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The effect of acute α-methyldopa administration on catecholamine levels in anterior hypothalamic-preoptic and medullary nuclei in rat brain
THE EFFECT OF ACUTE ~-METHYLDOPA ADM~NrSTRATION ON CATECHOLAMINE LEVELS IN ANTERIOR HYPOTHALAMIC-PREOPTIC AND MEDULLARY NUCLEI IN RAT BRAIN E. L. CONWAY,W. J. LOUIS and B. JARROTT University of Melbourne, Clinical Pharmacology and Therapeutics Unit, Austin Hospital. Heidelberg 3084. Victoria, Australia (Accrptvd
8 Noovmhrr
1978)
Summary-E-Methyldopa administration to rats (200 mg kg- ’ s.c.) produced a marked reduction in levels of noradrenaljne and dopamine in anterior hypo~alamic-pre~ptic nuclei implicated in catecholamine-mediated cardiovascular-inhibitory functions. Similar effects were observed in medullary nuclei, including the n. iractus solifmius which has been postulated as a site for the hypotensive action of r-methyldopa. The metabolite, %-methyldopamine, accumulated rapidly and 4 hr after drug administration levels were higher in most nuclei than in either the wdulla ohlonyara or hypothalamus as a whole, suggesting greater uptake of r-methyldopa into these specific areas. This was also reflected by higher levels of I-methylnoradrenaline in the nuclei than in the gross regions. The time course of accumulation and disappearance of z-methylnoradrenaline in the n. WUCIUS sofitarius differed from that in the anterior hypothalamic-preoptic nuclei and more closely correlated with the reported time course of the hypotensive effect of x-methyldopa. However. the action of this metabolite in both areas may contribute to the fall in blood pressure.
L-c+Methyidopa is now thought to produce a major part of its antihy~rtensive effect through an action on the central nervous system (Henning, 1975). In rats, the fall in blood pressure following acute administration of this drug is probably due to the action of a metabolite, r-methylnoradrenaline, acting on central a-adrenoreceptors (Day, Roach and Whiting, 1973; Finch and Haeusier, 1973). Ahhough the exact involvement of catecholaminecontaining neuron systems in the central control of blood pressure is not completely understood, evidence in recent years suggests that the anterior hypothalamic region and the A2 region of the nucleus (n.) tractus solitarius in the ~~d~l~a o~~o~gafa (see Dahlstrijm and Fuxe, 1964; Fuxe, 1965) may be involved in catecholamine-mediated cardiovascular-inhibitory functions (Struyker Boudier, Smeets, Brouwer and Van Rossum, 1974; De Jong, Nijkamp and Bohus, 1975; Philippu and Schartner, 1976; Nathan and Reis, 1977; Palkovits, De Jong, Zandberg, Verstecg, Van Der Gugten and Leranth, 1977). Further evidence indicates that both these regions respond to local injection of x-methylnoradrenaline by producing a fall in blood pressure in anaesthetized rats (De Jong et al., 1975; Struyker Boudier, Smeets, Brouwer and Van Rossum, 1975: Zandberg and De Jong, 1977). However, these studies are limited in that they take no account of the possible additive or synergistic effects of this amine acting simultaneously at different levels of the brain, Key words: z-methyldopa, r-methylnoradrenaline, ~-methyldopamine, cardiovascular inhibitory centres. 279
In a previous study it has been shown that acute administration of ~-methyldopa feads to a widespread depletion of dopamine and noradrenaline in ail areas of the rat brain, and to the replacement of these amines with r-methylnoradrenaline and n-methyldopamine (Conway, Louis and Jarrott, 1978). In the present study the effect of acute administration of ~-methyldopa on catecholamine levels has been examined in specific nuclei from the anterior hypothalamic-preoptic region which have been implicated as sites of the hypotensive action of rx-methylnoradrenaline (Struyker Boudier et ul., 1975) and in the n. tractus solitarius and adjacent nuclei in the medulla oblo~gata. The aim of this study was to characterize the effects of this drug in the catecholaminergic pathways which are most likely to be involved in mediating its hypotensive action,
METHODS Groups of male Sprague-Dawley rats (1%200g) were injected with 200 mg kg-’ of a-methyldopa as a single dose and killed 4 and 12 hr later. The drug was ground finely and administered subcutaneously as a suspension in 0.9% sodium chloride [W/V). One group of rats treated with vehicle was included as a control. The killing times were based on a previous study in different brain regions which indicated that maximal effects on the levels of dopamine and r-methyldopamine occurred at 4 hr whereas maximal effects on the levels of noradrenaline and r-methylnoradrenaline were seen at approximately 12 hr.
280
E. L.
CONWAY.
W. J. Louis and B.
Nuclei were dissected freehand from 4OOpm thick coronal sections of fresh brain tissue from the treated rats as described by Zigmond and Ben Ari (1976). Ideutific~~tion of the hypothalamic nuclei was based on the rat brain atlas of KGnig and Klippel (1970) and the descriptions of Palkovits, Brownstein, Saavedra and Axelrod (1974b). In the rnrdulla oblongam. identification of the nuclei was also based on the descriptions of Palkovits, Brownstein and Saavedra f1974a). Only the rostra1 sections astern in length) of the n. tractus spirxdis nervi tri~e}i~ilti and the dorsal and ventral n. reticularis medulla ohlongatae were removed. The n. tractus soliturius refers to that part of the nucleus just under the fourth ventricle surface and in the vicinity of the area postrema (A2 region). Nuclei from 3 rats were pooled and placed in small round-bottomed glass test-tubes (2.5 x OSem internal diameter, Samco). A plunger moulded to fit these tubes from Silastic J/RTV (Dow Corning Corp., Michigan, U.S.A.) was used to homogenize the tissue samples in 100~1 of 0.1 mol I- ’ perchloric acid. An aliquot of the homogenate (520~1) was taken for protein determination (Lowry, Rosebrough, Farr and Randall, 1951) and the remainder was centrifuged for 10,000 g min and the supernatant stored at -20°C until assayed. Noradrenaline. dopamine. x-methylnoradrenaline and ~-methyldo~mine were determined in the tissue supernatants by the following m~i~cation of the enzyme radiochemicai method of Van Der Gugten, Palkovits, Wijnen and Versteeg (1976). The endogenous amines and their x-methylated analogues were O-methylated by incubation with the substrate mixture containing catechol-Q-methyltransferase and S-adenosyl-L-~methyl-3H) methionine as described by these authors. The labeled 0-(3H)-methylated products were then extracted and separated by paper chromatography using the procedure of Cuello, Hiley and Iversen (1973). Under these conditions there was no significant cross-contamination between the methoxylated amines (Rf; normetanephr~ne~ 0.23; 3-OmethyI-~-methylnoradrenaline, 0.31; 3-methoxytyramine, 0.55 and 3-0-methyl-l-methyldopamine, 0.68). Tissue concentrations used in these determinations were low and inhibition by cellular components was negligible. Catecholamine levels in the samples were therefore determined by reference to external standards. This assay detected all four amines with a sensitivity of S@lOOpg. Data have been expressed as pg catecholamine/pg protein (mean + S.E.M.). Significance of the difference between values of control and treated groups was determined using student’s t--test. A P value of less than 0.05 was considered significant.
statistically
Materials
S-adenosyl-t_-(methyl-3H)-methionine, sp. act. 5 to 10 Ci mmol- ‘, was obtained from The Radiochemical Centre. Amersham. L-~-Methyldopa, (S, +)-a-methyl-
JARROTT
dopamine, I-(4-hydroxy-3-methoxyphenyl)-2-amino propane hydrochloride (3-O-methyl-a-methyldopamine) and erythro( ~)-~I-aminoethyl~4-hydroxy-3methoxy-benzyl alcohol hydrochloride (3-O-methyl-~methylnoradrenaline) were donated by Merck, Sharp & Dohme Research Lab., NJ. Initial supplies of 3-0methyl-z-methylnoradrenaline were donated by Dr A. A. Manian, Psychopharmacology Research Branch, National Institute of Mental Health, U.S.A. ~-Methylnoradrenaline hydrochloride was purchased from Calbiochem (U.S.A.). All additional chemicals used were reagent grade. RESULTS
fltrT&or ir~pothulffnric-preoptic
nuclei
Catecholamine levels in the anterior ~~pot~u~~~i7jcnuclei studied are shown in Table 1. Noradrenaline levels were highest in the n. prropticus medialis and the n. interstitialis striae terminalis. Dopamine levels were considerably lower than noradrenaline levels in all nuclei except the n. j~7terstjtiu~j.~ preoptic
striae
tert~l~~alis.
After renaline of 12 hr reduced
acute administration of g-methyldopa, noradlevels declined in all nuclei over a period (Fig. 1). At this time noradrenaline levels were to 16”/, of control values in the n. interstiticrlis striae ter~~i~u~is and to approximately 20% in the other nuclei. Levels of r-methytnoradrenaline increased in inverse proportion to the decrease in noradrenaline. At I2 hr the levels of this amine were approximately twice those at 4 hr and in all but one of the nuclei combined levels of z-methylnoradrenaline and noradrenaline were signifi~ntly elevated above control levels of noradrenaline alone (Table I). These results contrast with those obtained in the hypothalamus as a whole where the combined levels of noradrenaline plus r-methylnoradrenaline at 12 hr were significantly reduced from control noradrenaline levels (Table I). The hypothalamic nuclei studied comprise approximately 16% of the whole hypothalamus. Dopamine levels also declined after a-methyldopa injection although the low initial levels of this amine in many of the nuclei made it impossible to determine the maximum depletion (Fig. 1). r-Methyldopamine. on the other hand, accumulated rapidly in all nuclei and 4 hr after ~-methyldopa administration levels of this amine were at least 10 times control dopamine levels in all nuclei except the n. interstitialis striae terminalis (1.5 times) and the n. preopticus lateralis (2.3 times). The combined levels of dopamine and x-methyldopamine at 4 hr were significantly elevated above control levels of dopamine in all nuclei except the n. interstitialis striae terminalis (Table I), and ranged from 250% of control values in the n. preopticus lateralis to 1.560% in the n. anterior (hypothalami). This accumulation was in most cases higher than that found in the hypothalmus as a whole (3COo/,) (Table 1).
2.8 & 0.94 3.1 rf: 0.42
2.1 + 0.21 6.5 + 1.41 4.4 k 0.83 4.0 + i.71
15.4 f 3.08 13.7 f 2.48
12hr
__ _ _ _
__ _
_ r. - _” -
I
-
-~
.
.
_
,,,
_
_
_
_
.
_
5.7 + 0.38
.
-
.
-.
-
_.
,,
1.9 + 1.20 1.4 + 0.70 5.4 + 2.45
N.D. N.D.
4 hr
37.8 + 2.36 28.7 & 3.38
34.7 + 3.84 23.6 & 1.27 39.9 * 3.71
8.8 & 0.34
r-MDA (pg/fig protein) 0.4 I: 0.03
DA
(200 mg kg-’ s.c.)
DA-- dopamine: z-MDA-r-methyldopamine.
2.4 ? 0.96 2.9 + 0.61
3.3 f 0.15 10.2 _+ 3.25 27.5 f 8.08
2.7 f 0.20
r-MNA-rr-methylnoradrenaline:
21.8 rtr 1.69 21.0 + 1.33
36.7 + 3.76 24.5 t_ 1.68 30.5 & 7.03
administration
Control DA (pg/lrg protein)
nuclei 0. 4 and 12 hr after r-methyldopa
r-MNA (pg/~g protein)
14.8 k 0.74
NA
27.6 + 3.50 19.8 + 2.60 25.3 f 1.70
Control NA (pg/flg protein)
levels in anterior hypothalamic-preoptic
N.D. indicates no amine was detected (~0.2 pg/pg protein). n = 5: NA-noradrenaline:
n. Anterior (hypothalami) n. Latercrlis (hypothalami)
n. Preopticus lateralis n. Ittferstitjff1i.s srriue tertnit~alis
Hypothalamus n. Preopticus media/is
Area
Table 1. Catecholamine
282
E. L.
0
CONWAY,
6
4
45L
W. J. LOUISand B. JARROTT
0
12
r
porn
6
I
30
.12
PO1
0
6
I
12
Time, hr
40. ..:
. .:’ b
I 20-
T : ....
___._-L...
:
..,‘X._ . ,,*-J
....
"'....*J
iKL*<* .:' ,, ,..' ,s
o
NA a-MNA
_.---
DA .._..._..........&M,,A * P (0.05
,.c' 0
4
Time,
6
$2
hr
Fig. 1. Effect of acute administration of z-methyldopa (200 mg kg-’ s.c.) on levels of noradrenaline (NA). dopamine (DA). r-methylnoradrenaline (a-MNA) and a-methyldopamine (z-MDA) in nuclei from the anterior hypothalamic-preoptic region of the rat brain. Control levels of noradrenaline (4) and dopamine (#) are shown just adjacent to the vertical axis. Values represent means & S.E.M. (n = 5). Asterisks indicate a significant reduction in catecholamine levels compared with control values. ha-n. anterior (hypothalami); hl-n. lateralis (hypothalami); porn-n. preopricus rdialis; pol-n. preopticus /atrralis: St-n. interstitialis srriae terminalis.
At 12 hr after a-methyldopa injection, the levels of r-methyldopamine had dropped markedly in all the nuclei, representing less than 40% of the levels at 4 hr (Fig. I ). Nuclei
in the medulla
oblongata
Catecholamine levels in the medullary nuclei studied are shown in Table 2. The catecholamine levels of these nuclei were lower, than those of the anterior hypothalamic-preoptic nuclei. Noradrenaline levels were highest in the reticular nuclei in which no dopamine could be detected. Only trace amounts of dopamine were detected in the n. tractus solitarius (0.4 pg/pg protein) and the n. tractus spinalis nervi triyrnlini (0.2 pg/pg protein). Four hours following the administration of a single dose of r-methyldopa, noradrenaline levels were significantly lower than control values in all nuclei
except the n. tractus solitarius, and after 12 hr the levels were even lower or not detectable in all 4 nuclei (Fig. 2). a-Methylnoradrenaline had accumulated in all nuclei 4 hr following a-methyldopa injection in quantities at least equal to control noradrenaline levels. However, only in the n. tractus solitarius was the combined level of noradrenaline and a-methylnoradrenaline significantly elevated above control levels of noradrenaline (Table 2). The results in these nuclei, which constitute approximately 3% of the m&//a ohlongata, contrast with those obtained in the whole region where combined levels of noradrenaline and r-methylnoradrenaline at 4 hr were reduced from control noradrenaline levels (Table 2). In both the n. tractus solitarius and the n. tractus spinalis nervi trigemini a-methylnoradrenaline levels were lower at 12 hr than at 4 hr. whereas in the n. reticularis medulla ohlonga-
trace
1.1 * 0.67 2.0 2 0.88
8.0 i: 2.35
7.0 & 1.43
N.D. 3.4 5 1.28
5.5 If: 0.43 4.7 -f. 0.48 2.6 & 0.55
N.D.
9.0 +_0.8 I
x-MNA--r-mcthylnoradrenaline:
N.D.
N.D. trace trace
9.9 & 1.17
12.6 4 2.39
4.0 4 0.35 17.1 & 1.55 11.0 4 1.73
x-MDA (pg/pg protein1
4 hr
~-MDA-~-m~thyldopam~n~.
N.D.
N.D.
N.D. N.D. N.D.
DA
(2OOmg kg-’ s.c.)
DA-dopamine;
Control
8.0 5 1.45
2.8 & 0.05 10.4 & 1.01 7.2 _t- 1.74
r-MNA @g/p6 protein)
4hr
N.D. indicates no amine was detected (co.2 pg/hcg protein): II = 5: NA---noradrenaline:
Area
NA
levels in meduilary nuclei 0 and 4 hr after ~-methy~do~a administration
Control NA (pg/bcg protein)
Table 2. Catecholamine
284
E. L. CONWAY, W. J. Louis and B. JARUOTT
18.
JT
nts
.:’ ‘..... ,: ......
1
0
Time, -
hr
.------
4 12
Time,
hr
NA ci-MNA
P(O.05
*
Fig. 2. Eflect of acute administration of n-methyldopa (200mg kg-’ s.c.) on levels of noradrenaline (NA). r-methylnoradrenatine (c(-MNA) and a-methvidouamine &MDA) in nuclei fram the rriedrrlla ob~ongat~of ihe rat brain. n&-n. rraclus solifarius: tsv-n. tractus spinalis newi tri~~~f?lini;nrd-n. r~t~e~larist17~~uliu obiongata~(dorsal); nrv-n. reticu~ari~~?edul~aoblo~~atue(ventral). See Figure 1 legend for further details. I
tar (dorsal) they were unchanged and in the n. reticufuris t~edu~~uub~o~gu~~e(ventral) they had increased (Fig. 2). Although little or no endogenous dopamine could be detected in the medullary nuclei, 4 hr after a-methyldopa administration high levels of the metabolite, a-methyldopamine, were present in all 4 nuclei (Fig. 2). As in the hypothalamic nuclei, the accumulation of this amine was more pronounced in the medullary nuclei than in the gross medulla ohlongata region (Table 2). At 12 hr the levels of a-methyldopamine in all nuclei had fallen quite markedly.
DISCUSSION In this study the effect of acute administration of r-methyldopa has been examined on levels of both the endogenous catecholamines, dopamine and noradrenaline, and their a-methylated analogues in those nuclei from the anterior hypothalamic-preoptjc region and the medulla ob~o~guru believed to be involved in cardiovascular control. The values reported here for levels of noradrenaline and dopamine in these nuclei do not differ markedly from those of other reports (Palkovits et al., 1974b; Versteeg, Van Der Gugten, De Jong and Palkovits, 1976), although the levels of
.
noradrenaline in the n. tractus solitnrius are somewhat lower than reported by Versteeg et al., (1976) and could be related to strain differences in the rats. It is known from histofluorescence studies that the anterior hypothalamic-preoptic nuclei are innervated by catecholaminergic nerve terminals (Fuxe, 1965; Jacobowitz and Palkovits, 1974) whereas in the rnedu~l~obio~g~~a~the n. tractus soiitarius at the A2 level contains both catecholaminergic cell bodies and nerve terminals, and the other medullary nuclei studied are innervated only sparsely by catecholaminergic nerve terminals (Fuxe, 1965; Palkovits and Jacobowitz, 1974). In most of these nuclei noradrenaline is the predominant catecholamine. In the n. inters&i& striue terrn~~~alis, however, dopamine and noradrenaline levels are similar and this is consistent with the innervation of the dorsal part of this nucleus by the dopaminergic nigrostriatal pathway (Lindvall and Bjiirklund, 1974). In the nuclei studied from both the anterior hypothalamic~preoptic area and the med~llu obiungar~ acute administration of a-methyldopa produced a marked reduction in levels of the endogenous catecholamines, noradrenaline and dopamine, and an accumulation of both g-methyldopamine and a-methylnoradrenaline. The depletion of the endogenous amines has been attributed to inhibition of the rate-
Acute r-methyldopa
and amines in hypothalamic
limiting enzyme, tyrosine hydroxylase (Dominic and Moore, 197 I ; Uretsky. Chase and Lorenzo, 1975). In addition, competition between dopamine and r-methyldopamine for vesicular uptake sites and for the dopamine /?-hydroxylase enzyme may influence the depletion of noradrenaline (Conway et al., 1978). A striking feature of the results observed in all nuclei was the rapid accumulation of the metabolite, r-methyldopamine. to quite high levels considering the normal catecholamine content of these areas. The amount accumulated was such that at 4 hr combined levels of dopamine and r-methyldopamine were significantly elevated above control levels of dopamine in all but one of the anterior hypothalamic-preoptic nuclei. This accumulation was much higher than was found in the hypothalamus as a whole. It seems likely, therefore. that a-methyldopamine accumulates preferentially in these anterior hypothalamic-preoptic nuclei. The accumulation of r-methylnoradrenaline in the anterior hypothalamic-preoptic and medullary nuclei was also such that relatively high levels were reached when compared to the normal endogenous noradrenaline concentration of these areas. These results contrast with those obtained when both the hypothalamus and medulla ohlongara as a whole were studied. Here the levels of noradrenaline plus r-methylnoradrenaline were significantly reduced from control levels of noradrenaline both 4 and 12 hr after a-methyldopa injection (Conway rt a/., 1978). These differences may indicate that r-methylnoradrenaline is more readily accumulated in the nuclei investigated in this study (those implicated as cardiovascular inhibitory centres) than in other nuclei within the same uptake of regions. This could reflect differential x-methyldopa into nerie terminals in different neuronal pathways, or alternatively, an increased turnover of the z-methylamines in these areas. It is of interest that in the anterior hypothalamicpreoptic nuclei, levels of sr-methylnoradrenaline accumulated slowly after r-methyldopa administration and were higher at 12 hr than at 4 hr. By contrast, in the medulla ohlongata maximal levels of a-methylnoradrenaline were recorded in the n. tractus solitarius and n. tractus spinalis nerve trigemini at 4 hr after s-methyldopa administration and at this time the combined levels of noradrenaline and a-methvlnoradrenaline were significantly above control noradrenaline levels in the n. tractus solitarius. The more rapid accumulation and disappearance of r-methylr&adrenaline in the medullarv nuclei comnared to the anterior hypothalamic nuclei suggests that LYmethylnoradrenaline turnover (and possibly also noradrenaline turnover) is more rapid in these areas even though dopamine fi-hydroxylase activity is lower in the brain stem than in the hypothalamus @hen and Ganong, 1976). No studies of-dopamine /?-hydroxylase activity in these medullary nuclei are available and it may be that within the nuclei activity of this enzyme is higher than in the anterior hypothalamic
and mcdullary
nuclei
285
nuclei. It should also be noted that the time for maxima1 accumulation of r-methylnoradrenaline in the n. tractus solitarius and n. tractus spinalis nervi trigemini coincided with the time of the maximal hypotensive effect of acutely injected a-methyldopa (Day et al., 1973). Although the very high levels of r-methyldopamine that accumulated at 4 hr. particularly in the anterior hypothalamic-preoptic nuclei, may also suggest a role for this metabolite in the blood pressure response, this seems unlikely since inhibition of dopamine /?-hydroxylase in the central nervous system abolishes the acute antihypertensive effect of systemitally administered r-methyldopa (Day et al., 1973). r-Methylnoradrenaline injected directly into these anterior hypothalamic nuclei and the n. tractus solitarius in the rat produces a fall in blood pressure (see Introduction). Thus, the fall in blood pressure after systemic administration of a-methyldopa may be due to the accumulation and subsequent release of this amine from noradrenergic nerve terminals in these nuclei. The most likely site for this action would appear to be in the n. tractus solirarius since the accumulation and disappearance of r-methylnoradrenaline in this nucleus correlates with the time course of the antihypertensive effect of a-methyldopa in rats (Day et a/., 1973). By contrast, in the other medullary nuclei studied and in the anterior hypothalamic nuclei. r-methylnoradrenaline levels continued to rise or plateaued during recovery of the blood pressure. It is possible though. that freshly synthesized r-methylnoradrenaline in the anterior hypothalamic-preoptic nuclei contributes to the hypotensive effect since this amine is more potent than noradrenaline on anterior hypothalamic r-adrenoreceptors (De Jong et al., 1975). Thus. the hypotensive effect of a-methyldopa may reflect the combined actions of z-methylnoradrenaline in both the anterior hypothalamic-preoptic region and the n. tractus solitarius. Acknow/rdyc,rll~nts-This work was supported by grants from the National Health and Medical Research Council and National Heart Foundation of Australia. REFERENCES
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Zandberg. P. and De Jong. W. (1977). r-Methylnoradrenaline-induced hypotension in the nucleus tractus solitarii of the rat; a localization study. Neuropharmacology 16: 219-222.
Zigmond, R. E. and Ben-Ari. Y. (1976). A simple method for the serial sectioning of fresh brain and the removal of identifiable nuclei from stained sections for biochemical analysis. J. Neurochem. 26: 1285-1287.
8 Noovmhrr
1978)
Summary-E-Methyldopa administration to rats (200 mg kg- ’ s.c.) produced a marked reduction in levels of noradrenaljne and dopamine in anterior hypo~alamic-pre~ptic nuclei implicated in catecholamine-mediated cardiovascular-inhibitory functions. Similar effects were observed in medullary nuclei, including the n. iractus solifmius which has been postulated as a site for the hypotensive action of r-methyldopa. The metabolite, %-methyldopamine, accumulated rapidly and 4 hr after drug administration levels were higher in most nuclei than in either the wdulla ohlonyara or hypothalamus as a whole, suggesting greater uptake of r-methyldopa into these specific areas. This was also reflected by higher levels of I-methylnoradrenaline in the nuclei than in the gross regions. The time course of accumulation and disappearance of z-methylnoradrenaline in the n. WUCIUS sofitarius differed from that in the anterior hypothalamic-preoptic nuclei and more closely correlated with the reported time course of the hypotensive effect of x-methyldopa. However. the action of this metabolite in both areas may contribute to the fall in blood pressure.
L-c+Methyidopa is now thought to produce a major part of its antihy~rtensive effect through an action on the central nervous system (Henning, 1975). In rats, the fall in blood pressure following acute administration of this drug is probably due to the action of a metabolite, r-methylnoradrenaline, acting on central a-adrenoreceptors (Day, Roach and Whiting, 1973; Finch and Haeusier, 1973). Ahhough the exact involvement of catecholaminecontaining neuron systems in the central control of blood pressure is not completely understood, evidence in recent years suggests that the anterior hypothalamic region and the A2 region of the nucleus (n.) tractus solitarius in the ~~d~l~a o~~o~gafa (see Dahlstrijm and Fuxe, 1964; Fuxe, 1965) may be involved in catecholamine-mediated cardiovascular-inhibitory functions (Struyker Boudier, Smeets, Brouwer and Van Rossum, 1974; De Jong, Nijkamp and Bohus, 1975; Philippu and Schartner, 1976; Nathan and Reis, 1977; Palkovits, De Jong, Zandberg, Verstecg, Van Der Gugten and Leranth, 1977). Further evidence indicates that both these regions respond to local injection of x-methylnoradrenaline by producing a fall in blood pressure in anaesthetized rats (De Jong et al., 1975; Struyker Boudier, Smeets, Brouwer and Van Rossum, 1975: Zandberg and De Jong, 1977). However, these studies are limited in that they take no account of the possible additive or synergistic effects of this amine acting simultaneously at different levels of the brain, Key words: z-methyldopa, r-methylnoradrenaline, ~-methyldopamine, cardiovascular inhibitory centres. 279
In a previous study it has been shown that acute administration of ~-methyldopa feads to a widespread depletion of dopamine and noradrenaline in ail areas of the rat brain, and to the replacement of these amines with r-methylnoradrenaline and n-methyldopamine (Conway, Louis and Jarrott, 1978). In the present study the effect of acute administration of ~-methyldopa on catecholamine levels has been examined in specific nuclei from the anterior hypothalamic-preoptic region which have been implicated as sites of the hypotensive action of rx-methylnoradrenaline (Struyker Boudier et ul., 1975) and in the n. tractus solitarius and adjacent nuclei in the medulla oblo~gata. The aim of this study was to characterize the effects of this drug in the catecholaminergic pathways which are most likely to be involved in mediating its hypotensive action,
METHODS Groups of male Sprague-Dawley rats (1%200g) were injected with 200 mg kg-’ of a-methyldopa as a single dose and killed 4 and 12 hr later. The drug was ground finely and administered subcutaneously as a suspension in 0.9% sodium chloride [W/V). One group of rats treated with vehicle was included as a control. The killing times were based on a previous study in different brain regions which indicated that maximal effects on the levels of dopamine and r-methyldopamine occurred at 4 hr whereas maximal effects on the levels of noradrenaline and r-methylnoradrenaline were seen at approximately 12 hr.
280
E. L.
CONWAY.
W. J. Louis and B.
Nuclei were dissected freehand from 4OOpm thick coronal sections of fresh brain tissue from the treated rats as described by Zigmond and Ben Ari (1976). Ideutific~~tion of the hypothalamic nuclei was based on the rat brain atlas of KGnig and Klippel (1970) and the descriptions of Palkovits, Brownstein, Saavedra and Axelrod (1974b). In the rnrdulla oblongam. identification of the nuclei was also based on the descriptions of Palkovits, Brownstein and Saavedra f1974a). Only the rostra1 sections astern in length) of the n. tractus spirxdis nervi tri~e}i~ilti and the dorsal and ventral n. reticularis medulla ohlongatae were removed. The n. tractus soliturius refers to that part of the nucleus just under the fourth ventricle surface and in the vicinity of the area postrema (A2 region). Nuclei from 3 rats were pooled and placed in small round-bottomed glass test-tubes (2.5 x OSem internal diameter, Samco). A plunger moulded to fit these tubes from Silastic J/RTV (Dow Corning Corp., Michigan, U.S.A.) was used to homogenize the tissue samples in 100~1 of 0.1 mol I- ’ perchloric acid. An aliquot of the homogenate (520~1) was taken for protein determination (Lowry, Rosebrough, Farr and Randall, 1951) and the remainder was centrifuged for 10,000 g min and the supernatant stored at -20°C until assayed. Noradrenaline. dopamine. x-methylnoradrenaline and ~-methyldo~mine were determined in the tissue supernatants by the following m~i~cation of the enzyme radiochemicai method of Van Der Gugten, Palkovits, Wijnen and Versteeg (1976). The endogenous amines and their x-methylated analogues were O-methylated by incubation with the substrate mixture containing catechol-Q-methyltransferase and S-adenosyl-L-~methyl-3H) methionine as described by these authors. The labeled 0-(3H)-methylated products were then extracted and separated by paper chromatography using the procedure of Cuello, Hiley and Iversen (1973). Under these conditions there was no significant cross-contamination between the methoxylated amines (Rf; normetanephr~ne~ 0.23; 3-OmethyI-~-methylnoradrenaline, 0.31; 3-methoxytyramine, 0.55 and 3-0-methyl-l-methyldopamine, 0.68). Tissue concentrations used in these determinations were low and inhibition by cellular components was negligible. Catecholamine levels in the samples were therefore determined by reference to external standards. This assay detected all four amines with a sensitivity of S@lOOpg. Data have been expressed as pg catecholamine/pg protein (mean + S.E.M.). Significance of the difference between values of control and treated groups was determined using student’s t--test. A P value of less than 0.05 was considered significant.
statistically
Materials
S-adenosyl-t_-(methyl-3H)-methionine, sp. act. 5 to 10 Ci mmol- ‘, was obtained from The Radiochemical Centre. Amersham. L-~-Methyldopa, (S, +)-a-methyl-
JARROTT
dopamine, I-(4-hydroxy-3-methoxyphenyl)-2-amino propane hydrochloride (3-O-methyl-a-methyldopamine) and erythro( ~)-~I-aminoethyl~4-hydroxy-3methoxy-benzyl alcohol hydrochloride (3-O-methyl-~methylnoradrenaline) were donated by Merck, Sharp & Dohme Research Lab., NJ. Initial supplies of 3-0methyl-z-methylnoradrenaline were donated by Dr A. A. Manian, Psychopharmacology Research Branch, National Institute of Mental Health, U.S.A. ~-Methylnoradrenaline hydrochloride was purchased from Calbiochem (U.S.A.). All additional chemicals used were reagent grade. RESULTS
fltrT&or ir~pothulffnric-preoptic
nuclei
Catecholamine levels in the anterior ~~pot~u~~~i7jcnuclei studied are shown in Table 1. Noradrenaline levels were highest in the n. prropticus medialis and the n. interstitialis striae terminalis. Dopamine levels were considerably lower than noradrenaline levels in all nuclei except the n. j~7terstjtiu~j.~ preoptic
striae
tert~l~~alis.
After renaline of 12 hr reduced
acute administration of g-methyldopa, noradlevels declined in all nuclei over a period (Fig. 1). At this time noradrenaline levels were to 16”/, of control values in the n. interstiticrlis striae ter~~i~u~is and to approximately 20% in the other nuclei. Levels of r-methytnoradrenaline increased in inverse proportion to the decrease in noradrenaline. At I2 hr the levels of this amine were approximately twice those at 4 hr and in all but one of the nuclei combined levels of z-methylnoradrenaline and noradrenaline were signifi~ntly elevated above control levels of noradrenaline alone (Table I). These results contrast with those obtained in the hypothalamus as a whole where the combined levels of noradrenaline plus r-methylnoradrenaline at 12 hr were significantly reduced from control noradrenaline levels (Table I). The hypothalamic nuclei studied comprise approximately 16% of the whole hypothalamus. Dopamine levels also declined after a-methyldopa injection although the low initial levels of this amine in many of the nuclei made it impossible to determine the maximum depletion (Fig. 1). r-Methyldopamine. on the other hand, accumulated rapidly in all nuclei and 4 hr after ~-methyldopa administration levels of this amine were at least 10 times control dopamine levels in all nuclei except the n. interstitialis striae terminalis (1.5 times) and the n. preopticus lateralis (2.3 times). The combined levels of dopamine and x-methyldopamine at 4 hr were significantly elevated above control levels of dopamine in all nuclei except the n. interstitialis striae terminalis (Table I), and ranged from 250% of control values in the n. preopticus lateralis to 1.560% in the n. anterior (hypothalami). This accumulation was in most cases higher than that found in the hypothalmus as a whole (3COo/,) (Table 1).
2.8 & 0.94 3.1 rf: 0.42
2.1 + 0.21 6.5 + 1.41 4.4 k 0.83 4.0 + i.71
15.4 f 3.08 13.7 f 2.48
12hr
__ _ _ _
__ _
_ r. - _” -
I
-
-~
.
.
_
,,,
_
_
_
_
.
_
5.7 + 0.38
.
-
.
-.
-
_.
,,
1.9 + 1.20 1.4 + 0.70 5.4 + 2.45
N.D. N.D.
4 hr
37.8 + 2.36 28.7 & 3.38
34.7 + 3.84 23.6 & 1.27 39.9 * 3.71
8.8 & 0.34
r-MDA (pg/fig protein) 0.4 I: 0.03
DA
(200 mg kg-’ s.c.)
DA-- dopamine: z-MDA-r-methyldopamine.
2.4 ? 0.96 2.9 + 0.61
3.3 f 0.15 10.2 _+ 3.25 27.5 f 8.08
2.7 f 0.20
r-MNA-rr-methylnoradrenaline:
21.8 rtr 1.69 21.0 + 1.33
36.7 + 3.76 24.5 t_ 1.68 30.5 & 7.03
administration
Control DA (pg/lrg protein)
nuclei 0. 4 and 12 hr after r-methyldopa
r-MNA (pg/~g protein)
14.8 k 0.74
NA
27.6 + 3.50 19.8 + 2.60 25.3 f 1.70
Control NA (pg/flg protein)
levels in anterior hypothalamic-preoptic
N.D. indicates no amine was detected (~0.2 pg/pg protein). n = 5: NA-noradrenaline:
n. Anterior (hypothalami) n. Latercrlis (hypothalami)
n. Preopticus lateralis n. Ittferstitjff1i.s srriue tertnit~alis
Hypothalamus n. Preopticus media/is
Area
Table 1. Catecholamine
282
E. L.
0
CONWAY,
6
4
45L
W. J. LOUISand B. JARROTT
0
12
r
porn
6
I
30
.12
PO1
0
6
I
12
Time, hr
40. ..:
. .:’ b
I 20-
T : ....
___._-L...
:
..,‘X._ . ,,*-J
....
"'....*J
iKL*<* .:' ,, ,..' ,s
o
NA a-MNA
_.---
DA .._..._..........&M,,A * P (0.05
,.c' 0
4
Time,
6
$2
hr
Fig. 1. Effect of acute administration of z-methyldopa (200 mg kg-’ s.c.) on levels of noradrenaline (NA). dopamine (DA). r-methylnoradrenaline (a-MNA) and a-methyldopamine (z-MDA) in nuclei from the anterior hypothalamic-preoptic region of the rat brain. Control levels of noradrenaline (4) and dopamine (#) are shown just adjacent to the vertical axis. Values represent means & S.E.M. (n = 5). Asterisks indicate a significant reduction in catecholamine levels compared with control values. ha-n. anterior (hypothalami); hl-n. lateralis (hypothalami); porn-n. preopricus rdialis; pol-n. preopticus /atrralis: St-n. interstitialis srriae terminalis.
At 12 hr after a-methyldopa injection, the levels of r-methyldopamine had dropped markedly in all the nuclei, representing less than 40% of the levels at 4 hr (Fig. I ). Nuclei
in the medulla
oblongata
Catecholamine levels in the medullary nuclei studied are shown in Table 2. The catecholamine levels of these nuclei were lower, than those of the anterior hypothalamic-preoptic nuclei. Noradrenaline levels were highest in the reticular nuclei in which no dopamine could be detected. Only trace amounts of dopamine were detected in the n. tractus solitarius (0.4 pg/pg protein) and the n. tractus spinalis nervi triyrnlini (0.2 pg/pg protein). Four hours following the administration of a single dose of r-methyldopa, noradrenaline levels were significantly lower than control values in all nuclei
except the n. tractus solitarius, and after 12 hr the levels were even lower or not detectable in all 4 nuclei (Fig. 2). a-Methylnoradrenaline had accumulated in all nuclei 4 hr following a-methyldopa injection in quantities at least equal to control noradrenaline levels. However, only in the n. tractus solitarius was the combined level of noradrenaline and a-methylnoradrenaline significantly elevated above control levels of noradrenaline (Table 2). The results in these nuclei, which constitute approximately 3% of the m&//a ohlongata, contrast with those obtained in the whole region where combined levels of noradrenaline and r-methylnoradrenaline at 4 hr were reduced from control noradrenaline levels (Table 2). In both the n. tractus solitarius and the n. tractus spinalis nervi trigemini a-methylnoradrenaline levels were lower at 12 hr than at 4 hr. whereas in the n. reticularis medulla ohlonga-
trace
1.1 * 0.67 2.0 2 0.88
8.0 i: 2.35
7.0 & 1.43
N.D. 3.4 5 1.28
5.5 If: 0.43 4.7 -f. 0.48 2.6 & 0.55
N.D.
9.0 +_0.8 I
x-MNA--r-mcthylnoradrenaline:
N.D.
N.D. trace trace
9.9 & 1.17
12.6 4 2.39
4.0 4 0.35 17.1 & 1.55 11.0 4 1.73
x-MDA (pg/pg protein1
4 hr
~-MDA-~-m~thyldopam~n~.
N.D.
N.D.
N.D. N.D. N.D.
DA
(2OOmg kg-’ s.c.)
DA-dopamine;
Control
8.0 5 1.45
2.8 & 0.05 10.4 & 1.01 7.2 _t- 1.74
r-MNA @g/p6 protein)
4hr
N.D. indicates no amine was detected (co.2 pg/hcg protein): II = 5: NA---noradrenaline:
Area
NA
levels in meduilary nuclei 0 and 4 hr after ~-methy~do~a administration
Control NA (pg/bcg protein)
Table 2. Catecholamine
284
E. L. CONWAY, W. J. Louis and B. JARUOTT
18.
JT
nts
.:’ ‘..... ,: ......
1
0
Time, -
hr
.------
4 12
Time,
hr
NA ci-MNA
P(O.05
*
Fig. 2. Eflect of acute administration of n-methyldopa (200mg kg-’ s.c.) on levels of noradrenaline (NA). r-methylnoradrenatine (c(-MNA) and a-methvidouamine &MDA) in nuclei fram the rriedrrlla ob~ongat~of ihe rat brain. n&-n. rraclus solifarius: tsv-n. tractus spinalis newi tri~~~f?lini;nrd-n. r~t~e~larist17~~uliu obiongata~(dorsal); nrv-n. reticu~ari~~?edul~aoblo~~atue(ventral). See Figure 1 legend for further details. I
tar (dorsal) they were unchanged and in the n. reticufuris t~edu~~uub~o~gu~~e(ventral) they had increased (Fig. 2). Although little or no endogenous dopamine could be detected in the medullary nuclei, 4 hr after a-methyldopa administration high levels of the metabolite, a-methyldopamine, were present in all 4 nuclei (Fig. 2). As in the hypothalamic nuclei, the accumulation of this amine was more pronounced in the medullary nuclei than in the gross medulla ohlongata region (Table 2). At 12 hr the levels of a-methyldopamine in all nuclei had fallen quite markedly.
DISCUSSION In this study the effect of acute administration of r-methyldopa has been examined on levels of both the endogenous catecholamines, dopamine and noradrenaline, and their a-methylated analogues in those nuclei from the anterior hypothalamic-preoptjc region and the medulla ob~o~guru believed to be involved in cardiovascular control. The values reported here for levels of noradrenaline and dopamine in these nuclei do not differ markedly from those of other reports (Palkovits et al., 1974b; Versteeg, Van Der Gugten, De Jong and Palkovits, 1976), although the levels of
.
noradrenaline in the n. tractus solitnrius are somewhat lower than reported by Versteeg et al., (1976) and could be related to strain differences in the rats. It is known from histofluorescence studies that the anterior hypothalamic-preoptic nuclei are innervated by catecholaminergic nerve terminals (Fuxe, 1965; Jacobowitz and Palkovits, 1974) whereas in the rnedu~l~obio~g~~a~the n. tractus soiitarius at the A2 level contains both catecholaminergic cell bodies and nerve terminals, and the other medullary nuclei studied are innervated only sparsely by catecholaminergic nerve terminals (Fuxe, 1965; Palkovits and Jacobowitz, 1974). In most of these nuclei noradrenaline is the predominant catecholamine. In the n. inters&i& striue terrn~~~alis, however, dopamine and noradrenaline levels are similar and this is consistent with the innervation of the dorsal part of this nucleus by the dopaminergic nigrostriatal pathway (Lindvall and Bjiirklund, 1974). In the nuclei studied from both the anterior hypothalamic~preoptic area and the med~llu obiungar~ acute administration of a-methyldopa produced a marked reduction in levels of the endogenous catecholamines, noradrenaline and dopamine, and an accumulation of both g-methyldopamine and a-methylnoradrenaline. The depletion of the endogenous amines has been attributed to inhibition of the rate-
Acute r-methyldopa
and amines in hypothalamic
limiting enzyme, tyrosine hydroxylase (Dominic and Moore, 197 I ; Uretsky. Chase and Lorenzo, 1975). In addition, competition between dopamine and r-methyldopamine for vesicular uptake sites and for the dopamine /?-hydroxylase enzyme may influence the depletion of noradrenaline (Conway et al., 1978). A striking feature of the results observed in all nuclei was the rapid accumulation of the metabolite, r-methyldopamine. to quite high levels considering the normal catecholamine content of these areas. The amount accumulated was such that at 4 hr combined levels of dopamine and r-methyldopamine were significantly elevated above control levels of dopamine in all but one of the anterior hypothalamic-preoptic nuclei. This accumulation was much higher than was found in the hypothalamus as a whole. It seems likely, therefore. that a-methyldopamine accumulates preferentially in these anterior hypothalamic-preoptic nuclei. The accumulation of r-methylnoradrenaline in the anterior hypothalamic-preoptic and medullary nuclei was also such that relatively high levels were reached when compared to the normal endogenous noradrenaline concentration of these areas. These results contrast with those obtained when both the hypothalamus and medulla ohlongara as a whole were studied. Here the levels of noradrenaline plus r-methylnoradrenaline were significantly reduced from control levels of noradrenaline both 4 and 12 hr after a-methyldopa injection (Conway rt a/., 1978). These differences may indicate that r-methylnoradrenaline is more readily accumulated in the nuclei investigated in this study (those implicated as cardiovascular inhibitory centres) than in other nuclei within the same uptake of regions. This could reflect differential x-methyldopa into nerie terminals in different neuronal pathways, or alternatively, an increased turnover of the z-methylamines in these areas. It is of interest that in the anterior hypothalamicpreoptic nuclei, levels of sr-methylnoradrenaline accumulated slowly after r-methyldopa administration and were higher at 12 hr than at 4 hr. By contrast, in the medulla ohlongata maximal levels of a-methylnoradrenaline were recorded in the n. tractus solitarius and n. tractus spinalis nerve trigemini at 4 hr after s-methyldopa administration and at this time the combined levels of noradrenaline and a-methvlnoradrenaline were significantly above control noradrenaline levels in the n. tractus solitarius. The more rapid accumulation and disappearance of r-methylr&adrenaline in the medullarv nuclei comnared to the anterior hypothalamic nuclei suggests that LYmethylnoradrenaline turnover (and possibly also noradrenaline turnover) is more rapid in these areas even though dopamine fi-hydroxylase activity is lower in the brain stem than in the hypothalamus @hen and Ganong, 1976). No studies of-dopamine /?-hydroxylase activity in these medullary nuclei are available and it may be that within the nuclei activity of this enzyme is higher than in the anterior hypothalamic
and mcdullary
nuclei
285
nuclei. It should also be noted that the time for maxima1 accumulation of r-methylnoradrenaline in the n. tractus solitarius and n. tractus spinalis nervi trigemini coincided with the time of the maximal hypotensive effect of acutely injected a-methyldopa (Day et al., 1973). Although the very high levels of r-methyldopamine that accumulated at 4 hr. particularly in the anterior hypothalamic-preoptic nuclei, may also suggest a role for this metabolite in the blood pressure response, this seems unlikely since inhibition of dopamine /?-hydroxylase in the central nervous system abolishes the acute antihypertensive effect of systemitally administered r-methyldopa (Day et al., 1973). r-Methylnoradrenaline injected directly into these anterior hypothalamic nuclei and the n. tractus solitarius in the rat produces a fall in blood pressure (see Introduction). Thus, the fall in blood pressure after systemic administration of a-methyldopa may be due to the accumulation and subsequent release of this amine from noradrenergic nerve terminals in these nuclei. The most likely site for this action would appear to be in the n. tractus solirarius since the accumulation and disappearance of r-methylnoradrenaline in this nucleus correlates with the time course of the antihypertensive effect of a-methyldopa in rats (Day et a/., 1973). By contrast, in the other medullary nuclei studied and in the anterior hypothalamic nuclei. r-methylnoradrenaline levels continued to rise or plateaued during recovery of the blood pressure. It is possible though. that freshly synthesized r-methylnoradrenaline in the anterior hypothalamic-preoptic nuclei contributes to the hypotensive effect since this amine is more potent than noradrenaline on anterior hypothalamic r-adrenoreceptors (De Jong et al., 1975). Thus. the hypotensive effect of a-methyldopa may reflect the combined actions of z-methylnoradrenaline in both the anterior hypothalamic-preoptic region and the n. tractus solitarius. Acknow/rdyc,rll~nts-This work was supported by grants from the National Health and Medical Research Council and National Heart Foundation of Australia. REFERENCES
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