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THE DYNAMICS OF NOREPINEPHRINE IN THE

NEURONAI. CELL BODIES AND TERMINALS

Thesis for the Degree of Ph. D.

MICHIGAN STATE UNIVERSITY
RANBIR KRISHNA BHATNAGAR

1971

 

        

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THE DYNAMICS OF NOREPINEPHRINE IN THE NEURONAL

CELL BODIES AND TERMINALS

presented by

RANBIR K. BHATNAGAR

has been accepted toWards fulfillment
of the requirements for

Ph.D. degree in PharmacoIogy

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Kennet E. Moore

 

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ABSTRACT

THE DYNAMICS OF NOREPINEPHRINE IN THE NEURONAL
CELL BODIES AND TERMINALS

BY

Ranbir Krishna Bhatnagar

Steady state concentrations of norepinephrine are
maintained in peripheral tissues by synthesis and by
reuptake of released norepinephrine despite variable and
prolonged sympathetic stimulation. The relative
importance of these two mechanisms during periods of
varying activity of noradrenergic neurons is not well
understood, but it appears that the properties of storage,
uptake, and metabolism of norepinephrine in the terminals
are different from those in the cell bodies. The purpose
of the present study was to investigate the factors that
regulate the content of norepinephrine in cell bodies and
terminals of noradrenergic nerves during stimulation and
post-stimulation periods.

The contents of norepinephrine in superior cervical
ganglia, which represent cell bodies, and in submaxillary
salivary glands and nictitating membranes, which contain
terminals of postganglionic sympathetic neurons, were
determined in cats following various treatments. The

synthesis of norepinephrine was inhibited by

Ranbir Krishna Bhatnagar

a-methyltyrosine infused through both common carotid
arteries,and the reuptake of norepinephrine was blocked
by intravenous injections of desmethylimipramine.
Decentralized preganglionic fibers were stimulated uni-
laterally at 2 or 10 hz for l or 3 hours. The eXperiments
were designed so that contralateral tissues served as the
apprOpriate controls.

In decentralized, nonstimulated preparations, 3
hours of o-methyltyrosine infusion reduced the norepinephrine
content in the cell bodies but not in the terminals.
Desmethylimipramine did not alter the norepinephrine
content of cell bodies or terminals.

Low or high frequencies of preganglionic stimulation,
alone or in combination with desmethylimipramine or a-
methyltyrosine, did not alter the norepinephrine content
in the cell bodies. On the other hand, 1-3 hours of
stimulation at 10 hz partially depleted norepinephrine in
the terminals. a-Methyltyrosine enhanced the stimulus-
induced depletion at both low and high frequencies,
whereas desmethylimipramine increased the depletion of
norepinephrine only at high frequencies. The a-
methyltyrosine-induced depletion of norepinephrine did
not result from replacement by a-methylnorepinephrine.

Preganglionic stimulation had little effect upon
the conversion of tyrosine-14C to norepinephrine-14C in

cell bodies but markedly increased the formation of

Ranbir Krishna Bhatnagar

norepinephrine-14C in terminals. a-Methyltyrosine
reduced the synthesis of norepinephrine-14C in all tissues.

To examine the dynamics of norepinephrine in cell
bodies and terminals following periods of intense
stimulation,the contents and rates of synthesis of this
amine were determined immediately after,and at 2 and 6
hours after,the cessation of 3 hours of preganglionic
stimulation. In the cell bodies, neither the endogenous
norepinephrine contents nor the formation of norepinephrine-
14C from tyrosine-14C was altered at any time. In the
terminals, partial restoration of the norepinephrine
content occurred within 2 hours after cessation of
stimulation,but did not return to control values during
the next 4 hours. Partial restoration of norepinephrine
was prevented by a-methyltyrosine but not by desmethyl-
imipramine. The rate of formation of norepinephrine-14C
was accelerated during the 30 minute period immediately
after cessation of stimulation,but thereafter decreased
progressively as the endogenous norepinephrine concen-
trations increased. At 6 hours after cessation of
stimulation, when the endogenous concentrations of
norepinephrine were still reduced, the rate of conversion
of tyrosine-14C to norepinephrine-14C was not different
from nonstimulated controls.

These results suggest that in cell bodies synthesis

of norepinephrine proceeds at a rapid rate that is

Ranbir Krishna Bhatnagar

independent of nerve activity, and concentrations of
norepinephrine are maintained only by synthesis. In
terminals, synthesis of norepinephrine proceeds at a

slow rate in the absence of nerve activity; with low
frequencies of stimulation concentrations of norepinephrine
are partially maintained by synthesis,and at higher
frequencies by both synthesis and reuptake. When the
norepinephrine contents of terminals are extensively
depleted by stimulation, synthesis only partially
restores the norepinephrine concentrationsgsuggesting
that,if the rate of norepinephrine synthesis is regulated
by a feedback control mechanism,only part of the neuronal

stores of norepinephrine participate in this regulation.

THE DYNAMICS OF NOREPINEPHRINE IN THE NEURONAL

CELL BODIES AND TERMINALS

BY

Ranbir Krishna Bhatnagar

A THESIS

Submitted to
Michigan State University
in partial fulfillment of the requirements

for the degree of

DOCTOR OF PHILOSOPHY

Department of Pharmacology

1971

ACKNOWLEDGMENTS

The author is extremely indebted to Dr. Kenneth E.
Moore for his unceasing encouragement, guidance,
unfailing patience and assistance in all phases of this
study.

Grateful thanks are due to Drs. Theodore M. Brody,
Gerard L. Gebber, John H. McNeill and John 1. Johnson, Jr.
for their advice and constructive criticism during the
preparation of this thesis.

He wishes to thank Mrs. Barbara J. VonVoigtlander and
Mrs. Mirdza Gramatins for their excellent technical

assistance.

ii

TABLE OF CONTENTS

INTRODUCTION

METHODS

A. Surgical and EXperimental Methods
B. Biochemical Methods

1.
20

Assay of a-methyltyrosine in plasma
Assay of endogenous norepinephrine
content of superior cervical ganglia
Assay of endogenous norepinephrine
in salivary glands and nictitating
membranes 14

Assay of norepinephrine- C in
superior cervical gangli

Assay of norepineprhine- C in
salivary glands and nictitating
membranes

Assay of radioagtive o-methyl-
norepinephrine- H

Calculations of radioactivity in
tissues

C. Administration of Drugs and IsotOpes
D. Statistical Methods

RESULTS

A. The Dynamics of Norepinephrine in the
Neuronal Cell Bodies During the Resting
State

1.

Norepinephrine concentrations in
tissues from the right and left side

of the same cat

Plasma and tissue concentrations of
a-methyltyrosine

Tissue norepinephrine concentration
after infusion of a-methyltyrosine
Effects of desmethylimipramine and
a-methyltyrosine on the contents of
norepinephrine in nonstimulated tissues

iii

Page

33
33
37
37

38

40

41

43
44
47
47
48

49

49

49
51

56

S8

Page

B. The Dynamics of Norepinephrine in the
Neuronal Cell Bodies and Terminals During
Electrical Stimulation 62
1. Effects of stimulation, a-methyl-
tyrosine and desmethylimipramine on tissue

contents of norepinephrine 62
a. Effect of stimulation 62
b. Effects of stimulation and o-
methyltyrosine 63
c. Effects of stimulation and
desmethylimipramine 63

d. Effects of stimulation, a-
methyltyrosine and desmethyl-
imipramine 82
2. Effects of preganglionic stimulation
and a-methyltyrosine on the conversion
of tyrosine-1 C to norepinephrine-l C 83
3. Effects of stimulation and cycloheximide
on the tissue contents of norepinephrine
and on the conversion of tyrosine- 4C

to norepinephrine-14C 89
4. Possible fate of norepinephrine in

cell bodies 93
5. Possible formation of a-methyl-

norepinephrine from a-methyltyrosine 95

6. Norepinephrine contents and the con—
tractile responses of nictitating
membranes 97
a. Effects of a-methyltyrosine and
continuous stimulation at low
frequency (2 hz) 97
b. Effects of a-methyltyrosine and
continuous stimulation at frequen—
cies of 2 hz, 10 hz and 16 hz 101
c. Effects of a-methyltyrosine,
desmethylimipramine and intermittent
stimulation at low (2 hz) and high
(10 hz) frequencies 103
C. The Dynamics of Norepinephrine in the
Neuronal Cell Bodies and Terminals During
the Post-stimulation Period 106
1. Restoration of norepinephrine after
cessation of stimulation and the
effects of desmethylimipramine and
a-methyltyrosine 14 106
2. Conversion If depamine- C to nore-
pinephrine- C during the post-
stimulation period 14 114
3. Conversion Ti tyrosine— C to nore-
pinephrine- C during and at various
times after the cessation of
stimulation 115

iv

DISCUSSION

A. Regulation of Norepinephrine Content in
the Neuronal Cell Bodies and Terminals
During Stimulation

B. Regulation of Norepinephrine Content in
the Neuronal Cell Bodies and Terminals
After Stimulation

C. The Noradrenergic Neuron:A Single
Functional Unit

SUMMARY AND CONCLUSIONS

BIBLIOGRAPHY

Page

127

127

140
145
150

154

Table

10

LIST OF TABLES

Page

Norepinephrine concentration of tissues from
right and left side of individual untreated
cats 50

Tissue norepinephrine (NE) content after
three hours of a-methyltyrosine infusion 57

Tissue norepinephrine (NE) content after

three hours of a-methyltyrosine (aMT)

infusion in presence of desmethylimipramine

(DMI) 61

Effects of stimulation and 5 1/4 hours of
a-methyltyrosine (aMT) infusion on tissue
contents of norepinephrine (NE) 64

Effects of a-methyltyrosine (aMT), des-
methylimipramine (DMI) and electrical

stimulation at 2 and 10 hz on tissue con-

tents of norepinephrine (NE) 68

Effects of continuous and intermittent
stimulation on tissue contents of nor-
epinephrine (NE) 70

Effects of a-methyltyrosine (aMT), des-
methylimipramine (DMI), and stimulation for

one and three hours on tissue contents of
norepinephrine (NE) 72

Effects of stimulation and desmethylimi-
pramine (DMI) on tissue contents of
norepinephrine (NE) 76

Effect of stimulation and desmethylimi-
pramgne on uptake of norepinephrine—H
(NE-H 77
Effect of stimulation and a-methyl—
tyrosine (aMT) on the fqnversion of i.a.
inistez ed tyrosine- C to norepinephrine-
C (NE-J 84

vi

Table
11

12

l3

14

15

16

17

18

19

20

21

22

Effect of stimulation on the conversion of
i.v. administered t rosine-14C to norep-
inephrine-14C (NE-1 C)

Effect of cycloheximide on tissue contents
of norepinephrine

Effects of stimulation and cycloheximide on
tissue contents of endogenous norepinephrine

Effect of stimulation and cycloheximide on
the conversion of i.a. administered 14
tyrosine-14C to norepinephrine-l C (NE— C)
Effects of pheniprazine and three hours of
a-methyltyrosine (aMT) infusion on the
tissue contents of norepinephrine (NE)

Effects of a-methyltyrosine (aMT), phenip-
razine and tying of postganglionic fibers
on the norepinephrine (NE) content of
ganglia

Formatiqn of Wap arent a-methylnorepine-
phrine- H (aMNE-EH H)" af er infusion of a-
methyltyrosine- H (aMT- H)

Effects of stimulation and o-methyltyrosine
(aMT) on the norepinephrine (NE) contents
and contractile response of nictitating
membranes

Effects of desmethylimipramine (DMI) and
a-methyltyrosine (aMT) on the recovery of
tissue contents of norepinephrine (NE)
following stimulation-induced depletion

Formation of npiepinephrine-14C (NE-14C)
from dopamine- C six hours after the
cessation of stimulation

ConversipR of tyr sine-14C to norepin-
ephrine- C (NE-1 C) in superior cervical
ganglia during and at various times after
cessation of stimulation

Conversiog of tyrosine-14C to norepin-
ephrine-1 C (NE-14C) in salivary glands
during and at various times after cessation
of stimulation

vii

Page

88

9O

91

92

94

96

98

102

109

113

117

118

Table

23

24

Conversio 20f tyigsine-14C to norepin-
ephrine-Y 2C (NE- C) 1n nictitating
membranes during and at various times after
cessation of stimulation

Effect of desmethylimipramine (DMI) on the

conversion of i. a. administered tyrosine-
C to norepinephrine-14C (NE—14C) six

hours after the cessation of stimulation

viii

Page

120

122

Figure

10

LIST OF FIGURES

Page

Pathway for the biosynthesis of
norepinephrine 8

Schematic drawing of the surgical
preparation 35

Determination of the purity of a-methyl-
tyrosine—3H (aMT—3H) 46

Plasma concentrations of a-methyl-
tyrosine (aMT) following i.a. infusion
of the drug 53

The content of norepinephrine (NE) in
nictitating membranes and superior

cervical ganglia after the infusion of
a-methyltyrosine (aMT) 55

Tissue contents of norepinephrine (NE)
following administration of desmethyl-
imipramine (DMI) and/or a-methyltyrosine

(aMT) in nonstimulated preparations 60

Tissue contents of norepinephrine (NE)
following the administration of desmethyl-
imipramine (DMI) and/or a-methyltyrosine

(aMT) in stimulated preparations 67

Effect of desmethylimipramine (DMI) on
the contractile response of nictitating
membranes 75

Effects of a-methyltyrosine (aMT), des-
methylimipramine (DMI) and stimulation on

the norepinephrine (NE) contents of

ganglia, salivary glands and nictitating
membranes 80

Effects of stimulation and a-methyl-

tyrosine (aMT) on the contractile responses
of the nictitating membranes 100

ix

Figure

11

12

13

Contractile responses of the nictitating
membranes following the administration
of desmethylimipramine (DMI) and/or
a-methyltyrosine (aMT) in stimulated
preparations

Effects of desmethylimipramine (DMI) and
a-methyltyrosine (aMT) on the post-
stimulation recovery of tissue contents
of norepinephrine (NE)

Norepinephrine igntentl and formation of
norepinephrine- C (NE-14C) from tyrosine-

4C following stimulation and during
post-stimulation periods

Page

105

108

125

INTRODUCTION

The hypothesis of chemical mediation of synaptic
transmission and the role of the cell body in the homeo-
stasis of nerve function was formulated by Scott in 1906:

"I put forward the hypothesis that in the
body of the nerve cell a substance is formed
from the nucleus and Nissl bodies which
gradually passes into the nerve fibres;

and also that stimulation of other cells by
a nerve fibre is brought about by the
passage of some of this substance into the
cells on which the fibre acts... The nerve
cells secrete a substance the passage of
which from the nerve endings is necessary
to stimulation."

It is now recognized that norepinephrine functions
as a chemical transmitter at terminals of the peripheral
sympathetic nervous system (Euler, 1956). Much of the
knowledge of the pharmacological, biochemical and physio-
logical aSpects of the noradrenergic function has been
derived by altering either the affinity of post synaptic
receptors for norepinephrine or the storage, metabolism,
release and uptake of norepinephrine at the nerve
terminals (Goodman and Gilman, 1970). Less is known
about the dynamics of norepinephrine in other parts of

the neuron although some effort has been made to examine

the properties of this amine in the cell body (Costa et al.,

1961; Reinert, 1963; Norberg, 1967; Csillik et al.,
1967; Jacobowitz and Woodward, 1968; Van Orden et al.,
1970a) and the axon (Roth et al., 1967; Livett et al.,
1969; Dahlstrom and Haggendal, 1970).

The diSposition and dynamics of norepinephrine in
cell bodies appear to be quite different from that in
the terminals of noradrenergic neurons. For example, in
the cell bodies norepinephrine is located in the super-
natant of cell homogenates or immature vesicles, whereas
in the terminals most of the norepinephrine is contained
in well defined vesicles (Fischer and Snyder, 1965; Costa,
1970). The turnover of norepinephrine in ganglia (cell
bodies) is much faster than in the terminals (Fischer and
Snyder, 1965; Brodie et aZ., 1966) and cocaine blocks the
uptake of norepinephrine at noradrenergic nerve terminals
but not at cell bodies (Fischer and Snyder, 1965). Most
of the protein synthesis takes place in the cell bodies;
little RNA is found in the axons and nerve terminals
(Hyden, 1958). The functional significance of these
differences is not fully understoodimlralways appreciated.
For example, these differences can complicate the inter-
pretation of the action of drugs on norepinephrine
dynamics in those tissues (brain, gut, vas deferens,
uterus) that contain both cell bodies and terminals
(Costa, 1970). Furthermore, many efforts to demonstrate

a functional role of norepinephrine in ganglionic

transmission have been contradictory and inconclusive
(Costa, 1961; Reinert, 1963; Weir and McLennan, 1963;
Eccles, 1964; DeGroat, 1967). The purpose of the present
study was to compare the effects of neuronal activity on

the dynamics of norepinephrine in cell bodies and

terminals of the same neuron, and evaluate the signifi-
cance of the differential characteristics in the disposition

of norepinephrine at these two sites of the neuron.

Anatomical Considerations

 

The cell bodies of the preganglionic nerve fibers
of the peripheral sympathetic nervous system are located
in the central nervous system. The multipolar cell bodies
of postganglionic noradrenergic neurons are generally
located in autonomic ganglia (superior cervical, stellate,
coeliac, inferior mesentric). In some regions, gut,
vas deferens, urinary bladder, uterus, they are located
intramurally. Estimates of the number of neurons in the
superior cervical ganglia range from 20,000-30,000 in the
cat, 63,625 in the squirrel monkey and 1,041,652 in the
human to 35,528 cells/mm3 in monkey (Ebbeson, 1968;
Giacobini, 1970). Long or short unmyelinated axons (0.2-
l u in diameter) emanate from cell bodies to innervate
smooth muscle (nictitating membranes, iris, gut, uterus),
cardiac tissue and glands. The axons terminate in the
effector tissue as a "ground-plexus" of knobbed enlarge-

ments or varicosities (Ranson and Clark, 1959; Norberg,

1967; Dahlstrom and Haggendal, 1970). The distance
between the axon terminal and the effector cell is 200-
500 A (Van Orden et al., 1967). Some axons terminate on
the cell bodies of their origin (axon collaterals) along
with the terminals of the interneurons (Norberg, 1967;

Jacobowitz and Woodward, 1968; Van Orden et al., 1970a).

Localization and Storage

 

Using bioassay, chemical and histochemical techniques,
sympathetic ganglia have been shown to contain high
contents of norepinephrine (Vogt, 1954; Kirpekar et al.,
1962, Norberg, 1967). The norepinephrine content of
superior cervical ganglia in dog ranges from 3-12 ug/g
and that of stellate ganglia from 3.8-5.5 pg/g (Vogt,
1954). Autonomic ganglia of the cat contain 6-25 ug/g
norepinephrine (Norberg and Hedqvist, 1966). When a
ganglia homogenate is placed on a sucrose density-gradient
and subjected to ultracentrifugation most of the nor-
epinephrine is recovered in the supernatant fraction
(Fischer and Snyder, 1965). The norepinephrine recovered
in the supernatant fraction represents "soluble" or
"free" norepinephrine. The advent of fluorescence micro-
sc0py supported this conclusion as norepinephrine in cell
bodies was found to be diffusely distributed (Norberg,
1967). Nevertheless, in most autonomic ganglia the nerve
terminals of axon collaterals or interneurons that surround

the cell bodies contain characteristic dense core vesicles

which are presumably filled with norepinephrine
(Norberg and Hedqvist, 1966; Phillipu et aZ., 1967;
Jacobowitz and Woodward, 1968; Van Orden et al., 1970a).
The vesicular proteins could be free in the ax0p1asm or
be present in mature or immature vesicles or in mito-
chondria (Grafstein, 1969). Therefore, norepinephrine
could be present in cell bodies both in "granular" and
”agranular" forms. Nevertheless, it is not clear as to
what proportion of ganglionic norepinephrine is "granular"
and whether the various storage forms of norepinephrine
in cell bodies represent a morphological or functional
entity. The disposition of norepinephrine in ganglia is
further complicated by the presence of norepinephrine-
containing "chromaffin" cells (Norberg and Hedqvist,
1966; Van Orden et al., 1970a).

The norepinephrine contents of postganglionic nerves
which contain the axons of neuronal cell bodies range
from 3.6 to 15 ug/g (Euler and Hillarp, 1956; Geffen and
Rush, 1968). This norepinephrine is largely located
and bound within dense core storage vesicles (Euler and
Hillarp, 1956; Potter, 1966; Van Orden et al., 1966).

The norepinephrine contents of tissues innervated
by postganglionic nerves (Spleen, heart, salivary glands)
range from 1 to 4 ug/g (Euler and Hellner—ijrkman, 1955;
Euler and Hillarp, 1956; Iversen, 1967). The estimate

for the concentration of norepinephrine in the nerve

terminals is 1000 to 3000 ug/g or greater (Dahlstrom
and Haggendal, 1970).

Blaschko and Welch (1953) first identified granules
in the adrenal medulla which could store norepinephrine.
Since the relative amounts of norepinephrine in the
noradrenergic terminals were many fold greater than those
present in the axon or cell bodies, it was postulated
that norepinephrine must be protected and stored in the
nerve terminals in a manner similar to that in the
adrenals. Subsequently norepinephrine containing
particles were identified in the bovine Splenic nerves
(Euler and Hillarp, 1956) and in the nerve terminals of
the tissues using density gradient centrifugation techni—
ques (Potter, 1966), electron microsc0py (Wolfe et al.,
1962; Kapeller and Mayor, l967; Hékfelt, 1969) autoradio-
graphy (Wolfe et al., 1962) and fluorescence microsc0py
(Dahlstrom and Haggendal, 1970; Van Orden et al., 1970a).
The storage vesicles are about 500 A in diameter and
osmOphilic in nature; they contain norepinephrine which
is complexed with adenine nucleotides, Mg ions and a
Specific protein (Douglas, 1968). The vesicles presumably
contain dOpamine-B-hydroxylase (Kaufman and Friedman,
1965) and can concentrate norepinephrine (Iversen, 1967).
The association of the norepinephrine—concentrating
vesicles with the nerve terminals was confirmed by the

inability of the nerve terminals to concentrate

norepinephrine after denervation (Strémblad and Nickerson,
1961), chemical sympathectomy (Porter et al., 1963) or
immunosympathectomy (Levi-Montalcini and Angeletti, 1966).
In addition to their ability to concentrate
norepinephrine, the storage vesicles can retrieve dOpamine,
which in turn is oxidized to norepinephrine. Thus,
these vesicles prevent the oxidative deamination of both
catecholamines by intraneuronal monoamine oxidase. The
storage vesicles are thought to originate in cell bodies
from where they are transported somatofugally (Dahlstrom
and Haggendal, 1970). The Tl/Z of the vesicles has been
estimated to be about 3 weeks (Dahlstrom and Haggendal,

1970).

Biosynthesis and Catabolism

 

The biosynthesis of norepinephrine from tyrosine is
catalyzed by three enzymatic steps (Nagatsu et al., 1964).
This is illustrated in Figure 1.

The formation of dihydroxyphenylalanine (DOPA) is
considered to be the rate limiting step in the formation
of norepinephrine from tyrosine (Levitt et al., 1965).
This assumption is based on the following observations:

5M and

The Km of tyrosine hydroxylase is about 1-2 x 10-
the tissue concentration of tyrosine is 5 x lO-SM so that
the enzyme is normally saturated; little DOPA and

dOpamine are present in peripheral noradrenergic tissues;

the rate of norepinephrine synthesis is saturated only

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with tyrosine as a precursor; rates of formation of DOPA
are the same as that of norepinephrine; the activity of
tissue tyrosine hydroxylase is much lower than that of
DOPA decarboxylase and dOpamine-B-hydroxylase; agents

that markedly inhibit DOPA decarboxylase and dOpamine-B-
hydroxylase activity do not markedly lower tissue
norepinephrine contents, whereas inhibition of tyrosine
hydroxylase activity markedly lowers tissue norepinephrine
contents.

Some controversy still exists over the subcellular
localization of enzymes required for the synthesis of
norepinephrine. Dopamine-B-hydroxylase is associated with
the particulate fraction of tissue homogenates and is
present in the storage vesicles in synaptosomes (Kaufman
and Friedman, 1965). On the other hand tyrosine
hydroxylase and DOPA decarboxylase have been identified
in the cytoplasm (Laduron and Belpaire, 1968; Mussachio,
1968; Livett et al., 1969; Mueller et al., 1969a) and in
association with "granular" vesicles (Spector et al.,
1967; Stjarne and Lishajko, 1967). All three enzymes
are present in cell bodies (Fischer and Snyder, 1965;
Mueller et al., 1969a), axons (Stjarne and Lishajko,
l967; Geffen and Rush, 1968; Laduron and Belpaire, 1968;
Livett et al., 1969) and nerve terminals (Sedvall and

KOpin, l967a; Weiner, 1970).

10

Norepinephrine is enzymatically degraded by two
major pathways: oxidative deamination by monoamine
oxidase and methylation of the meta hydroxyl group by
catechol-O-methyltransferase (Axelrod et al., 1958;
KOpin, 1964). Monoamine oxidase is present both intra-
neuronally and extraneuronally. Intraneuronal monoamine
oxidase is located in mitochondria. Catechol-O—methyl—
transferase is recovered in the cytOplasmic fraction of
tissue homogenates, but some may be associated with
synaptic membranes (Alberici et al., 1965). Both monoamine
oxidase and catechol-O-methyltransferase are present in
cell bodies (Fischer and Snyder, 1965; Cervoni, 1969)
and nerve terminals (KOpin, 1964; Cervoni, 1969). The
route of norepinephrine metabolism is dependent upon the
site of metabolism. Intraneuronal norepinephrine is
deaminated and extra neuronal norepinephrine is O-
methylated to form normetanephrine. Normetanephrine
might be deaminated, in turn, to form 3-methoxy-4-
hydroxymandelic acid. However, enzymatic degradation of
norepinephrine does not seem to be the major pathway for
the inactivation of physiologically released nor-
epinephrine. Inhibition of catechol-O-methyltransferase
(e.g., by pyrogallol) does not elevate the tissue contents
of norepinephrine. Inhibition of both monoamine oxidase
(e.g., by pheniprazine or iproniazid) and catechol-O-

methyltransferase fails to potentiate noradrenergic

11

function (Crout, 1961). There appear to be Species
differences in the metabolism of norepinephrine. Mono-
amine oxidase inhibitors have little effect on the

tissue contents of norepinephrine in the cat but increase

the amine contents in the rat (Spector et al., 1963).

Axonal Transport

 

Weiss and Hiscoe (1948) demonstrated the somato-
fugal transport of cytoplasmic proteins in mouse retinal
ganglion cells. Similar somatofugal transport of
norepinephrine and proteins has been confirmed for
different species in both sensory and motor neurons and
in the central nervous system (Grafstein, 1969;
Dahlstr6m and Haggendal, 1970). In sciatic and Splenic
nerves norepinephrine is transported somatofugally; in
Splenic nerves dopamine-B-hydroxylase is also transported
in this manner (Geffen and Rush, 1968; Laduron and
Belpaire, 1968; Livett et al., 1969; Dahlstrom and
Haggendal, 1970).

There is a slow and a fast component for axonal
transport of protein (Grafstein, 1969). The slow
component might serve in the maintenance and renewal of
structural elements of the neuron and the fast component
as a communication mechanism between cell bodies and the
nerve terminals. The fast axonal transport presumably
occurs through the neurotubules. Estimates for the rate

of transport of the slow component vary from 0.2 to

12

1 mm/day and for the fast component from 100 to 930 mm/
day (Grafstein, 1969). The rate of somatofugal transport
of norepinephrine and newly synthesized protein is
identical (5 mm/hour) in noradrenergic nerves (Kapeller
and Mayor, l967; Livett et al., 1968).

The assembly of neurosecretory granules is initiated
in the cell bodies. Proteins that are synthesized in
cell bodies might be directly incorporated into cell
organelles or vesicles for movement through the axonal
matrix or be free in the axoplasm and mitochondria
(Grafstein, 1969; Dahlstrom and Haggendal, 1970). The
membranes, granules and synaptic vesicles carried by
ax0plasmic stream tend to settle at all terminal cul-de-
sacs of axons (Lubinska, 1964).

The axonal transport of norepinephrine and proteins
is independent of somatic influences and nerve impulse
flow. Inhibition of protein synthesis (Peterson et aZ.,
1967) or electrical stimulation (Geffen and Rush, 1968)
does not alter the rate of axonal tranSport of nor-
epinephrine or proteins. Disruption of neurotubules by
colchicine prevents the axonal transport of norepinephrine
(Dahlster and Haggendal, 1970). Although norepinephrine
in the cell bodies and axons does not appear to contribute
to the minute to minute requirements of this amine at
nerve terminals, the norepinephrine synthesis in the

nerve terminals seems to be dependent upon a continual

l3

supply of enzymes required for the biosynthesis of this

amine (Livett et al., 1968).

Release

Transmission of excitation from noradrenergic nerves
to cardiac tissue, smooth muscle and glands is the
result of depolarization of the effector membrane caused
by norepinephrine released from the nerve terminals on
arrival of the action potentials. Uptake of Ca++ is
thought to be associated with the coupling of excitation
and release of norepinephrine. An estimate of release
has been obtained by measuring the responses of effector
organs and by collecting and analyzing the venous
effluents for norepinephrine following nerve stimulation
of heart, Spleen, liver and intestines. Both endogenous
and exogenously administered norepinephrine are released
from the nerve terminals following electrical stimulation
(Blakeley et al., 1964; Gillespie and Kirpekar, 1965).
In those preparations where venous effluent was not
collected the reduction in tissue contents of nor-
epinephrine was considered as an indirect measure of
release of norepinephrine (Fredholm and Sedvall, 1966).

Much of the knowledge about neuronal release of
norepinephrine has been obtained from studies with the
adrenal medulla (Douglas, 1968) and with nerve terminals
in the spleen (Brown and Gillespie, 1957). Little is

known about the release of norepinephrine from the ganglia.

14

Small amounts of norepinephrine were released in the
venous effluent of isolated perfused superior cervical
ganglia following preganglionic nerve stimulation
(Bfilbring, 1944). Norepinephrine was detected in the
venous outflow from intact and chronically decentralized
ganglia (Reinert, 1963). Orthodromic and antidromic
stimulation of ganglia increased the norepinephrine
content in the venous outflow but this increase was not
consistently obtained (Reinert, 1963). Electrical
stimulation of isolated Splenic nerves did not cause the
release of norepinephrine (Roth et al., 1967).

Release of small amounts of norepinephrine occurs
Spontaneously from the nerve terminals as indicated by
the excitatory junction potentials. Spontaneously
released norepinephrine presumably occurs in "quantal
packets” (Burnstock and Holman, 1966). The basal rate
of norepinephrine release could be increased many fold
on stimulation of noradrenergic nerves supplying the
tissues; the stimulus-induced release is frequency
dependent (Brown, 1960; Folkow et aZ., 1967). At low
frequencies of stimulation (0.5-2 hz) less norepinephrine
is released than at higher frequencies (5-10 hz)
(Haefely et al., 1965; Folkow et al., 1967).

Stimulus-induced release of norepinephrine in
isolated perfused Spleen at 0.5 hz is 1.5 ng/stimulus

and at 1-2 hz, 4.5 to 5.5 ng/stimulus. With higher

15

stimulation rates (4—8 hz) no further increase is found
in the release of norepinephrine (Haefely et al., 1965).
At low frequencies of stimulation the output of nor-
epinephrine from the nerve terminals could be sustained
during prolonged periods of stimulation (Euler and
Hellner-Bjorkman, 1955; Folkow et al., 1967). With
frequencies higher than 10 hz norepinephrine overflows
into circulation, responses of effector tissue reach a
maximum, and the tissue is partially depleted of
norepinephrine. Therefore, it is thought that physiological
frequency of afferent input is around 2 hz with 8-10 hz
being the upper limit of tonic sympathetic discharge
(Brown, 1960; Folkow et al., 1967).

In adrenal glands both norepinephrine storage
vesicles and d0pamine-8-hydroxy1ase are intimately
associated with ATP and Mg ions. The contents of the
storage vesicles are extruded by exocytosis (Douglas,
1968; Viveros et al., 1969). Evidence for the release
of storage vesicles from noradrenergic terminals is
controversial (Stjarne et al., 1970) although stimulus-
induced release of synaptic vesicles and dOpamine-B-
hydroxylase from spleen has been reported (Geffen et al.,

1969; Gewirtz and KOpin, 1970).

l6

Disposition of Released Norepinephrine

 

Studies of the disposition of released norepinephrine
have been conducted with sympathetically innervated
tissues (Spleen, heart, salivary glands and intestines)
(Iversen, 1967). Little is known about the fate of
norepinephrine which is supposedly released from the
cell bodies. The norepinephrine released at the nerve
terminals can be disposed in at least three ways:
reuptake into the nerve terminals, diffusion into the
circulation, enzymatic degradation.

Sympathetically innervated tissues can concentrate
circulating norepinephrine (Str6mblad and Nickerson,
1961; Whitby et al., 1961); the rate of accumulation
depends on the blood flow and density of noradrenergic
innervation (Kopin et al., 1965). The uptake of
norepinephrine into the nerve terminals is an active
process (Trendelenburg, 1969). Norepinephrine in the
synpatic cleft is transported across the neuronal
membrane and then into the storage vesicles. The
transport of norepinephrine at the neuronal membrane is
blocked by cocaine, imipramine, desmethylimipramine and
phenoxybenzamine (Brown, 1960; Dengler et al., 1961;
Axelrod et al., 1962; Sigg et aZ., 1963; Fischer and
Snyder, 1965). Desmethylimipramine and reserpine also
block the transport of intraneuronal norepinephrine into

the storage vesicles (Brodie et al., 1968). Blockade of

17

uptake of norepinephrine into the nerve terminals

results in an elevation in the norepinephrine contents

in the perfusate of isolated Spleen following stimula-
tion of Splenic nerves (Brown, 1960), a decrease in the
uptake of norepinephrine and norepinephrine-3H by a
variety of tissues (Axelrod et al., 1961) and an

increase in the contraction of nictitating membranes
following electrical stimulation of sympathetic nerves
(Sigg et al., 1963). Surgical denervation (Stromblad

and Nickerson, 1961) or chemical sympathectomy (Jonsson
and Sachs, 1970) greatly reduce the uptake of exogenously
administered norepinephrine. The results of the above
studies indicate that most of the norepinephrine released
at nerve terminals is reincorporated into the nerve
terminals. This mechanism is believed to be the major
means for inactivating neuronally released norepinephrine
(Axelrod et al., 1961). Norepinephrine which is retrieved
into the nerve terminals is also available for release
(Blakeley et al., 1964).

The fate of norepinephrine released from ganglia is
unknown but the ganglia can retrieve circulating
norepinephrine (Fischer and Snyder, 1965). However,
cocaine does not block the uptake of norepinephrine into
the ganglia (Fischer and Snyder, 1965).

Some of the norepinephrine that is released into

the synaptic cleft diffuses into the blood. The amount

18

of diffusion is also frequency dependent (Brown, 1960).
Within the physiological range of stimulation fre-
quencies little norepinephrine diffuses Coverflows") into
the circulation (Brown, 1960; Haefely et al., 1965; Folkow
et al., 1967). At "supraphysiological" frequencies
(>12-l4 hz) or after the blockade of reuptake of nor-
epinephrine considerable amounts of norepinephrine
overflow into the circulation. Reuptake mechanisms do
not compensate for the excess release of norepinephrine
at higher frequencies indicating that this process is
saturable (Folkow et al., 1967). Approximately 70—80%

of released norepinephrine is reincorporated into the
terminals.

The balance between release and uptake of
norepinephrine can be sustained indefinitely if the
norepinephrine released per minute is below 1-1.5% of
total tissue norepinephrine contents (Folkow et al.,

1967; Iversen, 1967). The possibility remains,

however, that during depolarization uptake of nor-
epinephrine is reduced (Palaic and Panisset, 1969;

Folkow et al., 1967), and therefore at high frequencies

of stimulation less norepinephrine might be reincorporated
into the nerve terminals.

The disposition of released norepinephrine by
enzymatic degradation is negligible (Kopin, 1964; Jonason,
1969). Monoamine oxidase is present both intraneuronally

and extraneuronally. Perhaps some norepinephrine that is

l9

reincorporated into the neuron is deaminated by intra-
neuronal monoamine oxidase before it is transported into
and stored in the vesicles (Kopin, 1964). Part of the
norepinephrine is O-methylated in presence of catechol-
O-methyltransferase. Inhibition of monoamine oxidase
and catechol-O-methyltransferase, however, does not
potentiate the effector tissue (e.g., heart) reSponses

to norepinephrine (Crout, 1961).

Turnover

Most of the studies of noradrenergic function during
the last decade have focused on the alteration of nor-
epinephrine contents of tissues. It has become apparent
now that turnover or renewal of norepinephrine is of
major importance in the study of noradrenergic function
because turnover and not the norepinephrine contents of
tissue signifies the functional status of the neuron
(Costa, 1970). The turnover rate implies that a steady
state exists such that the synthesis and tranSport of
norepinephrine into a metabolic pool equals its catabolism
and release (Neff et aZ., 1969). Various methods have
been used to determine the turnover rate of norepinephrine
in both neuronal cell bodies and terminals by measuring:
a) the rate of decline of exogenously administered
norepinephrine-3H of high Specific activity (Montanari
et al., 1963; Fischer and Snyder, 1965); b) the rate of

decline of neuronal norepinephrine contents after

20

inhibition of the biosynthesis of this amine by a—methyl
tyrosine (Spector et aZ., 1965; Brodie et al., 1966);
c) the rate of decline in Specific activity of norepin-
ephrine after administration of labelled precursors of
norepinephrine (Burack and Draskéczy, 1964); d) the rate
of recovery of neuronal norepinephrine after its depletion
(Spector et al., 1962); e) the rate of increase of
neuronal norepinephrine contents after inhibition of
monoamine oxidase (Costa, 1969); f) the rate of conversion
of tyrosine-14C to norepinephrine-14C (Neff et al., 1969).
The turnover of norepinephrine appears to be
dependent on neuronal activity and on the neuronal
pOpulation of tissues; it varies in different parts of
the neuron. The estimate of the rates of synthesis of
norepinephrine in cell bodies (Brodie et aZ., 1966;
Costa, 1969), axons (Roth et al., 1967) and nor-
adrenergic terminals are 1.53, 3.0 and 0.12 to 0.22
ug/g/hr respectively. The Tl/Z of norepinephrine in
peripheral noradrenergic neuroeffector organs is
approximately 12-18 hours whereas the Tl/Z of ganglionic
norepinephrine is about 2 hours (Fischer and Snyder,
1965; Brodie et aZ., 1966; Costa, 1969). Decentrali-
zation slows the disappearance of norepinephrine-3H from
ganglia; decentralization for two weeks also results in
an elevated ganglionic content of norepinephrine

(Kirpekar et al., 1962; Fischer and Snyder, 1965). The

21

turnover of norepinephrine in the neuronal terminals of
heart, salivary glands and Spleen is also reduced after

chronic decentralization.

Regulation of Neuronal Content of Norepinephrine

 

Despite variable and prolonged sympathetic stimula—
tion, steady state concentrations of norepinephrine
remain constant at a level that is characteristic for
each individual tissue. Stimulation results in the neu-
ronal release of norepinephrine but does not cause a
significant change in the concentration of norepinephrine
in the cell bodies (Reinert, 1963) or nerve terminals
(Luco and Gofii, 1948; Euler and Hellner-Bjdrkman, 1955;
Folkow et aZ., 1967). Decentralization slows the
disappearance of norepinephrine in neuronal cell bodies
and terminals and with time elevates the norepinephrine
concentration of soma (Kirpekar et aZ., 1962; Fischer
and Snyder, 1965). These results suggest that changes
in sympathetic activity influence the neuronal
contents of norepinephrine by altering the rate of
synthesis of norepinephrine. The regulation of synthesis
of neuronal norepinephrine has been studied after acute
or chronic alterations in sympathetic nerve activity
following stress, exercise, eXposure to cold, a-
receptor blockade, electrical stimulation of noradrenergic
nerves, thyroidectomy, denervation and hypertension

(Dairman and Udenfriend, 1970; Weiner, 1970). Three

22

major mechanisms have been suggested for the regulation
of neuronal contents of norepinephrine: (1) rapid
changes in the norepinephrine content resulting from the
removal of tyrosine hydroxylase from product inhibition,
(2) gradual changes resulting from alterations in the
amount of tyrosine hydroxylase and (3) reuptake of
norepinephrine from the synaptic cleft.

Electrical stimulation has been frequently used to
alter the sympathetic discharge in order to study the
noradrenergic mechanism. Stimulation of the stellate
ganglion in viva results in an increased formation of
norepinephrine in the rat heart from labelled tyrosine
but not from labelled DOPA (Gordon et aZ., 1966).

During the in vivo stimulation of decentralized pre-
ganglionic fibers the rat submaxillary gland accumulates
S-times more norepinephrine-14C than the contralateral
decentralized nonstimulated gland following the adminis-
tration of tyrosine—14C but not after DOPA-14C (Sedvall
and KOpin, 1967). In vitro stimulation of hypogastric
nerves results in an increased conversion of tyrosine-3H
to norepinephrine-3H in the guinea pig vas deferens
(Alousi and Weiner, 1966; Roth et aZ., 1966). Acute
increase in noradrenergic activity resulting from

a -receptor blockade (by phenoxybenzamine) or cold
eXposure elevates the synthesis of norepinephrine in the

rat heart and adrenal glands (Dairman and Udenfriend,

23

1970). Since the conversion of DOPA to norepinephrine
was not accelerated the results of the above studies
support the concept that tyrosine hydroxylase is the
rate limiting step in the biosynthesis of norepinephrine
(Levitt et aZ., 1965) and that the stimulus-induced
increase in the synthesis of this amine is the result
of increased tyrosine hydroxylase activity. The
mechanism of increased synthesis, however, is not well
understood (Gordon et aZ., 1966).

Several attempts were made to eXplain the mechanism
of increase in the synthesis of norepinephrine
following the increase in afferent input. The increase
in the norepinephrine synthesis did not appear to be due
to the effector tissue activity (Roth et aZ., 1966)
or an increase in the amount of tyrosine hydroxylase
(Sedvall and Kopin, 1967). Cycloheximide, an inhibitor
of protein synthesis, did not block the increased
synthesis of norepinephrine following exercise (Gordon
et aZ., 1966). Electrical stimulation did not alter the
axonal transport of protein (Peterson et aZ., 1967) or
of norepinephrine (Roth et aZ., 1967). An increase in
the transport of tyrosine to the site where it could be
more readily hydroxylated or an increase in cofactors,
however, could not be excluded (Weiner and Rabadjija,
1968). Since the catechols, including norepinephrine,

inhibit the activity of purified tyrosine hydroxylase it

24

was prOposed (Nagatsu et aZ., 1964) and later demonstrated
(Weiner and Rabadjija, 1968) that norepinephrine exerts
end-product inhibition of the initial and rate-limiting
step in the biosynthesis of norepinephrine, that is, the
hydroxylation of tyrosine. Norepinephrine presumably
competes with pteridine co-factors to inhibit tyrosine
hydroxylase (Nagatsu et al., 1964). However, contrary
hypotheses have been prOposed. Considering that nor-
epinephrine is stored and protected in the synaptic
vesicles which do not contain tyrosine hydroxylase this
amine could not exert inhibition of tyrosine hydroxylase
(Costa, 1970); therefore, DOPA and dopamine might
regulate the tissue norepinephrine contents by feedback
inhibition of tyrosine hydroxylase. DOPA and dopamine
are readily formed in the cytoplasm and tyrosine hydroxy-
lase would be expected to be more accessible to these
catechols than to norepinephrine (Costa, 1970). Costa's
prOposal does not include the possibility that small
amounts of intraneuronal "free" norepinephrine could
inhibit synthesis (Weiner and Rabadjija, 1968). Indeed,
all norepinephrine which is retrieved into the nerve
terminals from synaptic cleft is not incorporated into
the synaptic vesicles, and part of it is free to be
deaminated by monoamine oxidase (KoPin, 1964). Con-
ceivably this norepinephrine could exert end-product

inhibition of tyrosine hydroxylase.

25

The acute augmentation of sympathetic discharge
did not alter the amount of tyrosine hydroxylase in the
nerve terminals (Sedvall and KOpin, 1967a; Dairman and
Udenfriend, 1970). Most studies on the influence of
nerve activity on the neuronal contents of tyrosine
hydroxylase have been conducted after the augmentation
of sympathetic discharge; little is known about the
effects of diminished neuronal activity. Decentralization
slows the turnover of norepinephrine in the nerve
terminals (Fischer and Snyder, 1965), but it does not
alter the tyrosine hydroxylase activity in the adrenal
glands (Thoenen et aZ., 1969). However, in Spontaneously
hypertensive rats, in which there is a reflex reduction
in the sympathetic activity, the amount of tyrosine
hydroxylase is decreased in the blood vessels (Tarver and
Spector, 1970). Again most studies on the interrelation
of nerve activity and norepinephrine contents were con-
ducted in tissues containing either the nerve terminals
(e.g., salivary glands) or a mixture of cell bodies and
terminals of peripheral noradrenergic neurons (e.g., vas
deferens).

There is no systematic study on the regulation of
norepinephrine contents in cell bodies. A few studies
indicate that acute changes in neuronal activity do not
alter either the activity of tyrosine hydroxylase or the

norepinephrine content in ganglia (Reinert, 1963; Sedvall

26

and KOpin, l967a; Dairman and Udenfriend, 1970).
Tyrosine hydroxylase activity in ganglia is many fold
higher than in the salivary glands (Sedvall and KOpin,
l967a). Synthesis of norepinephrine in the cell bodies
may be Operating at a maximal rate irrespective of the
degree of neuronal activity. Indeed, electrical
stimulation of Splenic nerves, where the rate of
synthesis of norepinephrine is 15-20 times greater than
in the vas deferens, did not alter the synthesis of
norepinephrine (Roth et aZ., 1967). Since the protein
synthesis in cell bodies adapts to functional demands
(Hydén, 1958) the lack of change in neuronal contents
of tyrosine hydroxylase was puzzling.

Recent investigations indicate that prolonged
increase in afferent input increases the synthesis of
norepinephrine by mechanisms other than the removal of
inhibition of tyrosine hydroxylase. The accelerated
synthesis of norepinephrine that is obtained in the vas
deferens during electrical stimulation of hypogastric
nerve could be blocked by exogenous norepinephrine but
not by puromycin, an inhibitor of protein synthesis.
The accelerated synthesis of norepinephrine, however,
continued after the cessation of stimulation of hypo-
gastric nerves despite little change in the norepinephrine
contents of tissue. This post-stimulation increase in

the synthesis of norepinephrine was blocked by puromycin

27

but not by norepinephrine. It was postulated that
stimulation caused an increase in the amount of tyrosine
hydroxylase in the vas deferens (Weiner and Rabadjija,
1968). These investigators did not measure the amount

of the enzyme, but it appears that acute changes in
neuronal activity do not cause an increase in the amount
of tyrosine hydroxylase (Dairman and Udenfriend, 1970).
Prolonged periods of drug-induced increases in sympathetic
discharge (with reserpine and 6-hydroxyd0pamine) increase
the amount of tyrosine hydroxylase in the rat superior
cervical ganglia and adrenal glands (Mueller et aZ.,
1969a). This increase in tyrosine hydroxylase is blocked
by cycloheximide and actinomycin-D (Mueller et aZ.,
1969b) or by sectioning the splanchnic nerves and
preganglionic sympathetic fibers (Thoenen et aZ., 1969).
Chronic increases in reflex sympathetic discharge caused
by repeated injections of phenoxybenzamine also increase
the conversion of tyrosine-14C to norepinephrine-14C in
the nerve terminals of heart. Part of the increase in
the formation of norepinephrine-14C could be attributed
to an increase in the amount of tyrosine hydroxylase.
Thus, a prolonged increase in the afferent input might
result in a compensatory induction of the rate limiting
enzyme in the biosynthesis of norepinephrine in both
neuronal cell bodies and terminals in order to maintain

transmitter requirements.

28

It would appear that the induction of tyrosine
hydroxylase is initiated in the cell bodies. The
labelled soluble proteins associated with the synaptic
vesicles appear first in the cell bodies and then in
the nerve terminals (Barondes, 1968). The neuronal
increase in the amount of tyrosine hydroxylase that is
obtained after drug-induced augmentation of sympathetic
discharge appears two days sooner in the cell bodies than
in the nerve terimals (Axelrod et aZ., 1970). After the
depletion of neuronal norepinephrine contents with
reserpine norepinephrine fluorescence appears first in
the cell bodies and then in the nerve endings (Dahlstrom
and Haggendal, 1970).

Of the two major mechanisms for the regulation of
neuronal norepinephrine contents, end-product inhibition
and inductive changes in enzyme contents, it would appear
that immediate functional demands of the neurotransmitter
are met and regulated primarily by the mechanism of
removal of product inhibition of tyrosine hydroxylase.
Prolonged increases in neuronal activity, however,
might result in inductive changes in the biosynthesis
of norepinephrine. The inductive changes are gradual.

It takes 12 hours for the increase in the amount of
tyrosine hydroxylase to become detected after a chronic
augmentation in sympathetic discharge (Mueller et aZ.,

1969a, b). On the other hand the increase in norepinephrine

29

synthesis from tyrosine as a result of the stimulus-
induced release of this amine, and hence, removal of
end-product inhibition of tyrosine hydroxylase, is

rapid. The importance of the mechanism of end-product
inhibition in the regulation of biosynthesis of nor-
epinephrine is further evidenced by the following
observations: Tl/Z of tyrosine hydroxylase (Mueller

et aZ., 1969a, b) and that of norepinephrine storage
vesicles (Dahlstrom and Haggendal, 1970) are estimated

to be 8 and 22 days respectively; electrical stimulation
and inhibition of protein synthesis does not alter the
axonal transport of protein (Peterson et aZ., 1967) and
the rate of axonal transport of enzymes and norepinephrine
storage vesicles (Livett et aZ., 1968; Dahlstrom and
Haggendal, 1970) is not fast enough to fulfill the
immediate transmitter requirements at the terminals.
Furthermore, in those tissues where turnover of nor-
epinephrine is Slow (heart and salivary glands) following
depletion by drugs (Spector et aZ., 1962) or electrical
stimulation (Fredholm and Sedvall, 1966) the amine con—
tents return to control levels rapidly. It appears

there is no correlation between the reduction of neuronal
contents of norepinephrine and the elevation of tyrosine
hydroxylase (Mueller et aZ., 1969a). Accordingly, the
terminals can independently maintain their ability to
synthesize norepinephrine for a relatively long period of

time.

30

Among the factors that regulate the neuronal con-
tents of norepinephrine the reuptake mechanism is
important, particularly at the nerve terminals. A series
of events take place in noradrenergic nerve terminals
upon the arrival of action potentials: norepinephrine
is released, much of the released norepinephrine is
retrieved and norepinephrine synthesis is accelerated.
It appears that steady state levels of norepinephrine
in the nerve terminals are maintained by local synthesis
and by reuptake. The relative importance of these two
processes in resting and active neurons, however, is
still controversial (Malmfors, 1969; Hedqvist and Stjarne,
1969). For example, in nerve terminals, synthesis of
norepinephrine is believed to be of prime importance at
low frequencies of stimulation while reuptake of this
amine predominates at higher frequencies (Bhagat and
Friedman, 1969).

Clearly, the localization and the processes of
storage, release and uptake of norepinephrine differ in
the cell bodies and terminals of noradrenergic neurons.
It appears that mechanisms for synthesis and storage of
neuronal norepinephrine are initiated in the cell body
and acquire different characteristics along the axon
and within the nerve terminals. Although there is a
distinct requirement of intact innervation for normal

turnover of neuronal norepinephrine the mechanism of

31

regulation of norepinephrine synthesis differs in the
cell bodies and terminals. Furthermore, there is a lack
of systematic studies of the effect of neuronal activity
on the disposition of norepinephrine in neuronal cell
bodies. The differences in the prOperties and diSposition
of norepinephrine in the neuronal cell bodies and
terminals is not always appreciated. Tissues that are
rich in cell bodies (e.g., vas deferens, brain, gut)
Should not be viewed as a system of identical functional
units Operating in parallel with those tissues (e.g.,
heart, Spleen, salivary glands) that are rich in nerve
terminals (Costa, 1970).

In the present studies some attempt was made to
understand the factors that control the dynamics of
norepinephrine in both cell bodies and terminals of
noradrenergic neurons during and after transient augmen-
tation of nerve impulses. The cell bodies were represented
by the superior cervical ganglia and the terminals by the
submaxillary salivary glands and the smooth muscle of
the nictitating membranes of cat. It will be demonstrated
that in the cell bodies only synthesis contributes to
the norepinephrine content; synthesis proceeds at a rapid
rate and is not altered by acute electrical stimulation
and for up to 6 hours after the cessation of stimulation.
In the terminals, during stimulation both synthesis and

reuptake of norepinephrine increase. The synthesis is

32

increased by removal of end—product inhibition; but the
norepinephrine concentrations are not exclusively
regulated by this mechanism, Since the synthesis rate
of norepinephrine returns to control values at a time

when the tissue norepinephrine content is still reduced.

METHODS

A. Surgical and Experimental Methods

 

EXperiments were performed on cats of either sex
weighing 1.6-3.5 kg. They were anesthetized with an
intraperitoneal injection of Dial Urethane (sodium
diallylbarbiturate, 70 mg/kg; urethane, 280 mg/kg;
monoethylurea, 280 mg/kg). The surgical preparation was
essentially the same as described by Volle (1962). The
trachea was eXposed and intubated at the level of the
clavicles. The rest of the trachea, larynx and
oeSOphaguS were either retracted through the oral cavity
or sectioned at the level of mandibles. The cervical
sympathetic trunks were dissected free from the vagi
and common carotid arteries. Unless mentioned otherwise,
the preganglionic fibers of both sides were sectioned
2-3 cms caudal to the superior cervical ganglia and the
cranial end was tied with thin thread to assist with
placement on electrodes. Skin flaps of neck were tied
to a metal frame to form a cervical well which was filled
with mineral oil. The final surgical preparation is

diagrammatically illustrated in Figure 2.

33

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35

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36

Supramaximal (15 v) unilateral electrical stimulation
(0.2 msec duration) of preganglionic fibers was effected
with platinum electrodes using a square wave generator
(Grass Model S4). The stimulation schedule was 2 hz or
10 hz for 30 seconds of each minute (intermittent) for
one or three hours. In a few eXperimentS stimulation was
conducted at various frequencies for 60 seconds of each
minute (continuous) for 1 to 3 hours. Effectiveness of
ganglionic transmission was constantly monitored by
recording nictitating membrane contractions on Grass
Model 7 polygraph using a strain gauge. The initial
tension on both nictitating membranes was set at 7—7.5
gms. The contralateral preganglionic fibers were placed
on platinum electrodes without being stimulated. A 27
gauge needle was inserted into each common carotid
artery for infusion of drugs. The needle was connected
to polyethylene tubing and fitted to a holder which was
clamped to the frame. Drugs were also infused into the
femoral vein. Both arterial and intravenous infusions
were made using a compact Harvard infusion pump (Model
975). Blood pressure was constantly monitored from a
femoral artery. Sodium chloride 0.9% solution was
infused i.a. or i.v. at the rate of 12 ml/hour to prevent
dehydration. The body temperature of the cat was main—
tained between 37-38°C using an electric lamp. At the

end of each eXperiment the ganglia, nictitating membranes

37

(smooth muscle, connective tissue and all orbital
attachments), and salivary glands were dissected out

and frozen until analyzed for norepinephrine. In
eXperiments where radioactive isotopes were used the
chest was opened and the cat was perfused with one liter
of cold 0.9% sodium chloride through a cannula inserted
into the aorta before the tissues were dissected out;

the right auricle was excised for drainage of blood.

B. Biochemical Methods

 

1. Assay of a-methyltyrosine in plasma

 

Plasma a-methyltyrosine concentrations were
determined fluorometrically according to the method of
Porter et a1. (1966) as modified by Carr and Moore
(1968). Aliquots (0.1 or 0.2 m1) of plasma and known
concentrations of a-methyltyrosine (6 ug and 2 ug) were
mixed with 1 m1 of 6% trichloroacetic acid in plastic
centrifuge tubes. Samples were centrifuged at 10,000 x
g for 5 min. The supernatant was transfered to 15 m1
glass centrifuge tubes containing 0.1 ml of pyridine
and 0.5 m1 of 1% ninhydrin. The tubes were heated in
boiling water bath for 10 min and cooled immediately
thereafter. One-tenth ml of concentrated HCl was added
to each tube and contents were mixed thoroughly. Two ml
of ethylacetate were added to each tube. The tubes were
stOppered and Shaken for 2 min. The organic layer was

discarded by aspiration. One ml of the remaining solution

38

was transfered to 15 m1 glass centrifuge tubes and the
following reagents were added: 0.5 m1 of nitric acid

reagent (1 ml 2.5% NaNo + 49 m1 HNO (1:5 dilution of

2 3
concentrated HNO3) and 0.5 m1 of 0.1% nitroso-napthol
in 95% ethanol. The mixture was heated for 30 min at
55°C and allowed to cool to room temperature. Ethylene
dichloride (2.5 ml) was then added, tubes were shaken
for 5 min and centrifuged. The fluorescence of the
supernatant was determined in an Aminco-Bowman Spectro-
photofluorometer at activation-fluorescent wave lengths
of 456-560 mu. Reagent blanks were determined from
samples that contained 0.2 ml water instead of plasma.
Tissue blanks were determined from blood that was
removed before the administration of a-methyltyrosine.

The unknown concentrations of a-methyltyrosine were

determined from known standards.

2. Assay of endogenous norepinephrine content of

 

superior cervical ganglia

 

The ganglia were cleaned on an ice—cooled Petri
dish under 3x magnifying lens, weighed, and homogenized
in 3 ml cold 0.4 N perchloric acid using an all glass
homogenizer. The homogenizers were washed with an
additional 2 ml 0.4 N perchloric acid. Washings and
tissue homogenate were combined, allowed to stand for
15 min and then centrifuged at 27,000 x g for 15 min.

The supernatant was transfered to beakers containing

1 m
oxi
prc
alt

21‘

had
lee

ads

C0.
tr

ti

0f

5&1

39

1 m1 of 0.2 M EDTA and approximately 200 mg of aluminum
oxide (Woelm). Alumina was washed according to the
procedure described by Moore and Rech (1967). The
alumina-tissue extracts were adjusted to pH 8.6 with

2 N, 0.2 N and 0.02 N sodium hydroxide under constant
stirring with a motor driven glass rod. After the pH

had stabilized at 8.6 the stirring was continued for at
least five additional minutes to ensure complete
adsorption of norepinephrine on alumina. The supernatant
was decanted and the alumina transferred to 15 ml conical
glass centrifuge tubes containing 10 ml of distilled
water. The tubes were Shaken for 2 min and centrifuged
at 900 x g for 1 min. Water was then aspirated and the
alumina washed again three more times with 5 m1 portions
of distilled water. After the last wash 3 ml 0.05 N
perchloric acid was added to each centrifuge tube. Tubes
were shaken for 15 min and centrifuged for one minute.
Two 5 m1 samples of 0.4 N perchloric acid containing known
concentrations of norepinephrine (.05 pg and .10 ug) were
treated with alumina exactly as described above for
tissue extracts. Two ml aliquots of 0.05 N perchloric
acid eluates from tissue and standard samples were
transferred to test tubes and the remainder of the
eluates from tissue samples were pooled for determination
<>f blanks. Similarly, the remaining eluates from standard

ESamples were pooled separately for determination of their

40

blanks. The fluorescent product of norepinephrine was
developed as follows. To one 2 ml aliquot were added,

1 m1 of 0.5 M potassium phosphate buffer, pH 7.0, and
0.05 ml of 0.25% potassium ferricyanide. Two min

after the addition of potassium ferricyanide, 0.3 ml

of freshly prepared alkaline ascorbate was added (1 m1
of 2% ascorbic acid + 9 m1 of 5 N NaOH). After each
addition the mixture was agitated on Vortex Genie Mixer.
Fluorescence was determined 10 min later in an Aminco-
Bowman spectrOphotofluorometer at activation-fluorescent
wave lengths of 390-510 mu. Blanks were determined by
adding to the 2 ml aliquots of pooled alumina eluates
from tissue and standard samples 1.0 ml 0.5 M potassium
phoSphate buffer, pH 7.0, and 0.3 m1 alkaline ascorbate.
Fifteen minutes later 0.06 ml potassium ferricyanide was
added. The recovery of the norepinephrine standards

was 73.5 i 1% (mean i l S.E., n = 25). The unknown
norepinephrine concentrations were determined from these

standards.

3. Assay of endogenous norepinephrine in salivary

 

glands and nictitating membranes

 

The salivary glands were cleaned on an ice-cooled
Petri dish, weighed, frozen inastainless steel mortar
withuliquid nitrogen and pulverized with a pre-cooled
Stainless steel pestle. The pulverized tissues were

honlog'enized in 8.0 ml 0.4 N perchloric acid using all

41

glass homogenizers. The homogenizers were washed with
an additional 4 ml 0.4 N perchloric acid. The tissue
homogenate and washings were pooled, kept on ice for
30 min, centrifuged at 27,000 x g for 15 min, and the
supernatant frozen until analyzed for norepinephrine.

The nictitating membranes were carefully trimmed,
cut into small pieces, and kept overnight in 10 ml
0.4 N perchloric acid at 4°C. The suspension of tissue
in perchloric acid was centrifuged at 27000 x g for
15 min, and the supernatant was filtered through glass
wool. The glass wool was washed with 2.0 ml of 0.4 N
perchloric acid. The combined supernatant-wash was
then analyzed for norepinephrine.

The perchloric acid extracts from the salivary
glands and nictitating membranes were analyzed for
norepinephrine content as described by Moore and Rech
(1967); recovery of norepinephrine standard was 75 i 2%
(mean i l S.E., n = 34). The norepinephrine concentra—
tions of the unknown samples were calculated from these

standards.

4. Assay of norepinephrine-14C in superior cervical
ganglia

The ganglia were homogenized in 2 ml of 0.4 N

perchloric acid and the homogenizers were washed with

1 m1 of 0.4 N perchloric acid. The tissue homogenate

and washings were combined, allowed to stand for 15 min,

42

and centrifuged at 27,000 x g for 15 min. A 100 ul
aliquot was withdrawn from the extract for the deter-
mination of total radioactivity. One pg of norepinephrine
standard and 0.1 m1 of 2% freshly prepared ascorbic acid
was added to each sample. Two standards of norepinephrine
(1.0 ug) containing known amounts of norepinephrine-14C
and 0.1 ml of 2% ascorbic acid were analyzed concurrently.
The procedure for alumina extraction was similar to that
described above for estimation of endogenous norepinephrine
content with the exception that elution from alumina was
performed with 3.0 ml of 0.2 N acetic acid instead of
perchloric acid. The pH of the alumina eluates was
adjusted to 6.0 using 2 N, 0.2 N and 0.02 N sodium
hydroxide under constant stirring. Samples were then
applied to columns of Dowex 50W-X4 (Na+, 28 mm2 x 40 mm)
buffered with 0.1 M sodium phosphate buffer, pH 6.5,
containing 0.1% EDTA. The columns were washed with 5 ml
of distilled water. The first 5 m1 of 1 N HCl eluate

was discarded. Norepinephrine-14C was eluted with
additional 12 ml 1.0 N HCl. The eluates were collected

in vials and dried under a stream of air. The residue
was dissolved in 1.0 ml 0.1 N HCl. Ten m1 of modified
Bray's solution (6 g of 2,5-diphenyloxazole and 100 g of
naphthalene/liter of dioxane) was added to each vial and
radioactivity determined infBeckman Model 100

Spectrometer with 87% efficiency. Recovery of the

43

norepinephrine-14C standard was 53 i 2% (mean t 1 S.E.,

n = 27). No correction was made for this recovery.

5. Assay of norepinephrine—14C in salivary glands

 

and nictitating membranes

 

The procedure followed for alumina extraction of
norepinephrine-14C was similar to the one described
above for the determination of total norepinephrine in
these tissues. Two norepinephrine standards (1.0 ug)
with known amounts of radioactivity of norepinephrine-14C
were analyzed concurrently. No carrier norepinephrine
was added to tissue samples. The procedure for separation
of norepinephrine-14C on Dowex columns was similar to
that described for ganglia with the exception that
ascorbic acid was not added to the alumina eluates as
it interfered with the determination of the total
norepinephrine content. Out of 12 ml of the 1 N HCl
eulates from Dowex columns, 3 ml were withdrawn for the
estimation of the endogenous norepinephrine content,
and 9 ml dried in scintillation vials under a stream of
air. The pH of a 2 ml aliquot of 1 N HCl eluates was

adjusted to 6.5-6.8 with saturated, 1 M and 0.5 M K CO

2
and 0.4 ml of potassium phosphate buffer, pH 6.5, was

3'

added. The fluorescent product was formed according to
the procedure described above for these tissues.

Recovery of norepinephrine standard was 55 i 2% (mean i

14

l S.E., n = 18) and of norepinephrine- C 53 i 2% (mean i

Cd

as

t)"

a"

de

fjfi

44

1 S.E., n = 27). Norepinephrine content of tissues was

calculated from these standards.

6. Assay of radioactive d-methylnorepinephrine-3H
a-Methyltyrosine was checked for purity by

ascending co-chromatography with authentic a-methyl-
tyrosine on cellulose coated thin layer plates using
butanol, glacial acetic acid, water (5:1:4) as the
developing solvent; only one major peak was detected.
This is illustrated in Figure 3. This peak coincided
with that of authentic a-methyltyrosine and represented
more than 99% of total radioactivity recovered from the
plate; the remaining 1% represented an unidentified peak.
The conversion of a-methyltyrosine-BH to a-methyl-
norepinephrine—3H was determined in all tissues according
to the procedure outlined above for measurement of
norepinephrine-14C. It was found that a-methylnorepinephrine
has the same elution pattern on the Dowex column as
norepinephrine. a-Methylnorepinephrine in the eluates
from Dowex columns was analyzed according to the procedure
described above for norepinephrine with the exception
that 2 ml aliquots were heated in a boiling water bath
for 50 min before assay for norepinephrine. Heating
increases the fluorescence intensity of a—methylnor-

epinephrine (Dominic and Moore, 1971).

45

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Eoum cmcflEuwump mums 825 no mocmomeOMHm paw wufl>fluomowpmu mza .Hom z a.o

mo HE H nuwz mcwxmnm an pmusaw 828 can #50 mumz mmumam Hmwma carp mo mmwuum

Eu wco Hmflucmsvmm .ucm>H0m mcflmoam>mc 0:» mm Aquaumv nouns .pflom owumom

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loo mumz mmuaza no on m.o maoumfiwxowmmm can 525 owucmnusm Mo on m>flm

.Aazdv mmnmcflmoumuawnumela mo wuwusm can no coaumcflEHmumo .m musmflm

46

FLUORESCENCE UNITS

0..

«—

0N

mu

0”

.m mnsmflm

 

 

.ABZSV mmnocflmouxuamnumE|a mo mufluom 030 m0 coflumcflaumumo
mcuhmcfih-‘uo
o— 3 up 0 p a o c 0
1 4 1 d
a .0

T

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1

 

 

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000—

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ALIAILOVOIO VB

(HdO)

47

7. Calculation of radioactivity in tissues

In all those eXperiments where isotOpes were used
radioactivity in the nictitating membranes is eXpressed
as dpm per membrane whereas radioactivity in the ganglia
and salivary glands is reported on the basis of wet
weight. The weights chosen, 10 mg for superior cervical
ganglia and l g for salivary glands, approximate the
actual weights of these tissues (ganglia--10.9 1 0.35 mg;
n = 36; salivary glands-~1.32 i 0.06 g; n = 17). Because
of variation in total radioactivity the amount of nor-
epinephrine-14C formed is eXpressed as an absolute amount

and as a percent of total radioactivity.

C. Administration of Drugs and Isotopes
Desmethylimipramine hydrochloride (supplied by
Dr. R. C. Ursillo of Lakeside Laboratories, Milwaukee,
Wis.), cycloheximide (Sigma Chemical Company) and
pheniprazine (Catron; JB 516; Lakeside Laboratories) were
injected i.v., and L-a-methyltyrosine (supplied by
Dr. C. A. Stone of Merck Institute for Therapeutic
Research, West Point, Pa.) was infused through each common
carotid artery at the rate of 0.1 mg/min. All drugs were
dissolved in 0.9% sodium chloride; a-methyltyrosine
required heating under constant stirring to dissolve.
L-tyrosine-14C (uniformly labelled, New England Nuclear
Corporation; specific activity, 395 mc/mmol) was either

infused through each common carotid artery or through the

48

femoral vein. L-a-methyltyrosine-BH (supplied by

Dr. C. Rosenblum of Merck, Sharp and Dohme,Rahway, N. J.)
(Specific activity 5.2 mc/mmol) was infused through each
common carotid artery. 3,4-diphdroxypheny1ethylamine-l-
14C:HBr (dOpamine) (New England Nuclear Corporation;
Specific activity, 6.28 mc/mmol) was infused through
each common carotid artery. All isotopes were checked

for purity by ascending co-chromatography with authentic

compounds on cellulose coated thin layer plates.

D. Statistical Methods

 

Statistical analysis was carried out with Student's
t test (Goldstein, 1964). In most of the experiments

a paired comparison was made.

RESULTS

A. The Dynamics of Norepinephrine in the Neuronal Cell

 

Bodies and Terminals During the Resting State.

 

1. Norepinephrine concentrations in tissues from

 

the right and left Side of the same cat

 

As summarized in Table 1, there was less
variation in the tissue concentrations of norepinephrine
between the right and left sides of the same cat than
there was between individual cats. For example, the
norepinephrine content in ganglia and nictitating
membranes varied from 6.78 to 13.96 ug/g and 0.18 to
1.60 ug/membrane respectively, but there was good
correspondence between the right and left sides of the
same cat. The same pattern was seen with the salivary
glands; the norepinephrine content ranged from 1.27 ug/g
to 2.40 ug/g with mean values of 1.74 i 0.34 ug/g for
the right side and 1.70 i 0.20 ug/g for the left side.
Because of the close correspondence of norepinephrine
concentrations in tissues from the left and right side
of the same cat, most eXperiments were designed so that

the contralateral tissues served as apprOpriate controls.

49

50

 

 

 

 

ma. ma. om. vm. ms. nu. .m.m a

“mm.o wam.o “on.a HSS.H “on.m “mm.m “cam:

om.a om.a oa.~ o¢.~ mm.m mo.aa

mm.o ~m.o mv.a RN.H Hm.m m4.n

ma.a ~m.o om.a «m.a «H.m mm.m

~m.o oo.o -- ..... mm.n mm.m

mH.o om.o -- ----- ma.m «o.m

mv.o m¢.o -- ----- om.ma H¢.ma

mm.o ~m.o -- ----- mv.h m~.o

qw.o mm.o -- ----- m~.n mm.n

new; unmwm puma unmwm poms unmflm
AmCMHnEmE\mnV Am\mnv Am\mnv maamcmm

mmSMuQEmE mcflumufluoflz

mocmam >Hm>wamm

H80fl>umo Hoflummsm

 

.mumo omummuuas Acapfl>fl©cw mo
moan puma can unmau Eouw mmSmmHu m0 coflumuucmocoo mcwunmmcflmwuoz .H manna

51

2. Plasma and tissue concentrations of a-methyltyrosine

 

Plasma contents of a-methyltyrosine at various
times after the start of the infusion of this drug into
both common carotid arteries are illustrated in Figure
4. The a-methyltyrosine content in plasma from the
femoral vein approached equilibrium fairly rapidly and,
as would be eXpected, was lower than that observed in
jugular vein plasma. At 1, 3 and 5 1/2 hours the
a-methyltyrosine concentrations in plasma from the
right and left jugular veins were the same. If the
jugular vein content of a-methyltyrosine represents the
concentrations of this drug in tissues of the head
regions, it could be predicted that the synthesis of
norepinephrine would be inhibited by at least 70% within
one hour (Udenfriend et aZ., 1966). The tissue concen-
trations of a-methyltyrosine were in fact higher than
those in the plasma. For example, at 1 hour the a—
methyltyrosine content in the salivary glands was 174 i
31 ug/g (mean i l S.E., N of 5). This concentration
is in excess of that calculated to inhibit norepinephrine
synthesis by 90% (Udenfriend et aZ., 1966). That the
administration of a-methyltyrosine did indeed block the
synthesis of norepinephrine is illustrated by the
exponential depletion of this amine in the superior
cervical ganglia (Figure 5), and by blockade of the
conversion of tyrosine-14C to norepinephrine-14C in ganglia,

nictitating membranes, and salivary glands (Table 10).

52

.m.m H H

ucmmmummn mwcfla Hmofiuum> may cam mucmefluwmxm ma on m Eonm omcHEMmqu mm

928 m0 coaumuucmocoo some may mucmmmummn ucflom zoom .SOHmsmcfl can mo uwmum

mfi umumm mmeu msowumtw um mcflmtw Hmasmsn A! puma can 2V ”Ewan mnu Eowm can

an: cflm> ammoEmm may Eoum c3muo£ufl3 mums mmHmEMm ooon .musoz m\H mud mom
SHE\mE H.o mo mum“ m um muwuum UHuOHmo :oEEoo comm oucw pom5mafi mm: 925

.msuo 050 m0 cowmsmcw
.m.H mcflsoaaom ABSSV mchouhuawnumfila mo macaumuucmocoo mammam .v onsmflm

53

.msuo may no cowm9mcfl
.m.w mcflsoHHOm Aezgv mcflmoumuamsumela mo mcoflumuucmocoo mammam .v madman

manor
0 n v m N _ o

L - d- u . .

 

 

 

00

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mpflw wmnuo may no omcofluomm mums mumnflm macofiamcmmmum map can .moflm mco Eowm
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. €23
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can mmcmunEmE mcfiumufluoa: cw Amzv mcflwnmmcammnoc mo unmuaoo mna .m mnsmflm

55

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mcflmouxua>£umEIo mo commswcfi may umumm mflamcmm Hmow>umo HowquSm

 

   
  

 

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56

3. Tissue norepinephrine concentration after
infusion of a-methyltyrosine

The effects of a-methyltyrosine on the norep-
inephrine content of nonstimulated decentralized
nictitating membranes and ganglia are depicted in
Figure 5. The norepinephrine content in the ganglia
declined in an eXponential fashion with a Tl/Z of
2 1/2 hours, whereas three hours of a-methyltyrosine
infusion did not significantly alter the norepinephrine
content in the nictitating membranes. These data
confirm previous reports that the rate of turnover of
norepinephrine is faster in cell bodies than in
terminals (Brodie et aZ., 1966). Similar results were
obtained in intact or nondecentralized preparations;
that is, three hours of a-methyltyrosine infusion
significantly reduced the norepinephrine levels in
ganglia but not in salivary glands or nictitating
membranes (Table 2). Therefore, it appears that in the
anesthetized cat resfing neuronal activity does not
markedly alter the rate of turnover of norepinephrine
in either the cell bodies or terminals Since the
effects of a-methyltyrosine on norepinephrine contents

were the same in intact and decentralized tissues.

57

.Aao.vmv ooa Eouw usmumMMHp maucmoHMHcmHm mum umnu mmmmucmoumm mmoau mmumofiocfl

.m.m H H mz mo ucmucoo SmmE mnu ucmmmummu mmsam> HH<

M

.mucmeflummxm mumummmm m 0» v cw omcHEumumo mm

.coflm5mcfl 628 map mo pumum

mnu muommn ©m>oEmH mum3 umsu mmSmmHu mo ucmucoo mz map ucmmmummu mmSHm> Honucou

 

 

Am\mnv

-- -- -- m as. om. moamam

“mm “mm.o “mm.o mum>wamm

AmcmunEmE\mnv

6 ma. ma. m ma. mH. mcmunSme

HOHH Hoo.a “ma.o Hem “mm.o “hm.o mawumufluofiz

m «m. mo.a 4 mm. as. Am\m:e

«Hem “on.m qu.oa mwmm “Hm.~ HSS.G mwamcmo
Houucou mo w 928 Houucoo Houucom mo w 928 Houucou mammfla

 

Amy pmuwamuucmomn

mchouwuahnumEIo Mo muse:

Avv pmuwamuucmomvcoz

.Smesmcw ABZSV
mmunu Hmumm Amzv mcfiunmmcwmmuoc mammws

.m manna

58

4. Effects of desmethylimipramine and a-methy14

 

tyrosine on the contents of norepinephrine in

 

nonstimulated tissues

 

Three hours after the i.v. injection or infusion
of desmethylimipramine to nonstimulated preparations,
the norepinephrine content was not altered in the
superior cervical ganglia, nictitating membranes, or in
the salivary glands (Figure 6 and Table 3). In addition,
desmethylimipramine did not alter the effects of a-
methyltyrosine; that is, desmethylimipramine plus
a-methyltyrosine produced the same effect as a—methyl-
tyrosine alone. For example, three hours of
a-methyltyrosine infusion alone reduced the ganglionic
norepinephrine contents to 35% of control (Table 2 and
Figure 6) and in presence of low (2 mg/kg) or high
(10 mg/kg) doses of desmethylimipramine, the ganglionic
norepinephrine content was 31 and 25% of control
respectively (Table 3). These data indicate that in
nonstimulated preparations norepinephrine is synthesized
at a rapid rate in cell bodies but not in terminals;
furthermore, in a resting neuron reuptake does not
play a major role in maintaining norepinephrine

concentrations in cell bodies or in terminals.

59

Figure 6. Tissue contents of norepinephrine (NE)
following administration of desmethyl-
imipramine (DMI) and/or a-methyltyrosine
(aMT) in nonstimulated preparations.

The eXperimentS were carried out in cats in which
the preganglionic fibers were sectioned but not stim-
ulated. aMT was infused through both common carotid
arteries at a rate of 0.1 mg/min for three hours.

DMI (2 mg/kg) was injected intravenously over a 5
minute period concomitant with the start of the aMT
infusion; another injection of DMI (1 mg/kg) was made
two hours later. The height of each bar represents
the mean content of NE eXpressed as a percentage of
the NE content in the tissues that were removed prior
to the administration of aMT and DMI. Vertical lines
projected on each bar represent 1 S.E. as determined
from 3 eXperiments. Asterisks mark those values that
are significantly different from 100% (P<.01). SCG -
superior cervical ganglia; NM - nictitating membranes.

60

DM

*MT \\\\ -<-MT+DMI Cl

100’

60-

40-

[NE] PER CENT OF CONTROL

 

 

 

SCG NM

Figure 6. Tissue contents of norepinephrine (NE)
following administration of desmethyl-
imipramine (DMI) and/or a-methyltyrosine
(aMT) in nonstimulated preparations.

61

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.SOHmswcH 928 m0
unmum map QHHB ucmuHEoocoo SHE\mE mmo.o mo mumu m2» um musos m How .>.H Ummswcfl was H2QQ

.conSmcH 928 m0 unmum may mHmeQ SHE om .>.H pmuommSH mos H2om

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m Eouw pmcHEHmumo mm .m.m H H came msu ucmmmummu mmSHm> .coHumuuchHEom H20 cam 928 may
no Humum mru mHOan pm>OEmH mums umnu mmSmmHu mo ucmucoo mz may Hammmummu mmsam> Houucou

 

 

 

m mo. «0. m mm. ma. 5 em. on.
Hana Hmm.o HHS.o HHOH HmH.H HHS.H onm HHS.H Hmn.m nmx\me OH
5 va. NH. m 80. ma. m av. mN.H
Hkoa HNH.H HHS.H “Hm HmS.H HMH.H oHHm HHH.~ Hum.m mmx\ma N
HZQ
Houucoo AmSMHQEmE\mnv Houucoo Am\mnv Houucou Am\mnv
m0 w B28 Houucou m0 w B28 Houucou m0 w B28 Houucou
mmcmunEmE mcHumuHuon mpcmam mHm>HHmm mHHmcmo unmEummHB

 

.AH2ov mcHEMHmHEHawsumEmmp mo mocmmmum SH SOHmnmcH
2928v mchouwuaanumEI8 mo muson mmuzu Hmuwm ucmucoo Amzv mcwusmmcflmmuo: mommwa .m manna

62

B. The Dynamics of Norepinephrine in Neuronal Cell Bodies
and Terminals During Electrical Stimulation.
1. Effects of stimulation, a-methyltyrosine and
desmethylimipramine on tissue contents of

norepinephrine

 

In this series of eXperiments the tissues of both
Sides received 0.9% sodium chloride, a-methyltyrosine,
and/or desmethylimipramine but the sympathetic trunk of
only one side was stimulated preganglionically. The
nonstimulated tissues served as the apprOpriate controls.
a. Effect of stimulation

In the absence of drugs the norepinephrine
contents in cell bodies were not altered by continuous
(60 sec/min) or intermittent (30 sec/min) preganglionic
nerve stimulation for 1-3 hours at low (2 hz) or at high
(10 hz) frequencies (Tables 4-6; Figures 7 and 9).

In the terminals, the norepinephrine contents
were not significantly altered by continuous (Table 4) or
intermittent (Table 5) stimulation at low frequencies,
but at high frequencies of intermittent stimulation for
1 hour the norepinephrine contents in the salivary glands
and nictitating membranes were reduced to 36% and 78% of
control respectively (Table 6). Similar reductions of
norepinephrine content were obtained when continuous
Stimulation was utilized (Table 6). The reduced concen-
trations of norepinephrine were maintained for 3 hours

0f Stimulation (Table 7; Figure 9).

63

b. Effects of stimulation and a-methyltyrosine

In the cell bodies, the norepinephrine after
infusions of two concentrations (0.05 mg/ml and
0.1 mg/ml) of a-methyltyrosine was not altered by
continuous pre- or postganglionic stimulation at low
frequencies (Table 4). Similar results were obtained
with high frequencies of intermittent stimulation for
l or 3 hours (Figure 9) or with low frequencies for
3 hours (Table 5). That is, a—methyltyrosine alone
reduced the norepinephrine concentration of cell bodies;
the magnitude of this reduction was the same in the
presence or absence of stimulation.

In salivary glands, a-methyltyrosine enhanced
the reduction in norepinephrine contents following 3
hours of intermittent stimulation at 2 and 10 hz (57%
and 12% of control reSpectively; Table 5 and Figure 7).
The reduction in the concentrations of norepinephrine
was obtained after 1 hour of stimulation at 10 hz and
was maintained for 3 hours of stimulation (Figure 9;
Table 7).

c. Effects of stimulation and desmethylimipramine

In the cell bodies, the norepinephrine con-
tents were not altered by desmethylimipramine in the
'presence or absence of preganglionic stimulation
(Tables 5, 7 and 8).

In salivary glands, desmethylimipramine

‘flfiianced the stimulus-induced depletion of norepinephrine

 

 

 

no. no. me. an.
em Hmm.o Hmo.a moa Hmh.m Hah.m cHa\ma oa.o
828
coaumas
IEaHm oacoaamcmmumom
ma. mm. mm.a ma.a
an Ham.o Hmm.a Hm Hom.a Hmo.~ cHe\me oa.o
mm. av. wm.a mm.
am Hmm.o Hma.a mma Hum.s Hmm.m cHs\ma mo.o
.528
ma. va. mm. mp.
om Hmo.a Hma.a aaa Hmm.m Hum.m mcaamm
GOaHmao
IEaum oacoaamcmmmum
I. we
ooa x m mz ooa x m m mz
AmcmunEmE\mnv Am\mnv
mmGMHQEmE mcaumuauoaz maamcmw Hcmaummua

 

.Amzv mcaunmmcammuoc mo mucmucoo mammau co scamsmca

2928v mcamoumuamnumal8 mo muson v\a m paw coaumaSEaum mo muommmm .v magma

65

.m.m a

H ammE nammmummu mmSam> aad .SOanMaSEanm mo nnmnm man mnowmn usoa a pmnumnm
was mmnmn pmnmoapaa man nm 828 no aoamsmaa .aoanmaSEanm no numnm man Hmnmm
mnsoa v\a v ammmm mz How om>oamu mumz mmSmmaB .mnsoa m mm3 poaumm aoanmaDEanm
amnon man nman 0m mmEan mnoE m Umnmsnnmnaa mmz ceanMaDEanm mas .pOaHmm

aaE mv nmanoam now pmsaanaoo paw medmmu mm: mpam amnamEHHmmxm mo GOHnMasEanm
amumnmaaao .mmcommmu maanomnnaoo nmmn m camnno on aaE a MOM pmnmasaanm

mums mmamunEmE amnamEaHmmxm can aounaoo anon Am m.hv GOamamn mo mam>ma

ammmn on pmausnmn oma mmamnnEmE mcanmnanoaa man Hmnma .caE ma no GOaHmm 8 How
HmnMa nae mv pmnmsunmnca mm3 coanmaSEanm .aonnaoo Amzv pmnmasaanmaoc mm Um>umm
mpam amnmnmamunaoo mas .Na N nm hamsoscanaoo amv pmnmaDEanm mumB mpam mac

:0 mnmnam oacoaamamqmnm mo cam amnmap man no mnmnau oacoaamammnmom .Umcoanomm
mums mumnam macoaamammmum man aoaas ca mnmu aa n50 Umaunmo mum: mnamEaummxm may

AU.naOUV v maQMB

66

Figure 7. Tissue contents of norepinephrine (NE)
following the administration of desmethyl-
imipramine (DMI) and/or a-methyltyrosine
(aMT) in stimulated preparations.

The eXperiments were carried out in cats in which the
preganglionic fibers were sectioned and the distal end
stimulated at 10 hz for 3 hours. aMT and DMI were
administered according to the schedule described in the
legend to Figure 3 beginning at the start of the electrical
stimulation. The height of each bar represents the mean
content of NE eXpressed as a percentage of the NE content
in the corresponding tissue on the unstimulated side.
Asterisks indicate the values that are significantly
different from those obtained from animals receiving only
saline (P<.01). Vertical lines projected on each bar
represent 1 S.E. as determined from 4 to 6 eXperiments.
SCG-—superior cervical ganglia; SG--salivary glands; NM--
nictitating membranes.

160-

m 140}
Q
U)

@120-

gm.
;C
U)
Z

‘3 80-
n
O

N 60"
TJ‘

_z_ 4o-

20'

0L

 

Figure 7.

 

67

SALINE I
DMI

°(:MT %
“(:MT +DM| E]

 

 

S<3C5 5(3

Tissue contents of norepinephrine (NE) following
the administration of desmethylimipramine (DMI)
and/or a-methyltyrosine (aMT) in stimulated
preparations.

68

Table 5. Effects of a-methyltyrosine (aMT), desmethyl-
imipramine (DMI) and electrical stimulation at
2 and 10 hz on tissue contents of norepinephrine

 

 

 

 

 

 

(NE).
Treatment Frequency Ganglia (ug/g)
of
Stimulation
NS S S
K x 100
Saline 2 hz 5.96: 6.92: 116
.97 .93
10 hz ' 8.25: 7.50: 91
.81 .34
GMT 2 hz 3.32: 3.50: 105
.73 .34
10 hz 3.75: 3.37: 90
.39 .52
DMI 2 hz 6.59: 6.40: 97
1.14 1.35
10 hz 9.26: 8.38: 90
1.08 1.00
aMT 2 hz 2.57: 2.60: 101
and .54 .77
DMI 10 hz 2.06: 2.82: 137
.12 .66

 

The eXperimentS were carried out in cats in which the pre-
ganglionic fibers were sectioned and the distal end stim—
ulated intermittently (30 sec/min) for 3 hours. aMT was
infused through both common carotid arteries at a rate of
0.1 mg/min for 3 hours. DMI (2 mg/kg) was injected i.v.
over a period of 5 min concomitant with the start of aMT
infusion; another injection of DMI (1 mg/kg) was made two
hours later. Values represent the mean content of NE :

l S.E. determined in 3 to 7 separate experiments. NS =
nonstimulated; S = stimulated.

aSignificantly different from corresponding saline controls
(P<.05).

bSignificantly different from 100% (P<.05).

69

Table 5 (Cont'd)

 

Salivary Glands (pg/g) Nictitating Membranes

 

 

 

 

 

(pg/membrane)

NS 5 5 NS 5 s

E x 100 N X 100
1.27: 1.02: 81 1.37: 1.28: 93
.16 .09 .22 .25
1.70: 0.72: 42b 1.22: 0.90: 74b
.30 .03 .08 .09
1.89: 1.07: 57‘3"b 1.01: 0.76: 75
.23 .17 .17 .17
1.29: 0.16: 12"“b 1.08: 0.52: 48a'b
.10 .05 .13 .03
1.47: 1.04: 71B 1.24: 1.04: 84
.31 .22 .12 .18
1.63: 0.39: 24a"b 1.63: 0.98: 60a'b
.16 .05 .27 .19
1.30: 0.58: 45‘3"b 0.92: 0.80: 87
.17 .08 .12 .18
1.70: 0.04: za'b 1.26: 0.47: 37a'b
.18 .00 .11 .10

 

70

Table 6. Effects of continuous and intermittent stim-
ulation on tissue contents of norepinephrine

 

 

 

 

(NE).
Stimulation Ganglia (Hg/g)
NS S S/NS x 100

Continuous

1 hour 6.89: 9.17: 132:
.25 1.28 14

3 hours 9.58: 9.36: 98:
.90 .82 5

Intermittent

1 hour 10.35: 11.56: 113:
.95 .84 7

3 hours 8.25: 7.50: 94:
.81 .34 8

 

The eXperiments were carried out in cats in which the
preganglionic fibers were sectioned and the distal end
stimulated at 10 hz either continuously (60 sec/min)
or intermittently (30 sec/min). All values represent
the mean content of NE : l S.E. determined in 3 to 5
separate eXperiments. NS = nonstimulated; S =
stimulated.

aSignificantly different from 100% (P<.05).

71

Table 6 (Cont'd)

 

 

 

 

Salivary Glands (ug/g) Nictitating Membranes
(pg/membrane)
NS 5 S/NS x 100 NS 5 S/NS x 100
0.92: 0.32: 36:a 1.45: 1.15: 78:a
.22 .07 3 .25 .24 4
1.81: 0.63: 46:a 1.15: 0.82: 71.:a
.19 .08 4 .08 .08 3
1.55: 0.65: 42:a 1.74: 1.44: 84:
.27 .15 4 .22 .15 5
1.70: 0.72: 45:a 1.22: 0.72: 74:a

.30 .03 6 .08 .09 4

 

72

Table 7. Effects of a-methyltyrosine (aMT), desmethyl-
imipramine (DMI) and stimulation for one and
three hours on tissue contents of norepinephrine

 

 

 

 

 

(NE).
Saline aMT
1 hr (4) 3 hr (5) 1 hr (4) 3 hr (4)
Ganglia
(HQ/9)
NS 10.35: 8.25: 4.01: 3.75:
.95 .81 .93 .39
8 11.56: 7.50: 5.03: 3.37:
.84 .34 1.34 .52
S/NS x 100 113: 94: 127: 88:
7 8 19 8
Salivary
glands
(09/9)
NS 1.55: 1.70: 1.49: 1.29:
.27 .30 .18 .10
S 0.65: 0.72: 0.35: 0.16:
.15 .03 .10 .05
b a,b a!
S/NS x 100 42: 45: 22: 12:
. 4 6 5 3
Nictitating
membranes
(pg/memb)
NS 1.74: 1.22: 1.57: 1.08:
.22 .08 .12 .13
S 1.44: 0.72: 1.18: 0.52:
.15 .09 .10 .03
S/NS x 100 84: 74¢b 75:b 49:a'b
5 4 3 4

 

The experiments were carried out in cats in which the pre-
ganglionic fibers were sectioned and the distal end stimulated
intermittently (30 sec/min) at 10 hz. NS = nonstimulated;

S = stimulated. aMT was infused through both common carotid
arteries at a rate of 0.1 mg/min for l or 3 hours. DMI

1'.
C):

73

Table 7 (Cont'd)

 

 

 

 

 

DMI aMT and DMI
1 hr (3) 3 hr (5) 1 hr (5) 3 hr (6)
9.07: 9.26: 6.16: 2.06:
.63 1.08 1.12 .12
10.30: 8.38: 6.64: 2.82:

.49 1.00 1.38 .66

114i 92: 106: 138:

4 6 5 32
1.50: 1.63: 1.62: 1.70:
.14 .16 .18 .18
0.27: 0.39: 0.10: 0.04:
.07 .05 .02 0

18ia,b 25ta,b 6:arb 3ia,b
4 4 l 0
1.04: 1.63: 1.21: 1.26:

.05 .27 .13 .11
0.70: 0.98: 0.88: 0.47:
.08 .19 .10 .10

69:b 58:5:b 72:a 37:arb

11 4 l 6

 

Q mg/kg) was injected i.v. over a 5 min period concomitant
81?}: the start of the aMT infusion and stimulation; another
1nJ€3ction of DMI (1 mc/kg) was made 2 hours later. Values
represent the mean : l S.E., figures in parentheses = N.
Values that are significantly different from saline controls
and: 100% (P<.05).

VHIUUes that are significantly different from 100% (P<.05).

74

.muswmmnm pooan man mnammmummn

amamm Eonnon mas .mamnnEmE maanmnanoac naman mo aoamamn maaammmn

man mnammmummu amamm maopae mas .Bonnm an UmmeE mEan man no amnomnaa mm3

A.>.a .mx\me my H2o .mpaoomm om mnm>m Na oa nm a0anma5Eanm mascaamammmum
on mamunama maanmnanoaa nmma man mo mmaommmu man mnammmummn amamm mos

.mmamunEmE maanmnanoaa mo

mmaommmn maanomunaoo man no Aazov maaEmnmaEaahanmEmmp mo nomwmm .m musmam

75

.mmamHnEmE maanmnanoaa no

 

 

 

 

 

 

 

mmaommmn maanomnnaoo man no AH2QI maaEmnmaEaamanmEmmp mo nommmm .m mnnmam
:20
-o
w 9
00.
.d
H
Lcow 6
14-»- --------------- 4 --------------------- “.041- ------- 4 141P11-11-1- -
i / } {1"
7 7 7/ 7 7.. 7, H116
. , ,I . r .. .., ._ I. _ . In

 

(LUE) )
NOISNBJ.

76

Table 8. Effects of stimulation and desmethylimipramine
(DMI) on tissue contents of norepinephrine (NE).

 

 

Treatment Ganglia Salivary glands Nictitating
membranes
Saline (5) 94:8 41:4 74:4
DMI a a
3 mg/kg (5) 91:6 25:4 59:4
6 mg/kg (4) 110:4 20:5a 67:4
12 mg/kg (3) 94:8 24:8 64:10

 

Two-thirds of the indicated dose of DMI was injected i.v.
concomitant with the start of stimulation and the
remaining one-third of the dose was administered two hours
later. The values are the mean : S.E. eXpressed as the
percent of the unstimulated control content of NE. NE
values were determined 3 hours after the start of inter-
mittent preganglionic stimulation at 10 hz. Figures

in parentheses represent the number of eXperiments.

aValues are significantly lower than in saline-treated
preparations (P<.05).

77

.Amo.vmv
namEnmmun mcaamm Hmnwm pmaamnno nman Eonw namnmmmap wanamoamaamam ma mdam> maaam

.mnamEanmmxm mnmummmm m Eonm UmaaEHmnmU

mm .m.m a H amme man nammmnmmn mmsam> .umnMa usoa a Umumnmaaaaom mms ma\mE a
nmanoam cam SOanmasEanm no nnmnm man ana3 namnanoaoo .>.a pmnomflaa mm3 amx\mE NI
H2Q .pOanmm a0anmasaanm usoa-m 8 mo Hsoa mama-mac nmma man maausa mnmnum panonmo
aoEEoo aomm amsonan pmmsmaa max 2 umz no 0: ca .UmnmaoEanmnm .UmnMaSEanmaoanmz
.Na oa nm IaaE\omm omI 2anamnna Hmnaa pmnma95anm mam amnmaa man cam pmGOanomm
mnmz mnmnam oaaOaamammmnm man aoaa3 ca mnmo ca n50 Umauumo mums mnamEaummxm maa

 

 

 

 

«a mam.a ovm.m NH oma.a~ omq.m~ Imcmunems\emcv
Hma Hoov.m Homm.m~ Hm: Hmmm.mm H¢a~.mv mcmnneme mannmnnnonz
mm oo~.mma . ono.-a a: oao.mmm on~.ooa am\emoe
608m nmm~.mva “moo.am~ Hmom Hamm.aoo Hmmm.mma magma: >H6>namm
4mm om: Noo.a Nan mvm.v mmo.m :85 oa\emov
Hmma “moa.~ “mom.a Hmoa Homm.m Hamm.m «Hamcmo
ooa.x_mm m mz ooa x_mm m mz
H20 maaamm mammaa
.Ammlmzv mmlmaauammaammwoa
no mxmnms co AHEQI maaEmumaEaamanmEmmn mam a0anmasEanm mo nommmm .m manna

78

only at the high frequency (24% of control; Table 5).
The responses obtained with 3, 6 and 12 mg/kg of des-
methylimipramine were the same (Table 8). That is, a
maximum response was obtained at the lowest dose used
(3 mg/kg). As was the case with a-methyltyrosine, the
stimulus-induced reduction in norepinephrine content
was obtained as early as 1 hour after the start of
stimulation and was maintained for 3 hours of stimulation
(Figure 9). Both a-methyltyrosine and desmethyl-
imipramine enhanced the stimulus-induced reduction to
approximately the same extent (20% of control;

Figure 9).

In these eXperimentS desmethylimipramine was utilized
to block the retrieval of norepinephrine. It is possible
that the observed effects may have resulted as a
consequence of some other actions of the drug (Brodie
et aZ., 1968). Nevertheless, as illustrated in Figure
8 and Table 9, desmethylimipramine blocked the uptake
of stimulus-induced release of norepinephrine into the
noradrenergic terminals.

Norepinephrine-3H accumulated in both neuronal cell
bodies and terminals (Table 9). In saline-treated cats,
stimulation alone did not increase the uptake of
norepinephrine-3H in the ganglia, but caused a 3-fold
increase in salivary glands. There was a marked
decrease in uptake of norepinephrine-3H in the nictitating

membranes; this was probably due to the reduced blood

79

Figure 9. Effects of a-methyltyrosine (aMT), desmethyl-
imipramine (DMI) and stimulation on the
norepinephrine (NE) contents of ganglia,
salivary glands and nictitating membranes.

Decentralized preganglionic fibers were stimulated
unilaterally for 1 or 3 hours. Contralateral nonstimulated
tissues served as controls. 0.9% sodium chloride solution
(0) was infused i.a. at a rate of 0.1 ml/min; DMI (I)

2 mg/kg was injected i.v. concomitant with the start of
stimulation. When stimulation was continued for 3 hours,
an additional dose of DMI (1 mg/kg) was injected 2 hours
after the initial dose. aMT (A) alone or in combination
with DMI (0) was infused through both common carotid
arteries for l or 3 hours at a rate of 0.1 mg/min. Each
point represents the mean of 3 to 6 eXperiments. The
vertical bars represent : 1 S.E. of the mean. The collated
NE contents of nonstimulated ganglia, salivary glands and
nictitating membranes were 8.98 : .63 ug/g, 1.57 : .06 ug/g
and 1.35 : .06 09/ membrane reSpectively. Single asterisks
indicate values that are Significantly different from
appropriate saline controls and 100% (P<.05). Double
asterisks indicate values that are Significantly less than
100 (P<.05).

80

150
GANG-LIA

    
 

100.

U!
0

 

8338

 

 

 

 

 

 

 

 

 

(NE) 96 OF NONSTIMULATED
:-
O

 

 

 

 

20
o l
‘00 mcnunuc MEMBRANES
3“) b .\. ‘fi
60 . \‘\ ii
‘4‘, _ ‘Ii*
20 .—
o L l l
0 'I 2 3
HOURS

Fbgure 9. Effects of a-methyltyrosine (aMT), desmethyl-
imipramine (DMI) and stimulation on the
norepinephrine (NE) contents of ganglia,
salivary glands and nictitating membranes.

81

flow resulting from contraction of vascular smooth
muscles during stimulation periods. Desmethylimipramine
did not alter the uptake of norepinephrine-3H in the
stimulated ganglia, but caused a 5-fold reduction in the
incorporation of norepinephrine in stimulated salivary
glands and nictitating membranes.

The effect of desmethylimipramine on the stimulus-
induced contractile response of the nictitating mem-
branes is illustrated in Figure 8. Immediately following
the administration of desmethylimipramine there was a
slight increase in the contractile reSponse of the
nictitating membranes and a marked slowing in the rate
of relaxation. From Figure 8, it appears that the
initial rate of relaxation is not altered by desmethyl-
imipramine and that the resting tension is increased.
Nevertheless, if the membranes were allowed to relax
after a single stimulus the tension returned to pre-
stimulus levels within 3 min in saline-treated cats,
but it took approximately 9 min in desmethylimipramine-
treated cats. These results suggest a more prolonged
contact of released norepinephrine with the receptor as
a result of reduced uptake of this amine. Similar
results have been reported by Fischer and Snyder (1965)
using cocaine, and by Sigg et al. (1963) using

desmethylimipramine to block norepinephrine uptake.

82

d. Effects of stimulation, a-methyltyrosine and
desmethylimipramine

In the presence of a combination of a—
methyltyrosine and desmethylimipramine the effects of
various schedules of stimulation on the norepinephrine
content of cell bodies were the same as those obtained
in the presence of d-methyltyrosine alone (Table 5).
That is, the a-methyltyrosine-induced reduction in the
ganglionic contents of norepinephrine was not altered
by desmethylimipramine in the presence or absence of
stimulation.

In the salivary glands, the combination of
desmethylimipramine and a—methyltyrosine enhanced the
norepinephrine depletion resulting from 3 hours of
preganglionic stimulation at 2 hz to a greater extent
than that produced by either one of these drugs alone
(Table 5). Stimulation at 10 hz in the presence of
a-methyltyrosine and desmethylimipramine, almost totally
depleted norepinephrine within 1 hour (Tables 5 and 7;
Figure 7).

The effects of stimulation, a-methyltyrosine
and desmethylimipramine in the nictitating membranes
were qualitatively similar to those seen in the
salivary glands, with the changes in norepinephrine
concentrations being less pronounced; the effects were

Sigflificant only at higher frequency of stimulation.

83

For example, stimulation for 1 hour alone, or in the
presence of a-methyltyrosine and/or desmethylimipramine,
did not cause a marked reduction in the norepinephrine
content of nictitating membranes (Figure 9; Table 7);
the effects of stimulation, a-methyltyrosine and
desmethylimipramine were not evident until after 3 hours
of stimulation.

These results suggest that in cell bodies norep-
inephrine concentrations are maintained by synthesis
independent of the frequency of neuronal activity. In
nerve terminals, stimulation reduces the norepinephrine
concentrations to new steady state levels; synthesis
partially maintains the norepinephrine concentrations
during high and low frequencies of stimulation. Reuptake,
on the other hand, appears to play a Significant role
only at higher frequencies of stimulation.

2. Effects of preganglionic stimulation and a-

 

methyltyrosine on the conversion of tyrosine-14C

 

to norepinephrine-14C

 

The formation of norepinephrine-14C in cell
bodies and terminals of stimulated and nonstimulated
neurons was determined following i.a. administration of
tyrosine-14C (Table 10).

Following i.a. infusions of tyrosine-14C
relatively more of this amino acid was converted to
norepinephrine in resting or nonstimulated cell bodies

than in the corresponding terminals. Thus, in ganglia

 

 

 

 

 

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mm mm mm 6 62
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o -mz 6n
6n
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.m.a mo c0amnm>aoo man no A928I maamonmnahanmE-8 cam a0anmaseanm mo nommmm .oa manna

85

.Amo.vmv a Eonw namnmwmap wanamoamaamam mum 6m5am> mmmaem

uanmmxm mnmummmm m Eonm pmaaeumnma mm .m.m a +
u m .pmaSEanmaoa n mz
Imnm pmuaamnnamomo
man ana3 namnanoaoo pmmSmaa 663 928
nmma man maausa wnmnnm panonmo aoafioo aomm amsonan ammsmaa 663 U

«a

.mnamE

- ammE man nammmnmmn mmsam> .pmnMasEanm
.Na oa n8 manamnnaanmnaa pmnMasEanm mums mnmnam oaaoaamamm
.mnsoa m now aaE\mE a.o no mnmn man nm aoanmasEanm mo nnmnm
.UOaHmm aoanMaSEanm naoa-m 8 mo ndoa mama-mao

Imaamonhn no on mm

AU.naOUI oa manna

86

approximately 17% of the total radioactivity was
represented by norepinephrine whereas in salivary
glands and nictitating membranes norepinephrine
accounted for only 0.1% of total radioactivity. These
results would be eXpected if the turnover of norep-
inephrine is faster in the cell bodies. Preganglionic
stimulation markedly increased the formation of
norepinephrine-14C in tissues containing nerve terminals;
the norepinephrine-14C content in stimulated salivary
glands was 22 times greater, and in nictitating mem-
branes was 3 times greater than in the respective
nonstimulated tissues. This increase is also apparent
when the norepinephrine-14C is eXpressed as a percent
of total radioactivity. Stimulation caused an approxi-
mate 12 fold increase in the percent norepinephrine—14C
in salivary glands and a 5 fold increase in nictitating
membranes. On the other hand, there was no difference
between the amount of norepinephrine-14C in terms of
absolute amount or percent of total radioactivity, in
stimulated and nonstimulated ganglia, when tyrosine-14C
was infused i.a. (Table 10).

Treatment with a-methyltyrosine did not alter total
radioactivity but reduced the conversion of tyrosine-14C
to norepinephrine-14C in all tissues (Table 10). In
nonstimulated preparations the effect of a-methyltyrosine

was more pronounced in cell bodies (a 10 fold reduction)

87

than in terminals (a 3 fold reduction in both salivary
glands and nictitating membranes). In salivary glands
and nictitating membranes a-methyltyrosine caused a
relatively greater inhibition of norepinephrine-14C
formation in stimulated than in nonstimulated tissues;
this was not true in ganglia. Interpretation of these
latter results, however, Should be tempered by the fact
that after c—methyltyrosine the radioactivity of norep—
inephrine in all tissues was very low, less than twice
the background.

To avoid possible errors that might result from the
i.a. administration of tyrosine-14C (e.g., unequal
distribution resulting from changes in blood flow due to
surgical manipulations in the neck region) eXperimentS
were performed using i.v. administration of this amino
acid. The results obtained with i.v. and i.a. infusion
of tyrosine-14C were similar (compare Tables 10 and 11).
Following i.v. tyrosine-14C more norepinephrine-14C
accumulated in the nonstimulated ganglia (9.8% of total
radioactivity) than in nonstimulated salivary glands
and nictitating membranes (approximately 0.1% of total
radioactivity). Stimulation markedly increased the
formation of norepinephrine-14C in the latter two
tissues but did not significantly influence the amount

of this radioactive amine in the ganglia.

88

.Amo.vmv a Eonw namnmmmaa manamoawacmam mum mmsam> mmmaam

.mnamEanmmxm mnmnmmmm v Eonw pmaaenmnma mm .m.m a H

same man nammmnmmn mmSam> .Aomnmasaanm n m NomnMaSEanmaoa u sz Na oa no wanamnnaEnmnaa
pmnmasaanm mums mumnam oaaoaamammmnm amuaamnnamomo .aoanmm aOanmaSEanm nsoa-m 6 no
Hsoa mama-mao nmma man maaHSp aam> amnoEmm m amsouan pmmsmaa 663 Ova-maamonmn no on ooa

 

 

 

 

16665\8666
6n. no. 66 6 ooo.o 6n6.6 mmomnneme
6oo.o 666.o 66o.o 666.6 HmmN Hn6 66.o HoN6.N6 6on6.66 oonnmnnnonz
noxsaoo
6N. o N6N on 6no.6 666.6 moomnm
666.Nn H66.n nnn.o 6n6.on 6666 H66 666.n Hon6.o6 HnNN.66 666>nn66
6.n 6.n o6 6n Nan noN :66 on\emov
66.n 6o6.6n Ho6.6 6NN.n H6nN H6Nn 6n.n n6o6.n 6666.n 6nnoo66
mm mm mm 6 6z
6 6 62 6 6 62 6
oon x Nun>nnomonmmm nmnoa ovn-mz ann>nnomonomn nmnoa

ovn

.a06a-mzv Omfilmaauammaammuoa

on Ova-maamonwn Umnmnmaaaeam .>.a mo a0amnm>aoo man so aoan sEanm mo nommmm .aa manna

89

3. Effects of stimulation and cycloheximide on the

 

tissue contents of norepinephrine and on the

 

conversion of tyrosine—14C to norepinephrine-14C

 

Previous results indicated that stimulation does
not modify the norepinephrine contents or the synthesis
of norepinephrine in cell bodies but increases the
synthesis of this amine in nerve terminals. In cell
bodies, synthesis of norepinephrine may proceed at a
maximal rate which cannot be modified by acute
stimulation. In the terminals, part of the stimulus-
induced augmentation in norepinephrine synthesis could
be due to an increase in catecholamine synthesizing
enzymes. These possibilities were tested with the
administration of cycloheximide, a drug which blocks
protein synthesis. The results are summarized in
Tables 12-14.

Cycloheximide increased the contents of norep-
inephrine in nonstimulated ganglia and nictitating
membranes (Table 12). This compound did not modify the
stimulus-induced decline of norepinephrine in salivary
glands and nictitating membranes (Table 13). In salivary
glands the norepinephrine contents were 45 and 41% of
control and in the nictitating membranes 74 and 88% of
control in the absence and presence of cycloheximide
respectively. When tyrosine-14C was infused the total
radioactivity in all tissues was doubled in the presence

of? cycloheximide (Table 14). Because of the variability,

90

.Amo.vmv wooa Eonm namnmmwaa wanamoamaamam mum mmSam>m

.mnamEanmmxm mnmnmmmm m Eonm omaaenmnma mm .m.m a H namnaoo mz amme man nammmummn mmsam>
.nmnma musoa v Um>OEmu mnm3 mmsmman amnmmnn “a.m.a .mx\mE oaI mpaaaxmaanwo mo a0anmnnm
uaaaEUm man mnOan Um>OEmn mum; nman mmSmman mo namnaoo mz man nammmnmmn mmsam> aounaoo

 

 

 

6a ma. ha. Na ma. om. v om. an.
mvaa Hmv.a Hmm.a Hmaa Hom.a Hmh.a mHava Hmm.oa Hmm.>
aonnaou manaxma aonncou aonnaoo manaxma aonnaov aounaou mpaEaxma aonnaou
no 6 -0n66o no 6 -0n06o no 6 -0noso
AmamnnEmE\m:I am\m:I Am\m:I
mmamnnEm2 maanmnanoaz mannao mnm>aamm maamamo

 

.Isz mannammaammnoa mo mnamnaoo mamman a0 mUaEaxmaoaomo mo nommwm .Na manme

91

.amo.vmv ooa Eonw namnmmmaa wanamoamaamam mum nman mmaam>m

.mnamEanmmxm mnmnmmmm mmnan

Eonw UmaaEHmnma mm .m.m a H ammE man nammmnmmu mmsam> .a0anmaaEanm no nnmnm

man mn0mmn nsoa mao .m.a Umnomnaa 663 ama\me oav manaxmaan>U

.26666n62666 u 6

NamasEanmaoa n mzv mnsoa m MOM Na oa nm omnmaSEanm cam amnmao man can UmGOanomm
mums mnmnam mascaamammmnm man aoaaz aa mnmo an nso Umannmo mnm3 mnamEaHmmxm may

 

 

 

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namEnmmnB
AmamunEmE\mnv am\mnv

mmamnnEmz maanmnanoaz

mannaw >Hm>aamm

 

no mnamnaoo mamman ao

.maanammaammnoa msocm00©am

666266626n02u 6:6 oonn6n22666 66 6606666 .6n 6nn66

92

nammmnmmn mmsam>
manaxmaoao>U

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.mnamEanmmxm mnmnmmmm mmnan Eonm omaaenmnmp mm .m.m a H some man

.amnmaSEanm

m Namnmaseanmaoa
mnmz mnmnan oaaoaamammmnm amnaamnnamomo
Imao nmma man maansp mnmnnm panonmo aoEEoo aomm amaonan ammnmaa 663 0

m2

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.62 on n6 2n666666266666 6666n62n66

.poanmm aoanMaSEanm nsoa-m 6 mo nsoa mama

Imaamonwn mo 0: mm

 

 

 

 

 

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m2 mm mm
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Umnmnmaaaapm .m.a no aOamnm>aoo man aw maaEaxwaoaomo cam a0anmasEanm mo nommmm .6a manms

93

total radioactivity and the absolute amounts of nor-
epinephrine-14C were not statistically different in the
stimulated and nonstimulated sides. Norepinephrine—14C,
reported as a percent of total radioactivity, was the
same in stimulated and nonstimulated ganglia, but was
increased 3 and 2 fold in stimulated salivary glands
and nictitating membranes respectively. This increase
was much less than that obtained in the absence of
cycloheximide (compare Tables 10 and 14). The
variability in these experiments prohibited definite
conclusion, but it appears that cycloheximide does not
affect the formation of norepinephrine-14C in cell
bodies but reduces the stimulus-induced increase in
the formation of norepinephrine-14C in the nerve
terminals.

4. Possible fate of norepinephrine in cell bodies

Norepinephrine in the cell bodies may be

released, metabolized by monoamine oxidase, and/or
transported down the axon. Pretreatment with a
monoamine oxidase inhibitor (pheniprazine) did not
alter Significantly the a-methyltyrosine-induced
depletion of norepinephrine in the ganglia (Table 15).
Pheniprazine (5 mg/kg, i.v.) was administered to two
cats. One hour later the ganglia on one side were
excised and a-methyltyrosine was then infused through
the common carotid arteries (0.1 mg/min for 3 hours).

The norepinephrine content declined to 41% of control

94

.mmmmanamnmm man aa pmnmoaoaa mnamEaummxm mo nmnfida man Eonm omaaEnmnmo
66 .m.m a H same man nammmnmmn 6m5am> .COamsmaa 928 no nnmnm man mn0mmn nsoa a
.>.a pmnomhaa 663 amx\me mI maaumnmaamam .mmsna no aoanmnnmaaaeam man no nnmnm
man mHOmmn pm>o€mn mnmz nman mmsmman mo namnaoo mz man nammmnmmn mmSam> aonnaou

 

 

 

n N6. 66 N N6. 6n.n

6onn 66n.n 666.n 6n6 666.6 666.6 2N6 666666666666
6 6n. 6N. 6 6N. n6.

666 666.6 666.6 666 6n6.N 666.6 266 mcoz

aonnaou B28 aonnaoo aonnaou B28 aonnaou
no 6 mo 6
26\6:6 26\666 666266666

mannao wnm>aamm waamamw

 

.asz mannammaammnoa mo mnamnaoo momman man no a0amswaa
2828I meamouanamanmsna no musoa mmnan can maanmnmaamam no mnommmm .ma manna

95

during the d-methyltyrosine infusion. This reduction is
similar to that obtained in the absence of a monoamine
oxidase inhibitor (see Table 2).

Norepinephrine is transported centrifugally from
the cell body. This transport appears to contribute
to the rapid decline of norepinephrine in the ganglia
following a-methyltyrosine administration. When the
postganglionic fibers were tied the depletion of
norepinephrine was reduced (Table 16). This eXperiment
was repeated after the administration of pheniprazine;
the results were essentially the same as those obtained
in the absence of pheniprazine. The interruption of
somatofugal tranSport of axonal constituents for 3 hours
did not alter the norepinephrine content of salivary
glands (1.06 : .39 and 1.09 : .32 ug norepinephrine/g in
salivary glands on intact and tied sides respectively).

5. Possible formation of a-methylnorepinephrine from

 

a-methyltyrosine

 

Some a-methyltyrosine is biotransformed to a-
methylnorepinephrine (Udenfriend et aZ., 1966; Maitre,
1965; Van Orden et aZ., 1970) which in turn could
diSplace endogenous norepinephrine (Dominic and Moore,
1971). a-Methyltyrosine-induced depletion of norep-
inephrine in terminals in the presence of a-
methyltyrosine, might result from the formation of

d-methylnorepinephrine. Therefore, experiments were

96

Table 16. Effects of a-methyltyrosine (aMT), pheniprazine
and tying of postganglionic fibers on the
norepinephrine (NE) content of ganglia.

 

 

Treatment Postganglionic Fibers Percent
Intact Tied of Intact

dMT, 1 hr (3) 4.85: 7.30: 150:
1.03 1.93 20

GMT, 3 hrs (5) 1.87: 3.77: 202:a
0.97 1.57 25

GMT, 3 hrs + 3.63: 5.38: 157:a

Pheniprazine (3) 1.20 1.20 15

 

Postganglionic fibers of both Sides were eXposed and those
of one side were tied immediately cranial to the ganglia
with a silk thread. aMT was then infused through both
common carotid arteries at the rate of 0.1 mg/min.
Pheniprazine (5 mg/kg) was injected i.v. one hour before
the start of the aMT infusion. Values represent the mean
: 1 S.E. of norepinephrine (pg/g) as determined from the
number of eXperiments indicated in the parentheses.

aThese values are Significantly greater than 100% (P<.05).

97

conducted to determine if the formation of a—methyl-
norepinephrine contributed to the actions of a-
methyltyrosine in cell bodies and terminals.
Twenty-five uc of a-methyltyrosine-BH was infused
through each common carotid artery during the last one-
half hour of a three hour stimulation period. The
results are summarized in Table 17. In nonstimulated
ganglia and salivary glands very little radioactivity
was detected in the a-methylnorepinephrine fraction.
The counts were too low (less than twice background)
to further identify this radioactivity. Furthermore,
stimulation did not increase the amount of radioactivity
in the a-methylnorepinephrine fraction. This is in
contrast to the marked increase in the conversion of
tyrosine-14C to norepinephrine-14C in stimulated
salivary glands (Table 10 and 11).

6. Norepinephrine contents and the contractile

 

responses of nictitating membranes

 

a. Effects of a-methyltyrosine and continuous

 

stimulation at low frequency (2 hz)

 

a—Methyltyrosine solutions (0.05 mg/ml or
0.1 mg/ml) were infused i.a. for 5 1/4 hours. One hour
after the beginning of a-methyltyrosine infusion
continuous stimulation of pre- or postganglionic fibers
was started. The stimulation was interrupted every

45 min to record a test contractile response of

98

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.npmnmasEanm - m NamnMaSEanmaoa u sz Na oa no wanamnnaenmnaa UmnMasEanm mum: mnmnam

mascaamammmnm amuaamnnamomo .UOnan aoanmasEanm nsoa-mmnan 6 mo nsoa mama-mac
n66a man maanSU wnmnnm panonmo coEEoo aomm amsonan pmmsmaa 663 am192-8 no on mm

 

 

 

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m

 

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nmnwm =Aam-mz28I am-maanammaammnoaa anmEn8 namnmmmm: mo acanmanom .ha manme

99

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m on m Eonm Umaaanmnma mm .m.m a nammmnmmn naaom aomm ao omnomflonm mmaaa

amoannm> .mmamnnEmE maanmnanoaa UmnmasEanmcoa man no aoHnmaSEanm nae a an

mmEan mnmanmonmmm no Umaamnno 663 nman a0anomnnaoo aonnaoo man no namonmm mm
ommmmnmxm aoanomnnaoo mamnnEmE maanmnanoaa ammE man mnammmnmmn naaom aomm

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m pmnmsnnmnaa 663 acanmaDEanm may .UOaHmm aaE mv nmanoam non omsaanaoo

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a0an8a58anm .Na N nm wamsosaanaoo pmnmaDEanm mums mnmnam oaaoaamammmnm mo aam

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nmnmm nsoa mao .mnsoa 6\a m now aaE\mE a.o no aaE\mE mo.o mo mnmn 8 nm

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oaaoaamammmnm man aoaa3 an mnmo an n50 amannmo mnm3 mncmEanmmxm mas

.mmamnnEmE meanmnanoaa man no 6mmaommmn maanomnnaoo
man so Aezov maamonmnawanmala cam a0anma58anm no mnommmm .oa mnsmam

100

.mmamnnEmE mannmnanoac man no 6mmaommmn maanomnnaoo
man so AB28I maamonwnawanmel8 cam a0anmasEanm no mnommmm .oa mnamam

mmbom
m m 8 m N a o

_ _ n _ n n n- 6

 

 

o:

  

TOHINDO JO % SV NOIiCVHINOO SNVHHWEN ONIIVIIlOIN

I. 8.

101

nonstimulated and stimulated nictitating membranes. The
results are summarized in Figure 10. The contractions
of the nictitating membranes progressively decreased in
saline-infused (control) cats. Pre- or postganglionic
stimulation in the presence of a-methyltyrosine appears
to reduce the contractile reSponses, but the effects
were variable and not Significantly different from
those obtained after stimulation alone. Similarly,
there was no significant difference in the norepinephrine
contents of nictitating membranes between saline- and
a-methyltyrosine-treated cats after pre- or
postganglionic stimulation (Table 4).

b. Effects of a-methyltyrosine and continuous

stimulation at frequencies of 2 hz, 10 hz,

and 16 hz.

 

Since the contractile reSponseS of nictitating
membranes were small and variable at low frequencies,
stimulation was carried out at higher frequencies. The
results are summarized in Table 18. The contractions
of nictitating membranes (eXpressed as % of initial
contractions) in a-methyltyrosine-treated cats at
stimulation frequencies of 10 hz and 16 hz were
Significantly less than those obtained at 2 hz. A
significant reduction in norepinephrine contents was
obtained only after stimulation at 16 hz. In saline-

treated cats, the norepinephrine contents and

102

Table 18. Effects of stimulation and a-methyltyrosine
(aMT) on the norepinephrine (NE) contents
and contractile response of nictitating
membranes.

 

 

 

Frequency of Contractile b
Treatment Stimulation Responsea NE
n n
GMT 2 hz 3 85: 3 76:
9 8
GMT 10 hz 3 56:C 2 74:
3 10
GMT 16 hz 3 45:C 3 69:C
8 6
Saline 16 hz 2 71$ 3 66:
ll 13

 

The eXperiments were carried out in cats in which the pre-
ganglionic fibers were sectioned. aMT was infused i.a. at
a rate of 0.1 mg/min for 75 min. Fifteen minutes after the
start of aMT infusion, decentralized preganglionic fibers
on one side were stimulated continuously for 1 hour at the
indicated frequencies.

aThe values are the mean : l S.E. eXpressed as the percent
of the initial contractile response.

bThe values are the mean : l S.E. eXpressed as the percent

of the nonstimulated control content of NE.

cThese values are significantly different from 100 (P<.05).

103

contractions of nictitating membranes were not signifi-
cantly different from controls after 1 hour of
stimulation at 16 hz but were reduced in the presence
of a-methyltyrosine.

c. Effects of a-methyltyrosine, desmethyl—

 

impramine and intermittent stimulation at

 

low (2 hz) and high (10 hz) freguencies.

 

To further test whether a correlation existed
between the norepinephrine contents and contractions of
nictitating membranes eXperiments were conducted after
the blockade of both synthesis and uptake of norep-
inephrine. The results are summarized in Figure 11.
After three hours of intermittent stimulation at 2 or
10 hz the contractions of nictitating membranes in
saline—treated preparations and in those that were
treated with a-methyltyrosine and/or desmethylimipramine
were not statistically different. On the other hand,
when stimulation was carried out at 10 hz for 1 hour
the contractile responses in preparations treated with
a-methyltyrosine or a—methyltyrosine and desmethyl-
imipramine were significantly less than those of
saline-treated preparations. Desmethylimipramine
alone did not modify the contractions of nictitating
membranes.

There does not appear to be any relationship
between the norepinephrine contents and the magnitude

of contraction of the nictitating membranes (compare

101+

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105

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106

Table 7 and Figure 11). For example, the norepinephrine
contents of nictitating membranes following stimulation
at 10 hz for 3 hours were reduced to 37% of pre—
stimulation values in the presence of a-methyltyrosine +
desmethylimipramine whereas in saline-treated
preparations the norepinephrine content was only

reduced to 74%. However, the contractions of nictitating
membranes in saline—treated and a-methyltyrosine +
desmethylimipramine-treated preparations were not
statistically different. Similar results were obtained
by Thoenen et a1. (1966) who proposed that there was no
functional relationship between the content of norep—

inephrine and the contractions of nictitating membranes.

C. The Dynamics of Norepinephrine in the Neuronal Cell

 

Bodies and Terminals During the Post—Stimulation

 

Period.

1. Restoration of norepinephrine after cessation of

 

stimulation and the effects of desmethylimipramine

 

and a—methyltyrosine

 

The results of eXperiments designed to determine
the recovery of norepinephrine during the post-stimulation
period are summarized in Figure 12 and Table 19. The
stimulation-induced depletion of norepinephrine observed
in previous eXperiments was presumably due to the
release of norepinephrine from noradrenergic terminals

until a new steady state was attained (Figure 9). This

107

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Amo.vmv ooflnmm coflumaosaum Hsoc m m mo coflummmmo mcu Hmumm hamumacmeefl
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108

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mo mucmccoo momma» mo aum>oomu cowumaoeflumumom mcu co

 

 

 

 

 

 

 

 

 

 

 

 

 

cazov mcflmoumuamcumEIo cam AHSQV mcHEmHmNEHamccmEmmo mo muommmm .NH musmflm
W _
250: I
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109

Table 19. Effects of desmethylimipramine (DMI) and a-
methyltyrosine (aMT) on the recovery of tissue
contents of norepinephrine (NE) following
stimulation-induced depletion.

 

Treatment and time

after cessation Ganglia (pg/g)

 

 

 

 

of stimulation NS S S/NS x 100
Saline
0 hours 8.25: 7.50: 94
.81 .34
2 hours 11.16: 10.21i 91
1.34 1.72
6 hours 8.61i 8.06i 94
1.70 1.16
DMI
0 hours 9.26i 8.38i 92
1.08 1.00 .
2 hours 11.33: 9.34i 82
1.24 1.06
6 hours 6.69i 7.20: 108
.97 1.13
DMI and
GMT
6 hours 1.55: 1.52: 98
.27 .07

 

The experiments were carried out in cats in which the pre-
ganglionic fibers were sectioned and the distal end
stimulated intermittently (30 sec/min) at 10 hz for 3 hours.
(NS = nonstimulated; S = stimulated). Two-thirds of the
total dose of DMI (3 mg/kg) was injected i.v. concomitant
with the start of stimulation and the remaining one-third
of the dose was injected 2 hours later. GMT was infused
through both common carotid arteries at the rate of 0.1 mg/
min for 6 hours simultaneously with the cessation of stimu-
lation. Tissue NE contents were determined immediately

(0 hour) and 2 and 6 hours after the cessation of stimulation.
The tissue contents of NE are reported as the mean i l S.E.
as determined from 3 to 5 experiments.

110

Table 19 (Cont'd)

 

 

 

 

 

Salivary Glands (ug/g) Nictitating membranes
(pg/membrane)

NS 8 S/NS x 100 NS 5 S/NS x 100
1.70: 0.72: 42b 1.22: 0.72: 74b
.30 .03 .08 .09
1.55: 0.98: 63b'C 1.02: 0.84: 82
.24 .13 .18 .14
1.68: 1.10: 65b'C 1.65: 1.47: 89
.24 .13 .31 .42
1.63: 0.39: zsa'b 1.63: 0.98: 58a
.16 .05 .27 .04
1.40: 0.69: 49b'C 1.58: 1.25: 79b
.11 .13 .18 .20
1.42: 1.04: 73k"C 1.58: 1.12: 71
.07 .04 .38 .10
1.35: 0.33: 24d 1.16: 0.80: 69
.33 .04 .18 .03

 

aThese values are significantly different from corresponding
saline controls (P<.05).

bThese values are significantly different from 100 (P<.05).

CThese values are significantly different from corresponding
0 hour values (P<.05).

dThis value is significantly different from that obtained

after 6 hours of cessation of stimulation in presence of

DMI (P<.05).

111

new steady state appears to be maintained by synthesis
and retrieval of released norepinephrine. However,
estimations of the ability of the terminals to
synthesize norepinephrine are complicated by the slow
rate of turnover of this amine in the resting state

and by release, uptake, and metabolism of norepinephrine
during stimulation. Therefore, the capacity of nor-
adrenergic terminals to synthesize norepinephrine was
studied by following the restoration of the tissue
contents of this amine after periods of stimulation.
Since norepinephrine presumably regulates its own
synthesis by feedback inhibition of tyrosine hydroXylase
the rate of recovery of norepinephrine would give an
estimate of the capacity of terminals to synthesize
norepinephrine. Decentralized preganglionic fibers were
stimulated unilaterally for 3 hours at 10 hz and
norepinephrine contents of tissues were determined
immediately and at 2 and 6 hours after the cessation

of stimulation.

The norepinephrine contents of ganglia were not
altered by stimulation and during the post-stimulation
period the ganglionic norepinephrine remained at control
levels. In salivary glands stimulation reduced the
norepinephrine content to 42% of control. Two hours
after the cessation of stimulation the norepinephrine
content increased to 65% of control and stabilized at

this level for an additional 4 hours. The

112

post-stimulation changes in the norepinephrine content
of nictitating membranes were similar to those of the
salivary glands but were less pronounced (Table 19).
Recovery of norepinephrine contents could be due,
in part, to the desmethylimipramine sensitive con-
centrating mechanism in noradrenergic terminals which
retrieves norepinephrine from the circulating blood
(Axelrod et aZ., 1961; Strbmblad and Nickerson, 1961;
Kopin and Gordon, 1963). In order to test this
possibility, and to accentuate the effects of stimulation
on the norepinephrine contents of nictitating membranes,
stimulation was carried out in the presence of des-
methylimipramine. After 3 hours of stimulation the
norepinephrine contents of salivary glands and
nictitating membranes decreased to 25 and 58% of controls
respectively. Two hours after cessation of stimulation
the norepinephrine contents increased to 49% of control
in salivary glands and to 78% of control in nictitating
membranes; again they tended to plateau at these levels.
Since the initial rates of recovery of norepinephrine
in salivary glands during the post-stimulation period
in the absence of desmethylimipramine seemed to parallel
the recovery of norepinephrine in the presence of this
drug, it seems unlikely that uptake of norepinephrine
from the circulation contributed to this recovery. The

restoration of norepinephrine appeared to be due

113

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.mucmEHummxm mumnmmmm m Eonm omcHEumump mm .m.m H H cmmE mcu ucmmmummu

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mo 0: m.N .cOHumHsEHum mo cOHummmmo mcu umumm musoc me .omumHDEHum u m «omwMHseHumcoc

u mz .musoc m How Nc OH um ACHE\omm omv mHucmuuHEHmuCH omumHsEHum cam HmumHo mcu 6cm
omQOHuomm mnm3 mumcHw oHcoHHmcmmmum mcu coHc3 2H mumo CH poo omHHumo mumz mucmEHnmmxm ch

 

 

 

 

 

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mm.~ om. 0mm hmm ommm mwms 66 an we
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mm.H mm.H OHsa emu coon mafia Amxsmmc
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umumm musoc me UVHImcHEmmoo Eoum AUVHImzv UanmcHHcmmchmuoc mo :oHumEuom .oN mHnmh

114

primarily to synthesis of norepinephrine since the
infusion of a-methyltyrosine, started immediately after
the cessation of stimulation and continued for 6 hours,
prevented the restoration of norepinephrine in salivary
glands and nictitating membranes.

2. Conversion of dOpamine-14C to norepinephrine-14C

 

during,the_post-stimu1ation_period

 

Following stimulation, the norepinephrine
contents of salivary glands and nictitating membranes
plateaued at levels that were markedly lower than
controls (Figure 12; Table 19). It is not clear what
factors limited the restoration of this amine. It has
recently been demonstrated (Geffen et aZ., 1969) that
dOpamine-B-hydroxylase is released into the venous
effluent of Spleen following stimulation of Splenic
nerves. Both storage vesicles and dOpamine-B-hydroxylase,
which are intimately associated, may be released
together (Viveros et aZ., 1969). Therefore, loss of
this enzyme and/or the storage vesicles may be respon-
sible for the failure of norepinephrine contents to
recover completely following prolonged periods of
stimulation. This possibility was tested by studying
the conversion of dOpamine-14C to norepinephrine-14C.

Dopamine-14C was infused i.a. 6 hours after the
Cessation of stimulation as described in Methods. The

results are summarized in Table 20. In nonstimulated

115

preparations the percentages of total radioactivity
incorporated into norepinephrine-14C in ganglia, salivary
glands, and nictitating membranes were 28%, 10% and 8%
reSpectively. These percentages of norepinephrine-14C
were greater than those found after the administration
of tyrosine-14C. This would be eXpected if the
hydroxylation of tyrosine to DOPA was the limiting

step in the synthesis of norepinephrine. There was no
difference in the formation of norepinephrine-14C in
stimulated and nonstimulated ganglia. However, the
formation of norepinephrine-14C in salivary glands and
nictitating membranes was significantly increased (1.9
and 1.6-fold respectively). These results suggest that
the capacity of the noradrenergic terminals for storage
and synthesis of norepinephrine from dOpamine is not
reduced following nerve stimulation. Indeed, the
capacity of the stimulated terminals to accumulate
norepinephrine-14C from dopamine-14C is enhanced.

3. Conversion of tyrosine-14C to norepinephrine-14C

 

during and at various times after the cessation

 

of stimulation

 

The results of studies on the conversion of
dOpamine-14C to norepinephrine-14C indicated that the
absence of complete restoration of norepinephrine
during post-stimulation period could not be due to a

deficiency of dOpamine-B-hydroxylase or storage sites

116

for norepinephrine. Therefore, the possibility that
DOPA, and hence dOpamine, could limit the complete
recovery of norepinephrine was considered. Previous
studies have demonstrated that electrical stimulation
increased the formation of radioactive norepinephrine
from tyrosine-14C but not from DOPA-3H (Sedvall and
Kopin, 1967). This would suggest that the stimulus-
induced increase in the formation of norepinephrine
occurs at or before the tyrosine hydroxylation step and
the formation of DOPA is the rate limiting step in the
biosynthesis of norepinephrine. Conversion of tyrosine-14C
to norepinephrine-14C was studied during the last one-
half hour of a 3 hour stimulation period, immediately
after, and at 2 and 6 hours after the cessation of the
3 hour stimulation period. The results are summarized
in Tables 21-24 and for clarity the results of the
studies with ganglia and salivary glands are graphically
depicted in Figure 13.

During stimulation and at all times after the end
of the stimulation period the formation of norepinephrine-
14C was similar in nonstimulated and stimulated ganglia.
On the other hand, stimulation resulted in approximately
12- and 6-fold increases in the formation of norep-
inephrine -14C in salivary glands and nictitating membranes
respectively. Immediately after the cessation of

stimulation the formation of radioactive amines in these

117

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vH

VH

eH

118

Table 22. Conversion of tyrosine-14C to norepinephrine-14C
(NE-14C) in salivary glands during and at
various times after cessation of stimulation.

 

Total radioactivity

 

 

Tyrosine-14C (dpm/g)
infused at S
NS 3 fig

-1/2 hour poststimulation 36,789: 74,215: 2.02a
(3) 8,663 16,014

0 hour poststimulation 54,874: 78,681: 1.43
(4) 16,117 6,792

2 hour poststimulation 36,032: 56,063: 1.56
(3) 9,079 24,377

6 hour poststimulation 96,810: 59,951: 0.62
(3) 44,833 36,809

 

The eXperiments were carried out in cats in which the pre—
ganglionic fibers were sectioned and the distal end
stimulated intermittently (30 sec/min) at 10 hz for 3 hours.
NS = nonstimulated; S = stimulated. 25 no of tyrosine-14C
was infused through each common carotid artery for one—half
hour during and at indicated periods after the cessation of
stimulation. Values represent the mean : l S.E. from the
number of eXperiments indicated in parentheses.

aThese values are significantly different from 1 (P<.05).

119

Table 22 (Cont'd)

 

 

 

 

NE-14C NE-l4c x 100
(dpm/g) Total radioactivity
s 3
NS 5 fig- NS 8 —§
55: 1,225: 22.27a .15: 1.73: 11.53a
15 153 .02 .18
89: 874: 9.82 .16: 1.09: 6.81a
31 252 .02 .29
141: 625: 4.43 .36: 1.01: 2.80
62 289 .13 .38
119: 138: 1.16 .14: .24: 1.71

43 80 .04 .03

 

120

Table 23. Conversion of tyrosine-14C to norepinephrine-14
(NE—1 C) in nictitating membranes during and at
various times after cessation of stimulation.

C

 

Total radioactivity

 

 

(dpm/membrane)
Tyrosine-14C infused at S

NS S -—-

NS

-1/2 hour poststimulation 64,760: 39,080: 0.60
(3) 11,772 5,904

0 hour poststimulation 69,600: 72,720: 1.04
(4) 8,858 10,243

2 hour poststimulation 44,460: 54,320: 1.22
(3) 4,140 13,140

6 hour poststimulation 103,880: 119,080: 1.15
(3) 18,243 22,722

 

The eXperiments were carried out in cats in which pre-
ganglionic fibers were sectioned and the distal end
stimulated intermittently (30 sec/min) at 10 hz for 3
hours. N§4= nonstimulated; S = stimulated. 25 no of
tyrosine- C was infused through each common carotid
artery for one-half hour during and at indicated periods
after the cessation of stimulation. Values represent the
mean : l S.E. from the number of eXperiments indicated in
parentheses.

aThese values are significantly different from 1 (P<.05).

Table 23 (Cont'd)

121

 

 

 

 

NE-14C NE—14C X 100
(dpm/membrane) Total radiéactivity
5
NS 5 fig- NS 5
61: 196: 3.21 0.09: 0.50: a
13 14 o .08
64: 207: 3.23 0.09: 0.29: a
14 44 0 .04
56: 122: 2.18a 0.09: 0.19:
34 82 .04 .09
73: 91: 1.25 0.07: 0.08:
12 20 0 0

 

122

Table 24. Effect of desmethylimipramine (DMI) on the con-
version of i.a. administered tyrosine—l C to
norepinephrine-l C (NE-14C) six hours after the
cessation of stimulation.

 

Total radioactivity

 

 

Tissue
S
NS S N-S-
Ganglia 3,795: 2,280: 0.60
(dpm/10 mg) 2,128 402
Salivary glands 22,864: 57,589: 2.52
(dpm/g) 5,908 25,466
Nictitating membranes 70,320: 42,840: 0.61
(dpm/membrane) 20,911 8,750

 

The experiments were carried out in cats in which pre-
ganglionic fibers were sectioned and the distal end
stimulated intermittently (30 sec/min) at 10 hz for 3
hours. NS = nonstimulated; S = stimulated. Two-thirds

of the dose of DMI (3 mg/kg) was injected i.v. concomitant
with the start of stimulation and another one-third of the
dose 2 hours later. 25 uc of tyrosine-14C was infused
through each common carotid artery for one-half hour 6
hours after the cessation of stimulation. Values repre-
sent the mean : l S.E. as determined from three separate
experiments.

123

Table 24 (Cont'd)

 

14

 

 

 

14 NE- C
NE- C Total radioactivity x 100
S S
NS S fi§ ~NS S fig
599: 344: 0.60 18.81: 14.21: 0.76
240 131 2.72 4.26
186: 983: 5.28 0.64: 1.08: 1.70
138 848 0.37 0.75
218: 65: 0.30 0.24: 0.14: 0.60

172 26 0.16 0.05

 

124

Figure 13. Norepinephrine content 12d formation of
norepinephrine-14C (NE- C) from tyrosine-
C following stimulation and during
poststimulation periods.

The eXperiments were carried out in cats in which
the preganglionic fibers were sectioned and the distal
end stimulated intermittently (30 sec/min) at 10 hz
for 1 hour (solid bar) or 3 hours (Open bar). Twenty-
five no of tyrosine-14C was infused through each
common carotid artery for one—half hour during the
last one-half hour of a 3 hour stimulation period,
immediately after and at 2 and 6 hours after the
cessation of the 3 hour stimulation period. In the
upper panel the NE contents of ganglia and salivary
glands is eXpressed as percent of nonstimulated
controls. Each point represents the mean from 3 to 5
exPeriments. Vertical bars at each point indicates
the : 1 S. E. of the mean. The height of each bar in
the lower panel represents the mean dpm of NE-14C
expressed as the difference between the stimulated
and nonstimulated tissues. Vertical lines projected
on each bar represents the : l S.E. as determined
from 3 to 4 experiments.

*Values are significantly different from 100%
(P<.05).

**Values are significantly different from 0
(P<.05).

125

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

c; 120
In F 1 '
IL 5 10° \ 1
O 3 80 ,. GANGLIA i
a? 5 * ak '
a 5 6° " #6 .1
z snuvnnv mumps
9; 4° +— ..
20 ,_ ..
o .- SALIVAIY GLANDD d
M
a 1250 f GANGLIA . q
III u:
l; ICKfii. I 4
5 750 _ ‘F .
0 E.
3 500 _ .
i. 250 '- I 11. -
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24 *1
0|- -250 r- .1
S
:,"50K’5y 1 1 1 1 1 1 1 1 1. H
E o I 2 3 4 s 6 7 8 9 10
5 - nouns .
" [ ]

 

Figure 13. Norepinephrine content and formation of
n3repinephrine-14C (NE-1 C) from tyrosine-
1 C following stimulation and during
poststimulation periods.

126

tissues continued at an accelerated rate. Two hours
later the formation of norepinephrine-14C was still
elevated but by 6 hours after the cessation of
stimulation the rate of norepinephrine-14C formation

was the same in stimulated and nonstimulated salivary
glands and nictitating membranes. The time course

of the increased formation of norepinephrine-14C did

not parallel the reduced endogenous norepinephrine.

That is, as the endogenous norepinephrine contents

began to increase the formation of norepinephrine-14C
progressively decreased. Six hours after the cessation
of stimulation when the endogenous norepinephrine
contents were well below (35%) control, the synthesis

of norepinephrine had returned to control levels.
Similar results were obtained in a series of eXperiments
where desmethylimipramine was administered to accentuate
the stimulus-induced depletion of norepinephrine in
nerve terminals (see Table 19). That is, 6 hours after
the cessation of stimulation, endogenous norepinephrine
contents plateaued at 35% below control (Figure 12),

but synthesis returned to control levels. These results
suggest that only part of the norepinephrine stores in
noradrenergic terminals participate in regulating the

norepinephrine synthesis in nerve terminals.

DISCUSSION

A. Regulation of Norepinephrine Content in Neuronal

 

Cell Bodies and Terminals During Stimulation

 

A series of events take place in the terminals of
noradrenergic neurons following electrical stimulation.
Stored norepinephrine is released (Brown and Gillespie,
1957), synthesis of norepinephrine is accelerated
(Weiner and Rabadjija,l968), and released norepinephrine
is recaptured (Iversen, 1967). Although the dynamics of
norepinephrine at nerve terminals have been studied
extensively little is known about the prOperties of
this amine in the cell body.

Small amounts of norepinephrine may be released from
autonomic ganglia following anti- or orthodromic electri—
cal stimulation (Bfilbring, 1944; Reinert, 1963), but
evidence for a modulating action of norepinephrine on
ganglionic transmission is weak (Costa et aZ., 1961).
Some drugs such as reserpine and a-methyltyrosine (Fischer
and Snyder, 1965; Reinert, 1963; Brodie et aZ., 1966),
deplete ganglia of norepinephrine whereas other drugs
are without effect (e.g., tyramine; Fischer and Snyder,
1965). Continuous electrical stimulation does not alter

the norepinephrine content of cat superior cervical

127

128

ganglia (Reinert, 1963). Similar results were obtained
in the present study; that is, a-methyltyrosine produced
a marked depletion of norepinephrine in the superior
cervical ganglia (Figure 5; Table 2) whereas 1 or 3

hours of preganglionic or postganglionic electrical
stimulation (Table 4; Table 5) at low or high frequencies
(Table 5) did not.

Although functions of ganglionic norepinephrine are
unknown the prOperties of this amine in cell bodies are
quite distinct from those in the terminals. For example,
the rate of turnover of norepinephrine is much greater
in ganglia than in neuroeffector organs (Brodie et aZ.,
1966). This is evidenced in the present study by the
fact that a-methyltyrosine caused a rapid decline in the
norepinephrine content in cell bodies but not in tissues
that contain terminals (Table 2; Figure 5). Chronic
decentralization is reported to reduce the turnover rate
and increase the content of norepinephrine in ganglia
(Kirpekar et aZ., 1962; Fischer and Snyder, 1965), but
in the present study acute decentralization (Table 2) or
3 hours of preganglionic stimulation (Figure 7) did not
alter the a-methyltyrosine—induced decline of norep-
inephrine.

It is likely that synthesis of norepinephrine in
the ganglia is proceeding at a maximal rate-~a rate
determined by innate activity of noradrenergic neurons.

The activity of tyrosine hydroxylase is much higher in

129

cell bodies and axons than it is in terminals and cannot
be altered by short term changes in neuronal activity
(Roth et aZ., 1967; Sedvall and Kopin, 1967). In the
present study, cycloheximide, an inhibitor of protein
synthesis, increased the ganglionic-content of norep-
inephrine (Table 12). This is contrary to what would

be eXpected if the high turnover rates of norepinephrine
were due to relatively high turnover rates of tyrosine
hydroxylase. The accumulation of norepinephrine in cell
bodies in presence of cycloheximide could not have been
due to block of axonal transport of norepinephrine
because inhibition of protein synthesis does not effect
the rates of axonal transport (Peterson et aZ., 1967).
Furthermore, an increase in norepinephrine concentration
was also obtained in the noradrenergic terminals of
nictitating membranes (Table 12). The effects of
cycloheximide on the tissue contents of norepinephrine
probably result from some nonspecific, and as yet unknown,
mechanism which is secondary to inhibition of protein
synthesis (Yeh and Shils, 1969). Indeed, after adminis-
tration of tyrosine-14C the total radioactivity in the
ganglia, presumably the non—metabolized amino acid, was
much higher in the presence than in the absence of cyclo-
heximide (compare Tables 10 and 14). No systematic
studies were conducted on the effect of inhibition of
protein synthesis on the turnover of ganglionic norep-

inephrine. The possibility cannot be excluded, however,

130

that elevated concentration of norepinephrine in the
ganglia following inhibition of protein synthesis may
have resulted from a decreased turnover of this amine.

Desmethylimipramine, in the presence or absence
of stimulation, did not alter the content of ganglionic
norepinephrine (Table 8; Figure 6) and did not affect
the uptake of norepinephrine-3H in the stimulated ganglia
(Table 9). In addition, desmethylimipramine did not
modify the effect of a-methyltyrosine. That is, the
decline of ganglionic norepinephrine after a-methyl-
tyrosine was the same in the presence or absence of
desmethylimipramine; therefore, reuptake does not appear
to play a major role in the maintenance of this amine
in cell bodies. A similar conclusion was reached by
Fischer and Snyder (1965) as a result of their studies
with cocaine.

What is the fate of norepinephrine in ganglia?
Dahlstrom and Haggendal (1970) have reported that nor-
epinephrine, supposedly in storage granules, is
transported down the axon. In the present studies some
of the norepinephrine in the ganglia appeared to be
transported somatofugally since a ligature around the
post-synaptic nerve bundle adjacent to the ganglia
partially prevented the a-methyltyrosine—induced decline
of norepinephrine. Nevertheless, the norepinephrine

content of the tied ganglia was reduced. This might be

131

due to incomplete blockade of axonal transport or Sparing
of some postganglionic fibers by the ligature, replace-
ment of the norepinephrine by a metabolite of a—
methyltyrosine (i.e., a-methylnorepinephrine), metabolism
of norepinephrine by monoamine oxidase, or release of
the norepinephrine into the blood. The latter possibility
was not examined, but metabolism by monoamine oxidase
does not appear to be an important factor. Although
this enzyme is found in the superior cervical ganglion
of the cat (Giacobini and Kerpel-Fronius, 1970) it
plays little role in the metabolism of norepinephrine
in this species (Spector et aZ., 1963). In the present
study, pretreatment with a monoamine oxidase inhibitor
did not prevent the reduction of the norepinephrine
content in the ganglia following a-methyltyrosine
administration. The increase in the ganglionic norep-
inephrine that was obtained after ligating the post-
ganglionic fibers was the same in the presence or
absence of a monoamine oxidase inhibitor (Table 16).
In addition, replacement of norepinephrine by a-
methylnorepinephrine did not appear to account for the
decline of norepinephrine stores since little of this
methylamine could be detected in the tissues after
infusions of a-methyltyrosine-3H (Table 17; see also
Van Orden et aZ., 1970b).

One hour of preganglionic stimulation at 10 hz

reduced the norepinephrine content of nerve terminals

132

in the salivary glands. This reduced concentration

of norepinephrine was maintained when stimulation was
continued for 3 hours (Figure 9). Although, a-methyl-
tyrosine did not significantly alter the norepinephrine
content in resting nerve terminals it increased the
stimulus-induced reduction of norepinephrine. The lack
of a significant effect of a-methyltyrosine in the
resting state would be eXpected if the turnover of
norepinephrine in the terminals is slow (Figure 5; see
also Fischer and Snyder, 1965). On the other hand,
increased utilization of norepinephrine at the nerve
terminals following stimulation presumably results in a
decreased product inhibition of tyrosine hydroxylase and
consequently an acceleration of norepinephrine synthesis
(Weiner and Rabadjija, 1968) which is prevented by
a-methyltyrosine. Similar effects were obtained with
desmethylimipramine; that is, desmethylimipramine alone
or in combination with a-methyltyrosine did not
significantly alter the norepinephrine content in
resting nerve terminals (Table 3; Figure 6), but
increased the stimulus-induced reduction of norepinephrine
and decreased the uptake of norepinephrine-3H in
stimulated terminals (Figure 7; Table 9). In salivary
glands a combination of d-methyltyrosine and desmethyl-
imipramine almost totally depleted norepinephrine as
early as 1 hour after stimulation, indicating that

almost all the norepinephrine in this tissue can be

133

mobilized for release. This suggests that during periods
of increased nerve activity both synthesis and reuptake
are Operating in an effort to maintain the norepinephrine
contents. These two mechanisms, however, cannot main-
tain the norepinephrine concentrations at the normal
values and a new steady state concentration of norepine-
phrine is reached if stimulation is continued for 3

hours (Figure 9). Brown (1960) obtained a similar

rapid decline in the norepinephrine contents of spleen
following stimulation of the splenic nerves; the reduced
norepinephrine concentration was maintained over long
periods of stimulation. Indeed, in the absence of
synthesis and uptake all of the amine could be con-
ceivably depleted in less than 15 min after the start

of stimulation at 4 hz (Haefely et aZ., 1965).

The relative importance of the processes of synthesis
and reuptake in maintaining norepinephrine stores is,
however, still controversial. For example, in the nerve
terminals, either uptake of norepinephrine (Hedqvist
and Stjarne, 1969) or synthesis of this amine (Malmfors,
1964) have been proposed to be of prime importance
during periods of increased nerve activity. The relative
roles of synthesis and reuptake of norepinephrine appear
to be dependent on the frequency of stimulation (Table
5). At low frequencies only synthesis, and at high
frequencies both synthesis and reuptake of norepinephrine

contribute to the maintenance of norepinephrine in the

134

nerve terminals; similar results have been reported by
Bhagat and Friedman (1969). In addition, the
magnitude of stimulus-induced reduction in the
norepinephrine content of salivary glands in the
presence of a-methyltyrosine is similar at low and high
frequencies of stimulation (Table 5). That is, at a
frequency of 2 hz, a-methyltyrosine reduced the norep-
inephrine content by 24% (from 81% of control to 57% of
control) and at 10 hz by 30% (from 42% of control to
12% of control). The extent of stimulus-induced
reduction in the norepinephrine contents of salivary
glands in presence of a-methyltyrosine was similar at
2 hz and 10 hz even in combination with desmethylimipr—
amine. This suggests that increased nerve activity
triggers the synthesis of norepinephrine at a rate which
is independent of the frequency and the initial concen-
tration of norepinephrine. Reuptake of released
norepinephrine becomes of major importance for the
maintenance of norepinephrine for transmitter function
only at higher frequencies of stimulation.
Qualitatively, the results obtained with studies
of the nerve terminals in nictitating membranes are
similar to those obtained with the salivary glands.
Quantitative discrepancies between these two tissues are
apparently due to the less pronounced effects of

stimulation on the norepinephrine content of nictitating

135

membranes and are further complicated by crude dissection
of the membranes. Functional and morphological
characteristics of the smooth muscle of the membranes
may preclude a large release of transmitter following
stimulation. Nictitating membranes have a dense
noradrenergic innervation and the distance between nerve
endings and smooth muscle is less than 300 a (Van Orden
et aZ., 1967). Such an arrangement would favor an
efficient uptake process and thus conserve transmitter.
Another factor might be a decreased blood flow during
stimulation and consequently a decreased loss of
released amine into the circulation. Blood flow of
skeletal muscle is reduced 2 to 5—fold following stimula-
tion at 5 to 12 hz whereas norepinephrine is reduced
30% and 53% respectively (Kernell and Sedvall, 1964); even
stimulation at 0.5 hz results in an increase in
vascular resistance (Fredholm and Sedvall, 1966). The
quantitative differences in the diSposition of norep-
inephrine in the terminals of nictitating membranes and
salivary glands might also be eXplained if a substantial
amount of this amine in the membranes is extraneuronal
(Draskoczy and Trendelenburg, 1970) and thus is not
affected by stimulation.

Studies with radioactive tyrosine confirmed that
the rate of synthesis of norepinephrine in cell bodies

was faster than in the terminals. The results with the

136

radioactive isotopes also indicated that synthesis of
norepinephrine in ganglia proceeds independent of changes
in afferent input. That is, preganglionic stimulation
did not alter the rate of synthesis of norepinephrine

in the cell bodies but it markedly increased the rate

of formation of norepinephrine in salivary glands and
nictitating membranes (Tables 10 and 11).

The total radioactivity in the stimulated and non-
stimulated ganglia was the same, but in salivary glands
the total radioactivity on the stimulated side was
doubled. The converse was true in the nictitating
membranes where total radioactivity was reduced in the
stimulated side. Since catecholamines represent only a
small percentage of the total radioactivity in the
terminals (0.1-2%) it was assumed that most of this
radioactivity represents tyrosine-14C. The increased
total radioactivity in the stimulated salivary glands may
have resulted from compensatory vasodilation and increased
blood flow (Bhoola et aZ., 1965), increased tranSport
of tyrosine into the nerve terminals, or increased
incorporation of tyrosine into protein in the salivary
gland. It was quite evident, however, that the increase
in norepinephrine formation was greater than the increase
in total radioactivity (total radioactivity increased
2 fold while the formation of norepinephrine increased
22 fold). The reduced total radioactivity in stimulated

nictitating membranes may have resulted from

137

vasoconstriction and decreased blood flow. Despite
this, however, there was a marked increase in the
conversion of tyrosine-14C to norepinephrine—14C. The
effects with i.a. infusions of tyrosine-14C were
essentially the same as those obtained following i.v.
infusions of this amino acid. That is, the formation
of norepinephrine-14C was markedly increased in
stimulated terminals (salivary glands and nictitating
membranes) but not in the cell bodies.

It has been suggested that rapid alterations of
norepinephrine synthesis in nerve terminals accompanying
nerve stimulation are obtained without the necessity for
an increase in the enzyme protein (Sedvall and KOpin,
l967a). In the present studies cycloheximide decreased
the formation of norepinephrine-14C from tyrosine-14C
(Table 14). Although these results do not support the
contention that immediate acceleration of synthesis of
norepinephrine during stimulation is due to the mechanism
of product inhibition and not due to increased protein
synthesis, the effects of inhibition of protein synthesis
in present studies are equivocal. Cycloheximide does
not alter the stimulus-induced decline in the norepine-
phrine content of nerve terminals (Table 13). If the
synthesis of the amine was blocked, a greater reduction
in the norepinephrine content would be eXpected.
Cycloheximide could reduce the transport and incorporation

of tyrosine into various metabolic pools in the tissues.

138

Indeed the total radioactivity was much higher in all
tissues in the presence of cycloheximide (compare
Tables 10 and 14). The stimulus-induced synthesis of
norepinephrine, however, was still elevated in the nerve
terminals although to a lesser extent than it was in the
absence of an inhibitor of protein synthesis. It is
unlikely that a reduction in the transmitter synthesis
in the presence of cycloheximide reflects a reduction
in the enzyme proteins. Puromycin inhibits the stimulus-
induced increase in the synthesis of norepinephrine
(Weiner and Rabadjija,1968) at a time following stimula-
tion when the amount of tyrosine hydroxylase is not
altered (Weiner, 1970). Furthermore, when the salivary
glands were stimulated for 3 hours and the slices of this
tissue incubated in presence of tyrosine-14C the
formation of norepinephrine-14C increased approximately
5 fold as compared to nonstimulated glands. This
increase was markedly reduced in the presence of lO-SM
norepinephrine and almost completely inhibited in the
presence of 10-4M norepinephrine (Chieuh et aZ., 1971).
Many efforts have been made to correlate the
norepinephrine content and physiological responses of
noradrenergic effector tissues (Iversen, 1967).
Generally, the norepinephrine content of nerve terminals
can be markedly reduced without causing a comparable

reduction in the responses of tissues to drugs or

139

stimulation. Although the responses of salivary glands
and pupils following stimulation of the sympathetic
trunk were not quantified in the present study some
attempt was made to quantify the responses of the
nictitating membranes to nerve stimulation. In agree-
ment with Thoenen et a1. (1966) no consistent
relationship between the amount of norepinephrine and
the degree of contraction of the membranes was obtained
(Table 18; Figures 10 and 11). The results, however, are
complicated by the fact that long periods of stimulation
may injure nerve fibers or cause ischemia. The lack

of functional correlation with tissue contents of norep-
inephrine is not a characteristic of all noradrenergic
tissues. A good correlation was obtained between the
norepinephrine content and contraction of Splenic nerve
following stimulation and the administration of a-
methyltyrosine (Thoenen et aZ., 1966). Factors other
than the absolute amounts of transmitter are probably
important determinants of the pharmacology of noradrenergic
tissues; for example, the availability of newly
synthesized norepinephrine (KOpin et aZ., 1968) and the
morphological organization of noradrenergic synapse.

The latter may vary from one tissue to another

(Trendelenburg, 1969).

140

B. Regulation of Norepinephrine Content in the Neuronal

 

Cell Bodies and Terminals After Stimulation

 

One of the means of studying noradrenergic mechanism
has been to investigate the dynamics of neuronal
norepinephrine during and after periods of increased nerve
activity. Clearly, the amount of norepinephrine in the
neuron during stimulation depends upon a number of factors:
release, synthesis, uptake of released norepinephrine,
catabolism, and overflow into the circulation. The
mechanism of synthesis, uptake, and axonal tranSport
cannot maintain the normal norepinephrine concentration
in the nerve terminals in the presence of high frequency
stimulation so that the tissue is rapidly depleted of
this amine. It is not clear, what factors control the
replenishment of norepinephrine after its depletion and
in what manner the changes in stimulation—induced nerve
activity alter the dynamics of neuronal norepinephrine
once the source of the increased nerve activity is
eliminated.

In the cell bodies, although acute changes in the
sympathetic activity do not alter the norepinephrine
content chronic increases in functional demands increase
tyrosine hydroxylase in the cell bodies of both peripheral
and central nervous systems (Mueller et aZ., 1969a, b;
Thoenen et aZ., 1969; Thoenen, 1970). However, it takes

12 to 24 hours before this increase becomes apparent.

141

In the present study neither the content of norepinephrine
nor the formation of norepinephrine-14C from tyrosine—14C
or dOpamine-14C was altered in the cell bodies during

the 6 hour period following the cessation of 3 hours of
stimulation (Figure 13; Table 21). This suggests that

the changes in dynamics of norepinephrine in the cell
bodies are not apparent until many hours after stimulation,
and when they do occur they are a consequence of
adaptation to long term functional demands.

In the terminals, the norepinephrine content increases
rapidly after a stimulus-induced depletion (Figure 12;
Table 19). This increase is not due to the uptake of
circulating norepinephrine since similar results are
obtained in the presence of desmethylimipramine. The
increase appears to be largely due to synthesis because
it can be prevented by administration of a-methyltyrosine
(Figure 12). The norepinephrine content, however, does
not reach normal levels following stimulation. Instead,
it appears to plateau within 6 hours of cessation of
stimulation at a level that is significantly below
control. Following stimulation-induced depletion of
norepinephrine, synthesis of norepinephrine proceeds at
an accelerated rate for 2 hours, but returns to control
values 4 hours later when the endogenous norepinephrine
contents are still below control (Figure 13). These

results are contrary to those reported by other workers.

142

Fredholm and Sedvall (1966) noted a complete restoration
of norepinephrine contents of rat salivary glands in

less than 3 hours after cessation of stimulation; however,
they stimulated the sympathetic trunk for 30 min at 5

hz. The rate of synthesis of norepinephrine during the
post-stimulation period obtained by Fredholm and

Sedvall was 0.5 ug/g/hr and is about 4 times that
reported by Costa (1969) in resting rat salivary glands.
During the first 2 hours after cessation of stimulation
the norepinephrine content in cat salivary glands
increased at a rate of 0.10 ug/g/hr (Table 19; Figure
12). Thus, during the post-stimulation periods norep-
inephrine stores in peripheral tissues are restored
largely by synthesis which proceeds at a rate that far
exceeds the turnover rates of norepinephrine in the
resting state. The increased synthesis of norepinephrine
during the post-stimulation period is most likely due to
the lack of end-product inhibition since the increase

in the synthesis of the amine could be markedly reduced
or prevented when the slices of stimulated salivary
glands were incubated with norepinephrine (Chieuh et aZ.,
1971). In addition, the rate of restoration of
norepinephrine does not appear to be dependent on the
extent of the depletion of the amine. The rates of
restoration of norepinephrine in the salivary glands

after a depletion to 45% of control by stimulation alone

143

or to 25% of control by stimulation in the presence of
desmethylimipramine appear to be similar (Figure 12).

In the presence of desmethylimipramine the amine

contents also tended to plateau below control levels

and the formation of norepinephrine-14C from tyrosine-14C
returned to control values before endogenous stores of
norepinephrine were restored (Table 24).

Several possibilities may be considered to eXplain
the lack of complete restoration of norepinephrine in
the nerve terminals following stimulus-induced depletion.
In the nerve endings, norepinephrine is stored in
characteristic storage vesicles that are intimately
associated with dOpamine-B-hydroxylase (Van Orden et aZ.,
1966; Hékfelt, 1969; Viveros et aZ., 1969). It has
been reported that dOpamine-B-hydroxylase and storage
vesicles are released from the spleen and adrenal glands
during stimulation (Geffen et aZ., 1969; Viveros et aZ.,
1969; Gewirtz and KOpin, 1970), but the release of
dOpamine-B-hydroxylase from other tissues remains
controversial (Stjarne et aZ., 1970). If dopamine-B—
hydroxylase is lost from salivary glands, then even if
there is accelerated synthesis of dOpamine from
tyrosine, the endogenous norepinephrine content will
remain subnormal. In the present study, however, a
deficiency of dOpamine-B-hydroxylase does not appear

to prevent the restoration of norepinephrine content in

144

the terminals since the formation of norepinephrine-14C
from dOpamine-14C was increased 2-fold in the terminals
(Table 20). These results, however, do not preclude some
loss of the enzyme and storage vesicles. The increase
in the formation of norepinephrine-14C from radioactive
dopamingizesult from an increase in the membrane trans-
port of dopamine or from the induction of dOpamine—B-
hydroxylase. Kvethansky et al. (1971) reported an
increase in the amount of dOpamine-B-hydroxylase in the
adrenal glands 6 hours after a stress-induced increase
in the sympathetic discharge; this increase was blocked
by actinomycin D and cycloheximide.

Only part of the total norepinephrine stores in
the nerve endings may exert a product-inhibition of
tyrosine hydroxylase. Multiple "compartments" of
norepinephrine in the nerve endings have been postulated
(KOpin, 1966) and only a "strategically" located com—
partment is thought to exert feedback inhibition of
tyrosine hydroxylase (Weiner and Rabadjija,1968). Since
most of the norepinephrine contents of salivary glands
can be mobilized for release (Figure 9), and only part
of the normally occurring norepinephrine contents are
restored by synthesis (Figure 12) more than one store
for norepinephrine probably exists in the nerve endings.
Norepinephrine is presumably mobilized for release

from all stores in the salivary glands during stimulation.

145

The release of the amine frees tyrosine hydroxylase
from product inhibition and the norepinephrine.synthesis
is accelerated (Figure 13; Tables 22 and 23). The
synthesis of norepinephrine returns to control levels
when the strategic compartment of norepinephrine is
restored (Figure 13). Since the residual storage
vesicles which have released their amine content might
still be empty, they can elicit an increased conversion
of dOpamine-14C to norepinephrine-14C (Table 20). This
suggests that formation of DOPA is limiting the
restoration of norepinephrine contents in the nerve
endings, or that the tissue contents of norepinephrine
are not exclusively regulated by the mechanism of feed-
back inhibition.

The results of this study confirm previous reports
which indicate that an increase in tyrosine hydroxylase
in terminals cannot be detected until many hours (at
least more than 6) after a period of increased sympathetic
discharge (Dairman and Udenfriend, 1970). Furthermore,
induced changes in the tyrosine hydroxylase appear in
the cell body before they are detected in terminals
indicating that inductive changes in enzyme protein

are initiated in the cell bodies (Axelrod et aZ., 1970).

C. The Noradrenergic Neuron:A Single Functional Unit
The results of degeneration studies suggest that
long and ramified neuronal processes are dependent upon

the soma for their integrity. The regulation of

146

adjustment of noradrenergic mechanism in cell bodies

and terminals to increased functional demands is not
well understood but both chemical and physical processes
may be involved. It would appear that cell bodies do
not contribute significantly to the dynamics of
norepinephrine in terminals during acute stimulation.
The total norepinephrine content of cell bodies of
noradrenergic neurons in one ganglion is approximately
90 nanograms whereas the terminals of these neurons in
the salivary glands and nictitating membranes contain
approximately 1000 nanograms. Only l7-l9 nanograms of
ganglionic norepinephrine were lost through axonal
transport in 3 hours (Table 16). The amount of stimulus-
induced release of norepinephrine from salivary glands
and nictitating membranes is not known but the amount
of amine released from spleen has been reported to be
14.68 and 12.19 ng/DNA-unit/min at 5 and 10 hz
reSpectively (Hedqvist and Stjarne, 1969). In the
present studies, if the rate of the initial rapid decline
in the norepinephrine content of salivary glands
following 1 hour of stimulation at 10 hz is taken as

an index of release, the estimate of the rate of
synthesis would approximate that obtained by Hedqvist
and Stjarne (1969) (Figure 9; Table 7). Thus, during
periods of acute stimulation the cell body could not

contribute significantly to the norepinephrine content

147

in the terminal. The same conclusion was reached as a
result of eXperiments in the cat spleen (Geffen and
Rush, 1968).

Although no function can be ascribed to norepinephrine
in ganglia it is known that synthesis of this amine in
cell bodies proceeds at a rapid rate independent of
neuronal activity (Table 5). Roth et al. (1967) have
reported similar results in bovine splenic nerve. In
cell bodies, norepinephrine forms an aggregate with,
protein (Lubinska, 1964; Dahlstrém and Haggendal, 1970).
These aggregates slowly mature, as evidenced by
differential staining characteristics during their
somatofugal transport and concentrate in the nerve
terminals where they are referred to as storage granules.
Once in the terminals the granules are available for
release, storage, uptake and synthesis of norepinephrine.
In the terminals the amine is released, tyrosine
hydroxylase is freed of feedback inhibition, and
synthesis of norepinephrine is thereby enhanced.

Since norepinephrine does not appear to be released

from cell bodies or axons, a similar control of synthesis
is probably not necessary in these parts of the neuron.
The amine may be present as a biological redundancy,

only because all the enzymes and substrates necessary for
its synthesis are available (Fischer and Snyder, 1965;

Laduron and Belpaire, 1968). The turnover rate of

148

tyrosine hydroxylase (Mueller et aZ., 1969a, b) and the
half life of norepinephrine storage granules (Dahlstrbm
and Haggendal, 1970) has been estimated to be 8 and 22
days reSpectively. Accordingly, the terminals can
maintain their ability to synthesize, recapture, and
store norepinephrine for relatively long periods of
time. Thus, in terminals, unlike in the cell bodies,
minute to minute regulation of the norepinephrine
content is accomplished by synthesis and by retrieval
of released norepinephrine. All of the normally
occurring tissue contents of norepinephrine, however,
may not exert a product inhibition and the transmitter
may be located in more than one morphological entity.
Indeed, newly synthesized norepinephrine is preferentially
released following stimulation of splenic nerves (Kopin
et aZ., 1968).

In addition to the immediate changes in the end-
product inhibition of tyrosine hydroxylase (Weiner and
Rabadjija, 1968) the neuronal norepinephrine contents
can be modulated by gradual alterations in the amount of
tyrosine hydroxylase and dOpamine-B- hydroxylase
(Thoenen, 1970; Kvethansky et aZ., 1971) that are induced
by prolonged sympathetic discharge. The induction of
tyrosine hydroxylase in adrenal glands and ganglia
following a drug—induced chronic increase in afferent
input takes at least 12 hours (Thoenen et aZ., 1969;
Mueller et aZ., 1969a, b). Induction of tyrosine

hydroxylase in the cell bodies of hind brain following

149

a stress-induced increase in the afferent input is
obtained within 24 hours of the stress (Thoenen, 1970).
The amount of tyrosine hydroxylase also increases in
the terminals following compensatory increases in the
sympathetic activity (Dairman and Udenfriend, 1970),
but the inductive changes in the enzyme protein are
initiated in the cell body and precede the enzymic
alterations in the terminals (Axelrod, et aZ., 1970).
Thus, the neuronal processes of a noradrenergic
neuron might be considered in a dynamic state in which
the nerve terminals are reformed and refashioned to
suit the physiological demands whereas the soma appears
to function as a source of enzymes and assembly of the
storage mechanism for the transmitter. The short term
regulation of norepinephrine concentrations in terminals
and cell bodies are maintained independent of one
another. Accordingly, the dynamics of norepinephrine
in various parts of the neuron are different. These
differences must be considered when interpreting the
results of studies involving norepinephrine turnover,
metabolism and uptake, etc. in tissues such as brain,
gut, vas deferens, uterus, etc., that contain both cell

bodies and terminals.

SUMMARY AND CONCLUSIONS

The purpose of the present study was to investigate
the factors that regulate the norepinephrine content in
superior cervical ganglia, which represent cell bodies,
and in submaxillary salivary glands and nictitating
membranes, which contain nerve terminals of post-
ganglionic sympathetic neurons, during stimulation and
post-stimulation periods.

In decentralized, nonstimulated noradrenergic
neurons, 3 hours of a-methyltyrosine infusion reduced
the norepinephrine content in the cell bodies but not
in the terminals. The a-methyltyrosine—induced
depletion of norepinephrine in cell bodies was retarded
if a ligature was tied around the axon. Desmethyl-
imipramine did not alter the norepinephrine content of
cell bodies or terminals.

Low or high frequencies of stimulation, alone or
in combination with desmethylimipramine or a-methyl-
tyrosine, did not alter the norepinephrine content in
the cell bodies. On the other hand, 1 hour of stimulation
alone at 10 hz partially depleted norepinephrine in the
terminals. The reduced concentration of norepinephrine

was maintained if stimulation was continued for 3 hours.

150

151

The administration of a—methyltyrosine enhanced the
stimulus-induced depletion at both low and high frequen-
cies, whereas desmethylimipramine increased the depletion
of norepinephrine only at high frequences. The com-
bination of a-methyltyrosine and desmethylimipramine
further enhanced the norepinephrine depletion at high
frequencies of stimulation. The a-methyltyrosine-induced
depletion did not result from the replacement of
norepinephrine by a-methylnorepinephrine.

Preganglionic stimulation at 10 hz had little
effect upon the conversion of tyrosine-14C to norepine-
phrine-14C in the cell bodies but increased the formation
of norepinephrine-14C in the terminals. a-Methyltyrosine
reduced the synthesis of norepinephrine-14C in all
tissues.

Decentralized preganglionic fibers were stimulated
at 10 hz for 3 hours and the tissue contents of
norepinephrine were determined immediately after and
at 2 and 6 hours after the cessation of stimulation.

In the cell bodies neither endogenous norepinephrine
content nor the formation of norepinephrine-14C from
tyrosine-14C was altered. In the terminals,
stimulation alone reduced the norepinephrine content;
the concentrations of norepinephrine increased rapidly
within 2 hours after cessation of stimulation but then
plateaued and during the next 4 hours did not return to

control values. Partial restoration of norepinephrine

152

was prevented by a-methyltyrosine but not by desmethyl-
imipramine. The rate of formation of norepinephrine-14C
was accelerated during the 30 minute period immediately
after cessation of stimulation but thereafter decreased
progressively as the endogenous norepinephrine concen-
trations increased. At 6 hours after cessation of
stimulation, when the endogenous concentrations of
norepinephrine were below control, the rate of conversion
of tyrosine-14C to norepinephrine-14C was not different
from nonstimulated controls whereas the conversion of
dopamine-14C to norepinephrine-14C was increased 2-fold.

The results suggest that in cell bodies synthesis
of norepinephrine proceeds at a rapid rate that is
independent of nerve activity and concentrations of
norepinephrine are maintained only by synthesis. In
terminals, synthesis of norepinephrine proceeds at a
slow rate in the absence of nerve activity; with low
frequencies of stimulation concentrations of norepinephrine
are partially maintained by synthesis and at higher
frequencies by both synthesis and reuptake.

All immediate requirements for the transmission
process are fulfilled locally at the terminals of nor-
adrenergic nerves. When the norepinephrine contents of
terminals are extensively depleted by stimulation, synthesis
only partially restores the norepinephrine concentrations.

This suggests that if the rate of norepinephrine synthesis is

153

regulated by a feedback control mechanism, only part of
the neuronal stores of norepinephrine participate in

the regulation.

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