Brain catecholamines during development of DOCA-salt hypertension in rats

Brain Research, 179 (1979) 121-127 © Elsevier/North-HollandBiomedicalPress

121

BRAIN CATECHOLAMINES DURING DEVELOPMENT OF DOCA-SALT HYPERTENSION IN RATS

JUAN M. SAAVEDRA Section on Pharmacology,Laboratory of Clinical Science, NationalInstitute of Mental Health, Bethesda, Md. 20205 (U.S.A.)

(Accepted April 5th, 1979) Key words: brain catecholamines - - DOCA - - salt hypertension

SUMMARY The activity of the adrenaline-forming enzyme, phenylethanolamine-N-methyltransferase (PNMT) and the levels of the catecholamines dopamine, noradrenaline and adrenaline were determined during the development of the DOCA-salt hypertension in selective areas of the rat brain stem and hypothalamus. Increases in PNMT activity were restricted to the A1 area and locus coeruleus after 2 weeks of DOCA-salt treatment and were extended to the A2 area after 9 weeks of treatment. Adrenaline concentrations were higher in these areas only after 9 weeks of treatment. Noradrenaline levels did not change, except in the nucleus tractus commissuralis. Dopamine levels were unchanged at all times and in all structures studied. These results implicate brain stem adrenaline neurons in the central response which occurs during the DOCA-salt experimental hypertension. INTRODUCTION Evidence of the involvement of catecholamines in hypertension has been recently reported 1. A commonly studied form of experimental hypertension, that induced by the combined administration of deoxycorticosterone (DOCA) and sodium chloride to unilaterally nephrectomized rats, resulted in a progressive elevation of the blood pressure4,5. The study of peripheral catecholamines in such a model indicated that both peripheral nerves and the adrenal medulla were involved in the production of the hypertensione,7. Experimental evidence was also presented indicating that the central nervous system was involved in the increased blood pressure and reduced peripheral noradrenaline turnover in DOCA-salt hypertensiona. Additional proof was found when the turnover of noradrenaline was shown to be reduced in the hypothalamus and brain

122 stem~2 and when centrally injected 6-hydroxydopamine resulted in the prevention of the development of the hypertensionL Recently, small groups of neurons containing the adrenaline-forming enzyme, phenylethanolamine N-methyltransferase (PNMT), have been found localized in brain stem areas involved in cardiovascular controlX0,20. These neurons are probably able to form adrenaline from its precursor noradrenaline, since the adrenaline levels in these areas are the highest in brain19, ~z. After 4 weeks of treatment, the PNMT activity in the DOCA-salt rats has been found to be increased in one of the adrenaline-rich areas mentioned, the A1 area ~7. These findings suggested the participation of central PNMT-containing adrenaline-forming neurons in DOCA-salt hypertension, and prompted us to study the activity of PNMT and the levels of adrenaline during the development of the DOCA-salt hypertension in selective areas of the rat brain. METHODS Animals Uninephrectomized and sham-operated male Sprague-Dawley rats, weighing 150 g, were obtained from Zivic-Miller Laboratories, Allison Park, Pa., and kept under diurnal lighting conditions with the lights on from 06.00 to 18.00 h. Hypertension was produced by administration of deoxycorticosterone pivalate (DOCA) (Percorten, Ciba Geigy Corp., Summit, New Jersey) subcutaneously, at the dose of 25 mg (1 ml) per kilogram of body weight, once a week, and a 1 ~ solution of sodium chloride to drink as desired. The systolic blood pressure was measured at weekly intervals by a tail cuff plethysmograph method using a pneumatic pulse transducer and a programmed electrosphygmomanometer (Narco Biosystems, Houston, Texas, Model PE-500). The animals were killed by decapitation, between 09.00 and 12.00h, after 2, 4 and 9 weeks of treatment, and 24 h after the last DOCA injection. Tissue dissection The brains were removed immediately after killing and rapidly frozen on microtome specimen holders on dry ice. Serial sections of 300 #m thickness were cut in a cryostat at a temperature o f - - 1 0 °C. Specific brain stem areas and nuclei were located under a dissecting microscope and dissected by the use of a needle with an internal diameter of 0.5 mm, as described elsewhere s,t3.19,21,~3. For the measurement of phenylethanolamine N-methyltransferase, the regions dissected were the At and A2 areas, area postrema, locus coeruleus, and the median eminence and paraventricular nuclei of the hypothalamus. Catecholamines were measured in these areas as well as in the nucleus commissuralis (NCO) and in the anterior part of the nucleus tractus solitarius (NTS) 19,23. Biochemical assays Phenylethanolamine N-methyltransferase activity was assayed as previously reported ~0.

123 Adrenaline, noradrenaline and dopamine were measured by a modification of recently described isotopic radioenzymatic assaysa,le,19. Results were expressed per mg of proteinlL RESULTS

Phenylethanolamine N-methyltransferase activity Phenylethanolamine N-methyltransferase activity was measured during the development of the hypertension in DOCA-salt rats, in a number of brain stem and hypothalamic nuclei. After two weeks of treatment with DOCA and salt, the experimental group showed statistically significant increases in brain phenylethanolamine N-methyltransferase restricted to the A1 area artd the locus coeruleus. After 4 weeks of treatment, there was a higher increase in blood pressure in the DOCA-salt animals (40 mm Hg when compared to control animals). The changes in brain phenylethanolamine N-methyltransferase were still restricted to the A1 area and locus coeruleus, although a non-significant increase in enzyme activity was noted in the A2 area (Table I). After 9 weeks of treatment, there was a still higher increase in the blood pressure of the treated group (Table I). In the brain, significantly increased phenylethanolamine N-methyltransferase activity was detected in the A~ area, the locus coeruleus, and also in the A2 area. No change in enzyme activity was noted in the hypothalamic areas studied, nucleus paraveatricularis and median eminence, at any time during the DOCA-salt treatment (Table I). TABLE I Phenylethanolamine N-methyltran#brase activity in discrete brain regions during development of the DOCA-salt hypertension in rats Results represent mean 4- S.E.M. for groups of 10 animals assayed individually(pmol/mg protein/h). Weeks of treatment Region

2 Control

Brain stem Alarea A~area Area postrema Locuscoeruleus Hypothalamus Paraventricular nucleus Median eminence Blood pressure§

4 DOCA

9

Control

DOCA

Control

DOCA

55.84-7.9 107.34-21.7" 80.14-8.4 77.34-6.4 29.0-4-1.8 36.84-5.2 18.6±1.7 27.04-1.2"

44.54-6.8 72.0-t-6.0 46.24-4.5 13.84-1.1

60.04-4.1" 88.44-8.3 48.8±4.7 19.5±1.5"

46.44-2.0 49.44-6.8 30.64-3.1 12.74-2.4

97.24-5.5* 77.04-5.4* 31.44-7.5 19.5+1.5"

31.04-2.0 18.14-0.9 118+6

18.9-/-1.2 20.14-1.8 18.04-1.3 20.54-1.7 1224-4 1624-10"

23.24-3.2 16.14-2.1 1384-5"

21.04-4.4 19.14-3.6 16.74-3.2 15.84-1.9 1254-3 1914-16"

§ Blood pressure is expressed in mm Hg. * Statistically significant (P < 0.05) DOCA vs. control group (Student's t-test).

124 C a t e c h o l a m i n e levels

The catecholamines dopamine, noradrenaline and adrenaline were measured at 2, 4 and 9 weeks of treatment with D O C A and salt. In the nucleus paraventricularis of the hypothalamus, and in the median eminence, no differences were detected in the levels of the three catecholamines. In the brain stem, dopamine levels were found to be unchanged in all brain areas studied and at all times during the development of the hypertension. Noradrenaline and adrenaline levels of DOCA-salt hypertensive rats after 2 and 4 weeks of treatment wete similar to those of matched controls in the brain stem areas studied (Tables II and 1II). After 9 weeks of DOCA-salt treatment, however, significant changes were found in restricted brain stern nuclei (Tables II and II1). Noradrenaline levels were only significantly increased in the nucleus commissuralis (Table I1). The changes in adrenaline levels, however, were not only localized to the nucleus commissuralis but also to the area A2, area A1, and the locus coeruleus (Table III). TABLE II Noradrenaline levels in discrete brain stem areas during development o f the DOCA-salt hypertension in rats

Results represent mean ± S.E.M. for groups of 10 animals assayed individually (ng/mg/protein). Region

Weeks o f treatment

2

Areapostrema Nucleuscommissuralis AreaA2 Nucleustractussolitarius AreaA1 Locuscoeruleus

4

9

Control

DOCA

Control

DOCA

45.13:4.1 31.5~4.3 38.7:k4.0 20.4±2.0 17.6:k2.2 28.6±2.1

46.23:3.0 35.1~3.6 39.03:1.1 24.0±2.8 19.75-3.0 27.1:k2.5

43.1:k4.7 46.1:L3.0 30.0~z4.0 39.2±5.1 35.7~3.9 37.0kl.2 19.1~2.5 25.0:L3.9 15.8~:2.2 16.8:E3.0 22.9:f_3.1 26.0-3:4.1

Control

DOCA

42.8±2.2 47.4&-4.5 30.0dc4.1 43.7dc4.3' 37.15-2.5 43.5:k3.7 22.23:2.5 23.63:1.9 16.7~1.2 18.1-~-1.4 29.6±3.6 32.9d~2.8

* Statistically significant (P < 0.05) DOCA-salt vs. control group (Student's t-test). TABLE III Adrenaline levels in discrete brain stem areas during development o f the DOCA-salt hypertension in rats

Results represent mean J: S.E.M. for groups of 10 animals assayed invidually (ng/mg protein). Region

Weeks o f treatment 2

Area postrema Nucleus commissuralis Area As Nucleus tractus solitarius Area A1 Locus coeruleus

4

9

Control

DOCA

Control

DOCA

Control

DOCA

2.6:~0.2 1.93:0.3 1.0&0.3 1.5:k0.2 0.9:k0.3 1.0~0.2

2.8~0.4 2.1±0.4 1.2±0.1 1.43:0.4 0.8±0.2 1.2~0.1

2.63:0.4 1.7i0.3 1.2~0.2 1.6:k0.3 1.05_0.3 0.8~0.1

2.93:0.5 2.05-0.2 1.5~:0.2 1.5:k0.3 1.2:~0.1 0.9~z0.1

2.93:0.3 1.6~0.2 1.03:0.1 1.1 d:0.2 0.75-0.1 0.55-0.1

3.8±0.4 3.35-0.5* 2.1 :E0.3* 1.6:~0.l 1.45-0.2" 1.35-0.1"

* Statistically significant (P < 0.05) DOCA-salt vs. control group (Student's t-test).

125 DISCUSSION An earlier report t7 demonstrated an elevation of the adrenaline-forming enzyme, PNMT, in the At area of DOCA-salt hypertensive rats, after 4 weeks of treatment. In this series of experiments we have investigated further the changes in PNMT activity in discrete brain areas during the development of the DOCA-salt hypertension, and the corresponding changes in the steady-state level of the catecholamines adrenaline, noradrenaline and dopamine. These parameters of central catecholamine~gic activity were studied after 2, 4 and 9 weeks of DOCA-salt treatment (Table I). As noted in Table I, increases in PNMT activity were present as early as 2 weeks after treatment, in the area A1 and the locus coeruleus, and persisted in these nuclei throughout the treatment. After a longer period of DOCA-salt administration (9 weeks) the increases in enzyme activity were also present in the A2 area of the brain stem, which partially corresponds to the nucleus tractus solitarius 2. At this later time in the development of the hypertension, the increases in the activity of the adrenalineforming enzyme coincided with increases in the steady state levels of adrenaline itself. The changes in adrenaline content were also localized to the same specific brain stem areas involved in cardiovascular control, and were quite specific for this catecholamine, since dopamine levels remained unchanged throughout the treatment and the noradrenaline levels were only increased in the nucleus commissuralis. The significance of this isolated change in brain stem noradrenaline cannot be presently explained. It must be noted that other authors ~4 have failed to detect significant changes in adrenaline levels after 3--4 weeks of DOCA-salt treatment. We have detected changes in adrenaline levels only after 9 weeks of DOCA-salt treatment. These changes are likely not to be related to a stress reaction produced after a prolonged DOCA-salt treatment since stress produces a substantial decrease, rather than an increase, in the adrenaline content of the same areas 19. Thus, our results suggest the activation of an adrenergic pathway localized to the brain stem in DOCA-salt hypertensive rats, the late appearance of the increase in adrenaline content possibly representing a central adaptive mechanism to a prolonged elevation of the blood pressure. It is not possible to know whether the observed differences are present in terminals, or cell bodies, since the areas dissected contain both 10. Changes in central adrenaline neurons are not restricted to the DOCA-salt model of experimental hypertension. In another form of renal hypertension, the one kidney Goldblatt renovascular hypertension, Petty and Reid 14 demonstrated early changes in central catecholamine levels, and a later increased PNMT activity localized in similar brain stem areas as those reported here 15. In the spontaneously (genetic) hypertensive rat (SHR) early changes in PNMT activity have also been detected in the A1-A2 areas of the brain stem 17,1s. All these data strongly suggest the involvement of central brain stem adrenalineforming neurons in the control of the blood pressure and in the production of several

126 forms o f hypertension. The role o f adrenaline in the A1, A2 a n d locus coeruleus with respect to the r e g u l a t i o n o f b l o o d pressure is still speculative. The changes observed m a y be s e c o n d a r y to the rise in the b l o o d pressure, the b r a i n adrenaline system being in this case p a r t o f a v a s o d e p r e s s o r system whose activation represents a c o m p e n s a t o r y mechanism. The possibility remains, however, t h a t the b r a i n adrenaline n e u r o n s m a y be p a r t o f a s t i m u l a t o r y system which can be activated by still u n k n o w n central or p e r i p h e r a l mechanisms.

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