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Effect of arginine vasopressin and oxytocin on acetylcholine-stimulation of corticosteroid and catecholamine secretion from the rat adrenal gland perfused in situ
NcwopcpMo (1988) 12,265271 0 Longman Group UK Ltd 1988
Effect of Arginine Vasopressin and Oxytocin on Acetylcholine-Stimulation of Corticosteroid and Catecholamine Secretion from the Rat Adrenal Gland Perfused ln Situ I. D. PORTER*, 6. J. WHITEHOUSE*, A. H. TAYLORt and S. S. NUSSEYt *Department of Physiology, King’s College London, Campden Hill Road, London, W8 7AH, tDepartment of Medicine, St George’s Hospital Medical School, Cranmer Terrace, London, SW17 ORE, (Reprint requests to S.S.N.).
Abstract-The effects of acetylcholine, arginine vasopressin (AVP) and oxytocin (OXT) on both catecholamine and steroid secretion have been investigated using the isolated rat adrenal gland perfused in situ. Significant stimulation of steroid (aldosterone and corticosterone) secretion occurred with 1 kmol/l acetylcholine; the ED= was approximately 20-fold higher (circa 20 pmol/l) than that for catecholamine secretion. The highest concentration of acetylcholine used (100 tJ,moWI)stimulated aldosterone secretion eight-fold; corticosterone secretion four-fold; noradrenaline and adrenaline secretion three-fold. AVP at 100 nmol/l but not at 1 nmolll significantly stimulated the secretion of both steroids and catecholamines. OXT had no significant effect on corticosteroid or catecholamine secretion at either concentration. The effects on aldosterone secretion of simultaneous administration of acetylcholine and AVP were additive. No similar effect was seen on corticosterone or catecholamine secretion where the degree of stimulation was the same as for acetylcholine alone. OXT (100 nmol/l) inhibited acetylcholine-stimulated aldosterone secretion but had no effect on acetylcholine-stimulated catecholamine secretion. Carbachol was equipotent with acetylcholine in stimulating steroid secretion from the perfused gland. Our results support the hypothesis that acetylcholine may play a role in the control of steroid secretion by the rat adrenal cortex. They fail to support a role for AVP and OXT in modulating catecholamine secretion by the adrenal medulla except at high concentrations.
Introduction The classical view holds that the adrenal medulla is under the cholinergic control of the splanchnic
Date received 27 June 1988 Date accepted 1 August 1988
nerve while the adrenal cortex is regulated by humoral factors, principally adrenocorkotrophih and angiotensin II. There is, however, also evidence that nervous control is exerted on the adrenal cortex. Early studies demonstrated acetylcholine in the adrenal cortex (1) and histological staining for acetylcholine esterase has provided
265
NEUROPEPTIDES
266 evidence for choline@ nerves in the cortex (2). In stimulates steroid seaddition, acetylcholine cretion from the perfused bovine adrenal gland (3). Recently a number of peptides have been described both in the splanchnic nerve and within the adrenal cortex and medulla themselves. The possible functions of only a few of these peptides have been described in detail. We and others have described the presence of both arginine vasopressin (AVP) and oxytocin (OXT) together with their associated neurophysins in the adrenal cortex and medulla of several species, including the rat (4-10). We have previously shown that both AVP and OXT in micromolar concentrations inhibit acetylcholine-stimulated release of catecholamines from isolated bovine chromaffin cells in primary monolayer culture (10). There is also evidence that both AVP and OXT may have functional roles in the control of cortical steroid synthesis, particularly in the zona glomerulosa of the rat (11-15). In order to investigate further the role of AVP and OXT in the adrenal gland we have studied the effects of AVP and OXT on cholinergic stimulation of adrenal cortical and medullary function in the rat adrenal gland perfused in situ.
Materials and Methods Perfusion of the rat adrenal gland Male Sprague-Dawley rats (25Og-45Og; King’s College London animal unit) were anaesthetized with urethane (l.$g/Kg i.p.; Sigma Chemical Co., Poole, Dorset, UK). The left adrenal gland was prepared for perfusion by the method of Sibley et al. (16). This method allows orthograde perfusion of the undisturbed adrenal gland by an artificial medium. In all experiments, after a 30 min “recovery period”, 10 min samples were collected on ice and their volumes recorded prior to mixing and division for assays. Test substances were infused in the perfusion medium for 10 min, at a constant concentration, only after 50 min control period. Aliquots for catecholamine assay were acidified with 100 *l/tube of 1.6 mol/l perchloric acid. The perfusate used throughout was medium 199 (TC199; Gibco Ltd., Paisley, Scotland) plus 0.5%
BSA at 37”C, pH 7.2, gassed with 95% Ozl5% COZ. Because TC199 has a potassium ion concentration of 5.8 mm0l.A it was diluted 4:3 with KCl-free Krebs-Ringer-bicarbonate solution to give a final potassium ion concentration of 3.9 mmoV1. In experiments involving acetylcholine infusion physostigmine (BDH Chemicals, Poole, U.K.) at 1 pmol/l was added to inhibit acetylcholine esterase. Arginine vasopressin and oxytocin were obtained from Cambridge Research Biochemicals (Huntingdon, Cambridgeshire, UK). Assays Samples for steroid assay were washed with nhexane (HPLC grade; Fisons, Loughborough, U.K.) and extracted with ethyl acetate (HiPerSolv; BDH Chemicals). Aldosterone was measured by radioimmunoassay without chromatography using antibody raised to aldosterone3carboxymethyloxime-BSA (17). Corticosterone was measured by gas-liquid chromatography (glc) as previously described (18). Adrenaline and noradrenaline were estimated by high performance liquid chromatography with electrochemical detection (10). Preliminary studies showed that catecholamines could be assayed without extraction from the acidified perfusate. Synthetic nOTadrenaline and adrenaline were used as standards. All experiments were assayed on at least 2 occasions by an operator unaware of the test substances perfused. Analysis ofdata Results are expressed as a ‘Stimulation Ratio’ (R): R is defined as the mean of two sample secretion rates during the effect of the substance divided by the mean of two basal samples immediately before administration of the test substance. Results were analysed by analysis of variance (ANOVA) and Student’s t-test (two-tailed) and are shown as mean + SEM. ReSUltS
Effects of acetylcholine on adrenal function Acetylcholine stimulated steroids and catecholamines
secretion of both from the intact gland
ARGININE VASOPRESSIN AND OXYTOCIN ON ACETYLCHOLINE-STIMULATION
267
OF CORTICOSTEROID
pmolil, stimulated aldosterone secretion eightfold; corticosterone secretion four-fold; noradrenaline secretion three-fold; adrenaline secretion three-fold. Carbachol (10 pmol/l), a predominantly muscarinic cholinergic agonist resistant to cholinesterases, was equipotent with acetylcholine in stimulating corticosterone (R = 1.83 -+ 0.28; n = 6) and aldosterone secretion (R = 2.2 + 0.23; n = 5) from the perfused gland. The latency and duration of stimulation of steroid secretion by carbachol was similar to that for acetylcholine (lo-20 min).
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Fig 1 Left panel: Dose-response curve for effect of acetyl choline (ACh) on steroid secretion from the perfused gland. C-m aldosterone (Aldo), O- - -0 corticosterone (Cot?), (means f SEM). Right panel: Dose-response curve for effect of acetylcholine (ACh) on catecholamine secretion from the perfused gland. c3 noradrenaline (NA), n - - -m adrenaline (A), (means +7 SEM).
in a dose-related manner (Figure 1). Significant stimulation of aldosterone and corticosterone secretion occurred with 1 pmoY1 acetylcholine (Fig 1); the EDso was pmol/l for aldosterone secretion and 20.0 pmol/l for corticosterone secretion. Catecholamine secretion was more sensitive to acetylcholine than corticosteroid secretion. The highest concentration of acetylcholine used, 100
Effects of A VP and OXT alone Neither AVP nor OXT had any effect on catecholamine or steroid secretion from the perfused gland at the lower concentration tested (1 nmolk not shown). However, at the higher concentration (100 nmol/l) AVP stimulated the secretion of both steroids and catecholamines (Table) The time-course of a typical experiment is shown in Figure 2. In contrast, OXT when given as a constant infusion (100 nmol/l) , had no significant effect on either corticosteroid or catecholamine secretion (Table). Effects of concomitant administrationof acetylcholine and AVP or OXT Simultaneous
administration
of acetylcholine
(10
Table Comparison of the effects of acetycholine (ACh, 10 p.mol/l), vasopressin (AVP, 100 nmoV1) and oxytocin (OXT, 100 nmol/l) alone and in combination on adrenal cortical and medullary function in the isolated perfused rat adrenal gland. Tabulated values are mean stimulation ratios (R) I?ZSEM for the analyte shown; aldosterone (Aldo), corticosterone (Cort), noradrenaline (NA) and adrenaline (A). The number of experiments is shown in brackets together with the value of p from Student’s t-test for comparison with basal values of the analyte tort
Aldo ACh OXT AVP ACh + OXT ACh + AVP
3.42 + (4, p c 1.12 f (4, p > z.49 k (4, p < 1.55 * (4, p > 7.21 + (4, p <
0.45 O.OL) 0.2 0.05) 0.39 0.05) 0.18’ 0.05) 0.73’ 0.01)
1.64 + (4, p < 0.67 + (4, p > 3.30 + (4, p < 1.51 + (4, p < 3.69 + (4, p <
tp < 0.01 Student’s t-test compared with values for ACh.
0.05 0.01) 0.17 0.05) 0.65 0.05) 0.16
0.05) 1.11 0.05)
NA
A
2.55 k 0.35 (11, p c 0.01) 0.8 zk 0.15 (6, p > 0.05) 1.66 + 0.15 (5, p < 0.02) 2.82 * 0.66 (4, p > 0.05) 2.71 + 0.48 (5, p < 0.05)
2.23 -+ 0.26 (13, p < 0.01) 0.96 + 0.13 (6, p > 0.05) 1.95 + 0.24 (6, P <: 0.02) 2.25 + 0.09 (4, p < 0.01) 1.69 + 0.05 (4, p < 0.01)
268
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100 l#
IL
% =
140
I
It0 100
Igi 0
Fig 2 Typical effects of 100 mnoyl arginine-vasopressin (AW) on aldosterone (Aldo) and corticosterone (Cort) (Left panel) and noradrenaline (NA) and adrenaline (A) (Right panel) secretion from the perfused rat adrenal gland in situ. The solid horizontal bars indicate the period of perfusion.
tr.mol/l) with AVP (100 nmol/l) markedly stimulated aldosterone secretion; the effects of the two stimulators were additive (Table and Fig 3). No similar effect was seen on corticosterone or catecholamine secretion where the stimulation ratio was the same as for AVP or acetylcholine alone. AVP (100 nmol/l) has no effect on acetylcholine-stimulated catecholamine secretion. OXT (100 nmol.4) inhibited acetylcholine-stimulated aldosterone secretion (Figure 3) but had no effect on acetylcholine-stimulated catecholamine secretion. Discussion
Immunohistochemical studies have demonstrated the presence of AVP and OXT in both the adrenal cortex and the medulla of several species (9). There is considerable inter-species variation in the medulla; in the rat groups of medullary cells positive for both peptides are found though the association with particular chromafhn cell-types has not been elucidated. The physiological role of AVP and OXT in the adrenal medulla remains unknown. Gibbs (19) has demonstrated an inhibitory effect of OXT at micromolar concentrations
on catecholamine secretion from the superfused rat adrenal gland. We have previously shown that both peptides inhibit acetylcholine-stimulated noradrenaline and adrenaline secretion from primary monolayer cultures of bovine adrenal chromaffin cells (10). However, the concentrations required were much greater than the dissociation constants (&) of the receptors studied on extracted crude membranes or the chromaffin cells in culture (10). The discrepancy was not due to breakdown of the added peptides by the cells in culture (10). The present data from the isolated perfused rat adrenal support these previous results and show that a modulatory role for either AVP or OXT on noradrenaline and adrenaline secretion by the perfused rat adrenal may only be demonstrated at concentrations much higher than the receptor K+ AVP alone fails to affect catecholamine secretion by bovine chromaffin cells but at high doses stimulates catecholamine secretion in the perfused rat adrenal; OXT alone is without effect in either system. Neither AVP nor OXT had an effect on acetylcholine-stimulated catecholamine secretion in the perfused gland. The discrepancy between receptor & and the Ki for the inhibition of acetylcholine-stimulated catechol-
ARGININE VASOPRESSIN AND OXYTOCIN ON ACEnLCHOLINE-STIMULATION
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from the intact gland in a dose-related manner. The fact that carbachol also stimulated steroid production in the intact gland suggests that cholinergic stimulation of corticosteroids in the rat is also mediated by muscarinic receptors. Although acetylcholine directly stimulates bovine adrenal cortical cells in culture in indirect effect in the perfused gland perhaps by the realease of another 4. 3 stimulant is not excluded. 2. Immunohistochemical studies demonstrate that there is less interspecies variability in the distribu1. tion of AVP and OXT in the adrenal cortex than in Othe medulla (8,9). In the adult adrenal gland of the Moo A cow, hamster, guinea pig and rat, AVP and OXT are present in all three zones with more intense Fig 3 Comparison of the effects of (acetylcholine) alone 0 and in combination with oxytocin (OXT) q or arginine staining in the zona glomerulosa (9). In fetal vasopressin (AVP) B on the secretion of aldosterone (Aldo), human cortex AVP is also present in the outer corticosterone (Cort), adrenaline (A) and noradrenaline (NA) zone while OXT is found in the cytoplasm of fetal from the perfused rat adrenal gland. zone cells (8). In the present studies AVP at 100 nmol/l but not 1 nmoY1 stimulated secretion of corticosterone and aldosterone from the perfused amine secretion from the adrenal medulla is also rat adrenal gland and, when infused with acetylnoted for other peptides including the opioids and choline, had an additive effect on aldosterone substance P (20-23). Indeed, it is possible to secretion. In a previous study using the same delineate two groups of peptides acting in the preparation (15) significant stimulation of both adrenal medulla: those with effects on catecholaldosterone and corticosterone secretion was amine secretion at nanomolar concentrations - noted using lower doses of AVP (50 pmol/l): this angiotensin II (24), corticotrophin releasing factor may have been due to the use of bolus adminis(25), bombesin and gas&in releasing peptide (26) tration (rather than infusion) of peptides in this and those which require micromolar concentrastudy to which this preparation seems particularly tions - opioids, vasoactive intestinal peptide, sensitive (29). Nevertheless, in the present study, substance P, neurotensin, somatostatin (27). The AVP alone and in combination with acetylcholine reason for this difference is unknown. For the stimulated steroid production in the perfused rat latter group, which as we have shown includes adrenal at concentrations which are within the AVP and OXT, further studies are required into range which might be expected of a paracrine other possible actions of these peptides. mediator or neuromodulator. The additivity of the The presence of acetylcholine and also acetyleffects of acetylcholine and AVP on aldosterone choline esterase positive fibres and plexuses has secretion is consistent with previous observations been adduced by previous workers as evidence for that both agonists stimulate polyphospoinositide a choline@ innervation of the adrenal cortex in metabolism (12-14, 30). several species including man (2). The effects of In the present study OXT infusion (1 nmoY1 and actylcholine on steroid secretion from the adrenal 100 nmoV1) had no effect on steroid secretion gland have mainly been studied using bovine tissue although previously it was found that low doses of where it has been found to stimulate cortisol and OXT given as a bolus stimulated aldosterone aldosterone production via muscarinic receptors secretion to a small extent (15). However, infusion and changes in polyphosphoinositide metabolism of OXT (100 nmol/l) significantly reduced the (28,29). In the present study, using the rat adrenal aldosterone response to acetylcholine; the mechagland, we found that acetylcholine stimulated nism involved requires further investigation. In secretion of both aldosterone and corticosterone preliminary experiments using isolated rat adreno?-
r:1
270 cortical cells we have been unable to reproduce this phenomenon. There may be parallels with the inhibitory effects of OXT on chorionic gonadotrophin stimulated steroid secretion by cultures of mixed testicular cells (31) which are not seen with purified Leydig cells (32). Thus, it is possible that the effects of OXT may be mediated via an interaction with other cell types in these tissues, an intraglandular mechanism for control of steroidogenesis, which is lost when the architecture of the gland is destroyed. In conclusion, our results support the hypothesis that acetylcholine may play a role in the control of steroid secretion by the rat adrenal cortex. The present results are in agreement with previous reports demonstrating that AVP may be involved in the regulation of zona glomerulosa cell function. The results of the prsent study together with results previously obtained using cultured bovine chromaffin cells fail to support a role for AVP and OXT in modulating catecholamine secretion by the adrenal medulla. Further studies are required to elucidate other possible roles for these peptides in the adrenal gland.
Acknowledgements SSN was a Wellcome Trust Lecturer. We thank the Medical Research Council and the Wellcome Trust for financial support. We are grateful to Professor G. P. Vinson and Dr J. P. Hinson of St Bartholomew’s Medical College for help with gas chromatography.
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contains vasopressin. Nature 310: 64-66. D., Kruip, Th. A. M. and Koll, R. (1985). Oxytocin determination in steroid producing tissues and in vitro production in ovarian follicles. Acta Endocrinol. 109: 530-536. Ravid, R., Oosterbaan, H. P., Hansen, B. L. and Swaab, D. F. (1986). Localisation of oxytocin, vasopressin and parts of precursors in the neonatal adrenal. Histochemistry 84,401-407. Hawthorn, J., Nussey, S. S., Henderson, J. R. and Jenkins, J. S. (1987). Immunohistochemical localization of oxytocin and vasopressin in the adrenal glands of rat, cow, hamster and guinea pig. Cell Tissue Res. 250: 1-6. Nussey, S. S., Prysor-Jones, R. A., Taylor, A. H., Ang, V. T. Y. and Jenkins, J. S. (1987). Arginine vasopressin and oxytocin in the bovine adrenal gland. J. Endocrinol. 115: 141-149. Payet, N. and Lehoux, J-G. (1982). Aldosterone and corticosterone stimulation by ACTH in isolated rat glomerulosa cells: interaction with vasopressin. J. Physiol. (Paris) 78: 317-321. Baila, T., Enyedi, P., Spat, A. and Antoni, F. A. (1985). Pressor-type vasopressin receptors in the adrenal cortex: properties of binding, effects on phosphoinositide metabolism and aldosterone secretion. Endocrinology 117: 421-423. Woodcock, E. A., McLeod, J. K. and Johnston, C. I. (1986). Vasopressin stimulates phosphatidylinositol tumover and rddosterone synthesis in rat adrenal glomerulosa cells: comparison with angiotensin II. Endocrinology 118: 2432-2436. Gallo-Payet, N., Guillon, G., Balestre, M. N. and Jard, S. (1986). Vasopressin induces breakdown of membrane phosphoinositides in adrenal glomeruiosa and fasciculata cells. Endocrinology 119: 1042-1047. Hinson, J. P., Vinson, G. P., Porter, I. D. and Whitehouse, B. J. (1987). Oxytocin and arginine vasopressin stimulate steroid secretion by the isolated perfused rat adrenal gland. Neuropeptides 10: 1-7. Sibley, C. P., Whitehouse, B. J., Vinson, G. P., Goddard, C. and McCredie, E. (1981). Studies on the mechanism of secretion of corticosteroids by the isolated perfused adrenal of the rat. J. Endocrinol. 91: 313-323. Vazir, H., Whitehouse, B. J., Vinson, G. P. and McCredie, E. (1981). Effects of prolonged ACTH treatment on adrenal steroidogenesis and blood pressure in rats. Acta Endocrinol. 97: 533-542. Vinson, G. P., Goddard, C. and Whitehouse, B. J. (1978). Corticosteroid production in vitro by adrenal tissue from rats with inherited hypothalamic diabetes insipidus (Brattleboro strain). J. Steroid Biochem. 9: 657665. Gibbs, D. M. (1986). Oxytocin inhibits ACTH and peripheral catecholamine secretion in the urethane-anesthetized rat. Regul. Pept. 14: 125-132. Lemaire, S., Lemaire, I., Dean, D. M. and Livett, B. G. (1980). Opiate receptors and adrenal medullary function. Nature 288: 303-304. Boksa, P., St-Pierre, S. and Livett, B. G. (1982). Characterization of substance P and somatostatin receptors on
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adrenal chromaffin cells using structural analogues. Brain Res. 245: 275-283. Saiani, L. and Guidotti, A. (1982). Opiate receptormediated inhibition of catecholamine release in primary cultures of bovine adrenal chromaffin cells. J. Neurochem. 39: 1669-1676. Livett, B. G., Boksa, P., Dean, D. M., Mizobe, F. and Lindenbaum, M. H. (1983). Use of isolated chromaffin cells to study basic release mechanisms. J. Auton. Nerv. sys. 7: 59%. Feldberg, W. and Lewis, G. P. (1964). The action of peptides on the adrenal medulla. Release of adrenaline by bradykinin and angiotensin. J. Physiol. (London) 171: 98-108. Udelsman, R., Harwood, J. P., Millan, M. A., Chrousos, G. P., Goldstein, D. S., Zimlichman, R., Catt, K. J. and AguiIIera, G. (1986). Functional corticotrophin releasing factor receptors in the primate peripheral sympathetic nervous system. Nature 319: 147-150. Ok&o, M., Kaku, K., Kaneko, T. and Yanaihara, N. (1985). Effects of bombesin and gastrin releasing peptide
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on catecholamine secretion from rat adrenal gland, in vitro. Endocrinol. Jpn. 32: 21-27. Livett, B. G. (1984). Adrenal medullarychromaffin cells in vitro. Physiol. Rev. 64, 1103-1161. Hadjian, A. J., GuidiceIIi, C. and Chambaz, E. M. (1982). Cholinergic muscarinic stimulation of steroidogenesis in bovine adrenal cortex fasciculata cell suspensions. Biochim. Biophys. Acta 714: 156-163. Hinson, J. P., Vinson, G. P. and Whitehouse, B. J. (1986). J. Endocrinol. 108 (Suppl): 124. Kojima, I., Kojima, K., Shibata, H. and Ogata, E. (1986). Mechanisms of cholinergic stimulation of aldosterone secretion in bovine adrenal glomerulosa cells. Endocrinology 119: 284-291. Adashi, E. Y., Tucker, E. M. and Hsueh, A. J. W. (1984). Direct regulation of rat testicular steroidogenesis by neurohypophysial hormones. J. Biol. Chem. 259: 5440-5446. Sharpe, R. M. and Cooper, I. (1987). Comparison of the effects on purified Leydig cells of four hormones (oxytocin, vasopressin, opiates and LHRH) with suggested paracrine roles in the testis. J. Endocrinol. 113: 89-96.
Effect of Arginine Vasopressin and Oxytocin on Acetylcholine-Stimulation of Corticosteroid and Catecholamine Secretion from the Rat Adrenal Gland Perfused ln Situ I. D. PORTER*, 6. J. WHITEHOUSE*, A. H. TAYLORt and S. S. NUSSEYt *Department of Physiology, King’s College London, Campden Hill Road, London, W8 7AH, tDepartment of Medicine, St George’s Hospital Medical School, Cranmer Terrace, London, SW17 ORE, (Reprint requests to S.S.N.).
Abstract-The effects of acetylcholine, arginine vasopressin (AVP) and oxytocin (OXT) on both catecholamine and steroid secretion have been investigated using the isolated rat adrenal gland perfused in situ. Significant stimulation of steroid (aldosterone and corticosterone) secretion occurred with 1 kmol/l acetylcholine; the ED= was approximately 20-fold higher (circa 20 pmol/l) than that for catecholamine secretion. The highest concentration of acetylcholine used (100 tJ,moWI)stimulated aldosterone secretion eight-fold; corticosterone secretion four-fold; noradrenaline and adrenaline secretion three-fold. AVP at 100 nmol/l but not at 1 nmolll significantly stimulated the secretion of both steroids and catecholamines. OXT had no significant effect on corticosteroid or catecholamine secretion at either concentration. The effects on aldosterone secretion of simultaneous administration of acetylcholine and AVP were additive. No similar effect was seen on corticosterone or catecholamine secretion where the degree of stimulation was the same as for acetylcholine alone. OXT (100 nmol/l) inhibited acetylcholine-stimulated aldosterone secretion but had no effect on acetylcholine-stimulated catecholamine secretion. Carbachol was equipotent with acetylcholine in stimulating steroid secretion from the perfused gland. Our results support the hypothesis that acetylcholine may play a role in the control of steroid secretion by the rat adrenal cortex. They fail to support a role for AVP and OXT in modulating catecholamine secretion by the adrenal medulla except at high concentrations.
Introduction The classical view holds that the adrenal medulla is under the cholinergic control of the splanchnic
Date received 27 June 1988 Date accepted 1 August 1988
nerve while the adrenal cortex is regulated by humoral factors, principally adrenocorkotrophih and angiotensin II. There is, however, also evidence that nervous control is exerted on the adrenal cortex. Early studies demonstrated acetylcholine in the adrenal cortex (1) and histological staining for acetylcholine esterase has provided
265
NEUROPEPTIDES
266 evidence for choline@ nerves in the cortex (2). In stimulates steroid seaddition, acetylcholine cretion from the perfused bovine adrenal gland (3). Recently a number of peptides have been described both in the splanchnic nerve and within the adrenal cortex and medulla themselves. The possible functions of only a few of these peptides have been described in detail. We and others have described the presence of both arginine vasopressin (AVP) and oxytocin (OXT) together with their associated neurophysins in the adrenal cortex and medulla of several species, including the rat (4-10). We have previously shown that both AVP and OXT in micromolar concentrations inhibit acetylcholine-stimulated release of catecholamines from isolated bovine chromaffin cells in primary monolayer culture (10). There is also evidence that both AVP and OXT may have functional roles in the control of cortical steroid synthesis, particularly in the zona glomerulosa of the rat (11-15). In order to investigate further the role of AVP and OXT in the adrenal gland we have studied the effects of AVP and OXT on cholinergic stimulation of adrenal cortical and medullary function in the rat adrenal gland perfused in situ.
Materials and Methods Perfusion of the rat adrenal gland Male Sprague-Dawley rats (25Og-45Og; King’s College London animal unit) were anaesthetized with urethane (l.$g/Kg i.p.; Sigma Chemical Co., Poole, Dorset, UK). The left adrenal gland was prepared for perfusion by the method of Sibley et al. (16). This method allows orthograde perfusion of the undisturbed adrenal gland by an artificial medium. In all experiments, after a 30 min “recovery period”, 10 min samples were collected on ice and their volumes recorded prior to mixing and division for assays. Test substances were infused in the perfusion medium for 10 min, at a constant concentration, only after 50 min control period. Aliquots for catecholamine assay were acidified with 100 *l/tube of 1.6 mol/l perchloric acid. The perfusate used throughout was medium 199 (TC199; Gibco Ltd., Paisley, Scotland) plus 0.5%
BSA at 37”C, pH 7.2, gassed with 95% Ozl5% COZ. Because TC199 has a potassium ion concentration of 5.8 mm0l.A it was diluted 4:3 with KCl-free Krebs-Ringer-bicarbonate solution to give a final potassium ion concentration of 3.9 mmoV1. In experiments involving acetylcholine infusion physostigmine (BDH Chemicals, Poole, U.K.) at 1 pmol/l was added to inhibit acetylcholine esterase. Arginine vasopressin and oxytocin were obtained from Cambridge Research Biochemicals (Huntingdon, Cambridgeshire, UK). Assays Samples for steroid assay were washed with nhexane (HPLC grade; Fisons, Loughborough, U.K.) and extracted with ethyl acetate (HiPerSolv; BDH Chemicals). Aldosterone was measured by radioimmunoassay without chromatography using antibody raised to aldosterone3carboxymethyloxime-BSA (17). Corticosterone was measured by gas-liquid chromatography (glc) as previously described (18). Adrenaline and noradrenaline were estimated by high performance liquid chromatography with electrochemical detection (10). Preliminary studies showed that catecholamines could be assayed without extraction from the acidified perfusate. Synthetic nOTadrenaline and adrenaline were used as standards. All experiments were assayed on at least 2 occasions by an operator unaware of the test substances perfused. Analysis ofdata Results are expressed as a ‘Stimulation Ratio’ (R): R is defined as the mean of two sample secretion rates during the effect of the substance divided by the mean of two basal samples immediately before administration of the test substance. Results were analysed by analysis of variance (ANOVA) and Student’s t-test (two-tailed) and are shown as mean + SEM. ReSUltS
Effects of acetylcholine on adrenal function Acetylcholine stimulated steroids and catecholamines
secretion of both from the intact gland
ARGININE VASOPRESSIN AND OXYTOCIN ON ACETYLCHOLINE-STIMULATION
267
OF CORTICOSTEROID
pmolil, stimulated aldosterone secretion eightfold; corticosterone secretion four-fold; noradrenaline secretion three-fold; adrenaline secretion three-fold. Carbachol (10 pmol/l), a predominantly muscarinic cholinergic agonist resistant to cholinesterases, was equipotent with acetylcholine in stimulating corticosterone (R = 1.83 -+ 0.28; n = 6) and aldosterone secretion (R = 2.2 + 0.23; n = 5) from the perfused gland. The latency and duration of stimulation of steroid secretion by carbachol was similar to that for acetylcholine (lo-20 min).
VJ ,’
,’
I’
__--
.
/’
Fig 1 Left panel: Dose-response curve for effect of acetyl choline (ACh) on steroid secretion from the perfused gland. C-m aldosterone (Aldo), O- - -0 corticosterone (Cot?), (means f SEM). Right panel: Dose-response curve for effect of acetylcholine (ACh) on catecholamine secretion from the perfused gland. c3 noradrenaline (NA), n - - -m adrenaline (A), (means +7 SEM).
in a dose-related manner (Figure 1). Significant stimulation of aldosterone and corticosterone secretion occurred with 1 pmoY1 acetylcholine (Fig 1); the EDso was pmol/l for aldosterone secretion and 20.0 pmol/l for corticosterone secretion. Catecholamine secretion was more sensitive to acetylcholine than corticosteroid secretion. The highest concentration of acetylcholine used, 100
Effects of A VP and OXT alone Neither AVP nor OXT had any effect on catecholamine or steroid secretion from the perfused gland at the lower concentration tested (1 nmolk not shown). However, at the higher concentration (100 nmol/l) AVP stimulated the secretion of both steroids and catecholamines (Table) The time-course of a typical experiment is shown in Figure 2. In contrast, OXT when given as a constant infusion (100 nmol/l) , had no significant effect on either corticosteroid or catecholamine secretion (Table). Effects of concomitant administrationof acetylcholine and AVP or OXT Simultaneous
administration
of acetylcholine
(10
Table Comparison of the effects of acetycholine (ACh, 10 p.mol/l), vasopressin (AVP, 100 nmoV1) and oxytocin (OXT, 100 nmol/l) alone and in combination on adrenal cortical and medullary function in the isolated perfused rat adrenal gland. Tabulated values are mean stimulation ratios (R) I?ZSEM for the analyte shown; aldosterone (Aldo), corticosterone (Cort), noradrenaline (NA) and adrenaline (A). The number of experiments is shown in brackets together with the value of p from Student’s t-test for comparison with basal values of the analyte tort
Aldo ACh OXT AVP ACh + OXT ACh + AVP
3.42 + (4, p c 1.12 f (4, p > z.49 k (4, p < 1.55 * (4, p > 7.21 + (4, p <
0.45 O.OL) 0.2 0.05) 0.39 0.05) 0.18’ 0.05) 0.73’ 0.01)
1.64 + (4, p < 0.67 + (4, p > 3.30 + (4, p < 1.51 + (4, p < 3.69 + (4, p <
tp < 0.01 Student’s t-test compared with values for ACh.
0.05 0.01) 0.17 0.05) 0.65 0.05) 0.16
0.05) 1.11 0.05)
NA
A
2.55 k 0.35 (11, p c 0.01) 0.8 zk 0.15 (6, p > 0.05) 1.66 + 0.15 (5, p < 0.02) 2.82 * 0.66 (4, p > 0.05) 2.71 + 0.48 (5, p < 0.05)
2.23 -+ 0.26 (13, p < 0.01) 0.96 + 0.13 (6, p > 0.05) 1.95 + 0.24 (6, P <: 0.02) 2.25 + 0.09 (4, p < 0.01) 1.69 + 0.05 (4, p < 0.01)
268
NEUROPEPTIDES
100 l#
IL
% =
140
I
It0 100
Igi 0
Fig 2 Typical effects of 100 mnoyl arginine-vasopressin (AW) on aldosterone (Aldo) and corticosterone (Cort) (Left panel) and noradrenaline (NA) and adrenaline (A) (Right panel) secretion from the perfused rat adrenal gland in situ. The solid horizontal bars indicate the period of perfusion.
tr.mol/l) with AVP (100 nmol/l) markedly stimulated aldosterone secretion; the effects of the two stimulators were additive (Table and Fig 3). No similar effect was seen on corticosterone or catecholamine secretion where the stimulation ratio was the same as for AVP or acetylcholine alone. AVP (100 nmol/l) has no effect on acetylcholine-stimulated catecholamine secretion. OXT (100 nmol.4) inhibited acetylcholine-stimulated aldosterone secretion (Figure 3) but had no effect on acetylcholine-stimulated catecholamine secretion. Discussion
Immunohistochemical studies have demonstrated the presence of AVP and OXT in both the adrenal cortex and the medulla of several species (9). There is considerable inter-species variation in the medulla; in the rat groups of medullary cells positive for both peptides are found though the association with particular chromafhn cell-types has not been elucidated. The physiological role of AVP and OXT in the adrenal medulla remains unknown. Gibbs (19) has demonstrated an inhibitory effect of OXT at micromolar concentrations
on catecholamine secretion from the superfused rat adrenal gland. We have previously shown that both peptides inhibit acetylcholine-stimulated noradrenaline and adrenaline secretion from primary monolayer cultures of bovine adrenal chromaffin cells (10). However, the concentrations required were much greater than the dissociation constants (&) of the receptors studied on extracted crude membranes or the chromaffin cells in culture (10). The discrepancy was not due to breakdown of the added peptides by the cells in culture (10). The present data from the isolated perfused rat adrenal support these previous results and show that a modulatory role for either AVP or OXT on noradrenaline and adrenaline secretion by the perfused rat adrenal may only be demonstrated at concentrations much higher than the receptor K+ AVP alone fails to affect catecholamine secretion by bovine chromaffin cells but at high doses stimulates catecholamine secretion in the perfused rat adrenal; OXT alone is without effect in either system. Neither AVP nor OXT had an effect on acetylcholine-stimulated catecholamine secretion in the perfused gland. The discrepancy between receptor & and the Ki for the inhibition of acetylcholine-stimulated catechol-
ARGININE VASOPRESSIN AND OXYTOCIN ON ACEnLCHOLINE-STIMULATION
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OF CORTKOSTEROID
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from the intact gland in a dose-related manner. The fact that carbachol also stimulated steroid production in the intact gland suggests that cholinergic stimulation of corticosteroids in the rat is also mediated by muscarinic receptors. Although acetylcholine directly stimulates bovine adrenal cortical cells in culture in indirect effect in the perfused gland perhaps by the realease of another 4. 3 stimulant is not excluded. 2. Immunohistochemical studies demonstrate that there is less interspecies variability in the distribu1. tion of AVP and OXT in the adrenal cortex than in Othe medulla (8,9). In the adult adrenal gland of the Moo A cow, hamster, guinea pig and rat, AVP and OXT are present in all three zones with more intense Fig 3 Comparison of the effects of (acetylcholine) alone 0 and in combination with oxytocin (OXT) q or arginine staining in the zona glomerulosa (9). In fetal vasopressin (AVP) B on the secretion of aldosterone (Aldo), human cortex AVP is also present in the outer corticosterone (Cort), adrenaline (A) and noradrenaline (NA) zone while OXT is found in the cytoplasm of fetal from the perfused rat adrenal gland. zone cells (8). In the present studies AVP at 100 nmol/l but not 1 nmoY1 stimulated secretion of corticosterone and aldosterone from the perfused amine secretion from the adrenal medulla is also rat adrenal gland and, when infused with acetylnoted for other peptides including the opioids and choline, had an additive effect on aldosterone substance P (20-23). Indeed, it is possible to secretion. In a previous study using the same delineate two groups of peptides acting in the preparation (15) significant stimulation of both adrenal medulla: those with effects on catecholaldosterone and corticosterone secretion was amine secretion at nanomolar concentrations - noted using lower doses of AVP (50 pmol/l): this angiotensin II (24), corticotrophin releasing factor may have been due to the use of bolus adminis(25), bombesin and gas&in releasing peptide (26) tration (rather than infusion) of peptides in this and those which require micromolar concentrastudy to which this preparation seems particularly tions - opioids, vasoactive intestinal peptide, sensitive (29). Nevertheless, in the present study, substance P, neurotensin, somatostatin (27). The AVP alone and in combination with acetylcholine reason for this difference is unknown. For the stimulated steroid production in the perfused rat latter group, which as we have shown includes adrenal at concentrations which are within the AVP and OXT, further studies are required into range which might be expected of a paracrine other possible actions of these peptides. mediator or neuromodulator. The additivity of the The presence of acetylcholine and also acetyleffects of acetylcholine and AVP on aldosterone choline esterase positive fibres and plexuses has secretion is consistent with previous observations been adduced by previous workers as evidence for that both agonists stimulate polyphospoinositide a choline@ innervation of the adrenal cortex in metabolism (12-14, 30). several species including man (2). The effects of In the present study OXT infusion (1 nmoY1 and actylcholine on steroid secretion from the adrenal 100 nmoV1) had no effect on steroid secretion gland have mainly been studied using bovine tissue although previously it was found that low doses of where it has been found to stimulate cortisol and OXT given as a bolus stimulated aldosterone aldosterone production via muscarinic receptors secretion to a small extent (15). However, infusion and changes in polyphosphoinositide metabolism of OXT (100 nmol/l) significantly reduced the (28,29). In the present study, using the rat adrenal aldosterone response to acetylcholine; the mechagland, we found that acetylcholine stimulated nism involved requires further investigation. In secretion of both aldosterone and corticosterone preliminary experiments using isolated rat adreno?-
r:1
270 cortical cells we have been unable to reproduce this phenomenon. There may be parallels with the inhibitory effects of OXT on chorionic gonadotrophin stimulated steroid secretion by cultures of mixed testicular cells (31) which are not seen with purified Leydig cells (32). Thus, it is possible that the effects of OXT may be mediated via an interaction with other cell types in these tissues, an intraglandular mechanism for control of steroidogenesis, which is lost when the architecture of the gland is destroyed. In conclusion, our results support the hypothesis that acetylcholine may play a role in the control of steroid secretion by the rat adrenal cortex. The present results are in agreement with previous reports demonstrating that AVP may be involved in the regulation of zona glomerulosa cell function. The results of the prsent study together with results previously obtained using cultured bovine chromaffin cells fail to support a role for AVP and OXT in modulating catecholamine secretion by the adrenal medulla. Further studies are required to elucidate other possible roles for these peptides in the adrenal gland.
Acknowledgements SSN was a Wellcome Trust Lecturer. We thank the Medical Research Council and the Wellcome Trust for financial support. We are grateful to Professor G. P. Vinson and Dr J. P. Hinson of St Bartholomew’s Medical College for help with gas chromatography.
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10.
11.
12.
13.
14.
15.
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