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Endogenous and α-methylated catecholamine levels in anterior hypothalamic-preoptic and medullary nuclei in rat brain after chronic α-methyldopa administration
N,,l,r,lphsrlll,,~,,l,,~,~ Vol. IX. pp. 287 to 290 0 Pergemon Press Ltd 1979. Printed in Great Britann
002X-390X 79.0301~02X7$02,CK!n
ENDOGENOUS AND a-METHYLATED CATECHOLAMINE LEVELS IN ANTERIOR HYPOTHALAMIC-PREOPTIC AND MEDULLARY NUCLEI IN RAT BRAIN AFTER CHRONIC a-METHYLDOPA ADMINISTRATION E. L. CONWAY,W. J. LOUIS and B. JARROTT University
of Melbourne, Austin Hospital.
Clinical Pharmacology and Therapeutics Heidelberg, 3084, Victoria, Australia
(Accepted
31 October
Unit.
1978)
Summary-Chronic administration of z-methyldopa to rats (40 mg kg-’ s.c., twice daily for 5 days) produced a profound depletion of noradrenaline and dopamine levels in nuclei from the anterior hypothalamic-preoptic region and the medulla oblongata. such that these amines were virtually undetectable 4 hr after cessation of the drug treatment. At this time r-methylnoradrenaline was the predominant amine present in all the nuclei, at levels always higher than the control noradrenaline levels: much lower levels of z-methyldopamine were also detected. Twenty-four hours after the last injection of z-methyldopa levels of all amines had started to recover, although z-methylnoradrenaline concentrations were still between 50 and 80% of 4 hr values and noradrenaline levels still less than 45% of control values. These data support the contention that r-methylnoradrenaline is involved in mediating the hypotensive effect of r-methyldopa through actions in the anterior hypothalamic-preoptic nuclei and the n. tractus solitarius in the medulla oblongata. The slow recovery of amine levels after cessation of r-methyldopa treatment may explain the persistence of the hypotensive effect of this drug reported in both rats and man.
Despite the fact that clinical usage of the hypotensive agent, L-x-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 following r-methyldopa administration is dependent on the formation of r-methylnoradrenaline centrally and the activation of a-adrenoreceptors by this metabolite is largely derived from acute studies (Day, Roach and Whiting 1973; Finch and Haeusler, 1973). In addition, the possible central sites of action have been suggested by reports that local injections of r-methylnoradrenaline into the anterior hypothalamic-preop tic region, and the nucleus (n.) tractus solitarius produce a fall in blood pressure in anaesthetized rats (De Jong, Nijkamp and Bohus, 1975; Struyker Boudier, Smeets, Brouwer and Van Rossum, 1975). It is possible that similar central mechanisms are involved in producing the hypotensive response during chronic administration of r-methyldopa. Certainly, it has been shown that chronic administration of this drug causes a reduction in spontaneous sympathetic outflow from the central nervous system in both rats and cats (Tauberger and Kuhn, 1971; Baum, Shropshire and Varner, 1972) and in man, administration of a peripheral decarboxylase inhibitor for periods of up to 28 days does not alter the hypotensive action of a-methyldopa (Sjoerdsma, Vendsalu Key words: z-methyldopa. z-methylnoradrenaline. diovascular-inhibitory centres.
car-
287
and Engelman, 1963; Louis, Vajda, McNeil, Doyle and Jarrott, 1978). Chronic administration of !x-methyldopa has also been reported to cause a marked depletion of central noradrenaline levels in the rat with an associated accumulation of large amounts of r-methylnoradrenaline both in whole brain (Brunner, Hedwall, Maitre and Meier, 1967) and in individual brain regions including the hypothalamus and the medulla oblongata (Conway, Louis and Jarrott, 1978a). There are no reports of the chronic effects of r-methyldopa in individual brain nuclei, although previous studies following the acute administration of r-methyldopa have demonstrated depletion of noradrenaline and dopamine with a rapid accumulation of +methyldopamine and a slower accumulation of r-methylnoradrenaline in individual anterior hypothalamic and medullary nuclei (Conway, Louis and Jarrott, 1978b). In this report these investigations have been extended to examine the effects of chronic administration of a-methyldopa on levels of both the endogenous and a-methylated catecholamines in these same nuclei which have been implicated as sites of the acute hypotensive action of this drug. METHODS Groups of male Sprague-Dawley rats (15@250 g) were injected with 40 mg kg-’ of a-methyldopa (subcutaneously as a suspension in 0.9% sodium chloride) twice daily for 5 days. This regime was chosen because it had been previously used in studies of the
n. n. n. n. n.
27.6 f 3.50 19.8 k 2.60 15.4 4 3.08 13.7 & 2.48 25.3 rf: 1.70
3.3 f 10.2 k 2.4 + 2.9 5 27.5 f
NA DA (pg/ng protein) 0.75 3.25 0.96 0.61 8.08
N.D. 0.8* It 0.50 N.D. N.D. N.D.
NA
4 he
51.1 29.2 32.7 25.1 55.3
f 6.16 N.D. + 4.86 3.5* & 1.70 rL:5.54 N.D. & 3.04 ND. & 10.61 6.1* 2 1.88
r-MNA DA (pg/pg protein)
* Significantly reduced from control catecholamine values (P < 0.05; Students t-test). N.D. indicates no amine was detectable (co.2 pg/ng protein).
Preopticus medialis Preopticus lateralis Anterior (hypothalami) Lateralis (hypothalami) Interstitialis striae terminalis
Nucleus
Control NA
4.3 2 1.83 1.0* & 0.40 4.8 + 1.70 4.2* f 1.34 4.4 * 1.30 N.D. 6.0 I: 2.66 l.O* f 0.56 11.4 _+3.36 3.3 i 2.78
r-MDA
34.9 _+ 3.00 23.1 + 4.61 23.3 + 4.84 13.1 + 3.18 38.2 + 3.69
0.9 5 0.40 2.3* & 0.65 N.D. 0.9 f 0.57 10.6 & 2.86
Y-MNA DA (pgllrg protein)
24 hr
0.5 f 0.35 I.1 + 0.36 1.6 f 0.67 3.2 & 2.59 4.4 k 2.57
x-MDA
Table 1. Effect of chronic administration of x-methyldopa (40 mg kg-’ s.c., twice daily for 5 days) on levels of noradrenaline (NA). dopamine (DA), x-methylnoradrenahne (r-MNA) and ~-methylopamine (z-MDA) in nuclei from the anterior hypothalamic-preoptic region. Animals were killed 4 and 24 hr after the final injection. Values represent means + SE. mean (n = 5)
Chronic r-methyldopa
effects of r-methyldopa in whole brain regions (Conway rt al., 1978a) and had been shown to lower blood pressure in the rat (Baum et ai., 1972). One group of rats treated with vehicle alone was included as a control. The killing times were based on a previous study in brain regions which indicated that maximal effects on catecholamine levels occurred at 4 hr and although dopamine and cc-methyldopamine levels had usually recovered by 12 hr, noradrenaline and a-methylnoradrenaline levels had only partially recovered by 24 hr. The dissection of nuclei and assay of catecholamines was carried out as previously described (Conway et al., 1978b). RESULTS Anterior
289
and amines in hypothalamic and medullary nuclei
hypotka~~~c-Freoptic nuciei
The catecholamine levels in the anterior hypothalamic-preoptic nuclei after chronic administration of Ix-methyldopa are shown in Table 1. Levels of noradrenaline in all of the nuclei 4 hr after the last administration of cr-methyldopa were virtually undetectable. At 24 hr the levels had begun to recover but were still less than 25% of controi values in all nuclei. a-Methylnoradrenaline was the predominant amine present 4 hr after the last dose of a-methyldopa and the levels were signi~cantly (P < 0.05) higher than control levels of noradrenaline in all nuclei except the n. preopticus lateralis. At 24 hr the levels of ~-methylnorad~naline had begun to decline in all nuclei but were still between 50 and 80% of 4 hr values. In the n. interstitialis striae terminalis the combined levels of ~-methylnoradrena~ne and noradrenaline were still significantly (P < 0.05) elevated above control levels of noradrenaline at 24 hr. In control nuclei dopamine levels were close to the limits of sensitivity of the assay (0.2 pg amine/pg protein) in the n. anterior .(hypothalami), n. lateralis (hypothalami) and the n. preopticus medialis and it was not possible accurately to determine levels in these nuclei after a-methyldopa administration. How-
ever, in the n. preopticus lateralis and the n. interstitialis striae terminalis dopamine levels were significantly reduced both 4 and 24 hr after the last administration of a-methyldopa. ~-Methyldopamine was present in only small amounts in each of the nuclei and at neither 4 hr nor 24 hr were the combined levels of dopamine and a-methyldopamine significantly elevated above control dooming levels. ~-~ethyldopamine was still present 24 hr after the last administration of a-methyldopa but levels had begun to decline. Nuclei in the meduiia ohiongata
Catecholamine levels in the medullary nuclei following chronic administration of ~-methyldopa are shown in Table 2. There were no detectable levels of noradrenaline 4 hr after the last a-methyldopa injection in any of the nuclei except the n. reticularis medulla oblongatae (ventral) where the levels were 5% of control. At 24 hr noradrenaline levels had begun to recover in all nuclei except the n. reticularis medulla oblongatae (dorsal), but were still less than 45% of control. ~-Methy~oradrena~ne had accumu~ted in at least equal amounts to control levels of noradrenaline 4 hr after the last dose of a-methyldopa. In the n. tractus solitarius and the n. reticularis medulla oblongatae (ventral) at this time, ~-methylnoradrenaline levels were significantly (P < 0.02) higher than control levels of noradrenaline. At 24 hr the levek of ~-methylnoradrenaline had begun to decline slightly but in the n. tractus solitarius the combined level of noradrenaline and ol-methylnoradrenaline was still significantly (P c 0.02) elevated above the control level of noradrenaline. Again only small amounts of ~-methyldopamine were found in all nuclei both 4 and 24 hr after the final administration of a-methyldopa. DISCUSSION The chronic administration of a-methyldopa in a dose of 80 mg kg-’ day-’ has been reported to sub-
Table 2. Effect of chronic administration of z-methyldopa (40 mg kg-’ s.c., twice daily for 5 days) on levels of noradrenaline (NA). a-methylnoradrenaline (r-MNA) and x-methyldopamine (r-MDA) in nuclei from the medulla oblongata. Animals were killed 4 and 24 hr after the final injection. Values represent means + S.E. mean (n = 5) Control Nucleus n. Tractus solitarius n. Tractus spinalis nervi trigemini n. Reticularis medulla oblongatae (dorsal) n. Reticularis medulla oblongatae (ventral)
(p&g
NA protein)
4.7 rt 0.48 2.6 kO.55 8.0 k2.35 7.0 + 1.43
4hr NA
z-MNA (pg/I*cgprotein)
N.D.
15.6 & 3.62 7.8 + 2.60 11.8 It 3.27 .14.8 & 1.68
N.D. ND. 0.32* kO.32
24 hr Y-MDA
2.3 _tl,O4 1.3 +0.73 2.8 50.90 0.8 &OS3
NA
trace trace N.D. I .9* ,058
* Si~ificantly reduced from control cate~hoiam~ne values (P < 0.05; Students r-test). N.D. indicates no amine was detectable (co.2 pg,$g protein).
I-MNA (Pg/,ug protein) 10.7 & 1.35 4.8 kO.63 6.1 & 1.45 12.8 If:2.79
a-MDA
4.2 f 1.79 0.7 i 0.05 2.2 &I.42 I.2 rtO.66
290
E. L. CONWAY.W. J. LOUISand B. JARROTT
stantially lower blood pressure in renal hypertensive rats (Baum et al., 1972). The results presented in this paper demonstrate that administration of this same dose of a-methyldopa (40 mg kg- ‘, twice daily for 5 days) has pronounced effects on catecholamine levels in nuclei in the anterior hypothalamic-preoptic region and the n. tractus solitarius in the medulla oblongata which are areas suggested as sites of the centrally mediated hypotensive action of r-methyldopa (see Introduction). The profound depletion of noradrenaline and the accumulation of high levels of r-methylnoradrenaline observed in all nuclei agree with results in larger brain regions (Conway et al., 1978a) and in the whole brain (Brunner et al., 1967) following similar chronic treatment with a-methyldopa. By comparison with the results reported following the administration of a single injection of 200 mg kg-’ of a-methyldopa (Conway et al.. 1978b) a-methylnoradrenaline levels were always higher and a-methyldopamine levels were very much lower in all nuclei in these chronic studies. The data previously reported in the acute investigations indicated that the formation and disappearance of r-methyldopamine was more rapid than cr-methylnoradrenaline. Thus, the larger accumulation of r-methylnoradrenaline reported here is probably a result of the longer half life of this amine. This longer half-life could reflect a significant intravesicular storage of r-methylnoradrenaline as well as its storage in the soluble compartment of the neuron, since it is not a substrate for monoamine oxidase. It has been suggested that the metabolite. a-methyldopamine, is involved in producing the hypotensive effect of a-methyldopa since intraventricular administration of this amine produced a fall in blood pressure in conscious spontaneously hypertensive rats (Finch, Hersom and Hicks, 1975). Although this response was abolished by cc-adrenoreceptor antagonists and by dopamine fi-hydroxylase inhibition, Waldmeier, Hedwall and Maitre (1975) found a high degree of correlation between the magnitude of the decrease in blood pressure and a-methyldopamine levels after acute systemic administration of s-methyldopa to renal hypertensive rats. They therefore suggested that only x-methylnoradrenaline newly synthesized from a-methyldopamine was involved in producing the fall in blood pressure and thus was dependent on the concentration of the precursor amine. The results reported here indicate that this is unlikely to be the case during chronic administration of a-methyldopa. The levels of a-methyldopamine were very low both 4 and 24 hr after the last administration of a-methyldopa, in contrast to the high levels previously reported after acute administration (Conway et al., 1978b). It would appear that a-methylnoradrenaline, independent of a-methyldopamine. is important in mediating the fall in blood pressure during chronic administration of r-methyldopa. Twenty four hours after the last injection of
a-methyldopa, a-methylnoradrenaline levels were still very high (over 50% of the 4 hr values). It is of interest that Brunner et al. (1967) found very little loss of antihypertensive effect 22 hr after cessation of similar chronic a-methyldopa therapy in rats and likewise, a slow loss of antihypertensive effect has been reported in man (Goldberg, Raftery and Wilkinson, 1977). These data, therefore, also are consistent with the idea that a-methylnoradrenaline is the active amine which mediates the antihypertensive action of a-methyldopa. Acknowlrdg~~mts-This work was supported by grants from the National Health and Medical Research Council and National Heart Foundation of Australia. REFERENCES
Baum, T., Shropshire, A. T. and Varner, L. L. (1972). Contribution of the central nervous system to the action of several antihypertensive agents (methyldopa, hydralazine and guanethidine). J. Pharmac. exp. Thrr. 182: 135-144.
Brunner. H., Hedwall, P. R., Maitre, L. and Meier. M. (1967). Antihypertensive effects of alpha-methylated catecholamine analogues in the rat. Br. J. Pharmac. 30: 108-I 22.
Conway, E. L.. Louis. W. J. and Jarrott. B. (1978a). Acute and chronic administration of r-methyldopa: regional levels of endogenous and z-methylated catecholamines in rat brain. Eur. J. Pharmac. In press. Conway, E. L.. Louis, W. J. and Jarrott. B. (1978b). The effect of acute z-methyldopa administration on catecholamine levels in anterior hypothalamic-preoptic and medullary nuclei in rat brain. NruropharmacoIogy 18: 279-286. Day, M. D., Roach, A. G. and Whiting, R. L. (1973). The mechanism of the antihypertensive action of r-methyldopa in hypertensive rats. Eur. J. Pharmac. 21: 271-280. De Jong, W., Nijkamp, F. P. and Bohus, B. (1975). Role of noradrenaline and serotonin in the central Control of blood pressure in normotensive and spontaneously hypertensive rats. Archs. int. Pharmacodyn. Thlr. 213: 272-284.
Finch, L. and Haeusler, G. (1973). Further evidence for a central hypotensive action of x-methyldopa in both rat and cat. Br. J. Pharmac. 47: 217-228. Finch, L., Hersom. A. and Hicks, P. (1975). Studies on the hypotensive action of r-methyldopamine. Br. J. Pharmac. 54
: 44545 1.
Goldberg, A. D.. Raftery. E. B. and Wilkinson, P. (1977). Blood pressure and heart rate and withdrawal of antihypertensive drugs. Br. Med. J. I: 1243-1246. Louis, W. J.. Vajda, F. J. E.. McNeil, J. J., Doyle. A. E. and Jarrott, B. (1978). The combined use of L-r-methyldopa hydrazine and methyldopa in the treatment of hypertension. Clin. exp. Pharmac. Physiol. Suppl. 4: 17-22. Sjoerdsma. A.. Vendsalu. A. and Engelman, K. (1963). Studies on the metabolism and mechanism of action of methyldopa. Circulation 28: 492-502. Struyker Boudier, H., Smeets, G., Brouwer. G. and Van Rossum. J. M. (1975). Central nervous system r-adrenergic mechanisms and cardiovascular regulation in rats. Archs. int. Pharmacodyn. Thir. 213: 285-293.
Tauberger, G. and Kuhn, P. (1971). Untersuchungen der zentralnerviisen sympathicusdampfenden wirkungen von a-methyl-dopa. Naunyn-Schmiedehergs Arch. exp. Path. Pharmak. 268: 33-43.
Waldmeier, P., Hedwall, P. R. and Maitre, L. (1975). On the role of a-methyldopamine in the antihypertensive effect of a-methyldopa. Naunyn-Schmiedebergs exp. Path. Arch. Pharmak. 289
: 303-3 14.
002X-390X 79.0301~02X7$02,CK!n
ENDOGENOUS AND a-METHYLATED CATECHOLAMINE LEVELS IN ANTERIOR HYPOTHALAMIC-PREOPTIC AND MEDULLARY NUCLEI IN RAT BRAIN AFTER CHRONIC a-METHYLDOPA ADMINISTRATION E. L. CONWAY,W. J. LOUIS and B. JARROTT University
of Melbourne, Austin Hospital.
Clinical Pharmacology and Therapeutics Heidelberg, 3084, Victoria, Australia
(Accepted
31 October
Unit.
1978)
Summary-Chronic administration of z-methyldopa to rats (40 mg kg-’ s.c., twice daily for 5 days) produced a profound depletion of noradrenaline and dopamine levels in nuclei from the anterior hypothalamic-preoptic region and the medulla oblongata. such that these amines were virtually undetectable 4 hr after cessation of the drug treatment. At this time r-methylnoradrenaline was the predominant amine present in all the nuclei, at levels always higher than the control noradrenaline levels: much lower levels of z-methyldopamine were also detected. Twenty-four hours after the last injection of z-methyldopa levels of all amines had started to recover, although z-methylnoradrenaline concentrations were still between 50 and 80% of 4 hr values and noradrenaline levels still less than 45% of control values. These data support the contention that r-methylnoradrenaline is involved in mediating the hypotensive effect of r-methyldopa through actions in the anterior hypothalamic-preoptic nuclei and the n. tractus solitarius in the medulla oblongata. The slow recovery of amine levels after cessation of r-methyldopa treatment may explain the persistence of the hypotensive effect of this drug reported in both rats and man.
Despite the fact that clinical usage of the hypotensive agent, L-x-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 following r-methyldopa administration is dependent on the formation of r-methylnoradrenaline centrally and the activation of a-adrenoreceptors by this metabolite is largely derived from acute studies (Day, Roach and Whiting 1973; Finch and Haeusler, 1973). In addition, the possible central sites of action have been suggested by reports that local injections of r-methylnoradrenaline into the anterior hypothalamic-preop tic region, and the nucleus (n.) tractus solitarius produce a fall in blood pressure in anaesthetized rats (De Jong, Nijkamp and Bohus, 1975; Struyker Boudier, Smeets, Brouwer and Van Rossum, 1975). It is possible that similar central mechanisms are involved in producing the hypotensive response during chronic administration of r-methyldopa. Certainly, it has been shown that chronic administration of this drug causes a reduction in spontaneous sympathetic outflow from the central nervous system in both rats and cats (Tauberger and Kuhn, 1971; Baum, Shropshire and Varner, 1972) and in man, administration of a peripheral decarboxylase inhibitor for periods of up to 28 days does not alter the hypotensive action of a-methyldopa (Sjoerdsma, Vendsalu Key words: z-methyldopa. z-methylnoradrenaline. diovascular-inhibitory centres.
car-
287
and Engelman, 1963; Louis, Vajda, McNeil, Doyle and Jarrott, 1978). Chronic administration of !x-methyldopa has also been reported to cause a marked depletion of central noradrenaline levels in the rat with an associated accumulation of large amounts of r-methylnoradrenaline both in whole brain (Brunner, Hedwall, Maitre and Meier, 1967) and in individual brain regions including the hypothalamus and the medulla oblongata (Conway, Louis and Jarrott, 1978a). There are no reports of the chronic effects of r-methyldopa in individual brain nuclei, although previous studies following the acute administration of r-methyldopa have demonstrated depletion of noradrenaline and dopamine with a rapid accumulation of +methyldopamine and a slower accumulation of r-methylnoradrenaline in individual anterior hypothalamic and medullary nuclei (Conway, Louis and Jarrott, 1978b). In this report these investigations have been extended to examine the effects of chronic administration of a-methyldopa on levels of both the endogenous and a-methylated catecholamines in these same nuclei which have been implicated as sites of the acute hypotensive action of this drug. METHODS Groups of male Sprague-Dawley rats (15@250 g) were injected with 40 mg kg-’ of a-methyldopa (subcutaneously as a suspension in 0.9% sodium chloride) twice daily for 5 days. This regime was chosen because it had been previously used in studies of the
n. n. n. n. n.
27.6 f 3.50 19.8 k 2.60 15.4 4 3.08 13.7 & 2.48 25.3 rf: 1.70
3.3 f 10.2 k 2.4 + 2.9 5 27.5 f
NA DA (pg/ng protein) 0.75 3.25 0.96 0.61 8.08
N.D. 0.8* It 0.50 N.D. N.D. N.D.
NA
4 he
51.1 29.2 32.7 25.1 55.3
f 6.16 N.D. + 4.86 3.5* & 1.70 rL:5.54 N.D. & 3.04 ND. & 10.61 6.1* 2 1.88
r-MNA DA (pg/pg protein)
* Significantly reduced from control catecholamine values (P < 0.05; Students t-test). N.D. indicates no amine was detectable (co.2 pg/ng protein).
Preopticus medialis Preopticus lateralis Anterior (hypothalami) Lateralis (hypothalami) Interstitialis striae terminalis
Nucleus
Control NA
4.3 2 1.83 1.0* & 0.40 4.8 + 1.70 4.2* f 1.34 4.4 * 1.30 N.D. 6.0 I: 2.66 l.O* f 0.56 11.4 _+3.36 3.3 i 2.78
r-MDA
34.9 _+ 3.00 23.1 + 4.61 23.3 + 4.84 13.1 + 3.18 38.2 + 3.69
0.9 5 0.40 2.3* & 0.65 N.D. 0.9 f 0.57 10.6 & 2.86
Y-MNA DA (pgllrg protein)
24 hr
0.5 f 0.35 I.1 + 0.36 1.6 f 0.67 3.2 & 2.59 4.4 k 2.57
x-MDA
Table 1. Effect of chronic administration of x-methyldopa (40 mg kg-’ s.c., twice daily for 5 days) on levels of noradrenaline (NA). dopamine (DA), x-methylnoradrenahne (r-MNA) and ~-methylopamine (z-MDA) in nuclei from the anterior hypothalamic-preoptic region. Animals were killed 4 and 24 hr after the final injection. Values represent means + SE. mean (n = 5)
Chronic r-methyldopa
effects of r-methyldopa in whole brain regions (Conway rt al., 1978a) and had been shown to lower blood pressure in the rat (Baum et ai., 1972). One group of rats treated with vehicle alone was included as a control. The killing times were based on a previous study in brain regions which indicated that maximal effects on catecholamine levels occurred at 4 hr and although dopamine and cc-methyldopamine levels had usually recovered by 12 hr, noradrenaline and a-methylnoradrenaline levels had only partially recovered by 24 hr. The dissection of nuclei and assay of catecholamines was carried out as previously described (Conway et al., 1978b). RESULTS Anterior
289
and amines in hypothalamic and medullary nuclei
hypotka~~~c-Freoptic nuciei
The catecholamine levels in the anterior hypothalamic-preoptic nuclei after chronic administration of Ix-methyldopa are shown in Table 1. Levels of noradrenaline in all of the nuclei 4 hr after the last administration of cr-methyldopa were virtually undetectable. At 24 hr the levels had begun to recover but were still less than 25% of controi values in all nuclei. a-Methylnoradrenaline was the predominant amine present 4 hr after the last dose of a-methyldopa and the levels were signi~cantly (P < 0.05) higher than control levels of noradrenaline in all nuclei except the n. preopticus lateralis. At 24 hr the levels of ~-methylnorad~naline had begun to decline in all nuclei but were still between 50 and 80% of 4 hr values. In the n. interstitialis striae terminalis the combined levels of ~-methylnoradrena~ne and noradrenaline were still significantly (P < 0.05) elevated above control levels of noradrenaline at 24 hr. In control nuclei dopamine levels were close to the limits of sensitivity of the assay (0.2 pg amine/pg protein) in the n. anterior .(hypothalami), n. lateralis (hypothalami) and the n. preopticus medialis and it was not possible accurately to determine levels in these nuclei after a-methyldopa administration. How-
ever, in the n. preopticus lateralis and the n. interstitialis striae terminalis dopamine levels were significantly reduced both 4 and 24 hr after the last administration of a-methyldopa. ~-Methyldopamine was present in only small amounts in each of the nuclei and at neither 4 hr nor 24 hr were the combined levels of dopamine and a-methyldopamine significantly elevated above control dooming levels. ~-~ethyldopamine was still present 24 hr after the last administration of a-methyldopa but levels had begun to decline. Nuclei in the meduiia ohiongata
Catecholamine levels in the medullary nuclei following chronic administration of ~-methyldopa are shown in Table 2. There were no detectable levels of noradrenaline 4 hr after the last a-methyldopa injection in any of the nuclei except the n. reticularis medulla oblongatae (ventral) where the levels were 5% of control. At 24 hr noradrenaline levels had begun to recover in all nuclei except the n. reticularis medulla oblongatae (dorsal), but were still less than 45% of control. ~-Methy~oradrena~ne had accumu~ted in at least equal amounts to control levels of noradrenaline 4 hr after the last dose of a-methyldopa. In the n. tractus solitarius and the n. reticularis medulla oblongatae (ventral) at this time, ~-methylnoradrenaline levels were significantly (P < 0.02) higher than control levels of noradrenaline. At 24 hr the levek of ~-methylnoradrenaline had begun to decline slightly but in the n. tractus solitarius the combined level of noradrenaline and ol-methylnoradrenaline was still significantly (P c 0.02) elevated above the control level of noradrenaline. Again only small amounts of ~-methyldopamine were found in all nuclei both 4 and 24 hr after the final administration of a-methyldopa. DISCUSSION The chronic administration of a-methyldopa in a dose of 80 mg kg-’ day-’ has been reported to sub-
Table 2. Effect of chronic administration of z-methyldopa (40 mg kg-’ s.c., twice daily for 5 days) on levels of noradrenaline (NA). a-methylnoradrenaline (r-MNA) and x-methyldopamine (r-MDA) in nuclei from the medulla oblongata. Animals were killed 4 and 24 hr after the final injection. Values represent means + S.E. mean (n = 5) Control Nucleus n. Tractus solitarius n. Tractus spinalis nervi trigemini n. Reticularis medulla oblongatae (dorsal) n. Reticularis medulla oblongatae (ventral)
(p&g
NA protein)
4.7 rt 0.48 2.6 kO.55 8.0 k2.35 7.0 + 1.43
4hr NA
z-MNA (pg/I*cgprotein)
N.D.
15.6 & 3.62 7.8 + 2.60 11.8 It 3.27 .14.8 & 1.68
N.D. ND. 0.32* kO.32
24 hr Y-MDA
2.3 _tl,O4 1.3 +0.73 2.8 50.90 0.8 &OS3
NA
trace trace N.D. I .9* ,058
* Si~ificantly reduced from control cate~hoiam~ne values (P < 0.05; Students r-test). N.D. indicates no amine was detectable (co.2 pg,$g protein).
I-MNA (Pg/,ug protein) 10.7 & 1.35 4.8 kO.63 6.1 & 1.45 12.8 If:2.79
a-MDA
4.2 f 1.79 0.7 i 0.05 2.2 &I.42 I.2 rtO.66
290
E. L. CONWAY.W. J. LOUISand B. JARROTT
stantially lower blood pressure in renal hypertensive rats (Baum et al., 1972). The results presented in this paper demonstrate that administration of this same dose of a-methyldopa (40 mg kg- ‘, twice daily for 5 days) has pronounced effects on catecholamine levels in nuclei in the anterior hypothalamic-preoptic region and the n. tractus solitarius in the medulla oblongata which are areas suggested as sites of the centrally mediated hypotensive action of r-methyldopa (see Introduction). The profound depletion of noradrenaline and the accumulation of high levels of r-methylnoradrenaline observed in all nuclei agree with results in larger brain regions (Conway et al., 1978a) and in the whole brain (Brunner et al., 1967) following similar chronic treatment with a-methyldopa. By comparison with the results reported following the administration of a single injection of 200 mg kg-’ of a-methyldopa (Conway et al.. 1978b) a-methylnoradrenaline levels were always higher and a-methyldopamine levels were very much lower in all nuclei in these chronic studies. The data previously reported in the acute investigations indicated that the formation and disappearance of r-methyldopamine was more rapid than cr-methylnoradrenaline. Thus, the larger accumulation of r-methylnoradrenaline reported here is probably a result of the longer half life of this amine. This longer half-life could reflect a significant intravesicular storage of r-methylnoradrenaline as well as its storage in the soluble compartment of the neuron, since it is not a substrate for monoamine oxidase. It has been suggested that the metabolite. a-methyldopamine, is involved in producing the hypotensive effect of a-methyldopa since intraventricular administration of this amine produced a fall in blood pressure in conscious spontaneously hypertensive rats (Finch, Hersom and Hicks, 1975). Although this response was abolished by cc-adrenoreceptor antagonists and by dopamine fi-hydroxylase inhibition, Waldmeier, Hedwall and Maitre (1975) found a high degree of correlation between the magnitude of the decrease in blood pressure and a-methyldopamine levels after acute systemic administration of s-methyldopa to renal hypertensive rats. They therefore suggested that only x-methylnoradrenaline newly synthesized from a-methyldopamine was involved in producing the fall in blood pressure and thus was dependent on the concentration of the precursor amine. The results reported here indicate that this is unlikely to be the case during chronic administration of a-methyldopa. The levels of a-methyldopamine were very low both 4 and 24 hr after the last administration of a-methyldopa, in contrast to the high levels previously reported after acute administration (Conway et al., 1978b). It would appear that a-methylnoradrenaline, independent of a-methyldopamine. is important in mediating the fall in blood pressure during chronic administration of r-methyldopa. Twenty four hours after the last injection of
a-methyldopa, a-methylnoradrenaline levels were still very high (over 50% of the 4 hr values). It is of interest that Brunner et al. (1967) found very little loss of antihypertensive effect 22 hr after cessation of similar chronic a-methyldopa therapy in rats and likewise, a slow loss of antihypertensive effect has been reported in man (Goldberg, Raftery and Wilkinson, 1977). These data, therefore, also are consistent with the idea that a-methylnoradrenaline is the active amine which mediates the antihypertensive action of a-methyldopa. Acknowlrdg~~mts-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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