Effects of Methyldopa Metabolites on Amine
Transmitters and Adrenergic Receptors in Rat Brain
WILLIAM J. LOUIS, ELIZABETH CONWAY, ROGER SUMMERS, PHILIP BEART, AND BEVYN JARROTT
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SUMMARY Studies of catecholamine concentrations in defined nuclei from the anterior hypothalamic-preoptic regions and the medulla oblongata, known to contribute to cardiovascular control,
were measured following acute or chronic methyldopa administration. These studies indicated that
methyldopa was enzymatic-ally converted to methyldopamine and methylnorepinephrine, and in some
areas to methylepinephrine which replaced endogenous epinephrine. The predominant metabolite
was methylnorepinephrine, which accumulated in concentrations higher than endogenous norepinephrine levels. ( —)Methylnorepinephrine was found to be 6 times more potent and 75 times more
selective for «2-adrenergic receptors than ( —) norepinephrine, and it is suggested that this a2adrenergic receptor action, particularly in the nucleus tractus solitarius, contributes to a major part
of the antihypertensive effect of methyldopa. (Hypertension 6 [Suppl II]: 11-40-11-44, 1984)
KEY WORDS • methyldopa • a-methyldopamine • a-methylnorepinephrine
«2-adrenergic receptors • radioligand receptor assays • rat brain
T
HE central nervous system contains a variety
of amine, amino acid, and peptide transmitters including dopamine, norepinephrine,
epinephrine, and 5-hydroxytryptamine, acetylcholine,
glycine, gamma-aminobutyric and glutamic acids.
Other possible neurotransmitters include substance P,
enkephalin, adenosine, histamine, and aspartic acid as
well as various peptides.1 2 It appears that a balance
between these transmitter systems in the central nervous system is necessary for proper regulation of brain
function, and there is increasing evidence that in
pathological disease states such as hypertension imbalances may occur and contribute to or be responsible for
the elevated blood pressure.
The most readily identified and perhaps the most
important of these transmitters in blood pressure regulation is norepinephrine, which mediates its action
through both types of a- as well as /3-adrenergic receptors. Methyldopa, which interferes with central nervous system norepinephrine levels, has been an extremely effective antihypertensive agent since its
introduction in 1960. Studies of its mode of action
have contributed to the understanding of central noradrenergic control of blood pressure, although its
•
mode of action has been the subject of considerable
dispute. Methyldopa initially was introduced as a decarboxylase inhibitor and a derivative carbidopa has
been used widely in the treatment of Parkinson's disease to prevent the peripheral decarboxylation of Ldopa. Because this mechanism did not satisfactorily
explain the antihypertensive effect of methyldopa or its
action on central nervous system transmitters, the false
transmitter hypothesis was developed. 3 Current knowledge indicates that methyldopa must be enzymatically
converted to methylnorepinephrine, which has a highly selective action that is mediated through a,-adrenergic receptors.
Catecholamine Levels After Chronic
Methyldopa Administration
Although clinical usage of the hypotensive agent
methyldopa involves chronic administration, most
studies on its mechanism of action have involved acute
systemic or intracerebral injection of either the parent
compound or its metabolites. Thus the evidence that
the hypotensive response that follows methyldopa administration depends on the formation of methylnorepinephrine centrally and the activation of a-adrenergic receptors by this metabolite is derived largely from
acute studies. 4 5 In addition, the possible central site of
action has been suggested by reports that local injections of methylnorepinephrine into the anterior hypothalamic-preoptic region and the nucleus tractus solitarius (NTS) produce a fall in blood pressure in
anesthetized rats. 6 7
From the University of Melbourne. Clinical Pharmacology and
Therapeutics Unit. Austin Hospital. Heidelberg. V i c . 3084. Australia.
Address for reprints: University of Melbourne. Clinical Pharmacology and Therapeutics Unit. Austin Hospital. Heidelberg. Victoria. 3084. Australia.
11-40
MODE OF ACTION OF METHYLDOPA/Louw el al.
In our studies (Figure 1) chronic administration of
methyldopa to rats (40 mg/kg subcutaneously b.i.d.
for 5 days) produced a profound depletion of norepinephrine and dopamine levels in nuclei in the anterior
hypothalamic-preoptic region and in the medulla oblongata such that these amines were virtually undetectable 4 hours after cessation of drug treatment.8 At this
time methylnorepinephrine was the predominant
amine present in all of the nuclei and the levels were
always higher than control norepinephrine levels in
untreated rats. Much lower levels of methyldopamine
also were present. In some nuclei the levels of methylnorepinephrine were as much as 6 times greater than
the endogenous norepinephrine levels in untreated animals. Twenty-four hours after the last injection of
methyldopa, methylnorepinephrine levels were still
between 50% and 80% of four-hour values and norepinephrine levels were still less than 45% of control
values.
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1*
i
•
nts
These data support the contention that methylnorepinephrine is involved in mediating the hypotensive
effect of methyldopa through actions in either the anterior hypothalamic-preoptic nuclei or the NTS in the
medulla oblongata or through a combination of actions.8 The slow disappearance of methylnorepinephrine after cessation of the methyldopa treatment in part
reflects the fact that methylnorepinephrine is not a
substrate for monoamine oxidase9 and may explain the
persistence of the hypotensive effect of this drug reported in both rats and humans.
M^jor Site of Action of Methyldopa
In acute studies (Figure 1) methyldopa administration to rats (200 mg/kg subcutaneously) produced a
marked reduction in levels of norepinephrine and dopamine in anterior hypothalamic and preoptic nuclei
implicated in catecholamine-mediated cardiovascular
inhibitory functions.l0 Similar effects were observed in
it
\
12
11-41
I.
nts
12
--J
t
t
t
*
•
\:-T
\T
It
o
a
/
12
12
I
pol
24
pol
J
2«
10
•v
T I M " hr
NA
ot-MNA
Contro1
a-MDA
FIGURE I. Effect of acute administration of methyldopa (200 mg/kg s.c; left-hand panel,) and chronic administration (40 mg/kg s.c. twice daily for 5 days; right-hand panel; on le\els of norepinephrine (
) . dopamine
(——), methylnorepinephrine (
J, and methyldopamine (
; in nuclei from the nucleus preopticus lateralis
(pol) in the hxpothalamus and the nucleus tractus soliiarius (nts) in the medulla. Control levels of norepinephrine
and dopamine in the chronic studies are shown adjacent to the vertical axis in the right-hand panel. Values
represent means ± SEM (n = 5). Asterisks indicate a significant reduction (p < 0.05) in catecholamine le\'els
compared with control values.l0
11-42
HYPERTENSION
SUPPLII VOL6, NO
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medullary nuclei including the NTS, which has been
postulated as a site for the hypotensive action of methy l d o p a . 6 " 1 2 The metabolite methyldopamine accumulated rapidly and 4 hours after drug administration
levels were higher in most nuclei than in either the
medulla oblongata or hypothalamus as a whole, which
suggests greater uptake of methyldopa into these specific areas.13 This was also reflected in high levels of
methylnorepinephrine in these nuclei. In most regions,
however, methylnorepinephrine levels were even
higher 12 hours after cessation of methyldopa administration; thus the levels of methylnorepinephrine in
medullary nuclei bore little relationship to the known
time course of the antihypertensive response in these
animals. '4 By contrast the time course of accumulation
of methylnorepinephrine in the NTS (Figure 1) closely
correlated with the reported time course of the hypotensive effect of methyldopa, which suggests that this
site is the major mediator of at least the acute antihypertensive effect of this drug. It is still possible, however, that the action of this metabolite in other areas
may contribute to the fall in blood pressure during
chronic administration.
Methylnorepinephrine
In recent years the understanding of a-adrenergic
receptors has been facilitated both by in vitro and in
vivo pharmacological studies and by the development
of appropriate ligands. Adrenergic receptors exist as
two pharmacologically distinct types designated a,
and a 2 , and it has been suggested that these receptors
mediate selectivity by different mechanisms: a, effects
are due to elevation of intracellular calcium ions and a,
effects are caused by inhibition of adenylate cyclase.
The development in recent years of selective radioligands for a,- and a 2 -adrenergic receptors has provided
tools that have facilitated the examination of the properties of adrenergic receptors at a molecular level. a2Adrenergic receptors can exist in two affinity states
designated a 2 H (high affinity) and a,L (low affinity).
In contrast to antagonist ligands, agonist ligands labeled predominantly the fraction of a,-adrenergic receptors in the high-affinity state. The size of that fraction is governed by the ionic environment and by the
presence of guanyl nucleotides.15
5, SEPTEMBER-OCTOBER 1984
We have examined the relative potency for a2adrenergic receptors of a series of clonidinelike compounds in membranes from rat cerebral cortex (Table
I).16 This and subsequent studies have demonstrated
that drugs like prazosin and labetalol are highly ar
selective whereas drugs like clonidine have a high selectivity for a2-adrenergic receptors. An important observation was that guanfacine and CP14304-18 were
even more selective for a 2 -adrenergic receptors than
was clonidine.17-l8 We have also used [3H]-guanfacine
to identify a2-binding sites in rat brain areas. The highest levels of binding, in descending order, were obtained in membranes prepared from cerebral cortex,
hippocampus, hypothalamus, medulla pons, spinal
cord, striatum, and cerebellum.
Table 2 compares inhibition constant (Ki) values for
norepinephrine and methylnorepinephrine with a number of clinically important compounds that act on aadrenergic receptors. (— )Methylnorepinephrine had a
Ki value for [3H]-guanfacine similar to that of clonidine and was 7 times more potent than (— )norepinephrine. In addition, (-)methylnorepinephrine had much
less effect on [3H]-prazosin binding than did ( - )norepinephrine. When selectivity for a,-adrenergic receptors was expressed as the ratio of |3H]-prazosin to |'H]guanfacine binding, methylnorepinephrine had a selectivity of more than 4000 and was the most selective compound tested. This selectivity contrasted with
(-)norepinephrine's selectivity of 65. Thus methylnorepinephrine was 7 times more potent and 70 times
more selective for brain a,-adrenergic receptors.19
Given the extremely high levels of methylnorepinephrine formed in cardiovascular inhibitory regions such
as the anterior hypothalamus and NTS during chronic
methyldopa administration, it seems likely that the
major mechanism of its antihypertensive effect is mediated through its selective a,-adrenergic receptor actions in these regions.
Methylepinephrine
We previously have reported the presence of epinephrine and phenylethanolamine N-methyltransferase (PNMT) in various medullary and hypothalamic
nuclei in rats.20 More recently, in vitro studies in homogenates of hypothalamus and medulla indicated that
TABLE I. Displacement of [3Hj-Prazosin and ['Hj-Clonidine Binding from Membranes Prepared from Ral Cerebral
Cortex by Clonidinelike Drugs
K, (3H]-Prazosin
Drug
K, |3H]-Prazosin
K, [3H]-Clonidine*
K, [3H]-Clonidine
l.9±0.3
6177±1 310
3220
Guanfacine
2627
CPI4,304-18
0.8±00l
2102±173
2.2 + 0.2
Clonidine
593 ±102
269
2.3±0.2
Lofexidine
181 ±49
79
40±2
1.8±0.3
22
44-549
Inhibition constant (K|) values have been calculated from the (ICJO) for inhibition of binding with Cheng and Prusoff16
equation IC50 = K| (1 + D/KD). IC50 = concentration inhibiting specific binding by 50%. Values given are means ±
SEM for four to seven experiments conducted in duplicate.
•Modified from Summers, Jarrott, and Louis.17
11-43
MODE OF ACTION OF METHYLDOPA/Low/s et al.
TABLE 2. Relative Potencies of a-Adrenergic Receptor Drugs at a,-Adrenergic Receptors Labeled by [3HJ-Pra:osin
and a2-Adrenergic Receptors Labeled b\ [3H]-Guanfacine in Membranes from Rat Cerebral Cortex
Drug
K, [3H]-Guanfacine
(nM)
K, [3H]-Prazosin
(nM)
K, [3H]-Prazosin
K, [3H]-Guanfacine
Guanfacine
1.8
6210
Clomdine
2.1
593
280
( - )a-Methylnorepinephrine
2.6
11.450
4400
Phentolamine
6.9
( ± )o-Methylnorepinephrine
( - ) Norepinephrine
( + ) Norepinephrine
Prazosin
7 2
3400
1.04
8.6
15.280
1777
14.7
960
65
39.580
99
400
12.000
0.11
0.000009
Source: Louis et al.
Downloaded from http://hyper.ahajournals.org/ by guest on June 17, 2017
methylnorepinephrine was transformed to methylepinephrine by enzyme preparations from both regions
but that methylnorepinephrine was a less effective substrate than norepinephrine.21
In further experiments Sprague-Dawley rats were
treated subcutaneously with methyldopa and the formation of methylepinephrine was studied in vivo.21
Freshly dissected hypothalami and medullae oblongatae were analyzed for the presence of methylepinephrine with a high-performance liquid chromatography
electrochemical detection system. These experiments
indicate that methylepinephrine can be formed from
methyldopa within the central nervous system. Moreover, formation was not significantly altered by the
administration of the PNMT inhibitor SKF 64139,
which suggests that the in vivo synthesis of methylepinephrine after the administration of methyldopa
probably occurs by the action of nonspecific methyltransferases.22-23
At this stage the functional significance of methylepinephrine is uncertain, but this amine may be involved in the depletion of epinephrine from medullary
and anterior hypothalamic nuclei seen after chronic
administration of methyldopa.24 Moreover, the work
of Dampney,23 Blessing et al., 26 and Reis et al.27 suggests that the Cl region, which is important in cardiovascular control, contains high concentrations of
epinephrine. More recently Robertson et al.28 have
demonstrated that administration of methylepinephrine into either the lateral ventricle or the NTS produces a prolonged antihypertensive effect.
Conclusion
It is apparent that methyldopa administration produces marked alterations in the levels of catecholamines in all regions of the central nervous system. In
the corpus striatum methyldopamine has been documented to replace the endogenous neurotransmitter dopamine in dopaminergic nerve terminals and is released in a manner similar to dopamine.29 Similarly
methylnorepinephrine replaces norepinephrine as a
neurotransmitter in noradrenergic nerve terminals in
other areas of the brain. Because it is not a substrate for
monoamine oxidase, methylnorepinephrine accumulates both in the synaptic vesicles and in the soluble
fraction of the cell, which explains in part the very high
levels of methylnorepinephrine relative to control values of norepinephrine. In addition, binding studies
indicate that methylnorepinephrine is considerably
more potent and much more selective than norepinephrine for a,-adrenergic receptors in the central nervous
system. Other reports indicate that methylnorepinephrine is more potent than norepinephrine in producing a
fall in blood pressure after direct injection into the
anterior hypothalamic-preoptic area and the NTS.
The mechanism by which the a2-agonists mediate
their effect in the central nervous system is uncertain,
however. The concept that presynaptic a-adrenergic
receptors modulate the amount of neurotransmitter released per pulse (see reviews by Langer30 and Starke31)
is largely derived from studies in peripheral tissues but
Rand et al.32 have demonstrated such an action for
clonidine in high concentrations in guinea pig hypothalamic slices, and similar results have been reported in
rat cerebral cortex.33-M It should also be noted, however, that clonidine still reduces sympathetic outflow and
enhances the vagally mediated cardiodepressor reflex
after catecholamine depletion with reserpine and amethyl-p-tyrosine.35-M These latter results suggest that
presynaptic effects in noradrenergic neurons cannot be
the only mechanisms involved in the hypotensive response to clonidine and perhaps to other central a2agonists.
More recently interest has focused on the presence
of adrenergic neurons in the Cl region of the medulla
oblongata. 26 -" It is now apparent that methyldopa can
be converted to methylepinephrine in both medullary
and hypothalamic regions.21 The significance of this
observation is uncertain, in part because there are no
clear data on the relative amounts of methylnorepinephrine and methylepinephrine formed and on whether methylnorepinephrine and methylepinephrine have
different actions in these regions. Methyldopa administration also may affect other neurotransmitter pathways in the brain; for example, there is evidence that it
can deplete serotonin pathways by replacing serotonin
with methyldopamine.37
11-44
HYPERTENSION
SUPPLII V O L 6 ,
Acknowledgments
The authors acknowledge the support from the National Health
and Medical Research Council of Australia and Merck, Sharp and
Dohme International for research carried out in their laboratories.
References
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1. Palkovits M. Topography of chemically identified neurons in
the central nervous system: a review. Acta Morphol Acad Sci
Hung 1978;26:211-290
2. Palkovits M. Topography of chemically identified neurons in
the central nervous system: progress in 1977-1979. Med Biol
1980;58:188-227
3. Day MD, Rand MJ. A hypothesis for the mode of action of amethyldopa in relieving hypertension. J Pharm Pharmacol
1963;15:221-224
4. Day MD, Roach AG, Whiting RL. The mechanism of the
antihypertensive action of a-methyldopa in hypertensive rats.
Eur J Pharmacol 1973;21:271-280
5. Finch L, Haeusler G. Further evidence for a central hypotensive action of a-methyldopa in both rat and cat. Br J Pharmacol
1973;47:2l7-228
6. De Jong W, Nijkamp FP, Bohus B. Role of noradrenaline and
serotonin in the central control of blood pressure in normotensive and spontaneously hypertensive rats. Arch Int PharmacodynTher 1975;213:272-284
7. Struyker Boudier H, Smeets G, Brouwer G, Van Rossum JM.
Central nervous system a-adrenergic mechanisms and cardiovascular regulation in rats. Arch Int Pharmacodyn Ther 1975;
213:285-293
8. Conway EL, Louis WJ, Jarrott B. Endogenous and a-methylated catecholamine levels in anterior hypothalamic-preoptic
and medullary nuclei in rat brain after chronic a-methyldopa
administration. Neuropharmacology 1979; 18:287-290
9. Louis WJ, Jarrott B, Conway EL. Methyldopa and catecholamines. In Villarreal H, ed. Proceedings of methyldopa symposium, Mexico. New York: BMI, 1980, 53
10. Conway EL, Louis WJ, Jarrott B. The effect of acute a-methyldopa administration on catecholamine levels in anterior hypothalamic-preoptic and medullary nuclei in rat brain. Neuropharmacology 1979; 18:279-286
11. Struyker Boudier HAJ, Smeets GWM, Brouwer GM, Van
Rossum JM. Hypothalamic alpha adrenergic receptors in cardiovascular regulation. Neuropharmacology 1974; 13:837846
12. Zandberg P, De Jong W. a-Methylnoradrenaline-induced hypotension in the nucleus tractus solitarius of the rat; a localization study. Neuropharmacology 1977; 16:219-222
13. Conway EL, Louis WJ, Jarrott B. Acute and chronic administration of a-methyldopa: regional levels of endogenous and amethylated catecholamines in rat brain. Eur J Pharmacol
I978;52:27I-28O
14. Day MD, Roach AG, Whiting RL. The mechanism of the
antihypertensive action of a-methyldopa in hypertensive rats.
Eur J Pharmacol 1983;21:271-280
15. Hoffman BB, Lefkowitz RJ. Radioligand binding studies of
adrenergic receptors: new insights into molecular and physiological regulation. Annu Rev Pharmacol Toxicol 1980;20:
581-608
16. Cheng YC, Prusoff WH. Relationship between the inhibition
constant (K|) and the concentration of inhibitor which causes
50 percent inhibition (I50) of an enzymatic reaction. Biochem
Pharmacol 1973;22:3099-3108
17. Summers RJ, Jarrott B, Louis WJ. Displacement of 3H cloni-
No 5, SEPTEMBER-OCTOBER 1984
dine binding by clonidine analogues in membranes from rat
cerebral cortex. Eur J Pharmacol 1980;66:233-241
18. Summers RJ, Jarrott B, Louis WJ. Selectivity of a series of
clonidine like drugs for a, and a 2 adrenoceptors in rat brain.
Neurosci Lett 1980;20:347-350
19. Louis WJ, Summers RJ, Dynon M, Jarrott B. New developments in a-adrenoceptor drugs for the treatment of hypertension. J Cardiovasc Pharmacol 1982;4(Suppl. 1):S168—S171
20. Beart PM, Prosser D, Louis WJ. Adrenaline and phenylethanolamine-N-methyltransferase in rat medullary and anterior
hypothalamic-preoptic nuclei (short communication). J Neurochem 1979;33:947-950
21. Beart PM, Rowe PR, Louis WJ. a-Methyladrenaline is a central metabolite of a-methyldopa. J Pharm Pharmacol 1983;
35:519-520
22. Saavedra JM, Coyle JT, Axelrod J. The distribution and properties of non-specific N-methyltransferase in brain. J Neurochem 1973;20:743-752
23. Fuller RW, Hemrick-Luecke S. Methylation of norepinephrine
and a-methylnorepinephrine in brain. Biochem Pharmacol
1982;31:3I28-313O
24. Beart PM, Prosser D, Louis WJ. Adrenaline depletion in the
hypothalamus of the rat after chronic a-methyldopa. Clin Exp
Pharmacol Physiol 1981;8:25-31
25. Dampney RAL. Functional organization of central cardiovascular pathways. Clin Exp Pharmacol Physiol 1981 ;8:241—259
26. Blessing WW, Goodchild AK, Dampney RAL, Chalmers JP.
Cell groups in the lower brain stem of the rabbit projecting to
the spinal cord with special reference to catecholamine-containing neurons. Brain Res 1981;221:35-55
27. Reis DJ, Ross CA, Granata AR, Ruggiero DA. Some neurochemical substrates in the brain stem regulating arterial pressure. Hypertension 1984
28. Robertson D, Tung C-S, Hollister AS, Goldberg MR, Oates
JA. The antihypertensive metabolites of alpha-methyldopa.
Hypertension 1984
29. Conway EL, Jarrott B, Louis WJ. Effect of a-methyldopa on
dopaminergic transmission in the corpus striatum. Neuropharmacology 1978; 17:355-361
30. Langer SZ. Presynaptic regulation of the release of catecholamines. Pharmacol Rev 1981;81:337-362
31. Starke K. Presynaptic receptors. Annu Rev Pharmacol 1981;
21:7-30
32. Rand MJ, McCulloch MW, Story DF. Prejunctional modulation of noradrenergic transmission by noradrenaline, dopamine
and acctylcholine. In: Davies DS, Reid JL, eds. Central action
of drugs in blood pressure regulation. London: Pitman Medical, 1975:94-132
33. Farnebo LO, Hamberger B. Drug-induced changes in the release of 3H-noradrenaline from field stimulated rat brain slices.
Acta Physiol Scand 197l;Suppl 371:35-44
34. Starke K, Montel H. Involvement of a-receptors in clonidineinduced inhibition of transmitter release from central monoamine neurons. Neuropharmacology 1973;12:1073-1080
35. Haeusler G. Clonidine-induced inhibition of sympathetic
nerve activity: no indication for a central presynaptic or an
indirect sympathomimetic mode of action. Naunyn Schmiedebergs Arch Pharmacol 1974; 286:97-111
36. Kobinger W, Pichler L. Centrally induced reduction in sympathetic tone — a postsynaptic a-adrenoceptor stimulating action
of imidazolines. Eur J Pharmacol 1976;40:311-320
37. Chalmers J, Minson JB, Choy V. Bulbospinal serotonin pressure pathways and hypotensive action of methyldopa in rat.
Hypertension 1984;6(5 Pt2): 16-21
Effects of methyldopa metabolites on amine transmitters and adrenergic receptors in rat
brain.
W J Louis, E Conway, R Summers, P Beart and B Jarrott
Hypertension. 1984;6:II40
doi: 10.1161/01.HYP.6.5_Pt_2.II40
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