INVOLVEMENT OF PROTEIN KINASE-A IN THE REGULATION OF INTRACELLULAR FREE CALCIUM AND PHOSPHOINOSITIDE TURNOVER IN RAT MYOMETRIUM

Preincubation of Fura 2-loaded rat myometrial cells with H-8, an inhibitor of protein kinase A, for 1 h reversed the inhibitory effects of 8-(4-chlorophenylthio)-cAMP (CPTcAMP) on the oxytocin-stimulated increase in (Ca2+)(i) (intracellular free calcium), with an EC50 of 47 μM. H-8 also prevented the inhibition by relaxin and isoproterenol of the oxytocin-induced increase in (Ca2+)(i). The EC50 of H-8 in reversing the relaxin effect was 42 μM. H-8 reversal of the effect of relaxin on (Ca2+)(i) was evident both in the absence of extracellular calcium and in cells pretreated with pertussis toxin. H-8 also reversed the inhibitory effects of relaxin and CPTcAMP on the oxytocin-induced increase in [3H]inositol phosphate formation and [3H]phosphoinositide hydrolysis. Preincubation of myometrial cells for 1 h with H-7, another protein kinase inhibitor, only partially attenuated the inhibition by relaxin and CPTcMP of the oxytocin-induced increase in (Ca2+)(i) and [3H]inositol phosphate formation at concentrations 4-5 times greater than those of H-8. Acute (15-min) exposure to phorbol myristate acetate (1.0 μM) did not affect basal (Ca2+)(i) or the oxytocin-stiimulated increases in (Ca2+)(i) or inositol phosphate formation. These results imply a regulatory role for protein kinase A in the inhibition of the oxytocin-induced increase in (Ca2+)(i) and inositol phosphate formation by relaxants.