Calcium dependence of the stimulatory action of hypertonicity on renal medullary prostaglandin synthesis

Abstract

Previous studies have shown that hypertonic mannitol or NaCl increases the release of [ 3H]arachidonate and immunoreactive prostaglandin E in inner medullary slices incubated in Ca 2+-free media containing EGTA. By contrast, the stimulation of these parameters by ionophore A23187 and by arginine-vasopressin are abolished in Ca 2+-free media plus EGTA. In the present study, the effects of Ca 2+ deprivation and the intracellular Ca 2+ antagonist TMB-8 ((8- N, N-diethylamino)octyl-3,4,5-trimethoxybenzoate-HCl) were further examined to asses the Ca 2+ dependence of the actions of different stimuli of prostaglandin E synthesis in rat renal inner medulla. Ca 2+-free media without EGTA abolished increases in [ 3H]arachidonate and immunoreactive prostaglandin E release induced by ionophore A23187, but not those induced by arginine-vasopressin, suggesting that different pools of Ca 2+ subserve expression of the actions of these two stimuli. At low concentrations, TMB-8 (10–25 μM) inhibited increases in [ 3H]arachidonate and immunoreactive prostaglandin E release induced by arginine-vasopressin, but did not influence effects of Ca 2+ plus ionophore A23187 or hypertonicity on these parameters. At higher concentrations (100–500 μM), TMB-8 suppressed effects of ionophore A23187, hyperosmolar NaCl and mannitol on immunoreactive prostaglandin E and [ 3H]arachidonate release from slices. The effects of a sub-optimal inhibitory concentration of TMB-8 on ionophore A23187 actions were overcome by increasing Ca 2+ in the media from 1.5 to 5 mM. Ca 2+ deprivation, or concentrations of EGTA or TMB-8, that were effective in suppressing increases in immunoreactive prostaglandin E induced by ionophore A23187, arginine-vasopressin or hypertonicity, did not modify increases in immunoreactive prostaglandin E induced by exogenous arachidonate. Moreover, in microsomal fractions of inner medulla, TMB-8 suppressed Ca 2+-dependent increases in phospholipase A 2 and C activities, an effect which was competitive with Ca 2+. Thus, Ca 2+ deprivation and TMB-8 act at a step in the immunoreactive prostaglandin E synthetic pathway proximal to cyclooxygenase activity, and probably at the level of Ca 2+-dependent acyl hydrolase activity. The results with TMB-8 indicate that an intracellular pool of Ca 2+ is involved in expression of the actions of hypertonicity to increase [ 3H]arachidonate release and immunoreactive prostaglandin E in inner medulla.