Endocrinology 96: 1063, 1975 Release of Vasopressin by Angiotensin TI L.C. Keil, J. Suirany-Long, and W.B. Severs2
Blom2.dU.caZ Rue.an.ck division, NASA, Amu Rue.an.ck Ce.nteA, Mo^eXt Fle.ld, Q.ati^oK.ni.a 94035 and Ve.pasitme.nt o^ Pharmacology, Pe.nnt>ylvania State. UniveJiA*£y Cotte.ge. o^ Me.cLLdnz, HeA&ke.y, Pennsylvania 17033 Abstract. To test the hypothesis that angiotensin II releases antidiuretic hormone (ADH) after injection into ventricular cerebrospinal fluid, conscious adult male SpragueDawley rats with a lateral cerebroventricular cannula received an intraventricular injection of 0, 10, 50, or 100 ng angiotensin II. Trunk blood was collected 90 seconds later for radioimmunoassay of ADH. Plasma ADH, pg/ml (mean ± S.E.), for the four dose levels were 2.8 ± 0.7, 9.6 ± 2.5, 22.6 ± 5.6 and 25.0 ± 5.0, respectively. The increases produced by angiotensin were statistically significant (p < 0.05). Plasma ADH of the 10 ng group was intermediate between control and the two highest angiotensin doses (p < 0.05), suggesting a dose-response relationship. These data provide direct evidence that angiotensin releases ADH by central mechanisms. Introduction. An experiment was designed to determine whether a central injection of angiotensin into conscious rats increases radioimmunoassayable vasopressin (ADH) in plasma. The results obtained provide direct evidence for ADH release by angiotensin. Although Malvin (1) states that angiotensin, among its other actions, is a direct stimulus to some area of the hypothalamic-pituitary axis, causing release of ADH, most evidence that angiotensin releases ADH in vivo by a central, mechanism is indirect (2-7). Few investigators assayed plasma levels of ADH after angiotensin administration jLn. vivo. Mouw et^ al. (8) reported that plasma ADH of dogs is elevated after intracarotid infusion or cerebroventricular perfusion of angiotensin. Shimizu et_ al. (9) were unable to observe an effect of intracarotid angiotensin on
plasma ADH in dogs but observed that the peptide potentiated ADH release by an osmotic stimulus. Two jLn_ vitro experiments with rat posterior pituitaries showed that angiotensin released ADH into the incubation medium (10,11). One report specified that intact hypothalamo-hypophysial tissue must be used (11). Methods. Adult male Sprague-Dawley rats (325-375 g) were anesthetized with sodium pentobarbital (40 mg/kg i p ) to allow implantation of a cannula (6) into the left lateral cerebroventricle. Three days later, between 8 and 10 A.M., the rats were given an intraventricular (IVT) injection containing 0, 10, 50 or 100 ng of val5-angiotensin II amide. The injection (5 yl) was made with a Hamilton syringe fitted with a stop so that the needle did not penetrate beyond the ventricular cannula. Angiotensin was dissolved in freshly prepared Merlis solution (12). Laboratory noise and animal handling were kept to a minimum. Doses were given in a random sequence. No water was allowed after the injection. Ninety seconds^ (by stopwatch) after the IVT injection the rats were sacrificed by decapitation. Trunk blood was collected into heparinized siliconized glass centrifuge tubes kept in ice water. Plasma was separated in a refrigerated centrifuge (150 x £, 20 min), transferred to siliconized glass containers and
Submitted January 23, 1975. Reprint requests to Dr. W.B. Severs, Hershey, Pennsylvania. 3 90 sec was selected as the sacrifice time based on analysis of the pressor response after IVT angiotensin (6) and a report that ADH half-life in rat plasma is about 3 min (13).
2
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Endo • 1975 Vol 96 • No 4
RAPID COMMUNICATIONS
frozen. Confirmation of the ventricular cannula placement was obtained post-mortem. These procedures were performed at the Hershey laboratory. On the following day the plasma samples were packed in dry ice and shipped by air express to the NASA facility at Moffett Field for ADH determination. On arrival, the frozen samples were kept at -20 C until assayed. There was no communication between laboratories concerning animal treatment or assay procedures until the results were obtained. Plasma ADH was determined by radioimmunoassay. Antibodies to vasopressin were produced in New Zealand white rabbits by repeated injections of a mixture of Freund's complete adjuvant and synthetic arginine vasopressin coupled to bovine serum albumin (14). Vasopressin was extracted from 1 ml aliquots of plasma with Bentonite (15). Each extract was reconstituted to 0.5 ml with buffer, and two 0.2 ml aliquots were then assayed. The method of vasopressin iodination and other details of the assay were similar to those reported by Husain et_ al. (16). Synthetic arginine vasopressin (262 ± 20 U/mg) obtained from Schwarz Mann Laboratories was used for antibody production, standards and iodination. The ADH concentration was expressed as pg of vasopressin per ml of plasma. Only 65 to 70% of added vasopressin could be recovered from the Bentonite extracted samples. The values shown in Table 1 have not been corrected for the hormone lost during extraction. Results. The plasma ADH concentrations after intracerebroventricular angiotensin appear in Table 1. Basal (0 ng) ADH concentration is low and similar to values from plasma of normally hydrated rats after their overnight consummatory cycle (L.C.K., unpublished observation). All 3 angiotensin doses increased plasma ADH concentrations (p < 0.05, t test). The 10 ng group was significantly different from all groups (p < 0.05) showing that angiotensin-
induced ADH release was a graded phenomenon. The plasma ADH concentration of the rats receiving 50 or 100 ng angiotensin was similar, indicating a maximal response. This level of ADH was similar to that seen in plasma from rats deprived of water for 48 h (L.C.K., unpublished observation). Thus, intracerebroventricular angiotensin releases large amounts of ADH rapidly, in normally hydrated animals. Table 1 Plasma Vasopressin Concentration After Intraventricular Angiotensin II Dose (ng)
plasma ADH (pg/ml±S.E.)
(n)//
0 10 50 100
2.8±0.7 9.6±2.5* 22.6±5.6*t 25.0±5.0*t
13 14
14 12
* p < 0.05 vs 0 ng, t test t p < 0.05 vs 10 ng, t test // number of animals Discussion. Angiotensin may have access to ventricular cerebrospinal fluid (17,18) and choroid plexi (17, 19). When exogenous angiotensin is injected into ventricular cerebrospinal fluid of conscious rats, spontaneous drinking occurs with doses as low as 1 ng (6), an effect recently confirmed (18). It has been suggested that ADH release occurs along with the drinking behavior (see 20 for review). However, most of these reports are based on indirect indices, such as blockade of pressor effects by supraoptic lesions (6) or antidiuresis and natriuresis (6,7). The data obtained in the present report show that angiotensin injection into ventricular cerebrospinal * fluid does indeed release ADH. A recent abstract from Guyton's laboratory (21) suggests that regulation of plasma sodium is controlled to a large extent by the ADH-thirst mechanism; and angiotensin appears to participate in the regulation of the ADH-thirst mechanism (1,5,20).
The Endocrine Society. Downloaded from press.endocrine.org by [${individualUser.displayName}] on 31 August 2015. at 10:43 For personal use only. No other uses without permission. . All rights reserved.
RAPID COMMUNICATIONS Malvin (1) has suggested that the renin-angiotensin system is involved in the moment-to-moment control of ADH secretion. Our results support this hypothesis in general, but the physiologic role(s) of endogenous angiotensin remains to be established.
9.
10.
11. References 1. Malvin, R.L., in Scow, R.O., (ed.), Int. Cong, of Endocrinology, Ath, Washington, D.C., 1972. Endocrinology; Proceedings, Excerpta Medica, I.C.S. No. 273, Amsterdam, 1973, p. 717. 2. Sakai, K.K., B.H. Marks, J. George, and A. Koestner, Life Sci 14:1337, 1974. 3. Nicoll, R.A., and J.L. Barker, Nature New Biol 223:172, 1971. 4. Andersson, B., and K. Olsson, Cond Reflex 8:147, 1973. 5. Andersson, B., and K. Olsson, in Scow, R.O., (ed.), Int. Cong, of Endocrinology, 4th, Washington, D.C., 1972. Endocrinology; Proceedings, Excerpta Medica, I.C.S. No. 273, Amsterdam, 1973, p. 724. 6. Severs, W.B., J. Summy-Long, J.S. Taylor, and J.D. Connor, J Pharm Exp Ther 174:27, 1970. 7. Severs, W.B., A. Daniels-Severs, J. Summy-Long, and G.J. Radio, Pharmacology 6:242, 1971. 8. Mouw, D., J. Bonjour, R.L. Malvin, and A. Vander, Am J Physiol 220:
12. 13. 14.
15.
16.
17. 18. 19. 20.
21.
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239, 1971. Shimizu, K., L. Share, and J.R. Claybaugh, Endocrinology 93:42, 1973. Gagnon, D.J., D. Cousineau, and P.J. Boucher Life Sci 12:487, 1973. and R.L. Malvin, Gregg, C M . , Physiologist 17:232, 1974 (Abstract). Merlis, J.K., Am J Physiol 131: 67, 1940. Aziz, 0., Pflugers Arch 311:373, 1969. Goodfriend, T.L., L. Levine, and G.D. Fasman, Science 144:1344, 1964. Skowsky, W.R., A.A. Rosenbloom, and D.A. Fisher, J Clin Endocrinol Metab 38:278, 1974. Husain, M.K., N. Fernando, M. Shapiro, A. Kagan, and S.M. Glick, J Clin Endocrinol Metab 37:616, 1973. Volicer, L., and C.G. Loew, Neuropharmacology 10:631, 1971. Johnson, A.K., and A.N. Epstein, Brain Res, in press. Richardson, J.B., and A. Beaulnes, J Cell Biol 51:419, 1971. Severs, W.B., and A. DanielsSevers, Pharmacol Rev 25:415, 1973. Young, D.B., and A.C. Guyton, Physiologist 17:364, 1974 (Abstract) .
The Endocrine Society. Downloaded from press.endocrine.org by [${individualUser.displayName}] on 31 August 2015. at 10:43 For personal use only. No other uses without permission. . All rights reserved.
Blom2.dU.caZ Rue.an.ck division, NASA, Amu Rue.an.ck Ce.nteA, Mo^eXt Fle.ld, Q.ati^oK.ni.a 94035 and Ve.pasitme.nt o^ Pharmacology, Pe.nnt>ylvania State. UniveJiA*£y Cotte.ge. o^ Me.cLLdnz, HeA&ke.y, Pennsylvania 17033 Abstract. To test the hypothesis that angiotensin II releases antidiuretic hormone (ADH) after injection into ventricular cerebrospinal fluid, conscious adult male SpragueDawley rats with a lateral cerebroventricular cannula received an intraventricular injection of 0, 10, 50, or 100 ng angiotensin II. Trunk blood was collected 90 seconds later for radioimmunoassay of ADH. Plasma ADH, pg/ml (mean ± S.E.), for the four dose levels were 2.8 ± 0.7, 9.6 ± 2.5, 22.6 ± 5.6 and 25.0 ± 5.0, respectively. The increases produced by angiotensin were statistically significant (p < 0.05). Plasma ADH of the 10 ng group was intermediate between control and the two highest angiotensin doses (p < 0.05), suggesting a dose-response relationship. These data provide direct evidence that angiotensin releases ADH by central mechanisms. Introduction. An experiment was designed to determine whether a central injection of angiotensin into conscious rats increases radioimmunoassayable vasopressin (ADH) in plasma. The results obtained provide direct evidence for ADH release by angiotensin. Although Malvin (1) states that angiotensin, among its other actions, is a direct stimulus to some area of the hypothalamic-pituitary axis, causing release of ADH, most evidence that angiotensin releases ADH in vivo by a central, mechanism is indirect (2-7). Few investigators assayed plasma levels of ADH after angiotensin administration jLn. vivo. Mouw et^ al. (8) reported that plasma ADH of dogs is elevated after intracarotid infusion or cerebroventricular perfusion of angiotensin. Shimizu et_ al. (9) were unable to observe an effect of intracarotid angiotensin on
plasma ADH in dogs but observed that the peptide potentiated ADH release by an osmotic stimulus. Two jLn_ vitro experiments with rat posterior pituitaries showed that angiotensin released ADH into the incubation medium (10,11). One report specified that intact hypothalamo-hypophysial tissue must be used (11). Methods. Adult male Sprague-Dawley rats (325-375 g) were anesthetized with sodium pentobarbital (40 mg/kg i p ) to allow implantation of a cannula (6) into the left lateral cerebroventricle. Three days later, between 8 and 10 A.M., the rats were given an intraventricular (IVT) injection containing 0, 10, 50 or 100 ng of val5-angiotensin II amide. The injection (5 yl) was made with a Hamilton syringe fitted with a stop so that the needle did not penetrate beyond the ventricular cannula. Angiotensin was dissolved in freshly prepared Merlis solution (12). Laboratory noise and animal handling were kept to a minimum. Doses were given in a random sequence. No water was allowed after the injection. Ninety seconds^ (by stopwatch) after the IVT injection the rats were sacrificed by decapitation. Trunk blood was collected into heparinized siliconized glass centrifuge tubes kept in ice water. Plasma was separated in a refrigerated centrifuge (150 x £, 20 min), transferred to siliconized glass containers and
Submitted January 23, 1975. Reprint requests to Dr. W.B. Severs, Hershey, Pennsylvania. 3 90 sec was selected as the sacrifice time based on analysis of the pressor response after IVT angiotensin (6) and a report that ADH half-life in rat plasma is about 3 min (13).
2
1063
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1064
Endo • 1975 Vol 96 • No 4
RAPID COMMUNICATIONS
frozen. Confirmation of the ventricular cannula placement was obtained post-mortem. These procedures were performed at the Hershey laboratory. On the following day the plasma samples were packed in dry ice and shipped by air express to the NASA facility at Moffett Field for ADH determination. On arrival, the frozen samples were kept at -20 C until assayed. There was no communication between laboratories concerning animal treatment or assay procedures until the results were obtained. Plasma ADH was determined by radioimmunoassay. Antibodies to vasopressin were produced in New Zealand white rabbits by repeated injections of a mixture of Freund's complete adjuvant and synthetic arginine vasopressin coupled to bovine serum albumin (14). Vasopressin was extracted from 1 ml aliquots of plasma with Bentonite (15). Each extract was reconstituted to 0.5 ml with buffer, and two 0.2 ml aliquots were then assayed. The method of vasopressin iodination and other details of the assay were similar to those reported by Husain et_ al. (16). Synthetic arginine vasopressin (262 ± 20 U/mg) obtained from Schwarz Mann Laboratories was used for antibody production, standards and iodination. The ADH concentration was expressed as pg of vasopressin per ml of plasma. Only 65 to 70% of added vasopressin could be recovered from the Bentonite extracted samples. The values shown in Table 1 have not been corrected for the hormone lost during extraction. Results. The plasma ADH concentrations after intracerebroventricular angiotensin appear in Table 1. Basal (0 ng) ADH concentration is low and similar to values from plasma of normally hydrated rats after their overnight consummatory cycle (L.C.K., unpublished observation). All 3 angiotensin doses increased plasma ADH concentrations (p < 0.05, t test). The 10 ng group was significantly different from all groups (p < 0.05) showing that angiotensin-
induced ADH release was a graded phenomenon. The plasma ADH concentration of the rats receiving 50 or 100 ng angiotensin was similar, indicating a maximal response. This level of ADH was similar to that seen in plasma from rats deprived of water for 48 h (L.C.K., unpublished observation). Thus, intracerebroventricular angiotensin releases large amounts of ADH rapidly, in normally hydrated animals. Table 1 Plasma Vasopressin Concentration After Intraventricular Angiotensin II Dose (ng)
plasma ADH (pg/ml±S.E.)
(n)//
0 10 50 100
2.8±0.7 9.6±2.5* 22.6±5.6*t 25.0±5.0*t
13 14
14 12
* p < 0.05 vs 0 ng, t test t p < 0.05 vs 10 ng, t test // number of animals Discussion. Angiotensin may have access to ventricular cerebrospinal fluid (17,18) and choroid plexi (17, 19). When exogenous angiotensin is injected into ventricular cerebrospinal fluid of conscious rats, spontaneous drinking occurs with doses as low as 1 ng (6), an effect recently confirmed (18). It has been suggested that ADH release occurs along with the drinking behavior (see 20 for review). However, most of these reports are based on indirect indices, such as blockade of pressor effects by supraoptic lesions (6) or antidiuresis and natriuresis (6,7). The data obtained in the present report show that angiotensin injection into ventricular cerebrospinal * fluid does indeed release ADH. A recent abstract from Guyton's laboratory (21) suggests that regulation of plasma sodium is controlled to a large extent by the ADH-thirst mechanism; and angiotensin appears to participate in the regulation of the ADH-thirst mechanism (1,5,20).
The Endocrine Society. Downloaded from press.endocrine.org by [${individualUser.displayName}] on 31 August 2015. at 10:43 For personal use only. No other uses without permission. . All rights reserved.
RAPID COMMUNICATIONS Malvin (1) has suggested that the renin-angiotensin system is involved in the moment-to-moment control of ADH secretion. Our results support this hypothesis in general, but the physiologic role(s) of endogenous angiotensin remains to be established.
9.
10.
11. References 1. Malvin, R.L., in Scow, R.O., (ed.), Int. Cong, of Endocrinology, Ath, Washington, D.C., 1972. Endocrinology; Proceedings, Excerpta Medica, I.C.S. No. 273, Amsterdam, 1973, p. 717. 2. Sakai, K.K., B.H. Marks, J. George, and A. Koestner, Life Sci 14:1337, 1974. 3. Nicoll, R.A., and J.L. Barker, Nature New Biol 223:172, 1971. 4. Andersson, B., and K. Olsson, Cond Reflex 8:147, 1973. 5. Andersson, B., and K. Olsson, in Scow, R.O., (ed.), Int. Cong, of Endocrinology, 4th, Washington, D.C., 1972. Endocrinology; Proceedings, Excerpta Medica, I.C.S. No. 273, Amsterdam, 1973, p. 724. 6. Severs, W.B., J. Summy-Long, J.S. Taylor, and J.D. Connor, J Pharm Exp Ther 174:27, 1970. 7. Severs, W.B., A. Daniels-Severs, J. Summy-Long, and G.J. Radio, Pharmacology 6:242, 1971. 8. Mouw, D., J. Bonjour, R.L. Malvin, and A. Vander, Am J Physiol 220:
12. 13. 14.
15.
16.
17. 18. 19. 20.
21.
1065
239, 1971. Shimizu, K., L. Share, and J.R. Claybaugh, Endocrinology 93:42, 1973. Gagnon, D.J., D. Cousineau, and P.J. Boucher Life Sci 12:487, 1973. and R.L. Malvin, Gregg, C M . , Physiologist 17:232, 1974 (Abstract). Merlis, J.K., Am J Physiol 131: 67, 1940. Aziz, 0., Pflugers Arch 311:373, 1969. Goodfriend, T.L., L. Levine, and G.D. Fasman, Science 144:1344, 1964. Skowsky, W.R., A.A. Rosenbloom, and D.A. Fisher, J Clin Endocrinol Metab 38:278, 1974. Husain, M.K., N. Fernando, M. Shapiro, A. Kagan, and S.M. Glick, J Clin Endocrinol Metab 37:616, 1973. Volicer, L., and C.G. Loew, Neuropharmacology 10:631, 1971. Johnson, A.K., and A.N. Epstein, Brain Res, in press. Richardson, J.B., and A. Beaulnes, J Cell Biol 51:419, 1971. Severs, W.B., and A. DanielsSevers, Pharmacol Rev 25:415, 1973. Young, D.B., and A.C. Guyton, Physiologist 17:364, 1974 (Abstract) .
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