THE EFFECTS OF CORTICOTROPIN-RELEASING HORMONE, ARGININE-VASOPRESSIN AND THEIR ANTAGONISTS ON ACTH RELEASE FROM PERIFUSED HORSE ANTERIOR-PITUITARY-CELLS

Antagonists are useful for probing hormone action and receptor characteristics. In this study we have investigated the inhibitory effects of analogues of arginine vasopressin (AW) and corticotrophin-releasing hormone (CRH) on stimulated release of immunoreactive ACTH from perifused equine anterior pituitary cells in vitro. Our aims were to gain some insight into the characteristics of the CRH and AVP receptors of the horse pituitary and to establish whether the response induced by AVP and CRH together could be blocked by combining antagonists. Experimental design included 5-min pulses of AVP (12.5 nmol/l), CRH (0.3 nmol/l) or CRH plus AVP given every 40 min alternately with pulses of secretagogue(s) plus appropriate antagonist(s). The effect of combined antagonists on the response to lower secretagogue concentrations (CRH, 0.03 nmol/l plus AVP, 2.5 nmol/l) was also tested. Response in the presence of an antagonist was compared with the mean response to secretagogue in the immediately preceding and following pulse and was expressed as per cent expected ACTH. The ACTH response to AVP was inhibited over the dose range 0.4-50 mu mol/l by Phaa-D-Tyr(Et)(2)Lys(6)Arg(3)VP (P < 0.002; ANOVA) and by d(CH2)(5)[Tyr(Me)(2)] AVP (P < 0.001). Suppression of the expected ACTH response to AVP by these two antagonists was most effectively achieved by antagonist concentrations of 10 mu mol/l (to 28 +/- 2.1%) and 25 mu mol/l (to 22 +/- 5.1%) respectively. Inhibition was not improved by preinfusion compared with a bolus pulse. Aaa-D-Tyr(Et)(2)Val(4)Abu(6)Arg(8.9)VP and the non-peptide antagonist OPC-21268 had no inhibitory effect. Two alpha-helical (alpha-h) analogues of CRH, (alpha-hCRH(12-41) and alpha-hCRH(9-41)) tested over the dose range 0.5-5 mu mol/l, suppressed CRH-induced ACTH secretion (P < 0.001) but CRH(23-41) had no significant effect. The alpha-hCRH(12-41) achieved greater suppression of ACTH release than the (9-41) derivative (8.7 +/- 4.2% compared with 19.3 +/- 3.5% of the expected ACTH response). Combination of d(CH2)(5)[Tyr(Me)(2)]AVP (25 mu mol/l) plus alpha-hCRH (12-41) (5.0 mu mol/l) achieved suppression to -0.5 +/- 1.3% and 0.8 +/- 1.5% of the expected response to CRH+AVP at 0.3 + 12.5 nmol/l and 0.03 + 2.5 nmol/l respectively These effects were seater than seen by the individual antagonists alone. The antagonist effects suggest that the CRH and AVP receptors of the equine pituitary have similar properties to those from other species and are consistent with the pituitary AVP receptor being unlike the V-2 receptor and resembling but not being identical to the V-1 type. We also conclude that alpha-hCRH(12-41) and d(CH2)(5)[Tyr(Me)(2)]AVP can together block the ACTH response to CRH plus AVP and suggest that these antagonists should provide a means of investigating additional secretagogues involved in ACTH release in the horse.