BiochemicalPharmacology, Vol. 32, No. 6, pp. 1113-1118, 1983. 0006-2952/83/061113--06 $03.00/0 Printed in Great Pritain. ~ 1983 Pergamon Press Ltd. ROLE OF CALCIUM AND PROSTAGLANDINS IN THE ANTIDIURETIC HORMONE RESPONSE EFFECT OF IONOPHORE A23187 THOMAS YORIO,* SHERYL L. HENRY, DONALD H. HODGES and JAMES L. CAFFREY Departments of Pharmacology and Physiology, Texas College of Osteopathic Medicine, Fort Worth, TX 76107, U.S.A. (Received 7 May 1982; accepted 8 September 1982) Abstract--The calcium ionophore A23187 (IP) inhibited the antidiuretic hormone (ADH)-stimulated hydro-osmotic response in toad urinary bladder but had no effect on the osmotic transfer of water in the absence of hormone. Extracellular calcium was necessary for this effect at lower but not at higher IP concentrations. The hydro-osmotic response to exogenous cyclic AMP was unaltered by IP, but the same response produced by inhibition of phosphodiesterase was reduced significantly. Cyclic AMP concentrations in isolated toad bladder epithelial cells were reduced by 50% with IP or exogenous prostaglandin E2 (PGE2). Indomethacin, a prostaglandin synthesis inhibitor, prevented the inhibitory actions of the IP on the ADH-mediated response. Collectively, these observations suggest a key role for cellular calcium in modulating the actions of antidiuretic hormone and are consistent with the hypothesis that the ionophore, through increasing intracellular calcium, stimulates the synthesis of prostaglandins which have a negative feedback on adenylcyclase. This effect would terminate the action of the hormone. The biochemical steps leading to the action of anti- diuretic hormone (ADH) are not fully understood but are believed to involve a series of complex steps that include ADH binding to its membrane receptor [1], activation of adenylcyclase, the production of adenosine 3'-5'-cyclic monophosphate (cyclic AMP) [2], and the activation of protein kinase [3] followed by an increase in membrane permeability and an increase in osmotic water flow. Recent observations have also implicated a role for microtubules and microfilaments in ADH actions [4]. The ADH response may be modified in part by a change in cellular calcium metabolism. The fol- lowing observations are consistent with this hypoth- esis. In man, chronic hypercalcemia can impair the maximal urine-concentrating ability [5], and acute hypercalcemia diminishes the renal responsiveness to exogenous ADH. In isolated epithelia, elevated extracellular calcium inhibits the ADH and theo- phylline mediated hydro-osmotic response but not that induced by exogenous cyclic AMP [6--8]. Low calcium concentrations, however, augment the ADH hydro-osmotic response [6]. Recently, calcium ionophores have been reported to inhibit as well as enhance the in vitro hydro- osmotic response to ADH [9, 10]. A preliminary report has suggested that the inhibitory effects of the calcium ionophore are mediated by endogenous prostaglandins (PG) [11]. Prostaglandins inhibit the hormone induced increase in osmotic water flow and, * Address all correspondence to: Dr T. Yorio, Depart- ment of Pharmacology, Texas College of Osteopathic Med- icine, Camp Bowie at Montgomery, Fort Worth, TX 76107, U.S.A. like calcium, do not modulate exogenous cyclic AMP-mediated effects [12, 13]. Recently, Berl [14] has suggested that prostaglandins attenuate the ADH response by primarily interfering with mem- brane calcium transport. The present study further probes the relationships among calcium, prostaglan- dins, and the action of antidiuretic hormone in an isolated epithelium, the toad urinary bladder, util- izing the calcium ionophore A23187. METHODS Toads, Bufo marinus, were obtained from a com- mercial supplier (NASCO, Fort Atkinson, WI) and kept in terraria at 25 ° . Toad urinary bladder preparation. Toad urinary bladder "sacs" were set up and incubated in Ringer's solution as described previously [15]. For the measurement of the hydro-osmotic response, blad- der sacs were filled with 4 ml of 1/10 Ringer's solution and immersed so that the serosal (blood) side was bathed in 30 ml of Ringer's solution. The size of the bladder sacs was kept constant so as to minimize variations in surface area. Osmotic water flow was measured gravimetrically by weighing the bladder sacs before and after a 30-min or 60-min period of incubation in the presence of an osmotic gradient [15]. The experimental design is described where appropriate in the text and tables. The Ringer's solution was constituted as follows (miUimolar concentrations): NaC1, 111; KC1, 3.35; CaC12, 2.7; MgC12, 0.5; NaHCO3, 4.0; and glucose, 5. This solution was aerated throughout, and the pH was 8.0 at the start of the experiment and did not fall below 7.8 by the end of the incubation period. 1113 sP 32:6 - K