamental differences between sympathetic neuroeffector
transmission in mesenteric arteries (MA) and veins (MV) from healthy rats.

Using these two tools, studies were performed to learn about the ftmctional differences
between NE release, reuptake and the elicited contractile response of arteries and veins
from healthy animals. Using the oxidation current or charge recorded for endogenous NE
along with the various vasoactive agents, knowledge was gained about factors controlling

NE release and clearance at sympathetic neuroeffector junctions.

Specific aim 3 : Identify hypertension-associated alteration in sympathetic
neuroeffector transmission in MA and MV in the DOCA-salt animal model for
hypertension. Deoxycorticosterone acetate (DOCA)-salt hypertension is a well-
characterized model of experimental hypertension, which is associated with altered
sympathetic nerve activity. In Vitro continuous arnperometry and video microscopy,

along with various vasoactive substances, were used to learn what changes in the neural

control mechanism of MA and MV occur in salt-sensitive hypertension in terms of the

mechanisms of release, reuptake and effector cell physiological response.

1.2 Hypertension

Hypertension is an increasingly important disease state because high blood pressure
is a common finding in and is one of the most important modifiable risk factors for
cardiovascular disease [1, 2]. Hypertension is defined as a sustained systolic and/or
diastolic blood pressure greater than 140/90 mmHg. 50 million or more Americans have
high blood pressure and, worldwide, estimates suggest as many as 1 billion individuals
suffer from the disease. Approximately 7.1 million deaths worldwide per year are
attributable to hypertension. In 90-95 % of these cases, the causation is unclear and this is
referred to as “essential hypertension”. A number of physiological mechanisms are
involved in the maintenance of normal blood pressure, and their derangement may play a
part in the development of essential hypertension [3]. It is probable that a great many
interrelated factors, such as obesity, salt intake, insulin resistance, genetics, low birth
weight, endothelial dysfunction, altered renin-angiotensin and sympathetic nervous
system (SNS) activity, contribute to the elevated blood pressure in hypertensive patients.
The relative influence of these factors may differ between individuals. The mechanisms
underlying hypertension development are poorly understood, so rather than curing the
disease, current therapeutic approaches simply serve to lower blood pressure and
hopefiilly prevent other cardiovascular diseases secondary to hypertension.
Understanding the causation factors could lead to better treatments and even a cure for

essential hypertension. In the remaining cases, high blood pressure results from a

identifiable underlying renal, adrenal disease or endocrine disorders as the cause for the
raised blood pressure [4]. This kind of hypertension is called “secondary hypertension”

and can often be cured by correcting its original cause.

Regulation of blood pressure. The mean arterial blood pressure is determined by a
balance between cardiac output (CO) and total peripheral vascular resistance (TPR).
Peripheral resistance is mainly dependent on small arteries and arterioles, whereas
cardiac output depends on the volume of blood being pumped by the heart (heart rate)
and cardiac contractility [5]. The roles of CO and TPR are still controversial but most
patients and animals with established essential hypertension have a normal CO and
elevated TPR [6, 4]. However, TPR in the initial stages of hypertension is not raised or
reduced so the blood pressure elevation is caused by increased CD, which is related to
increased venous tone and caused, at least in part by, sympathetic overactivity [7, 4]. The
subsequent rise in peripheral arteriolar resistance might, therefore, develop in a
compensatory manner to prevent the raised pressure by the increased CO.

The homeostasis of blood pressure involves a complex interplay between the
nervous, endocrine, renal and cardiovascular systems. As examples, the central nervous
system controls blood pressure by adjusting the autonomic outflow to the cardiovascular
system and the kidney is important for maintaining soditun and water balance and
controlling the intravascular blood volume [8]. Various hormonal and vasoactive factors,
such as angiotensin II, vasopressin, nitric oxide and endothelin, also act on the
cardiovascular system to regulate blood pressure [9, 10]. The above systems interact with

one another, forming a delicate blood pressure controlling mechanism.

1.3 Blood Vessel

The blood vessels are the plumbing of the circulatory system and function to
transport blood throughout the body; a process is that is essential for life. Blood flows to
or from cells and organs delivering oxygen and nutrients and removing carbon dioxide
and waste products. Blood flow also maintains the optimum pH and the mobility of the
proteins and cells of the immune system. The most important vessels, arteries and veins,
are so termed because they carry blood away from or towards the heart, respectively. The
walls of arteries and veins are composed of three layers (Figure 1.1): the innermost

intima layer, the media layer and the outermost adventitia layer.

Elasticai tema
"v Endothelial

    

' ' Intima
, Media
1 Adventitia
Figure 1.1. A part of cross section of blood vessel.

The innermost layer, which is in direct contact with the flow of blood, is the tunica
intima, commonly called the intima. The intima is made up of mainly a single layer of
endothelial cells. The endothelial cells are capable of releasing vasodilators, such as nitric
oxide and adenosine, and vasoconstrictors, such as endothelin, which influence vascular
tone. Outside this layer is the tunica media or media, which is made up of smooth muscle
cells and elastic tissue [1 1]. Blood flow is regulated by contraction of the smooth muscle

cells, which alters the diameter and resistance of the small blood vessels. The number of

smooth muscle layers varies from 2 to 10 among the vessels and is largely in proportion
to blood vessel wall thickness [12]. The outermost layer is known as the tunica adventitia
or the adventitia, which is the most variable layer and contains the nerves that innervate

the smooth muscular cell layer.

Arterial system. There are several types of arteries according to their fimction and size.
The aorta is the root and largest systemic artery that carries blood away from the heart.
Branches of the aorta are called arteries, such as the carotid and mesenteric arteries.
Pulmonary arteries carry deoxygenated blood that has just returned from the body to the
lung, where carbon dioxide is exchanged for oxygen. Smaller than arteries are the
arterioles followed by the smallest vessels, the capillaries. The capillaries connect the
arterial to the venous circulation. Systemic arteries deliver blood to the arterioles and then
to the capillaries, where nutrients and gases are exchanged. Capillaries consist of little
more than a layer of endothelium and occasionally connective tissue. The small arteries
(150-250 um), arterioles (<100 um), and capillaries account for 90% of the vascular
resistance. The larger arteries have a low resistance to blood flow and function primarily
as conduits. However, as arteries approach an organ they divide into many small arteries

both just outside and within the organ, these increasing the vascular resistance.

Venous system. There are also several types of veins according to their function and size.
A venule is a small blood vessel that allows deoxygenated blood to return from the
capillary beds to the larger blood vessels called veins. The largest vein, venae cavae
returns the deoxygenated blood away from the body into the heart. The pulmonary veins

carry oxygen-rich blood from the lungs to the left atrium of the heart. They are the only

veins in the postal-fetal human body that carry oxygenated blood. The portal vein drains
blood from the digestive system and its associated glands, such as mesenteric and
splanchnic veins, to the liver. Although the venous system has a similar structure to that
of the arterial system, there is less smooth muscle and connective tissue. This makes the
walls of veins thinner than those of arteries, which is related to the fact that blood in the
veins is under less pressure than in the arteries [11]. In addition, veins have a larger cross-
sectional area (lumen diameter) than their arteriole counterparts. Therefore, veins are up

to 60 times more distensible than arterioles due to these structural differences and have i
the capacity to store 3-4 times more blood than arterioles. Thus, veins and venules are
often described as the capacitance section of the vascular bed and usually contain
approximately 2/3 of the body’s total blood volume [13]. Although multiple factors
regulate vascular capacitance, the structure and smooth muscle constrictor activity
(venomotor tone) of veins entirely determines the capacitance.

Hypertension is also associated with altered structural, mechanical and
pathophysiological properties of resistance arteries and capacitance veins [14, 15].
Structural changes in vascular smooth muscle cells in hypertension consist of two forms:
remodeling and growth. Remodeling is a rearrangement of normal sized cells whereas
growth is either an increased smooth muscle cell mass due to increased protein synthesis
(hypertrophy) or cell replication (hyperplasia) [16]. In essential hypertension, the
increased media/lumen ratio of small arteries due to remodeling could contribute to
increased vascular resistance [17]. Also, research with hypertension animal models has
revealed altered vascular structure by hyperplasia or hypertrophy. These structural

changes, like thickening of the vascular wall, include narrowing of the lumen and

external diameters, and increases in media width and media cross-sectional area [18, 19,
15]. However, whether the altered vascular structure is a cause or a consequence of the

increased blood pressure is unclear.

1.4 Sympathetic Nervous System (SNS)

The autonomic nervous system regulates bodily functions and the activity of
specific organs. One of the its functions is to regulate the cardiovascular system by
controlling vascular tone and cardiac strength through feedback with the central nervous
system [20]. Thus, the autonomic nervous system has an important role in maintaining a
normal blood pressure [4]. The autonomic nervous system originates from the brain stem
and is divided into two large subsystems: sympathetic and parasympathetic. The
sympathetic and parasympathetic systems have contrasting function in regulating the
internal environment [21]. The sympathetic nervous system (SNS) has classically been
divided into pre- and postganglionic fibers. Preganglionic neurons of the sympathetic
division originate in the thoracic (T1 to T12) and upper lumbar (L1 to L3) region of the
spinal cord (Figure 1.2).

Postganglionic sympathetic neurons consist of the axons of the ganglionic neurons
and innervate the heart and resistance vessels help to control cardiac output, arterial blood
pressure and regional vascular conductance, thus ensuring the proper perfiision of vital
organs [22]. Moreover, SNS is the primary mediator of acute changes in blood pressure
and also contributes to long-terrn blood pressure regulation. Peripheral sympathetic
nerves travel along arteries and veins and are present near the adventitial-medial border

to make close contact with vascular smooth muscle cells (Figure 1.3).

—— Sympathetic preganglionic fibers
— — — , Sympathetic postganglionic fibers

MW 2

 

Brain Stem

 

Cervical

Thoracic

 

Lumbar

 

 

Figure 1.2. Sympathetic divisions of the autonomic nervous system.

This is the site of sympathetic neuroeffector transmission. The nerves communicate with
the effector cells of the resistance and capacitance vessels and venous system through the
release of norepinephrine (NE), a vasoconstrictor neurotransmitter, onto the surface of
smooth muscle cells in vessel walls. This is the typical pattern for the sympathetic
innervation of blood vessels [23]. Only the smooth muscle cells located at the
adventitial/medial border are directly innervated [24, 25]. The appearance and
arrangement of sympathetic nerves associated with the veins is similar to that of arteries
although nerve density in veins is generally less than that observed in the adjacent arteries

[26, 12].

  
 

Adventitia

Media

Intima Blood Vessel

Figure 1.3. Sympathetic nerves on a blood vessel.

Nerve-mediated constriction of arteries and veins increases peripheral resistance
and cardiac output, respectively [27, 28]. Thus, the SNS plays an important role in blood
pressure regulation by controlling the two functional parameters of blood pressure: TPR
and CO. Although SNS has a central function in homeostasis, in general, and in
circulatory adaption, in particular, it is paradoxical that so little is known about the

possible contribution of disturbed sympathetic nervous firnction to the development of

human diseases [29]. The diseases related to sympathetic nervous system dysfunction
include essential hypertension, cardiac failure, coronary artery spasm, cirrhosis, mitral

valve prolapse and Raynaud’s syndrome.

SNS and mesenteric vasculature. Sympathetic nerves have the greatest quantitative
importance in minute-to-minute regulation of vasoconstriction, especially in the
splanchnic circulation. The splanchnic circulation receives up to 25 % of cardiac output
at rest and is a major reservoir of blood in the body (the largest capacitance bed) [30, 12].
Sympathetic nerve stimulation can reduce intestinal blood volume by up to 60%, leading
to a significant redistribution of blood from the splanchnic veins. This change in volume
distribution can have a profound effect on overall cardiovascular fitnction including
blood pressure [30, 31]. Therefore, the splanchnic vascular bed hemodynarnically
represents one of the most important vascular regions [12]. The blood flow to the
splanchnic organs, such as the gastrointestinal tract, liver, spleen, and pancreas, is derived
from three main arterial trunks: the celiac, the superior mesenteric and the inferior
mesenteric artery. The superior mesenteric artery supplies the small intestine and part of
the large intestine [32]. The mesenteric circulation refers specifically to the vasculature of
the intestines, whereas the splanchnic circulation provides blood flow to the entire
abdominal portion of the digestive system. The superior mesenteric artery, the major
inflow vessel of the mesenteric circulation, delivers about 12 % of the cardiac output and
supplies the entire small intestine [33]. The mesenteric vascular bed originating from the
superior mesenteric artery consists of arcades of blood vessels with a gradient of vessel
caliber in which the largest blood vessels is most distal to (first order) and the smallest is

most proximal to the intestinal wall (third or fourth order).

11

1.5 Sympathetic Neuroeffector Transmission in Arteries and Veins
Chemical neurotransmission is initiated by an electrical signal, the action potential.
Action potentials are sometimes called spikes or units. They are ca. 100 mV in amplitude
and have a duration 1-2 ms [34]. When the action potential fires down to the sympathetic
nerve terminal, it causes an influx of Ca2+ into the nerve terminal with subsequent fusion
of vesicles with the plasma membrane, followed by exocytosis of NE and co-transmitters,
adenosine 5'-triphosphate (ATP) and neuropeptide Y (NPY), into the junctional cleft. The
transmitter substances, by convective or diffusive mass transport, reach receptor sites on

the postjunctional membrane.

Pre-junction (Sympathetic Nerve)

  
   
   
 
 

Action Potential 3"

Diffusion

J unctional
Cleft
<100 nm)

Figure 1.4. Sympathetic neuroeffector transmission to blood vessels. P: purinergic
receptor, or: adrenergic receptor, ATP: adenosine 5'-triphosphate, and NE:
norepinephrine.

The transmitters then activate adrenergic and/or purinergic receptors in the effector
cell to indirectly trigger specific second messenger pathways through adrenergic
receptors and/or directly activate Ca2+ ion channels by purinergic receptors (Figure 1.4).
This results in an increased intracellular Ca2+ concentration and vasoconstriction. This

neurotransmission process will be discussed in more detail in the following sections.

NE and co-transmitters. All blood vessels are innervated by sympathetic nerves that
release the neurotransmitters NE, ATP and NPY. Neurotransmitters have been studied
extensively since their discovery in 1921 because of their important role in the body.
They are the key communication link between neurons. A lack or excess of even one of
these neurotransmitters may result in serious disease. NE is one catecholamine, along
with epinephrine (EE) and dopamine (DA), found in mammalian tissue. Although NE is
present in the adrenal medulla, it functions mainly as a neurotransmitter in the SNS and
generally acts locally on effector cells. Abnormal levels of NE are a cause for depression
and attention deficit hyperactivity disorder (ADHD). NE is also a powerful medicine
used in critically-ill patients as vasopressor. As mentioned, ATP and NPY are
neurotransmitters that are co-released with NE from sympathetic nerves and mediate
vasoconstriction [35, 36]. ATP serves as the major energy source within the cell for a
number of biological processes, such as photosynthesis, muscle contraction and protein
synthesis. It is produced by various cellular processes under aerobic conditions with the
majority of the synthesis occurring in the mitochondria during oxidative phosphorylation,
which is catalyzed by ATP synthase. The main fuels for ATP synthesis are glucose and
triglycerides. Extracellularly, ATP has been found to act as a neurotransmitter in the

arteries, intestines and bladder [37, 38]. NPY is also localized in sympathetic nerve

13

terminals [39]. NPY in porcine brain was discovered by K. Tatemoto in the early 19805
and is known to be one of the most abundant and widely distributed neuropeptides in the
central and peripheral nervous systems [40, 41]. NPY modulates numerous physiological
processes including regulation of cardiovascular and renal functions, intestinal motility,
memory, anxiety and nociception. NPY is synthesized and co-stored with ATP and NE in
peripheral neurons, particularly in the nerve endings surrounding blood vessels.
Interestingly, while NPY is primarily co-stored in the large, dense-core vesicle, the
smaller vesicles contain essentially only ATP and NE [42, 38]. This peptide is thought to
predominantly mediate responses at higher frequencies of stimulation and, therefore, play
a critical role during high levels of sympathetic nerve activity [43]. NPY is known to
cause constriction of some vascular smooth muscle cells, such as those from the human
coronary artery and cerebral circulation. However, some research has shown that NPY
does not contribute to vascular constriction in the mesenteric vascular bed [35, 36]. NPY
does not have a direct action on postjunctional cells but rather acts as a pre- and
postjunctional modulator of both the release and the action of NE and ATP [44, 45].
Therefore, NPY is more likely to function as a neuromodulator in mesenteric vascular

beds.

Synthesis and metabolism of NE. NE is synthesized by a series of enzymatically
catalyzed steps from the precursor amino acid, tyrosine (Figure 1.5). The first reaction is
the oxidation of tyrosine into dihydroxyphenylalanine (DOPA) by the amino acid
tyrosine hydroxylase (TH); the rate-limiting enzyme in the synthesis. This is followed by
decarboxylation to form dopamine through the action of DOPA carboxylase. Finally,

dopamine is converted to NE by side-chain hydroxylation through the action of

14

dopamine-B-hydroxylase (DBH) in the synaptic vesicles where NE is stored and released
via exocytosis [12]. NE can be further methylated by phenylethanolamine N-

methyltransferase to epinephrine.

 

NHz HO

m COOH HOWNHCE
HO HO

Tyrosine EE (epinephrine)

Tyrosine hydroxylase
Phenylethanolamiue
NH; N-methyl transferase H
HO
DOPA (dihydroxyphenylalanine) NE (norepinephrine)
DOPA carbo MA DA - h drox lase
HO
IDA (dopamine)

Figure 1.5. Major steps in norepinephrine synthesis.

Released NE in the junctional clefi is rapidly degraded to various metabolites via two
main pathways, one involving deamination by monoarnine oxidase (MAC) and the other
O-methylation by catechol-O-methyltransferase (COMT). The principle metabolites are
norrnetanephrine (NMN) via the enzyme COMT, and 3,4-dihydroxymandelic acid
(DMAE), 3-methoxy-4-hydroxymandelic acid (MHMA) and 3-methoxy-4-

hydroxyphenylglycol (MHPG) via MAO. However, NE in tissue undergoes a

15

quantitatively different metabolic fate. The NE present in sympathetic nerves is
metabolized intracellularly mainly by MAO. The physiologically inactive deaminated

product is excreted from the neuron and is then O-methylated extracellularly [46, 47].

Fate of released NE. NE released from sympathetic nerves endings is rapidly cleared by
several mechanisms: 1) a portion is removed by diffusion away from the release sites
eventually passing into the circulatory system (overflow), 2) a portion is metabolically
degraded by COMT or MAO and 3) the major portion is taken back up into the nerve
terminal by the norepinephrine transporter (NET) through a process called “reuptake”.
Once returned to the neuron, it is either repackaged into the synaptic vesicles for re-

release or metabolized by MAO and excreted [48].

0.2-adrenegic receptors and NE uptake transporters in prejunctional sites.
Prejunctional a2-adrenergic receptors located at the sympathetic nerve terminals are
called “autoreceptors” and are activated by the exocytotic release of NE. They function
by a feedback mechanism to inhibit further release of NE [49-51]. All adrenergic
receptors, such as a2 subtypes, are heterotrimeric G protein-coupled receptors.
Activation of prejunctional a2-adrenergic receptors inhibit Ca2+ entry, which leads to
inhibition of NE release [52]. Some studies have also shown that a2-adrenergic receptors
modulate the release of ATP [53-56]. After its release into the junctional cleft,
approximately 95% of the NE is transported back into the nerve terminal by the NET.

NE reuptake, mediated by a neuronal pump, is frequently the most important of the
clearance processes. The uptake probably occurs in the periods between transmitter

release, and serves to rid the neuromuscular space of transmitter in order to quickly

l6

terminate its vascular action [12]. The NET protein is thought to have 12 transmembrane-
spanning domains with intracellularly oriented N- and C- termini [47]. There are two
types of NET, one located in the junctional cleft and the other outside of it. Highly
efficient NET, referred to as uptake 1, is located in the plasma membrane of sympathetic
neurons and is responsible for clearing about 90% of the released NE. Approximately 5%
of total released NE penetrates into extraneuronal NET, so-called uptake 2, and is rapidly
inactivated by COMT to NMN. The uptake 2 transporter is much more diversely
localized in vascular endothelium, smooth muscle, glial and other cells. The remainder of
the released NE is either transported into the smooth muscle cell, metabolized by
enzymes, or escaped the junction into the extracellular fluid by diffusion.

The activity of released ATP is mainly regulated extracellularly by enzymatic
degradation and not by reuptake. There are at least two kinds of nucleotidase mediating
the degradation of ATP: membrane-bound ecto-nucleotidase particulate and releasable
nucleotidase [51]. There is no conclusive evidence as to how the NPY process is

regulated.

Adrenergic and purinergic receptors in postjunctional sites. NE is a potent
vasoconstrictor, acting through al- and a2-adrenergic receptors located in vascular
smooth muscle cells. The adrenergic receptors are G protein-coupled metabotropic
receptors and the binding affinity of NE for al-adrenergic receptors is higher than a2-
adrenergic receptors [57]. NE binding activates the associated Gq protein, which is linked
to phospholipase C (PLC). Subsequently increased PLC activity produces a second
messenger, inositol 1,4,5-triphosphate (1P3) and diacylglycerol (DAG). 1P3 causes

increased intracellular Ca2+ levels from intracellular Ca2+ stores by their binding to 1P3

l7

receptors on the stores, which promote smooth muscle contraction [58]. DAG activates
membrane-associated protein kinase C (PKC) and regulates several intracellular
substrates involved in Ca2+ handling, such as the Ca2+ channel [59]. NE mediates all
components of sympathetic vasoconstriction in rat mesenteric vein (MV) associated with
sympathetic nerves mainly by postjunctional a1 and/or a2-adrenergic receptors, whereas
ATP plays a more important role than NE does in neurally-mediated vasoconstriction in
rat mesenteric artery (MA) with their binding to postjunctional P2X purinergic receptors.
The P2X receptors, one of the P2 receptor subtypes, are ligand-gated ionotropic protein
exhibiting calcium permeability that contribute to vascular smooth muscle contractions
[60]. Vasoconstriction, mediated via P2X-receptors, is mainly dependent upon influx of
extracellular calcium ions through voltage-dependent calcium channels and the response

to nerve-released ATP is faster while the response to NE is more gradual [3 7, 61].

1.6 Experimental Hypertension Animal Models

A number of animal models of hypertension have been developed to study the role
of these various blood pressure regulating systems in the development of secondary and
essential hypertension. Since each animal model has a different etiology, it is conceivable
that the choice of a model significantly influences the outcome of the experiments
performed. Both spontaneous hypertensive rat (SHR), generated from the Wistar-Kyoto
(WKY) normotensive strain, and Dahl salt-sensitive rats, generated from normotensive
Sprague-Dawley, are the most widely used genetic strain of hypertensive animals [62,
63]. The SHR model is relatively insensitive to changes in sodium intake in the

developmental stages of hypertension, whereas the development of hypertension in Dahl

18

salt-sensitive depends on sodium intake. Therefore, Dahl salt-sensitive rats are used to
study not only genetic but also environmental factors [63]. The two-kidney, one-clip
(2K1C) and the one-kidney, one-clip (lKlC) hypertension models are also commonly
used to study for renal-dependent human secondary hypertension. Renal models of
hypertension use blockade of renal blood flow via stenosis of the supplying arteries with
a silver clip. This causes a partial occlusion of blood flow and /or reduction of renal mass.
In 2K1C, one renal artery supplying on one kidney is clipped but both kidneys are left
intact, while in lKlC, one kidney is removed and the renal artery supplying another
kidney is clipped. The deoxycorticosterone acetate (DOCA)-salt model, with suppressed
renin activity due to water and sodium retention is also most widely used to examine
neural and hormonal contributions to hypertension, which are relevant to the

understanding of essential hypertension [64].

DOCA-salt hypertension. This model is a relatively cheap and easy to employ, and the
control state is easy to define unlike many genetic states of hypertension [64]. The
DOCA-salt hypertension model is produced by surgical uninephrectomization followed
by implantation of the mineralocorticoid, called DOCA, and the administration of excess
salt, NaCl, in the drinking fluid. Arterial blood pressure will significantly rise in a few
weeks after such a treatment. If left untreated, the animal will eventually lose weight and
develop end-organ damage [65, 64]. DOCA salt-treatment causes significant changes in
the cardiovascular system that contribute to the overall pathology of this model. The
administration of mineralocorticoids changes hormonal and neural pressor mechanisms
[64]. The main role of mineralocorticoids is to affect ion transport, such as the Nair/H+

exchange and the Na+ pump in kidney epithelial cells, which conserves Na+ in the body.

19

Therefore, mineralocorticoids increase the rate of Na+ reabsorption by the kidneys. Na+
imbalance may be related to alternations in the central and peripheral control of
autonomic nervous system function [64]. An increased reabsorption of Na+ will increase
water retention, causing increased blood volume and finally increased blood pressure
[66]. The sequence of events in the pathogenesis of DOCA-salt induced hypertension is
not entirely clear but a number of consistent features have been observed allowing a
generalized picture to be formed [64]. Several mechanisms have been suggested to
contribute to the pathogenesis of DOCA-salt hypertension. Relevant prehypertensive
changes would include elevated sympathetic activity, altered neural angiotensin II and
vasopressin activity, and distorted baroreflex response [67, 64, 68]. In addition, numerous
peripheral effects of DOCA-salt hypertension induce characteristic changes in vascular
structure enhanced vascular reactivity, and altered ion transport that contribute to the

overall pathophysiology of DOCA-salt state [64].

1.7 Hyperactivity of the SNS in Hypertension

Alterations in the activity and function of sympathetic nerves are associated with
cardiovascular disease [69]. Although the precise causal mechanisms leading to
sympathetic augmentation in hypertension are still poorly understood and controversial,
growing evidence suggests that essential hypertension and hypertensive animal models
are initiated and sustained by overactivity of the sympathetic nervous system [70].
Abnormality of peripheral sympathetic outflow and catecholamine metabolism have been
described in the development of hypertension in humans and the animal hypertension

models, SHR and DOCA-salt rats [71, 48, 72]. Overactivity of the sympathetic nervous

20

system is commonly present in younger patients with essential hypertension. Early
hypertension is characterized by increased cardiac output as a result of hyperactivity of
cardiac sympathetic nerves. Therefore, it is generally agreed that hyperactivity of the
SNS is a critical initiating factor in hypertension development. Elevated cardiac and renal
sympathetic activity were also demonstrated in patients with essential hypertension [73,
74]. In the longer-term, hyperactivity of the SNS may cause increased renal
vasoconstriction and renal sodium retention, thickening of blood vessel walls, etc. These
observations support an ongoing role of the SNS in chronic hypertension. Overactivity of
the SNS is an attractive candidate mechanism to explain human and animal hypertension,
however, it is unclear whether the SNS plays a substantial role in long-tenn blood

pressure control in humans [6].

1.8 Increased NE Release in Hypertension

The development of hypertension in human and experimental hypertensive models
has been associated with increased basal sympathetic tone, as was suggested by the
observation of higher NE concentrations or its synthesis rats in the heart and other highly
vascularized organs [75-77]. Increased NE release are seen in some disease states,
including cardiac failure, cirrhosis, depressive illness, and essential hypertension [29].
The level of sympathetic drive differs between vascular beds and plasma NE is not
consistently elevated in hypertension. Esler’s study showed that total NE spillover to
plasma is increased in essential hypertension but is less than noted in patients with
cardiac failure or cirrhosis. Approximately 50% of the increase in total NE spillover in 50
patients with untreated essential hypertension was explicable in terms of increased

overflow of NE from the kidney and heart. However, there was no increase in NE

21

overflow from lungs, skeletal muscle or hepatomesenteric circulation in hypertensive
patients [78]. This may reflect, in part, selective sympathetic activation to specific
vascular beds, such as the kidney and heart, but a regional pattern of increased
sympathetic nerve activation to the kidney and heart might have originated remains
unclear. Increased NE release in hypertension suggests that such alteration can take place
at the level of the sympathetic nerve terminal. It has been generally hypothesized that
abnormalities of NE release in hypertension were due to impaired prejunctional a2-
adrenergic receptor function and/or neuronal reuptake, and elevated nerve firing rates.
However, few studies have shown that there was alteration in purinergic transmission
associated with ATP in hypertension. The possible implication of neuropeptide Y, which
was also found to exert presynaptic inhibitory influence on the liberation of NE by

sympathetic nerves also needs to be explored in the hypertensive mechanism.

Alteration in local adrenergic modulation mechanisms on sympathetic nerves in
hypertension. Data from several studies indicate that the increased NE overflow in
hypertensive animals is due, at least in part, to impaired prejunctional a2-adrenergic
autoreceptor function [79-81]. As examples, NE release was increased in vascular
sympathetic nerves of the isolated mesenteric vasculature of DOCA-salt rats and in the
portal vein and caudal artery of SHRs due to impaired a2-adrenergic autoreceptors [82,
83, 68]. However, controversial results have been also reported indicating that there was
no difference in inhibitory function of prejunctional a2-adrenergic receptors in the cadual
artery, or the perfused mesenteric bed of young SHRs and in the isolated mesenteric
artery of DOCA-salt rats [28, 82]. Although prejunctional otZ-adrenergic receptors

associated with sympathetic nerves also regulate NE release in veins [84, 85], only a few

22

studies of theese function in hypertension and the results have been controversial [77,
68]. Another possibility is that other modulatory mechanisms mediated by a variety of
presynaptic or local heteroreceptors can also be altered in hypertension but this remains

to be investigated [28].

Alteration of NE uptake in hypertension. Neuronal uptake of NE is the activity
mechanism responsible for removing NE from the synaptic cleft once released from the
nerve terminal. Previous studies have shown that there is impairment in total and cardiac
neuronal NE reuptake in patients with essential hypertension and in hypertension animal
models [86, 87, 67, 48, 70]. However, the evidence is inconclusive and controversial.
Some studies revealed that neuronal NE reuptake impairment may facilitate increased NE
spillover from sympathetic nerves to plasma due to decreased clearance of NE from
plasma for the body as a whole and in individual organs, such as the heart and brain [88,
89, 70]. Studies with a variety of hypertensive animal models, including SHR and
DOCA-salt rats, have also shown that NE reuptake was reduced. As examples, reuptake
was reduced in the skeletal muscle and kidney of chronic hypertensive SHR and the
mesenteric artery of hypertensive dogs after the development of hypertension [90, 91,
72]. On the other hand, neuronal NE uptake was significantly increased in isolated
subcutaneous resistance vessels of human essential hypertensive patients, in SHR
mesenteric and tail arteries, and in DOCA-salt rat mesenteric veins [92, 93, 17, 31].
Moreover, there was no difference in uptakegactivity in DOCA-salt rat [94, 67].
Therefore, some pertinent questions remain to be answered such as whether or not

impaired prejunctional a2-adrenergic receptors and/or alteration of NET in sympathetic

23

nerves are directly involved in the increased NE release, altered contractile responses of

blood vessels and pathogenesis of essential hypertension.

1.9 Assessment of SNS Activity

Clinical methods have been devised to assess SNS activity based on measurement
of NE levels in tissue, spillover into plasma and urinary excretion. Also, analysis of the
metabolite levels in patients with essential hypertension provides an assessment of the
activity of sympathetic nerves innervating the organ [86, 95]. Therefore, NE overflow is
an index of sympathetic activity [29]. However, great caution must be used when
attempting to relate plasma or urine NE levels to increased nerve activity or to a specific
disease state because the sympathetic outflow to all organs is not uniform. The level of
circulating NE depends not only on the release of NE from the nerve terminals but also
on plasma clearance of NE [96, 97]. Local and organ-specific increases or decreases in
sympathetic activity can also occur with different reflexes and in different disease states.
Moreover, only a small fraction of the NE released by sympathetic nerves throughout the
body escapes into the blood stream and urine. Therefore, the release of NE from
perivascular nerve has primarily been investigated using regional tissues, such as the
heart and kidney, labeled with tritiated NE instead of measuring NE in the whole body
[98, 99]. In a smaller number of studies, the release of endogenous NE overflow in
tissues by electrical nerve stimulation has been also measured by hi gh-perfonnance liquid
chromatography [100, 85]. However, due to the relatively low concentration of NE, it is
usually necessary to measure NE release elicited by long trains of electrical stimuli; a

condition that dose not closely mimic normal nerve firing [85]. Moreover, short stimulus

24

trains (5 3 8) may mimic in vivo sympathetic nerve activity, which occurs in short bursts
(< 2 s) [101]. Although the increased NE release from tissue or spillover to plasma is also
undoubtedly attributable, at least in part, to increased sympathetic nerve activity, NE
release cannot be measured directly with these methods [70]. It turns out that little is
known prejunctional or local modulatory mechanisms controlling NE release and action
from altered sympathetic nerves in hypertension. The electrochemical recording approach
provides simple and extremely sensitive approaches and makes it possible to monitor,
near release rate, the real time rise and fall in the concentration of released endogenous
NE in blood vessels on an impulse-by-impulse basis during low and short fi'equency
trains of stimulation. Learning about the characteristics of NE release on an irnpulse-by-
impulse basis is important for understanding how the sympathetic nervous system
regulates blood pressure, and more importantly, what alterations in the regulatory

mechanism are associated with hypertension and cardiovascular disease.

Electroanalytical techniques. Electrochemical measurements with solid electrodes were
introduced more than 50 years, but there was little interest in them by biologists until the
19703. A breakthrough occurred in the 19703 when Ralph Adams and his colleagues
showed that it was possible to implant a small working electrode in a rat brain and detect
electroactive biogenic amines and related substances in the extracellular fluid (ECF) in
vivo using voltamrnetry [34]. Their work demonstrated that it was possible to study the
release of neurotransmitters in intact animals, without the use of complicated radioactive
labeling techniques [102]. These, electrochemical measurements with microelectrodes
provide a sensitive, selective and rapid detection method for chemical events occurring in

tissue.

25

Since that time, other electroanalytical techniques, including fast scan voltammetry,
continuous amperometry and various pulsing methods, have been used with
microelectrodes to provide unique information on changes in the local concentration of
neurotransmitters and related substances. Easily oxidized or reduced species can be
detected, both in vivo and in Vitro, in the mammalian central nerve system, in single-cell
preparations, brain slices and other tissues [103-105]. Fast scan cyclic voltammetry
(FSCV) and continuous amperometry are the tools commonly used in recent work, which
has focused primarily on the biogenic amine neurotransmitters, dopamine,
norepinephrine, serotonin and metabolites of these, and ascorbic acid [106-109]. Both
electroanalytical techniques offer a degree of selectivity, temporal resolution and
sensitivity in the study of neurochemical events [110]. In continuous amperometry, a
microelectrode is held at a constant potential sufficient to oxidize or reduce in an analyte
of interest at a mass transfer limited rate and the resulting electrochemical current is
monitored with time. Therefore, amperometry has excellent temporal resolution and
allows detailed real-time analysis of the kinetic processes involved in the release of
vesicular content into the extracellular space. Using amperometry, the contents of very
small vesicles and small concentrations of transmitter released from a single cell, such as
a chromaffin cell, can be quantified [111, 112]. The charge (Q) in coulombs is related to
the number of moles of analyte detected by Faraday’s law, Q = nFN, where in is the
number of electrons involved in the electrochemical reaction (n = 2 for catecholamines)
and F is Faraday’s constant (96,485 C/equivalent). Recent studies using amperometry
have allowed for the detection and quantification of attomole and zeptomole amounts of

neurotransmitter upon release from single cells [113]. Such sensitive measurements using

26

amperometry are enabled by the development of low noise instrumentation and enhanced
filtering techniques [110]. Therefore, amperometry is an excellent technique for
monitoring rapid transient changes associated with neurochemical signals of known
origin. However, it provides no qualitative information due to the poor selectivity from
interferences, other oxidizable neurotransmitters and their metabolites, ascorbic acid and
uric acid, that are present in brain extracellular fluid at very high concentration [34, 105].

For FSCV, electroactive species can be detected at very high scan rates (> 100 V/s)
with the scan lasting only a few milliseconds. This reduces the extent of electrocatalysis
[114] and allows for more frequent measurements. Based on the potential of the oxidation
and reduction peaks as well as the peak current ratio, FSCV provides qualitative as well
as quantitative information about redox analyte. At high scan rates, a relatively large but
constant charging current is observed. Smaller faradaic currents can only be detected by
background subtraction. The resulting faradaic current provides a ‘fingerprint’ for the
molecules detected at the electrode that represent changes in the local concentration of
these molecules [110]. A major difference between amperometry and FSCV is the
selectivity of FSCV possesses in vivo and in vitro for different electroactive molecules of
interest.

Most of the work to date using these methods has been to study neurogenic
processes in the central nervous system. However, there have been some reports on the
use of continuous amperometry to study NE overflow from peripheral sympathetic nerve
terminals [115-118]. Electrical measurements with a microelectrode have been used since
the 1990's to study neurogenic processes in the peripheral nervous system, such as the

release of NE from the sympathetic nerve terminals of isolated organs [115, 119-121,

27

118]. Although NE, along with co-transmitters ATP and NPY, are released from
sympathetic nerves innervating blood vessels, NE is the only one that can be detected
directly electrochemically. NE is oxidatively detected with a microelectrode via a 2H’72e'
oxidation reaction. Mermet, Gonon and Stj time were the first to study NE release from
the sympathetic nerve terminals innervating the smooth muscle cells of the rat tail artery
[115, 119, 122]. In fact, the rat tail artery has been the organ of focus in most
investigations of the peripheral nervous system because it is densely innervated
exclusively by sympathetic nerve fiber, that form a two-dimensional plexus at the
external surface [115, 119, 122].

In a series of more recent papers, Brook and coworkers studied the characteristic
features of NE release from postganglionic sympathetic nerves in rat mesenteric artery
mesenteric artery [123, 121, 124]. The release of endogenous NE was measured by
continuous amperometry using a carbon fiber microelectrode coated with the
permselective ionomer, Nafionm. The group found that NE release is regulated by
activation of prejunctional a2-adrenergic receptors and that clearance of the released NE

in this tissue depends, in part, on neuronal reuptake.

1.10 Microelectrodes

Microelectrodes can be fabricated with such small size (0.5 to 5 pm diam) that they can
be used to probe chemical events inside a single biological cell. The extremely small size
of the recording electrode can be placed in close proximity to the site of release or to a
synapse without appreciable perturbation of the surrounding environment [125]. Its use

enables the discrete sampling of small nuclei within the brain with a spatial resolution of

28

10-100 pm. Most electrodes used for in vivo or in vitro measurements have diameters of
10-30 pm and sample from a region of comparable size. Therefore, microelectrodes have

a key role for detection of neurotransmitters.

Advantages of microelectrodes. Their very small size provides the advantage of high
temporal resolution and the ability to make spatially resolved chemical measurements in
tissue [103]. Due to their small size (10-30 pm in diameter, 30-500 pm in length), the
current at these electrodes is extremely low, so some sophisticated monitoring electronics
are needed. Small electrodes are suitable for use in tissue with minimal peripheral
damage to the surrounding environment. Moreover, since electrochemical currents at
microelectrodes are 4 to 5 orders of magnitude smaller than those seen at electrodes of
conventional size, they can be used to make measurements in solutions of high resistance
(i.e., less iR loss). The low capacitance of the small area electrode allows the electrode
potential to be changed rapidly. Therefore, microelectrodes are exclusively useful for fast
measurements, like FSCV (300V/s) [126, 127]. This rapid measurement enables
monitoring of millisecond and microsecond chemical events (e.g., exocytosis) and kinetic
processes (e.g., reuptake). As a consequence of the reduced charging current and
increased faradaic current (enhanced mass transport), microelectrodes exhibit excellent
signal-to-noise (S/N) characteristics, which enhances sensitivity. Depending on the time
scale of the experiment, voltammograms obtained at microelectrodes may differ from
those obtained at electrodes of conventional size. Since the large diffusion layer relative
to the microelectrode dimensions and the volume from molecules diffusing to suppdrt the
current are relatively large by the radial diffusion, steady-state (time independent)

response is observed. Contrarily, because the dimension of the diffusion layer is smaller

29

than that of the conventional size and the ratio of the number of molecules diffusion
perpendicular (planar diffusion) to those diffusion in at the edges (radial diffusion) is
much greater at conventional size, the peak shape response (time dependent) is observed
under these conditions. The steady-state limiting current is directly proportional to the
analyte concentration, which provides a direct means of quantitating electroactive

substances [125].

1.11 Carbon Fiber Microelectrode

Carbon is the material of choice for fabricating microelectrodes for use in the brain.
The response of different materials is differentiational selectivity, sensitivity, stability and
reproducibility [125]. Noble metals, such as Pt and Au, and carbon fiber have been used
as microelectrodes. However, Pt and Au are unstable and their surface is deactivated
immediately upon exposure to brain tissue [128, 129]. Carbon fiber differs from metals in
both its electronic properties and surface chemistry [130]. Another major difference
between carbon and metal electrodes is the nature of the surface oxides. Metals form
oxides and hydroxides easily and reversibly in aqueous solution, whereas carbon forms a
richer variety of surface oxides, such as carboxyl and hydroxyl functional groups [131].
The surface oxides can have a variety of effects on the electron-transfer rates and
adsorption of catecholamines and interferences. The presence of oxide functional groups
raises the possibility of specific chemical interactions between the surface and a molecule
in solution. Such interactions can catalyze redox reactions, and promote or inhibit

adsorption.

30

Carbon surface oxides can also greatly increase the background voltammetric
current, in part, due to interactions between charged surface groups and electrolyte ions
and the psudocapacitance associated with redox-active functionalities (e. g.,
quinone/hydroquinone). Carbon fiber types differ significantly in the degree of
microstructural orientation, crystallite size, and surface chemistry, and these parameters
are important for electrochemical performance. Most carbon fibers are made with
polyacrylonitrile (PAN) or petroleum pitch and formed during high temperature pyrolysis
of the materials. PAN fibers have low tensile strength and more microstructural defects in
the graphite sheets. Pitch type carbon fibers tend to have high tensile strength and
modulus due to a greater level of microstructural ordering.

Carbon fibers are traditionally used as strengthening materials and because of their
good electrical conductivity, non-toxicity and small size, they have been applied for both
the electrochemical detection of oxidizable compounds and extracellular single unit
recording [132]. Moreover, because of their resistance to drifi when exposed to biological
tissue and easy of surface modification, carbon fibers have become popular and are now
widely employed for in vivo and in vitro monitoring of oxidizable compounds in the

living tissue [133, 103, 134-136].

Limitations of carbon fibers for in vivo and in vitro measurements. One
limitation of carbon fibers is their fragility and response deactivation over time during
exposure to physiological environments due to fouling by biomolecule adsorption. The
adsorption is linked to the presence of the extended 7! electron system and the carbon-
oxygen functional groups on the surface. A 30 — 50% decrease in the short-tenn electrode

response sensitivity is not uncommon following implantation into brain tissue. The

31

response generally levels off after ca. 2 hours [137, 131]. For in vivo measurements,
electrode response stability is required for long periods of time, at least 8b in most
experiments [126]. Therefore, the bare carbon fibers are unsuitable for chronic
implantation in tissue [138]. A wide range of surface carbon-oxygen functional group
exist on the electrode that can affect the electrode kinetics of various classes of redox
systems in different ways.

Quinone-like functional groups are known to be present and exhibit pH-dependent
electroactivity. It is the oxidation/reduction of these functional groups that gives rise to
background voltammetric features. The shifting of voltammetric background current with
pH leads to complication in the background-subtracted voltammetric responses because
these groups are oxidative at the same potentials as the catecholamine neurotransmitters.
Simultaneous changes in the catecholamine concentration and pH frequently occur in
biological tissue as a result of the coupling of respiration and energy production to pH
[139, 140]. Several electrode pretreatrnents have been developed over the years to
improve the response sensitivity, stability and selectivity especially for catecholamines.
These including coating the surface with the parmselective polymer, NafionTM [134].
Coating with NafionTM improves selectivity and minimizes electrode fouling from
biomolecule adsorption by repelling anions from SO3' groups but allowing cations
through. However, polymer coatings increase the background current and decrease the
electrode response time, which results in decreased sensitivity [141, 104, 134].

Another perfidious pitfall is that some drugs alter the oxidation reaction, and the
response time to evoke release is slow (10-30 s) at treated electrodes. Therefore, the real

kinetics of the evoked release cannot be directly recorded. Therefore, a more stable

32

microelectrode material would enable more widespread application of this informative

technique.

1.12 Properties of Diamond and Its Application

Diamond offers exceptional material properties including mechanical strength,
thermal conductivity, optical transparency, and electrical conductivity when doped
compared with any material. Another important property for electrochemical application
is the microstructural stability and corrosion resistance. These properties are a
consequence of the small interatomic distance between tetrahedrally-bonded carbon
atoms in the diamond lattice and the hydrogen surface termination. Synthetic diamond
can be produced by high-pressure, high—temperature techniques or by low-pressure
chemical vapor deposition (CVD). CVD diamond became a reality in the early 19705 and
affords the possibility of producing thin film diamond on a variety of substrates at
relatively low cost. To date, hot filament and microwave-assisted CVD are the most

popular deposition methods with a CH4/H2 source gas mixture routinely used for growth.
The high concentration of H2 produces a high flux of atomic hydrogen, which is known

to be critical for high quality diamond growth. It is possible to manipulate the physical
and chemical properties of diamond by proper control over the source gas composition,
system pressure and substrate temperature. The crystalline diamond produced is used for
a wide range of industrial applications that require hardness and wear resistance. Other
specialized applications also exist or are being developed including its use as active and
passive materials, semiconductor. Diamond is naturally an excellent electrical insulator

but it can be made electrically conducting by controlled doping with impurities, such as

33

boron. CVD allows for in situ doping with boron, phosphorus and sulfur, rendering the
diamond film p-type or n-Iype semiconductors [142]. The boron impurity serves as an
electron acceptor with an activation energy of ca. 0.37 eV [143]. Boron is the most
prominent dopant because of its small covalent radius, which leach to easy incorporated
into substitutional sites within the diamond without causing lattice distortion. In order to
have sufficient electrical conductivity for electroanalytical measurements (_<. 0.1 0 cm),

polycrystalline diamond films must be doped with boron at a concentration of l x 10'9

cm"3 or higher. This produces a carrier concentration in the high 1 x 1020 to low 1 x 1019

cm'3 and a carrier motility between 0.5 and 10 cm2 V'ls'l [144]. At present, no

appropriate method exists for preparing highly conducting n-type diamond electrodes.
Well-known donors, such as nitrogen and phosphorus, have too high an activation energy
(1.7 and 0.6 eV, respectively) and thus, cannot impart reasonable room temperature
conductivity. However, recently, sulfur was reported to be a donor impurity for diamond
with an acceptable activation energy (0.36 - 0.38 eV) [145]. The advent of low-pressure
diamond synthesis along with the facility of dopant incorporation, has prompted a
growing number of investigations into its use as an electrode material. Diamond thin
films with sufficient electrical conductivity undoubtedly are promising candidates and
attractive electrode material. The use of diamond in electrochemistry is a relatively new
field of research that has only begun to blossom in recent years since the early 19803

[146-150].

34

1.13 Boron-Doped Diamond Electrodes

A new electrode material, boron-doped diamond thin-film, with an sp3-bonded
carbon microstructure and little surface oxygen (< 0.02 atomic %) due to a hydrogen
surface termination, offers advantages for electroanalytical measurements in terms of
linear dynamic range, limit of quantitation, response time, response precision, and

response stability over other electrodes, especially commonly used sp2 carbon material

with extended n-electron systems, such as glassy carbon, pyrolytic graphite or carbon
paste [146, 151]. The relative absence of electroactive carbon-oxygen fimctionalities on
the hydrogen-terminated diamond surface (hydrophobic, non-polar, and chemically
stable) introduces superb electrochemical properties [152]. First, diamond possesses wide
working potential window (3-4 V) and excellent response stability. These properties
enable diamond’s use in measurements that otherwise would be difficult or impossible
with spz-bonded carbon electrodes. Second, diamond electrodes give improved signal-to-
background (S/B) and signal-to-noise (S/N) ratios in electroanalytical measures due to a
low double layer capacitance (l to 8 uF/cmz) compared with sp2 carbon electrodes (30 to
40 uF/cmz), and the absence of background current due to redox-activity surface carbon-
oxygen functional groups [153]. The slightly lower density of surface electronic states
near the Fermi level caused by the semiconductor nature of boron-doped diamond
contributes to the low capacitance of the material [153, 154]. A lower surface charge
carrier density at a given potential leads to a reduced accumulation of counter-balancing
ions and water dipoles on the solution side of the interface, thereby a lowering of the
background current and capacitance. Third, diamond exhibits high resistance to surface

fouling due to the weak adsorption of polar molecules and a pH-independent background

35

voltammetric response due to the hydrogen surface termination [153]. The majority of the
research conducted so far has involved the use of a planar thin film (macroelectrodes).
The fabrication, characterization, and application of diamond microelectrodes have not
yet been extensively studied. There are only a few reports describing the preparation and
basic electrochemical characterization of diamond microelectrodes [155-157]. Diamond
microelectrodes have been found to provide superior detection figures of merit, as
compared to carbon fibers in terms of linear dynamic range, limit of detection, response

variability and stability [158].

1.14 Outline

This work described in this dissertation focused on the application of diamond
microelectrodes for measurements in biological environments. Specifically, these new
microelectrodes were used to record in vitro NE overflow from sympathetic nerves
innervating mesenteric arteries (MA) and veins (MV) in normotensive (sham) and
hypertensive (DOCA-salt) rats. We sought to accomplish these research goals: (i)
fabricate and characterize diamond microelectrodes in comparison with conventional
carbon fibers, (ii) use continuous amperometry and video microscopy in vitro to learn
more about how the sympathetic nervous system (SNS) regulates the tone of MA and
MV and (iii) use the same two tools to better understand hypertension-associated changes
in NE overflow and adrenergic vasoconstriction mechanisms in MA and MV. An
overview of each chapter is given below along with a synopsis of the findings.

Chapter 1 introduces the background of and motivation for the research. Chapter 2

is referred to throughout as the experimental section, as it contains details of all

36

experimental procedures, tissue preparation and microelectrode fabrication. In Chapter 3,
the characterization of microcrystalline boron-doped diamond microelectrodes is
described in comparison with carbon fibers. The purpose of this work was to investigate
the fundamental differences between carbon fiber and diamond and learn about the
physicochemical properties and electrochemical responses of the microelectrodes during
measurement in biological tissue. Chapter 4 describes the applications of a diamond
microelectrode coupled with video imaging for simultaneous monitoring endogenous NE
overflow at the surface of a MA and its effect on the contractile response in vitro. The use
of various pharmacological agents to better understand the neurogenic response is also
described. This study shows that there is a correlation between the NE overflow, as
elicited by electrical stimulation, and the dynamics and extent of vascular constriction.
Several observations indicate that the bare diamond microelectrode provides superior
sensitivity, reproducibility and stability coupled with a bare carbon fiber microelectrode.
Chapter 5 presents the fundamental differences in sympathetic neuroeffector
transmission to rat MA and MV, which contribute to their different hemodynamic
functions. These differences include the neurotransmitters released from sympathetic
nerves, the time course of NE action, the arrangement of perivascular nerves, and the
function of pre- and postjunctional receptors on MA and MV. Prej unctional a2-
adrenergic autoreceptors and NE uptake play a greater role in regulating NE availability
at the neuroeffector junction in MA than in MV, and NE overflow in rat MV exceed that
in MA. The differences contribute to, at least in part, the greater sensitivity of MV to the
constrictor effects of sympathetic nerve stimulation compared to MA. Chapter 6 presents

alterations in sympathetic neuroeffector transmission to MA and MV in DOCA-salt

37

hypertensive rats. Increased NE availability in MA due to impaired a2-adrenoceptors
contribute to increased peripheral resistance in DOCA-salt rats. There is also an increase
in NE uptake in DOCA-salt hypertensive rat MA. The increased uptake offsets the
vasoconstrictor effects of elevated NE overflow in these tissues. Finally, a summary of all

the results, and the conclusions is given in Chapter 7.

38

Chapter 2

Experimental Section

2.1 Boron-Doped Diamond Film Growth

Boron-doped diamond thin film was deposited on a sharpened Pt wire by
microwave-assisted chemical vapor deposition (CVD) [155, 150, 159]. The Pt wire was
electrochemically etched in l M KOH. An etching solution containing 7.0 g of
CaClz-Hzo in a mixture of 20 mL of ultrapure water and 20 mL of acetone also
functioned well [160, 159]. A 1.4 cm long piece of Pt wire (99.99%, Aldrich Chemical,
76 um diam) was used. Both ends of the wire were sharpened so that two electrodes
could be made from one wire. The wire end was immersed to a depth of l to 2 mm and
positioned in the center of four connected carbon rod counter electrodes during the
etching. An AC polarization of 12 V (60 Hz) was applied between the wire and the
counter electrodes using a variable autotransfonner (Staco Energy Products, Dayton,
OH). The etching procedure lasted for approximately 5 3 until gas evolution visibly
ceased at the tip.

The etched wire was conically-shaped near the end and cylindrically-shaped above.
A thin film of boron-doped diamond was deposited on the sharpened wire using a
commercial CVD system (1.5 kW, 2.54 GHz, ASTeX, Woburn, MA)[158, 159]. Prior to
deposition, the sharpened wire was ultrasonically cleaned in acetone (5-10 min) and
ultrasonically seeded (30 min) in a diamond powder suspension (5 nm particles, ca. 20
mg in 100 mL of ethanol, Tomai Diamond Co., Tokyo, Japan). In order to prevent

damage to the sharpened end, the wire was cleaned and seeded while vertically

39

suspended in the agitated solution. During the seeding process, the surface is scratched by
the diamond particles with some getting embedded. Both the scratches and the embedded
particles serve as the initial nucleation sites for film growth. A high instantaneous
nucleation density is desired because this leads to the formation of a continuous film in
the shortest time and at a low nominal thickness. The pretreated Pt wire (3-4 in parallel
per deposition run) was mounted horizontally on top of a quartz plate (10X10X1 mm) in
the reactor. The quartz plate was placed in the center of the reactor’s molybdenum
substrate stage and served to thermally isolate the wire. The thin film of boron-doped
diamond was deposited from a 0.5% CH4/H2 (v/v) source gas mixture with 10 ppm of
diborane (0.1 % B2H6 diluted in H2) added for doping.

All source gases were ultrahigh purity grade (99.999%). The system pressure was
45 Torr, the substrate temperature was ca. 700 °C, or less, (as estimated with an optical
pyrometer), the microwave power was 400 W and the total gas flow was 200 standard
cubic centimeters per min (sccm) during growth. The deposition time was 10 h. After
deposition, the CH4 and B2H6 gas flows were shut-off and the films cooled in the
presence of atomic hydrogen (H2 plasma) by slowly reducing the plasma power and
system pressure over a 30 min period. This post-growth treatment was necessary for
removing adventitious sp2 carbon impurity, minimizing dangling bonds and ensuring full
hydrogen termination. Under these conditions, the nominal growth rate was estimated to
be about 0.3 um/h based on the final film thickness of 3-5 pm. Once coated, the diameter

of the cylindrical portion was approximately 80 um [155, 160, 159].

40

2.2 Diamond Film Characterization

The electrode surface morphology and microstructure were investigated by

scanning electron microscopy (SEM) and visible-Rarnan spectroscopy.

Scanning electron microscopy (SEM). SEM was performed with either a JEOL 6400V
or a JEOL 6300F field-emission electron microscope. Figure 2.1A shows SEM images; a
sharpened wire coated Pt wire (top) and a Pt wire coated with a boron-doped diamond
film (bottom). The wire is covered with polycrystalline diamond as no major cracks or
areas void of diamond were seen [155, 159]. Moreover, electrochemical tests described
below in section 2.4 revealed no evidence for exposed Pt, consistent with complete film
coverage. A higher magnification image of the coated wire is presented in Figure 2.18
The polycrystalline film consists of randomly oriented, well-faceted crystallites ranging
in diameter from 0.5 to 3 um. There is a wide crystallite size distribution with smaller

secondary crystallites existing on the larger primary crystallite facets.

 

Figure 2.1. SEM images of (A) an electrochemically sharpened Pt wire (top) and a Pt
wire coated with a polycrystalline boron-doped diamond film (bottom). (B) An expanded
view of the end of the diamond-coated Pt wire.

41

This wire is not the only substrate that can be coated with diamond as smaller diameter Pt
wires (25 and 10 um), tungsten (W) wire and quartz rod (ca. 270 um) have also been
coated in our laboratory. The most conformal coating and reproducible electrode
properties, however, were obtained with the 76 um diam Pt wire. Therefore, this substrate
was used for all microelectrode preparation in this work. It should be noted that the
diameter of this wire is large in comparison with microelectrode diameters commonly

used (<30 um diam), but was nonetheless useful for our measurements.

Raman Spectroscopy. Raman spectra were acquired at room temperature using a
RAMAN 2000 spectrograph (Chromex, Inc., Albuquerque, NM) that consisted of a
diode-pumped, frequency-doubled continuous wave (CW) Nd:YAG laser (500 mW at
532 nm, COHERENT), a Chromex 500 is spectrometer (f/4, 600 grooves/mm
holographic grating), and a therrnoelectrically cooled 1024 x 256 element charge-coupled
device (CCD) detector (ANDOR Tech., Ltd., South Windsor, CT). Spectra were
collected with an incident power density of ca. 500 lecm2 (100 mW at the sample and 3
pm diameter spot size) and a 10 3 integration time. A white-light spectrum was collected
under the same conditions and served as the background for spectral correction. The
spectrometer was calibrated (wavelength position) with 4-acetamidophenol
(CH3CONHC6H40H). Figure 2.2 shows a series of Raman spectra for diamond films
deposited on the 76 um diam Pt wire using different CH4/H2 ratios. All spectra possess an
intense one-phonon diamond line at 1332 cm". The fact that there is no up-shift or down-
shift of the peak means that the diamond film is not under significant tensile or

compressive stress [161]. There is also some scattering intensity near 1580 cm",

42

particularly for the 1% CH4/H2 film, which is attributable to amorphous spz-bonded

carbon impurity.

 

1332 cm"

      

1530 cm"

Intensity

 

7..
0.5%
1600 ' 1500 2000
Raman Shift (cm")

Figure 2.2. Raman spectra for diamond thin film deposited on Pt wire with different
methane-to-hydrogen ratios.

 

 

 

The scattering intensity in this region increases with the CH4/l-Iz ratio used in the
source gas indicating that more sp2 carbon is formed at the higher source gas carbon
levels [162, 163]. We suppose that much of the impurity responsible for the signal is
located at the interface between the Pt and diamond film and not on the film surface. The
1332 cm'1 line intensity decreases and the linewidth (full width at half maximum,
FWHM) increases with increasing CH4/H2 ratio. For example, the one-phonon mode
linewidths are 7, 11 and 17 cm '1 for the films deposited with 0.5, 0.75, and 1.0% CH4/H2,
respectively. By way of comparison, the line width for a piece of single crystal diamond

using the same instrumental settings was ca. 2 cm'l

. The linewidth, to a first
approximation, is inversely related to the phonon lifetime. The larger linewidth for the

films on Pt is caused by increased phonon scattering as a result of the grain boundaries

and defects in the polycrystalline film [164, 163]. Based on the Raman spectral features,

43

the films deposited from 0.5% CH4/H2 are of good quality and were used throughout this

work.

2.3 Microelectrode Preparation

The carbon fiber was a heat-treated (3000 0C) pitch-base type (Textron Speciality)
with a nominal diameter of 35 pm. The resulting microcylinder electrode was disk-
shaped at the end. For the electroanalytical measurements, both the carbon fiber and
diamond microelectrodes were attached to a Cu wire with conductive epoxy. Each
electrode was then sealed in polypropylene [165, 166]. This was accomplished by
inserting the microelectrode into a pipet tip and heating the tapered end using the heating
coil of the micropipet puller.

The assembly is shown in Figure 2.3A. Electrodes insulated with polypropylene are
non-toxic to tissue and chemically resistant to the isopropyl alcohol (IPA) used for
electrode cleaning. This insulating procedure was found to be great for sealing the rough
diamond surface. SEM images of the polypropylene-coated insulated diamond
microelectrode are presented in Figures 2.3B and C. The conical shape of the exposed
microelectrode is seen in the middle image with an arrow pointing to the edge of the
polypropylene insulation. The polymer layer appears thin and uniform over the wire.
Figures 2.3D and E show top view images of the diamond film with and without the
polypropylene coating. The microelectrode diameter at the narrowest point was 10-20 pm
and at the widest point was about 80 pm. The length of the exposed electrode was 100-

200 pm.

44

A Conductive Epoxy Tip of Diamond

200 um

 

Figure 2.3. (A) Diagram of the conically shaped diamond microelectrode insulated with
polypropylene. SEM images of the polypropylene-insulated diamond microelectrode at
(B) lower and (C) higher magnification. Top-view SEM images of the diamond film
morphology without (D) and with (E) the polypropylene insulation layer.

45

Application of this polypropylene insulation method is quite reproducible in terms of
coating the diamond microelectrode with a thin and continuous polymer propylene film,

but precisely control of the exposed electrode length is difficult to achieve.

2.4 Electrochemical Measurement

Cyclic voltammetric measurements were made with a CS-1200 computer-
controlled potentiostat (Cypress Systems Inc., Lawrence, KS) in a three-electrode
configuration using a single compartment glass cell [153]. The cell volume was
approximately 5 mL. A carbon rod served as the counter electrode and a commercial
Ag/AgCl electrode (3 M KCl, Cypress Systems Inc.) was used as the reference. All
measurements were made in a Faraday cage at room temperature and all solutions were
deoxygenated with N2 for at least 10 min prior to a measurement. Both microelectrode
types were cleaned by rinsing and soaking with distilled isopropyl alcohol [167]. The in
vitro continuous amperometric measurements were made using an Omni 90 analog
potentiostat (Cypress Systems Inc.). An analog-to-digital converter (Labmaster 125) and
a computer running Axotape software (version 2.0, Axon Instruments, Foster City, CA)

were used to record the current-time profiles.

Cyclic voltammetry. Cyclic voltammetry provides information not only on the
thermodynamics of redox reactions but also on the kinetics of heterogeneous electron-
transfer reactions and coupled chemical reactions. Routinely, voltammetric experiments
are performed using a stationary working electrode in a quiet (unstirred) solution
containing a dissolved redox-active molecule. A three electrode set-up is most often used

consisting of a working, auxiliary and reference electrode. The working electrode is the

46

one at which the reaction of interest is studied. The properties and placement of the
auxiliary and reference electrodes are critical and must be carefully considered for
appropriate experiment design. The working electrode serves as a source or sink for
electrons. Starting from an initial potential, Bi, 3 linear potential sweep (potential ramp) is
applied to the working electrode. After reaching a switching potential, E1, the sweep is
reversed and the potential returns linearly to its original value. The potential of the
working electrode is controlled versus an appropriately placed reference electrode such as
a silver/silver chloride electrode (Ag/AgCl). The value of this potential relative to the
standard reduction potential, E°, for the redox molecule represents the driving force for
Faradaic charge-transfer reactions. If the potential is negative of E° then the reduced form
of the redox-active molecule is stable at the interface, and if the potential is positive of E°
then the oxidized form is favored. The controlled potential can be considered the
excitation signal in the measurement. The Faradaic current that flows at any time between
the working and auxiliary electrodes is a direct measure of the rate of the electrochemical
reaction taking place at the electrode surface. In cyclic voltammetry, mass transport of
the redox-active molecule generally occurs solely by diffusion. At conventionally-sized
electrodes, a peak current will be seen in the i-E curve for the forward cycle if the
reaction rate is limited by diffusional mass transport. The peak current is given by the

Randles-Sevcik equation
1', = (2.69 x 105) nmADl/ZC u‘”

where ip is the peak current (A), n is the electron stoichiometry, A is the electrode area

(cmz), D is the diffusion coefficient (cmz/s), C is the redox molecule concentration

47

(mol/cm3) and u is the potential sweep rate (V/s). Clearly, ip varies proportionally with A,
C and pm.

In contrast, a steady-state current is observed in the i-E curve for microelectrodes
with a curve shape that depends on the geometry of the exposed microelectrode. In other
words, the shape of the electrode determines the reactant flux (molecules/cmz-s), which,
in fact, determines the current profile. The current at the carbon fiber is described by
simply a cylindrical term. On the other hand, since the diamond microelectrode has both
a cylindrical and conical shape exposed, the current is described by two terms according

to the equations below [155, 159]

iCylinder _ anADC

SS

r In t
if“ = 4nFDcr(l + qH”)

where n is the number of electrons, F is the Faraday constant, A is the electrode surface
area, D is diffusion coefficient, c is bulk concentration of a compound, r is cylinder or
cone radius, r = 4Dt/r2 , H is the aspect ratio h/ r , where h is height of the cone, q =
0.30661 and p = 1.14466. The aspect ratio was determined from SEM measurements. The

exposed microelectrode area was calculated according to the equations.

Electrochemical characterization of the diamond microelectrode. Cyclic voltammetry
was used to evaluate the basic electrochemical properties of the diamond microelectrode.
Comparison measurements were made with a conventional carbon fiber microelectrode.
Figure 2.4A shows background cyclic voltammetric i—E curves in 1.0 M HClO4 for a

typical diamond and carbon fiber microelectrode, and for a Pt wire incompletely coated

48

with a diamond overlayer. The area exposed to the solution was different for each
electrode (and not measured), which makes direct comparison of the current magnitudes
impossible. In general, though, diamond exhibits a lower background current and a wider
working potential window than does the carbon fiber [155, 159]. For example, the
background current density for diamond is typically a factor of 5—10 lower than that for a
carbon fiber. This inherently leads to improved signal-to-background ratios in
electroanalytical measurements.

The background current for diamond results primarily from the charging of the
electric double layer (i.e., potential dependent solvent dipole reorientation and ion
movement at the interface). There is little contribution to this current from electroactive
surface carbon-oxygen functionalities, as is the case for most sp2 carbon electrodes [104,
168, 139]. For the most part, the voltammogram for diamond is flat and featureless over
the potential range, and is stable in shape with extended cycling. If defect (cracks and
pinholes) exist in the diamond overlayer, then the solution will eventually reach the Pt
substrate and electrochemical features characteristic of this metal will appear in the
voltammogram. An example of this is the voltammetric i—E curve for the incompletely
covered Pt wire. Pt is an active electrode for hydrogen evolution and this reaction is
evidenced by the sharp increase in cathodic current at —250 mV. The fact that little
current for hydrogen evolution flows until ca. —1500 mV for the diamond microelectrode
indicates that the diamond film completely covers the Pt substrate. The kinetic
overpotential for this reaction on diamond microelectrode is similar to what has been
reported for high-quality and well characterized microcrystalline diamond thin-film

electrode formed on Si [146, 153, 148].

49

B

 

—L
C?

 

 

. Current (nA)
O

 

 

 

 

 

 

 

10- .14?
D!
’“.
' r l f r ' IL? I - r w I '
-1.5 -1.0 -0.5 0.0 0.5 1.0 1.5
WNW-Adm)
3(1)
. —Dianond
200. --Ca'bonFibar
<
5
3100
2
'5
0 0.
4(1) 3.........-.
-0.4 -0.2 0.0 0.2 0.4 0.6 0.8
WNvaAdAfi)

Figure 2.4. Background cyclic voltammetric i —E curves in (A) 1.0 M HClO4 for a
diamond (—), carbon fiber (— —) and Pt-exposed diamond microelectrode (w). Cyclic

voltammetric i —E curves for a diamond and carbon fiber microelectrode in (B) 1.0 mM
Fe(CN) (3H in 1 M KCl. Scan rate=100 mV/s.

50

Another interesting and important feature of the diamond microelectrode response
is the absence of peaks between the working potential limits. In the case of the carbon
fiber microelectrode, an oxidation peak is present at about 400 mV and a corresponding
reduction peak is seen at 250 mV. These peaks are associated with redox-active surface
carbon—oxygen functionalities, specifically, the quinone/hydroquinone type [169, 166,
167]. Carbon electrodes, particularly 3p2 materials, possess a variety of carbon—oxygen
functional groups at the exposed edge plane sites. Some of these functional groups are
electroactive (e.g., quinone/hydroquinone) and some are not [170]. Other types are acidic
and ionizable (e.g., R—COOH). In the case of the electroactive type, these functional
groups undergo electron-transfer in the —0.2 to 0.6 V vs. Ag/AgCl potential window, at
this pH, according to 2H+/2e’ redox reaction. The potential region in which this charge—
transfer reaction takes place, as well as the extent of functional group deprotonization,
depends on the solution pH, which can change during an in vitro or in vivo measurement
[139, 140]. Both types of functional groups can be problematic in in vitro or in vivo
electroanalytical measurements because (i) they affect the electrode surface charge,
which influences the concentration of catechol and catecholamine molecules (cationic,
neutral and anionic) at the surface, and (ii) they undergo electron-transfer in a potential
region that overlaps that for the catecholamine neurotransmitters being detected. In vitro
and in vivo voltammetric measurements often require background subtraction to enable
the identification and quantification of endogenous catecholamine neurotransmitters. The
presence of electroactive surface functional groups complicates this background

correction [139]. The diamond microelectrode does not possess surface oxides when

51

hydrogen-terminated and thus is ideal for these measurements because the background
voltammetric signal is featureless and independent of pH [155, 159].

Figure 2.4B shows cyclic voltammetric i—E curves for 1 mM Fe(CN)6‘3/‘4 in l M

KCl at both a diamond and carbon fiber microelectrode. Fe(CN)(,'3/’4 is a good redox
system to use for testing the electrochemical behavior of both diamond and 3p2 carbon
electrodes [171-173]. In general, the heterogeneous electron-transfer rate constant for this
redox system is highly sensitive to the surface cleanliness, exposed microstructure and
the density of electronic states near the formal potential [174, 175, 148]. The
voltammetric i—E curves for Fe(CN)6’3/‘4 are nearly identical in shape for both electrode
types. The half-wave potentials (Em) are 267 and 269 mV, respectively, for the diamond
and carbon fiber microelectrode. In general, the Em values for multiple diamond and
carbon fiber microelectrodes tested were quite similar indicating that both electrodes are
active for this redox system. This result indicates that (i) the response of diamond toward
this redox system is similar to that for the carbon fiber, (ii) the diamond surface is
effectively cleaned, when needed, by the alcohol soak and (iii) the diamond electrode
possesses a high density of electronic states in this potential region enabling relatively

rapid electron-transfer kinetics.

2.5 Animals

The University Committee on Animal Use and Care at Michigan State University
approved all procedures for handling and caring for the laboratory test animals. All rats

were allowed 2-3 days of acclimatization prior to entry into any experimental protocol.

Pelleted rat chow (Harlan/Teklad 8640 Rodent Diet; Harlan/Teklad) and water were

52

provided. Rats were housed in temperature and humidity controlled rooms using a 12-h

on/off light cycle.

Preparation of DOCA-salt and sham Rats. Male Sprague-Dawley rats (175-200 g,
Charles River Inc., Portage, M1) were exteriorized and the left kidney was removed under
anesthesia with sodium pentobarbital (50 mg/kg i.p.(intraperitoneal)) after ligation of the
renal artery, vein and ureter with 4-0 silk sutures. DOCA implantation was made between
the back shoulder blades of the animal using a 1 cm incision. DOCA implants (600
mg/kg) were prepared by mixing deoxycorticosterone acetate in silicone rubber resulting
in a dose of 200 mg/kg. DOCA-implanted rats received standard pelleted rat chow and
salt water (1% NaCl + 0.2% KCl). Sham, normotensive rats also underwent left kidney
removal but no DOCA pellet implantation and received unsalted tap water. The surgery
was performed on a heated pad and rats recovered in a heated box. Antibiotics
(enrofloxacin, 5 mg/kg s.c.(subcutaneous)) and an analgesic (butorphanol tartrate, 2
mg/kg, so.) were administered immediately after the surgery. After recovery, the rats
were housed under the above listed conditions for 4 weeks. Systolic blood pressure was

measured using the tail-cuff method four weeks after surgery. Rats with mean systolic

blood pressure of 2 150 mmHg were considered hypertensive [176, 31].

2.6 In Vitro Electrochemical and Diameter Measurement System

The combined use of continuous amperometry with diamond and carbon fiber
microelectrodes and video imaging were used for the simultaneous in Vitro measurement
of NE release from sympathetic nerves innervating a rat mesenteric artery (MA) and vein

(MV) and the evoked contractile response.

53

These techniques, along with various drugs, were employed to show the
relationship between the oxidation current associated with endogenous NE overflow and

blood vessel constriction. A block diagram of the experimental set-up is shown in Figure

2.5.

Mesentery (Small Intestine)

. 3" i'i Blood Vessel
’° Diameter
I 180-330 um

 
   
 

 

    

Computer

Q

 

Buffer

°nunl

 

35-37 °C
Oxygenated Krebs’ Buffer
Flow rate: 5~6 mL/min

 

 

 

 

I

Video Camera

Figure 2.5. Block diagram of the experimental set-up

Tissue preparation. The sham and DOCA-salt rats (300 — 430 g,) were euthanized with
a lethal pentobarbital injection (50 mg, i.p.). The abdomen was surgically opened, and the
small intestine carefully removed and placed in an oxygenated (95% 02, 5% C02) Krebs’
buffer solution (pH 7.4) of the following composition: 117 mM NaCl, 4.7 mM KCl, 2.5
mM CaClz, 1.2 mM MgC12, 25 mM NaHCO;, 1.2 mM NaH2P04, and 11 mM glucose. A

section of the mesentery close to the ileal wall was carefully cut free from the intestine

54

and transferred to a small silicone elastomer-lined, Teflon flow bath (5 mL volume).
Secondary or tertiary arteries and veins (180-330 um outside diameter) were isolated for
in vitro study by carefully removing the surrounding adipose and connective tissue under
a dissecting microscope using fine scissors and forceps. The Krebs’ buffer solution
entered the bath at one end (center) and flowed out the opposite end (center). The bath,
containing the fixed tissue preparation, was attached to the stage of an inverted
microscope (Olympus CK-2) and superfused continuously with 37 °C Krebs’ buffer at a
flow rate of 1.6 mL/min (Chapter 3 and 4) and 5-6 mL/min (Chapter 5 and 6). The

solution flow was controlled by a peristaltic pump.

Assessment of electrode performance. The diamond and carbon fiber microelectrodes
were cleaned by soaking in distilled isopropyl alcohol (IPA) for at least 15 min prior to a
measurement [167]. The microelectrode was affixed to a micromanipulator (MP-1,
Narishige Instruments, Japan), which was used to position the electrode on the surface of
the MA and MV. The microelectrode was positioned in the center of the bath containing
the fixed tissue preparation, equidistant from the inlet and outlet solution ports. A Pt wire
counter and a commercial “no leak” Ag/AgCl (3 M KCl, model EE009, Cypress Systems
Inc.) reference electrode were also mounted in the bath to complete the electrochemical
cell. All electrochemical measurements were made with an Omni 90 analog potentiostat
(Cypress Systems Inc.), an analog-to-digital converter (Labmaster 125), and a computer
running Axotape software (version 2.0, Axon Instruments, Foster City, CA). The
software was used to record the continuous amperometric current-time profiles and blood
vessel diameter changes. The analog output current from the potentiostat was low pass

filtered (5 Hz or 200 ms RC time constant). All data were digitized using a sampling rate

55

of 100 Hz. The Krebs’ buffer flowed over the electrode and the tissue sample for 30 min

prior to the start of a series of measurements.

Focal stimulation of perivascular nerves. Short trains of electrical stimulation were
used to trigger NE release. Perivascular nerves were stimulated using a bipolar focal
stimulating electrode positioned on the surface of the blood vessel at a distance of ca. 200
pm from the tip of the carbon fiber. This positioning minimized the noise introduced into
the current recordings. The focal stimulating electrode consisted of two AgCl-coated Ag
wires inserted into double barrel capillary glass (tip diameter = 180 pm). The wires were
connected to a stimulus isolation unit and a Grass Instruments stimulator (S88, Quincy,
MA). Trains of 60 stimuli (0.3 ms duration, 30-70 V) at frequencies ranging between 0.5

and 30 Hz were used.

In vitro video monitoring of vasoconstriction. The output of a black and white video
camera (KP-111, Hitachi, Yokohama, Japan) attached to the microscope was fed to a PC
Vision Plus frame-grabber board (Imaging Technology, Wobum, MA) mounted in a
personal computer. Changes in blood vessel diameter of 0.1 pm could be resolved.
Video images were analyzed using Diamtrak software (http://www.diamtrgk.com,
Adelaide, Australia). The digitized signal was converted to an analog output (DAC-02
board, Keithley Metrabyte, Tauton, MA) for subsequent processing by an analog-to-
digital converter (Labmaster 125) and analysis in a second computer running Axotape for
a permanent recording of blood vessel diameter as function of time. Analog signals were
sampled at 100 Hz and data were stored on the computer’s hard drive for subsequent

analysis and display.

56

2.7 Fluorescence Histochemistry

Tissue preparation. Second or third-order veins and arteries from the ileal mesentery,
were isolated by carefully clearing away the surrounding adipose and connective tissues.
Red blood cells were flushed out of the blood vessel lumen with phosphate buffer (0.1 M,
pH 7.2) using a 30 ga. hypodermic needle and syringe, and tissues were cut to 1 cm
lengths. For these experiments, we used more than 30 MA and MV segments from 13

different rats.

Gyloxylic acid fluorescence histochemistry. Catecholamine fluorescence was revealed
after incubating the prepared tissues in a 2% glyoxylic acid/0.2 M phosphate buffer (pH
7.0) solution for 5 minutes at room temperature. The blood vessels were stretched onto
glass slides and then heated at 80 °C for 5 min. Preparations were mounted in mineral oil
and observed using a fluorescence microscope (Nikon Eclipse TE 2000-U) equipped with

a filter set, UV2E/C (excitation filter, 340-380 nm and emission filter, 435-485 nm).

2.8 Chemical and Drug Application

All chemicals were reagent-grade quality, or better, and used without additional
purification. The chemicals and drugs used for electrode performance testing were
postassiurn ferrocyanide (II) trihydrate, epinephrine (EE), dopamine (DA),
norepinephrine (N E), catechol (CA), t-butyl catechol (TBC), 4-methyl catechol (4MC)
and 3,4-dihydroxyphenylacetic acid (DOPAC). Tetrodotoxin (TTX, 0.3 uM), pyridoxal-
phosphate-6-azophenyl-2',4'-disulfonic acid (PPADS) (10 uM), prazosin (0.1 uM),
yohimbine (1.0 uM), UK 14,304 (1.0 uM) and cocaine (10 uM) were used for in Vitro

study. The drugs were added to the superfusing Krebs’ buffer solution. Drugs were

57

applied for 20 min prior to assessing their effects on nerve-mediated responses and were
applied after a series of control measurements were made. All chemicals and drugs were
obtained from Sigma Chemical Company (Saint Louis, MO). Ultrapure water (distilled,
deionized, and passed over activated carbon, 17-18 MQ, Barnstead E-PureSystem) was

used for all solution preparation, and glassware and electrode cleaning.

2.9 Data Analysis

Data were obtained from >150 sham rats and >150 DOCA-salt rats for the research.
Mean systolic blood pressure from sham and DOCA-salt rats was 125 mm Hg and 194
mm Hg, respectively. The mean weight of sham rats was 416 g, and the mean weight of
DOCA-salt rats was 336 g. Each series of measurements required at least 3 rats.
pCLAMP 8.1 software (Axon Instruments, Foster City, CA) was used to analyze all data.
The rise time and decay of NE signals (S/N > 3) or blood vessel constriction were fitted
using a standard exponential model in Clarnpfit 8.1. Data are presented as mean i S.E.M;
where SEM is standard error of the mean. Means were compared by using Student’s t-
test and P < 0.05 was regarded as significant. MA and MV constrictions are expressed as
a percentage of the initial resting diameter (in pm) of the blood vessel. For nerve
stimulation frequency-response curves, the half maximal effective stimulation frequency
(Sso) and maximum constriction (Emu) were calculated from curves obtained in
individual tissues using the following function:

Y=EmaxX/(S50+X)

where X is the stimulation frequency tested and Y is the peak response amplitude.

58

Chapter 3

Comparison of Electrochemical Properties of Diamond and
Carbon Fiber Microelectrodes During Exposure to Biological
Environments

3.1 Introduction

An electrochemical electrode used in biological media should possess several
properties: good sensitivity for the target analyte, rapid response time, a stable
background response that is unaffected by changes in the surrounding solution
environment (e.g., pH) and resistance to biomolecule adsorption (deactivation and
fouling). Carbon fibers are useful for monitoring small quantities of various electroactive
catecholamines because of their small size (<30 um) and temporal response [103, 177,
178, 139, 140]. However, a drawback with their use for measurements in vitro and in vivo
is the lost response sensitivity and poor stability that typically occurs during exposure to
physiological environments. Specifically, background current features (e.g., redox active
functional groups) and drift, sensitivity to local pH changes and response attenuation due
to surface fouling caused by biomolecule adsorption are complications that arise. The
cause for these complications is linked to the presence of carbon—oxygen functional
groups on the electrode surface [104, 139]. Conductive diamond is a new electrode
material with an sp3-bonded carbon microstructure with no extended n-electron system,
and little surface oxygen due to a hydrogen termination. As will be shown in this
Chapter, the diamond microelectrode has properties that make it useful for measurements
in complex biological environments. Ideally, one would like to have a microelectrode that

is void of surface carbon—oxygen functional groups and that possesses a surface on which

59

weak molecular adsorption occurs. Planar diamond thin-film electrodes have attracted
considerable interest in recent years due to some superb electrochemical properties [179,
153, 180, 148]. However, so far, there have been only a few reports describing the
electrochemical properties and application of diamond microelectrodes [155, 156]. Such
electrodes are typically prepared by coating a thin layer of conducting diamond on a
small diameter metal wire. The diamond microelectrode is attractive for electroanalytical
measurements in complex media, like biological environments, because of its (i) hard and
lubricious nature that enables easy penetration into tissue with minimal peripheral
damage, (ii) low and stable background current over a wide potential range, (iii) good
chemical and microstructural stability, (iv) low surface oxygen content (when H-
terrninated) - a property that leads to minimal change in the background current with
variation in solution pH, (v) chemical inertness and (iv) a non-polar, hydrophobic surface
that renders it resistant to molecular adsorption (i.e., deactivation and fouling). Another
purported property of diamond is biocompatibility. Diamond electrodes are presumed to
be highly resistant to deactivation and fouling when exposed to complex biological
environments; however, to the best of our knowledge, there has been no systematic study
of the response sensitivity, precision and stability in such environments. How stable and
resistant to deactivation this new microelectrode is during exposure to biological
environments and how useful it is for in vitro electroanalytical measurements are two
questions this work sought to answer.

An objective for this work was to evaluate the diamond microelectrode response
for endogenous NE in vitro and to compare the response with that of a conventional

carbon fiber microelectrode [181, 34, 104]. Preparations of rat mesenteric arteries

60

maintained in vitro were used as a test system to make this comparison. It has previously
been shown that electrical stimulation of these tissues causes the perivascular nerves to
release NE [119, 121]. We report presently on the response sensitivity, precision and
stability of a diamond microelectrode for (i) NE and Fe(CN)6’3/‘4 before and after
exposure to the laboratory atmosphere and to biological tissue, and (ii) NE during in vitro

release measurements at the surface of a test animal’s mesenteric artery.

3.2 Results and Discussion

Effect of pH on the background voltammetric response. Figures 3.1A and B show
background voltammetric i—E curves for a diamond and carbon fiber microelectrode in
phosphate buffer solutions ranging in pH from 3 to 7. The i—E curves for the diamond
microelectrode between —300 and 500 mV are all featureless and the current magnitude is
the same at all pH values. The current beyond 600 mV is presumably due to the onset of
oxygen evolution, which increases as expected with increasing solution pH. The
calculated geometric areas of the diamond and carbon fiber microelectrode were 6.2 x
10'5 and 1.5 x 10’5 cm2, respectively. Even though the diamond microelectrode area is
approximately four times larger, it exhibits a lower background current. The low
background current is a characteristic feature of diamond and leads to improved signal-
to-background ratios in electroanalytical measurements [179, 153, 180, 148]. The
electrode capacitance was not measured directly but the values calculated from the cyclic
voltammetric anodic current at 0 V were 11 and 200 uF/cmz, respectively, for the

diamond and carbon fiber.

61

 

 

 

 

 

Q:

04 am
-03 pI-Iso
gag: #450
Eat mm
5°". -
o0m “’

.02.

3‘ an ' I ' r v r v u v r

400 -200 0 200 400 am 800

mart/unmet)

 

B

p
9 -

 

Current (nA)
is .

 

04-

 

 

 

400-200 0 "zin'400'a'n'800
warmqugo)

Figure 3.1. Background cyclic voltammetric i—E curves for a (A) diamond and (B)

carbon fiber microelectrode in 0.1 M phosphate buffer solutions of different pH ranging
from 3 to 7.2. Scan rate = 100 mV/s.

62

The assumption in this calculation is that the background current is all capacitive in
nature. For the diamond electrode, this is likely the case, but for the carbon fiber, the
background current contains a significant Faradaic contribution from the electroactive
surface oxide functionalities. Therefore, the electrode capacitance for the carbon fiber
calculated in this manner is much larger than the true value (expected to be
approximately 30 uF/cmz). The voltammetric i—E curves for the carbon fiber have redox
waves in the 200 — 600 mV range that shift with pH. Specifically, the peaks shift by —59
mV/pH, consistent with an equal number of electrons and protons being transferred in the
redox reaction. These peaks are attributed to redox-active carbon—oxygen functionalities
on the electrode surface that exist at edge plane and defect sites. It is generally accepted
that the redox-active groups are of the quinone/hydroquinone type [170, 130].

Carbon electrode surfaces, particularly 3p2 materials possess a variety of oxygen
functional groups, some electroactive and some not. A non-electroactive carbon—oxygen
firnctional group that is sensitive to pH is the carboxylic acid type. Negative excess

surface charge will exist on the electrode when these groups are deprotonated at pH
values above the pKa, which is near 4.5. Increasing the excess surface charge will alter

the ionic charge stored in the electric double layer and is reflected by an increasing
background voltammetric current. These redox peaks and changes in the background
D ,

rrent with pH are problematic in in vitro and in vivo electroanalytical measurements,
particularly for catecholamine analysis. This is because they occur in a potential range
that overlaps the oxidation and reduction peaks for the catecholamines. The fact that
background current varies with pH complicates background subtraction/correction [104,

139, 140]. The pH changes occur in biological tissue as a result of coupling respiration

63

and energy production to pH [104, 139, 140]. The diamond microelectrode does not
possess surface oxides when hydrogen-terminated, therefore, there are no redox waves
and the background is relatively insensitive to changes in solution pH at constant ionic

strength.

Catecholamine voltammetric response. Figures 3.2A and B show cyclic voltammetric
i—E curves for 10 uM norepinephrine (NE) and epinephrine (BE) in 0.1 M phosphate
buffer (pH 7.2) at the two microelectrode types. The calculated geometric areas of the
diamond and carbon fiber microelectrode were 2.4 x 10'3 and 1.3 x 10‘3 cm2,
respectively. Both curves have some peak-shape character at this scan rate, which reveals
that a steady-state flux of analyte to the electrode surface is not fully achieved on the time
scale of this measurement. Rather, a thin depletion layer develops leading to a time-
dependent flux. We estimate, based on the 80 um diamond microelectrode diameter, that
scan rates less than 50 mV/s would be needed to observe a steady state oxidation current.
There may also be some molecular adsorption particularly on the carbon fiber, which
would contribute to the peak-shape character. We did not conduct any electroanalytical
measurements to probe for molecular adsorption though. The maximum oxidation peak
currents for both NE and EE at diamond are 13 nA and at the carbon fiber are 8 nA. The
factor of 1.6 difference in current is comparable to the factor of 1.8 difference in the
calculated area for the two microelectrodes. The oxidation half-wave potentials (El/2) for

NE and EE at the diamond microelectrode are approximately 200 mV more positive than

the values for the carbon microelectrode.

64

 

$-23-“

Current (nA)
- 5

 

-9.‘."

-s
400200 0 '200'400'600'80011m0
marmvuwgcr)

 

 

 

 

$3238

 

Current (nA)

9-9‘-

 

 

 

2m0m2014006008t'l)
meantime/rumor)

Figure 3.2. Cyclic voltammetric i—E curves for a diamond and carbon fiber

microelectrode in (A) 10 M NE and (B) 10 uM EE, both in 0.1 M phosphate buffer, pH
7.2. Scan rate = 100 mV/s.

65

This is a common observation for catecholamine electrochemistry at diamond with
the more positive Em being attributed to more sluggish reaction kinetics, not ohmic
resistance effects [146, 153, 180, 154]. The reason for the more sluggish kinetics at
diamond is still under study but one postulate is that molecular adsorption is not involved
in the reaction mechanism on the hydrogen-terminated, sp3-bonded carbon surface. Of
particular relevance here is the fact that McCreery and DuVall have shown that
catecholamines adsorb strongly on GC and have correlated the adsorption with more
rapid electrode reaction kinetics [182].

Importantly from an electroanalytical point of view, the more positive detection
potential for diamond does not appear to be a major drawback as the background current
remains low and the physicochemical properties remain unchanged. A summary of the
cyclic voltammetric Em values for several catechols and catecholamines at the two
microelectrode types (n 2 4) is presented in Table 3.1. The analyte concentrations were
all 0.1 mM. Consistently for all the analytes, the Em for diamond is 150—250 mV more
positive than for the carbon fiber. The response precision is quite good for both electrode
types with a relative standard deviation (RSD) of l—4%. The results indicate that the

diamond microelectrode can be formed with reproducible electrochemical properties.

Diamond electrode response stability during exposure to the laboratory atmosphere.

A series of measurements was made to assess the stability of the diamond
microelectrode response during exposure to the laboratory atmosphere. This is a good test
of stability because carbon electrodes are notoriously prone to deactivation during

exposure to air [170].

66

Table 3.1. Summary of cyclic voltammetric El,2 data for diamond and carbon
microelectrodes.

 

 

 

Analyte Electrode
Diamond E1 /2 (mV) Carbon Fiber Em (mV)

Dopamine 421 d: 17 188 i 3
Norepinephrine 456 i 12 236 :l: 10
Epinephrine 472 i 18 266 :t 14
Catechol 543 i 8 291 :1: 12
t-Butyl catechol 497 :l: 7 218 :t 5
4-Methyl catechol 495 d: 6 241 :E 8
DOPAC 614 :t 19 352 i 11

 

The solutions were 0.1 mM norepinephrine (NE), epinephrine (EE), 3,4-dihydroxy-
phenylacetic acid (DOPAC), dopamine (DA), catechol (CA), t-butyl catechol (TBC), and
4-methyl catechol (4MC) all in 0.1 M phosphate buffer (pH 7.2). Scan rate = 20 mV/s.

Furthermore, the electrode response sensitivity and reproducibility can vary considerably
depending on the electrode preparation conditions, the storage conditions and the past
history of use. Figure 3.3A and B show cyclic voltammetric i—E curves for a diamond

and carbon fiber microelectrode in 1 mM Fe(CN)(,"3/'4 and 0.1 M KCl before and after a
multiple-day exposure to the laboratory atmosphere.

Fe(CN)t,-’3/’4 is a good redox system for this kind of test because the electrode
reaction kinetics are quite sensitive to the surface cleanliness of carbon electrodes [174].
The kinetics are also sensitive to the surface microstructure of 3p2 carbon electrodes [174,
17 5] and to the surface oxygen content on diamond [172]. The electrode reaction kinetics
are influenced by the surface oxygen content on sp2 carbon electrodes, but mainly when a
thick oxide layer exists [175]. Both electrodes were initially cleaned by rinsing and

soaking in distilled isopropanol for 20 min [167].

67

 

Current (nA)

 

 

 

 

 

Current(nA)
9.9.8833ggg

 

 

 

 

 

400-2110 0 2000400600 80001000
warms. range)

Figure 33. Cyclic voltammetric i-E curves for 1 mM Fe(CN)6’3/'4 in 0.1 M KC1 at (A)

diamond and (B) carbon fiber microelectrode before and after laboratory atmosphere
exposure. Scan rate = 100 mV/s.

68

It is important to note that while this treatment was applied to diamond for consistency, it
was seldom needed as the “as grown”, untreated electrode typically exhibited an active
response for this surface-sensitive redox system [153, 154].

The calculated geometric areas for the diamond and carbon fiber microelectrodes
were 1.8 x 10'4 and 9.7 x 10'4 cm2, respectively. Well-defined oxidation peaks are seen
for both electrodes with the initial Em of 260 and 282 mV, respectively, for the diamond
and carbon fiber microelectrode. After a two-week exposure to the laboratory
atmosphere, the diamond microelectrode response was largely unaltered. No pretreatment
(e.g., cleaning with alcohol) was applied to either electrode after the air exposure. A
pseudo-sigrnoidal oxidation current is still observed with the maximum current remaining
about the same and Em shifting positive by only 7 mV. This indicates that the relatively
non-polar, hydrogen-terminated diamond surface is resistant to deactivation in the
laboratory atmosphere (e.g., surface oxidation and adsorption of airborne contaminants).
A pseudo-sigrnoidal oxidation current is also observed for the carbon fiber
microelectrode, but the Em is shifted positive by 138 mV after a one-week exposure. The
positive shift was progressive with time and reflects more sluggish electrode reaction
kinetics, presumably because of deactivation by contaminant adsorption [170]. The
maximum oxidation current of 52 nA for diamond and 240 nA for the carbon fiber differ
by a factor of approximately 5, and this is consistent with the factor of 5 difference in
geometric area. Clearly, the diamond microelectrode is more resistant to deactivation in
the air than is the carbon fiber.

The extent to which the physical, chemical, and electronic properties affect the

electrochemical response of 3p2 (e.g., carbon fiber) and 3p3 (e.g., diamond) carbon

69

electrodes depends on the mechanism for the particular redox test system [175, 146, 182,
153]. Some redox systems are more outer-sphere than others in terms of their electron-

+3l+2 , methyl viologen, and IrClrg‘ZJ'3 fall into

transfer mechanism. For example, Ru(NH3)6
this category for both GC and diamond electrodes [175, 146, 182, 153]. For such redox
systems, the apparent heterogeneous electron-transfer rate constant, k°app, is relatively
unaffected by the physical and chemical properties of the electrode surface. The primary
factor influencing k°app is the electronic properties of the electrode — the density of
electronic states near the formal potential of the redox system. On the other hand, some

_ .4 . . .
3’ , catecholamines, ascorbic acrd, and 02, are more

redox systems, such as Fe(CN)6
inner sphere in terms of their electron-transfer mechanism [175, 146, 182, 153]. For such
systems, k°app is highly sensitive to the surface cleanliness, microstructure and chemistry,
as well as the density of electronic states near the formal potential. As mentioned above,
F e(CN)6"3/ '4 is particularly sensitive to the surface cleanliness and exposed
microstructure (i.e., fraction of clean edge plane) of 3p2 carbon electrodes [175, 146, 182,
153], and to the presence of surface oxygen functionalities on diamond electrodes [172].

Therefore, F e(CN)6‘3’ 7‘ is a very good probe of a carbon electrode’s response sensitivity

and stability.

Diamond electrode response stability during exposure to biological tissue. Another
series of measurements was performed to learn how exposure to biological tissue affects
the diamond microelectrode response. A concern with any electroanalytical measurement
in biological environments is response attenuation caused by the irreversible adsorption
of biomolecules (e.g., proteins, lipids). Fouling of oxygenated sp2 carbon electrodes can

occur in biological media because of strong molecular adsorption on the polar,

70

hydrophilic surface. One of the ways fouling of 3p2 carbon fiber microelectrodes can be
minimized is by coating with the ionomer, NafionTM [183].

While this treatment improves the electrode sensitivity for cationic
neurotransmitters and renders the electrode more resistant to fouling, it also causes an
increase in the electrode response time to concentration changes brought about by a

'3/"4 was again used as the redox test

neuronal event (in vivo or in vitro) [131]. Fe(CN)6
system to probe the bare diamond electrode response sensitivity and stability. The test
involved first recording a cyclic voltammetric i—E curve for 1 mM Fe(CN)6‘3/“4 in 0.1 M
KCl. The microelectrode was then removed from the standard electrochemical cell and
transferred to the flow bath containing a mesenteric artery preparation in which most of
the surrounding adipose tissue had not been removed. The microelectrode was inserted
into the tissue for 10 min before being removed and transferred back to the

electrochemical cell containing the Fe(CN)6‘3"4

solution. The cyclic voltammetric
response was then re-recorded.

A bare carbon fiber microelectrode was also tested for comparison. The geometric
areas for the diamond and carbon fiber microelectrode were 2.2 x 10"4 cm2 and 1.5 x 10’3
cm2, respectively. No soak in isopropyl alcohol was used to clean the electrode surfaces
after the tissue exposure. Observation of the implanted microelectrodes with an optical
microscope revealed that the hardness and lubricious nature of diamond allowed it to
smoothly penetration the tissue without causing significant peripheral damage. On the
other hand, the flexibility of the carbon fiber microelectrode made insertion difficult

without bending or breaking. In these measurements, the carbon fiber microelectrode did

not actually penetrate the tissue but merely was in contact with it on the outside surface.

71

Figures 3.4A and B show cyclic voltammetric i—E curves for 1 mM Fe(CN)6_3/T1 in 0.1 M
KCl at both microelectrode types, before and after the tissue exposure. The curves for
both electrodes before exposure possess some peak-shape character with E”; values of
262 and 333 mV, respectively, for the diamond and carbon fiber. The maximum
oxidation current was 60 nA for diamond and 360 nA for the carbon fiber. The factor of 6
difference in current is consistent with the factor of 7 difference in the calculated
geometric area. After exposure to the tissue sample, the response for the diamond
microelectrode was slightly affected as there was only a 21 mV positive shift in Em with
the oxidation current maximum remaining about the same. In contrast, a 205 mV positive
shift in Em was seen for the carbon fiber along with a slight decrease in the maximum
current. It appears that biomolecule adsorption occurs to a greater extent on the polar,
oxygenated carbon fiber surface even though the electrode did not actually penetrate the
tissue. Molecular adsorption deactivates the electrode for this redox system by partially
blocking some specific surface sites involved in the reaction. Furthermore, it was

'3” could not be regained

observed that the original carbon fiber response for Fe(CN)6
after soaking in clean isopropanol for 20 min. This indicates that the biomolecule
adsorption is very strong on the it-bonded, oxygen-containing carbon fiber surface. In
contrast, the original diamond microelectrode response, which was only weakly affected
by the tissue exposure anyway, was regained after a 20 min isopropanol soak. This is
consistent with weak biomolecule adsorption, as expected. The diamond and carbon fiber

microelectrode response sensitivity and stability after tissue exposure was also probed

using NE.

72

 

5-8-8

Current (nA)
9- B -

 

1'3-

 

 

 

4001200'0'200'400'600'800
Whit/BMW)

 

Current (nA)
¢. 8 - § § §

 

I
E

I

l

 

 

 

500 ' 0 ' 500 ' 10'00
murmnmmgo)

Figure 3.4. Cyclic voltammetric i—E curves for 1 mM Fe(CN)6'3/'4 in 0.1 M KCl at a (A)

diamond and (B) carbon fiber microelectrode before and after exposure to biological
tissue. Scan rate = 100 mV/s.

73

The diamond microelectrode is being used for the in vitro measurement of NE released in
the vasculature of normal and hypertensive test animals. Therefore, it was important to
learn how tissue exposure affects the short and long-term electrode response for this
neurotransmitter.

Measurements of NE were made by continuous amperometry using the previously
described flow bath and involved first placing the microelectrode in the bath above the
tissue. An NE solution was then flowed through the bath with the electrode poised at a
potential positive enough to oxidize the catecholamine at a mass-transfer limited rate.
The optimum detection potentials were determined from hydrodynamic voltammetric
measurements to be 0.8 and 0.4 V, respectively, for the diamond and carbon fiber
microelectrode. The electrode was inserted into the tissue for at least 10 min. The
electrode was then removed from the tissue and a NE-containing solution injected into
the flow bath. Figures 3.5A and B show the results of such a test. The calculated
geometric areas were 6.4x 10’4 cm2 and 4.3x 10‘4 cm2 for the diamond and carbon fiber
microelectrode, respectively. Again, the biological sample contained the blood vessels
with the surrounding adipose tissue intact. The responses shown are for a 3 ml injection
of 0.1 uM NE at a flow rate of 1.6 ml/min. Both bare electrodes showed some
deactivation during the tissue exposure with the diamond microelectrode being less
affected. For diamond, the nominal current response before exposure was 65.0 i 0.8 pA
and after 8 h decreased to 36.3 i 0.7 pA (n 2 5). In contrast for the carbon fiber, the
nominal current response was 41.4 i 1.6 pA before exposure and after 7 h decreased to
14.3 :t 0.5 pA (n _>. 5). This corresponds to a response attenuation of 44% for diamond

and 66% for the carbon fiber microelectrode.

74

 

A At beginning [Diamond I

120 pA 8 h later

  
  

   

1'91?

 

B At beginning [Carbon Fiber I

1:10 PA 7 h later

 

 

c 1°". '\ imam»
804 i\{>
a 60: \¢\§
t» l + \t
:3 40 \+

 

 

 

TM (1‘)

Figure 3.5. Continuous amperometric i—t responses for a (A) diamond and (B) carbon
fiber microelectrode during multiple injections of 0.1 uM NE in Krebs’ buffer, pH 7.4
before and after exposure to tissue. The measurements were made in the flow bath.
Injection volume = 3 ml. Flow rate = 1.6 mL/min. Detection potential = +0.8 V
(diamond) and +0.4 V (carbon fiber) vs. Ag/AgCl. (C) Plot of the nominal current
response as a function of time for the diamond and carbon microelectrodes.

75

The time dependence of the response attenuation is shown in Figure 3.5C. Greater and
more rapid response attenuation is seen for the carbon fiber. Even though less extensive,
the diamond microelectrode is deactivated some during exposure to the biological tissue,
particularly when the adipose tissue is present. A major difference between the two
electrode types, however, is the fact that the diamond response could be almost fully
regained after a short soak in distilled isopropyl alcohol, while the carbon fiber response
could not. This observation again indicates that weak biomolecule adsorption occurs on

the diamond surface.

Application of the diamond microelectrode for the in vitro detection of NE release
from a mesenteric artery. It is possible to directly measure endogenous NE released
from perivascular nerve fibers in real time when a microelectrode is positioned near a
blood vessel. Such NE overflow from rat tail and small mesenteric arteries has been
measured using carbon fiber microelectrodes [119, 121, 118]. In this work, a diamond
microelectrode was used for the first time to measure NE release from a mesenteric artery
after electrical stimulation. Much of the surrounding adipose tissue, in this case, was
carefully removed from the blood vessel prior to electrode insertion. This permitted
placement of the electrodes directly on the blood vessel surface in close proximity to the
perivascular nerve fibers. The tissue was bathed with warm Krebs’ buffer for 1 h prior to
the continuous amperometric measurements. Multiple diamond microelectrodes were
used in these measurements with geometric areas ranging from 5.3 to 9.0 x 10‘4 cm2.
Figure 3.6A shows a current—time profile before and after an electrical stimulation with
the electrode potential poised at 0 mV. Clearly, no NE oxidation current is detected as the

electrode potential is not positive enough to oxidize the released neurotransmitter. More

76

importantly, the electrical stimulation does not introduce noise into the electrode signal.
Figure 363 shows a current—time profile before and after an electrical stimulation event,
which reveals that the NE-release can be detected at 0.8 V; a potential that is sufficiently
positive to oxidize NE at a mass-transfer limited rate. A series of NE-release events
evoked by a train of 60 pulses at 20 Hz with a interval of 20 s between each train is
shown in Figure 3.6C.

Another series of release events with an interval of 10 min between each pulse train
is presented in Figure 3.6D. Using a series of agonists and antagonists, we have
determined that this current is associated primarily with the oxidation of NE released
from sympathetic neurons. There could also be a contribution from the oxidation of the
three major NE metabolites (norrnetanephrine, 3,4-dihydroxy-phenylethyleneglycol
(DOPEG) and vanillyl mandelic acid (VMA)) as we have observed that all three undergo
oxidation at the diamond microelectrode in the same potential range as NE.

Clearly, the diamond microelectrode exhibits excellent reproducibility for the in
vitro detection of NE. The nominal current response was 9.8 i 0.3 and 8.2 i 0.6 pA (n =
4) in Figures 3.6C and D, respectively. The response precision was good with RSD
values ranging from 3 to 7%. Figure 3.7A and B show examples of the bare diamond and
carbon fiber microelectrode response over several hours of in vitro NE release
measurements. Current time profiles for a diamond (0.8 V, Fig. 3.7A) and a carbon fiber

(0.4 V, Fig. 7B) microelectrode are presented. The calculated electrode areas were 9.0 x

10‘4 cm2 for diamond and 6.1 x 10‘4 cm2 for the carbon fiber.

77

A B

 

0mV mOmV
Bewiedstimiaim
10 A
[UH—Ina- 10 PAjw

l—-l

103 103

5min
103

Figure 3.6. In vitro continuous amperometric i—t responses for a diamond microelectrode
during measurement of NE-release from the mesenteric artery of a laboratory rat. The
electrode response at (A) 0 V after electrical stimulation of the artery and (B) +0.8 V
after electrical stimulation of the artery. The electrode response at +0.8 V after multiple
electrical stimulation events with a time interval between each of (C) 20 s and (d) 10 min.
The blood vessels were stimulated with the same pulse number and frequency (60 pulses
at 20 Hz). Flow rate = 1.6 mL/min.

78

For diamond, the nominal current response at the beginning of the measurements
was 16.9 i 0.4 pA and after 3 h the response was nearly unchanged at 16.1 i 0.5 pA (n =
3). This improved response stability, as compared to that shown in Figure 3.5, is due to
the removal of the adipose and connective tissues surrounding the artery. In contrast, the
nominal current response for the carbon fiber was 8.0 :I: 0.3 pA at the beginning of the
measurements and after 1 h decreased to 7.1 :l: 0.3 pA (n = 3). The peak-to-peak noise for
the diamond microelectrode remained constant over time (approximately 2.4—2.6 pA, Fig.
3.7(A)) while it varied somewhat for the carbon fiber ranging between 2.9 and 3.4 pA.
The response attenuation for the diamond microelectrode was 5% over 3 h and for the
carbon fiber was is 11% after just] h. The factor of 2.1 difference in nominal current for
the two microelectrode types is slightly larger than the factor of 1.5 difference in
calculated geometric area, therefore, we are probably underestimating the actual area.

An additional reason for the difference in current could be the density of neurons,
something that is variable along a blood vessel. The response stability of the two
microelectrode types over a 4 h period of in vitro use is presented in Figure 3.7C. The NE
oxidation current for the bare diamond decreased by only 8% over the period while the
NE current for the bare carbon fiber decreased by about 30%. Clearly, the diamond
microelectrode response is stable over several hours without the need for a protective

ionomer coating (e.g., NafionTM).

Diamond microelectrode response sensitivity for NE in vitro Calibration of a
microelectrode response during an in vitro electrochemical measurement is a critical

issue in neurochemical studies [131, 184].

79

 

C

100» .\e*—O\

90 \ <>
_ 8° §\§
a 7°. \t
.3 m
o\° 50‘ —o—|jamnd

« —O—Ca‘bon
40
30' . . . . .

 

 

 

 

TII'I‘B (h)
Figure 3.7. In vitro continuous amperometric i—t curves for a (A) diamond and (B)
carbon fiber microelectrode during NE-release from a mesenteric artery as a function of
time. The NE-release was evoked by electrical stimulation consisting of 60 pulses at 20
Hz. (C) Plots of the nominal current response as a function of time for the diamond and
carbon fiber microelectrodes. Flow rate = 1.6 mL/min. Detection potential = +0.8 V
(diamond) and +0.4 V (carbon fiber) vs. Ag/AgCl.

80

Calibration curves for NE were constructed and used to quantitate the amount of
NE overflow detected a mesenteric artery adventitia. Calibration curves were generated
for an electrode before and after a series of NE-release measurements [184]. Figures 3.8A
and B show continuous amperometric i-t responses for a diamond and carbon fiber
microelectrode, respectively, for different injected NE concentrations (dose—response
curves). The NE was introduced through a non-metallic syringe needle (id. 250 um,
MicroFilTM) that was fixed in position adjacent to the microelectrode at a distance of
approximately 10 pm. The injected volume was approximately 2 mL and flow rate of
Krebs’ buffer in the bath was 1.6 mL/min. Figures 3.8C and D demonstrate that both
electrode types exhibit an oxidation current that increases proportionally with the NE
concentration injected. The steady-state current for both microelectrodes varied linearly
with the NE concentration between 0.01 and 1.0 uM (r2 > 0.99, n = 3, not tested above
1.0 uM). The difference in sensitivity for the two electrodes (slopes differ by 1.5) is
attributed to differences in the electrode geometric area. Importantly, the response
sensitivity was largely unchanged by exposing the diamond microelectrode to the tissue.
The minimum detected concentration was 10 nM (SfN > 3) for the diamond

microelectrode.

3.3 Conclusion

It has been shown for the first time that the diamond microelectrode yields a
sensitive, reproducible and stable response for the catecholamine neurotransmitter,

norepinephrine, during in vitro electrochemical measurements.

81

 

  

 

 

 

 

 

    

 

1400
00 pA] -1200? f/
$1000d
20 PA1 :g eooi
l g 500: /
r T o 400' Area:5.4x10'4cm2
_.,—..—-—--—-'—’—" 200‘ Slope = 1.18 (pA/nM)
|.___. 0‘ f2=0.999
1003
0.0 0.2 0.4 0.6 0.8 1.0
Concentratiompfl)
1000
800- ,
‘2 ‘ //V
:600- , //
5 ‘ .
5400-
0 ‘ Area=6.0x10'4om2
2001 Slope = 0.78 (pA/nM)
‘ r2: .
Oi 099’

 

 

 

0T0 ' 0T2 ' 0T4 ' ofe ' 0:8 ' 1T0
Concentration (all)

 

Figure 3.8. Dose—response curves for NE at (A, C) diamond and (B, D) carbon fiber
microelectrodes. Measurements with 0.01—1.0 uM NE were made. Flow rate — 1.6

mL/rnin. Detection potential = +0.8 V (diamond) and +0.4 V (carbon fiber) vs. Ag/AgCl.

82

This new microelectrode is useful for electrochemical measurements in biological
environments, providing comparable response sensitivity and precision, and superior
response stability as compared to a carbon fiber microelectrode. The superior diamond
response stability was achieved without a protective ionomeric coating and results
because of the hydrophobic, sp3-bonded carbon surface on which weak adsorption of
polar biomolecules and contaminants occurs. Furthermore, it was demonstrated that the

diamond microelectrode response for Fe(CN)?”—4

is unaltered during exposure to the
laboratory atmosphere. It is postulated that the same surface properties which lead to the
superb diamond response precision and stability (i.e., the absence of it bonding and
surface oxides) are also the cause for the more sluggish electrode reaction kinetics for
catechols and catecholamines. Additionally, it was shown that the diamond
microelectrode, when H-terrninated, is devoid of detectable electroactive surface carbon—

oxygen functionalities and exhibits a low and stable background current that is

independent of the solution pH.

83

Chapter 4

Monitoring Endogenous Norepinephrine Release and Its Effect
on the Contractile Response of a Rat Mesenteric Artery Using
Continuous Amperometry and Video Microscopy

4.1 Introduction

Electroanalytical methods have proven useful over the years for the in vitro and in
vivo monitoring of electroactive catecholamine neurotransmitters, like dopamine (DA)
and norepinephrine (N E), in the central and peripheral nervous systems. The
microelectrode is usually 5-10 pm in diameter and must be prepared appropriately for use
in biological environments in order to maximize the response sensitivity and stability
[185, 104, 112]. For example, carbon electrode preparation can involve electrochemical
pretreatment for activation. However, while useful for improving sensitivity, such
pretreatment can introduce surface toughening, surface oxide formation and
microstructural damage [34, 141]. The magnitude of these physical and chemical changes
and the extent to which they affect the carbon electrode response for the neurotransmitter
depend on the pretreatment conditions and the initial electrode microstructure.
Pretreatment that causes such physiochemical change has the benefit of increasing the
carbon electrode response sensitivity for the catecholamine but the drawbacks of
increasing the background current and decreasing the electrode response time. Also,
protective polymer coatings, like Nafionm, can be applied to improve the response
sensitivity and selectivity for the catecholamine and to minimize fouling in the biological

environment by protecting the electrode from biomolecule adsorption [141, 104].

84

Application of the polymer coating has the drawback of increasing the electrode response
time.

Electroanalytical methods with carbon fiber microelectrodes have been used
extensively for catecholamine monitoring in the brain and the central nervous system
since the pioneering work of Gonon and Adams [131, 115, 177, 119-121, 118]. By
comparison, there have been fewer reports of in vitro or in vivo electrochemical
monitoring of neurogenic processes in the peripheral nervous system, such as the release
of NE from the sympathetic nerve terminals of isolated organs [115, 119-121, 118]. As
stated in Chapter 1, the sympathetic nervous system regulates blood pressure by
controlling the tone of muscular resistance arteries and capacitance veins. At sympathetic
neuroeffector junctions with smooth muscle cells, the electrical response is mediated by
neurotransmitter release. A triad of neurotransmitters can be released including NE, ATP
(adenosine 5’-triphosphate) and neuropeptide Y. Once released, the neurotransmitters
diffuse across the synaptic cleft, bind briefly to receptor proteins in the effector cell
membrane and, if present in an adequate amount, elicit a specific physiological response
(e.g., contraction). However, in many respects, the pathophysiological mechanisms of
neural control in arteries and veins are incompletely understood [50, 54, 118]. Long-term,
we seek a better understanding of the functional differentiation of noradrenergic
transmission in arteries and veins and how these control mechanisms are altered in
cardiovascular disease states (e. g., hypertension).

Of the neurotransmitters released from sympathetic nerves, NE is the only
compound that is easily electrooxidizable, thus, it can be monitoring electrochemically.

To date, published reports have demonstrated that electrically or chemically elicited NE

85

release can be electrochemically monitored in vitro in vasculature preparations [123, 121,
124, 118]. For example, continuous amperometry with a carbon fiber microelectrode has
been employed to record NE release, as an overflow from sympathetic nerve endings at
rat tail and mesenteric arteries [123, 121, 124, 118]. In the majority of these studies, rat
tail artery was used because of its dense sympathetic innervation that forms a two-
dimensional plexus at the external surface [120, 122, 121, 186, 124]. The carbon fiber
microelectrode used in these studies was prepared for use by either anodic polarization
[120, 122, 121, 186] or coating with NafionTM [124, 187, 118]. Using the oxidation
current or charge recorded for endogenous NE along with the effect of various vasoactive
agents, researchers have learned about factors controlling release, receptor binding and
clearance at sympathetic neuroeffector junctions. As stated above, we seek to better
understand the functional differentiation of noradrenergic transmission in arteries and
veins and how these control mechanisms are altered in hypertension.

In Chapter 3, the fabrication and electrochemical characterization of the diamond
microelectrode were described, along with initial results from its application in
electroanalytical measurements in biological tissue [155, 159, 157]. This new carbon
electrode provides a greater level of response (i.e., oxidation) sensitivity, reproduciblility
and stability than does a bare carbon fiber for monitoring exogenous and endogeous NE
in tissue [159, 157]. This new electrode has three characteristics beneficial for in vitro
electrochemical measurement: (i) conventional pretreatment is not required to prepare the
electrode for use, (ii) the electrode surface is relatively oxygen-free so that the
background voltammetric current is low and independent of the solution pH, and there

are no features present associated with redox-active carbon-oxygen functional groups,

86

and (iii) a high level of response sensitivity and stability are achieved without the use of a
protective polymer film, like NafionTM.

In this Chapter, we report on the combined use of in vitro continuous amperometry
with a diamond microelectrode and video microscopy to simultaneously measure
endogenous NE released from sympathetic nerves innervating a rat mesenteric artery and
the resulting contractile response. The NE released was measured as an overflow at blood
vessel surface. These two techniques, along with various vasoactive agents, were
employed to study the functional relationship between the oxidation current associated
with endogenous NE released at neuroeffector junctions near the electrode and the extent

of arterial constriction from sympathetic nerve fibers.

4.2 Results and Discussion

Simultaneous recording of NE release and vascular constriction evoked by electrical
stimulation. Figure 4.1A is a video micrograph showing the placement of the diamond
microelectrode (right) and the bipolar stimulator electrode (left) at the surface of a
mesenteric artery. The separation between the two was maintained at about 200 pm. The
artery is visible in the image and has a rest diameter of 228 um. Figure 4.1B is a video
micrograph of the same artery after electrical stimulation, which shows the contracted
vessel with a diameter decrease of about 28%. The NE that is released can be oxidatively
detected according to the redox reaction shown in Figure 4.1C. The contractile (top) and
NE oxidation current (bottom) response transients to a 20 Hz stimulus are presented in
Figure 4.1D. Maximal responses were found for this frequency, as discussed later in this

Chapter.

87

 

 

 

 

 

 

 

 

 

H OH
HO NH? O\ NH»
—> + 2HJr + 26'
HO 0
NE NE-o-quinone
Tl 1‘ TC Era TR ‘1
Constriction
. Tso
A
IIIIIX
Current
W T50
Tr TD 7'

Figure 4.1. Video micrographs showing a mesenteric artery (A) before and (B) after a 20
Hz electrical stimulation (60 pulses with a 0.3 ms pulse width). The bipolar stimulator
electrode and the diamond microelectrode, both positioned at the artery surface, are
evident in (A). The NE oxidation reaction mechanism is shown in (C). Characteristic
contractile (top) and NE oxidation current (bottom) response transients in response to a
20 Hz stimulation are presented in (D). Detection potential = 800 mV. Flow rate = 1.6
mL/min. Several numerical parameters are obtained from these curves including: the
maximum oxidation current, 1",“; the current rise time, T,; the time required for current
decay to 50% of the maximum, T50; the time required for full current decay, TD; the
percent constriction, Co/,; the time required to reach full constriction, Tc; the time required
for full vessel relaxation, TR; and the latency period prior to the onset of constriction, TL.

88

The oxidation current recorded with the diamond microelectrode poised at a
detection potential of 800 mV rapidly increased after application of the stimulus. In our
methodology, the oxidation current is a measure of the concentration of endogenous NE
released at multiple varicosities in the vicinity of the microelectrode. The measurement
does not provide a direct measure of NE from an individual release site, rather it provides
a temporal profile of the nerve stimulation-induced rise in NE levels at the surface of the
artery. The magnitude of the measured current (i.e., NE flux) depends on a balance
between the amount released from the nearby varicosities and diffusion from the
junctional cleft to the microelectrode, and clearance by neuronal reuptake and
extracellular enzymatic degradation. The time from the current onset to the point the
maximum is reached is described by the rise time, T,. The latency in the onset of the
current after application of the stimulus was less than 30 ms. After reaching a maximum,
the current decreased slowly over time back to the prestimulation level due to a gradually
reduced flux of NE to the electrode. The decrease in current is characterized by a delay
time, TD, which in this case was about 30 s. The decreased current reflects a reduction in
the NE concentration at the artery surface caused by a combination of diffusion away and
junctional clearance mechanisms. The contractile response follows similar temporal
dynamics consistent with there being a connection between the oxidation current
magnitude and the extent of vasoconstriction.

After release, NE diffuses across the junction and binds to al-adrenoreceptors on
vascular smooth muscle cells eliciting the contractile response. As it is cleared from the
junction, less and less NE is available to activate the 011 receptors, the smooth muscle

cells relax and the artery dilates. As with all neuroeffector junctions, the spatio-temporal

89

dynamics of neurotransmitter action at sympathetic varicosities are influenced by
diffusion, uptake, enzymatic degradation and receptor desensitization.

Noteworthy is the fact that the onset of the oxidation current appears ca. 300 ms
earlier than the onset of constriction after stimulation at 20 Hz.This observation is similar
to that of Gonon and coworkers who reported that the onset of the oxidation current
occurs 10x faster than the onset of arterial constriction [115]. A cause for this may be the
longer time required for NE to diffuse into the smooth muscle cell layers and reach a
concentration necessary to elicit the contractile response.

As mentioned earlier, the constriction did not influence the magnitude of the
oxidation current. This was confirmed by simultaneously recording the oxidation current
and the evoked contractile response with and without added pyridoxal-phosphate-6-
azophenyl-2’,4’-disulfonic acid (PPADS, 10 uM). PPADS is a P2X receptor antagonist
that blocks the constrictor effects of ATP released from sympathetic nerves. ATP and NE
are both released from sympathetic nerves at mesenteric arteries but ATP is the dominant
vasoconstrictor, particularly at low stimulation frequencies. Blocking the P2X receptor
largely abolished constriction under the electrical stimulation conditions used in this
work. The oxidation current measured for NE release in the presence of PPADS was 12.1

i 1.7 pA and was unchanged at 12.2 i 1.0 pA in the absence of the drug.

Potential dependence of the NE oxidation current. Confirmation that the electrically
evoked oxidation current reflects endogenous NE is provided by the following data.
Figure 4.2A shows an in vitro hydrodynamic voltammetric i-E curve for the oxidation of
NE released from sympathetic nerves innervating a mesenteric artery. The oxidation

current was measured as a function of the electrode potential using a 20 Hz electrical

90

stimulation. The oxidation current begins to increase at ca. 300 mV and reaches a limiting
value at 800 mV. The half-wave potential, Em, is ca. 500 mV and the limiting current,
itim, is 12.5 pA. Based on this curve and others like it, a detection potential of 800 mV
was selected for the in vitro continuous amperometric recordings described herein.
Evidence that the oxidation current results from endogenous NE is the fact that this E1 ,2 is
identical to that for a standard solution of NE. Contractile (top) and oxidation current
(bottom) response transients for a mesenteric artery electrically stimulated at 20 Hz are
shown for a detection potential of (B) 0 and (C) 800 mV. A reproducible contractile
response was observed for both stimuli but oxidation current was only detected when the
electrode potential was poised at 800 mV; a potential at which NE is oxidized at a mass
transfer limited rate. It should be noted that E1 /2 for NE at diamond is some 200-400 mV
more positive than the value for a carbon fiber microelectrode [155, 159].

This positive shift reflects more sluggish electrode reaction kinetics; a characteristic
of diamond for this class of redox molecules. Importantly, this shift is not due to ohmic
resistance (iR loss). It is supposed that the more sluggish kinetics arise because of the
absence of strong molecular adsorption on the H-terrninated sp3-bonded surface [182,
155, 159]. McCreery and coworkers have studied the relationship between the adsorption
of catechols and the observed electron-transfer kinetics on glassy carbon, and concluded
that an adsorbed layer acts as an electrocatalyst for solution phase redox components
[182]. This is explained in Chapter 3. A mechanism of “self-catalysis” was proposed.
Adsorption apparently lowers the activation barrier for the reaction leading to an increase

in the overall redox reaction rate.

91

>

.3
- «h

 

A
'9

  
   
 

.3
-93-

Current (pA)
'9 - «P - 9’ 9°

 

 

 

B C
0 mV 800 mV

l98um W.
25ml] f“
10pA W 10mm
H
53

lng

stimulation

Figure 4.2. (A) An in vitro hydrodynamic voltammetric i-E curve recorded for NE
released from the sympathetic nerves innervating a mesenteric artery. Contractile (top)
and NE oxidation current (bottom) response transients for a mesenteric artery in response
to a 20 Hz stimulation (60 pulses and a 0.3 ms pulse width) at detection potentials of (B)
0 and (C) 800 mV. The oxidation current was measured with a diamond microelectrode.
Flow rate =1.6 mL/min of Krebs' buffer.

92

Effect of TTX on NE release and vascular constriction. Additional evidence that the
oxidation current arises from endogenous NE and that the constriction is neurogenically
mediated came from studies using various vasoactive substances. The sodium channel
antagonist, tetrodotoxin (TTX), was first tested at stimulation frequencies from 1 to 20
Hz. This potent neurotoxin blocks action potentials in nerves by binding to voltage-gated
sodium channels in the cell membrane, thereby inhibiting neurotransmitter release and
vascular constriction. It is routinely used to verify that changes in vascular tone are
neurogenically mediated [115, 186]. Figure 4.3A-C shows contractile response (top) and
oxidation current (bottom) transients recorded in response to a 20 Hz stimulation before,
after bathing the tissue with TTX, and after washing out the drug. Prior to adding the
TTX, reproducible contractile responses and oxidation currents were seen (Figure 4.3A).
The nominal maximum current and percent constriction were 17.3 :t 0.2 pA and 29.7 i
0.4 % (n = 3), respectively. Clearly, the response precision for both was good. The
latency, TL, in the onset of the oxidation current and the constriction after application of
the stimulus was 40 and 500 ms, respectively (about a factor of 10 difference). After the
addition of TTX (0.3 uM) for 5 min, no oxidation current or vasoconstriction was seen
(Figure 4.3B). There was some constriction evident after the 3rd and 4th stimulation events
because the process of washing out the TTX from the tissue had begun. After completely
washing out the TTX, though, the elicited oxidation current and contractile response were
fully recovered (Figure 4.3C). The results indicate that the contractile response of the
mesenteric artery is neurogenically mediated and that NE release is a sodium-dependent

process.

93

 

 

25 um]:

 

Figure 4.3. (A) Typical contractile and NE oxidation current response transients for a
mesenteric artery in response to a 20 Hz stimulation. Contractile and NE oxidation
current response transients (B) in the presence of TTX (0.3 11M) and (C) after washing
out the drug. The dotted circles indicate the time the electrical stimulation was applied.
The oxidation current was measured with a diamond microelectrode poised at 800 mV.
Flow rate = 1.6 lemin of Krebs’ buffer.

94

Effect of yohimbine on NE release and vasoconstriction. Nerves that communicate
using NE have prejunctional a2-adrenergic autoreceptors that regulate release of the
neurotransmitter when activated during electrical stimulation [100, 85, 68]. The receptor
antagonist, yohimbine, was tested to learn its effect on the oxidation current and the
dynamics of vascular tone [115, 119, 186]. It was hypothesized that blocking this
autoreceptor would cause an increase in NE overflow at the neuroeffector junction in
response to an electrical stimulus. As a consequence, a larger oxidation current was
expected. Furthermore, it was hypothesized that the increased junctional NE would
increase the magnitude of the contractile response, particularly at low stimulation
frequencies. It was previously shown in tissue over-flow measurements that yohimbine
(1.0 uM) increases the amount of NE released from the sympathetic nerves innervating a
rat mesenteric artery [100, 85, 68]. Other researchers, like Brook and coworkers, have
used (la-adrenergic receptor antagonists, like yohimbine and idazoxan, to study
noradrenergic transmission in arteries and electrochemically measured increased NE
oxidation currents [115, 119, 121, 186, 124]. Figure 4.4 shows contractile response (top)
and oxidation current (bottom) transients elicited by 3 and 20 Hz stimulations with (A)
being the control data set and (B) being the data set recorded with yohimbine. In the
presence or absence of yohimbine, both the oxidation current and contractile response
magnitudes were greatest for the 20 Hz stimulation. In fact, the oxidation current in
response to the 3 Hz stimulation, particularly in the absence of the drug, was difficult to
resolve from the background. However, in both cases, a broad contractile response was
seen. As expected, both the oxidation current and the contractile response magnitudes

increased in the presence of yohimbine.

95

 

 

 

 

 

 

 

 

 

 

 

 

254 —o—W 40 _.—W
. -o—Yotirrflm(1uM) 30] —o—Youttum(1ttm)
s . / § 3 l /i t/
:15‘ / 5 m.
3 030/ 0/ ° 10 0+
5. ./’ 32 /
.o/ ‘9
0 5 10 15 20 0 5 10 15 20
Well) Fmrcya-Iz)

Figure 4.4. Effect of yohimbine (1.0 M) on the contractile (top) and NE oxidation
current (bottom) response transients recorded at a mesenteric artery. The transients were
elicited by electrical stimulation at 20 and 3 Hz (60 pulses and a 0.3 ms pulse width) (A)
without and (B) with added yohimbine. The dotted circles indicate the time the 3 Hz
electrical stimulation was applied. Plots of the oxidation current for NE, with and
without (i.e., control) added yohimbine, versus the stimulation frequency are shown in
(C). Plots of the contractile response, with and without added yohimbine, versus the
stimulation frequency are shown in (D). The data are presented as mean values with the
bars reflecting the standard error of the mean. The oxidation current was measured with a
diamond microelectrode poised at 800 mV. Flow rate = 1.6 mL/min of Krebs' buffer.

96

This trend is more clearly revealed in the plots presented in Figures 4.4C and D. An
increase in the stimulation frequency up to about 10 Hz resulted in an increase in both the
NE oxidation current and the percent constriction. More oxidation current was recorded
at higher stimulation frequencies because each stimulus during a pulse train causes NE
release. Given the fact there is less time for clearance between each pulse, a sustained
increase in the oxidation current results because the signals evoked by each successive
stimulus sum with the junctional NE remaining from the previous ones. The contractile
response curve in the presence of yohimbine is left-shifted from the curve without the
drug at frequencies up to about 10 Hz, which reflects the presence of greater junctional
levels of NE. Above 10 Hz, the contractile response became constant even though more
junctional NE was present. Under these conditions, there is simply more than enough NE
present at the surface of the effector cells to activate all of the Oil-adrenergic receptors,
hence the unchanging contractile response.

Table 4.1 presents a summary of the oxidation current and contractile response data
for the 20 Hz stimulation, with and without yohimbine. As noted before, both the
oxidation current and contractile responses were quite reproducible. In the presence of
yohimbine, the nominal oxidation current increased by 74% from 14.8 to 25.8 pA but
there was not much difference in the rise time, T,, or the half-life, T50, of the oxidation
current. The decay time, TD, was lengthened as more time was required to clear the high
junctional concentration. Within statistical variance, the contractile response, Co/,, was
about the same with and without the drug, as were the latency period, TL, and the
recovery time, TR. What did increase with yohimbine was the constriction time, Tc, from

a nominal value of 5.32 to 6.48 s, and the half-life, T50, from 6.94 to 11.11 s.

97

Table 4.1. Numerical parameters measured from the oxidation current and contractile
response transients recorded in the presence and absence of yohimbine (1.0 pM).

 

 

 

20 Hz Control (n = 3) Yohimbine (n = 3)

NE release (1mm pA) 14.8 i 0.2 25.8 i 03*—
Rise Time (T,, s) 2.78 i 0.05 2.86 i 0.03
Half-life (T50, s) 5.43 i 0.16 4.46 i 0.28

Decay Time (TD, 3) 21.28 ;|: 0.95 26.99 i 1.72*
Constriction (Co/,) 33.58 :t 1.27 29.92 i 1.53
Latency (TL, ms) 420 j: 12 490 i 51

Constriction Time (Tc, 3) 5.32 i 0.24 6.48 i 0.31
Half-life (T50, s) 6.94 i 0.43 11.11 i 0.81 *
Relaxation Time (TR, 3) 30.0 :I: 3.9 31.07 i 2.98

 

The oxidation current and contractile response were elicited by electrical stimulation at
20 Hz. Data shown are means i SEM. The values were compared by paired t-tests. *P <
0.05.

Even though TR was unchanged, the dynamics of full vessel relaxation were altered
by yohimbine. The larger Tc and T50 values were caused by the higher junctional NE

concentration and the longer time required for clearance. These results further indicate

that the oxidation current measured resulted from endogenous NE.

Effect of cocaine on NE clearance and vasoconstriction. In another series of
measurements, the NE reuptake inhibitor, cocaine, was added and its effect on the evoked
oxidation current and contractile response was investigated [115, 119, 120]. Reuptake of
NE back into the nerve terminal by the norepinephrine transporter protein (NET) is

inhibited by cocaine. This reduces the clearance rate of junctional NE. It was

98

hypothesized that its presence would cause an increase in the NE oxidation current and a
greater contractile response.

Figures 4.5A and B show oxidation current and the contractile response transients
for 3 and 20 Hz stimulations, with and without added cocaine. For both stimulation
frequencies, the oxidation current increased in the presence of cocaine with a larger
increase seen for the lower frequency. The contractile response at 20 Hz was unaffected
by the presence of cocaine while the response at 3 Hz was significantly increased. It is
supposed that this results because the junctional concentration of NE at the higher
frequency is sufficient to saturate the nearby smooth muscle cell receptor sites.
Interestingly, the increased contractile response at 3 Hz has a biphasic character to it.
Sufficient data are not in-hand at present to confirm the origin of this transient response.
Figures 4.5C and D show plots of the oxidation current and the contractile responses as a
function of the stimulation frequency. At all frequencies, the oxidation current was larger
in the presence of cocaine. At frequencies less than 10 Hz, a point at which nearby
receptor site saturation occurs, increased constriction was seen in the presence of the drug
as evidenced by the leftward shift in the response curve.

Table 4.2 presents a summary of the oxidation current and contractile response
transient data for the 3 and 20 Hz stimulations, with and without added cocaine. In the
presence of cocaine, the nominal oxidation current, imax, increased by 23% from 15.1 to
18.6 pA at 20 Hz and by 222% fi'om 5.06 to 16.3 pA at 3 Hz. Minor changes in T50 were
seen for the 20 Hz stimulation; however, TD increased significantly in the presence of

cocaine from 16.3 to 27.2 3 due to the lower rate of clearance.

99

 

Cocaine

 

25-

8

Current (pA)
a a:

DUI

«monitor
-o- Cocaine (10 an)

¢\¢

% Constriction

 

 

Figure 4.5. Effect of cocaine (10 uM) on the NE oxidation current (left) and contractile
(right) response transients recorded at a mesenteric artery. The transients were elicited by
electrical stimulation at (A) 20 and (B) 3 Hz (60 pulse and a 0.3 ms pulse width). Plots
of the oxidation current for NE, with and without (i.e., control) added cocaine, versus the
stimulation frequency are shown in (C). Plots of the contractile response, with and
without added cocaine, versus the stimulation frequency are shown in (D). The data are
presented as mean values with the bars reflecting the standard error of the mean. The
oxidation current was measured with a diamond microelectrode poised at 800 mV. Flow

 

//<>/
/ /r
O ,0/9
../."
0 '5 1'0 ' 1'5 I 20
Mll'k)

rate = 1.6 mL/min of Krebs' buffer.

100

 

 

—o-oonuor
—o— Coedne(10 uM)

 

 

o/ O/
04/
/

i

 

 

0

1'0 ' 1'5 '
WM)

5

2'0

 

For the 3 Hz stimulation, significant increases in both T50 and TD were seen; the latter by
400 % from 4.1 to 20.6 s. The contractile response increased by 115% from 11.6 to

24.9% in the presence of the drug.

Table 4.2. Ntunerical parameters measured from the oxidation current and contractile
response transients recorded in the presence and absence of cocaine (10 uM).

 

 

 

 

20 Hz Control (n = 3) Cocaine (n = 3)
NE release (ImaX, pA) 15.1 :t 0.2 18.6 i 0.2*
Half-life (T50, s) 5.08 i 0.10 6.60 i- 0.44
Decay Time (TD, 3) 16.3 i 0.3 27.2 i 2.2*
Constriction (Co/,) 33.9 :t 0.5 34.2 i- 0.5 %
3 Hz Control (n = 3) Cocaine (n = 3)
NE release (Imam, pA) 5.06 i: 0.2 16.3 i 0.4*
Half-life (T50, s) 1.09 i 0.16 5.10 i 0.37*
DecayTime (TD, 3) 4.1 -l_- 0.8 20.6 i 1.0*
Constriction (C%) 11.6 i 0.4 24.9 i 0.6 *

 

The oxidation current and contractile response were elicited by electrical stimulation at
20 and 3 Hz. Data shown are means i SEM. The values were compared by paired t-
tests. *P < 0.05.

These trends are all consistent with a reduced rate of junctional clearance. These

results further confirm the oxidation current is caused by endogenous NE and that the

contractile response is mediated to a significant extent by NE.

NE overflow and vascular constriction as a function of the stimulation conditions.
Since diamond is a new type of electrode for neurochemical studies, the electrode
response for NE was investigated as a function of the stimulation frequency and pulse
number. In blood vessels with only a few smooth muscle cell layers, all the cells are all in

close proximity to the nerve terminals. This condition produces a strong neurogenic

101

response during electrical stimulation [188, 189]. The frequency dependence of the
oxidation current and the contractile responses are presented in Figure 4.6. Increasing the
stimulation frequency from 3 to 20 Hz (60 pulse sequence with a 0.3 ms pulse width)
caused a proportional increase in both the NE oxidation current and contractile response.
Figure 4.6A displays contractile (top) and oxidation current (bottom) response transients
in response to several low frequency stimulations. Clearly, the oxidation current increases
with increasing frequency, reflecting a greater junctional NE concentration. The
increased levels of junctional NE elicit increased vascular constriction.

Figure 4.6B presents plots of the NE oxidation current and the percent constriction
as a function of the stimulation frequency up to 60 Hz. Both the NE oxidation current and
the percent constriction track one another and pass through a maximum at 20 Hz. It was
based on these results that measurements with the drugs (vide supra) were performed
using a 20 Hz stimulation. However, the oxidation current and constriction both decline
at higher stimulation frequencies. There are several dynamic events in nerve signaling
processes including (i) storage and release of the neurotransmitter from the neuron, (ii)
interaction of the neurotransmitter with the receptor sites on the vascular smooth muscle
cells and subsequent production of the post-junctional response, (iii) clearance of the
neurotransmitter from the junction by diffusion, reuptake and metabolism and (iv)
packaging NE in intemeuronal storage vesicles for release. One possible explanation for
the decrease in oxidation current and contractile response with increasing stimulation
frequency is rapid depletion of a readily releasable pool of NE-containing synaptic

vesicles.

102

A 3 5 7 10 20 Hz

{W5 [5 pA

 

   

 

18 4o

 

 

 

16: -o—IEOdddlonQnart _
A , —.—<W c
«12 ’ .g
C . / l on
it \ at
° 8‘ Xi °
. L $
6.
4; L10
21
0 80 4'0 60 80 100100100

Wall-I!)

Figure 4.6. Effect of pulse frequency (3, 5, 7, 10 and 20 Hz, all at 60 pulses with a 0.3
msec pulse width) on the contractile (top) and NE oxidation current (bottom) response
transients recorded at a mesenteric artery (A). Plots of the NE oxidation current and
contractile response versus the stimulation frequency are shown in (B). The data are
presented as mean values with the bars reflecting the standard error of the mean. The
oxidation current was measured with a diamond microelectrode poised at 800 mV. Flow
rate = 1.6 mL/min of Krebs' buffer.

103

In other words, both responses decline in magnitude because the stimulation frequencies
exceed the rate at which the stores of releasable NE can be replenished. A similar
frequency dependence of the excitatory junction currents (EJCs) for ATP-release has
been reported for sympathetic nerves innervating a rat tail artery [186].

Taken together, it would appear that the junctional levels of NE (and ATP) can be
depleted during trains of high frequency stimulation (>20 Hz). While this is one plausible
explanation, it is important to recognize that several other factors can influence the
frequency-response characteristics of signaling at a neuroeffector junction, such as the
distance between the nerve ending and the smooth muscle cells and the density of
innervation (e.g., neuroeffector junctions per length of vessel). Interestingly, not only did
the oxidation current and contractile response increase with increasing frequency up to
about 20 Hz, but the temporal shape of both responses was altered as well. This was most
notable for the low frequency stimulations (1, 3 and 7 Hz) for which multiple maxima are
seen in the transient responses. An example of this is presented for the 3 Hz stimulation
(dotted circle) in Figure 4.6A. Again, we do not have sufficient data at present to fully
explain these trends.

Figures 4.7A and B show the effect of the stimulation pulse number, at 20 Hz, on
the oxidation current and contractile responses. Figure 4.7A shows contractile response
(top) and oxidation current (bottom) transients as a function of the pulse number from 12
to 120 using a 4 min interval between each sequence. Measurable effects (SN 2 3) were
observed only after at least 3 pulses at 20 Hz. In other words, the NE released by

individual pulses was too low to be detected in our present arrangement.

104

12

30 60

I20 pm

90 120

 

2 min

 

 

 

 

25 50
1 —areouanauanut _
A20. -O-W _40=
< , ¢/<} _ .9.
5 / ‘6
#515- O/¢ 50%

o .//
t ‘ Q 0/’\ /9 ’ 5
310- );0/ i 202
. , . a-
5- ~10
010T 50 1 100 ' 1' ' 208
Edsanwflmr2%

Figure 4.7. Effect of pulse number (12, 30, 60, 90 and 120, all at 20 Hz) on the
contractile (top) and NE oxidation current (bottom) response transients recorded at a
mesenteric artery (A). Plots of the NE oxidation current and contractile response versus
the stimulation frequency are shown in (B). The data are presented as mean values with
the bars reflecting the standard error of the mean. The oxidation current was measured
with a diamond microelectrode poised at 800 mV. Flow rate = 1.6 mL/min of Krebs’

buffer.

105

Both the percent constriction and oxidation current increase with pulse number, but only
up to a point. Figure 4.78 shows the corresponding plots of these data. The oxidation
current increases with pulse number up to about 60. Beyond this, the increase in current
with increase in pulse number becomes less. On the other hand, the maximum contractile
response is reached after 12 pulses.

Unlike the current, the constriction does reach a constant value at higher pulse
numbers. It is supposed that this is caused by saturation of all the available smooth

muscle cell receptor sites being activated by NE.

Quantitative Determination of NE Release. Even though these measurements reflect
the concentration of NE at the artery surface and not the concentration released at an
individual neuroeffector junction, it is still important to know the concentration
corresponding to the measured current. Calibration of a microelectrode during in vitro
electroanalytical measurements is a critical issue in neurochemical studies [104, 190,
112]. Calibration curves for NE were constructed and used to determine the concentration
detected at the artery surface in response to the electrical stimulation. At the beginning
and the end of a series of in vitro measurements (ca. 4 h), the electrode, while remaining
in the tissue, was exposed to different injected concentrations of NE. The concentrations
were introduced through a nonmetallic (a combination of plastic and fused silica) syringe
needle (id. 250 um, MicroFilTM) positioned adjacent to the microelectrode at a distance
of about 10 um. Buffer was flowing continuously through the organ bath at 1.6 mL/min
during the measurement. This was the same flow rate used in the in vitro tissue
measurements described vide supra. A delay time of 1 min was used between injections.

The steady-state oxidation current was found to increase linearly with the injected NE

106

concentration between 10 and 100 nM (12> 0.999, n = 3). The electrode response to
higher concentrations of NE was not examined. The minimum concenuation detectable at
a S/N 2 3 was 10 nM. Importantly, only a slight decrease of 6% in the electrode response
sensitivity was observed after a 4 h period of continuous use in tissue. Using this
response curve, it was determined that the nominal oxidation current measured in
response to a 20 Hz stimulation corresponded to an NE concentration at the artery surface
of between 10 and 30 nM. The concentration detected depended on the particular artery.
When the nerves were stimulated continuously to achieve a steady-state level of
junctional NE, the concentration determined was approximately 70 nM. This value is in
good agreement with detected concentrations reported by Gonon et al. for the densely
innervated rat tail artery using an anodized carbon fiber [115, 119, 186]. Differences are
expected from artery to artery, and even with position along an artery, because the local
NE concentration at the electrode surface depends on the distance between
microelectrode and varicosities, density of varicosities in the vicinity of the
microelectrode, and the concentration of NE released at each varicosity after the

stimulation [77, 177].

4.3 Conclusion

In vitro continuous amperometry with a diamond microelectrode and video
imaging were used to simultaneously record NE released from sympathetic nerves at the
surface of a mesenteric artery and the evoked contractile response. Electrical stimulation
of the innervating sympathetic nerves elicited a transient oxidation current, as measured

with the microelectrode, that was attributed to endogenous NE. Several observations

107

support the conclusion that NE was the species being oxidatively detected and that the
contractile response was neurogenically mediated. First, the hydrodynamic voltammetric
E1 /2 for the endogenous NE was identical to that for a standard solution of the
neurotransmitter. Second, the evoked oxidation current exhibited the expected
characteristics of neurogenically-controlled release, that is, it was abolished by TTX.
Third, the oxidation current increased as did the contractile response, particularly at low
stimulation frequency, in the presence of the 012-adrenoreceptor antagonist, yohimbine.
Fourth, the oxidation current increased, the clearance rate decreased and the contractile
response increased at low frequency in the presence of the NE reuptake inhibitor,
cocaine. The methodology reported on herein represents an advancement for studies of
synaptic physiology in monoaminergic systems. The uncoated diamond microelectrode
provided a sensitive, reproducible and stable oxidation current response that varied with
the stimulation frequency and pulse number in a predictable manner, similar to that for a

carbon fiber microelectrode.

108

Chapter 5

Differences in Sympathetic Neuroeffector Transmission to Rat
Mesenteric Arteries and Veins as Probed by In Vitro
Continuous Amperometry and Video Imaging

5.1 Introduction

Veins in the splanchnic circulation, make important contributions to the regulation
of overall hemodynamics including blood pressure [191, 192]. Sympathetic nerve activity
plays a prominent role in control of the capacitance function of veins and the resistance
function of arteries. Veins differ significantly from arteries in the properties of their
contractile and electrical responses to sympathetic nerve stimulation or to drugs that
mimic the activity of sympathetic nerves [22, 193, 85]. One reason for these differences
is that arteries and veins may be innervated by different sympathetic neurons and these
neurons may communicate using different vasoconstrictor neurotransmitters. Adenosine
5’-triphosphate (ATP) and norepinephrine (N E) are the vasoconstrictor transmitters
released by sympathetic nerves supplying small (5 200 um) mesenteric arteries while NE
is the vasoconstrictor released by peri-venous nerves [193, 194, 50, 53]. In addition, the
dynamics of sympathetic neuroeffector transmission to arteries and veins may also differ
because the dynamics of vasomotor responses in arteries and veins are different. Arterial
diameter controls the delivery of oxygenated blood to tissues and, therefore, arterial
sympathetic neuroeffector transmission should permit moment-to-moment control of
blood pressure and flow to tissues [195]. Alternatively, veins are a reservoir of blood and

shifting blood from the veins to arteries in response to orthostatic adjustments would

109

require rapid and sustained activation of venous sympathetic neuroeffector transmission
[196].

Advantage was taken of the excellent time and spatial resolution provided by
continuous amperometry to monitor NE oxidation currents subsequent to nerve
stimulation in isolated rat mesenteric artery (MA) and vein (MV) preparations in vitro. A
carbon fiber was used in this work rather than a diamond microelectrode because it was
found that several of the drugs employed to study the pathophysiology were oxidized at
the positive detection potential needed to detect NE at the diamond. Detailed studies of
the dynamics of NE release in veins have not been reported in the literature because, at
least in part, it is a low pressure system that does not determine total peripheral resistance
and, thus, has been viewed as largely unimportant in acute or chronic changes in blood
pressure. Testing of the hypothesis that there are fundamental differences in sympathetic
neural control in mesenteric arteries (MA) and veins (MV) from a normotensive rat is
reported on herein. These studies also made use of video imaging, that permitted
simultaneous measurement of blood vessel diameter changes in response to nerve
stimulation. This allowed for a comparison of the relationship between NE release and

vasomotor dynamics in MA and MV.

5.2 Results

Distribution of sympathetic nerve fibers associated with MA and MV. Glyoxylic acid
(GA) converts NE into an intensely fluorescent 2-carboxymethyl-dihydroisoquinoline

derivative [197]. Therefore, the distribution of GA-induced fluorescence of neuronal

110

stores of NE was used to assess the disposition of periarterial and perivenous sympathetic
nerves.

Figures 5.1A and B show GA-induced fluorescence that revealed a network of
varicose nerve fibers around MA and MV. The periarterial nerve plexus consisted of
bundles of several axons arranged in a net-like manner. The perivenous plexus was less
dense than in arteries and consisted of individual varicose axons largely oriented
circumferentially around the vessel. hnages for both are shown in Figures 5.1C and D.
The difference in the arrangement of periarterial and perivenous sympathetic nerves was
quantitated by counting the number of nerve fibers crossing horizontal and vertical grids
superimposed on an image at a 400 X magnification. The number of vertical and
horizontal crossing nerve fibers was similar in MA (vertical = 9.7 i 1.2, horizontal = 9.6
i 1.2, n = 4 rats), whereas there were fewer vertical than horizontal nerve fiber crossings
in MV (vertical = 3.6 i 0.4, horizontal = 6.8 i 0.9, n = 4; P < 0.05). In addition, the
number of vertical crossings in MV was significantly less than the number in MA (P <

0.05).

In Vitro NE overflow and contractile responses in MA and MV. The previous study
revealed that an electrode potential of 400 mV is sufficient to oxidize NE at a carbon
fiber at a mass-transfer limited rate. Furthermore, the oxidation current measured at this
potential reflects the concentration of endogenous NE released from sympathetic
varicosities near the electrode tip [159]. A detection potential of 400 mV was used for all
measurements reported herein.

Preparations in which the electrically evoked constriction or oxidation current were

not blocked by tetrodotoxin (TTX) were discarded.

111

 

Figure 5.1. Glyoxylic acid-induced fluorescence of catecholamines in peri-vascular
sympathetic nerves. A and B show low magnification (100 X) images of the sympathetic
nerve plexus while C and D show high magnification images (400 X). The nerve plexus
in arteries has a mesh-like arrangement and nerve fibers are oriented in both the
longitudinal and circular axis of the blood vessel (C). The plexus in veins has a selective
circular arrangement with very few fibers oriented in the long-axis of the blood vessel
(D).

112

The oxidation current and vascular constriction require activation of TTX-sensitive
sodium channels and, therefore, these responses are neurogenically mediated. The
anatomical data presented above indicated that there is higher nerve density in MA
compared to MV. In all work reported on herein, several locations on a blood vessel were
probed for NE overflow in order to find a location of maximum current. Sites yielding the
largest oxidation current were then chosen for the comparative studies of NE overflow
from perivenous and periarterial nerves. It was not possible to reliably detect NE
overflow at arteries or veins in response to a single electrical stimuli. Rather, short trains
(60 pulses) of stimuli were needed to produce a frequency dependent (0.2 — 30 Hz) and
reliably detectable NE oxidation current.

The same trains of stimuli also produced frequency-dependent constrictions of MA
and MV. NE overflow, as evidenced by the oxidation current, and the evoked contractile
response were detected at a lower threshold frequency in MV (0.5 and 0.2 Hz,
respectively) than in MA (1 and 0.5 Hz, respectively). Figures 5.2A and B display
representative recordings of constriction (top) and NE oxidation current (bottom)
response transients from MA and MV at stimulation frequencies of 3 and 20 Hz. It can be
seen that at 20 Hz, a constriction of ca. 25% occurs in both vessels. The vessel constricts
rapidly in response to the stimulation followed by a slow relaxation to the rest diameter.
However, at 3 Hz, the range in arterial constriction was hardly detectable while a broad
contractile response was seen for the vein at 3 Hz. For both MA and MV, an intense
oxidation current is seen with maximum currents of 12.7 and 23.8 pA, respectively at 20
Hz. The oxidation current elicited by the 3 Hz stimulation is broad but detectable. The

current magnitude was larger for MV than for MA, 5.1 and 9.8 pA, respectively.

113

 

 

     

 

 

 

 

 

 

 

 

Artery Vein
3 Hz 20 Hz 3 Hz 20 112
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Figure 5.2. Contractile (top) and NE oxidation current (bottom) response transients for
(A) MA and (B) MV elicited by a 3 and 20 Hz stimulation. Plots of the (C) NE overflow
current and (D) constriction for MA and MV as a function of the stimulation frequency.
*Significantly different from MA and MV (P < 0.05). Data are mean i S.E.M.

114

Figures 5.2C and D contain summary plots for the oxidation current and contractile
response magnitudes for MA and MV. These data demonstrate a frequency-dependent
(0.2-30 Hz) increase in the NE overflow and constriction for both MA and MV. These
data also reveal that NE oxidation currents were greater for MV than for MA and that the
frequency-response curve for nerve stimulation-induced constriction in MV was shifted
to the left of the curve for MA (Figure 5.2D). The half maximum stimulation frequency
(S 50) of the constriction was 1.9 i 0.2 Hz in MV, this value that was significantly lower

(P < 0.05) than that for MA (4.8 i 0.3 Hz) (Figure 5.2D). The maximum constriction

(Ema) was similar in MV (28 i 2%) and MA (31 i 3%).

Quantitative detection of NE overflow. The carbon fiber microelectrodes used for
recording NE overflow were calibrated by generating a response curve using a series of
standard NE solutions. The NE solution was introduced through a nonmetallic
(combination of plastic and fused silica) needle (id. 250 um, MicroFilTM) that was fixed
in position adjacent to the microelectrode at a distance of ca. 10 um [159]. Linear
response curves were seen from 10 to 300 nM (r2>0.995), as shown in Figures 5.3A and
B. In all measurements, the NE overflow current dose not reflect release from an
individual varicosity but rather the NE concentration present at the vessel adventitia that
is released from multiple varicosities near the electrode.

In order to directly compare the sensitivity of MA and MV to the constrictor effects
of endogenous NE, the noradrenergic contribution to neurogenically-mediated
constriction was investigated in the both blood vessel types. This is discussed further in

the next section.

115

   
    

Norepinephrine (nM)

 

70

 

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oo
0

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A

.h
o

A

Current (pA)

N
O
4 j

 

 

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I ' I V I ' U

' 20 40 60 80 '100'120
Concentration (nM)

C

Figure 5.3. Calibration of a carbon fiber electrode for quantitative recording of NE
overflow. (A) Oxidation currents produced by injection of standard NE solution. (B)
Calibration plot showing how the NE oxidation current changes with concentration. Five
different electrodes were used to record the data set. Data are mean i S.E.M.

116

Influence of postjunctional receptor antagonists on NE release and vasoconstriction.
Along with NE, ATP acting at P2X; receptors contributes to the neurogenic constrictions
of arteries in the rat mesentery [31]. In order to study the noradrenergic constriction, the
P2X receptor antagonist, PPADS (10 uM) was used to block the constrictor effect of
ATP. Figure 5.4A shows that PPADS did not alter NE oxidation current but it did
substantially reduce the constriction of MA. In contrast, PPADS did not affect the
oxidation current or the constriction of MV as seen in Figure 5.4B.

This result indicates that ATP, at least at this stimulation frequency, mediates
arterial constriction while NE mediates venous constriction. The all-adrenergic receptor
antagonist, prazosin (0.1 uM), was also applied to the tissue individually and in
combination with PPADS. For MA, the presence of prazosin partially reduced the
contractile response mediated by NE while the presence of both antagonists almost totally
abolished constriction in Figure 5.4C. In contrast, adding PPADS had little effect on
either the constriction or NE overflow from MV in Figure 5.4D. The addition of prazosin
nearly completely abolished the constriction. Comparison of the endogenous NE-
concentration-constriction curves in the presence of PPADS for MA and MV indicates
that MA are relatively insensitive to endogenously released NE with a maximum
constriction of ca. 7% at 20 nM NE (Figure 5.4C). MV are much more sensitive to

endogenous NE as 20 nM caused a 3-fold greater constriction (ca. 23%) (Figure 5.4D).

Influence of a prejunctional 112-adrenergic receptor agonist and antagonist on NE
release and vasoconstriction. Prejunctional a2-adrenergic receptors located at the

sympathetic nerve terminals, also called an “autoreceptor”, are activated by NE and

function to inhibit further NE release [49-51].

117

PPADS

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Figure 5.4. Contribution of all—adrenergic and P2 receptors to neurogenic constrictions
of MA and MV. (A) Effects of PPADS (10 uM) on neurogenic constriction (top) and NE
oxidation current caused by a 20 Hz stimulus train in a MA. PPADS blocked the
constriction but not the NE oxidation current. (B) PPADS did not alter the neurogenic
constriction or NE oxidation current in a MV. (C) Concentration-constriction curve for
neurally-released NE in MA in the absence and presence of PPADS and prazosin (0.1
uM). Peak NE levels were measured during stimulus trains (1, 3, 7, 10 and 20 Hz) by
converting oxidation currents to NE levels using the calibration curve shown in Figure
SB. (D) Experiments similar to those shown in C except these studies were done in MV
during stimulus trains (0.5, 1, 3, 7, 10 and 20 Hz).

118

The influence of the a2-adrenergic receptor antagonist, yohimbine (1.0 uM), and the
agonist, UK 14,304 (1.0 uM), on the NE oxidation current and contractile response of
MA and MV was studied.

Figures 5.5 A-D show the NE oxidation current and the contractile responses
detected at the surface of MA and MV in the absence (control) and presence of
yohimbine and UK 14,304. Each datum represents the signal seen for a particular
stimulation frequency up to 20 Hz. In the presence of yohimbine, the a2-adrenergic
autoreceptor was inactivated and NE overflow for MA increased at all frequencies
(Figure 5.5A) while an increase in the contractile response was seen only at low
frequencies up to 10 Hz (Figure 5.5C). In contrast, for MV in the presence of yohimbine,
the NE overflow current increased with frequency up to 7 Hz, but generally had less
effect on NE overflow at high frequencies for MV than for MA (Figure 5.5B). The
contractile response was similar or lower than the control, particularly at 10 and 20 Hz
(Figure 5.5 D). For example, in the presence of yohimbine, the nominal oxidation current
for MA increased by 50 % fiom 8.0 to 12.0 after a stimulation, 7 Hz, whereas the current
for MV increased by 25 % from 11.8 to 15.8 pA. Interesting is the fact that the contractile
responses for MV decreased at high frequencies in magnitude with yohimbine at 10 and
20 Hz (Figure 5.5D).

Table 5.1 presents a statistical analysis of the NE oxidation current and contractile
response data with and without (control) added yohimbine. In the presence of yohimbine,
rate of rise of the oxidation current (slope) increased significantly at both 3 and 20 Hz for
MA but there were no significant differences in the half-life decay time (T50) of the

oxidation current.

119

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Figure 5.5. Contribution of 112-adrenergic receptor-mediated components to NE
overflow and neurogenic constrictions of sham mesenteric arteries (MA) and mesenteric
veins (MV). Frequency-response for NE oxidation current before (control) and after
application of yohimbine (1.0 nM) and UK 14,304 (1.0 nM) in MA (A) and MV (B).
F requency-response curves for contractile response before (control) and after application
of yohimbine and UK 14,304 in MA (C) and MV (D). *Indicates significantly different
from NE oxidation current in control MA (MV) and in yohimbine MA (MV). “Indicates
significantly different from NE oxidation current in control MA (MV) and in UK 14,304
MA (MV) (P<0.05). Data are mean i S.E.M.

120

Table 5.1. Numerical parameters obtained from the NE overflow oxidation current and
contractile response transients recorded in the presence and absence (control) of

yohimbine (1.0 uM).

 

 

 

 

 

 

 

Artery (n = 4) Vein (n = 4)
Hz Control Yohimbine Control Yohimbine
slope 3 0.05 i 0.02 0.09 :I: 0.01* 0.07 i- 0.01 0.15 i 0.02 *
(pA/s) 20 0.92i0.03 1.33:t0.17* 1.10i0.09 1.12:0.12
T50 (S) 3 2.80 i 0.55 4.52 i' 1.76 2.45 i 0.10 4.68 i 0.59 *
20 3.35 i 0.74 5.04 i 1.17 3.08 i 0.49 3.64 :l‘. 0.82
TL (S) 3 2.12 i 0.49 2.01 i' 0.54 1.78 i 0.34 2.69 i 0.56
20 0.49 i 0.06 0.53 :t 0.05 1.12 :t 0.12 1.45 i 0.24
RC 3 5.09 i 1.01 4.64 :l: 0.38 4.29 :I: 0.54 9.97 i 1.97*
(W’s) 20 3.68 a 0.48 3.93 i 0.70 2.60 a 0.19 2.85 1 0.30
TR (3) 3 7.52 i 1.67 8.86 i 3.29 28.5 i 8.5 38.7 i 7.7
20 10.7 i- l.0 12.1 i: 3.5 23.2 i 5.5 19.6 i 2.9

 

The oxidation current and contractile responses were elicited by electrical stimulation at 3
and 20 Hz. *Indicates significantly different from NE oxidation current and contractile
responses in control MA (MV) (P<0.05). Data are mean i S.E.M. Slope, rate of rise of
oxidation current; T50, half-life current decay time; TL, latency to onset of constriction;

RC, rate of constriction; TR, the time required for full vessel relaxation.

121

Interestingly, the slope of the current and T50 and the rate of constriction (RC) were
increased significantly only at 3 Hz for MV. Other responses, such as the latency period
to onset of constriction (TL) and the time required for full vessel relaxation (TR) were
changed little by the drug. While blocking of a2-adrenergic autoreceptors with antagonist
would be expected to increase the junctional NE levels, activating the 112-adrenergic
autoreceptors with an agonist would be expected to decrease the amount of transmitter
released. This was the case as the data reveal that both NE overflow and contractile
responses in MA were mostly blocked in the presence of UK 14,304 (1.0 uM) (Figure
5.5A and C). However, UK 14,304 reduced NE overflow and the contractile response
less for MV than for MA (Figures 5.5B and D). As an example, in the presence of the
agonist, the nominal oxidation current and contractile responses for MA decreased by
68% from 11.9 to 3.8 pA and by 71% from 27.8 to 8.1% at 20 Hz, respectively, whereas
the responses in MV decreased by 39 % from 15.0 to 9.1 pA and by 55% from 28.3 to
12.8% at 20 Hz, respectively. Table 5.2 shows TL and RC were significantly increased in
both MA and MV but other values were decreased due to decreased NE oxidation current
in the presence of UK 14,304. Interestingly, UK 14,304 preconstricted MV, 15.1% i 5.7

(n = 6) during addition of the drug.

Influence of cocaine and yohimbine + cocaine on NE overflow and vasoconstriction.
NE reuptake by the norepinephrine transporter protein (NET) can be blocked by cocaine.
Such inhibition will reduce the clearance rate of junctional NE and increase in NE

overflow and contractile responses.

122

Table 5.2. Numerical parameters obtained from the NE overflow oxidation current and
contractile response recorded in the absence (control) and presence of UK 14,304 (1.0

 

 

 

 

 

 

 

uM) treatment.
Artery (n = 3) Vein (n = 3)
Hz Control UK 14,304 Control UK 14,304
Slope 3 0.04 a 0.02 -* 0.12 a 0.02 0.02 a 001*
(pm) 20 0.97 i 0.22 0.11 a 005* 1.41 i 0.26 0.36 i 009*
T50 (s) 3 2.9 a 0.6 -* 3.0 a 0.6 1.0 a 05*
20 4.0 a 1.5 0.4 at. 02* 1.8 i 0.2 1.0 i 01*
1.C (s) 3 3.8 a 0.9 -* 1.9 :l: 0.4 5.6 i 04*
20 0.41 i006 1.34:013* 0.93 :01] 1.83 5015*
Re 3 4.9 a 0.7 -* 3.3 :t 0.5 13.3 a 2.5*
(“m/S) 20 3.8 i 0.5 3.4 .‘L' 0.2 2.1 a 0.2 2.8 :l: 02*
TR (3) 3 7.2 a 1.4 -* 20.7 i 2.6 4.4 i 07*
20 10.6 a 1.5 4.4 a 02* 45.6 a 14.9 15.2 i 5.4*

 

The oxidation current and contractile responses were elicited by electrical stimulation at 3
and 20 Hz. *Indicates significantly different from NE oxidation current and contractile
responses in control MA (MV) and in the drug treated MA (MV) (P<0.05). Data are
mean i S.E.M. Slope, rate of rise of oxidation current; T50, half-life current decay time;
TL, latency to onset of constriction; RC, rate of constriction; TR, the time required for full
vessel relaxation.

123

Figure 5.6 shows NE oxidation currents and the contractile responses for MA and
MV as a function of the stimulation frequency up to 20 Hz, before (control) and after the
addition of cocaine (10 uM) and yohimbine (1.0 uM) + cocaine (10 uM). In the presence
of cocaine, the NE oxidation current increased for MA (Figure 5.6A) at all frequencies
while the contractile response increased at low frequencies below about 7 Hz (Figure
5.6C). In contrast, the NE oxidation current and contractile responses for MV were
relatively unchanged except at 3 Hz (Figures 5.68 and D). As an example, in the
presence of cocaine, the nominal NE oxidation current for MA increased by 106 % from
6.4 to 13.2 pA at 3 Hz, whereas for MV the increase was 46 % from 7.9 to 11.5 pA.
Table 5.3 presents a statistical analysis of the NE oxidation current and contractile
response data before (control) and after addition of cocaine. In the presence of cocaine,
slope, T50, and other values significantly increased for MA while NE oxidation current
and contractile responses in MV are little changed.

In order to study an interaction between reuptake and the prejunctional 02-
adrenergic autoreceptor, cocaine (10 11M) and yohimbine (1.0 uM) were used in MA and
MV. Figures 5.6A and B show that combined drug treatment increased markedly the
oxidation current for MA compared with cocaine alone, whereas both drugs increased the
oxidation current only at low frequencies for MV. The contractile response was increased
at low frequencies (< 7 Hz) but not at high frequencies for MA (Figure 5.6C), whereas
decreased constriction was observed at all frequencies for MV (Figure 5.6D). Table 5.4
shows that the combined drugs significantly increased all values related to oxidation

current in MA at both 3 and 20 Hz but only increased at 3 Hz in MV.

124

 

 

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60- 1: Comol (n =10) 604:1 Como! (n = 10)
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Figure 5.6. Contribution of the NE reuptake to NE clearance and neurogenic constriction
of mesenteric arteries (MA) and mesenteric veins (MV). Frequency-response for NE
oxidation current before (control) and after application of cocaine (10 uM) and cocaine +
yohimbine in MA (A) and MV (B). Frequency-response curves for contractile response
before (control) and after application of cocaine (10 uM) and cocaine + yohimbine in
MA (C) and MV (D). *Indicates significantly different from NE oxidation current in
control MA (MV) and in cocaine treated MA (MV) (P<0.05). ”Indicates significantly
different from NE oxidation current in control MA (MV) and in cocaine + yohimbine
treated MA (MV) (P<0.05). Data are mean i S.E.M.

125

Table 5.3. Numerical parameters obtained from the NE overflow oxidation current and
contractile response transients recorded in the presence and absence (control) of cocaine

 

 

 

 

 

 

 

(10 11M).
Artery (n = 4) Vein (n = 4)
Hz Control Cocaine Control Cocaine
Slope 3 0.06 i 0.02 0.18 i 0.05”“ 0.20 i 0.11 0.24 i 0.09
(pA/s) 20 0.67 i 0.08 1.28 i 0.28“ 1.10 i 0.18 1.14 i 0.32
T50 (s) 3 3.2 i 0.2 6.9 i 1.2* 6.2 i 1.5 6.7 i 1.5
20 2.6 i 0.3 5.74 :t 2.0 3.2 i 1.0 2.5 i 0.5
LC (5) 3 3.1 i 0.7 1.7 i 0.4* 2.1 i 0.4 2.6 :1: 0.4
20 0.51 :t 0.03 0.58 i 0.06 1.01 i 0.08 1.01 i 0.06
RC 3 4.4 i 0.8 4.4 i 0.5 12.9 i 5.3 18.2 i 3.9
(“m/S) 20 4.0 i 0.2 3.6 i 0.2 2.8 i: 0.2 2.9 i 0.2
TR (5) 3 8.4 i 0.6 7.9 i- 2.5 106 i 40 116 i 38
20 20.3 i 1.6 20.7 i 3.2 88.5 i 29.8 79.9 i 36.4

 

The oxidation current and contractile response were elicited by electrical stimulation at 3
and 20 Hz. *Indicates significantly different from NE oxidation current in control MA
(MV) (P<0.05). Data are mean i SEM. Slope, rate of rise of oxidation current; T50, half-
life current decay time; TL, latency to onset of constriction; RC, rate of constriction; TR,
the time required for full vessel relaxation.

126

Table 5.4. Numerical parameters obtained from the NE overflow oxidation current and
contractile response transients recorded in the presence and absence (control) of cocaine

 

 

 

 

 

 

 

+ yohimbine (Co + Yo).
Artery (n = 4) Vein (n = 4)
Hz Control Co + Yo Control Co + Yo
Slope 3 0.04 i 0.01 0.19 i 005* 0.09 :0 .01 0.14 i 0.01 *
(pm) 20 0.88 i 0.08 1.89 i 032* 0.95 i 0.07 0.82 3: 0.16
T50 (s) 3 2.6 i 0.20 9.8 i 09* 2.8 i 0.2 6.3 3. 0.8 *
20 2.9 i 0.5 8.2 i 1.5* 3.1 i 0.5 4.7 i 0.9
LC (3) 3 3.2 i 0.4 2.3 i 05* 2.1 i 0.3 1.8 :1: 0.2
20 0.52 i 0.1 0.6 4: 0.06 0.99 i: 0.12 0.93 i 0.1
Re 3 4.6 i 0.6 4.5 :1: 0.6 4.5 i 0.6 11.7 i 2.9 *
(“m/S) 20 3.2 i 0.3 3.5 i 0.3 2.3 i 0.2 2.8 i 0.2
TR (5) 3 6.5 i 0.8 9.1 i 2.8 38.9 i 8.1 39.5 i 7.9
20 10.2 i 0.7 9.6 i 2.5 42.6 3: 8.4 47.6 i 9.6

 

The oxidation current and contractile response were elicited by electrical stimulation at 3
and 20 Hz. *Indicates significantly different from NE oxidation current in control MA
(MV) (P<0.05). Slope, rate of rise of oxidation current; T50, half-life current decay time;
TL, latency to onset of constriction; RC, rate of constriction; TR, the time required for full

vessel relaxation.

127

RC was only significantly increased at 3 Hz in MV in the presence of both drugs but other

contractile values were little changed in both MA and MV.

5.3 Discussion

The fluorescence images showed that MA and MV have networks of sympathetic
nerve fibers running over their adventitia] surfaces but there are fewer nerve fibers in MV
[26, 189]. This does not necessarily indicate that the density of innervation of MV is less
than MA. MV are thinner than MA so the number of axons per smooth muscle cell could
be similar in MA and MV. This suggestion is supported by the fact that ultrastructural
studies have shown that in guinea pigs the density of neuromuscular junctions was
similar for MA and MV [198]. The nerve plexus for MA consists of bundles of axons
arranged in a mesh-like network with nerve fibers equally likely to run parallel or
perpendicular to the longitudinal axis of the vessel. For MV, the plexus consisted of
single axons with more of a circumferential arrangement. This arrangement made optimal
detection of NE overflow quite dependent on the position of the recording microelectrode
on the MV surface, more so than for MA. Small microelectrode movements (5 50 um)
resulted a marked change in NE oxidation current for MV. Therefore, NE overflow
measurements were made with MV only after the electrode had been optimally
positioned to record the maximum current (i.e., positioned in the vicinity of multiple

varicosities).

Perivenous sympathetic nerves are more sensitive to nerve stimulation than as
periarterial nerves. Frequency-response curves for the NE oxidation current measured

at the surface of MV were left-shified from those for MA. Similar trends have been

128

observed for NE spillover from whole MA and MV segments by high-performance liquid
chromatography (HPLC) [100, 85]. Although the trends in the data from these previously
reported studies are similar to those reported herein, there is major difference in
pathophysiological interpretation. In the present work, short stimulus trains were used to
evoke measurable concentrations of NE at the recording microelectrode very close to the
release sites. Spillover measurements, on the other hand, require longer stimulus trains to
evoke detectable NE release and reflect NE concentration from large segments of
vasculature. Short stimulus trains (5 3 s) mimic in vivo sympathetic nerve activity, which
occurs in short bursts (< 2 s) [101].

MV are more sensitive to the constrictor effects of sympathetic nerve stimulation
than MA [22, 193, 31]. As MV are also more sensitive to the constrictor effects of
exogenous NE, post-junctional factors clearly contribute to this functional difference
between MA and MV. Additionally, there may be functional differences in periarterial
and perivenous sympathetic nerves. F requency-response curves for the NE overflow
oxidation currents for MV are shifted to the lefi of those for MA. These data are similar
to those obtained in previous work which revealed that NE overflow in canine MV
exceeded that in MA [54]. Increased NE overflow in MV relative to MA could be due to
several factors. Firstly, the mechanisms of NE release from perivenous and periarterial
sympathetic nerves could differ such that individual action potentials release more NE in
MV. There are no data available to address this issue; however, different populations of
sympathetic nerves are known to supply arteries and veins [199]. There may be
differential expression of calcium channels, autoreceptors or vesicular proteins in arterial

and venous sympathetic nerves, which contributes to the functional differentiation.

129

Continuous amperometric recording and traditional spillover measurements are sensitive
to the fraction of released NE that diffuses away fiom the neuroeffector junction. With
amperometric techniques, the distance from the release site to the recording
microelectrode is minimized as are the number of variables that could alter NE
measurement. However, differences in NE oxidation currents in MA and MV might be
due to differences in the efficiency with which neuroeffector junctions in MA and MV
clear NE. If little NE escapes the neuroeffector junction because of efficient recovery by
the norepinephrine transporter (NET) in MA for example, then less NE will be measured
outside the MA junction.

In this study, it was hypothesized that prejunctional modulation in rat MV and
MA regulates NE release differently. Multiple autoreceptors and heteroreceptors
localized at the nerve terminal modulate sympathetic nerve activity. These modulatory
receptors, such as 112-adrenergic autoreceptors, can either enhance or attenuate
transmitter release during sympathetic nerve stimulation. Secondly, it was hypothesized
that the functional properties of NET in rat MA and MV also differentially regulate the

rate of NE clearance from the neuroeffector junction.

Prejunctional 0.2-adrenergic receptors regulate NE release differently in MA and
MV. Prej unctional 112-adrenergic autoreceptors located at the sympathetic nerve
terminals are activated by exocytotic release of NE and inhibit further release [49].
Therefore, the autoreceptors mediate NE release through a feedback mechanism at the
axon terminal. The results demonstrate that blocking the neuronal aZ-adrenergic
receptors with yohimbine increased NE overflow oxidation current for MA at low and

high frequencies. However, the oxidation current for MV was increased only at low

130

frequencies. Conversely, UK 14,304 significantly decreased the NE oxidation current for
both MA and MV, but more significantly for MA. These results suggest that
prejunctional (112-adrenergic autoreceptors play more prominent role in regulating NE
release in MA than in MV. There may be quantitative (density) differences in 012-
adrenoceptor-mediated neuromodulation in the MA and MV. The aZ-adrenoceptor sites
may also be spatially separated from the sites of NE release in MV, which may
participate to differing NE release in the presence of aZ-adrenoceptors antagonist or

agonist in MA and MV.

Differential activity of norepinephrine transporter (NET) in MV and MA. Blocking
the neuronal 02-adrenergic autoreceptors caused an increase in the amount of NE
released, which leads to a higher junctional concentration. Inhibition of reuptake also
increased the junctional NE concentration because of reduced clearance. Therefore, the
junctional concentration of NE and its postjunctional effect is influenced not only by the
rate of NE release but also by reuptake, metabolism and diffusion [48]. Termination of
transmitter actions at the neuroeffector junction is dependent on both removal and
metabolic processes. The mechanism of NE clearance may vary between MA and MV.
After its released into the junctional region, approximately 95% of the released NE is
transported back into the nerve terminal via the NET. The neuronal NET (uptake 1)
accounts for the clearance of almost 90% of the total NE released. There is also an
extraneuronal transporter [47]. The extraneuronal transporter (uptake 2) is much more
diversely localized in the vascular endothelium and smooth muscle cells and has lower
affinity for NE than does neuronal NET. It is responsible for clearing only about 5% of

the total NE released [47]. The remainder of the NE is metabolized extraneuronally by

131

enzymes or simply diffuses away into the extracellular medium. It is then latter NE that is
oxidatively detected by the microelectrode. Inhibition of the NET was accomplished with
cocaine. In the present of cocaine, the endogenous NE concentration was significantly
increased for MA but not for MV. Interestingly, cocaine increased the half-life decay
time (T50) at 3 Hz for MA but not for MV, whereas yohimbine increased T50 at 3 Hz for
MV but not MA. This result suggests that NET play a more important role in the
clearance of released NE at MA than at MV. However, an uptake 2 blocker,
corticosterone (10 uM), did not significantly change the amplitude or time course of the
electrochemical signals evoked by electrical stimulation, indicating that uptake 2 does not
contribute significantly to the clearance of NE at either rat MA and MV (not shown). The
corticosterone result is similar to that reported for the rat mesenteric and tail arteries [200,
1211

The finding that the endogenous NE concentration was not altered by blockade of
neuronal uptake in MV can be explained if clearance of NE from its site of release is
primarily by diffusion and not by reuptake. One possible reason is for the reduced
reuptake is that the NET sites are spatially separated from the sites of NE release in the
rat MV, whereas the NET is more closely located to the sites of NE release in MA.
Another possibility is that strong feedback mechanism between uptake and the
autoreceptors may function more effectively in MV than MA. When higher extracellular
NE concentrations occur after cocaine treatment, this effect would be suppressed by the
activation of inhibitory autoreceptors in MV. Thus, the increased extracellular NE levels
resulting from cocaine uptake blockade might activate orZ-autoreceptors and shut down

subsequent NE release in MV. Alternatively, increased NE release by yohimbine

132

activates uptake transporter more in MV. Therefore, in the presence of either cocaine or

yohimbine in MV, NE overflow was less increased compared with MA.

Autocompensation system between prejunctional 0.2-receptors and NE uptake
transporters in MA but not in MV. The higher concentrations of NE in the region of
the nerve-ending limit release of the neurotransmitter by feedback inhibition via
prejunctional 012-adrenergic autoreceptors, thereby potentiation by uptake inhibition
could be masked to sympathetic stimulation. The results showed that NE overflow was
significantly increased in the presence of cocaine + yohimbine for MA but not for MV, as
compared with either cocaine or yohimbine alone. Also, the more significant decline in
the NE oxidation current for MA after repeated stimulation in the present of cocaine or
cocaine + yohimbine may result from exhaustion of prejunctional NE stores. The rate of
synthesis of NE may not compensate for the NE lost during a firing event [136]. The
present finding indicates that NE reuptake and autoinhibition play an important role in

maintaining available stores of NE in MA.

Different neurogenic contractile responses in MA and MV. Differences in the
transmitters released from periarterial and perivenous sympathetic nerves contribute to
functional differences in MA and MV. The al-adrenergic receptor antagonist, prazosin,
had little effect on neurogenic constriction of MA but it blocked venous responses.
Alternatively, the P2X receptor antagonist, PPADS, blocked constriction in MA but not
MV. Therefore, neurogenic constrictions in MV are mediated moreso by NE acting at

al-adrenergic receptors, while MA tone is mediated by ATP acting at P2X receptors

133

[100, 201, 36, 31]. These results confirm that in small MA, ATP is the dominant
neurotransmitter, whereas in MV, NE is the transmitter mediating constriction.

It was also showed that MA is relatively insensitive to the constrictor effects of
endogenous NE and this highlights the importance of ATP as a transmitter in MA. The
NE concentrations measured were undoubtedly lower than the junctional concentrations
[121]. Recovery of NE via NET will limit the amount of NE diffusing to the
microelectrode [47]. It can be seen from these data that change in vascular tone did not

influence the NE oxidation current recorded with the microelectrodes.

Postjunctional orZ-adrenergic receptors in MV. Although the NE overflow oxidation
current increased in the presence of yohimbine or cocaine, the contractile response
reached a maximal value and was the same magnitude with and without the drugs at high
frequencies (> 7 Hz). In some cases, constriction decreased with the combined drugs.
This may be because the postjunctional receptor sites on the vascular smooth muscle cells
were saturated at the concentrations of NE released from nearby varicosities and
maintained in the junction.

Another possibility is that the postjunctional Oil-adrenergic and P2X purinergic
receptors for MA were downregulated or desensitized by increased NE and ATP
concentrations in junctional clefi in the present of the drug. Interestingly, MV was
preconstricted in the presence of the orZ-agonist, UK 14,304 (1.0 uM), ca.15 % of their
original diameter. Also, MV contractile response in the presence of the orZ-antagonist,
yohimbine (1.0 uM), remained constant or decreased despite the increased NE overflow
at low frequencies of 1 and 3 Hz, and even decreased at high frequencies 10 and 20 Hz.

The results suggest that neurogenic constrictions in MV may also be mediated partly by

134

orZ-adrenergic receptors not only by or] -adrenergic receptors. However, other possibilities
are that high concentrations of aZ-adrenergic receptor antagonist and agonist influenced

on (rd-adrenergic receptors as partial al-adrenergic receptor antagonist and agonist in

MV.

5.4 Conclusion

In this Chapter, the contribution of NET and the prejunctional aZ-adrenergic
autoreceptor to the regulation NE overflow from sympathetic nerves in MA and MV was
evaluated. NE overflow was evaluated using continuous amperometry with carbon fiber
microelectrode by measuring the oxidation current. The results confirmed that the
mechanism of NE release and the corresponding vascular constriction in MV are
significantly different from that in MA. The different sympathetic neuroeffector
mechanisms in MV could be due to different functions of prejunctional NET and
autoreceptors, and different types of postjunctional receptors. NET and aZ-adrenergic
autoreceptors play a more prominent role in controlling NE clearance and release at the
neuroeffector junction in MA than in MV. It would contribute that NE overflow in rat
MV exceeded that in MA. Therefore, the greater NE overflow in veins compared to
arteries is not likely due to the difference in innervation density of arteries and veins.
Rather, it is due to the difference in functional properties of sympathetic nerves
innervating veins and arteries. This provides further evidence supporting the notion that
sympathetic nerves supplying veins and arteries are different [22, 193]. These differences
in neuroeffector transmission may contribute to the different hemodynamic functions of

arteries and veins.

135

Chapter 6

Alterations in Sympathetic Neuroeffector Transmission to
Mesenteric Arteries and Veins in DOCA-salt Hypertensive
Rats

6.1 Introduction

The sympathetic nervous system (SNS) influences importantly on the
cardiovascular hemodynamics through its control over the liberation of NE into the
circulation. Therefore, alternations in the peripheral sympathetic tone and reactivity
might facilitate the development of various pathologies of the cardiovascular system
including hypertension [69]. Since increased SNS activity increases arterial and venous
constriction and blood pressure, hyperactivity of the SNS is a critical factor for
hypertension. There is growing evidence that human essential hypertension and many
animal models for the disease are associated with overactivity of the SNS. However, the
precise causal mechanisms leading to sympathetic augmentation in hypertension are still
poorly understood because maintenance of normal blood pressure involves a complex
interplay among the nervous, endocrine, renal and cardiovascular systems.

The deoxycorticosterone acetate (DOCA)-salt hypertensive rat model is used
widely to study salt-sensitive hypertension, which involves neuronal and hormonal
mechanisms [64, 31]. The DOCA-salt rat model has been used to study the involvement
of both the central and peripheral components of the SNS in the development and/or
maintenance of hypertension [202, 203, 28, 80]. This animal model allows one to
evaluate the functional consequences of altered sympathetic regulation of the

cardiovascular system and providing insight into the defects that underlie the sympathetic

136

impairment [67]. Mesenteric arteries (MA) and veins (MV) are extensively used in
physiological and pharmacological experiments to understand the role of the SNS in
modulation of vascular smooth muscle tone [12]. Many studies have reported that
increased activity of the SNS increases plasma catecholamine level in MA and/or MV
[202, 28, 204-206]. The elevated NE concentrations might reflect elevated sympathetic
tone. Therefore, measurement of NE levels is an indirect index of sympathoadrenal
activity under certain standardized conditions [207, 208, 67]. The increased sympathetic
nerve activity and NE release observed in DOCA-salt hypertension suggest that there
may be alterations in the local mechanisms that modulate sympathetic neurotransmission
in arteries and veins. Altered NE reuptake, impaired prejunctional aZ-adrenergic
autoreceptors and/or increased sympathetic nerve firing rates have been proposed as
causes for elevate NE release or could be responsible for the increased plasma
catecholamine levels in hypertension animal models [86, 87, 67, 79, 70, 31, 68].
Increased NE release and or reactivity of postjunctional receptors may also result in
increased or decreased contractile responses [209, 31]. However, the results are still
controversial. Moreover, there have been few detailed studies of the mechanisms
underlying increases in sympathetic input to veins in hypertension [210, 176, 31, 68]. As
emphasized in this dissertation changes in neuroeffector transmission to veins in
hypertension is an important factor for blood pressure regulation [176].

Most previous studies have indirectly investigated endogenous NE release from
perivascular sympathetic nerve endings using tissues labeled with tritiated NE and high-

performance liquid chromatography [98-100, 85, 68]. Therefore, little is known about

137

altered characteristics of NE release from perivascular sympathetic nerves in
hypertension on an impulse-by-impulse basis [121].

It was hypothesized that there are dysfunctional 112-adrenergic autoreceptors and or
NET in DOCA-salt hypertensive rat MA and MV is a cause for the increased junctional
NE concentration and altered contractile responses compared to normotensive sham rat
MA and MV. Therefore, altered neuroeffector transmission is a cause for the increased
blood pressure in DOCA-salt hypertensive rats. To test the hypothesis, neurogenic NE
overflow and vasoconstriction were measured directly in the absence and presence of

reuptake blocker, prejunctional (112-adrenergic receptor antagonist and agonist,

postjunctional 011 -adrenergic receptor antagonist and P2X purinergic receptor antagonist.

6.2 Results

In Vitro measurement of neurogenic NE overflow and vasoconstriction in
normotensive and DOCA-salt MA and MV. The administration of DOCA and saline
water to rats for a period of 4 weeks results in increased blood pressure and decreased
body weight. Mean systolic blood pressure for sham and DOCA-salt rats was 125 i 2
mmHg (11 = 40) and 194 i 3 mmHg (n = 32), respectively (P < 0.05). The mean weight of
sham rats was 416 j: 5 g (n = 34) which the mean weight of DOCA-salt rats was 336 t 7g
(n = 35) (P < 0.05). NE overflow and vasoconstriction caused by electrical stimulation of
the nerve endings was investigated using MA and MV from sham and DOCA-salt
hypertensive rats. Short trains (60 pulses) of stimuli produced frequency dependent (0.2—
20 Hz) and reliably detectable NE oxidation current from periarterial and perivenous

nerves. NE overflow and contractile response were detected at lower threshold frequency

138

for MV (0.5 and 0.2 Hz, respectively) than for MA (1 and 0.5 Hz, respectively). Figures
6.1A and B display representative recordings of the contractile (top) and NE oxidation
current (bottom) response transients for a 3 Hz stimulation of sham and DOCA-salt MA
and MV.

As mentioned in Chapter 5, since there are more nerve fibers associated with MA
compared to MV, measurement of NE overflow at MV was critically dependent on the
electrode position. Several positions along a blood vessel were tested in order to find a
maximum current. It was at this location that all of the subsequent testing was done.
Figures 6.1C and D are plots of the oxidation current and percent (%) constriction as a
function of stimulation frequency for both vessel types. These data demonstrate a
frequency-dependent (0.2 -20 Hz) increase in the NE oxidation current (NE overflow)
and constriction for both sham and DOCA-salt MA and MV. The data reveal that NE
overflow from sham MV was greater than from sham MA but not significantly different
compared with DOCA-salt MV (Figure 6.1C). However, NE overflow in DOCA-salt MA
was significantly higher compared to that in sham MA.

The frequency-response curves for the contractile response of sham and DOCA-salt
MV were both shified to the left of the curves for sham and DOCA—salt MA (Figure
6.1D). The mean of the half maximum stimulation frequency (S50) were similar for sham
and DOCA-salt MV but the maximum constriction (Em) obtained from DOCA-salt MV
was significantly lower than that for sham MV (Table 6.1). No significant differences

were seen for sham and DOCA-salt MA in terms of the Em or S50 values.

139

A B
Artery Veln

273 1““ ww 309 pm 273 pm
I201mm 1%
I5 p A I5 pA

SS SS

Sham DOCA-calf Sham DOCA-salt

U

 

 

 

 

 

 

 

 

 

5
‘ -D-SlnnMA(n=21)
A c 25. -A-SlunMV(n=16) A if?
g- ,9 ‘ .. .
:I 33 20
c 1.1 1
E g 15
3 .
U U 10
°\"
5-
0
O.

 

   

Figure 6.1. Contractile (top) and NE oxidation current (bottom) response transients for
(A) MA and (B) MV from sham and DOCA-salt rats in response to a 3 Hz stimulation.
Comparison of the frequency response of (C) the oxidation current and (D)
vasoconstriction in sham and DOCA- salt MA and MV. *Significant difference between
sham and DOCA- salt MA (P < 0. 05). &Significant difference between sham MA and
sham MV (P < 0. 05). Data are presented as mean + S. E. M.

140

Table 6.1. Maximum constriction (Em) and half maximum stimulation frequency (S50)
for MA and MV from sham and DOCA-salt rats. All data are expressed as the mean i
S.E.M and “11” value refers to the number of animals from which the data were obtained.

 

 

 

Artery Vein
Sham (n=15) DOCA-salt (n=l6) Sham (n=13) DOCA-salt (n=14)
S50 (Hz) 5.8 a: 0.3 5.6 :1: 0.7 1.6 i 0.3 " 1.3 3. 0.4 ”
15......r (%) 31.3 i 3.4 29.8 :1: 2.9 28.0 i 2.2 21.6 i 1.9*

 

”Indicates significantly different from the S50 in mesenteric arteries (MA) (P<0.05).
*lndicates significantly different from the Em in sham mesenteric veins (MV) (P<0.05).

Effect of prejunctional 0.2-adrenergic receptors on NE release and constrictions in
DOCA MV and MA. To determine whether the 112-adrenergic autoreceptor that
regulates NE release is altered in DOCA-salt MA and MV, the effects of the 02-
adrenergic receptor antagonist, yohimbine (1.0 uM) and the agonist, UK 14,304 (1.0
uM), on NB overflow and the elicited contractile response were investigated. Figure 6.2A
Shows that yohimbine significantly increased NE overflow at all frequencies for sham
MA. However, yohimbine was less effective increasing NE overflow in DOCA-salt MA.
In the presence of yohimbine, the nominal oxidation current increased by 49.4 % from
14.0 to 21.0 pA at 20 Hz (11 = 5) and by 120.6 % from 5.8 to 12.7 pA at 3 Hz (11 = 5) for
sham MA, whereas the nominal oxidation current increased by 10.4 % from 16.8 to 18.5
pA at 20 Hz (11 = 6) and by 54.6 % from 7.3 to 11.3 pA at 3 Hz (11 = 6) for DOCA-salt
MA. The apparent increase in NE overflow caused elevated constrictions in sham and
DOCA-salt MA at low frequencies (1 and 3 Hz) and the contractile response curves in
the presence of yohimbine were lefi-shified from the curve without (control) the drug as

seen in Figure 6.2C and Table 6.2.

141

141 if}

 

 

 

       

     

        

        

35 35
11:181unMMn'5) I:ISIunMV(n-4)
30.-oocAMA(n=6) -DOCAMV(n-5)
A 1 Emmmmrfi) Eamwnmmmc)
é 25‘ -mCAMNYolintim(n*-6) at 2 -mmNMm(n-5)
:20, _# E L920 - ,
515‘ g 2: E15 xg 4%”
= 1 E a * 7g 5
10* g: g: 10* Z: Z:
1 = a 1 a 5
54 g; g; 54 g; f;

 

 

 

 

 

 

 

 

 

 

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O

 

 

1—A—ShunMV(n-6)
-o— oocnlmwme)

H—sIunMVIYotinumn-c)
.ro- DOCAMVIYdinumn-e)

 

%Constr1ctlon
991-593.818.913
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Figure 6.2. Contribution of aZ-adrenergic receptor-mediated components to NE
overflow and neurogenic constriction of sham and DOCA-salt mesenteric arteries (MA)
and mesenteric veins (MV). Frequency-response for NE oxidation current before
(control) and after application of yohimbine (1.0 uM) in sham and DOCA-salt MA (A)
and MV (B). Frequency-response curves for the contractile response before (control) and
after application of yohimbine in sham and DOCA-salt MA(C) and MV (D). *indicates
significantly different from NE oxidation current in control sham MA (MV) (P<0.05).
#indicates significantly different from NE oxidation current in control DOCA-salt
MA(MV) (P<0.05). Data are presented as mean 3: S.E.M.

142

Table 6.2. Maximum constriction (Em) and half maximum stimulation frequency (S50)
for MA and MV from sham and DOCA-salt rats in the absence (control) and presence of
yohimbine (Yo, 1.0 uM) and UK 14,304 (UK, 1.0 uM). All data are expressed as the

mean i S.E.M and “11” value refers to the number of animals from which the data were
obtained.

 

 

 

 

 

 

 

 

 

 

 

 

Sham Artery DOCA-salt Artery
Control (n=5) Yo (n=5) Control (n=6) Yo (n=6)
S50 (Hz) 5.4 a 1.1 2.4 i 07“ 5.6 :t 0.9 2.5 3. 03*
Em, (%) 33.9 :1: 1.9 30.0 :1: 2.3 33.0 i 2.7 29.4 d: 1.9
Control (n=5) UK (n=5) Control (n=6) UK (n=6)
S50 (Hz) 5.8 :1: 0.6 11.3 :t 07“ 6.5 :1 0.9 7.9 :1: 0.7 ”
5mm) 36.9 d: 2.3 13.5 i 2.3“ 33.5 d: 2.5 20.4 :1: 1.9 *"
Sham Vein DOCA-salt Vein
Control (n=5) Yo (n=5) Control (n=6) Yo (n=6)
S50 (Hz) 3.0 :1: 0.7 3.4 a: 0.8 1.5 :1: 0.4 1.4 i 0.7
Em, (%) 27.3 3: 2.3 21.9 a 2.9 21.7 :1: 1.9 20.0 3: 1.7
Control (n=5) UK (n=5) Control (n=4) UK (n=4)
S50(Hz) 1.9308 10.1 a 1.1“ 2,431.1 9.341.5*
15mm) 31.9 a 2.3 11.9 i 1.6“ 28.9 :1: 1.7 15.9 :1: 2.6*

 

&Indicates Significantly different from the S50 or EmaJlr in control sham MA(MV) (P<0.05).
*lndicates significantly different fi'om the S50 or Em, in control DOCA-salt MA(MV)

(P<0.05). ”Indicates significantly different from the S50 or Em in the UK 14,304 treated
sham MA (P<0.05).

143

However, there was no difference between the contractile responses for sham and
DOCA-salt MA. Yohimbine also significantly increased NE overflow in sham and
DOCA-salt MV at low frequencies (1 to 7 Hz) but not at high frequencies in Figure 6.2B.
In the presence of yohimbine, the elevated NE overflow for sham and DOCA-salt MV
was not Significantly different. The nominal oxidation current increased by 38.3 % from
12.5 to 17.3 pA at 7 Hz (11 = 5) and by 76.7 % from 3.8 to 6.6 pA at 1 Hz (11 = 6) for sham
MV, and by 39.6 % from 11.9 to 16.7 pA at 7 Hz (n = 6) and by 81.9 % from 4.2 to 7.6
pA at 1 Hz (n = 6) for DOCA-salt MV. Interestingly, the elevated NE overflow did not
alter neurogenic constriction for both sham and DOCA-salt MV as seen in Figure 6.2D
and Table 6.2.

Figure 6.3 shows that UK 14,304, an orZ-adrenergic receptor agonist significantly
inhibited NE overflow at all frequencies in sham and DOCA-salt MA and MV, which
resulted in decreased constriction. However, NE overflow and the elicited contractile
response for DOCA-salt MA were much less affected by UK 14,304 compared to the
responses for sham MA (Figure 6.3A and C). In the presence of UK 14,304, the nominal
oxidation current decreased by 70.0 % from 12.5 to 3.8 pA at 20 Hz for sham MA,
whereas the nominal oxidation current decreased by only 31.1 % from 17.6 to 12.1 pA
for DOCA-salt MA. The Ema, for DOCA-salt MA was also significantly greater than that
for sham MA in the presence of UK 14,304 but there were no differences between the
frequency response curves obtained for sham and DOCA-salt MV as seen in Figure 6.3D

and Table 6.2.

144

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Figure 6.3. Contribution of (112-adrenergic receptor-mediated components to NE
overflow and neurogenic constrictions of sham and DOCA-salt mesenteric arteries (MA)
and mesenteric veins (MV). F requency-response for NE oxidation current before
(control) and afier application of UK 14,304 (1.0 uM) in sham and DOCA-salt MA (A)
and MV (B). Frequency-response curves for contractile response before (control) and
after application of UK 14,304 in sham and DOCA-salt MA (C) and MV (D). *Indicates
significantly different from NE oxidation current in control Sham MA (MV) (P<0.05).
“Indicates significantly different from NE oxidation current in control DOCA-salt MA
(MV) (P<0.05). &Indicates significantly different from NE oxidation current in the drug
treated sham MA (P<0.05). Data are mean i S.E.M.

145

Effects of NET on NE clearance and vasoconstriction in DOCA-salt MA and MV.
Reuptake of NE by the norepinephrine transporter (NET) is the primary clearance
mechanism responsible for removing the neurotransmitter from the junctional sites. To
test whether NET was altered in DOCA-salt MA or MV, the effect of cocaine on NE
overflow and the contractile response was investigated.

Figures 6.4A and B show that cocaine (10 uM) increased the NE oxidation current
for sham and DOCA-salt MA but was less influence on NE overflow for sham and
DOCA-salt MV. Interestingly, the NE oxidation current of the DOCA-salt MA was
greater than that for sham MA after cocaine treatment. The nominal oxidation current
increased by 72.8 % from 13.7 to 23.7 pA at 20 Hz and by 73.4 % from 12.6 to 21.8 pA
at 10 Hz for DOCA-salt MA, whereas the nominal oxidation current increased by 32.2 %
from 12.7 to 16.8 pA at 20 Hz and by 41.2 % from 11.6 to 16.4 pA at 10 Hz for sham
MA in the presence of cocaine. For sham and DOCA-salt MA, the slower clearance of
NE from junctional sites in the presence of cocaine enhanced the contractile response,
particularly at low frequencies. The contractile response curves in the presence of cocaine
were left-shifted from the curve without the drug (control) (Figure 6.4C and Table 6.3).
However, there was little effect on the contractile response of sham and DOCA-salt MV,
since the negligible effect of cocaine on NB clearance of MV (Figure 6.4D and Table
6.3).

Corticosterone, an extraneuronal transporter (uptake 2) blocker, also had no
significant effect on NB overflow or the contractile response for either MA or MV (see

also Chapter 5).

146

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Figure 6.4. Contribution of reuptake transporter-mediated components to NE clearance
and neurogenic constrictions of sham and DOCA-salt mesenteric arteries (MA) and
mesenteric veins (MV). F requency-response for NE oxidation current before (control)
and after application of cocaine (10 11M) in sham and DOCA-salt MA (A) and MV (B).
Frequency-response curves for contractile response before (control) and after application
of cocaine in sham and DOCA-salt MA (C) and MV (D). *lndicates significantly
different from NE oxidation current in control sham MA (P<0.05). ”Indicates
significantly different from NE oxidation current in control DOCA-salt MA (P<0.05).
&Indicates Significantly different from NE oxidation current in the drug treated sham MA
(P<0.05) Data are mean i S.E.M.

147

Table 6.3. Maximum constriction (Em) and half maximum stimulation frequency (S50)
for MA and MV from sham and DOCA-salt rats in the absence (control) and presence of

cocaine (Co, 10 nM) and the combined drug (cocaine + yohimbine). All data are
expressed as the mean .+_ S.E.M and “11” value refers to the number of animals from which

 

 

 

 

 

 

 

 

 

the data were obtained.
Sham Artery DOCA-salt Artery
Control (n=5) Cocaine (n=5) Control (n=6) Cocaine (n=6)
S50 (Hz) 5.7 3 0.3 2.3 3 07“ 5.5 3 0.4 1.6 3 1.2*
Emax (%) 35.7 is 1.6 33.3 i 2.1 31.3 3: 1.3 27.9 3: 2.7
Control (n=5) Co + Yo (n=5) Control (n=5) Co + Yo (n=5)
S50 (Hz) 5.9 3 1.3 1.8 3 09“ 6.3 3 2.3 1.8 3 02*
Emax (%) 29.6 :1: 1.7 28.0 :t 2.9 25.9 :h 2.3 21.6 :1: 1.9
Sham Vein DOCA-salt Vein

 

Control (n=5) Cocaine (n=5)

Control (n=5) Cocaine (n=5)

 

 

 

S50 (Hz) 1.3 3 0.6 1.9 3 0.4 2.5 3 0.8 1.8 3 0.5
15...... (%) 24.6 3 1.3 24.8 3 1.9 23.9 3 1.7 20.6 3 2.1
Control (n=5) Co + Yo (n=5) Control (n=5) Co + Yo (n=5)
S50 (Hz) 1.8 3 0.6 2.4 3 0.6 1.6 3 0.9 —
Em...(%) 33.3 3 3.1 24.8 3 2.9“ 21.9 3 1.2 16.2 3 07*

 

&indicates significantly different from the S50 or Em in control Sham MA(MV) (P<0.05).
*Indicates Significantly different from the S50 or Emax in control DOCA-salt MA(MV)

(P<0.05).

148

Effect of combined application of cocaine and yohimbine. The interrelationship
between reuptake and prejunctional orZ-adrenergic receptors in DOCA-salt MA and MV
was studied using the combined drug, cocaine (10 11M) and yohimbine (1.0 uM). Figure
6.5A shows that the combined application of cocaine and yohimbine markedly increased
the oxidation current in sham and DOCA-salt MA compared with the effects of either of
the individual drugs. There were no differences between the elevated oxidation currents
obtained from sham and DOCA-salt MA. In the presence of the combined drug, the
nominal oxidation current increased by 74.3 % from 16.0 to 28.0 pA at 20 Hz and by 316
% from 6.4 to 25.6 pA at 3 Hz for sham MA, and by 102 % from 16.3 to 33.0 pA at 20
Hz and by 279 % from 7.8 to 29.6 pA at 3 Hz for DOCA-salt MA. The increased
junctional NE concentration caused elevated constrictions in sham and DOCA-salt MA,
particularly at low frequencies, with left-shifted from the curve without the drug (control)
(Figure 6.5C and Table 6.3). The elevated contractile responses for sham and DOCA-salt
MA were not significantly different. The S50 value from Sham and DOCA-salt MA were
5.9 i 1.3 Hz and 6.3 :1: 2.3 Hz, before the combined drug treatment and 1.8 i 0.9 Hz and
1.8 i 0.2 Hz, after the combined drug treatment, respectively (Table 6.3.)

However, the NE oxidation current increased significantly only at low frequencies
in sham and DOCA-salt MV after the drug treatment but there were no differences
between the elevated oxidation currents obtained from Sham and DOCA-salt MV.
Though the NE concentration was increased significantly at low frequencies in MV, the
contractile responses were significantly decreased in the presence of the drug (Figure

6.5D and Table 6.3).

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Figure 6.5. Contribution of uptake transporter/or2-adrenergic receptor-mediated
components to NE clearance/release and neurogenic constrictions of sham and DOCA-
salt mesenteric arteries (MA) and mesenteric veins (MV). Frequency-response for NE
oxidation current before (control) and after application of combined drug (cocaine (10
uM) + yohimbine (1.0 uM)) in sham and DOCA-salt MA (A) and MV (B). Frequency-
response curves for contractile response before (control) and after application of the
combined drug in sham and DOCA-salt MA (C) and MV (D). *Indicates significantly
different from NE oxidation current in control sham MA(MV) (P<0.05). I’Indicates
significantly different from NE oxidation current in control DOCA-salt MA(MV)
(P<0.05). Data are mean i S.E.M.

150

Purinergic and adrenergic contribution to neurogenic constriction in DOCA-salt
MA and MV. NE and ATP are major vasoconstrictor neurotransmitters released from
sympathetic nerves. For the study of contributions of ATP and NE to neurogenic
constrictions of DOCA-salt MA and MV, NE oxidation current and contractile response
curves in sham and DOCA-salt tissues were constructed in the absence (control) and
presence of prazosin (0.1 uM), an al-adrenergic receptor antagonist and pyridoxal-
phosphate-6-azophenyl-2',4'-disulfonic acid (PPADS) (10 HM), a P2X receptor
antagonist. Prazosin and PPADS block the constrictor effects of NE and ATP. Figures
6.6A and B show the evidence that the postjunctional receptor blockers did not alter NE
oxidation current in both sham and DOCA-salt MA and MV. Prazosin reduced part of
neurogenic constriction of sham MA but markedly inhibited the contractile response in
DOCA-salt MA (Figure 6.6C). In Table 6.4, the Em in sham and DOCA-salt MA was
31.6 i 2.1 % and 33.7 i 1.9 %, before prazosin treatment and 23.4 i 2.4 % and 13.7 i 3.4
% afier prazosin treatment, respectively. The S50 in sham and DOCA-salt MA was 5.7 i
0.4 Hz and 5.8 i 0.9 Hz, before prazosin treatment and 5.9 i 1.0 and 11.3 i 2.7 Hz after
prazosin treatment, respectively. Subsequent addition of PPADS completely blocked the
neurogenic response remaining in the presence of prazosin. PPADS alone greatly reduced
neurogenic constrictions in sham MA but PPADS had a little effect on responses in
DOCA-salt MA.

In contrast, prazosin mostly blocked constriction caused by NE during electrical
nerve stimulation in sham and DOCA-salt MV but PPADS alone had no effect on the

contractile responses in sham and DOCA-salt MV (Figure 6.6D).

151

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Figure 6.6. Contribution of oil-adrenergic receptor and P2X-mediated components to NE
release and neurogenic constrictions of sham and DOCA-salt mesenteric arteries (MA)
and mesenteric veins (MV). Frequency-response for NE oxidation current before
(control) and after application of prazosin (0.1 uM) or/and PPADS (10 pM) in sham and
DOCA-salt arteries (A) and veins (B). Frequency-response curves for contractile
response before (control) and after application of the drug in sham and DOCA-salt MA
(C) and MV (D). Data are mean t S.E.M.

152

Table 6.4. Maximum constriction (Em) and half maximum stimulation frequency (S50)
in MA and MV from sham and DOCA-salt rat in the absence (control) and presence of
prazosin (0.1 uM) and PPADS (10 uM) treatment. All data are expressed as the mean i
SEM and “n” values refer to the number of animals from which the data were obtained.

 

 

 

 

 

 

 

Sham Artery DOCA-salt Artery

Control (n=5) Prazosin (n=5) Control (n=5) Prazosin (n=5)
350012) 5.7 i 0.4 5.9 a: 1.0 5.8 :l: 0.9 11.7 :h 22*”
15mm) 31.6 :t 2.1 23.4 i 2.4“ 33.7 2!: 1.9 13.7 :h 3.4“

I Sham Vein DOCA-salt Vein

Control (n=5) Prazosin (n=5) Control (n=5) Prazosin (n=5)
S50 (Hz) 1.55:0.6 — 1.9105 11.5:t3.1*
Ew(%) 27.4 :t 1.7 5.7 :l: 3.1“ 24.9 d: 1.9 8.5 :t 1.7*

 

&Indicates significantly different from the S50 or Em in control sham MA(MV) (P<0.05).
*Indicates significantly different fiom the S50 or Em in control DOCA-salt MA(MV)
(P<0.05). ”Indicates significantly different from the S50 or Em, in the prazosin treated
sham MA (P<0.05).

153

6.3 Discussion

All NE overflow and evoked contractile responses for both MA and MV induced
by electrical stimulation were blocked completely by TTX; a voltage-gated sodium
channel blocker (see Chapter 4). This result indicates that NE overflow and contractile
response were nerve-mediated. In this Chapter, NE overflow and vasoconstriction for
DOCA-salt MA and MV were investigated in order to assess changes that might be
associated with hypertension. A carbon fiber was used to monitor NE in this work rather
than a diamond microelectrode because it was found that several of the drugs employed
were oxidized at the positive detection potential needed to detect NE at the diamond

micro electrode.

Increase NE overflow in DOCA-salt MA and altered neurogenic responses in
DOCA-salt MV. In the early stage of certain forms of experimental hypertension [69] as
well as in hypertensive patients [203, 17, 48], sympathetic traffic to the cardiovascular
system is elevated. Previous studies have shown that there was increased sympathetic
neuronal activity and NE overflow from sympathetic nerves in DOCA-salt rats [77, 69,
68]. However, NE overflow was not monitored in real time and not measured directly in
these studies. The present study revealed that NE overflow was significantly increased in
DOCA-salt MA compared with sham MA. However, little change was seen in the
overflow for DOCA-salt MV. This result is different from previous in vitro work that
showed an increased NE overflow in DOCA-salt MV [68]. This difference may be
accounted for by methodological differences. In this study, NE overflow was measured
locally at the vessel surface near the point of focal stimulation rather than as

accumulative overflow from an entire tissue bed.

154

The increased NE overflow in DOCA-salt MA could result from increased NE
concentrations released from nerve endings, reduced clearance by NE transporter (NET),
dysfunction of the prejunctional aZ-adrenergic autoreceptors, reduced extracellular
enzymatic metabolism or some contribution of these. It was hypothesized that
dysfunction of the prejunctional aZ-adrenergic autoreceptors and/or NET located at the

sympathetic nerve terminals is the cause for the increased NE overflow in DOCA-salt

MA.

Impaired prejunctional (1.2-adrenergic autoreceptors in DOCA-salt MA.
Prejunctional aZ-adrenergic autoreceptors mediate the inhibition of NE release from
sympathetic terminals, constituting an important autoregulatory system for sympathetic
tone [211, 52]. Previous studies showed that prejunctional regulation of transmitter
release by a2-adrenergic autoreceptor was altered in experimental rat models of
hypertension [77, 80]. However, either impaired [79, 68] or unaltered [28] a2-adrenergic
autoreceptors in the isolated MA of DOCA-salt rats have been reported. My study shows
that blockade of these receptors with an a2-adrenergic receptor antagonist, yohimbine,
resulted in increased NE overflow in both sham and DOCA-salt MA, because the
negative feedback system was interrupted by the drug. However, yohimbine was less
effective of increasing NE overflow in DOCA-salt MA compared to sham MA.
Moreover, an aZ-adrenergic receptor agonist, UK 14,304 inhibited NE overflow in sham
MA more than DOCA-salt MA. This result is consistent with studies showing a failure of
yohimbine to increase plasma NE level [80, 81] and nerve stimulation—induced NE

overflow in the mesenteric vasculature of DOCA-salt rats [49, 79, 68]. Taken together

155

these data suggest that aZ-adrenergic autoreceptors may be impaired in DOCA-salt MA.
However, yohimbine or UK 14,304 did affect NE overflow less in sham and DOCA-salt
MV compared with sham MA. Also, there were no differences in NE overflow in the
presence of the drugs between sham and DOCA-salt MV. These experiments indicate
that NE release and its regulation through the prejunctional a2-adrenergic autoreceptor
(prejunctional negative feedback system) does not seem to be altered in the isolated MV
of the DOCA-salt hypertensive rat. Westfall et al. also observed that there was no
significant elevated NE release from the portal vein and no attenuation of the yohimbine
effect in DOCA-salt hypertensive rats [77]. However, the portal vein may not be an
appropriate blood vessel to study hypertension associated with changes in the function of
peri-venous sympathetic nerves because the portal vein makes only a small contribution

to total vascular capacitance.

Upregulation of NE reuptake in DOCA-salt MA. NET activity is responsible for
removing NE from the synaptic cleft once released from the nerve terminal [72]. Reduced
uptake has been proposed to explain the elevated NE associated with DOCA-salt
hypertension [87]. However, unaltered neuronal reuptake of NE in DOCA-salt
hypertensive rats [67] or enhanced NE clearance rate in chronically hypertensive animals
[212, 92] and in human with essential hypertension [17] have been reported. The
unaltered or enhanced neuronal uptake reflected that elevated NE overflow was due to
increased NE secretion rate subsequent to attenuation of the a2-presynaptic or local
inhibitory mechanism [205, 69]. However, the explanation for reduced or increased NE
reuptake in smooth muscle of DOCA-salt blood vessels afier the development of

hypertension is still not clear. The present study showed that blockade of NET, by

156

cocaine, resulted in an increased NE overflow in both sham and DOCA-salt MA but the
magnitude of increase in DOCA-salt MA was bigger than sham MA at all frequencies
and significantly greater at high fiequencies (10 and 20 Hz). However, when 0L2-
adrenergic autoreceptor and NET were blocked by the combined drug (yohimbine +
cocaine), NE overflow markedly increased in both sham and DOCA-salt MA but not
significantly different between sham and DOCA-salt MA.

The results suggest that cocaine-sensitive reuptake mechanism seems to be
upregulated in DOCA-salt MA. Altered neuronal reuptake of NE in DOCA-salt MA may
be caused by increased NE release due to impaired a2-adrenergic autoreceptors. The
increased NE release may stimulate increased NET expression in DOCA-salt MA [31].
However, cocaine affected NE clearance less in sham and DOCA-salt MV. The
combined drug increased NE overflow at low frequencies in sham and DOCA-salt MV
due to blockade of autoinhibition by yohimbine not by cocaine. The result indicates that
NE clearance for seems to be mainly by diffusion instead of through NET for sham and

DOCA-salt MV.

Postjunctional (1.2-adrenergic receptors in sham and DOCA-salt MV. The contractile
responses of sham and DOCA-salt MA at low frequencies (< 10 Hz) in the presence of
yohimbine and/or cocaine increased due to the elevated NE overflow and slow rate of NE
clearance in junctional cleft by yohimbine and cocaine, respectively while the contractile
response at high frequency ( S 10 Hz) relatively was unaffected by the drug treatment in
both sham and DOCA-salt MA. This result suggests that the junctional NE concentration

at the higher frequency may be sufficient to saturate receptor sites.

157

Elevated NE overflow in the presence of yohimbine, had little effect on neurogenic
constriction or even decreased the maximum constriction (Em) in MV from sham and
DOCA-salt hypertensive rats. Moreover, UK 14,304 preconstricted over 10 % in both
sham and DOCA-salt MV. The result suggests that postjunctional a2-adrenergic
receptors may also regulate neurogenic constriction of MV in sham and DOCA-salt rats.
Previous studies have proposed that a2-adrenergic receptors also located postj unctionally
and mediate contractile response in blood vessels [213-215]. However, UK 14,304 did
not preconstrict sham and DOCA-salt MA, suggesting that a2-adrenergic receptors may
not be expressed by MA and are not involved in contractile response to NB. This was

agreement with previous studies in rat and mice MA [31, 216].

Alternation vasculature and/or postsynaptic adrenergic dysfunction in DOCA-salt
MA and MV. The present and previous work showed that first, MV were more sensitive
than MA to nerve stimulation, which was based on the observation that the S 50 was lower
in sham and DOCA-salt MV compared with sham and DOCA MA [22, 31]. Therefore,
changes in neuroeffector transmission to vein in hypertension are important because
veins are more sensitive to the vasoconstrictor effects of sympathetic nerve stimulation
than arteries [22, 31]. Second, contractile response in MA was mediated mainly by ATP
because PPADS, P2X receptor antagonist greatly reduced neurogenic constriction of
sham MA while NE makes a major contribution to neurogenic constriction of DOCA-salt
MA because the constriction of DOCA-salt MA was greatly reduced by prazosin. There
was an increase in the adrenergic contribution to neurogenic constrictions in DOCA-salt
MA compared to that occurring in sham MA. This result is consistent with other studies

showing that postjunctional al-adrenergic function become dominant in DOCA-salt and

158

SHR rats [80] and strikingly increased in density of or] binding sites in DOCA-salt MA
[217, 31]. Previous study suggested that the increased adrenergic transmission may be
due to increased NE release but there was no changes in postjunctional sensitivity of
DOCA-salt MA to NE [31]. The presence of an imbalance in postjunctional adrenoceptor
functions may be associated with a change in neuroeffector mechanisms in DOCA-salt
MA and promotes the pressor effects of the sympathetic system [80]. However, elevated
NE overflow did not alter the contractile response in DOCA-salt MA, though NE made a
major contribution to neurogenic constriction of DOCA-salt MA and even, there was no
change in reactivity of al-adrenergic receptors to NE in DOCA-salt MA. The elevated
NE overflow caused an increase in NE uptake in DOCA-salt MA, the increased uptake
may offset the vasoconstrictor effects of elevated NE release in DOCA-salt MA.

Prazosin alone mostly blocked neurogenic constriction in both sham and DOCA—
salt MV. Therefore, NE mediates neurogenic constriction of sham and DOCA-salt MV.
Interestingly, my study shows that the E,,m value was significantly lower in DOCA-salt
MV compared with sham MV, though NE overflow was not significantly different
between sham and DOCA-salt MV. Therefore, the reduced contractile response was not
due to the decreased NE overflow in DOCA-salt MV from my data. Luo et al. suggested
that Oil-adrenergic receptor sensitivity to NE was decreased in DOCA-salt MV due to
downregulation of Oil-adrenergic receptors in DOCA-salt veins caused by the increased
sympathetic nerve activity [80, 176, 31]. Also, similar decrease in NE sensitivity occurs
in hand veins taken from human hypertensive subjects [209]. Other mechanisms include
altered vasculature structure in DOCA-salt rats may also account for this result. The

decreased compliance of DOCA-salt MV may cause redistribution of blood toward the

159

heart, leads to increased cardiac output to arterial side and finally will result in increased

arterial blood pressure [218, 81].

6.4 Conclusion

The present study demonstrates that prej unctional a2-adrenergic autoreceptors and
NE reuptake play a greater role in regulating NE availability at the neuroeffector junction
in MA than in MV. The elevated NE overflow from perivascular sympathetic nerves in
DOCA-salt MA is likely due to altered a2-adrenergtic autoreceptors. The elevated NE
overflow caused an increase in NE reuptake in DOCA-salt MA and the increased uptake
may offset the vasoconstrictor effects of elevated NE release in these tissues.

NE mediated venous constriction by acting at postjunctional Oil-adrenergic
receptors in MV from sham and DOCA-salt rats but contractile response was decreased
in DOCA-salt MV due to desensitized al-adrenergic receptors. ATP, acting at P2
receptors, was the dominant vasoconstrictor transmitter in small MA from sham rats. In
contrast, NE made a major contribution to neurogenic constriction of DOCA-salt MA due
to an increase in the adrenergic contribution to neurogenic constrictions. However,
changed contractile response in DOCA-salt rat may be also due to the altered vasculature
in DOCA-salt rats. Therefore, there may be altered vasculature and/or postjunctional
dysfunction in DOCA-salt MA and MV.

All the observations suggest that there was differential neural control of MA and
MV and the neural control of MA artery and MV were altered in DOCA-salt rats. The
alternation in the local mechanisms modulating sympathetic neuroeffector mechanisms in

hypertension will impact overall hemodynamics.

160

Chapter 7

Conclusions

1. Advantages of Diamond Microelectrode for the In Vitro Electroanalytical
Measurement.

The fabrication, electrochemical characterization and application of boron-doped
diamond microelectrodes for the in vitro continuous amperometric measurement of the
vasoconstrictor neurotransmitter, norepinephrine (N E), in Chapters 2 - 4. The diamond
microelectrode is formed by depositing a high quality, thin film of electrically conducting
diamond on a sharpened 76 um diam Pt wire. The electrode exhibits a low and stable
background current over a wide potential range that is independent of the solution pH.
Additionally, the bare electrode provides high resistance to deactivation and fouling
during exposure to laboratory atmosphere and complex biological environments, at least
in part, because of the non-polar, low oxygen, sp3-bonded carbon surface on which weak
adsorption of polar biomolecules and other contaminants occurs. In contrast, the carbon
fiber microelectrode exhibits a pH-dependent background current response with evidence
for electroactive surface carbon-oxygen functional groups, and deactivates irreversibly
during laboratory air exposure and contact with tissue. Diamond clearly provides superior
response sensitivity precision and stability making it useful for sensitive and stable

electroanalytical measurements in complex biological environments.

161

II. In Vitro Monitoring of Endogenous NE Overflow with a Diamond Microelectrode
and Vasoconstriction.

Continuous amperometry with the diamond microelectrode and video imaging were
used the elicited for the first time to simultaneously record endogenous NE overflow and
the evoked contractile response of a rat mesenteric artery. Electrical stimulation of
sympathetic nerve endings elicited a transient oxidation current that was attributed to
endogenous NE. NE overflow was elicited by electrical stimulation at frequencies
between 1-60 Hz, with a maximum response seen at 20 Hz. Confirmation that the
oxidation current was in fact associated with endogenous NE came fi'om the results of
several drugs. Tetrodotoxin (TTX), a voltage-dependent sodium channel antagonist
which blocks nerve conduction, abolished both the oxidation current and the arterial
constriction. The a2-adrenergic autoreceptor antagonist, yohimbine, caused an increase
in the oxidation current and the corresponding constriction. The addition of cocaine, a
monoamine reuptake blocker, caused both the oxidation current and the contractile
response to increase. These results, combined with the fact that the hydrodynamic
voltammetric Em for endogenous NE was identical to that found for a standard solution
confirmed that the oxidation current was due to the neurotransmitter. The TTX result also
suggested that NE overflow and the contractile response were mediated neurogenically.
The uncoated diamond microelectrode provided a sensitive, reproducible and stable
oxidation current response that varied with the stimulation frequency and pulse number in
a predictable manner, similar to that for a carbon fiber microelectrode. Furthermore, this
study demonstrated that continuous amperometric monitoring of NE with a diamond

microelectrode and video imaging of vascular diameter allow for real time local

162

measurement of the temporal relationship between nerve stimulated NE overflow and

arterial constriction.

III. Differential Sympathetic Neuroeffector Transmission to Mesenteric Arteries
(MA) and Veins (MV) from Rats.

The dual measurement technique was applied to investigate differences in
sympathetic neuroeffector transmission in normal rat MV and MA. A better
understanding of the firnctional differences is important because arteries and veins make
different contributions to overall hemodynamics. The results indicated the NE transporter
(NET) and prej unctional a2-adrenergic autoreceptors play a more prominent role in
controlling NE release at neuroeffector junction in MA than in MV. Therefore, NE
overflow in MV exceeded that in MA. These conclusions are based on following results:
i) NE overflow in MA was more strongly enhanced by the a2-adrenergic receptor
antagonist, yohimbine and significantly attenuated by a2-adrenergic receptor agonist, UK
14,304, compared to MV; and ii) NE overflow in MA increased more significantly in the
presence of cocaine or the combined drug (cocaine + yohimbine) compared to MV.

Neurogenic constriction of MV was mediated by NE acting at al-adrenergic
receptors while that of MA was mediated by ATP acting at P2X receptors as well as NE.
The conclusion is based on the observation that the P2 receptor antagonist, PPADS,
reduced the contractile response in MA but not in MV while the al-adrenergic receptor
antagonist, prazosin, blocked the contractile response in MV but only slightly in MA.
Glyoxylic acid (GA)-induced fluorescence images also showed that there are differences

in the arrangement of sympathetic nerves in MA and MV. The perisympathetic nerve

163

plexus in MV consisted of individual varicose axons largely arranged circumferentially
along the vessel, denser, more mash-like network was observed for MA. Taken together,
these results suggest that there are fundamental differences in sympathetic neuroeffector
transmission to arteries and veins, which contribute to their different hemodynamic

functions in regulation of arterial and venous tone.

IV. Altered Sympathetic Neuroeffector Transmission to MA and MV in DOCA-salt
Hypertensive Rats
Small MA and MV in vitro preparations from DOCA-salt hypertensive rats were
used what junctional differences are associated with the disease state. NE overflow in
DOCA-salt MA was less attenuated by UK 14,304, an a2-adrenergic receptor agonist,
and less elevated by yohimbine while cocaine increased NE overflow to a greater extent
compared to sham MA. However, the combined drug (cocaine + yohimbine) produced a
greater increase in NE overflow in both sham and DOCA-salt MA and elevated NE
overflow by the drug was not different between sham and DOCA-salt MV. These results
suggest that sympathetic nerves associated with DOCA-salt MA released more NE than
sham MA due to impaired prejunctional a2-adrenergic autoreceptors. The elevated NE
overflow caused an increase in NE reuptake in DOCA-salt MA and the increased
clearance may offset the vasoconstrictor effects of elevated NE release in these tissues. In
contrast, NE overflow was unchanged between sham and DOCA-salt MV.
PPADS greatly reduced neurogenic responses in sham but not DOCA-salt MA,
whereas prazosin inhibited response more in DOCA-salt MA compared to sham MA. The

result suggests that NE made a major contribution to neurogenic constriction of DOCA-

164

salt MA because the elevated NE release increased the adrenergic contribution to the
constriction of DOCA-salt MA. Contractile response in sham and DOCA-salt MV was
mostly blocked by prazosin but not by PPADS. Neurogenic constriction in MV from
sham and DOCA-salt rat was more sensitive than that in sham and DOCA-salt MA.
However, the maximum contractile response was decreased in DOCA-salt MV compared
to sham MV. It may be due to desensitization of al-adrenergic receptors and/or due to
the altered vasculature in DOCA-salt MV. The observations suggest that the altered
sympathetic neural control of both arteries and veins in hypertension will impact overall

blood pressure.

165

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