Stimulation
of Growth Hormone and Corticotropin Angiotensin II in Man
E.C. degli Uberti,
G. Trasforini,
A. Margutti,
R. Rossi, M.R. Ambrosio,
Release by and R. Pansini
The intravenous (IV) infusion of angiotensin II (All) was administered to seven healthy male volunteers in a randomized placebo-controlled study. As expected, All induced a significant increase in blood pressure and plasma aldosterone concentrations. All caused a significant increase in corticotropin (ACTH) and growth hormone (GH) release, but had no effect on the release of thvrotropin . . (TSHI and prolactin (PRL). These findings suggest that peripherally circulating All might influence ACTH and GH secretion in humans. @ 1990 by W.B. Saunders Company.
A
NGIOTENSIN II (AH) is recognized as exerting a number of actions in the central nervous system (CNS) in addition to its well-defined mineralocorticosteroidstimulating and vasoconstrictor effects.’ The central actions of AI1 include effects on thirst and fluid intake,2 blood pressure regulation3 and release of pituitary hormones4-* The demonstration of the existence of the brain reninangiotensin system’ and of a relatively high concentration of AI1 receptors in both CNS and pituitary gland” has raised the possibility that the AH could have an important neuroendocrine role and be involved in the control of pituitary function. However, it is not yet clear whether the several actions of AI1 on pituitary function are only due to a direct effect of centrally generated octapeptide on the pituitary or may also be attributed to peripherally circulating AII. At present, the information concerning the effects of peripherally generated AI1 on adenohypophyseal hormone levels in humans is sparse. This study was designed to analyze in seven normal subjects the effects of AI1 infusion in different doses on plasma concentrations of growth hormone (GH), corticotropin (ACTH), thyrotropin (TSH), prolactin (PRL), cortisol (PC), and aldosterone (PA). In such a way it is possible to investigate whether elevations in plasma levels of AI1 able to induce significant increase in blood pressure and PA concentrations can influence the secretion of pituitary hormones. MATERIALS AND METHODS
Seven normal men, age 19 to 35 years, volunteered to participate in the studies. Informed consent was obtained. The subjects were within 12% of ideal body weight and were not receiving any medication. All were admitted to the Clinical Center at least 2 days before the study and were kept on a constant diet containing 90 mEq sodium and 60 mEq potassium per day. The use of alcohol, tobacco, and caffeine-containing food was prohibited. Each subject was tested receiving 60-minute infusions of AH (Aspl,IleS-angiotensin II, Hypertensin, Ciba-Geigy, Varese, Italy) in successively increasing doses of 4,8, and 16 ng/kg/min, each dose for 20 minutes (AI1 test), and a 60-minute 0.9% saline infusion (control test), in random sequence with a 4-day interval between the two tests. The subjects were studied after an overnight fast. They were in the supine position for at least 10 hours before the test and remained so until completion of the procedure. Beginning at 7:30 AM on the test day, a 19-gauge needle was placed in each antecubital vein and was kept patent with a slow infusion of 0.9% sodium chloride. One needle was for the infusion (AI1 or saline), and the other was used to obtain blood samples. At 8:30 AM a 60-minute infusion of AI1 or 0.9% saline
Metabolism, Vol39, No 10 (October). 1990: pp 1063-1067
solution was administered. Blood samples were drawn at - 15, 0, and 30,60,90, 120, 150, and 180 minutes after the beginning of the AI1 or saline infusion into tubes containing EDTA-2Na (1.5 mg/mL) and immediately centrifuged at 4OC for 10 minutes; tubes for ACTH also contained Trasylol (100 IU/mL, Bayer, Milano, Italy). Plasma was then frozen and stored at - 30°C until assay. All samples for each hormone from a single subject were processed in duplicate in the same assay. PRL, GH, TSH, ACTH, PC, and PA were measured by radioimmunoassay using commercial kits. The intraassay and interassay coefficients of variation were, respectively: PRL, 4.2% and 5.6%; GH, 6.5% and 11%; TSH, 5% and 4.3%; ACTH, 12.1% and 15%; PC, 6.2% and 9%; and PA, 8.3% and 12.7%. All infusions were performed with a volumetric infusion pump (IMED 922, Oxon, England). Blood pressure was recorded throughout the period of each study every 10 minutes by means of a quasiportable oscillometric instrument, model BP-203Y, manufactured by Nippon Cohn, Ltd (Komaki, Japan) provided with an automatically inflatable cuff. A nurse and physician were present for all infusions. The circulating concentration of each hormone was expressed as the mean (*SEM) values at various time intervals. Statistical evaluation of the data was achieved using both paired and unpaired Student’s t test and ANOVA, as applicable. RESULTS Responses of BP and PA to AII
During AI1 infusion, a slight, but significant, increase in systolic and diastolic blood pressure occurred (Fig I), thus confirming the biological activity of the peptide used. Mean systolic blood pressure increased from the basal level of 104 2 3.4 mm Hg to 126 + 1.2 mm Hg at 30 minutes and to 120 * 7 mm Hg at 60 minutes, with a statistically significant (P-c .Ol, P < .05) difference from the control values (observed during saline infusion). Mean diastolic blood pressure increased from the basal value of 62 ? 2.2 mm Hg to peak values of 75 f 1.8 mm Hg at 30 minutes (P < .05 v control value). As expected, PA increased in all the subjects from the mean basal value (mean of the values obtained at - 30 and 0 minutes) of 7.1 + 1.6 ng/dL to a peak of 43.5 + 4 ng/dL 60 minutes after the beginning of the AI1 infusion (Fig 2).
From the Instirute of Clinical Medicine, University of Ferrara, Ferrara, Italy. Address reprint requests to Professor E.C. degli Uberti. Institute of Clinical Medicine, University of Ferrara, I-44100 Ferrara, Italy. @ 1990 by W.B. Saunders Company. 00260495/90/391 O-001 I %3.00/O
1063
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[min]
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Fig 1. Changes in blood pressure during IV infusion of saline (O----O) and All at successively increasing doses of 4. 8, and 16 Means + SEM are shown. Significances: nglkg * min f-1. l*P c .Ol, lP -c .05 compared with saline control.
Fig 3. Changes in plasma ACTH and cortisol during IV infusion of saline (O---O) and All at various doses W--W. Means + SEM are shown. Significances: ***P -c .OOl. l*P -c .Ol, lP -c .05 compared with saline control.
Responses of ACTH and PC
Responses of GH, PRL, and TSH
The administration of AI1 resulted in a significant increase in mean ACTH levels compared with those after saline control infusion (Fig 3). ACTH levels increased from the mean basal value of 23 + 4.7 pg/mL to a maximum of 65 * 12 pg/mL at 150 minutes, and remained significantly elevated (P -c .05) up to 180 minutes. AI1 infusion induced a progressive and slight increase in PC, reaching significantly higher levels than those obtained during saline infusion at 90 (P < .05) and 120 minutes (P < .Ol) (Fig 3).
Infusion of AI1 did not change the plasma concentrations of both PRL and TSH (Fig 4B and C). Conversely, plasma GH concentrations increased from the mean basal value of 0.8 f 0.2 ng/mL to the peak value of 6.5 + 2.5 ng/mL at 60 minutes after the end of the AI1 infusion, remaining significantly higher than during saline infusion at 150 (P < .02) and 180 minutes (P c; .05), respectively. No subject complained of physical or mental discomfort during the entire procedure. DISCUSSION
56,
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Fig 2. Changes in plasma aldosterone during IV infusion of saline (O----O) and All at various doses (0-W. Means * SEM are shown. Significances: l**P < 001, l*P c .05, lP < .Q2 compared with saline control.
Our study shows that intravenous (IV) infusion of AI1 in man significantly increased plasma levels of ACTH. Previous in vivo and in vitro experimental studies have indicated that in animals, AI1 has the capacity to elicit ACTH release5~“-‘8 and have also suggested that in vivo central mechanisms are primarily involved in mediating the ACTH-stimulating activity of AII, probably increasing secretion of corticotropinreleasing hormone (CRH). I9 At present, there is little and controversial information on the effect of AI1 in the regulation of ACTH release in humans. In fact, both the stimulatory and inhibitory action of AI1 on ACTH release has been reported.2@22In addition, Gaillard et al,” with an IV infusion of 5 or 7 ng/kg/min of AI1 during 2 hours, did not find any effect on ACTH levels in normal male subjects. We are unable to account for such a discrepancy between our results and those of Gaillard et al.” A plausible explanation may be that the dose of AI1 infused IV in our study is higher than that in Gaillard’s study. This appears reasonable, because circulating AI1 is known to penetrate the blood-brain barrier to a limited degree, and although it has been shown that acute hypertension resulting
1065
GH AND ACTH RELEASE INDUCED BY All IN MAN
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The present investigation indicates that in man, IV AI1 administration is associated with a significant increment in plasma GH levels. There are a few studies concerning the effect of AI1 on GH secretion in animals, but the results are conflicting. In the ovariectomized rat, plasma levels of GH were reduced after intraventricular, as well as systemic administration of AII.7,8 On the other hand, whereas Aguilera et al6 did not find any effect of AI1 on GH release in cultured rat pituitary cells, recently Robberecht and Denef*’ reported that AI1 exerted both a stimulatory and an inhibitory effect on GH release in an vitro anterior pituitary cell preparation, suggesting that the dual action of AI1 seemed to be dependent on the developmental stage of the animal and the hormonal environment. Our data do not allow us to state the mechanism and the site of action of GH-releasing effect of AIL In addition to a direct pituitary site of action, a possible involvement of the CNS in mediating the effect of AI1 on GH secretion in man may be suggested. In this regard, there is a large body of evidence indicating numerous interaction between reninangiotensin system and the CNS.’ Peripheral administration of AI1 leads to increase in the serotonin content of the hypothalamus and brain stem of dogs28 and the activation of the sympathetic nervous system.29 These studies might support the suggestion that the GH-stimulating activity of AI1 in man can be attributed to an action of this octopeptide on the central neurotransmitter system involved in controlling GH release. Nevertheless, further investigations are needed to elucidate this point. In order to maintain a strict control on possible side effects, we administered scalar doses of AI1 to our subjects. The ensuing slow increase of plasma levels might have contributed to the delay observed in the response of GH and ACTH. The delay could also reflect the indirect kind of the regulatory action of AI1 on GH and ACTH secretion. In other terms, AI1 could be more a modulator of the multifactorial complex involved in regulating the release of GH and ACTH than a direct stimulator of GH and ACTH secretion. Furthermore, the pressor effect of AI1 can counteract and delay to some extent the secretory action of this peptide, as suggested previous studies.‘6,25.26 Since acute hypertension may represent a stressful stimulus for the conscious man, capable of inducing secretion of stress-sensitive hormones, we cannot rule out the possibility that the parallel increases in ACTH and GH levels observed after AI1 infusion are not specific and could be related to a possible physical or mental discomfort. However, the specificity of AI1 effect can be supported by the finding that no concomitant increase in PRL, another stress-sensitive hormone, was ever observed and that the subjects did not complain of any significant unpleasant side effect during AI1 infusion. Finally, it still remains to be determined whether peripheral changes in blood-borne AI1 may really contribute to modulating ACTH and GH secretion in humans, and whether the AII-induced effects are of any physiological significance since the AI1 circulating concentrations reached in our study may be too high and not within the physiological range. Therefore, further studies are needed to place the observed
1
-
1.5
c
0
1
0
30
60
90
120
150
180
TIME (min)
Fig 4. Changes in plasma GH, PRL, and TSH during IV infusion of saline (O---O) and All at various doses W--O). Means + SEM are shown. Significances: l*f < .02, lP -c .05 compared with saline control.
from AI1 injection can open the blood-brain barrier,23 only a small fraction of the IV-injected AI1 would reach the effective site to induce ACTH release. In fact, AI1 has been shown to induce a dose-related ACTH release in vitro and in vivo. ‘2~‘3~‘5~‘4 Thus, it would appear that the dose of AI1 employed in our study is close to the one which is sufficient to effectively induce changes in ACTH release. Our results do not allow us to comment on either the exact mechanism or the main site of action of AIL However, it is likely that systemically administered AI1 can elicit ACTH release also in humans primarily by a mechanism involving the CNS.“,‘6 The possibility that ACTH release might be mediated by the central hypertensive effect of AI1 cannot be completely excluded. However, it has been demonstrated in conscious dogs that ACTH secretion is inhibited by increases in blood pressure25,26 and that the pressor response to AI1 can counteract the AII-stimulated ACTH secretioni Furthermore, it has been shown that exogenous AI1 is capable of maintaining its ACTH-stimulating activity when the pressor action is eliminated with the simultaneous administration of the vasodilator nitroprusside.16 The possibility that circulating AI1 may increase ACTH secretion by acting through AI1 receptors on the circumventricular organs, areas where the blood-brain-barrier is deficient, cannot be excluded. However, the physiological role of these intracerebral receptors in the regulation of ACTH secretion remains still uncertain and awaits experimental elucidation in man.
1066
DEGLI UBERTI ET AL
increase in plasma ACTH and GH in humans in its proper biological perspective. In contrast to the positive effects on plasma ACTH and GH, no effect of AI1 on TSH was found. In this regard, our results are in keeping with those of Steele et al,’ who have shown that AI1 injected intraventricularly or IV into ovariectomized rats had no effect on plasma TSH levels. However, these findings are in contrast with the report3’ that AI1 IV or intraperitoneally injected in rat caused elevation of plasma TSH levels and significantly enhanced the plasma TSH responses to cold and TRH. At present, the reasons for these descrepancies are unclear. Nevertheless, Steele et al’ reported that the in vitro incubation of hemipituitaries with large doses of AI1 increased TSH levels. These researchers postulated that AI1 may have a stimulatory effect on TSH secretion by acting directly at the pituitary level and explained the lack of action of intraventricular and IV AI1 on TSH secretion by suggesting that in those cases a simultaneous release of somatostatin could have blunted the TSH response to AII. However, in our study, the finding of the GH response to AI1 argues against this hypothesis. Plasma PRL concentrations were not significantly affected by IV AD. This result confirms our previous report3’ that IV infusion of AI1 does not produce any consistent change in plasma PRL in normal women. These data taken together indicate that peripherally generated AI1 may not play an important role in modulating PRL secretion, even if AI1 reaches plasma levels able to induce a significant increase in
blood pressure and PA concentration. Dufy-Barbe et aL3* using the conscious ovariectomized rhesus monkey, reported increase of PRL release after intraventricular and IV administration of AII. Steele et a17.8have shown that in rats IV-injected AI1 caused a slight increase followed by a decrement in PRL concentrations, whereas intraventricularly injected AI1 depressed plasma levels of PRL. Furthermore, there still remains the controversial question of whether the action of AI1 on PRL might be attributed prevalently to a direct local effect of AI1 on the pituitary, rather than to a central effect, since in vitro studies indicate that AI1 may stimulate PRL release acting directly on anterior pituitary cells.6,7 It seem unlikely that in our study the AI1 failure to influence plasma PRL and TSH concentration can be attributed to the use of an inadequate dose of octapeptide or to the route of administration, since AI1 was able to stimulate ACTH and GH secretion with a similar protocol. However, we cannot exclude that the dose required at the CNS level to influence the secretion of both TSH and PRL must be higher than the one we used. In conclusion, we have shown that in man IV administration of AI1 produces a significant increase in plasma ACTH and GH levels, without modifying the plasma concentration of TSH and PRL. Nevertheless, more studies are necessary to clarify the involvement of AI1 in the modulation of pituitary hormone release, as well as its physiological significance.
REFERENCES
1. Phillips MI: Functions of angiotensin in the central nervous system. Ann Rev Physiol49:413-435, 1987 2. Simonnet G, Rodriguez R, Furnoux F, et al: Vasopressin release and drinking induced by intracranial injection of AI1 in monkey. Am J Physiol237:R20-R25,1979 3. Ferrario CM, Dickinson CJ, McCubbin JW: Central vasomotor stimulation by angiotensin II. Clin Sci 39:239-244, 1970 4. Keil LC, Summy-Long H, Severs WB: Release of vasopressin by angiotensin II. Endocrinology 96:1063-1065,1975 5. Ramsay DJ, Keil LC, Shape MC, et al: Angiotensin II infusion increases vasopressin, ACTH and 1I-hydroxycorticoid secretion. Am J Physio1234:R66-R7 1, 1978 6. Aguilera G, Hyde CL, Catt KJ: Angiotensin II receptors and prolactin release in pituitary lactotrophs. Endocrinology 111:10451050,1982 7. Steele MK, Negro-Vilar A, McCann SM: Effect of angiotensin II on in vivo and in vitro releases of anterior pituitary hormones in the female rat. Endocrinology 109:893-899, 1981 8. Steele MK, McCann SM. Negro-Vilar A: Modulation by dopamine and estradiol of the central effects of angiotensin II on anterior pituitary hormone release. Endocrinology 111:722-729, 1982 9. Ganten D, Fuxe K, Phillips MI, et al: The brain isoreninangiotensin system: Histochemistry, localization and possible role in drinking and blood pressure regulation, in Ganong WF, Ganten D (eds): Frontiers in Neuroendocrinology. New York, NY, Karger, 1978, pp 61-99 10. Hauger RL, Aguilera G, Baukal A, et al: Characterization of angiotensin II receptors in the anterior pituitary gland. Mol Cell Endocrino125:203-213, 1982 11. Maran HW, Yates EF: Cortisol secretion during intrapitu-
itary infusion of angiotensin II in conscious dogs. Am J Physiol 233:E273-E285,1977 12. Gaillard RC, Grossman A, Gillies G, et al: Angiotensin II stimulates the release of ACTH from dispersed rat anterior pituitary cells. Clin Endocrinol (Oxford) 15:573-578, 1981 13. Spinedi E, Negro-Vilar A: Angiotensin II and ACTH release: Site of action and potency relative to corticotropin releasing factor and vasopressin. Neuroendocrinology 37:446-453, 1983 14. Sobel DO: Characterization of angiotensin-mediated ACTH release. Neuroendocrinology 36:249-253, 1983 15. Gaillard RC, Favrod-Coune A, Capponi AM, et al: Corticotropin-releasing activity of the renin-angiotensin system peptides in rat and in man. Neuroendocrinology 41:511-517, 1985 16. Brooks VL, Reid IA: Interaction between angiotensin II and the baroreceptor reflex in the control of adrenocroticotropic hormone secretion and heart rate in conscious dogs. Circ Res 58:816-828, 1986 17. Murakami K, Ganong WF: Site at which angiotensin II acts to stimulate ACTH secretion in vivo. Neuroendocrinology 46:23 l235,1987 18. Schoenenberg P, Kehrer P, Muller AF, et al: Angiotensin II potentiates corticotropin-releasing activity of CRF-41 in rat anterior pituitary cells: Mechanism of action. Neuroendocrinology 45:86-90, 1987 19. Ganong WF, Murakami K: The role of angiotensin II in the regulation of ACTH secretion. Ann NY Acad Sci 512:176-186, 1987 20. Rayys SS, Horton R: Effect of angiotensin II on adrenal and pituitary function in man. J Clin Endocrinol Metab 32:539-546, 1971 21. Semple PF, Buckingham JC, Mason PA, et al: Suppression of
GH AND ACTH RELEASE INDUCED BY All IN MAN
plasma ACTH concentration by angiotensin II infusion in normal humans and in a subject with a steroid 17-alfa-hydroxylase defect. Clin Endocrinol (Oxford) 10:137-144, 1979 22. Mason PA, Fraser R, Semple PF, et al: The interaction of the ACTH and angiotensin II in the control of corticosteroid plasma concentration in man. J Steroid Biochem 10:235-239, 1979 23. Johansson B, Li CH, Olsson Y, et al: The effect of acute arterial hypertension on the blood brain barrier to protein tracers. Acta Neuropathol 16:117-124, 1970 24. Keller-Wood M, Kimura B, Shinsako J, et al: Interaction between CRF and angiotensin II in control of ACTH and adrenal steroids. Am J Physiol250:R396-R402, 1986 25. Lorenzen LC, Ganong WF: Effect of drugs related to L-ethyltryptamine stress-induced ACTH secretion in the dog. Endocrinology 80:889-892, 1967 26. Gann DS, Ward DG, Carlson DE: Neural control of ACTH: A homeostatic reflex. Ret Prog Hormone Res 34:357-400, 1978 27. Robberecht W, Denef C: Stimulation and inhibition of
1067
pituitary growth hormone release by angiotensin II in vitro. Endocrinology 122:1496- 1504, 1988 28. Haulica I, Petrescu G, Stratone A, et al: Possible functions of brain renin, in Ganten D, Printz M, Phillips MI, et al (eds): The Renin Angiotensin System in the Brain. Heidelberg, FRG, SpringerVerlag, 1982, pp 335-342 29. Sumners C, Phillips MI: Central injection of angiotensin II alters catecholamine activity in rat brain. Am J Physiol 244:R257R265,1983 30. Mannisto PT. Kokkonen J, Ranta T: Effects of acute hypotension and hypertension on serum TSH concentrations in male rats. Acta Physiol Stand 107:105-107, 1979 31. degli Uberti EC, Trasforini G, Margutti A, et al: Failure of angiotensin II to affect prolactin concentration in normal women. Neuroendocrinol Lett 6:163-167, 1984 32. Dufy-Barbe L, Rodriguez F, Arsaut J, et al: Angiotensin II stimulates prolactin release in the rhesus monkey. Neuroendocrinology 35:242-247,1982
of Growth Hormone and Corticotropin Angiotensin II in Man
E.C. degli Uberti,
G. Trasforini,
A. Margutti,
R. Rossi, M.R. Ambrosio,
Release by and R. Pansini
The intravenous (IV) infusion of angiotensin II (All) was administered to seven healthy male volunteers in a randomized placebo-controlled study. As expected, All induced a significant increase in blood pressure and plasma aldosterone concentrations. All caused a significant increase in corticotropin (ACTH) and growth hormone (GH) release, but had no effect on the release of thvrotropin . . (TSHI and prolactin (PRL). These findings suggest that peripherally circulating All might influence ACTH and GH secretion in humans. @ 1990 by W.B. Saunders Company.
A
NGIOTENSIN II (AH) is recognized as exerting a number of actions in the central nervous system (CNS) in addition to its well-defined mineralocorticosteroidstimulating and vasoconstrictor effects.’ The central actions of AI1 include effects on thirst and fluid intake,2 blood pressure regulation3 and release of pituitary hormones4-* The demonstration of the existence of the brain reninangiotensin system’ and of a relatively high concentration of AI1 receptors in both CNS and pituitary gland” has raised the possibility that the AH could have an important neuroendocrine role and be involved in the control of pituitary function. However, it is not yet clear whether the several actions of AI1 on pituitary function are only due to a direct effect of centrally generated octapeptide on the pituitary or may also be attributed to peripherally circulating AII. At present, the information concerning the effects of peripherally generated AI1 on adenohypophyseal hormone levels in humans is sparse. This study was designed to analyze in seven normal subjects the effects of AI1 infusion in different doses on plasma concentrations of growth hormone (GH), corticotropin (ACTH), thyrotropin (TSH), prolactin (PRL), cortisol (PC), and aldosterone (PA). In such a way it is possible to investigate whether elevations in plasma levels of AI1 able to induce significant increase in blood pressure and PA concentrations can influence the secretion of pituitary hormones. MATERIALS AND METHODS
Seven normal men, age 19 to 35 years, volunteered to participate in the studies. Informed consent was obtained. The subjects were within 12% of ideal body weight and were not receiving any medication. All were admitted to the Clinical Center at least 2 days before the study and were kept on a constant diet containing 90 mEq sodium and 60 mEq potassium per day. The use of alcohol, tobacco, and caffeine-containing food was prohibited. Each subject was tested receiving 60-minute infusions of AH (Aspl,IleS-angiotensin II, Hypertensin, Ciba-Geigy, Varese, Italy) in successively increasing doses of 4,8, and 16 ng/kg/min, each dose for 20 minutes (AI1 test), and a 60-minute 0.9% saline infusion (control test), in random sequence with a 4-day interval between the two tests. The subjects were studied after an overnight fast. They were in the supine position for at least 10 hours before the test and remained so until completion of the procedure. Beginning at 7:30 AM on the test day, a 19-gauge needle was placed in each antecubital vein and was kept patent with a slow infusion of 0.9% sodium chloride. One needle was for the infusion (AI1 or saline), and the other was used to obtain blood samples. At 8:30 AM a 60-minute infusion of AI1 or 0.9% saline
Metabolism, Vol39, No 10 (October). 1990: pp 1063-1067
solution was administered. Blood samples were drawn at - 15, 0, and 30,60,90, 120, 150, and 180 minutes after the beginning of the AI1 or saline infusion into tubes containing EDTA-2Na (1.5 mg/mL) and immediately centrifuged at 4OC for 10 minutes; tubes for ACTH also contained Trasylol (100 IU/mL, Bayer, Milano, Italy). Plasma was then frozen and stored at - 30°C until assay. All samples for each hormone from a single subject were processed in duplicate in the same assay. PRL, GH, TSH, ACTH, PC, and PA were measured by radioimmunoassay using commercial kits. The intraassay and interassay coefficients of variation were, respectively: PRL, 4.2% and 5.6%; GH, 6.5% and 11%; TSH, 5% and 4.3%; ACTH, 12.1% and 15%; PC, 6.2% and 9%; and PA, 8.3% and 12.7%. All infusions were performed with a volumetric infusion pump (IMED 922, Oxon, England). Blood pressure was recorded throughout the period of each study every 10 minutes by means of a quasiportable oscillometric instrument, model BP-203Y, manufactured by Nippon Cohn, Ltd (Komaki, Japan) provided with an automatically inflatable cuff. A nurse and physician were present for all infusions. The circulating concentration of each hormone was expressed as the mean (*SEM) values at various time intervals. Statistical evaluation of the data was achieved using both paired and unpaired Student’s t test and ANOVA, as applicable. RESULTS Responses of BP and PA to AII
During AI1 infusion, a slight, but significant, increase in systolic and diastolic blood pressure occurred (Fig I), thus confirming the biological activity of the peptide used. Mean systolic blood pressure increased from the basal level of 104 2 3.4 mm Hg to 126 + 1.2 mm Hg at 30 minutes and to 120 * 7 mm Hg at 60 minutes, with a statistically significant (P-c .Ol, P < .05) difference from the control values (observed during saline infusion). Mean diastolic blood pressure increased from the basal value of 62 ? 2.2 mm Hg to peak values of 75 f 1.8 mm Hg at 30 minutes (P < .05 v control value). As expected, PA increased in all the subjects from the mean basal value (mean of the values obtained at - 30 and 0 minutes) of 7.1 + 1.6 ng/dL to a peak of 43.5 + 4 ng/dL 60 minutes after the beginning of the AI1 infusion (Fig 2).
From the Instirute of Clinical Medicine, University of Ferrara, Ferrara, Italy. Address reprint requests to Professor E.C. degli Uberti. Institute of Clinical Medicine, University of Ferrara, I-44100 Ferrara, Italy. @ 1990 by W.B. Saunders Company. 00260495/90/391 O-001 I %3.00/O
1063
DEGLI
1064
140
-i ;
-
ET AL
66 -
602 4: 46 H : 20 0 :
Ys I E120
-
E w 5100 w c/J w
10 -
," 80
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+
a s &
UBERTI
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/
01
I
I
I
I
I
1
I
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60
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[min]
5 :
16-
d t
12-
5 u
6-
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60
90
‘1
120
150
160
TIME (Inin)
Fig 1. Changes in blood pressure during IV infusion of saline (O----O) and All at successively increasing doses of 4. 8, and 16 Means + SEM are shown. Significances: nglkg * min f-1. l*P c .Ol, lP -c .05 compared with saline control.
Fig 3. Changes in plasma ACTH and cortisol during IV infusion of saline (O---O) and All at various doses W--W. Means + SEM are shown. Significances: ***P -c .OOl. l*P -c .Ol, lP -c .05 compared with saline control.
Responses of ACTH and PC
Responses of GH, PRL, and TSH
The administration of AI1 resulted in a significant increase in mean ACTH levels compared with those after saline control infusion (Fig 3). ACTH levels increased from the mean basal value of 23 + 4.7 pg/mL to a maximum of 65 * 12 pg/mL at 150 minutes, and remained significantly elevated (P -c .05) up to 180 minutes. AI1 infusion induced a progressive and slight increase in PC, reaching significantly higher levels than those obtained during saline infusion at 90 (P < .05) and 120 minutes (P < .Ol) (Fig 3).
Infusion of AI1 did not change the plasma concentrations of both PRL and TSH (Fig 4B and C). Conversely, plasma GH concentrations increased from the mean basal value of 0.8 f 0.2 ng/mL to the peak value of 6.5 + 2.5 ng/mL at 60 minutes after the end of the AI1 infusion, remaining significantly higher than during saline infusion at 150 (P < .02) and 180 minutes (P c; .05), respectively. No subject complained of physical or mental discomfort during the entire procedure. DISCUSSION
56,
;pif
l:*
46 5 40
-
z = 32 r z
34 -
e $
16 -
9 4
6-
OJ
,
I
0
30
60
90
120
150
180
TIME (r&I>
Fig 2. Changes in plasma aldosterone during IV infusion of saline (O----O) and All at various doses (0-W. Means * SEM are shown. Significances: l**P < 001, l*P c .05, lP < .Q2 compared with saline control.
Our study shows that intravenous (IV) infusion of AI1 in man significantly increased plasma levels of ACTH. Previous in vivo and in vitro experimental studies have indicated that in animals, AI1 has the capacity to elicit ACTH release5~“-‘8 and have also suggested that in vivo central mechanisms are primarily involved in mediating the ACTH-stimulating activity of AII, probably increasing secretion of corticotropinreleasing hormone (CRH). I9 At present, there is little and controversial information on the effect of AI1 in the regulation of ACTH release in humans. In fact, both the stimulatory and inhibitory action of AI1 on ACTH release has been reported.2@22In addition, Gaillard et al,” with an IV infusion of 5 or 7 ng/kg/min of AI1 during 2 hours, did not find any effect on ACTH levels in normal male subjects. We are unable to account for such a discrepancy between our results and those of Gaillard et al.” A plausible explanation may be that the dose of AI1 infused IV in our study is higher than that in Gaillard’s study. This appears reasonable, because circulating AI1 is known to penetrate the blood-brain barrier to a limited degree, and although it has been shown that acute hypertension resulting
1065
GH AND ACTH RELEASE INDUCED BY All IN MAN
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The present investigation indicates that in man, IV AI1 administration is associated with a significant increment in plasma GH levels. There are a few studies concerning the effect of AI1 on GH secretion in animals, but the results are conflicting. In the ovariectomized rat, plasma levels of GH were reduced after intraventricular, as well as systemic administration of AII.7,8 On the other hand, whereas Aguilera et al6 did not find any effect of AI1 on GH release in cultured rat pituitary cells, recently Robberecht and Denef*’ reported that AI1 exerted both a stimulatory and an inhibitory effect on GH release in an vitro anterior pituitary cell preparation, suggesting that the dual action of AI1 seemed to be dependent on the developmental stage of the animal and the hormonal environment. Our data do not allow us to state the mechanism and the site of action of GH-releasing effect of AIL In addition to a direct pituitary site of action, a possible involvement of the CNS in mediating the effect of AI1 on GH secretion in man may be suggested. In this regard, there is a large body of evidence indicating numerous interaction between reninangiotensin system and the CNS.’ Peripheral administration of AI1 leads to increase in the serotonin content of the hypothalamus and brain stem of dogs28 and the activation of the sympathetic nervous system.29 These studies might support the suggestion that the GH-stimulating activity of AI1 in man can be attributed to an action of this octopeptide on the central neurotransmitter system involved in controlling GH release. Nevertheless, further investigations are needed to elucidate this point. In order to maintain a strict control on possible side effects, we administered scalar doses of AI1 to our subjects. The ensuing slow increase of plasma levels might have contributed to the delay observed in the response of GH and ACTH. The delay could also reflect the indirect kind of the regulatory action of AI1 on GH and ACTH secretion. In other terms, AI1 could be more a modulator of the multifactorial complex involved in regulating the release of GH and ACTH than a direct stimulator of GH and ACTH secretion. Furthermore, the pressor effect of AI1 can counteract and delay to some extent the secretory action of this peptide, as suggested previous studies.‘6,25.26 Since acute hypertension may represent a stressful stimulus for the conscious man, capable of inducing secretion of stress-sensitive hormones, we cannot rule out the possibility that the parallel increases in ACTH and GH levels observed after AI1 infusion are not specific and could be related to a possible physical or mental discomfort. However, the specificity of AI1 effect can be supported by the finding that no concomitant increase in PRL, another stress-sensitive hormone, was ever observed and that the subjects did not complain of any significant unpleasant side effect during AI1 infusion. Finally, it still remains to be determined whether peripheral changes in blood-borne AI1 may really contribute to modulating ACTH and GH secretion in humans, and whether the AII-induced effects are of any physiological significance since the AI1 circulating concentrations reached in our study may be too high and not within the physiological range. Therefore, further studies are needed to place the observed
1
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Fig 4. Changes in plasma GH, PRL, and TSH during IV infusion of saline (O---O) and All at various doses W--O). Means + SEM are shown. Significances: l*f < .02, lP -c .05 compared with saline control.
from AI1 injection can open the blood-brain barrier,23 only a small fraction of the IV-injected AI1 would reach the effective site to induce ACTH release. In fact, AI1 has been shown to induce a dose-related ACTH release in vitro and in vivo. ‘2~‘3~‘5~‘4 Thus, it would appear that the dose of AI1 employed in our study is close to the one which is sufficient to effectively induce changes in ACTH release. Our results do not allow us to comment on either the exact mechanism or the main site of action of AIL However, it is likely that systemically administered AI1 can elicit ACTH release also in humans primarily by a mechanism involving the CNS.“,‘6 The possibility that ACTH release might be mediated by the central hypertensive effect of AI1 cannot be completely excluded. However, it has been demonstrated in conscious dogs that ACTH secretion is inhibited by increases in blood pressure25,26 and that the pressor response to AI1 can counteract the AII-stimulated ACTH secretioni Furthermore, it has been shown that exogenous AI1 is capable of maintaining its ACTH-stimulating activity when the pressor action is eliminated with the simultaneous administration of the vasodilator nitroprusside.16 The possibility that circulating AI1 may increase ACTH secretion by acting through AI1 receptors on the circumventricular organs, areas where the blood-brain-barrier is deficient, cannot be excluded. However, the physiological role of these intracerebral receptors in the regulation of ACTH secretion remains still uncertain and awaits experimental elucidation in man.
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DEGLI UBERTI ET AL
increase in plasma ACTH and GH in humans in its proper biological perspective. In contrast to the positive effects on plasma ACTH and GH, no effect of AI1 on TSH was found. In this regard, our results are in keeping with those of Steele et al,’ who have shown that AI1 injected intraventricularly or IV into ovariectomized rats had no effect on plasma TSH levels. However, these findings are in contrast with the report3’ that AI1 IV or intraperitoneally injected in rat caused elevation of plasma TSH levels and significantly enhanced the plasma TSH responses to cold and TRH. At present, the reasons for these descrepancies are unclear. Nevertheless, Steele et al’ reported that the in vitro incubation of hemipituitaries with large doses of AI1 increased TSH levels. These researchers postulated that AI1 may have a stimulatory effect on TSH secretion by acting directly at the pituitary level and explained the lack of action of intraventricular and IV AI1 on TSH secretion by suggesting that in those cases a simultaneous release of somatostatin could have blunted the TSH response to AII. However, in our study, the finding of the GH response to AI1 argues against this hypothesis. Plasma PRL concentrations were not significantly affected by IV AD. This result confirms our previous report3’ that IV infusion of AI1 does not produce any consistent change in plasma PRL in normal women. These data taken together indicate that peripherally generated AI1 may not play an important role in modulating PRL secretion, even if AI1 reaches plasma levels able to induce a significant increase in
blood pressure and PA concentration. Dufy-Barbe et aL3* using the conscious ovariectomized rhesus monkey, reported increase of PRL release after intraventricular and IV administration of AII. Steele et a17.8have shown that in rats IV-injected AI1 caused a slight increase followed by a decrement in PRL concentrations, whereas intraventricularly injected AI1 depressed plasma levels of PRL. Furthermore, there still remains the controversial question of whether the action of AI1 on PRL might be attributed prevalently to a direct local effect of AI1 on the pituitary, rather than to a central effect, since in vitro studies indicate that AI1 may stimulate PRL release acting directly on anterior pituitary cells.6,7 It seem unlikely that in our study the AI1 failure to influence plasma PRL and TSH concentration can be attributed to the use of an inadequate dose of octapeptide or to the route of administration, since AI1 was able to stimulate ACTH and GH secretion with a similar protocol. However, we cannot exclude that the dose required at the CNS level to influence the secretion of both TSH and PRL must be higher than the one we used. In conclusion, we have shown that in man IV administration of AI1 produces a significant increase in plasma ACTH and GH levels, without modifying the plasma concentration of TSH and PRL. Nevertheless, more studies are necessary to clarify the involvement of AI1 in the modulation of pituitary hormone release, as well as its physiological significance.
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GH AND ACTH RELEASE INDUCED BY All IN MAN
plasma ACTH concentration by angiotensin II infusion in normal humans and in a subject with a steroid 17-alfa-hydroxylase defect. Clin Endocrinol (Oxford) 10:137-144, 1979 22. Mason PA, Fraser R, Semple PF, et al: The interaction of the ACTH and angiotensin II in the control of corticosteroid plasma concentration in man. J Steroid Biochem 10:235-239, 1979 23. Johansson B, Li CH, Olsson Y, et al: The effect of acute arterial hypertension on the blood brain barrier to protein tracers. Acta Neuropathol 16:117-124, 1970 24. Keller-Wood M, Kimura B, Shinsako J, et al: Interaction between CRF and angiotensin II in control of ACTH and adrenal steroids. Am J Physiol250:R396-R402, 1986 25. Lorenzen LC, Ganong WF: Effect of drugs related to L-ethyltryptamine stress-induced ACTH secretion in the dog. Endocrinology 80:889-892, 1967 26. Gann DS, Ward DG, Carlson DE: Neural control of ACTH: A homeostatic reflex. Ret Prog Hormone Res 34:357-400, 1978 27. Robberecht W, Denef C: Stimulation and inhibition of
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pituitary growth hormone release by angiotensin II in vitro. Endocrinology 122:1496- 1504, 1988 28. Haulica I, Petrescu G, Stratone A, et al: Possible functions of brain renin, in Ganten D, Printz M, Phillips MI, et al (eds): The Renin Angiotensin System in the Brain. Heidelberg, FRG, SpringerVerlag, 1982, pp 335-342 29. Sumners C, Phillips MI: Central injection of angiotensin II alters catecholamine activity in rat brain. Am J Physiol 244:R257R265,1983 30. Mannisto PT. Kokkonen J, Ranta T: Effects of acute hypotension and hypertension on serum TSH concentrations in male rats. Acta Physiol Stand 107:105-107, 1979 31. degli Uberti EC, Trasforini G, Margutti A, et al: Failure of angiotensin II to affect prolactin concentration in normal women. Neuroendocrinol Lett 6:163-167, 1984 32. Dufy-Barbe L, Rodriguez F, Arsaut J, et al: Angiotensin II stimulates prolactin release in the rhesus monkey. Neuroendocrinology 35:242-247,1982
