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Catecholamine synthesizing enzymes in brain stem and hypothalamus during the development of renovascular hypertension
Brain Research, 163 (1979) 277-288 © Elsevier/North-HollandBiomedicalPress
277
CATECHOLAMINE SYNTHESIZING ENZYMES IN BRAIN STEM AND HYPOTHALAMUS DURING THE DEVELOPMENT OF RENOVASCULAR HYPERTENSION
MARGARET A. PETTY* and JOHN L. REID Department of Clinical Pharmacology, Royal Postgraduate Medical School, London (Great Britain)
(Accepted June 29th, 1978)
SUMMARY The activities of tyrosine hydroxylase (TH) and phenylethanolamine N-methyltransferase (PNMT) have been measured in brain stem and hypothalamic nuclei during the development of renovascular hypertension. TH activity fell at 72 h in the posterior hypothalamic and peri- and paraventricular nuclei of the hypothalamus, but had returned to control levels by 7 days. PNMT activity was raised in the nucleus of the solitary tract at 7 days and was also elevated in the nucleus of the solitary tract, parahypoglossal nucleus, locus coeruleus and cerebellar cortex at 4 weeks. No change in PNMT was noted in hypothalamus. It appears from these results that both central noradrenergic and adrenergic pathways are involved in the development of this model of experimental hypertension.
INTRODUCTION Fluorescence histochemical studies have revealed a system of catecholaminergic pathways in the rat brain4,~,tl, t2. The highest concentrations of catecholamines were found in certain hypothalamic2z and brain stem nuclei. Several of these catecholaminergic containing areas are now considered to play important roles in the control of arterial pressureZ, a as well as being involved in the pathogenesis of some models of experimental hypertension2,1a, 20. Recent developments in biochemical micromethods, together with a precise microdissection technique 21, have enabled the measurement of catecholamine concentration and the activity of catecholamine synthesizing enzymes in these brain stem and hypothalamic nuclei during the development of renovascular hypertension. The onekidney Goldblatt model of renovascular hypertension has been used, since central * Medical ResearchCouncilPh.D. student
278 catecholaminergic neurones have been shown to be involved in the rise in blood pressure. Destruction of central catecholaminergic neurones with intracisternally administered 6-hydroxydopamine prevents the development of the hypertension 6. The rise in blood pressure is accompanied by an increase in plasma noradrenaline 6, and catecholamine turnover s, and the centrally acting antihypertensive agent clonidine lowers the pressureL In this model, concomitant with the rise in systolic blood pressure in the early stages of the hypertension, there is a reduction in the noradrenaline concentration in some of these selected nuclei 26. This initial fall in tissue noradrenaline may reflect either increased or decreased neuronal activity, hence tyrosine hydroxylase (TH) activity has been estimated in the same areas. TH is the rate-limiting enzyme in the synthesis of noradrenaline 1~, and therefore provides an index of catecholamine synthesis. Phenylethanolamine-N-methyltransferase (PNMT), the adrenaline synthesizing enzyme, has been demonstrated, by an immunohistochemical procedure, in cell body areas of the medulla oblongata and in nerve terminal regions in brain stem, spinal cord and hypothalamus 14. PNMT is thought to be a marker for adrenaline in the brain 3°, its regional distribution being consistent 32. Bolme et al. have postulated that such adrenaline-containing neurones might play a important role in vasomotor and respiratory control mechanismsL We have measured PNMT activity in these brain regions during the development of renovascular hypertension, since any change in PNMT activity might indicate the involvement of brain adrenaline in hypertension. METHOD AND MATERIALS Hypertension was induced in male Wistar rats (Anglia Laboratories, Huntingdon, Great Britain), weighing between 150 and 200 g, by the application of a silver clip of internal diameter 0.23 mm to the left renal artery with contralateral nephrectomy. Sham operations, carried out in litter mates, involved the application of a nonconstricting clip to the left renal artery together with contralateral nephrectomy. The rise in systolic blood pressure was measured by the indirect method of tail plethysmography using a programmed electrosphygomanometer (Narco Biosystems, Houston, Texas). The rats were killed by decapitation 72 h, 7 days and 4 weeks after operation and the brains were immediately removed and frozen with dry ice. The hypothalamic and brain stem nuclei were removed from 300 #m thick frozen serial sections of the brain, according to the method of Palkovits 21 with special stainless steel needles. For the assay of TH a modification of the method of Shiman et al. al was used. Tissue samples from two rats were pooled and homogenized in 75 #l of ice cold 0.5 M Tris.HC1 buffer, pH 6, containing 0.2 % Triton X-100 in a 0.1 ml microhomogenizer (Jencons, Hemel Hempstead, Great Britain). The homogenate was centrifuged at 2000 rpm (1250 × g) for 10 min at 4 °C and 3/~1 of the supernatant removed for protein estimation 16. TH activity was measured in a 40 #1 aliquot of supernatant. In preliminary experiments it was confirmed that the amount of tissue assayed (40-100 #g) was linearly related to TH activity. To the aliquot a 10/~1 solution containing 2.5
279 #1 [14C]tyrosine (specific activity 513 mCi/mmole, Radiochemical Centre, Amersham), 2.5/~1 (pH 5.8) sodium acetate, 1.0 #1 catalase (Boehringer Mannheim, Lewes, Great Britain), 2.5 #1 of a solution of 5.92 mg/ml DL-6-methyl-5,6,7,8-tetrahydropterine HC1 ,(Calbiochem, San Diego, Calif.) in 0.56 M mercaptoethanol (BDH Chemicals Ltd., Poole, Great Britain) and 1.5 /A distilled water, was added. The samples were incubated for 20 min at 37 °C. The rate of [14C]DOPA formation was linear up to 30 min of incubation. One ml of 0.4 N perchloric acid containing 10/zg of carrier LDOPA (Sigma, London) was used to terminate the reaction. After centrifugation at 2000 rpm (1250 × g) for 10 min, 6.5 ml of a solution containing 5 ml o f 2 ~ disodium EDTA and 1.5 ml of 0.35 M potassium phosphate was added. The pH of each sample was titrated to 8.6 with sodium hydroxide solution and then applied to an activated aluminium oxide column (Woelm neutral activity, grade 1). The [14C]DOPA produced during the reaction was eluted with 4 ml of 0.5 N acetic acid and after the addition of scintillation cocktail (Instagel, Packard, Reading, Great Britain) was counted in a Packard Tri-Carb liquid scintillation spectrometer with a counting efficiency of 71 ~o. [14C]DOPA recovery from alumina was calculated as a percentage of a known amount of [14C]DOPA (Radiochemical Centre, Amersham), recovered from each experiment. It was consistently 60--70~o. TH activity was expressed as nmoles of [14C]DOPA formed/rag protein/h and included a correction for column recovery. Samples which contained tissue and all constituents of the reaction mixture but were not incubated (zero time blanks) were used for blank values throughout. For PNMT estimation a modification of the method of Saavedra et al. 27 was used. Brain tissue was homogenized in 100 #1, 0.2 M Tris.HC1 buffer, pH 8.6 containing 0.2 ~ v/v Triton X-100. After the removal of 10 #1 homogenate for protein estimation, the samples were centrifuged at 2000 rpm (1250 × g) for 10 rain at 4 °C. Fifty/~1 supernatant was placed in ice cold 13 ml stoppered centrifuge tubes and after the addition of 92.5 #1 distilled water, 5 #1 of a 1 mg/ml solution of phenylethanolamine in water (Regis Chemical Co., Franklin, Chicago, Ill.) and 2.5 #1 [3H]methyl-Sadenosylmethionine (specific activity 12.6 Ci/mmole, Radiochemical Centre, Amersham), the samples were incubated for 30 min at 37 °C. Blanks were prepared by omitting the substrate from the incubation mixture and gave similar values to blanks obtained using tryptamine or phenylethylamine which are not substrates for this reaction ~7. The reaction was terminated by the addition of 0.5 ml of 0.5 M borate (pH 10) and 6 ml of 3 ~ amyl alcohol in toluene was added. After a back extraction, which involved 0.1 N hydrochloric acid, l M borate (pH 12.5) and further 3 ~ amyl alcohol in toluene, scintillation cocktail (Instagel) was added a/id the product counted in a Packard Tri-Carb liquid scintillation spectrometer. PNMT activity was calculated as pmoles N-methylphenylethanolamineformed~mg proi~in/h. In some instances partially purified bovine adrenal PNMT was used as an internal standard. The experimental values for the nuclei involved ranged from 2 to 15 times blank (100-200 cpm), using both these assays. All values used in analysis represent the average of 10 determinations of each nucleus. The significance of differences between the nuclei of hypertensive animals as compared to shams were calculated using a Student's t-test for unpaired data 8. There
280 was a marked interindividual variation in levels of enzyme activity between different groups of rats 17. In all experiments, litter mates were sham operated, sacrificed and assayed at the same time for comparison with hypertensive groups. RESULTS
Table I shows the rise in systolic blood pressure and Table II the body weights, in different groups of rats 72 h, 7 days and 4 weeks after operation. At 72 h after operation the blood pressure had risen from 126 -+- 4 to 154 ~k 5 mm Hg. The pressure continued to rise, being 167 -4- 4 on the 7th day and 213 ± 7 mm Hg at 4 weeks. At none of the time intervals examined during this development stage was there any difference between body weights of the clipped animals as compared to shams (Table II).
Tyrosine hydroxylase In sham-operated rats, T H activity ranged from 1315.17 ± 175.11 nmole/mg protein/h in the locus coeruteus to 38.3 ± 5.53 nmole/mg protein/h in cerebellar cortex (Table III) 72 h after operation; although there was an increase in the TH activity of certain brain stem nuclei, especially in the nucleus of the solitary tract and the parahypoglossal nucleus, this did not achieve statistical significance when tested by Student's t-test. In the hypothalamus, however, there was a reduction in TH activity TABLE I
Systolic blood pressure (ram Hg) in groups of hypertensive rats and sham-operated controls (mean ± S.E.) There are 20 animals in each group at each time interval.
Hypertensive rats Sham-operated rats
Before operation
After operation 72 h
7 days
4 weeks
126 d- 4 127 ± 2
154 i 5** 130 ± 2
167 4- 4** 134 zk 3
213 5- 7** 133 4- 2
** P < 0.01 when hypertensive and sham operated groups compared by unpaired Student's t-test. TABLE II
Body weights (g) of groups of hypertensive rats with sham-operated controls (mean i S.E.) during the development of renovascular hypertension There are 20 rats in each group at each time interval. There were no significant differences between hypertensive and sham-operated groups when compared by unpaired Student's t-test
Hypertensive rats Sham-operated rats
Before operation
After operation 72 h
7 days
4 weeks
188 ± 2 194 ± 2
200 ± 4 207 zk 3
206 ± 3 211 ± 2
365 ± 8 366 zk 6
281
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Fig. 1. Mean (~_ S.E.) tyrosine hydroxylase activity in brain stem and hypothalamic regions in onekidney renovascular hypertensive rats, expressed as a percentage of sham-operated controls. Groups of rats (n = 10) were sacrificed at 72 h after renal artery clipping or sham operation. Hypertensive and sham-operated groups were compared by Student's unpaired t-test.
which was significant in the periventricular (P < 0.05), paraventricular (P < 0.01) and posterior hypothalamic nuclei (P < 0.05), levels being reduced to 68.67 4- 2.6 ~, 59.58 4- 2.28 ~ and 60.39 4- 3.28 ~ respectively of sham-operated litter mates (Fig. 1). Seven days after operation the TH activity of the hypertensive animals had returned to control values (Table III). Four weeks after operation again there was no significant difference between the hypertensive and control groups in any region investigated, with the exception of the parietal cortex where levels were reduced to 60.29 ~ 2.08~o of shams (P < 0.01).
Phenylethanolamine- N-methyltransferase In sham rats PNMT activity ranged from 68.5 4- 15.84 pmoles N-methylphenylethanolamine/mg protein/h in locus coeruleus to 6.94 4- 0.86 pmoles/mg protein/h in cerebellar cortex (Table IV). At 72 h after operation there was no detectable change in PNMT levels in any of the regions investigated. Seven days after operation the activity was significantly elevated to 197.7 4- 12.26 ~ (P < 0.05) in the
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Fig. 2. Mean (4- S.E.) phenylethanolamine N-methyltransferase activity in brain stem areas in onekidney renovascular hypertensive rats, expressed as a percentage of sham-operated controls. Groups of rats (n = 10) were sacrificed 7 days and 4 weeks after renal artery clipping or sham operation. Hypertensive and sham-operated groups were compared by Student's unpaired t-test.
nucleus of the solitary tract (Fig. 2), and at 4 weeks after operation PNMT activity was increased in the majority of brain stem regions investigated in the hypertensive group as compared to shams. At this time the level in the nucleus of the solitary tract was increased further to 327.29 ~ 31.1 ~ (P < 0.01) and was also significantly raised in the parahypoglossal nucleus (P < 0.05), the locus coeruleus (P < 0.05) and cerebellar cortex (P < 0.05), being 184.71 ~. 11.78~, 240.61 ± 8.56~o and 223.18 -t- 10.94~o of shams, respectively. At no time interval investigated did the PNMT activity in hypothalamic nuclei of the hypertensive animals differ significantly from controls. DISCUSSION
In this series of experiments we have investigated further the changes in noradrenaline concentrations which occur in discrete areas of brain during the development of the one-kidney Goldblatt model of renovascular hypertension. Decreases in noradrenaline levels were found in selected regions of both brain stem and hypothalamus 72 h after renal artery constriction. In this study we have measured both TH and PNMT activities in these same nuclei during the development of
285 hypertension, at 72 h, 7 days and 4 weeks after operation, when the systolic blood pressure was still rising. During this period there was no difference between the body weights of the hypertensive and sham group, suggesting that malignant hypertension had not developed. Although 72 h after operation TH activity was not significantly changed in the nucleus of the solitary tract and parahypoglossal nucleus of the brain stem, in the hypothalamus there was a significant reduction in the periventricular, paraventricular and posterior hypothalamic nuclei. At this time no change in PNMT activity was observed. Seven days after operation there was no difference between the TH levels of the hypertensive and sham group. PNMT activity was now significantly raised in the nucleus of the solitary tract of the brain stem. At 4 weeks the only change in TH activity observed was in the parietal cortex where a reduction occurred. PNMT levels were elevated in several brain stem regions at this time, being significant in the nucleus of the solitary tract, parahypoglossal nucleus, locus coeruleus and cerebellar cortex. No change was observed in any of the hypothalamic nuclei. The reduction in noradrenaline concentration is, therefore, accompanied by a decrease in the activity of TH in certain hypothalamic nuclei which are sites of noradrenergic nerve terminals. In some brain stem regions in which are located noradrenergic cell bodies as well as terminals, there was a non-significant elevation in enzyme activity. These changes may be due to a localized increase in noradrenaline turnover, resulting in a reduction in amine concentration and enzyme activity in the nerve endings and a compensatory increase in enzyme synthesis in the cell bodies. Alternatively, a reduction in noradrenaline turnover may lead to a temporary decrease in enzyme activity due to end product inhibition, observed in the hypothalamic areas. Perhaps direct determination of enzyme protein by immunotitration would assist in confirming these observations. Previous studies have been carried out in whole brain or large areas of brain, and it is unlikely that they could reflect changes in the activity of catecholaminergic neurones in the small areas of brain that participate in cardiovascular control. TH activity has been found to be elevated in whole hypothalamus of 3-week-old spontaneously hypertensive rats 19, although in 4-month-old animals no change has been observed in whole brain stems. In DOCA salt hypertensive rats a reduction in the noradrenaline content has been reported in whole medulla oblongata 20. The hypertension induced in these adult rats has been shown to be accompanied by a rise in whole brain TH z6. In isolated brain nuclei decreases in noradrenaline levels have been found in some hypothalamic nuclei and surrounding regions (areas that receive innervation from the locus coeruleus) in young and adult spontaneously hypertensive rats 29. Recently, Wijnen et al. 33 reported no significant changes in noradrenaline content of several brain nuclei in renovascular hypertension at 8 days and 3.5 weeks after renal artery constriction without contralateral nephrectomy (two-kidney Goldblatt model). These results are, at these times, in agreement with our previous observations 2~, although it is unlikely that the one- and two-kidney models are comparable at these times 7.
286 The elevation of P N M T activity at 7 days and 4 weeks in brain stem regions suggests that adrenaline formation in these neurones is increased in this model of renovascular hypertension. Similar changes in P N M T activity in brain stem areas have been observed in 4-week-old spontaneously hypertensive rats and rats with established D O C A salt hypertension 2s. The administration of P N M T inhibitors results in a decrease in pressure to normal levels 30. Although adrenaline was increased in some brain stem areas of spontaneously hypertensive rats 2s, the amine was unchanged in the D O C A salt and two-kidney renovascular models 33. It appears from these results that both central noradrenergic and adrenergic pathways are involved in the development of renovascular hypertension, although they present a different time course. The exact roles of the two catecholamines are still speculative: the changes may be primary causes of the hypertension, or secondary results of the raised pressure. It is possible that the noradrenaline changes reflect attempts of the arterial baroreflex arc to compensate for the hypertension. Since they occur in the early stages of the hypertension and are short-lived, they may possibly coincide with the mechanism responsible for resetting of the baroreceptor reflex. There was no significant difference from controls 7 days after operation. The largest changes occurred in nuclei participating in the baroreflex arc. The first synapse in the arc and the origin of the secondary neurones is the nucleus of the solitary tract. The fibres of the secondary neurones terminate in various medullary nuclei, such as the lateral reticular nucleus, as well as terminating in hypothalamic nuclei by means of direct or multisynaptic pathways, some of which synapse in the locus coeruleus. Descending pathways to the spinal cord appear to follow the same route ~3. The changes in P N M T activity are much slower in onset and more persistent, becoming obvious 7 days after operation and affecting most of the brain stem nuclei by 4 weeks. The time course of the rise in the nucleus of the solitary tract at 7 days, with later increases in other areas, in particular the locus coeruleus, suggests activation of an adrenergic pathway, having its cell bodies in the nucleus of the solitary tract and projecting to the locus coeruleus. Such a pathway, which has been proposed on the basis of histochemical data 14, may have a functional role in cardiovascular regulation. Boime et al. 1 have postulated that the brain adrenaline system is vasodepressor but this view conflicts with the observation that P N M T inhibitors lower the blood pressure of spontaneously hypertensive rats 30. However, these drugs may have other pharmacological actions which possibly contribute to the fall in blood pressure 9'4. Further studies will be required to define the role of brain adrenaline and these pathways in cardiovascular control.
REFERENCES 1 Bolme, P., Corrodi, M., Fuxe, K., Hbkfelt, T., Lidbrink, P. and Goldstein, M., Possible involvement of central adrenaline neurones in vasomotor and respiratory control. Studies with clonidine and its interactions with piperoxane and yohimbine, Europ. J. Pharmacol., 28 (1974) 89-94. 2 Chalmers, J., Brain amines and models of experimental hypertension, Circular. Res., 36 (1975) 469-480. 3 Cochran, E. and Cox, G., Experimental Designs, Wiley, New York, 1953.
287 4 Dahlstr~Sm, A. and Fuxe, K., Evidence for the existence of mon0amine-containing neurones in the central nervous system. I. Denervation of monoamines in the cell bodies of brain stem neurones, Acta physiol, scand., 62, Suppl. 232 (1964) 1-55~ 5 Dahlstr6m, A. and Fuxe, K., Evidence for the existence 0f monoamine-containing neurones in the central nervous system. II. Experimentally induced changes in the intraneuronal amine levels of bulbospinal neurone systems, ActaphysioL scand., 64, Suppl. 247 (1964) 1-36. 6 Dargie, H., Franklin, S. and Reid, J., Plasma noradrenaline concentrations in experimental renovascular hypertension in the rat, Clin. Sci. molec. Med., 52 (1977) 447-483. 7 Dargie, H., Reid, J. and Dollery, C. T., Central and peripheral mechanisms in renovascular hypertension in the rat, Fed. Proc., 36 (1977) 1155. 8 De Champlain, J. and van Ameringen, M., Regulation of blood pressure by sympathetic nerve fibres and adrenal medulla in normotensive and hypertensive rats, Circulat. Res., 31 (1972) 617-628. 9 De Champlain, J. and van Ameringen, M., Role of sympathetic fibres and of adrenal medulla in the maintenance of cardiovascular homeostasis in normotensive and hypertensive rats. In E. Usdin and S. Snyder (Eds.), Frontiers in Catecholamine Research, Pergamon Press, Oxford, 1973, p. 859. 10 Frohlich, E., The adrenergic nervous system and hypertension: state of the art, Mayo Clin. Proc., 52 (1977) 361-368. 11 Fuxe, K., Evidence for the existence of monoamine-containing neurones in the central nervous system. III. The monoamine nerve terminal, Z. Zellforsch., 65 (1965) 573. 12 Fuxe, K., Evidence for the existence of monoamine-containing neurones in the central nervous system. IV. Distribution of monoamine nerve terminals in t he central nervous system, Actaphysiol. scand., 64, Suppl. 247 (1965) 38-85. 13 Haeusler, G., Finch, L. and Thoenen, H., Central adrenergic neurones and the initiation and development of experimental hypertension, Experientia (Basel), 28 (1972) 1200-1203. 14 H6kfelt, T., Fuxe, K., Goldstein, M. and Johansson, O., Immunohistochemical evidence for the existence of adrenaline neurones in the rat brain, Brain Research, 66 (1974) 235-251. 15 Levitt, M., Spector, S., Sjoerdsma, A. and Udenfriend, S., Elucidation of the rate-limiting step in norepinephrine biosynthesis in the perfused guinea-pig heart, J. Pharmacol. exp. Ther., 148 (1965) 1-8. 16 Lowry, O., Rosebrough, N., Farr, A. and Randal, R., Protein measurement with Folin phenol reagent, J. bioL Chem., 193 (1951) 265-275. 17 Lovenberg, W., Yamabe, H., de Jong, W. and Hansen, C. T., Genetic variation ofthe catecholamine biosynthetic enzyme activities in various strains of rats including the spontaneously hypertensive rat. In E. Usdin and S. Snyder (Eds.), Frontiers in Catecholamine Research, Pergamon Press, Oxford, 1973, pp. 891-895. 18 Nagatsu, T., Mizutani, K., Umezawa, H., Matsuzaki, M. and Takeuchi, T., Adrenal tyrosine hydroxylase and dopamine beta hydroxylase in spontaneously hypertensive rats, Nature (Lond.), 230 (1971) 381-382. 19 Nagatsu, T., Kato, T., Numata, Y., Ikuta, K., Umezawa, H. and Takechi, T., Catecholamine synthesising enzymes in hypertension. In E. Usdin, R. Kuetnansky and I. Kopin (Eds.), Catecholamines and Stress, Pergamon Press, Oxford, 1975, pp. 251-255. 20 Nakamura, K., Gerold, M. and Thoenen, H., Experimental hypertension of the rat: reciprocal changes of norepinephrine turnover in heart and brainstem, Naunyn-Schmiedeberg's Arch. exp. Path. Pharmak., 268 (1971) 125-139. 21 Palkovits, M., Isolated removal of hypothalamic or other brain nuclei of the rat, Brain Research, 59 (1973) 449-450. 22 Palkovits, M., Brownstein, M., Saavedra, J. and Axelrod, J., Norepinephrine and dopamine content of hypothalamic nuclei of the rat, Brain Research, 77 (1974) 137-149. 23 Palkovits, M. and Zaborszky, L., Neuroanatomy of central cardiovascular control. Nucleus tractus solitarii: afferent and efferent neuronal connections in relation to the baroreceptor reflex arc. In W. de Jong, A. P. Provoost and A. P. Shapiro (Eds.), Hypertension andBrain Mechanisms, Progr. Brain Res., Vol. 47, Elsevier, Amsterdam, 1977, pp. 9-34. 24 Pendleton, R., Kaiser, C., Gessner, G., Finlay, E. and Rreen, H., Studies on S K + F 7698; an inhibitor of phenylethanolamine N-methyltransferase, J. Pharmacol. exp. ther., 190(1974) 551-562. 25 Petty, M. and Reid, J., Changes in noradrenaline concentration in brain stem and hypothalamic nuclei during the development of renovascular hypertension, Brain Research, 136 (1977) 376-380. 26 Rylett, P., Dean, H. and Lee, M., Brain tyrosine hydroxylase activity and systolic blood pressure in rats treated with deoxycorticosterone and salt or angiotensin, J. Pharm. Pharmacol., 28 (1976) 559-562.
288 27 Saavedra, .I., Palkovits, M., Brownstein, M. and Axelrod, J., Localisation of phenylethanolamine N-methyltransferase in the rat brain nuclei, Nature (Lond.), 248 (1974) 695-696. 28 Saavedra, J., Grobecker, H. and Axelrod, J., Adrenaline forming enzyme in brainstem: elevation in genetic and experimental hypertension, Science, 191 (1976) 483-484. 29 Saavedra, J., Grobecker, H. and Axelrod, J., Biochemical and morphologic study of catecholamine metabolism in spontaneously hypertensive rats, Mayo Clin. Proc., 52 (1977) 391-394. 30 Sauter, A., Lew, J., Baba, Y. and Goldstein, M., Effect of phenylethanolamineN-methyltransferase and dopamine-beta hydroxylase inhibition on epinephrine levels in the brain, Life Sci., 21 (1977) 261-266. 31 Shiman, R., Akino, M. and Kaufman, S., Solubilization and partial purification of tyrosine hydroxylase from bovine adrenal medulla, J. Biol. Chem., 246 (1971) 1330-1340. 32 Van de Gugten, J., Palkovits, M., Wijnen, H. and Versteeg, D., Regional distribution of adrenaline in rat brain, Brain Research, 107 (1976) 171-175. 33 Wijnen,H., Versteeg, D., Palkovits, M. and De Jong, W., Increased adrenaline content ofindividual nuclei of the hypothalamus and the medulla oblongata of genetically hypertensive rats, Brain Research, 135 (1977) 186-189.
277
CATECHOLAMINE SYNTHESIZING ENZYMES IN BRAIN STEM AND HYPOTHALAMUS DURING THE DEVELOPMENT OF RENOVASCULAR HYPERTENSION
MARGARET A. PETTY* and JOHN L. REID Department of Clinical Pharmacology, Royal Postgraduate Medical School, London (Great Britain)
(Accepted June 29th, 1978)
SUMMARY The activities of tyrosine hydroxylase (TH) and phenylethanolamine N-methyltransferase (PNMT) have been measured in brain stem and hypothalamic nuclei during the development of renovascular hypertension. TH activity fell at 72 h in the posterior hypothalamic and peri- and paraventricular nuclei of the hypothalamus, but had returned to control levels by 7 days. PNMT activity was raised in the nucleus of the solitary tract at 7 days and was also elevated in the nucleus of the solitary tract, parahypoglossal nucleus, locus coeruleus and cerebellar cortex at 4 weeks. No change in PNMT was noted in hypothalamus. It appears from these results that both central noradrenergic and adrenergic pathways are involved in the development of this model of experimental hypertension.
INTRODUCTION Fluorescence histochemical studies have revealed a system of catecholaminergic pathways in the rat brain4,~,tl, t2. The highest concentrations of catecholamines were found in certain hypothalamic2z and brain stem nuclei. Several of these catecholaminergic containing areas are now considered to play important roles in the control of arterial pressureZ, a as well as being involved in the pathogenesis of some models of experimental hypertension2,1a, 20. Recent developments in biochemical micromethods, together with a precise microdissection technique 21, have enabled the measurement of catecholamine concentration and the activity of catecholamine synthesizing enzymes in these brain stem and hypothalamic nuclei during the development of renovascular hypertension. The onekidney Goldblatt model of renovascular hypertension has been used, since central * Medical ResearchCouncilPh.D. student
278 catecholaminergic neurones have been shown to be involved in the rise in blood pressure. Destruction of central catecholaminergic neurones with intracisternally administered 6-hydroxydopamine prevents the development of the hypertension 6. The rise in blood pressure is accompanied by an increase in plasma noradrenaline 6, and catecholamine turnover s, and the centrally acting antihypertensive agent clonidine lowers the pressureL In this model, concomitant with the rise in systolic blood pressure in the early stages of the hypertension, there is a reduction in the noradrenaline concentration in some of these selected nuclei 26. This initial fall in tissue noradrenaline may reflect either increased or decreased neuronal activity, hence tyrosine hydroxylase (TH) activity has been estimated in the same areas. TH is the rate-limiting enzyme in the synthesis of noradrenaline 1~, and therefore provides an index of catecholamine synthesis. Phenylethanolamine-N-methyltransferase (PNMT), the adrenaline synthesizing enzyme, has been demonstrated, by an immunohistochemical procedure, in cell body areas of the medulla oblongata and in nerve terminal regions in brain stem, spinal cord and hypothalamus 14. PNMT is thought to be a marker for adrenaline in the brain 3°, its regional distribution being consistent 32. Bolme et al. have postulated that such adrenaline-containing neurones might play a important role in vasomotor and respiratory control mechanismsL We have measured PNMT activity in these brain regions during the development of renovascular hypertension, since any change in PNMT activity might indicate the involvement of brain adrenaline in hypertension. METHOD AND MATERIALS Hypertension was induced in male Wistar rats (Anglia Laboratories, Huntingdon, Great Britain), weighing between 150 and 200 g, by the application of a silver clip of internal diameter 0.23 mm to the left renal artery with contralateral nephrectomy. Sham operations, carried out in litter mates, involved the application of a nonconstricting clip to the left renal artery together with contralateral nephrectomy. The rise in systolic blood pressure was measured by the indirect method of tail plethysmography using a programmed electrosphygomanometer (Narco Biosystems, Houston, Texas). The rats were killed by decapitation 72 h, 7 days and 4 weeks after operation and the brains were immediately removed and frozen with dry ice. The hypothalamic and brain stem nuclei were removed from 300 #m thick frozen serial sections of the brain, according to the method of Palkovits 21 with special stainless steel needles. For the assay of TH a modification of the method of Shiman et al. al was used. Tissue samples from two rats were pooled and homogenized in 75 #l of ice cold 0.5 M Tris.HC1 buffer, pH 6, containing 0.2 % Triton X-100 in a 0.1 ml microhomogenizer (Jencons, Hemel Hempstead, Great Britain). The homogenate was centrifuged at 2000 rpm (1250 × g) for 10 min at 4 °C and 3/~1 of the supernatant removed for protein estimation 16. TH activity was measured in a 40 #1 aliquot of supernatant. In preliminary experiments it was confirmed that the amount of tissue assayed (40-100 #g) was linearly related to TH activity. To the aliquot a 10/~1 solution containing 2.5
279 #1 [14C]tyrosine (specific activity 513 mCi/mmole, Radiochemical Centre, Amersham), 2.5/~1 (pH 5.8) sodium acetate, 1.0 #1 catalase (Boehringer Mannheim, Lewes, Great Britain), 2.5 #1 of a solution of 5.92 mg/ml DL-6-methyl-5,6,7,8-tetrahydropterine HC1 ,(Calbiochem, San Diego, Calif.) in 0.56 M mercaptoethanol (BDH Chemicals Ltd., Poole, Great Britain) and 1.5 /A distilled water, was added. The samples were incubated for 20 min at 37 °C. The rate of [14C]DOPA formation was linear up to 30 min of incubation. One ml of 0.4 N perchloric acid containing 10/zg of carrier LDOPA (Sigma, London) was used to terminate the reaction. After centrifugation at 2000 rpm (1250 × g) for 10 min, 6.5 ml of a solution containing 5 ml o f 2 ~ disodium EDTA and 1.5 ml of 0.35 M potassium phosphate was added. The pH of each sample was titrated to 8.6 with sodium hydroxide solution and then applied to an activated aluminium oxide column (Woelm neutral activity, grade 1). The [14C]DOPA produced during the reaction was eluted with 4 ml of 0.5 N acetic acid and after the addition of scintillation cocktail (Instagel, Packard, Reading, Great Britain) was counted in a Packard Tri-Carb liquid scintillation spectrometer with a counting efficiency of 71 ~o. [14C]DOPA recovery from alumina was calculated as a percentage of a known amount of [14C]DOPA (Radiochemical Centre, Amersham), recovered from each experiment. It was consistently 60--70~o. TH activity was expressed as nmoles of [14C]DOPA formed/rag protein/h and included a correction for column recovery. Samples which contained tissue and all constituents of the reaction mixture but were not incubated (zero time blanks) were used for blank values throughout. For PNMT estimation a modification of the method of Saavedra et al. 27 was used. Brain tissue was homogenized in 100 #1, 0.2 M Tris.HC1 buffer, pH 8.6 containing 0.2 ~ v/v Triton X-100. After the removal of 10 #1 homogenate for protein estimation, the samples were centrifuged at 2000 rpm (1250 × g) for 10 rain at 4 °C. Fifty/~1 supernatant was placed in ice cold 13 ml stoppered centrifuge tubes and after the addition of 92.5 #1 distilled water, 5 #1 of a 1 mg/ml solution of phenylethanolamine in water (Regis Chemical Co., Franklin, Chicago, Ill.) and 2.5 #1 [3H]methyl-Sadenosylmethionine (specific activity 12.6 Ci/mmole, Radiochemical Centre, Amersham), the samples were incubated for 30 min at 37 °C. Blanks were prepared by omitting the substrate from the incubation mixture and gave similar values to blanks obtained using tryptamine or phenylethylamine which are not substrates for this reaction ~7. The reaction was terminated by the addition of 0.5 ml of 0.5 M borate (pH 10) and 6 ml of 3 ~ amyl alcohol in toluene was added. After a back extraction, which involved 0.1 N hydrochloric acid, l M borate (pH 12.5) and further 3 ~ amyl alcohol in toluene, scintillation cocktail (Instagel) was added a/id the product counted in a Packard Tri-Carb liquid scintillation spectrometer. PNMT activity was calculated as pmoles N-methylphenylethanolamineformed~mg proi~in/h. In some instances partially purified bovine adrenal PNMT was used as an internal standard. The experimental values for the nuclei involved ranged from 2 to 15 times blank (100-200 cpm), using both these assays. All values used in analysis represent the average of 10 determinations of each nucleus. The significance of differences between the nuclei of hypertensive animals as compared to shams were calculated using a Student's t-test for unpaired data 8. There
280 was a marked interindividual variation in levels of enzyme activity between different groups of rats 17. In all experiments, litter mates were sham operated, sacrificed and assayed at the same time for comparison with hypertensive groups. RESULTS
Table I shows the rise in systolic blood pressure and Table II the body weights, in different groups of rats 72 h, 7 days and 4 weeks after operation. At 72 h after operation the blood pressure had risen from 126 -+- 4 to 154 ~k 5 mm Hg. The pressure continued to rise, being 167 -4- 4 on the 7th day and 213 ± 7 mm Hg at 4 weeks. At none of the time intervals examined during this development stage was there any difference between body weights of the clipped animals as compared to shams (Table II).
Tyrosine hydroxylase In sham-operated rats, T H activity ranged from 1315.17 ± 175.11 nmole/mg protein/h in the locus coeruteus to 38.3 ± 5.53 nmole/mg protein/h in cerebellar cortex (Table III) 72 h after operation; although there was an increase in the TH activity of certain brain stem nuclei, especially in the nucleus of the solitary tract and the parahypoglossal nucleus, this did not achieve statistical significance when tested by Student's t-test. In the hypothalamus, however, there was a reduction in TH activity TABLE I
Systolic blood pressure (ram Hg) in groups of hypertensive rats and sham-operated controls (mean ± S.E.) There are 20 animals in each group at each time interval.
Hypertensive rats Sham-operated rats
Before operation
After operation 72 h
7 days
4 weeks
126 d- 4 127 ± 2
154 i 5** 130 ± 2
167 4- 4** 134 zk 3
213 5- 7** 133 4- 2
** P < 0.01 when hypertensive and sham operated groups compared by unpaired Student's t-test. TABLE II
Body weights (g) of groups of hypertensive rats with sham-operated controls (mean i S.E.) during the development of renovascular hypertension There are 20 rats in each group at each time interval. There were no significant differences between hypertensive and sham-operated groups when compared by unpaired Student's t-test
Hypertensive rats Sham-operated rats
Before operation
After operation 72 h
7 days
4 weeks
188 ± 2 194 ± 2
200 ± 4 207 zk 3
206 ± 3 211 ± 2
365 ± 8 366 zk 6
281
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Fig. 1. Mean (~_ S.E.) tyrosine hydroxylase activity in brain stem and hypothalamic regions in onekidney renovascular hypertensive rats, expressed as a percentage of sham-operated controls. Groups of rats (n = 10) were sacrificed at 72 h after renal artery clipping or sham operation. Hypertensive and sham-operated groups were compared by Student's unpaired t-test.
which was significant in the periventricular (P < 0.05), paraventricular (P < 0.01) and posterior hypothalamic nuclei (P < 0.05), levels being reduced to 68.67 4- 2.6 ~, 59.58 4- 2.28 ~ and 60.39 4- 3.28 ~ respectively of sham-operated litter mates (Fig. 1). Seven days after operation the TH activity of the hypertensive animals had returned to control values (Table III). Four weeks after operation again there was no significant difference between the hypertensive and control groups in any region investigated, with the exception of the parietal cortex where levels were reduced to 60.29 ~ 2.08~o of shams (P < 0.01).
Phenylethanolamine- N-methyltransferase In sham rats PNMT activity ranged from 68.5 4- 15.84 pmoles N-methylphenylethanolamine/mg protein/h in locus coeruleus to 6.94 4- 0.86 pmoles/mg protein/h in cerebellar cortex (Table IV). At 72 h after operation there was no detectable change in PNMT levels in any of the regions investigated. Seven days after operation the activity was significantly elevated to 197.7 4- 12.26 ~ (P < 0.05) in the
283
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Fig. 2. Mean (4- S.E.) phenylethanolamine N-methyltransferase activity in brain stem areas in onekidney renovascular hypertensive rats, expressed as a percentage of sham-operated controls. Groups of rats (n = 10) were sacrificed 7 days and 4 weeks after renal artery clipping or sham operation. Hypertensive and sham-operated groups were compared by Student's unpaired t-test.
nucleus of the solitary tract (Fig. 2), and at 4 weeks after operation PNMT activity was increased in the majority of brain stem regions investigated in the hypertensive group as compared to shams. At this time the level in the nucleus of the solitary tract was increased further to 327.29 ~ 31.1 ~ (P < 0.01) and was also significantly raised in the parahypoglossal nucleus (P < 0.05), the locus coeruleus (P < 0.05) and cerebellar cortex (P < 0.05), being 184.71 ~. 11.78~, 240.61 ± 8.56~o and 223.18 -t- 10.94~o of shams, respectively. At no time interval investigated did the PNMT activity in hypothalamic nuclei of the hypertensive animals differ significantly from controls. DISCUSSION
In this series of experiments we have investigated further the changes in noradrenaline concentrations which occur in discrete areas of brain during the development of the one-kidney Goldblatt model of renovascular hypertension. Decreases in noradrenaline levels were found in selected regions of both brain stem and hypothalamus 72 h after renal artery constriction. In this study we have measured both TH and PNMT activities in these same nuclei during the development of
285 hypertension, at 72 h, 7 days and 4 weeks after operation, when the systolic blood pressure was still rising. During this period there was no difference between the body weights of the hypertensive and sham group, suggesting that malignant hypertension had not developed. Although 72 h after operation TH activity was not significantly changed in the nucleus of the solitary tract and parahypoglossal nucleus of the brain stem, in the hypothalamus there was a significant reduction in the periventricular, paraventricular and posterior hypothalamic nuclei. At this time no change in PNMT activity was observed. Seven days after operation there was no difference between the TH levels of the hypertensive and sham group. PNMT activity was now significantly raised in the nucleus of the solitary tract of the brain stem. At 4 weeks the only change in TH activity observed was in the parietal cortex where a reduction occurred. PNMT levels were elevated in several brain stem regions at this time, being significant in the nucleus of the solitary tract, parahypoglossal nucleus, locus coeruleus and cerebellar cortex. No change was observed in any of the hypothalamic nuclei. The reduction in noradrenaline concentration is, therefore, accompanied by a decrease in the activity of TH in certain hypothalamic nuclei which are sites of noradrenergic nerve terminals. In some brain stem regions in which are located noradrenergic cell bodies as well as terminals, there was a non-significant elevation in enzyme activity. These changes may be due to a localized increase in noradrenaline turnover, resulting in a reduction in amine concentration and enzyme activity in the nerve endings and a compensatory increase in enzyme synthesis in the cell bodies. Alternatively, a reduction in noradrenaline turnover may lead to a temporary decrease in enzyme activity due to end product inhibition, observed in the hypothalamic areas. Perhaps direct determination of enzyme protein by immunotitration would assist in confirming these observations. Previous studies have been carried out in whole brain or large areas of brain, and it is unlikely that they could reflect changes in the activity of catecholaminergic neurones in the small areas of brain that participate in cardiovascular control. TH activity has been found to be elevated in whole hypothalamus of 3-week-old spontaneously hypertensive rats 19, although in 4-month-old animals no change has been observed in whole brain stems. In DOCA salt hypertensive rats a reduction in the noradrenaline content has been reported in whole medulla oblongata 20. The hypertension induced in these adult rats has been shown to be accompanied by a rise in whole brain TH z6. In isolated brain nuclei decreases in noradrenaline levels have been found in some hypothalamic nuclei and surrounding regions (areas that receive innervation from the locus coeruleus) in young and adult spontaneously hypertensive rats 29. Recently, Wijnen et al. 33 reported no significant changes in noradrenaline content of several brain nuclei in renovascular hypertension at 8 days and 3.5 weeks after renal artery constriction without contralateral nephrectomy (two-kidney Goldblatt model). These results are, at these times, in agreement with our previous observations 2~, although it is unlikely that the one- and two-kidney models are comparable at these times 7.
286 The elevation of P N M T activity at 7 days and 4 weeks in brain stem regions suggests that adrenaline formation in these neurones is increased in this model of renovascular hypertension. Similar changes in P N M T activity in brain stem areas have been observed in 4-week-old spontaneously hypertensive rats and rats with established D O C A salt hypertension 2s. The administration of P N M T inhibitors results in a decrease in pressure to normal levels 30. Although adrenaline was increased in some brain stem areas of spontaneously hypertensive rats 2s, the amine was unchanged in the D O C A salt and two-kidney renovascular models 33. It appears from these results that both central noradrenergic and adrenergic pathways are involved in the development of renovascular hypertension, although they present a different time course. The exact roles of the two catecholamines are still speculative: the changes may be primary causes of the hypertension, or secondary results of the raised pressure. It is possible that the noradrenaline changes reflect attempts of the arterial baroreflex arc to compensate for the hypertension. Since they occur in the early stages of the hypertension and are short-lived, they may possibly coincide with the mechanism responsible for resetting of the baroreceptor reflex. There was no significant difference from controls 7 days after operation. The largest changes occurred in nuclei participating in the baroreflex arc. The first synapse in the arc and the origin of the secondary neurones is the nucleus of the solitary tract. The fibres of the secondary neurones terminate in various medullary nuclei, such as the lateral reticular nucleus, as well as terminating in hypothalamic nuclei by means of direct or multisynaptic pathways, some of which synapse in the locus coeruleus. Descending pathways to the spinal cord appear to follow the same route ~3. The changes in P N M T activity are much slower in onset and more persistent, becoming obvious 7 days after operation and affecting most of the brain stem nuclei by 4 weeks. The time course of the rise in the nucleus of the solitary tract at 7 days, with later increases in other areas, in particular the locus coeruleus, suggests activation of an adrenergic pathway, having its cell bodies in the nucleus of the solitary tract and projecting to the locus coeruleus. Such a pathway, which has been proposed on the basis of histochemical data 14, may have a functional role in cardiovascular regulation. Boime et al. 1 have postulated that the brain adrenaline system is vasodepressor but this view conflicts with the observation that P N M T inhibitors lower the blood pressure of spontaneously hypertensive rats 30. However, these drugs may have other pharmacological actions which possibly contribute to the fall in blood pressure 9'4. Further studies will be required to define the role of brain adrenaline and these pathways in cardiovascular control.
REFERENCES 1 Bolme, P., Corrodi, M., Fuxe, K., Hbkfelt, T., Lidbrink, P. and Goldstein, M., Possible involvement of central adrenaline neurones in vasomotor and respiratory control. Studies with clonidine and its interactions with piperoxane and yohimbine, Europ. J. Pharmacol., 28 (1974) 89-94. 2 Chalmers, J., Brain amines and models of experimental hypertension, Circular. Res., 36 (1975) 469-480. 3 Cochran, E. and Cox, G., Experimental Designs, Wiley, New York, 1953.
287 4 Dahlstr~Sm, A. and Fuxe, K., Evidence for the existence of mon0amine-containing neurones in the central nervous system. I. Denervation of monoamines in the cell bodies of brain stem neurones, Acta physiol, scand., 62, Suppl. 232 (1964) 1-55~ 5 Dahlstr6m, A. and Fuxe, K., Evidence for the existence 0f monoamine-containing neurones in the central nervous system. II. Experimentally induced changes in the intraneuronal amine levels of bulbospinal neurone systems, ActaphysioL scand., 64, Suppl. 247 (1964) 1-36. 6 Dargie, H., Franklin, S. and Reid, J., Plasma noradrenaline concentrations in experimental renovascular hypertension in the rat, Clin. Sci. molec. Med., 52 (1977) 447-483. 7 Dargie, H., Reid, J. and Dollery, C. T., Central and peripheral mechanisms in renovascular hypertension in the rat, Fed. Proc., 36 (1977) 1155. 8 De Champlain, J. and van Ameringen, M., Regulation of blood pressure by sympathetic nerve fibres and adrenal medulla in normotensive and hypertensive rats, Circulat. Res., 31 (1972) 617-628. 9 De Champlain, J. and van Ameringen, M., Role of sympathetic fibres and of adrenal medulla in the maintenance of cardiovascular homeostasis in normotensive and hypertensive rats. In E. Usdin and S. Snyder (Eds.), Frontiers in Catecholamine Research, Pergamon Press, Oxford, 1973, p. 859. 10 Frohlich, E., The adrenergic nervous system and hypertension: state of the art, Mayo Clin. Proc., 52 (1977) 361-368. 11 Fuxe, K., Evidence for the existence of monoamine-containing neurones in the central nervous system. III. The monoamine nerve terminal, Z. Zellforsch., 65 (1965) 573. 12 Fuxe, K., Evidence for the existence of monoamine-containing neurones in the central nervous system. IV. Distribution of monoamine nerve terminals in t he central nervous system, Actaphysiol. scand., 64, Suppl. 247 (1965) 38-85. 13 Haeusler, G., Finch, L. and Thoenen, H., Central adrenergic neurones and the initiation and development of experimental hypertension, Experientia (Basel), 28 (1972) 1200-1203. 14 H6kfelt, T., Fuxe, K., Goldstein, M. and Johansson, O., Immunohistochemical evidence for the existence of adrenaline neurones in the rat brain, Brain Research, 66 (1974) 235-251. 15 Levitt, M., Spector, S., Sjoerdsma, A. and Udenfriend, S., Elucidation of the rate-limiting step in norepinephrine biosynthesis in the perfused guinea-pig heart, J. Pharmacol. exp. Ther., 148 (1965) 1-8. 16 Lowry, O., Rosebrough, N., Farr, A. and Randal, R., Protein measurement with Folin phenol reagent, J. bioL Chem., 193 (1951) 265-275. 17 Lovenberg, W., Yamabe, H., de Jong, W. and Hansen, C. T., Genetic variation ofthe catecholamine biosynthetic enzyme activities in various strains of rats including the spontaneously hypertensive rat. In E. Usdin and S. Snyder (Eds.), Frontiers in Catecholamine Research, Pergamon Press, Oxford, 1973, pp. 891-895. 18 Nagatsu, T., Mizutani, K., Umezawa, H., Matsuzaki, M. and Takeuchi, T., Adrenal tyrosine hydroxylase and dopamine beta hydroxylase in spontaneously hypertensive rats, Nature (Lond.), 230 (1971) 381-382. 19 Nagatsu, T., Kato, T., Numata, Y., Ikuta, K., Umezawa, H. and Takechi, T., Catecholamine synthesising enzymes in hypertension. In E. Usdin, R. Kuetnansky and I. Kopin (Eds.), Catecholamines and Stress, Pergamon Press, Oxford, 1975, pp. 251-255. 20 Nakamura, K., Gerold, M. and Thoenen, H., Experimental hypertension of the rat: reciprocal changes of norepinephrine turnover in heart and brainstem, Naunyn-Schmiedeberg's Arch. exp. Path. Pharmak., 268 (1971) 125-139. 21 Palkovits, M., Isolated removal of hypothalamic or other brain nuclei of the rat, Brain Research, 59 (1973) 449-450. 22 Palkovits, M., Brownstein, M., Saavedra, J. and Axelrod, J., Norepinephrine and dopamine content of hypothalamic nuclei of the rat, Brain Research, 77 (1974) 137-149. 23 Palkovits, M. and Zaborszky, L., Neuroanatomy of central cardiovascular control. Nucleus tractus solitarii: afferent and efferent neuronal connections in relation to the baroreceptor reflex arc. In W. de Jong, A. P. Provoost and A. P. Shapiro (Eds.), Hypertension andBrain Mechanisms, Progr. Brain Res., Vol. 47, Elsevier, Amsterdam, 1977, pp. 9-34. 24 Pendleton, R., Kaiser, C., Gessner, G., Finlay, E. and Rreen, H., Studies on S K + F 7698; an inhibitor of phenylethanolamine N-methyltransferase, J. Pharmacol. exp. ther., 190(1974) 551-562. 25 Petty, M. and Reid, J., Changes in noradrenaline concentration in brain stem and hypothalamic nuclei during the development of renovascular hypertension, Brain Research, 136 (1977) 376-380. 26 Rylett, P., Dean, H. and Lee, M., Brain tyrosine hydroxylase activity and systolic blood pressure in rats treated with deoxycorticosterone and salt or angiotensin, J. Pharm. Pharmacol., 28 (1976) 559-562.
288 27 Saavedra, .I., Palkovits, M., Brownstein, M. and Axelrod, J., Localisation of phenylethanolamine N-methyltransferase in the rat brain nuclei, Nature (Lond.), 248 (1974) 695-696. 28 Saavedra, J., Grobecker, H. and Axelrod, J., Adrenaline forming enzyme in brainstem: elevation in genetic and experimental hypertension, Science, 191 (1976) 483-484. 29 Saavedra, J., Grobecker, H. and Axelrod, J., Biochemical and morphologic study of catecholamine metabolism in spontaneously hypertensive rats, Mayo Clin. Proc., 52 (1977) 391-394. 30 Sauter, A., Lew, J., Baba, Y. and Goldstein, M., Effect of phenylethanolamineN-methyltransferase and dopamine-beta hydroxylase inhibition on epinephrine levels in the brain, Life Sci., 21 (1977) 261-266. 31 Shiman, R., Akino, M. and Kaufman, S., Solubilization and partial purification of tyrosine hydroxylase from bovine adrenal medulla, J. Biol. Chem., 246 (1971) 1330-1340. 32 Van de Gugten, J., Palkovits, M., Wijnen, H. and Versteeg, D., Regional distribution of adrenaline in rat brain, Brain Research, 107 (1976) 171-175. 33 Wijnen,H., Versteeg, D., Palkovits, M. and De Jong, W., Increased adrenaline content ofindividual nuclei of the hypothalamus and the medulla oblongata of genetically hypertensive rats, Brain Research, 135 (1977) 186-189.