0013-7227/90/1261-01l8$02.00/0 Endocrinology Copyright© 1990 by The Endocrine Society
Vol. 126, No. 1 Printed in U.S.A.
Differential Central Effects of Mineralocorticoid and Glucocorticoid Agonists and Antagonists on Blood Pressure* D. T. W. M. VAN DEN BERG, E. R. DE KLOET, H. H. VAN DIJKEN, AND W. DE JONG Rudolf Magnus Institute for Pharmacology, University of Utrecht, 3521 GD Utrecht, The Netherlands
ABSTRACT. Systolic blood pressure was measured, using an indirect tail method, in conscious male rats at several time intervals after the intracerebroventricular injection of mineraloand glucocorticoid agonists and antagonists. Intracerebroventricular administration of the antimineralocorticoid RU 28318 (10 ng) decreased blood pressure, while the antiglucocorticoid RU 38486 (10 ng) caused an increase, which was slower in onset and of longer duration. The effect of the antimineralocorticoid was maximal at 8 h and had disappeared after 24 h. The antiglucocorticoid had a significant effect 24 and 48 h after injection. Neither antagonist was effective when administered sc at the same dose (10 ng). Intracerebroventricular administration of aldosterone (10 ng) and the selective glucocorticoid agonist RU 28362 (10 ng) increased and decreased blood pressure, respectively. Corticosterone given intracerebroventricullarly (10-100 ng) did not affect blood pressure unless the dose was increased to 1 ng. Two weeks after adrenalectomy a decrease
A
in blood pressure was observed when the rats were given 0.9% saline instead of water to drink. Replacement therapy with corticosterone (12.5-mg steroid pellet, sc) restored blood pressure to the level in the sham-operated controls. The chronically elevated level of circulating corticosterone produced by a 100mg sc corticosterone pellet increased blood pressure. The 12.5and 100-mg sc corticosterone pellets resulted in plasma corticosterone levels of approximately 3 and 20 Mg/100 ml, respectively. Intracerebroventricular administration of the glucocorticoid and mineralocorticoid antagonists (10 ng) increased and decreased, respectively, the blood pressure of the adrenalectomized rats receiving corticosterone substitution. From these data we conclude that corticosteroids can affect the central regulation of blood pressure. The mineralo- and glucocorticoids have opposite effects, which differ in onset and duration. The mineralocorticoids increased blood pressure, whereas the glucocorticoid decreased it. {Endocrinology 126: 118-124, 1990)
of adrenal steroids, as encountered in patients with Cushing's syndrome and hyperaldosteronism (8, 9). The actions of adrenal steroids on the kidney, vascular smooth muscle, and central nervous system are recognized to be of importance in cardiovascular control (2, 3, 6). The steroids are proposed to act in coordination with a number of vasoactive agents {e.g. vasopressin, angiotensin-II, atrial natriuretic factor, and catecholamines). In this respect, adrenal steroids may interact at the level of the target cell and also affect the production of some of these agents (2, 3, 5, 6, 10, 11). The present study focusses on the significance of the central nervous system in steroid-regulated cardiovascular homeostasis. Recent studies have shown that chronic infusion of mineralocorticoids in the lateral cerebral ventricle increases blood pressure (12,13) and may change the responsiveness to vasopressin (14). Other studies have shown that lesioning of the anteroventral part of the hypothalamus (AV3V) prevents the development of mineralocorticoid-dependent hypertension (2, 3, 11). It is thought that mineralocorticoids may change the intracellular sodium content of these hypothalamic cells
DRENAL steroids are of critical importance for cardiovascular homeostasis (1-3). This can be inferred from the effects of chronic and acute changes in the circulating level of corticosteroids on blood pressure. Hypotension develops as a result of bilateral adrenalectomy (ADX) (4) Furthermore, replacement therapy with corticosteroids restores the blood pressure of ADX rats (5). Chronically elevated levels of glucocorticoids and mineralocorticoids in animals are associated with hypertension (6). In addition, a separate class of hypertensinogenic steroids has been proposed, which includes 17a,20-dihydroxyprogesterone and 17a-hydroxyprogesterone. The latter steroids were found to increase the blood pressure of sheep (7). High blood pressure is a pathological concomitant of endogenous hypersecretion Received May 18,1989. Address all correspondence and requests for reprints to: Dr. E. R. de Kloet, Rudolf Magnus Institute for Pharmacology, University of Utrecht, Vondellaan 6, 3521 GD Utrecht, The Netherlands. * Presented in part at the symposium The Adrenal and Hypertension: From Cloning to Clinic, Satellite Symposium to the VIII International Congress of Endocrinology, July 25 and 26, 1988, Tokyo, Japan and supported by the Dutch Heart Foundation. 118
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BRAIN CORTICOSTEROID RECEPTORS AND BLOOD PRESSURE and affect the release of agents that stimulate drinking or influence vascular responsiveness (3). Furthermore, a differential responsiveness to various vasoactive agents has been observed in ADX rats receiving either mineralocorticoids or glucocorticoids (5, 15). Here we report the acute effect of intracerebroventricularly (icv) administered selective mineralo- and glucocorticoid receptor agonists and antagonists on the systolic blood pressure of conscious male rats. The data show that adrenal steroids can affect central cardiovascular control differentially via mineralo- and glucocorticoid receptors.
Materials and Methods
119
10 mm Hg the measurement was repeated. The difference between the four measurements was less then 4% (see also Table 1). As assessed with a direct method, the mean arterial pressure of conscious rats was 111.8 ± 2.3 mm Hg (n = 12), which is in agreement with a previous report (20). It has been shown that the values obtained with the indirect method are not different from those measured directly in the carotid artery at the same moment (r = 0.99) (18). The rats received the steroids or vehicle icv slowly over a period of 30 sec in a volume of 0.2 /A. The mineralocorticoid and glucocorticoid agonists and antagonists were administered in doses of 1 and 10 ng; corticosterone was administered over a dose range of 1-1000 ng. The systolic blood pressure and heart rate were measured at the time intervals indicated after the injection.
Animals
Statistical analysis
Male Wistar rats (140-160 g BW) were used. The animals were housed individually in cages under standard conditions, with the lights on from 0600-2000 h. All animals received food and water ad libitum. ADX was performed bilaterally under ether anesthesia 2 weeks before the start of the experiments at 0800-1000 h. The ADX animals received 0.9% NaCl as their drinking solution. Groups of ADX animals were implanted sc with pellets of corticosterone or cholesterol immediately after ADX. The pellets, which were prepared according to the method of Meyer et al. (16), consisted of 100 mg corticosterone (100%) or 12.5 mg of the steroid mixed with 87.5 mg cholesterol (12.5%). Permanent polyethylene canulas were implanted under Hypnorm (Janssen Pharmaceutica BV, Tilburg, The Netherlands) anesthesia in the right lateral ventricle as described by Brakkee et al. (17).
The data are expressed as the mean of the absolute systolic blood pressure (mm Hg) ± SEM. Significant differences were assessed by using a multivariate analysis of variance (MANOVA) with a repeated measure design (SPSS computer program) followed by a multiple range test according to the method of Tukey B for assessing significance between groups at a specific time point. Differences were considered to be significant at P < 0.05.
Steroids Steroids for icv administration were dissolved in vehicle (2% ethanol-saline). The antiglucocorticoid RU 38486, the antimineralocorticoid RU 28318, and the glucocorticoid agonist RU 28362 were kindly donated by Roussel-UCLAF (Romainville, France). Corticosterone and aldosterone were gifts from Organon International B.V. (Oss, The Netherlands).
Results Figure 1A shows the effect of the antimineralocorticoid RU 28318 (10 ng), and Fig. IB the effect of the antiglucocorticoid RU 384861 (10 ng) on systolic blood pressure. RU 28318 (antimineralocorticoid) caused a transient decrease in blood pressure at 2 and 8 h (values at 8 h were 143 mm Hg for vehicle-injected controls us. 129.8 mm Hg for RU 28318-treated animals; P < 0.05, by Tukey B test); no decrease was detectable after 24, 48, and 96 h. In contrast, RU 38486 (the antiglucocorticoid) caused an increase in blood pressure. This increase was signifiTABLE 1. Systolic blood pressure (BP) and heart rate (HR) of ADX rats and controls
Measurement of blood pressure and heart rate The systolic blood pressure was measured in conscious rats with the indirect tail sphygmographic method of Leenen and de Jong (18). Heart rate was obtained from the pulse by using a tachometer. The generally accepted procedure that is recommended to reduce the variability and increase the reliability of the systolic blood pressure measurements was followed (19). Briefly, all rats were trained for 2 weeks before experimentation. At the start of each training session (and of actual blood pressure measurement) the rats were placed for a maximum of 30 min in an environment with a temperature of 32-34 C. This is necessary to release the arterial sphincter at the base of the tail, so that higher flow levels in the tail result. The blood pressure was recorded four times, and the average of these measurements was taken as the measured blood pressure. In case the lowest and the highest values differed by more than
Control ADX cholesterol ADX + 12.5% CORT ADX + 100% CORT
20 14 15 14
Drinking solution
BP
HR
H2O 0.9% NaCl 0.9% NaCl 0.9% NaCl
141 ± 1.2 124 ± 0.8° 143 ± 0.8 153 ± 0.7°
434 ± 7 434 ± 4 448 ± 6 430 ± 6
Data are the mean ± SEM of 14-20 rats/group. Steroid pellets were implanted immediately after adrenalectomy. ADX rats received 0.9% NaCl as the drinking solution. CORT, Corticosterone. ° P < 0.01 vs. control. 1 The following trivial names are used: antiglucocorticoid RU 38486, 17(8-hydroxy-ll/3-(4-dimethylamino-phenyl)17a-(l-propynyl)estra4,9-diene-3-one; antimineralocorticoid RU 28318, 3,3-oxo-7-propyl-17hydroxy-androstan-4-en-17yl-propionic acid-lactone; glucocorticoid agonist RU 28362, ll/3,1718-dihydroxy-6-dimethyl-17a-(l-propynyl) androstan-l,4,6-triene-3-one.
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120
BRAIN CORTICOSTEROID RECEPTORS AND BLOOD PRESSURE 155-i
A JL
~ FIG. 1. Effect of 10 ng of the mineralocorticoid antagonist RU 28318 (A) and 10 ng of the glucocorticoid antagonist RU 38486 (B) on systolic blood pressure (BP) after injection into the lateral ventricle (icv) of male rats. Data are expressed as absolute systolic blood pressures (mm Hg) in the vehicle-treated control and steroid-treated animals and are given as the mean of 8-10 animals ±
145H
Q. CO
135-
125
hours; t = 0 indicates preinjection values. Results from MANOVA are as follows: A: treatment effect, F(l,13) = 28.73; P < 0.001; time effect, F(5,65) = 8.69; P < 0.001; time by treatment effect, F(5,65) = 3.32; P < 0.05; B: treatment effect, F(l,12) = 10.26; P < 0.05; time effect, F(5,60) = 9.49; P < 0.001; time by treatment effect, F(5,60) = 5.07; P < 0. 05. *, P V
48
72
hrs post-injection
*
B Z § 160 Q. QQ
i
150-
140
2
8
i
24
** • VEHICLE 0 10 ng RU38486
48
I
96
hrs post-injection cantly different from controls at 24 h (146.0 mm Hg for vehicle-injected controls vs. 158.8 mm Hg for RU 38486treated animals; P < 0.05, by Tukey B test) and 48 h (147.4 mm Hg for vehicle-injected controls vs. 161.1 mm Hg for RU 38486-treated animals; P < 0.05, by Tukey B test) postinjection. There was no effect of steroid treatment on the variation in the pressure measurements. The effect of 1 ng of the antagonists administered icv was not significantly different from the effect of icv administered vehicle (data not shown). No effects on heart rate were observed (data not shown). The blood pressure and heart rate of vehicle-treated animals were not different from preinjection values (t = 0 h). In a separate experiment the effect of sc administration of the antagonists was studied with the same protocol. Subcutaneous administration of the 10-ng doses of antagonists did not alter blood pressure or heart rate (data not shown). Figure 2 shows the effect of icv administered agonists at 24 h. Aldosterone (10 ng) increased blood pressure at 8 and 24 h (140.8 mm Hg for vehicle-injected controls
vs. 148.6 mm Hg for aldosterone-treated animals; P < 0.05, by Tukey B test). Corticosterone exerted a small but significant effect 24 h after a 100-fold higher dose of 1000 ng (143.0 mm Hg for vehicle-injected controls vs. 149.4 mm Hg for corticosterone-treated animals; P < 0.05, by Tukey B test). Finally, the selective glucocorticoid RU 28362 (10 ng) caused a decrease in blood pressure (143.1 mm Hg for vehicle-injected controls vs. 134.8 mm Hg for RU 28362-treated animals; P < 0.05, by Tukey B test), while doses of 0.1 and 1 ng were ineffective (data not shown). The effect of 10 ng RU 28362 lasted up to 7 days and ranged between —11 and —17 mm Hg. The onset of the hypotension was slow and was not observed within 24 h after the injection. The blood pressure of vehicle-treated animals was not different from preinjection values (t = 0 h). The heart rate (controls, 434 ± 7 beats/min) was not affected by any of the treatments. The effect of ADX and chronic corticosterone replacement on the systolic blood pressure is shown in Table 1. Fourteen days after ADX blood pressure was decreased
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BRAIN CORTICOSTEROID RECEPTORS AND BLOOD PRESSURE 160-1
D • O ED
I
140-
vehicle 1 ng 10 ng 1000 ng
130
ALDO
CORT
RU 2 8 3 6 2
FIG. 2. Effect of aldosterone (ALDO; 1-10 ng), corticosterone (CORT; 1,10, and 1000 ng), and the glucocorticoid agonist RU 28362 (1-10 ng) after administration into the lateral cerebral ventricle of male rats. Data are expressed as the mean of absolute systolic blood pressures (BP; mm Hg) in the vehicle-treated control and steroid-treated animals ± SEM measured 24 h after administration. Results from MANOVA are as follows: 10 ng ALDO: treatment effect, F(l,8) = 3.82; P < 0.05; time effect, F(5,40) = 2.79; P < 0.05; time by treatment effect, F(5,40) = 4.3; P < 0.05. 1000 ng CORT: treatment effect, F(l,12) = 1.63; P < 0.05; time by treatment effect, F(5,60) = 6.35; P < 0.05. 10 ng RU 28362: treatment effect, F(l,8) = 16.64; P < 0.05; time effect, F(5,40) = 23.67; P < 0.001; time by treatment effect, F(5,40) = 5.8; P < 0.001. *, P < 0.05 us. corresponding vehicle-treated animals (by Tukey B test).
(—17 mm Hg). Replacement therapy with the low dose of corticosterone (12.5-mg pellet) restored blood pressure to the level of the sham-operated control animals. A rise in blood pressure (+12 mm Hg) occurred after replacement therapy with the high dose of corticosterone (100mg pellet). No changes in heart rate were observed. The low and high replacement doses of corticosterone caused circulating plasma corticosterone levels of approximately 3 and 20 Mg/100 ml, respectively. Figure 3 shows the effect of RU 38486 (10 ng) and RU 28318 (10 ng) administered icv to ADX rats receiving corticosterone [12.5 mg (Fig. 3A) or 100 mg (Fig. 3B)] in an sc implanted pellet. Neither antagonist was effective in ADX animals (data not shown). In the ADX animals substituted with 12.5 or 100 mg corticosterone, icv RU 28318 (10 ng) decreased blood pressure, an effect that was measurable both 2 and 24 h postinjection, but not later (cf. Fig. 3, A and B). In contrast to the effect of RU 28318, icv RU 38486 (10 ng) caused a long-lasting elevation of blood pressure in the animals with the low and high replacement doses of corticosterone (Fig. 3, A and B).
Discussion The present study suggests that glucocorticoids and mineralocorticoids exert effects on blood pressure in the central nervous system. The steroid agonist and antagonists, at a dose of 10 ng, altered blood pressure when administered icv, but none was effective when given systemically. The central effects on blood pressure of the two classes of steroids appeared to be opposite in direc-
121
tion. The mineralocorticoids caused a rise in blood pressure after icv administration, and the glucocorticoids induced a decrease. The reverse was observed with the mineralo- and glucocorticoid antagonists administered icv to either intact animals or ADX animals receiving chronic replacement therapy with corticosteroids released from sc implanted steroid pellets. The steroid antagonists were not effective in the ADX animals. It is interesting to speculate that the observed effects of the treatments may reflect the possibility of an interaction between steroid treatment and stress. Some degree of arousal is present under the conditions of the indirect blood pressure measurement, as can be seen from the slightly elevated heart rate. It cannot be concluded from the present experiments whether the steroid effects are exerted on basal or stress-induced blood pressure. The pressor effect caused by a single icv dose of aldosterone extends recent findings on the effects of chronic icv infusion of mineralocorticoids (2, 12, 13). In the studies of Gomez Sanchez (12, 13), the continuous icv infusion of 5 ng/h aldosterone in uninephrectomized Sprague-Dawley rats maintained on a high salt intake produced hypertension after about 2 weeks. The hypertension was reversible upon cessation of the treatment. Although the blood pressure was unchanged after 2 days of icv aldosterone infusion, Janiak and Brody (14) reported, under similar conditions, a blunted pressor response to a high dose of vasopressin (400 ng, icv). The latter observation suggests that the mineralocorticoid induced desensitization of central vasopressin receptors. It should be added, however, that administration of vasopressin in the nucleus tractus solitarii causes a dosedependent (1-100 pg) decrease in the blood pressure and heart rate of anesthetized rats (19) and, thus, participates at that level in inhibitory cardiovascular control mechanisms. Mineralocorticoid-dependent hypertension is prevented by an AV3V ablation (2, 3, 11). The lesion may impair the release of vasopressin and other vasoactive agents (2, 3,11) and is thought to disturb the integrative control of cardiovascular function, thirst, and salt appetite (2, 3,11). Glucocorticoids block the stress-induced synthesis of AVP and affect the action of catecholamines, angiotensin, and atrial natriuretic peptide (21). Recent studies by Yagil and Krakoff (5, 15) in conscious rats have clearly demonstrated that during glucocorticoid deficiency these rats are more dependent on renin-angiotensin and vasopressin systems for the maintenance of blood pressure than are mineralocorticoid-deficient rats when both groups are on high salt intake. It would, therefore, be of interest to study the peptide-glucocorticoid interaction in the control of blood pressure after a single central administration of the compounds. Our study shows that replacement therapy of ADX
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BRAIN CORTICOSTEROID RECEPTORS AND BLOOD PRESSURE
Endo • 1990 Voll26-Nol
155FIG. 3. Effect of 10 ng of the antiglucocorticoid RU 38486 and 10 ng of the antimineralocorticoid RU 28318 on systolic blood pressure (BP) after injection into the lateral cerebral ventricle of normotensive male rats, ADX 2 weeks previously. ADX rats were replaced with 12.5 mg corticosterone (A) or 100 mg corticosterone (B) implanted sc and were given 0.9% NaCl to drink. Data are expressed as absolute systolic blood pressures (mm Hg) in the vehicle-treated control and steroid-treated animals and are given as the mean of 8-10 animals ± SEM. Time intervals are indicated in hours; t = 0 indicates preinjection values. Significant results from MANOVA are as follows: A: RU 38486: treatment effect, F(l,22) = 52.38; P < 0.001; time effect, F(4,88) = 29.96; P < 0.005; time by treatment effect, F(4,88) = 99.42; P < 0.001. RU28318: Treatment effect, F(l,16) = 3.31; P < 0.01; time effect, F(4,64) = 10.79; P < 0.001; time by treatment effect, F(4,64) = 17.67; P < 0.001. B: RU38486: treatment effect, F(l,16) = 16.77; P < 0.05; time effect, F(4,64) = 5.74; P < 0.001; time by treatment effect, F(4,64) = 8.28; P < 0.001. RU 28318: treatment effect, F(l,13) = 11.91; P < 0.05; time effect, F(4,52) = 9.68; P < 0.001; time by treatment effect, F(4,52) = 9.78; P < 0.001. *,P< 0.05 vs. corresponding vehicle-treated animals (by Tukey B test).
145-
w Q. CO
xi
L
135-
0 RU 38486 • VEHICLE E3 RU 28318
** 1
125
2
24
48
72
hrs post injection
165-
~
Q. CQ
155@ RU 38486 • VEHICLE M RU 28318 145-
135
2
rats with a low circulating concentration of corticosterone restores the reduced blood pressure to the level in sham-operated controls. The corticosterone concentration of 3 /ug/100 nil approaches the average plasma corticosterone level observed throughout the day and was found to normalize a large number of parameters, including the pituitary release of ACTH and thymus weight (22). The replacement regimen, resulting in high circulating corticosterone levels, increased blood pressure. While this effect is likely to occur via an action of the chronically elevated levels of corticosterone on vascular smooth muscle and kidney function, our data suggest that it also has a central component. This can be inferred from the observation that after icv injection the different steroid antagonists altered blood pressure in the same direction in the corticosterone-replaced ADX animals and intact animals. The present study suggests that brain receptors for mineralo- and glucocorticoids are involved in the central
24 48 72 144 192 hrs post injection regulation of blood pressure. Using radioligand binding, autoradiography, immunocytochemistry, and in situ hybridization, several researchers have detected mineralocorticoid receptors (type 1) in neurons of the hippocampus, septum, periventricular regions, and a number of discrete neuronal cell groups in the cortex and the brain stem (23-26). Glucocorticoid receptors (type 2) are widely distributed throughout the brain. These receptors occur in abundance in the paraventricular and supraoptic nuclei, arcuate nucleus, limbic neurons, cortical and thalamic neurons, and all neurons of the ascending aminergic projections (26-29). Some of the neurons containing a high density of mineralo- and glucocorticoid receptors are known to be involved in the central regulation of blood pressure (e.g. paraventricular nuclei and nucleus tractus solitarii). The effects observed after icv administration of the selective analogs suggest that the mineralocorticoid receptors mediate the pressor response, while the glucocorticoid receptors mediate inhibitory ste-
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BRAIN CORTICOSTEROID RECEPTORS AND BLOOD PRESSURE roid actions on blood pressure mechanisms. Corticosterone (icv) had little effect on blood pressure except at the highest (1000 ng) dose tested. An explanation could be that the steroid binds to both receptor types, which have opposite effects on blood pressure. However, the affinity of corticosterone at mineralocorticoid receptors is about 6- to 10-fold higher than its affinity at glucocorticoid receptors (27). Accordingly, one would expect a pressor response after very low doses of corticosterone, and this was not observed. An alternative explanation could be that corticosterone is converted to its 11-dehydrometabolite, which has low affinity at the receptors (30). The enzyme 110-hydroxysteroid dehydrogenase is particularly rich in mineralocorticoid tarqet tissues, such as the kidney medulla and the parotid glands. Although the enzyme is virtually absent in the hippocampus, it might be present in periventricular tissues such as the AV3V. Accordingly, such tissues will be relatively insensitive to corticosterone unless the dose exceeds the enzyme capacity (31, 32). The mineralo- and glucocorticoid analogs will bypass this enzyme modification. Furthermore, the selective control of salt appetite by aldosterone may be exerted via this mineralocorticoid specificity-conferring mechanism (33). The aldosterone effects on salt appetite were observed in hippocampectomized rats, suggesting an extrahippocampal site of action (34). Interestingly, other behavioral and biochemical studies in which the steroid was administered to ADX rats have shown that corticosterone exerts its effects with considerable specificity (35-38). Certain conditioned behaviors (39) as well as serotonergic neurotransmission (40) were disturbed after ADX and could only be restored after corticosterone replacement. These findings have led to a pharmacological distinction between corticosterone-preferring- in the hippocampal neurons and aldosteroneselective mineralocorticoid (type 2) receptors in the kidney (36, 38, 41-44) and some brain regions such as the AV3V. The classical glucocorticoid receptors are also termed type 2 according to the classification by Funder et al. (43, 45, 46) and mediate glucocorticoid actions on stress-induced molecular events in the brain (47). The present study shows that the effect of the glucocorticoid analogs is slower in onset and of longer duration than the effect observed after the mineralocorticoid treatment. The glucocorticoid agonists and antagonists affected blood pressure only 24 h after administration, and the effect lasted several days. In contrast, the mineralocorticoids exerted their action within 24 h. One of the factors involved in the slow onset of the (anti)glucocorticoid action may be related to administration of the hormones in the morning, when circulating corticosterone levels are low (48, 49). Taken together, our study has shown that the brain
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mineralo- and glucocorticoid receptor systems mediate opposing actions for corticosteroids on the same physiological end point, and that the effects are characterized by different time courses. This finding is consistent with our previous observation on the differential neurochemical, neuroendocrine, and behavioral responses mediated via the two receptor systems, which were also found to differ in direction and in the critical time of corticosterone requirement (37, 38). In our concept, tonic activating influences are mediated via the brain mineralocorticoid receptors regardless of their mineralocorticoid- or corticosterone-specific pharmacological identity. The glucocorticoid receptors restore disturbances in homeostasis induced by stress (38, 50). This concept of opposing mineralocorticoid and glucocorticoid actions reinforces the earlier notions of Selye (1). References 1. Selye H 1952 The Story of the Adaptation Syndrome. Acta Medical Publications, Montreal 2. Myers JH, Bohr DF 1985 Mechanism responsible for the pressure elevation in sodium-dependent mineralocorticoid hypertension. In: Mantero F, Biglieri EG, Funder JW, Scoggins BA (eds) The Adrenal Gland and Hypertension. Serono Symposia, Raven Press, New York, vol 27:131 3. Lohmeier TE, Carroll RG 1985 Adrenocortical hormones and their interactions with angiotensin II and catecholamines in the production of hypertension. In: Mantero F, Biglieri EG, Funder JW, Scoggins BA (eds) The Adrenal Gland and Hypertension. Serono Symposia, Raven Press, New York, vol 27:159 4. Imms FJ, Neame RI 1974 Circulatory changes following adrenalectomy in the rat. Cardiovasc Res 8:268 5. Yagil Y, Koreen R, Krakoff LB 1986 Role of mineralocorticoids
6. 7. 8. 9. 10. 11.
12. 13. 14. 15. 16.
and glucocorticoids in blood pressure regulation in normotensive rats. Am J Physiol 251:H1354 Bravo EL 1986 Aldosterone and other adrenal steroids. In: Zanchetti A, Tarazi RC (eds) Handbook of Hypertension. Elsevier, Amsterdam, vol 8:603 Scoggins BA, Coghlan JP, Denton DA 1984 ACTH-induced hypertension in sheep. In: De Jong W (ed) Handbook of Hypertension. Elsevier, Amsterdam, vol 4:107-135 Schalekamp MADH, Wenting GJ, Man in't Veld AJ 1981 Pathogenesis of mineralocorticoid hypertension. Clin Endocrinol Metab 10:397 Kaplan NM 1983 Cushing's syndrome and hypertension. In: Robertson JIS (ed) Handbook of Hypertension. Elsevier, Amsterdam, vol 2:208 Reid JL, Rubin PC 1987 Peptides and central neural regulation of the circulation. Physiol Rev 67:725 Brody MJ, Johnson AK 1980 Role of the anteroventral third ventricle region in fluid and electrolyte balance, arterial pressure regulation and hypertension. In: Martini L, Ganong WF (eds) Frontiers in Neuroendocrinology. Raven Press, New York, vol 6:249 Gomez-Sanchez EP 1986 Intracerebroventricular infusion of aldosterone induces hypertension in rats. Endocrinology 118:819 Gomez-Sanchez EP 1988 Dose-response studies of intracerebroventricular infusion of aldosterone in sensitized and non-sensitized rats. J Hypertension 6:437 Janiak P, Brody MJ 1988 Central interactions between aldosterone and vasopressin in cardiovascular systems. Am J Physiol 255:R166 Yagil Y, Krakoff LB 1988 The differential effect of aldosterone and dexamethasone on pressor responses in adrenalectomized rats. Hypertension 11:174 Meyer JS, Micco DJ, Stephenson B, Krey LC, McEwen BS 1979
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17. 18. 19. 20. 21. 22. 23. 24.
25.
26. 27. 28. 29.
30.
31.
32.
BRAIN CORTICOSTEROID RECEPTORS AND BLOOD PRESSURE
Subcutaneous implantation method for chronic glucocorticoid replacement therapy. Physiol Behav 22:867 Brakkee J, Wiegant VM, Gispen WH 1979 A simple technique for rapid implantation of a permanent canula into the rat brain ventricular system. Lab Animal Sci 29:78 Leenen FH, De Jong W 1971 A solid silver clip for induction of predictable of renal hypertension in the rat. J Applied Physiol 31:142 Lovenberg W 1987 Techniques for the measurement of blood pressure. Hypertension 9:1 Van Giersbergen P, De Jong W 1988 Antagonism by naltrexone of the hypotension and bradycardia induced by a-methyldopa in conscious normotensive rats. J Pharmacol Exp Ther 244:341 Brattstrom A, De Jong W, De Wied D 1988 Vasopressin microinjections into the nucleus tractus solitarii decreases heart rate. J Hypertension 6:S521 Dallman MF, Akana SF, Cascio CS, Darlington DN, Jacobson L, Levin N 1987 Regulation of ACTH secretion: variation on a theme of B. Recent Prog Horm Res 43:113 Anderson NS, Fanestil DD 1976 Corticoid receptors in rat brain: evidence for an aldosterone receptor. Endocrinology 98:676 DeNicola AK, Tornello S, Weisenberg L, Friedman O, Birmingham MK 1981 Uptake and binding of 3H-aldosterone by the anterior pituitary and brain regions in adrenalectomized rats. Horm Metab Res 13:103 Stumpf WE, Sar M 1979 Glucocorticoid and mineralocorticoid target sites in the brain. Autoradiographic studies with corticosterone, aldosterone, deoxycorticosterone. In: Jones MF, Gillham B, Dallman MF, Chattopadhyay S (eds) Interaction within the BrainPituitary Adrenocortical System. Academic Press, New York, p 137 Van Eekelen JAM, Jiang W, de Kloet ER, Bohn MC 1988 Distribution of the mineralocorticoid and the glucocorticoid receptor mRNA's in the rat hippocampus. J Neurosci Res 21:88 Reul JMHM, de Kloet ER 1985 Two receptor systems for corticosterone in the brain: microdistribution and differential occupation. Endocrinology 117:2505 Van Eekelen JAM, Kiss JZ, Westphal HM, de Kloet ER 1987 Immunocytochemical study of the intracellular localization of the Type 2 glucocorticoid receptor in the rat brain. Brain Res 436:120 Fuxe K, Wikstr8m AC, Okret S, Agnati LF, Harfstrand F, Yu ZY, Granholm L, Zoli M, Vale W, Gustafsson JA 1985 Mapping of glucocorticoid receptor immunoreactive neurons in the rat tel- and diencephalon using a monoclonal antibody against rat liver glucocorticoid receptors. Endocrinology 117:1803 Stewart PM, Wallace AM, Valentino R, Burt B, Shackleton CHL, Edwards CRW 1987 Mineralocorticoid activity of liquorice: 118hydroxysteroid dehydrogenase deficiency comes of age. Lancet 2:821 Edwards CRW, Stewart PM, Burt D, Brett L, Mclntyre MA, Sutanto de Kloet ER, Monder C 1988 Tissue localization of 11/3hydroxysteroid dehydrogenase: paracrine protector of the mineralocorticoid receptor. Lancet Oct 29:986 Funder JW, Pearce PT, Smith R, Smith Al 1988 Mineralocorticoid action: target tissue specificity is enzyme, not receptor, mediated.
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Science 242:583 33. McEwen BS, Lambdin LT, Rainbow TC, De Nicola AF 1986 Aldosterone effects on salt appetite in adrenalectomized rats. Neuroendocrinology 43:38 34. Magarinos AM, Coirini H, De Nicola AF, McEwen BS 1986 Mineralocorticoid regulation of salt intake is preserved in hippocampectomized rats. Neuroendocrinology 44:494 35. Bohus B, de Kloet ER, Veldhuis HD 1982 Adrenal steroids and behavioural adaptation: relationship to brain corticoid receptors. In: Ganten D, Pfaff DW (eds) Current Topics in Neuroendocrinology. Springer-Verlag, Berlin, p 107 36. McEwen BS, de Kloet ER, Rostene WH 1986 Adrenal steroid receptors and actions in the nervous system. Physiol Rev 66:1121 37. de Kloet ER, Ratka A; Reul JMHM, Sutanto W, Van Eekelen 1987 Corticosteroid receptor types in brain: regulation and putative function. Ann NY Acad Sci 512:351 38. de Kloet ER, Reul JMHM 1987 Feedback action and tonic influence of corticosteroids on brain function: a concept arising from heterogeneity of brain receptor systems. Psychoneuroendocrinology 12:83 39. Bohus B, de Kloet ER 1981 Adrenal steroids and extinction behaviour. Antagonism by progesterone, deoxycorticosterone, and dexamethasone of a specific effect of corticosterone. Life Sci 28:433 40. de Kloet ER, Sybesma H, Reul JMHM 1986 Selective control by corticosterone of 5HT, receptor capacity in raphe-hippocampal system. Neuroendocrinology 42:513 41. Veldhuis HD, Van Koppen C, Van Ittersum M, de Kloet ER 1982 Specificity of the adrenal steroid receptor system in the rat hippocampus. Endocrinology 110:2044 42. Krozowski ZS, Funder JW 1983 Renal mineralocorticoid receptors and hippocampal corticosterone binding species have identical steroid binding specificity. Proc Natl Acad Sci USA 80:6056 43. Funder JW 1987 Adrenal steroids: new answers and new questions. Science 237:236 44. McEwen BS, Weiss JM, Schwartz LS 1969 Uptake of corticosterone by rat brain and its concentration by certain limbic structures. Brain Res 16:227 45. Funder JW, Feldman D, Edelman IS 1973 The roles of plasma binding and receptor specificity in the mineralocorticoid action of aldosterone. Endocrinology 92:994 46. Funder JW, Sheppard K 1987 Adrenocortical steroids and the brain. Annu Rev Physiol 49:397 47. Nicols NR, Lerner SP, Masters JN, May PC, Millas SL, Finch CE 1988 Rapid corticosterone-induced changes in gene expression in rat hippocampus display Type II glucocorti-coid r-eceptors specificity. Mol Endocrinol 2:284 48. Moguilevski M, Philibert D 1984 Potent anti-glucocorticoid activity correlated with strong binding capacity to cytosolic glucocorticoid receptor followed by an impaired activation. J Steroid Biochem 20:271 49. Philibert D 1984 RU 38486: an original multifacetted anti-hormone in vivo. In: Agarwal MK (ed) Adrenal Steroid Antagonism, de Gruyter, Berlin, p 77 50. Munck A, Guyre PM, Holbrook NJ 1984 Physiological functions of glucocorticoids in stress and their relationship to pharmacological action. Endocr Rev 5:25
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Vol. 126, No. 1 Printed in U.S.A.
Differential Central Effects of Mineralocorticoid and Glucocorticoid Agonists and Antagonists on Blood Pressure* D. T. W. M. VAN DEN BERG, E. R. DE KLOET, H. H. VAN DIJKEN, AND W. DE JONG Rudolf Magnus Institute for Pharmacology, University of Utrecht, 3521 GD Utrecht, The Netherlands
ABSTRACT. Systolic blood pressure was measured, using an indirect tail method, in conscious male rats at several time intervals after the intracerebroventricular injection of mineraloand glucocorticoid agonists and antagonists. Intracerebroventricular administration of the antimineralocorticoid RU 28318 (10 ng) decreased blood pressure, while the antiglucocorticoid RU 38486 (10 ng) caused an increase, which was slower in onset and of longer duration. The effect of the antimineralocorticoid was maximal at 8 h and had disappeared after 24 h. The antiglucocorticoid had a significant effect 24 and 48 h after injection. Neither antagonist was effective when administered sc at the same dose (10 ng). Intracerebroventricular administration of aldosterone (10 ng) and the selective glucocorticoid agonist RU 28362 (10 ng) increased and decreased blood pressure, respectively. Corticosterone given intracerebroventricullarly (10-100 ng) did not affect blood pressure unless the dose was increased to 1 ng. Two weeks after adrenalectomy a decrease
A
in blood pressure was observed when the rats were given 0.9% saline instead of water to drink. Replacement therapy with corticosterone (12.5-mg steroid pellet, sc) restored blood pressure to the level in the sham-operated controls. The chronically elevated level of circulating corticosterone produced by a 100mg sc corticosterone pellet increased blood pressure. The 12.5and 100-mg sc corticosterone pellets resulted in plasma corticosterone levels of approximately 3 and 20 Mg/100 ml, respectively. Intracerebroventricular administration of the glucocorticoid and mineralocorticoid antagonists (10 ng) increased and decreased, respectively, the blood pressure of the adrenalectomized rats receiving corticosterone substitution. From these data we conclude that corticosteroids can affect the central regulation of blood pressure. The mineralo- and glucocorticoids have opposite effects, which differ in onset and duration. The mineralocorticoids increased blood pressure, whereas the glucocorticoid decreased it. {Endocrinology 126: 118-124, 1990)
of adrenal steroids, as encountered in patients with Cushing's syndrome and hyperaldosteronism (8, 9). The actions of adrenal steroids on the kidney, vascular smooth muscle, and central nervous system are recognized to be of importance in cardiovascular control (2, 3, 6). The steroids are proposed to act in coordination with a number of vasoactive agents {e.g. vasopressin, angiotensin-II, atrial natriuretic factor, and catecholamines). In this respect, adrenal steroids may interact at the level of the target cell and also affect the production of some of these agents (2, 3, 5, 6, 10, 11). The present study focusses on the significance of the central nervous system in steroid-regulated cardiovascular homeostasis. Recent studies have shown that chronic infusion of mineralocorticoids in the lateral cerebral ventricle increases blood pressure (12,13) and may change the responsiveness to vasopressin (14). Other studies have shown that lesioning of the anteroventral part of the hypothalamus (AV3V) prevents the development of mineralocorticoid-dependent hypertension (2, 3, 11). It is thought that mineralocorticoids may change the intracellular sodium content of these hypothalamic cells
DRENAL steroids are of critical importance for cardiovascular homeostasis (1-3). This can be inferred from the effects of chronic and acute changes in the circulating level of corticosteroids on blood pressure. Hypotension develops as a result of bilateral adrenalectomy (ADX) (4) Furthermore, replacement therapy with corticosteroids restores the blood pressure of ADX rats (5). Chronically elevated levels of glucocorticoids and mineralocorticoids in animals are associated with hypertension (6). In addition, a separate class of hypertensinogenic steroids has been proposed, which includes 17a,20-dihydroxyprogesterone and 17a-hydroxyprogesterone. The latter steroids were found to increase the blood pressure of sheep (7). High blood pressure is a pathological concomitant of endogenous hypersecretion Received May 18,1989. Address all correspondence and requests for reprints to: Dr. E. R. de Kloet, Rudolf Magnus Institute for Pharmacology, University of Utrecht, Vondellaan 6, 3521 GD Utrecht, The Netherlands. * Presented in part at the symposium The Adrenal and Hypertension: From Cloning to Clinic, Satellite Symposium to the VIII International Congress of Endocrinology, July 25 and 26, 1988, Tokyo, Japan and supported by the Dutch Heart Foundation. 118
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BRAIN CORTICOSTEROID RECEPTORS AND BLOOD PRESSURE and affect the release of agents that stimulate drinking or influence vascular responsiveness (3). Furthermore, a differential responsiveness to various vasoactive agents has been observed in ADX rats receiving either mineralocorticoids or glucocorticoids (5, 15). Here we report the acute effect of intracerebroventricularly (icv) administered selective mineralo- and glucocorticoid receptor agonists and antagonists on the systolic blood pressure of conscious male rats. The data show that adrenal steroids can affect central cardiovascular control differentially via mineralo- and glucocorticoid receptors.
Materials and Methods
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10 mm Hg the measurement was repeated. The difference between the four measurements was less then 4% (see also Table 1). As assessed with a direct method, the mean arterial pressure of conscious rats was 111.8 ± 2.3 mm Hg (n = 12), which is in agreement with a previous report (20). It has been shown that the values obtained with the indirect method are not different from those measured directly in the carotid artery at the same moment (r = 0.99) (18). The rats received the steroids or vehicle icv slowly over a period of 30 sec in a volume of 0.2 /A. The mineralocorticoid and glucocorticoid agonists and antagonists were administered in doses of 1 and 10 ng; corticosterone was administered over a dose range of 1-1000 ng. The systolic blood pressure and heart rate were measured at the time intervals indicated after the injection.
Animals
Statistical analysis
Male Wistar rats (140-160 g BW) were used. The animals were housed individually in cages under standard conditions, with the lights on from 0600-2000 h. All animals received food and water ad libitum. ADX was performed bilaterally under ether anesthesia 2 weeks before the start of the experiments at 0800-1000 h. The ADX animals received 0.9% NaCl as their drinking solution. Groups of ADX animals were implanted sc with pellets of corticosterone or cholesterol immediately after ADX. The pellets, which were prepared according to the method of Meyer et al. (16), consisted of 100 mg corticosterone (100%) or 12.5 mg of the steroid mixed with 87.5 mg cholesterol (12.5%). Permanent polyethylene canulas were implanted under Hypnorm (Janssen Pharmaceutica BV, Tilburg, The Netherlands) anesthesia in the right lateral ventricle as described by Brakkee et al. (17).
The data are expressed as the mean of the absolute systolic blood pressure (mm Hg) ± SEM. Significant differences were assessed by using a multivariate analysis of variance (MANOVA) with a repeated measure design (SPSS computer program) followed by a multiple range test according to the method of Tukey B for assessing significance between groups at a specific time point. Differences were considered to be significant at P < 0.05.
Steroids Steroids for icv administration were dissolved in vehicle (2% ethanol-saline). The antiglucocorticoid RU 38486, the antimineralocorticoid RU 28318, and the glucocorticoid agonist RU 28362 were kindly donated by Roussel-UCLAF (Romainville, France). Corticosterone and aldosterone were gifts from Organon International B.V. (Oss, The Netherlands).
Results Figure 1A shows the effect of the antimineralocorticoid RU 28318 (10 ng), and Fig. IB the effect of the antiglucocorticoid RU 384861 (10 ng) on systolic blood pressure. RU 28318 (antimineralocorticoid) caused a transient decrease in blood pressure at 2 and 8 h (values at 8 h were 143 mm Hg for vehicle-injected controls us. 129.8 mm Hg for RU 28318-treated animals; P < 0.05, by Tukey B test); no decrease was detectable after 24, 48, and 96 h. In contrast, RU 38486 (the antiglucocorticoid) caused an increase in blood pressure. This increase was signifiTABLE 1. Systolic blood pressure (BP) and heart rate (HR) of ADX rats and controls
Measurement of blood pressure and heart rate The systolic blood pressure was measured in conscious rats with the indirect tail sphygmographic method of Leenen and de Jong (18). Heart rate was obtained from the pulse by using a tachometer. The generally accepted procedure that is recommended to reduce the variability and increase the reliability of the systolic blood pressure measurements was followed (19). Briefly, all rats were trained for 2 weeks before experimentation. At the start of each training session (and of actual blood pressure measurement) the rats were placed for a maximum of 30 min in an environment with a temperature of 32-34 C. This is necessary to release the arterial sphincter at the base of the tail, so that higher flow levels in the tail result. The blood pressure was recorded four times, and the average of these measurements was taken as the measured blood pressure. In case the lowest and the highest values differed by more than
Control ADX cholesterol ADX + 12.5% CORT ADX + 100% CORT
20 14 15 14
Drinking solution
BP
HR
H2O 0.9% NaCl 0.9% NaCl 0.9% NaCl
141 ± 1.2 124 ± 0.8° 143 ± 0.8 153 ± 0.7°
434 ± 7 434 ± 4 448 ± 6 430 ± 6
Data are the mean ± SEM of 14-20 rats/group. Steroid pellets were implanted immediately after adrenalectomy. ADX rats received 0.9% NaCl as the drinking solution. CORT, Corticosterone. ° P < 0.01 vs. control. 1 The following trivial names are used: antiglucocorticoid RU 38486, 17(8-hydroxy-ll/3-(4-dimethylamino-phenyl)17a-(l-propynyl)estra4,9-diene-3-one; antimineralocorticoid RU 28318, 3,3-oxo-7-propyl-17hydroxy-androstan-4-en-17yl-propionic acid-lactone; glucocorticoid agonist RU 28362, ll/3,1718-dihydroxy-6-dimethyl-17a-(l-propynyl) androstan-l,4,6-triene-3-one.
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120
BRAIN CORTICOSTEROID RECEPTORS AND BLOOD PRESSURE 155-i
A JL
~ FIG. 1. Effect of 10 ng of the mineralocorticoid antagonist RU 28318 (A) and 10 ng of the glucocorticoid antagonist RU 38486 (B) on systolic blood pressure (BP) after injection into the lateral ventricle (icv) of male rats. Data are expressed as absolute systolic blood pressures (mm Hg) in the vehicle-treated control and steroid-treated animals and are given as the mean of 8-10 animals ±
145H
Q. CO
135-
125
hours; t = 0 indicates preinjection values. Results from MANOVA are as follows: A: treatment effect, F(l,13) = 28.73; P < 0.001; time effect, F(5,65) = 8.69; P < 0.001; time by treatment effect, F(5,65) = 3.32; P < 0.05; B: treatment effect, F(l,12) = 10.26; P < 0.05; time effect, F(5,60) = 9.49; P < 0.001; time by treatment effect, F(5,60) = 5.07; P < 0. 05. *, P V
48
72
hrs post-injection
*
B Z § 160 Q. QQ
i
150-
140
2
8
i
24
** • VEHICLE 0 10 ng RU38486
48
I
96
hrs post-injection cantly different from controls at 24 h (146.0 mm Hg for vehicle-injected controls vs. 158.8 mm Hg for RU 38486treated animals; P < 0.05, by Tukey B test) and 48 h (147.4 mm Hg for vehicle-injected controls vs. 161.1 mm Hg for RU 38486-treated animals; P < 0.05, by Tukey B test) postinjection. There was no effect of steroid treatment on the variation in the pressure measurements. The effect of 1 ng of the antagonists administered icv was not significantly different from the effect of icv administered vehicle (data not shown). No effects on heart rate were observed (data not shown). The blood pressure and heart rate of vehicle-treated animals were not different from preinjection values (t = 0 h). In a separate experiment the effect of sc administration of the antagonists was studied with the same protocol. Subcutaneous administration of the 10-ng doses of antagonists did not alter blood pressure or heart rate (data not shown). Figure 2 shows the effect of icv administered agonists at 24 h. Aldosterone (10 ng) increased blood pressure at 8 and 24 h (140.8 mm Hg for vehicle-injected controls
vs. 148.6 mm Hg for aldosterone-treated animals; P < 0.05, by Tukey B test). Corticosterone exerted a small but significant effect 24 h after a 100-fold higher dose of 1000 ng (143.0 mm Hg for vehicle-injected controls vs. 149.4 mm Hg for corticosterone-treated animals; P < 0.05, by Tukey B test). Finally, the selective glucocorticoid RU 28362 (10 ng) caused a decrease in blood pressure (143.1 mm Hg for vehicle-injected controls vs. 134.8 mm Hg for RU 28362-treated animals; P < 0.05, by Tukey B test), while doses of 0.1 and 1 ng were ineffective (data not shown). The effect of 10 ng RU 28362 lasted up to 7 days and ranged between —11 and —17 mm Hg. The onset of the hypotension was slow and was not observed within 24 h after the injection. The blood pressure of vehicle-treated animals was not different from preinjection values (t = 0 h). The heart rate (controls, 434 ± 7 beats/min) was not affected by any of the treatments. The effect of ADX and chronic corticosterone replacement on the systolic blood pressure is shown in Table 1. Fourteen days after ADX blood pressure was decreased
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BRAIN CORTICOSTEROID RECEPTORS AND BLOOD PRESSURE 160-1
D • O ED
I
140-
vehicle 1 ng 10 ng 1000 ng
130
ALDO
CORT
RU 2 8 3 6 2
FIG. 2. Effect of aldosterone (ALDO; 1-10 ng), corticosterone (CORT; 1,10, and 1000 ng), and the glucocorticoid agonist RU 28362 (1-10 ng) after administration into the lateral cerebral ventricle of male rats. Data are expressed as the mean of absolute systolic blood pressures (BP; mm Hg) in the vehicle-treated control and steroid-treated animals ± SEM measured 24 h after administration. Results from MANOVA are as follows: 10 ng ALDO: treatment effect, F(l,8) = 3.82; P < 0.05; time effect, F(5,40) = 2.79; P < 0.05; time by treatment effect, F(5,40) = 4.3; P < 0.05. 1000 ng CORT: treatment effect, F(l,12) = 1.63; P < 0.05; time by treatment effect, F(5,60) = 6.35; P < 0.05. 10 ng RU 28362: treatment effect, F(l,8) = 16.64; P < 0.05; time effect, F(5,40) = 23.67; P < 0.001; time by treatment effect, F(5,40) = 5.8; P < 0.001. *, P < 0.05 us. corresponding vehicle-treated animals (by Tukey B test).
(—17 mm Hg). Replacement therapy with the low dose of corticosterone (12.5-mg pellet) restored blood pressure to the level of the sham-operated control animals. A rise in blood pressure (+12 mm Hg) occurred after replacement therapy with the high dose of corticosterone (100mg pellet). No changes in heart rate were observed. The low and high replacement doses of corticosterone caused circulating plasma corticosterone levels of approximately 3 and 20 Mg/100 ml, respectively. Figure 3 shows the effect of RU 38486 (10 ng) and RU 28318 (10 ng) administered icv to ADX rats receiving corticosterone [12.5 mg (Fig. 3A) or 100 mg (Fig. 3B)] in an sc implanted pellet. Neither antagonist was effective in ADX animals (data not shown). In the ADX animals substituted with 12.5 or 100 mg corticosterone, icv RU 28318 (10 ng) decreased blood pressure, an effect that was measurable both 2 and 24 h postinjection, but not later (cf. Fig. 3, A and B). In contrast to the effect of RU 28318, icv RU 38486 (10 ng) caused a long-lasting elevation of blood pressure in the animals with the low and high replacement doses of corticosterone (Fig. 3, A and B).
Discussion The present study suggests that glucocorticoids and mineralocorticoids exert effects on blood pressure in the central nervous system. The steroid agonist and antagonists, at a dose of 10 ng, altered blood pressure when administered icv, but none was effective when given systemically. The central effects on blood pressure of the two classes of steroids appeared to be opposite in direc-
121
tion. The mineralocorticoids caused a rise in blood pressure after icv administration, and the glucocorticoids induced a decrease. The reverse was observed with the mineralo- and glucocorticoid antagonists administered icv to either intact animals or ADX animals receiving chronic replacement therapy with corticosteroids released from sc implanted steroid pellets. The steroid antagonists were not effective in the ADX animals. It is interesting to speculate that the observed effects of the treatments may reflect the possibility of an interaction between steroid treatment and stress. Some degree of arousal is present under the conditions of the indirect blood pressure measurement, as can be seen from the slightly elevated heart rate. It cannot be concluded from the present experiments whether the steroid effects are exerted on basal or stress-induced blood pressure. The pressor effect caused by a single icv dose of aldosterone extends recent findings on the effects of chronic icv infusion of mineralocorticoids (2, 12, 13). In the studies of Gomez Sanchez (12, 13), the continuous icv infusion of 5 ng/h aldosterone in uninephrectomized Sprague-Dawley rats maintained on a high salt intake produced hypertension after about 2 weeks. The hypertension was reversible upon cessation of the treatment. Although the blood pressure was unchanged after 2 days of icv aldosterone infusion, Janiak and Brody (14) reported, under similar conditions, a blunted pressor response to a high dose of vasopressin (400 ng, icv). The latter observation suggests that the mineralocorticoid induced desensitization of central vasopressin receptors. It should be added, however, that administration of vasopressin in the nucleus tractus solitarii causes a dosedependent (1-100 pg) decrease in the blood pressure and heart rate of anesthetized rats (19) and, thus, participates at that level in inhibitory cardiovascular control mechanisms. Mineralocorticoid-dependent hypertension is prevented by an AV3V ablation (2, 3, 11). The lesion may impair the release of vasopressin and other vasoactive agents (2, 3,11) and is thought to disturb the integrative control of cardiovascular function, thirst, and salt appetite (2, 3,11). Glucocorticoids block the stress-induced synthesis of AVP and affect the action of catecholamines, angiotensin, and atrial natriuretic peptide (21). Recent studies by Yagil and Krakoff (5, 15) in conscious rats have clearly demonstrated that during glucocorticoid deficiency these rats are more dependent on renin-angiotensin and vasopressin systems for the maintenance of blood pressure than are mineralocorticoid-deficient rats when both groups are on high salt intake. It would, therefore, be of interest to study the peptide-glucocorticoid interaction in the control of blood pressure after a single central administration of the compounds. Our study shows that replacement therapy of ADX
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BRAIN CORTICOSTEROID RECEPTORS AND BLOOD PRESSURE
Endo • 1990 Voll26-Nol
155FIG. 3. Effect of 10 ng of the antiglucocorticoid RU 38486 and 10 ng of the antimineralocorticoid RU 28318 on systolic blood pressure (BP) after injection into the lateral cerebral ventricle of normotensive male rats, ADX 2 weeks previously. ADX rats were replaced with 12.5 mg corticosterone (A) or 100 mg corticosterone (B) implanted sc and were given 0.9% NaCl to drink. Data are expressed as absolute systolic blood pressures (mm Hg) in the vehicle-treated control and steroid-treated animals and are given as the mean of 8-10 animals ± SEM. Time intervals are indicated in hours; t = 0 indicates preinjection values. Significant results from MANOVA are as follows: A: RU 38486: treatment effect, F(l,22) = 52.38; P < 0.001; time effect, F(4,88) = 29.96; P < 0.005; time by treatment effect, F(4,88) = 99.42; P < 0.001. RU28318: Treatment effect, F(l,16) = 3.31; P < 0.01; time effect, F(4,64) = 10.79; P < 0.001; time by treatment effect, F(4,64) = 17.67; P < 0.001. B: RU38486: treatment effect, F(l,16) = 16.77; P < 0.05; time effect, F(4,64) = 5.74; P < 0.001; time by treatment effect, F(4,64) = 8.28; P < 0.001. RU 28318: treatment effect, F(l,13) = 11.91; P < 0.05; time effect, F(4,52) = 9.68; P < 0.001; time by treatment effect, F(4,52) = 9.78; P < 0.001. *,P< 0.05 vs. corresponding vehicle-treated animals (by Tukey B test).
145-
w Q. CO
xi
L
135-
0 RU 38486 • VEHICLE E3 RU 28318
** 1
125
2
24
48
72
hrs post injection
165-
~
Q. CQ
155@ RU 38486 • VEHICLE M RU 28318 145-
135
2
rats with a low circulating concentration of corticosterone restores the reduced blood pressure to the level in sham-operated controls. The corticosterone concentration of 3 /ug/100 nil approaches the average plasma corticosterone level observed throughout the day and was found to normalize a large number of parameters, including the pituitary release of ACTH and thymus weight (22). The replacement regimen, resulting in high circulating corticosterone levels, increased blood pressure. While this effect is likely to occur via an action of the chronically elevated levels of corticosterone on vascular smooth muscle and kidney function, our data suggest that it also has a central component. This can be inferred from the observation that after icv injection the different steroid antagonists altered blood pressure in the same direction in the corticosterone-replaced ADX animals and intact animals. The present study suggests that brain receptors for mineralo- and glucocorticoids are involved in the central
24 48 72 144 192 hrs post injection regulation of blood pressure. Using radioligand binding, autoradiography, immunocytochemistry, and in situ hybridization, several researchers have detected mineralocorticoid receptors (type 1) in neurons of the hippocampus, septum, periventricular regions, and a number of discrete neuronal cell groups in the cortex and the brain stem (23-26). Glucocorticoid receptors (type 2) are widely distributed throughout the brain. These receptors occur in abundance in the paraventricular and supraoptic nuclei, arcuate nucleus, limbic neurons, cortical and thalamic neurons, and all neurons of the ascending aminergic projections (26-29). Some of the neurons containing a high density of mineralo- and glucocorticoid receptors are known to be involved in the central regulation of blood pressure (e.g. paraventricular nuclei and nucleus tractus solitarii). The effects observed after icv administration of the selective analogs suggest that the mineralocorticoid receptors mediate the pressor response, while the glucocorticoid receptors mediate inhibitory ste-
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BRAIN CORTICOSTEROID RECEPTORS AND BLOOD PRESSURE roid actions on blood pressure mechanisms. Corticosterone (icv) had little effect on blood pressure except at the highest (1000 ng) dose tested. An explanation could be that the steroid binds to both receptor types, which have opposite effects on blood pressure. However, the affinity of corticosterone at mineralocorticoid receptors is about 6- to 10-fold higher than its affinity at glucocorticoid receptors (27). Accordingly, one would expect a pressor response after very low doses of corticosterone, and this was not observed. An alternative explanation could be that corticosterone is converted to its 11-dehydrometabolite, which has low affinity at the receptors (30). The enzyme 110-hydroxysteroid dehydrogenase is particularly rich in mineralocorticoid tarqet tissues, such as the kidney medulla and the parotid glands. Although the enzyme is virtually absent in the hippocampus, it might be present in periventricular tissues such as the AV3V. Accordingly, such tissues will be relatively insensitive to corticosterone unless the dose exceeds the enzyme capacity (31, 32). The mineralo- and glucocorticoid analogs will bypass this enzyme modification. Furthermore, the selective control of salt appetite by aldosterone may be exerted via this mineralocorticoid specificity-conferring mechanism (33). The aldosterone effects on salt appetite were observed in hippocampectomized rats, suggesting an extrahippocampal site of action (34). Interestingly, other behavioral and biochemical studies in which the steroid was administered to ADX rats have shown that corticosterone exerts its effects with considerable specificity (35-38). Certain conditioned behaviors (39) as well as serotonergic neurotransmission (40) were disturbed after ADX and could only be restored after corticosterone replacement. These findings have led to a pharmacological distinction between corticosterone-preferring- in the hippocampal neurons and aldosteroneselective mineralocorticoid (type 2) receptors in the kidney (36, 38, 41-44) and some brain regions such as the AV3V. The classical glucocorticoid receptors are also termed type 2 according to the classification by Funder et al. (43, 45, 46) and mediate glucocorticoid actions on stress-induced molecular events in the brain (47). The present study shows that the effect of the glucocorticoid analogs is slower in onset and of longer duration than the effect observed after the mineralocorticoid treatment. The glucocorticoid agonists and antagonists affected blood pressure only 24 h after administration, and the effect lasted several days. In contrast, the mineralocorticoids exerted their action within 24 h. One of the factors involved in the slow onset of the (anti)glucocorticoid action may be related to administration of the hormones in the morning, when circulating corticosterone levels are low (48, 49). Taken together, our study has shown that the brain
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mineralo- and glucocorticoid receptor systems mediate opposing actions for corticosteroids on the same physiological end point, and that the effects are characterized by different time courses. This finding is consistent with our previous observation on the differential neurochemical, neuroendocrine, and behavioral responses mediated via the two receptor systems, which were also found to differ in direction and in the critical time of corticosterone requirement (37, 38). In our concept, tonic activating influences are mediated via the brain mineralocorticoid receptors regardless of their mineralocorticoid- or corticosterone-specific pharmacological identity. The glucocorticoid receptors restore disturbances in homeostasis induced by stress (38, 50). This concept of opposing mineralocorticoid and glucocorticoid actions reinforces the earlier notions of Selye (1). References 1. Selye H 1952 The Story of the Adaptation Syndrome. Acta Medical Publications, Montreal 2. Myers JH, Bohr DF 1985 Mechanism responsible for the pressure elevation in sodium-dependent mineralocorticoid hypertension. In: Mantero F, Biglieri EG, Funder JW, Scoggins BA (eds) The Adrenal Gland and Hypertension. Serono Symposia, Raven Press, New York, vol 27:131 3. Lohmeier TE, Carroll RG 1985 Adrenocortical hormones and their interactions with angiotensin II and catecholamines in the production of hypertension. In: Mantero F, Biglieri EG, Funder JW, Scoggins BA (eds) The Adrenal Gland and Hypertension. Serono Symposia, Raven Press, New York, vol 27:159 4. Imms FJ, Neame RI 1974 Circulatory changes following adrenalectomy in the rat. Cardiovasc Res 8:268 5. Yagil Y, Koreen R, Krakoff LB 1986 Role of mineralocorticoids
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