In:   The Neurosciences.  Third Study
          Program.  Schmitt, F.O. and
        Worden, F.G. (eds.), MIT Press,
mfl      Cambridge, Mass., 1974, pp. 863-876.

/b R egu 1 ation of the Neurotransmitter

Norepinephrine

JULIUS AXELROD

ABSTRACT  The catecholamines dopamine, norepinephrine, and
epinephrine are in a state of flux, yet they maintain a constant
level in nerves and glandular tissues. The level of these biogenic
amines is regulated by changes in activity of the biosynthetic
enzymes tyrosine hydroxylase, dopamine+hydroxylase, and
phenylethanolamine-N-methyltransferase. The minute-to-min-
ute regulation of the level of the neurotransmitter norepine-
phrine is controlled by rapid changes in tyrosine hydroxylase
activity caused by feedback inhibition of the enzyme by nor-
epinephrine and dopamine. There is no chave in the amount of
enzyme protein. Elevation in tyrosine hydroxylase occurs in the
cell body, nerve terminals, and adrenal medulla when there is an
increase in firing ofsympathetic nerves. This results in formation
of new enzyme protein by a transsynaptic process. A similar
transsynaptic induction by increased nerve firing occurs with
the enzyme dopamine-B-hydroxylase in nerves and adrenal
medulla. The induction of these enzymes appears to be initiated
by acetylcholine and possibly controlled by intracellular
concentrations of norepinephrine. The activity of tyrosine
hydroxylase and dopamine-b-hydroxylase and especially pheny-
lethanolamine N-methyltransferase in the adrenal medulla
is reduced by removal of the pituitary gland and induced by
ACTH.

Dopamine-B-hydroxylase is transported from cell body to
nerve terminals. When nerves are depolarized, dopamine-j?-
hydroxylase is released from the nerves, together with the
neurotransmitter norepinephrine, by a process of exocytosis.
The release of dopamine-/$hydroxylase requires Ca+ +,
microtubules, and microfilaments.

The biogenic amine serotonin undergoes a circadian change
in levels in the pineal gland. The level of serotonin is regulated by
the neurotransmitter norepinephrine released from sympathetic
nerves. Norepinephrine reduces the serotonin levels by stimula-
ting the enzyme that acetylates serotonin via cyclic AMP.
Changes in the rate of neuronal release of norepinephrine
markedly influence the activity of N-acetyltransferase.

THE ACTIVITY OF the sympathetic nervous system under-
goes rapid changes yet maintains a constant level of its
neurotransmitter, norepinephrine. This is made possible
by a variety of self-regulatory systems involving changes
in its biosynthesis, storage, release, and metabolism within
the neuron as well as modifications of the pre- and post-

JULIUS AXELROD Laboratory of Clinical Science, National
Institute of Mental Health, Bethesda, Maryland

synaptic membrane. The special morphology of the
sympathetic neuron also contributes to the maintenance
of the neurotransmitter. The sympathetic neuron con-
sists ofa cell with a considerable spatial separation from its
nerve terminals (Figure 1). The nerve terminals are highly
branched and have swellings or varicosities that are in
close proximity to the effector cells. Within the varicosity,
norepinephrine is stored in a dense core vesicle of about
500 A (Wolfe et al., 1962). This structural organization is
present in both the peripheral and central nervous systems.
The cell body of a sympathetic neuron synapses with a
preganglionic fiber, usually cholinergic, and the varicosity
of the nerve terminals innervates many thousand effector
cells en passant.

SYMPATHETIC
NERVE

NERVE
TERMINAL

EFFECTOR CELL

FIGURE 1  Sites at which the neurotransmitter norepinephrine
can be regulated (see text for explanation).

The levels of norepinephrine within the sympathetic
neuron can be regulated at several sites (Figure 1) : Cell
body via preganglionic nerves (areas 1, 2); the axon,
which transports the biosynthetic enzymes made in the
cell body (area 3); the cytoplasm and storage vesicle of
nerve terminal (area 4) ; the neuronal membrane (area 5) ;
and the postsynaptic membrane (area 6).

JULIUS AXELROD   863

           Reprinted by the
U.S. DEPARTMENT OF HEALTH, EDUCATION. AND WELFARE
         National Institutes of Health


Biosynthesis of norepinephrine and epinephrine

The enzymes involved in the formation of norepinephrine
are synthesized in the cell body of the sympathetic neuron.
These enzymes are tyrosine hydroxylase, which converts
tyrosine to dopa (Nagatsu et al, 1964), dopa decarbox-
ylase (Holtz et al., 1938), and dopamine-/Lhydroxylase,
the enzyme that j?-hydroxylates clopamine to norepine-
phrine (Friedman and Kaufman, 1965). Phenylethano-
lamine-N-methyltransferase, the enzyme that methylates
norepinephrine to epinephrine, is present mainly in
adrenal medulla of mammals and sympathetic nerves of
amphibians (Axelrod, 1962). Tyrosine hydroxylase is a
mixed function oxidase requiring tetrahydropteridine and
0, and Fe++. It is found in the cell body, axon, nerve
terminals, as well as the adrenal medulla, and is absent in
extraneural tissue. Tyrosine hydroxylase is found in
soluble and bound form. A molecular weight of 192,000
has been reported for the soluble enzyme, while the bound
enzyme after trypsin digestion has a mo1ecula.r weight of
50,000 (Wurzburger and Musacchio, 197 1). Tyrosine
hydroxylase can be inhibited by norepinephrine, which
serves as an important controlling mechanism for its
synthesis. Inhibition of the enzyme by catecholamines is
competitive for its pteridine cofactor in its reduced form
and not with its substrate, tyrosine (Ikeda et al., 1966).
Dopa decarboxylase is unspecific in that it can decarbox-
ylate a variety of L-aromatic amino acids. It requires
pyridoxal phosphate as a cofactor and is tightly bound to
the apoenzyme as a Schiff base. Dopa decarboxylase is
present both in neuronal and extraneuronal tissues. Using
an immunoassay, it was shown that aromatic acid de-
carboxylase is a single enzyme with a molecular weight of
109,000 (Christenson et al., 1972). Dopamine-fi-hydrox-
ylase hydroxylates dopamine on the beta carbon to form
norepinephrine. It is a mixed function oxidase containing

2 mole of Cu++, which is reduced by ascorbic acid
(Friedman and Kaufman, 1965). The enzyme lacks spec-
ificity and can ,!?-hydroxylate a variety of phenylethyla-
mines. The enzyme is highly localized in the sympathetic
neuron as well as the adrenal medulla. Within the neuron
dopamine-/Lhydroxylase is present in the cell body, axon,
and nerve terminal. It is highly localized in the norepine-
phrine storage vesicles of nerves (Potter and Axelrod,
1963a) and chromaffin granules of the adrenal medulla
(Kirshner, 1957).

The epinephrine-forming enzyme, phenylethanol-
amine-N-methyltransferase, is highly localized in the
cytoplasm of mammalian adrenal medulla (Axelrod,
1962) and is present in sympathetic nerves of amphibians
(Wurtmanet al., 1968b). It methylates norepinephrine as
well as j.l-hydroxylated phenylethanolamine derivatives;
S-adenosyhnethionine is the methyl donor. The enzyme
has been purified and its molecular weight has been found
to be about 30,000 (Connett and Kirshner, 1970).
Phenyiethanolamine-N-methyltransferase shows different
electrophoretic mobility on starch block, and multiple
forms of the enzyme have also been reported (Axelrod and
Vesell, 1970). The biosynthesis ofcatechoiamines isshown
in Figure 2.

Neural regulation of the catecholamine
biosynthetic enzymes

In a study to determine the intraneural localization of
tyrosine hydroxylase, 6-hydroxydopamine, a compound
that destroys sympathetic nerve terminals (Thoenen and
Tranzer, 1968), was administered to rats. There was an
almost complete disappearance of this enzyme in the heart
in 40 hr suggesting that it was highly localized in sympa-
thetic nerve t:rminals (Mueller et al., 1969a). When
tyrosine hydroxylase was measured in the adrenal gland

TYROSINE

Dopamine $ oridaso

864

                     NOREPINEPHRINE               EPINEPHRINE

   FIGURE 2  Biosynthesis of catecholamines. PNMT is phenylethanolamine N-methyltransferase.

REGULATION OF THE NEUROTRANSMITTER NOREPINEPHRINE


there was a marked increase in this enzyme and a smaller
elevation of phenylethanolamine-N-methyltransferase
about 1 day after the administration of 6-hydroxy-
dopamine. This was an unexpected finding, and it
appeared to be due to the ability of 6-hydroxydopamine
to lower blood pressure. This would cause a reflex increase
in sympathetic nerve activity, resulting in an increase in
enzyme activity in the adrenal gland. To examine this
possibility, reserpine and phenoxybenzamine, compounds
that lower blood pressure and increase sympathetic nerve
activity, were given, and their effects on tyrosine hydroxyl-
ase in the adrenal gland examined (Mueller et al., 1969b).
Both compounds elevated tyrosine hydroxylase activity,
not only in the adrenal gland but also in the superior cervi-
cal (Figure 3) and stellate ganglia. The maximal enzyme
activity observed in the adrenal gland and the ganglia
occurred 3 days after reserpine administration, indicating
a slow rise in enzyme activity. Reserpine was also found to
increase tyrosine hydroxylase activity in adrenal gland of
all mammalian species examined as well as the brainstem
of the rabbit. Increased tyrosine hydroxylase activity after
reserpine was also observed in the nerve terminal as well
as in the cell body. The increase in enzyme activity in the
nerve terminal was delayed and lagged behind the rise in
the ganglia by 2 days (Thoenen et al., 1970).

The elevation in tyrosine hydroxylase activity after the
increase in sympathetic nerve activity was shown phys-
iologically by the increased formation of [' 4C]catechol-

amine from [`4C]tyrosine after the administration of
phenoxybenzamine or 6-hydroxydopamine (Dairman
and Udenfriend, 1970 ; Mueller, 197 1). The rise in tyro-
sine hydroxylase activity in the ganglia and adrenal
medulla after reset-pine administration could be prevented
by the administration of cycloheximide or actinomycin
D (Mueller et al., 1969c), suggesting that this elevation
of enzyme activity is due to induction of new enzyme
protein. The Km for both the substrate and the pteridine
cofactor with the enzyme obtained from reserpine-
treated rats was not different from the untreated rats,
although there was a marked elevation in the V,,,., for
both substrate and cofactor. These results are consistent
with an increase in the number ofactivesiteson theenzyme
molecule caused by a drug-induced rise in sympathetic
nerve activity. Increased tyrosine hydroxylase activity
was found in the adrenal gland after cold (Thoenen et al.,
1969a), immobilization (Kvetnansky et al., 1970), and
psychosocial stress (Axelrod et al., 1970), in the ganglia
after administration of nerve growth factor (Thoenen,
1970)) and in brain after cold stress and administration of
reserpine (Segal et al., 1971).

To examine whether the induction in tyrosine hydrox-
ylase after reserpine was a transsynaptic event the pre-
ganglionic fibers to the superior cervical ganglia (Thoenen
et al., 1969a) and the splanchnic nerve to the adrenal
gland were cut unilaterally (Thoenen et al., 1969b).
When reserpine was administered, there was an elevation

TYROSINE
H,YDROXYLASE

O NORMAL

DOPAMINE
b-OXIDASE

la DECENTRALIZED

CONTROL RESERPINE     CONTROL RESERPINE

FXCIJRE 3 Transsynaptic induction of tyrosine hydroxylase
and dopamine-D-oxidase(hydroxylase) in sympathetic ganglia.
Superior cervical ganglion was decentralized unilaterally by
transection of the preganglionic trunk. Two to six days later

reserpine (5 mg/kg) was given for 1 day before tyrosine hydroxy-
lase was measured or 3 alternate days before dopamine-/I-
oxidase(hydroxylase) was measured (from Axelrod, 1971).

JULIUS AXELROD   865


of enzyme activity in the innervated side of the ganglia
and adrenal gland, but the increase on the denervated
side was completely blocked (Figure 3). All of these
observations indicate that tyrosine hydroxyriase activity is
increased in both the sympathetic nerve, cell body, bra-in,
and adrenal medulla by sympathoadrenal hyperactivity.
This elevation in tyrosine hydroxylase activity appears to
involve a transsynaptic induction of new enzyme mol-
ecules.

The presynaptic fibers that regulate tyrosine hydroxyl-
ase activity are cholinergic. Interrupting the rholinergic
splanchnic nerve blocks the drug-induced rise in tyrosine
hydroxylase (Thoenen et al., 1969a, 1969b). Ganglionic
blocking agents also inhibit the increase in this enzyme
(Mueller et al., 1970b), while acetylcholine causes an
elevation of tyrosine hydroxylase activity in the adrenal
(Patrick and Kirshner, 1971). In the superior cervical
ganglia of the newborn mouse, the development of tyro-
sine hydroxylase is prevented by cutting the pregan-
glionic cholinergic nerve (Black et al., 1971), suggesting
that presynaptic cholinergic terminals regulate the forma-
tion of tyrosine hydroxylase in the sympathetic nerve cell
body. The selective destruction of sympathetic nerves
chemically with 6-hydroxydopamine or immunologically
with antiserum to the nerve growth factor prevents the
normal development of choline acetyltransferase in pre-
synaptic nerve endings, indicating that postsynaptic
adrenergic neurons regulate the biochemical development
of presynaptic cholinergic nerves (Black et al., 197 1 j.

Breeding studies are being carried out utilizing re-
ciprocal F, and F, and dominant recessive backcross
generations with respect to the catecholamine bio-
syntheticenzymes (Ciaranello, unpublished observations),
Preliminary results suggest that the genes controlling
tyrosine hydroxylase, dopamine-fi-hydroxylase and
phenylethanolamine-N-methyltransferase are linked. It
is possible that the three genes are linked and that a single
regulatory locus is responsible for the activity of the three
biosynthetic enzymes.

The activity of dopamine-fl-hydroxylase is also affected
by nerve impulses. The development of a sensitive assay
for measuring dopamine-B-hydroxylase made it possible
to measure this enzyme in the celi body and nerve ter-
minals and to study changes after drugs that increase
sympathetic nerve firing (Molinoff et al., 1971). The
administration ofreserpine resulted in a marked elevation
of dopamine-/?-hydroxylase activity in sympathetic
ganglia (Figure 3), nerve terminals, and adrenal gland
but not in the brain (Molinoff et al., 1970). This increase
in enzyme activity in cell bodies is neuronally mediated,
because the reserpine could not elevate dopamine-/?-
hydroxylase activity in a denervated ganglia (Figure 3).
Pretreatment of animals with the protein synthesis in-

hibitor, cycloheximide, prevented the rise in enzyme
activity in the ganglia. Further evidence that new enzyme
protein was induced hy nerve impulses comes from the
use of an antibody for dopamine-,!Lhydroxylase. Reser-
pine caused an increase in the rate of incorporation of
[ 3H]leucine into dopamine-/?-hydroxylase measured by
immunoabsorption (Hartman et al., 197Oj.

It appears that nerve depolarizaticn is involved in the
induction of dopamine-fi-hydroxylase. An increase in the
potassium concentration 1-X the media containing rat
superior cervical ganglia maintained in organ culture
results in a marked increase in dopamine-P-hydroxylase
in ganglia (Silberstein et al., 1972). This increase in
enzyme activity is inhibited by cycloheximide. Nicotinic
antagonists block the induction ofdopamine-/I-hydroxyl-
ase in ganglia after reserpine, suggesting that a cholinergic
site is involved (T\lolinoff et al., 1972). Acetylcholine also
increases dopamine-P-hydroxylase activity in the de-
nervated adrenal gland (Patrick and Kirshner, 1971).
However, it does not appear that the cholinergic receptor
is essential for induction of the enzyme, at least in sympa-
thetic nerves, because elevated potassium concentration
can increase enzyme activity in the absence of neuronal
influences.

Another biosynthetic enzyme, phenylethanolamine-N-
methyltransferase, in the adrenal gland, is regulated by
neuronal influences. Increasing splanchnic nerve activity
with 6-hydroxydopamine, reserpine, or stress causes a
small elevation of phenylethanolamine-N-methyltrans-
ferase in the rat adrenal gland (Mueller et al., 1969a) and
a large increase in the mouse adrenal (Ciaranello et al.,
1972a). This increase ran be abolished by transection of
the nerve supplying the adrenal gland (Thoenen et al.,
1970).

Unlike the nt+r catecholamine biosynthetic enzymes,
dopa decarboxylase activity in the superior cervical
ganglia or adrenal gland is not induced by drug-mediated
increase in preganglionic neural activity (Black et al.,
197 1). These experiments suggest that tyrosine hydroxyl-
ase, dopamine-/J-hydroxylase, and phenylethanolamine-
N-methyltransferase are linked in a coordinate fashion.
Genetic studies also indicate that these enzymes are linked
(Ciaranello, unpublished observations).

Several experiments suggest that catecholamines are
implicated in the induction of dopamine-fl-hydroxylase
and tyrosine hydroxylase (Molinoff et al., 1972). Drugs
that elevate the level of catecholamines, such as IA-dopa,
monoamine oxidase inhibitors, and bretylium, inhibit the
induction of hoth tyrosine hydroxylase and dopamine-/I-
hydroxylase. On the other hand, reduction of catechol-
amines by sc-methyl-paratyrosine or high potassium
(Silberstein et al., 1972j results in an induction of dopa-
mine-fi-hydroxylase activity.

866    REGULATION OF THE NEUROTRANSMITTER NOREPINEPHRINE


The nerve terminal may have an important influence
on the induction of the catecholamine biosynthetic
enzymes in the cell body. Destruction of adrenergic nerve
terminals with 6-hydroxydopamine or surgical section of
the postganglionic axons causes a long-lasting decrease in
dopamine-/?-hydroxylase in the superior cervical ganglia
(Brimijoin and Molinoff, 1971). 6-Hydroxydopamine
administration or a postganglionic section also results in a
marked increase in the uptake of [ 3H]norepinephrine in
the sympathetic ganglia (Kopin and Silberstein, 1972).
The latter phenomena reflects growth of adrenergic mem-
brane surface. These experiments show that when nerve
terminals are destroyed the metabolic machinery of the
cell body changes its priorities from the production of
enzymes concerned with function to the formation of
structural elements required for the restoration of the
nerve ending.

Hormonal regulation

In addition to nervous inputs, the corticoids can also
infiuence the biosynthesis of catecholamines. The effects
of hormones are principally in the adrenal medulla, a
structure that can be considered analogous to the cell body
of the sympathetic nervous system. An examination of the
effects of hormones on catecholamine formation was
prompted by the observation that large amounts of
epinephrine are present in the adrenal gland of those
species in which the medulla is surrounded by a cortex
(Coupland, 1965). This suggested to us that corticoids

present in the cortex might be the compounds that
stimulate the methylation of norepinephrine to epine-
phrine. The experimental design to examine this possi-
bility was to reduce corticoids in adrenal cortex by
removal of the pituitary gland in rats and then to measure
the activity of phenylethanolamine-N-methyltransferase
in the adrenal gland (Wurtman and Axelrod, 1966).
When rats were hypophysectomized, there was a gradual
and steady decline in the norepinephrine methylating
enzyme. After about 7 days,only about 20% of the enzyme
activity remained in the adrenal medulla (Figure 4). The
administration of either dexamethasone, a  potent
glucocorticoid, or ACTH restored phenylethanolamine-
N-methyltransferase activity to the adrenal gland after
several days (Figure 4). Inhibition of protein synthesis
blocked the increase in enzyme activity after the ad-
ministration of dexamethasone. When dexamethasone or
ACTH was given repeatedly to normal rats there was no
increase in enzyme activity in the adrenal gland. All of
these experiments demonstrated that glucocorticoids in
the adrenal cortex are necessary to maintain phenyl-
ethanolamine-N-methyltransferase activity. There are
negligible amounts of phenylethanolamine-N-methyl-
transferase in sympathetic nerve cell body. When
dexamethasone is given to newborn rats, phenyletha-
nolamine-N-methyltransferase appears in the superior
cervical ganglia (Ciaranello, Jacobowitz, and Axelrod,
unpublished observations). The ability of dexamethasone
to induce the methylating enzyme in the ganglia is lost
after the rat is 2 weeks old. Dexamethasone also increased

0 Normal
m HYPOX
m Hypax +ACTH
654 Hypox + Dexamethasone

IOO-

PNMT     Tyrosine Hydroxylose  DoDamine-/3 Oxidose

FICWRE 4  Hormonal regulation of catecholamine biosynthetic enzymes in the adrenal. Rats were hypo-
physectomized for about 1 week and then given dexamethasone or ACTH (from Axelrod, 1973).

JULIUS AXELROD   867


the amounts of small, intensely fluorescent (SIF) cells in
the superior cervical ganglia. These cells are morpholog-
ically related to chromaffin cells. These findings indicate
that glucocorticoid hormones may be invoived in dif-
ferentiation of nerve cell to chromaffin type cell .

Removal of the pituitary also affected other catechol-
amine biosynthetic enzymes in the adrenal medulla. After
rat hypophysectomy the activity of tyrosine hydroxylase
(Mueller et al., 1970a) and dopamine-B-hydroxylase
fell (Weinshilboum and Axelrod, 1970) (Figure 4).
Repeated administration of ACTH restored the activity
of both enzymes in the adrenal gland (Figure 4). How-
ever, dexamethasone failed to elevate tyrosine hydroxyl-
ase or dopamine-b-hydroxylase (Figure 4). When mice
were subjected to psychosocial stimulation, there was
marked elevation of tyrosine hydroxylase and phenyl-
ethanolamine-N-methyltransferase in the adrenal gland
(Axelrod et al., 1970). When certain mouse strains were
exposed to cold stress for 3 to 6 hr, there was a small but
significant elevation of phenylethanolamine-N-methyl-
transferase in the adrenal (Ciaranello et al., 1972a).
Implantation of an ACTH-secreting tumor in rats
resulted in an elevation of phenylethanolamine-N-
methyltransferase, demonstrating that the enzyme can be
elevated under conditions of extreme pituitary-adreno-
cortical activation. Forced immobilization stress also
increases tyrosine hydroxylase and dopamine-p-hydrox-
ylase in adrenal to a considerable extent and phenyl-
ethanolamine-N-methyltransferase to a smaller degree
(Kvetnansky et al., 1970). These stress-induced elevations
are mediated by neuronal and hormonal influences.

Regulation of catecholamines at nerue terminals

There is a rapid regulation of the biosynthesis of the
adrenergic neurotransmitter in the nerve terminals, which
isdifferentfrom theslowerinductionofthecatecholamine-
forming enzymes described above. Stimulation of the
splanchnic nerve leads to a release of catecholamines. The
sum of the amount of catecholamines released and that
remaining in the gland is greater than the amount
initially present in the gland (Bydgeman and von Euler,
1958). Studies with the hypogastric nerve (Weiner, 1970)
and salivary gland (Sedvall and Kopin, 1967) indicate
that the rapid changes in the biosynthesis of norepine-
phrine are regulated by tyrosine hydroxylase. Stimulation
of the sympathetic nerve of the vas deferens in vitro or the
salivary gland in vivo (Table I) led to an increased con-
version of [`"Cl tyrosine to [ i4C]norepinephrine. How-
ever, there was no increase in the formation of [r 4C]dopa
to [' 4C]norepinephrine when the nerves were stimulated,
suggesting that tyrosine hydroxylase is the enzyme in-

fluenced by nerve activity. However, there was no
increase in the amount of tyrosine hydroxylase in the
stimulated salivary gland. In an in vitro study with the vas
deferens, it was found that addition of norepinephrine to
the bath can partially or completely prevent the increased
formation of [i 4C]norepinephrine from [i "C] tyrosine
(Weiner, 1970). It has been shown that ryrosine hydroxyl-
ase is inhibited by catecholamines such as dopamine and
norepinephrine, due to the competition between the
catecholamines and the pteridine cofactor (Ikeda et al.,
1966). Most of the norepinephrine in the nerve terminals
is present in vesicles with little access to tyrosine hydrox-
ylase that appears to be present in the cytoplasm. Thus,
there is a small compartment of free catecholamines in
the cytoplasm that is critical in the regulation of tyrosine
hydroxylase. This small compartment is rapidly de-
pleted during nerve stimulation and thus allows more
norepinephrine to be synthesized by increasing the con-
version of tyrosine to dopa.

TABLE I

EJect of sympathetic nerve impulses on norepinephrine synthesis and
tyroske hydroxylase activity in the rat salivary gland

Norepinephrine formed in
     vivo from

In vitro assay

      [`4C]Tyrosine [3H]Dopa of tyrosine
        (countlmin)  (count/min)  hydroxylase*

Decentralized      68        410       3190
Stimulated       358        396       3170
*Tyrosine hydroxylase is expressed as count/min [14C]dopa

formed from [i4C] tyrosine by an aliquot of homogenate of the
salivary glands. (From Sedvall and Kopin, 1967).

Norepinephrine is stored in the nerve terminal in more
than one compartment. After the administration of [`HI-
norepinephrine, the decrease in its specific activity in
tissues was found to be multiphasic (Axelrod et al., 1961),
and the specific activity of norepinephrine released by
tyramine was dependent on the time the sympathomi-
metic amine was administered (Potter and Axelrod,
1963b). Kopin et al. (1968) demonstrated that norepine-
phrine newly synthesized from tyrosine was more rapidly
released from the spleen after nerve stimulation. There
thus appears to be a relatively small available pool of
norepinephrine and a larger reserve store of the catechol-
amine. The more available pool might be present in the
vesicles closest to the synaptic cleft and, because of its
location with respect to the neuronal membrane, would
be more easily released (Figure 1). The major store of

868    REGULATION OF THE NEUROTRANSMITTER NOREPINEPHRINE


norepinephrine is located at a greater distance from the
neuronal membrane and is thus utilized at a slower rate.
This pool might serve as a reservoir for the more readily
releasable transmitter.

Axonal transport of catecholamine biosynthetic
enzymes

The cell body of the sympathetic neuron is separated from
the nerve terminals by long distances (Figure 1). The
protein-synthesizing apparatus of the neuron is confined
to the cell body, while the terminal is involved in nerve
function. The enzymes for the biosynthesis of catechol-
amines made in the cell body must be transported down
the axon to the nerve terminal where most of the neuro-
transmitter is synthesized. Weiss and Hiscoe (1948)
demonstrated that the axon is capable of transporting
substances from the cell body to the nerve terminal.
Axonal transport is a highly specialized process and
different constituents are transported in a proximodistal
direction at their own characteristic rate, rapidly (1 to
10 mm/hr) or slowly (1 to 3 mm/day) (Ochs, 1972).

Studies on axoplasmic transport are made by ligation
of nerves. When adrenergic axons are pinched, there is a
rapid accumulation of norepinephrine and dense core
vesicles proximal to the constrictions (Dahlstrom and
Haggendal, 1966). When two ligations are made on the
same nerve, no accumulation of norepinephrine is ob-
served above the more distal constriction. Colchicine and
vinblastine, compounds that cause a disaggregation of
microtubules, block the proximodistal transport of dense
core vesicles and norepinephrine in noradrenergic
neurons (Hokfelt and Dahlstrom, 1971) implicating
microtubules in the rapid axonal transport.

Biochemical and immunological studies indicate that
dopamine-fi-hydroxylase (Laduron and Belpaire, 1968)
and chromogranins, proteins associated with catechol-
amine binding, also rapidly accumulate proximal to a
constriction in peripheral noradrenergic neurons (Geffen
et al., 1969). Recently it has been found that dopamine-j-
hydroxylase, an enzyme localized in the storage vesicle,
and tyrosine hydroxylase, an enzyme not associated with
these vesicles, are both transported down the axon of the
rat sciatic nerve at an identical rate (1 to 5 mmjhr) (Coyle
and Wooten, 1972). Colchicine blocks the -transport of
both enzymes. These observations suggest that dopamine-
b-hydroxylase and tyrosine hydroxylase are transferred
from the cell body to the nerve terminal in close associa-
tion. Local application of colchicine or vinblastine to the
superior cervical ganglion of the rat causes a rapid increase
in the levels of dopamine-j&hydroxylase in the ganglia
and decrease in the salivary gland (Kopin and Silberstein,

1972). When protein synthesis is inhibited the levels
of dopamine-j?-hydroxylase in the ganglia are rapidly
decreased, indicating that the accumulation of the
enzyme is due to new synthesis and the decrease after
protein synthesis inhibition is the consequence of transport
of the enzyme out of the ganglion. Using this approach,
the rate of synthesis of dopamine-/I-hydroxylase has been
calculated to be 5% of the content per hour.

Release of norepinephrine from nerve terminals

The neurotransmitter, norepinephrine, is contained in a
membrane-bound vesicle (Wolfe et al., 1962). Thus its
discharge from the nerve after depolarization might occur
by release into the cytoplasm followed by rapid passage
through the neuronal membrane, or by fusion of vesicular
membrane with the neuronal membrane and then
liberation, or by an opening of the fused membrane and
discharge of norepinephrine into the exterior of the ter-
minal together with the soluble contents of the vesicle.
This latter process is called exocytosis. Evidence that
exocytosis occurs comes from studies with adrenal
medulla. Stimulation of the adrenal gland with acetyl-
choline or electrically results in the release ofATP, as well
as catecholamines (Douglas and Rubin, 1961). Acetyl-
choline can also cause the release of the soluble protein of
the chromaffin granule, including dopamine-fl-hydrox-
ylase (Viveros et al., 1968). The ratio of norepinephrine
to dopamine-/I-hydroxylase was found to be the same as
that present in the chromaffin granule of the adrenal
medulla (Viveros et al., 1969). These findings and micro-
scopic evidence indicate that catecholamines are released
from the adrenal medulla by a process of exocytosis. When
the sympathetic nerve to the spleen is stimulated, dopa-
mine-j?-hydroxylase is released together with norepine-
phrine (Smithetal., 1970). However, theratiooftheamine
to dopamine-fi-hydroxylase released was 100 times greater
than that found in the vesicles isolated from the splenic
nerve. Using a very sensitive assay for dopamine-j-hydrox-
ylase, together with the addition of albumin to protect the
enzyme, the ratio of dopamine-/I-hydroxylase to nor-
epinephrine released after electrical stimulation of the
hypogastric nerve of the vas deferens was found to be
similar to that in the soluble portion of the contents of the
synaptic vesicle (Weinshilbbum et al., 1971e). This data
indicates that norepinephrine and dopamine-B-hydrox-
ylase are released from the nerve by a process of exocytosis.
The absence of Ca+ + prevents the release of the enzyme
and neurotransmitter, while their discharge is enhanced
by increasing the concentration of Ca+ + to twice that used
normally (Johnson et al., 197 1). The increased release of
dopamine-P-hydroxylase with high Ca+ + concentration

JULIUS AXELROD      869


IS blocked by prostaglandin E,. The a-adrenergic blocking
agent, phenoxybenzamine, also increases the release of
norepinephrineanddopamin~:-P-hydri)xylaseinthestim.tl-
lated vas deferens, but this drug has no effect on the
unstimulated preparation. Prostaglandin also blocks the
effects of phenoxybenzamine (Johnson et al., 1971). The
enhanced release afdopamine-p-hydroxylase by phenoxy-
benzamine only when the nerve is stimulated suggests that
there is an a-adrenergic receptor on the nerve membrane,
and blocking this receptor keeps the nerve membrane in a
conformational state that allows larger molecules to be
secreted for a longer period of time. Prostaglandins may
act by interfering with the actions of Caf + and thus
reduce the Ca+ `-d ependent secretion of norepinephrine
and dopamine-fi-hydroxylase.

Microtubules have been shown to be involved in the
discharge of intracellular stored products such as the
release of t 311 from the thyroid gland (Williams and
Wolff, 1970), insulin from the beta cells of the pancreas
(Lacy et al., 1968), histamine from mast cells (Gillespie
et al., 1968), and catecholnmines from the adrenal medul-
la (Poisner and Bernstein, 197 1). These findings suggested
that microtubules might play a role in the release of
dopamine-/)-hydroxylase from sympathetic. nerve ter-
minals. Treatment of the vas deferens with colchicine and
vinblastine, compounds that disaggregate microtubules,
almost completely prevented the release of dopamine-j?-
hydroxylase and norepinephrine when the nerve is
stimulated (Thoa et al., 1972). These compounds, how-

              STIMUL.
o Dopamine-fl-Oxidase
o Norepinephrine

ever, have no effect on the spontaneous release of the
enzyme or transmitter. Cytochalasin B, a fungal metab-
olite that disrupts microfilaments (Carter, 1567), also
inhibits the release of norcpinephrine and dopamine-#l-
hvdroxylase. These findings suggest that both micro-
tubules and microfilaments are involved in release of
norepinephrine and dopamine-/3-hydroxylase by exocy-
tosis. Microtubules are presumed to function as a cyto-
skeleton, and nerve depolarization might affect the
microtubules in such a way as to direct the vesicles to the
proper site on the neuronal membrane where release occurs
(Figure 5). Ca+ + has also been reported to activate the
contractile microfilaments in nonmuscle cells (Wessells
et al., 197 1) . These findings would suggest the presence of
a contractile microfilament on the neuronal membrane
that is activated by Caf +, which makes an opening in
the membrane large enough to allow the soluble contents
of the vesicle to be released (Figure 5). Cyclic AMP might
also be involved since it has been demonstrated that
dibutyryl cyclic AMP and theophylline increase the
release of norepinephrine and dopamine-p-hydroxylase
after nerve stimulation (Wooten, Thoa, Kopin, and
Axelrod, unpublished observation).

Circulating dopamine+hydroxylase

The observation that dopamine-P-hydroxylase can be
released from the adrenal gland and the nerve terminals
prompted an examination of the blood for this enzyme.

STIMUL.

FIGURE 5 A possible mechanism for release of norepinephrine and dopamine-a-oxidase
(hydroxylase) by exocytosis (see text for explanation). (From Axclrod, 1973).

870    REGULATION OF THE NEUROTRANSMITTER NOREPINEPHRINE


Dopamine+hydroxylase was found to be present in
the plasma of man and other mammalian species
(Weinshilboum and Axelrod, 197lb; Goldstein et al.,
197 l).The enzyme in the plasma issimilar topurifieddopa-
mine /?-hydroxylase from the adrenal medulla; both have
the same requirements for ascorbic acid, fumarate, and
oxygen (Weinshilboum et al., 1971b). They also have
similar electrophoretic mobilities and the same Km with
respect to substrate.

The plasma dopamine-/?-hydroxylase could arise from
the sympathetic nerves or the adrenal gland. The ad-
ministration to rats of 6-hydroxydopamine, a compound
that destroys most of the sympathetic nerve terminals but
does not affect the adrenal medulla, markedly reduced
the level of the plasma dopamine-/?-hydroxylase
(Weinshilboum and Axelrod,`197lc). On the other hand
adrenalectomy did not affect the plasma enzyme levels.
The experiments indicate that plasma dopamine-b-
hydroxylase comes from sympathetic nerve terminals
and that levels of this enzyme in blood suggest a method
for measuring activity of these nerves.

In rats subjected to stress, there is an elevation of serum
dopamine-/?-hydroxylase (Weinshilboum et al., 1971d).
When humans were stressed by vigorous exercise or cold
pressor test, there was rapid elevation of plasma enzyme.
In familial dysautonomia a decrease in plasma dopamine-
b-hydroxylase (Weinshilboum and Axelrod, 197la) is
found while subjects with neuroblastoma have an in-
creased enzyme in plasma (Goldstein et al., 1972).
Removal of the pituitary gland causes a marked decrease
in enzyme activity, which can be prevented by the
administration of vasopressin (Lamprecht and Wooten,
1973). This suggests that hypophysectomy, which reduces
blood volume, increases sympathetic nerve activity and
increases the release of dopamine-/?-hydroxylase. Vaso-
pressin increases blood volume and thus results in a
reduced sympathetic nerve activity and blood enzyme
level.

Regulation of norefiinephrine at the neuronal
membrane

When norepinephrine is injected into animals it is
selectively taken up by sympathetic nerve terminals
(Axelrod, 1971; Hertting and Axelrod, 1961). The
norepinephrine is then bound in the synaptic vesicie and
retained in a physiologically inactive form. This uptake
and binding serves as a rapid and effective means of
terminating the action of the neurotransmitter. When
both monoamine oxidase and catechol-O-methyltrans-
ferase, enzymes involved in the metabolism of catechol-
amines, are inhibited in vivo, the physiological actions

of norepinephrine are only slightly prolonged (Crout,
1961). However, when the uptake of norepinephrine is
blocked by drugs (Whitby et al., 1960), or when the
sympathetic nerves are destroyed (Hertting et al., 196lb),
the response of norepinephrine is considerably increased.
These results indicate that uptake of norepinephrine
across the neuronal membrane and retention by storage
vesicles are a major mechanism for the rapid inactivation
of the neurotransmitter (Figure 6).

TERMINAL

FIGURE 6 Fate'of norepinephrine at the sympathetic nerve
terminal (see text for explanation). NAis norepinephrine; DBH
is dopamine-/?-hydroxylase; COMT is catechol-O-methyl-
transferase; and MAO is monoamine oxidase. (From Axelrod
and Weinshilboum, 1972).

The properties of the neuronal uptake mechanism were
studied in brain slices (Dengler et al., 1962) and isolated
from perfused heart (Iversen, 1963). Uptake of norepine-
phrine across the neuronal membrane obeys saturation
kinetics of the Michaelis-Menten type with high affinity.
It also*requires sodium iohs in the external medium, is
temperature dependent, and involves active transport.
The uptake process is stereoselective and can be utilized
by other phenylethylamine derivatives such as epine-
phrine, dopamine, tyramine, amphetamine, ol-methyl-
norepinephrine, and meteraminol (Iversen, 1971). High
affinity uptake processes similar to norepinephrine's have
been demonstrated for other putative neurotransmitters,

JULIUS AXELROD   871


serotonin, gamma amino butyric acid, glutamate, aspar-
tate, and glycine (Logan and Snyder, 1971).

Norepinephrine can also be taken up by an extra-
neuronal process (Iversen, 1965 ; Eisenfeld et al., 1967).
This extraneuronal uptake can be blocked by adrenergic
blocking agents, normetanephrine (Eisenfeld et al., 1967)
and corticosteroids (Iversen, 1971). Compounds such as
isoproterenol, which have a low affinity for the intra-
neuronal uptake and a high affinity for extraneuronal
uptake, may be inactivated by the latter process. Extra-
neuronal uptake may be an important mechanism for
removal of the norepinephrine in which the density of the
sympathetic innervation is very low or when the synaptic
cleft is wide.

Norepinephrine can be inactivated by a variety of
mechanisms (Figure 6) : Uptake into the neuron, removal
by the circulation, enzymatic 0-methylation and de-
amination by liver and kidney, 0-methylation and
deamination by effector cells, and extraneuronal uptake.
Although neuronal uptake is the major mechanism for
terminating the action of the sympathetic neurotrans-
mitter, other types of inactivation may predominate,
depending on the density of sympathetic innervation, size
of the synaptic cleft, blood supply and activity of catechol-
0-methyltransferase, and monoamine oxidase.

Several studies have shown that a-adrenergic blocking
agents inhibit the uptake of norepinephrine and cause an
increased overflow of the neurotransmitter on nerve
stimulation (Hertting et al., 196 1 b ; Brown and Gillespie,
1957). It has also been demonstrated that large amounts
of endogenous norepinephrine inhibit the discharge of
norepinephrine from nerves (Starke, 1971). These ob-
servations suggest another regulatory site on the neuronal
membrane. The neuronal membrane appears to have an
inhibitory a-adrenergic receptor (Starke, 1971), which
would cause an increased release of the neurotransmitter
when the receptor is blocked and a decreased release when
a large amount of norepinephrine is present in the syn-
aptic cleft.

Regulation at ttie postsynaptic efector cell

The sympathetic effector cell could influence the activity
of the sympathetic nerve, and conversely the presynaptic
cell could influence the activity of the postsynaptic
effector cell. When the response of postsynaptic sympa-
thetic effector cells is blocked by phenoxybenzamine,
there is a marked increase in tyrositie hydroxylase activity
in the adrenal medulla (Thoenen et al., 1969b) and much
greater conversion of [`4C]tyrosine to [i4C]catechol-
amine (Dairman and Udenfriend, 1970).

Denervation of the sympathetic nerves leads to an
increased response of the postsynaptic cell. One ex-

planation for the increased sensitivity is the removal of an
important inactivating mechanism; uptake by the
neuronal membrane. Another possible mechanism for
supersensitivity is an increased responsiveness of the post-
synaptic site. It has been shown that in the denervated
muscle, the area of binding of a-bungarotoxin, a com-
pound that binds irreversibly to acetylcholine receptors,
is increased (Miledi and Potter, 1971).

The pineal cell has been used to study the relationship
between the sympathetic nerves and postsynaptic cell,
This gland is richly innervated with sympathetic nerves
that regulate the synthesis of the hormone melatonin
(Wurtman et al., 1968a). Serotonin N-acetyltransferase is
an enzyme that N-acetylates serotonin to form the pre-
cursor of melatonin (Weissbach et al., 1960). It is present
in the postsynaptic pineal cell and is markedly stimulated
by norepinephrine and dibutyryl cyclic 3',5'-adcnosine
monophosphate in organ culture (Klein et al., 1970). In
the intact rat, the enzyme activity is also sharply increased
after administration of catecholamines  (Figure 7)
(Deguchi and Axelrod, 1972). The induction of pineal
serotonin N-acetyltransferase by catecholamines is pre-
vented by the B-adrenergic blocking agent, propranolol.
When the pineal is denervated the catecholamines cause
a superinduction (lOO-fold increase) of serotonin N-
acetyltransferase (Figure 7). Although other possibilities
have not been excluded, these findings suggest that the
increased responsiveness after denervation is due to
changes on the postsynaptic P-adrenergic receptor on the
pineal cell.

Conclusions

The formation and conservation of the sympathetic
neurotransmitt&, norepinephrine, is controlled at several
sites in the sympathetic neuron. Its synthesis can be
rapidly changed by feedback mechanisms on tyrosine
hydroxylase in the nerve terminals. Another regulatory
site occurs in the cell body and adrenal medulla whereby
sympathetic nerve activity changes the rate of formation
of various biosynthetic enzymes. Glucocorticoid hormones
also influence the synthesis of these enzymes in the adrenal
medulla. The biosynthetic enzymes are made in the cell
body and transported down the axon to the nerve ter-
minals via proximodistal flow. The transmitter is then
synthesized and stored in a vesicle in the nerve terminal.
Norepinephrine is released by a process of exocytosis.
Once released, the neurotransmitter can be taken up by
the nerve terminal and stored and reused again. The
presynaptic membrane can affect the activity of the post-
synaptic cell by rapidly removing the transmitter by
reuptake and influencing the responsiveness of the post-
synaptic adrenergic receptor.

872    REGULATION OF THE NEUROTRANSMITTER NOREPINEPHRINE


0

cl None

INNERVATED

FIGURE 7 Induction and superinduction of serotonin N-
acetyltransferase in the rat pineal. Rat pineals were denervated
by the bilateral removal of the superior cervical ganglia. Dopa

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876    REGULATION OF THE NEUROTRANSMITTER NOREPINEPHRINE