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Vasopressin release from rat medial basal hypothalamus induced in vitro by angiotensin
European Journal of Pharmacology, 80 (1982) 247-249
247
Elsevier Biomedical Press
Short communication VASOPRESSlN RELEASE FROM RAT MEDIAL BASAL HYPOTHALAMUS INDUCED IN VITRO BY AN GI OT EN S I N WILLHART KNEPEL, HENNING ANHUT, DORIS NUTTO, ANGELIKA HOLLAND and DIETER K. MEYER
Department of Pharmacology, University of Freiburg i. Br., Hermann-Herder-Str. 5, D- 7800 Freiburg i. Br., Federal Republic of Germany Received 11 March 1982, accepted 29 March 1982
W. KNEPEL~ H. ANHUT, D. NUTTO, A. HOLLAND and D.K. MEYER, Vasopressin release from rat medial basal hypothalamus induced in vitro by angiotensin, European J. Pharmacol. 80 (1982) 247-249. Rat medial basal hypothalami were superfused in vitro. The effect of angiotensin II on vasopressin outflow was investigated. Angiotensin II (10 nM or 1/xM, added to the superfusion medium) increased the veratridine-evoked vasopressin release. The higher concentration also slightly elevated the basal outflow. The effect of angiotensin II was blocked by saralasin. We conclude that angiotensin II can act on the median eminence and/or on the stump of the pituitary stalk to promote the release of vasopressin, which then may influence anterior pituitary hormone secretion.
Median eminence
Veratridine Vasopressinrelease
Angiotensin
I. Introduction
Peptide hormone release from the corticotrope cells of the anterior lobe of the pituitary gland is regulated by hypothalamic releasing factors which are secreted into the portal blood of the median eminence. In addition to catecholamines and a recently isolated ovine peptide, CRF-(1-41), vasopressin is also assumed to be one of these factors (see: Fink, 1981). Vasopressin is found in nerve terminals in the external layer of the median eminence adjacent to portal capillaries and possesses marked ACTH/fl-endorphin-releasing activity. Furthermore, several 'submaximum' stimuli for ACTH/fl-endorphin release in vivo have been shown to be mediated in part by vasopressin (Yates et al., 1971; Knepel et al., 1980). Although glucocorticoids (Stillman et al., 1977) and opiates (Knepel and Reimann, 1982) are assumed to participate, the control mechanisms of vasopressin release from the median eminence are poorly understood. The topographic correspondence of angiotensin II binding sites and immunoreactive vasopressin in the medial palisade zone of the zona externa (vanHouten et al., 1980) raises the possibility that vasopressinergic nerve 0014-2999/82/0000-0000/$02.75 © 1982 Elsevier Biomedical Press
terminals in this region are direct targets for angiotensin II. Therefore, we studied in vitro the effect of angiotensin II on vasopressin release from medial basal hypothalamus.
2. Materials and methods
The methods employed have been described in detail (Knepel and Reimann, 1982). Briefly, a slice of the basal hypothalamus (approximately 0.4 mm thick) of male Wistar rats (250-300 g) was cut. The so-called medial basal hypothalamus (MBH) was prepared from this slice by a frontal cut just rostral to the median eminence and one j u s t ~ u d a l to the pituitary stalk; the width was approximately 1.0 mm (wet weight 1.8 - 0 . 1 mg, vasopressin content 18.52-1.93 ng, n = 2 4 ) . Three MBH per chamber were superfused for 80 min (0.25 ml/min; 37°C) and 20 min samples collected for radioimmunoassay of vasopressin. The composition of the superfusion medium was as follows (mM): NaC1 118, KCl 4.8, CaCl 2 1.3, MgSO 4 1.2, NaHCO 3 25, KHEPO 4 1.2, glucose l l, bacitracin 0.07; 0.1% bovine serum albumin (saturated with 5% CO 2 in O:; pH 7.4). For stimulation of vasopressin re-
248
lease, veratridine (3/~M, Aldrich, U.S.A.) was added to the superfusion medium from the 60th to the 80th min of superfusion. The ceveratrum alkaloid veratridine is known to depolarize excitable cells by holding in an open state some of the sodium channels, which normally participate in the production of the action potential (see: Nordmann and Dyball, 1978, and references cited therein). Pilot studies demonstrated that vasopressin release by veratridine was reversible, related to the concentration used and depended on the presence of calcium (veratridine 3/~M - ED25). 5-11eangiotensin II was purchased from Serva, FRG.
3. Results
The results are shown in table 1. Vasopressin concentration in the superfusion medium decreased somewhat with time under control conditions, but was raised by veratridine ( P < 0 . 0 2 , paired Student's t-test). In the presence of angiotensin II 10 nM or 1/~M (added starting from the 40th rain of superfusion) veratridine was even more effective in elevating vasopressin outflow ( P < 0 . 0 2 or 0.05, respectively; unpaired Student's t-test). Angiotensin II 1 # M but not 10 nM also elevated the basal vasopressin concentration (P < 0.005 comparing the vasopressin outflow from the 40th to the 60th rain of superfusion with the outflow of the preced-
ing fraction by paired Student's t-test). The effect of angiotensin II was blocked by saralasin.
4. Discussion The results show that angiotensin II can increase both the evoked and, when added in higher concentrations, the basal vasopressin release from rat MBH in vitro, suggesting that activation of specific angiotensin receptors on terminals of vasopressin-containing neurones in the median enainence a n d / o r the stump of the pituitary stalk promotes vasopressin release. Similarly, presynaptic facilitatory angiotensin receptors on peripheral and central noradrenergic neurones have been described in the rabbit and dog (see: Zimmerman, 1981) and electrophysiological evidence has recently been presented for a direct action of angiotensin II on axons of rat oxytocin/vasopressincontaining neurones (Baertschi et al., 1981). The physiological function of the mechanism found in vitro in the present study remains to be defined. However, blood-borne angiotensin II induces the release of corticotropin and fl-endorphin from the anterior lobe of the pituitary gland (see: Simpson, 1981). And interestingly enough, the increase in plasma levels of fl-endorphin-like immunoreactivity elicited by intravenous angiotensin II was recently found to be published to be di-
TABLE 1 Vasopressin concentration (pg/fraction) in the superfusion medium of rat medial basal hypothalami. When indicated, veratridine (Vera, 3/~M) or 5-ile-angiotensin II (All) were added to the medium starting from the 60th or 40th rain of superfusion, respectively; saralasin (Sara) was present, when indicated, throughout superfusion. Values are means ± S.E.M. Min of superfusion 20-40 Controls Vera Vera+AII 10nM V e r a + A l I 1 ~M V e r a + A I l 1/~M + S a r a 10/~M
70.5 -+ 15.8 63.7±22.3 26.3± 5.3 42.5± 10.6 110.3±67.4
n 40-60
60-80
50.8 ± 24.3 61.2±22.0 17.9± 4.8 66.9-'- 9.1 b
43.6 ± 339.4± 777.5± 684.5±
52.9±22.4
349.4± 65.5
" P<0.02 compared to Vera (unpaired Student's t-test). b P<0.05 compared to the preceding fraction by paired Student's t-test. ¢ P<0.05 compared to Vera (unpaired Student's t-test).
21.2 83.6 121.6a 126.1 ~
4 6 8 6 6
249 minished b y a b o u t 35% in Brattleboro rats (which lack vasopressin) when c o m p a r e d to n o r m a l Long E v a n s rats suggesting the i n v o l v e m e n t of vasopressin. Access of circulating angiotensin II to the b r a i n is limited to those structures which lack the b l o o d - b r a i n barrier, i.e. the circumventricular organs i n c l u d i n g the m e d i u m eminence. Three of the circumventricular organs, the area postrema, the subfornical organ, a n d the o r g a n u m vasculosum, have already b e e n suggested to mediate various central effects of a n g i o t e n s i n (see: Simpson, 1981). The present results, coupled with the pertin e n t interest in vasopressin as a corticotropin-releasing factor (see: Fink, 1981), offer the m e d i a n e m i n e n c e as a site of action by which angiotensin m a y activate the h y p o t h a l a m o - p i t u i t a r y - a d r e n a l axis.
Acknowledgement Supported by DFG grant Me 541/3-1.
References Baertschi, A.J., H.H. Zingg and J.3. Dreifuss, 1981, Enkephalins, substance P, bradykinin and angiotensin II: d~fferential
sites of action on the hypothalamo-neurohypophysial system, Brain Res. 220, 107. Fink, G., 1981, Has corticotropin-releasing factor finally been found?, Nature 294, 511. Knepel, W., H. Anhut, D. Nutto and G. Hertting, 1980, Evidence that vasopressin is involved in the isoprenaline-induced fl-endorphin release, European J. Pharmacol. 68, 359. Knepel, W. and W. Reimann, 1982, Inhibition by morphine and/3-endorphin of vasopressin release evoked by electrical stimulation of the rat medial basal hypothalamus in vitro, Brain Res. (in press). Nordmann, J.J. and R.E.J. Dyball, 1978, Effects of veratridine on Ca fluxes and the release of oxytocin and vasopressin from the isolated rat neurohypophysis,J. Gen. Physiol. 72, 297. Simpson, J.B., 1981, The circumventricular organs and the central actions of angiotensin, Neuroendocrinology32, 248. Stillman, M.A., L.D. Recht, S.L. Rosario, S.M. Seif, A.G. Robinson and E.A. Zimmerman, 1977, The effects of adrenalectomy and glucocorticoid replacement on vasopressin and vasopressin-neurophysin in the zona externa of the median eminence of the rat, Endocrinology 101, 42. VanHouten, M., E.L. Schiffrin, J.F.E. Mann, B.I. Posner and R. Boucher, 1980, Radioautographic localization of specific binding sites for blood-borne angiotensin II in the rat brain, Brain Res. 186, 480. Yates, F.E., S.M. Russell, M.F. Dallman, G.A. Hedge, S.M. McCann and A.P.S. Dhariwal, 1971, Potentiation by vasopressin of corticotropin release induced by corticotropin-releasing factor, Endocrinology 88, 3. Zimmerman, B.G., 1981, Adrenergic facilitation by angiotensin: does it serve a physiological function?, Clin. Sci. 60, 343.
247
Elsevier Biomedical Press
Short communication VASOPRESSlN RELEASE FROM RAT MEDIAL BASAL HYPOTHALAMUS INDUCED IN VITRO BY AN GI OT EN S I N WILLHART KNEPEL, HENNING ANHUT, DORIS NUTTO, ANGELIKA HOLLAND and DIETER K. MEYER
Department of Pharmacology, University of Freiburg i. Br., Hermann-Herder-Str. 5, D- 7800 Freiburg i. Br., Federal Republic of Germany Received 11 March 1982, accepted 29 March 1982
W. KNEPEL~ H. ANHUT, D. NUTTO, A. HOLLAND and D.K. MEYER, Vasopressin release from rat medial basal hypothalamus induced in vitro by angiotensin, European J. Pharmacol. 80 (1982) 247-249. Rat medial basal hypothalami were superfused in vitro. The effect of angiotensin II on vasopressin outflow was investigated. Angiotensin II (10 nM or 1/xM, added to the superfusion medium) increased the veratridine-evoked vasopressin release. The higher concentration also slightly elevated the basal outflow. The effect of angiotensin II was blocked by saralasin. We conclude that angiotensin II can act on the median eminence and/or on the stump of the pituitary stalk to promote the release of vasopressin, which then may influence anterior pituitary hormone secretion.
Median eminence
Veratridine Vasopressinrelease
Angiotensin
I. Introduction
Peptide hormone release from the corticotrope cells of the anterior lobe of the pituitary gland is regulated by hypothalamic releasing factors which are secreted into the portal blood of the median eminence. In addition to catecholamines and a recently isolated ovine peptide, CRF-(1-41), vasopressin is also assumed to be one of these factors (see: Fink, 1981). Vasopressin is found in nerve terminals in the external layer of the median eminence adjacent to portal capillaries and possesses marked ACTH/fl-endorphin-releasing activity. Furthermore, several 'submaximum' stimuli for ACTH/fl-endorphin release in vivo have been shown to be mediated in part by vasopressin (Yates et al., 1971; Knepel et al., 1980). Although glucocorticoids (Stillman et al., 1977) and opiates (Knepel and Reimann, 1982) are assumed to participate, the control mechanisms of vasopressin release from the median eminence are poorly understood. The topographic correspondence of angiotensin II binding sites and immunoreactive vasopressin in the medial palisade zone of the zona externa (vanHouten et al., 1980) raises the possibility that vasopressinergic nerve 0014-2999/82/0000-0000/$02.75 © 1982 Elsevier Biomedical Press
terminals in this region are direct targets for angiotensin II. Therefore, we studied in vitro the effect of angiotensin II on vasopressin release from medial basal hypothalamus.
2. Materials and methods
The methods employed have been described in detail (Knepel and Reimann, 1982). Briefly, a slice of the basal hypothalamus (approximately 0.4 mm thick) of male Wistar rats (250-300 g) was cut. The so-called medial basal hypothalamus (MBH) was prepared from this slice by a frontal cut just rostral to the median eminence and one j u s t ~ u d a l to the pituitary stalk; the width was approximately 1.0 mm (wet weight 1.8 - 0 . 1 mg, vasopressin content 18.52-1.93 ng, n = 2 4 ) . Three MBH per chamber were superfused for 80 min (0.25 ml/min; 37°C) and 20 min samples collected for radioimmunoassay of vasopressin. The composition of the superfusion medium was as follows (mM): NaC1 118, KCl 4.8, CaCl 2 1.3, MgSO 4 1.2, NaHCO 3 25, KHEPO 4 1.2, glucose l l, bacitracin 0.07; 0.1% bovine serum albumin (saturated with 5% CO 2 in O:; pH 7.4). For stimulation of vasopressin re-
248
lease, veratridine (3/~M, Aldrich, U.S.A.) was added to the superfusion medium from the 60th to the 80th min of superfusion. The ceveratrum alkaloid veratridine is known to depolarize excitable cells by holding in an open state some of the sodium channels, which normally participate in the production of the action potential (see: Nordmann and Dyball, 1978, and references cited therein). Pilot studies demonstrated that vasopressin release by veratridine was reversible, related to the concentration used and depended on the presence of calcium (veratridine 3/~M - ED25). 5-11eangiotensin II was purchased from Serva, FRG.
3. Results
The results are shown in table 1. Vasopressin concentration in the superfusion medium decreased somewhat with time under control conditions, but was raised by veratridine ( P < 0 . 0 2 , paired Student's t-test). In the presence of angiotensin II 10 nM or 1/~M (added starting from the 40th rain of superfusion) veratridine was even more effective in elevating vasopressin outflow ( P < 0 . 0 2 or 0.05, respectively; unpaired Student's t-test). Angiotensin II 1 # M but not 10 nM also elevated the basal vasopressin concentration (P < 0.005 comparing the vasopressin outflow from the 40th to the 60th rain of superfusion with the outflow of the preced-
ing fraction by paired Student's t-test). The effect of angiotensin II was blocked by saralasin.
4. Discussion The results show that angiotensin II can increase both the evoked and, when added in higher concentrations, the basal vasopressin release from rat MBH in vitro, suggesting that activation of specific angiotensin receptors on terminals of vasopressin-containing neurones in the median enainence a n d / o r the stump of the pituitary stalk promotes vasopressin release. Similarly, presynaptic facilitatory angiotensin receptors on peripheral and central noradrenergic neurones have been described in the rabbit and dog (see: Zimmerman, 1981) and electrophysiological evidence has recently been presented for a direct action of angiotensin II on axons of rat oxytocin/vasopressincontaining neurones (Baertschi et al., 1981). The physiological function of the mechanism found in vitro in the present study remains to be defined. However, blood-borne angiotensin II induces the release of corticotropin and fl-endorphin from the anterior lobe of the pituitary gland (see: Simpson, 1981). And interestingly enough, the increase in plasma levels of fl-endorphin-like immunoreactivity elicited by intravenous angiotensin II was recently found to be published to be di-
TABLE 1 Vasopressin concentration (pg/fraction) in the superfusion medium of rat medial basal hypothalami. When indicated, veratridine (Vera, 3/~M) or 5-ile-angiotensin II (All) were added to the medium starting from the 60th or 40th rain of superfusion, respectively; saralasin (Sara) was present, when indicated, throughout superfusion. Values are means ± S.E.M. Min of superfusion 20-40 Controls Vera Vera+AII 10nM V e r a + A l I 1 ~M V e r a + A I l 1/~M + S a r a 10/~M
70.5 -+ 15.8 63.7±22.3 26.3± 5.3 42.5± 10.6 110.3±67.4
n 40-60
60-80
50.8 ± 24.3 61.2±22.0 17.9± 4.8 66.9-'- 9.1 b
43.6 ± 339.4± 777.5± 684.5±
52.9±22.4
349.4± 65.5
" P<0.02 compared to Vera (unpaired Student's t-test). b P<0.05 compared to the preceding fraction by paired Student's t-test. ¢ P<0.05 compared to Vera (unpaired Student's t-test).
21.2 83.6 121.6a 126.1 ~
4 6 8 6 6
249 minished b y a b o u t 35% in Brattleboro rats (which lack vasopressin) when c o m p a r e d to n o r m a l Long E v a n s rats suggesting the i n v o l v e m e n t of vasopressin. Access of circulating angiotensin II to the b r a i n is limited to those structures which lack the b l o o d - b r a i n barrier, i.e. the circumventricular organs i n c l u d i n g the m e d i u m eminence. Three of the circumventricular organs, the area postrema, the subfornical organ, a n d the o r g a n u m vasculosum, have already b e e n suggested to mediate various central effects of a n g i o t e n s i n (see: Simpson, 1981). The present results, coupled with the pertin e n t interest in vasopressin as a corticotropin-releasing factor (see: Fink, 1981), offer the m e d i a n e m i n e n c e as a site of action by which angiotensin m a y activate the h y p o t h a l a m o - p i t u i t a r y - a d r e n a l axis.
Acknowledgement Supported by DFG grant Me 541/3-1.
References Baertschi, A.J., H.H. Zingg and J.3. Dreifuss, 1981, Enkephalins, substance P, bradykinin and angiotensin II: d~fferential
sites of action on the hypothalamo-neurohypophysial system, Brain Res. 220, 107. Fink, G., 1981, Has corticotropin-releasing factor finally been found?, Nature 294, 511. Knepel, W., H. Anhut, D. Nutto and G. Hertting, 1980, Evidence that vasopressin is involved in the isoprenaline-induced fl-endorphin release, European J. Pharmacol. 68, 359. Knepel, W. and W. Reimann, 1982, Inhibition by morphine and/3-endorphin of vasopressin release evoked by electrical stimulation of the rat medial basal hypothalamus in vitro, Brain Res. (in press). Nordmann, J.J. and R.E.J. Dyball, 1978, Effects of veratridine on Ca fluxes and the release of oxytocin and vasopressin from the isolated rat neurohypophysis,J. Gen. Physiol. 72, 297. Simpson, J.B., 1981, The circumventricular organs and the central actions of angiotensin, Neuroendocrinology32, 248. Stillman, M.A., L.D. Recht, S.L. Rosario, S.M. Seif, A.G. Robinson and E.A. Zimmerman, 1977, The effects of adrenalectomy and glucocorticoid replacement on vasopressin and vasopressin-neurophysin in the zona externa of the median eminence of the rat, Endocrinology 101, 42. VanHouten, M., E.L. Schiffrin, J.F.E. Mann, B.I. Posner and R. Boucher, 1980, Radioautographic localization of specific binding sites for blood-borne angiotensin II in the rat brain, Brain Res. 186, 480. Yates, F.E., S.M. Russell, M.F. Dallman, G.A. Hedge, S.M. McCann and A.P.S. Dhariwal, 1971, Potentiation by vasopressin of corticotropin release induced by corticotropin-releasing factor, Endocrinology 88, 3. Zimmerman, B.G., 1981, Adrenergic facilitation by angiotensin: does it serve a physiological function?, Clin. Sci. 60, 343.