Evidence for inhibition by protein kinase A of receptor/Gα(q)/phospholipase C (PLC) coupling by a mechanism not involving PLC(β2)

Abstract

The effects of cAMP on the oxytocin-stimulated increase in phosphatidylinositide turnover and the possible pathways involved were investigated in a human myometrial cell line (PHM1-41) and in COS-M6 cells overexpressing the oxytocin receptor. Preincubation with chlorophenylthio- cAMP (CPT-cAMP), forskolin, or relaxin inhibited oxytocin-stimulated phosphatidylinositide turnover in PHM1-41 cells, and the inhibition was reversed by H-89, a relatively specific protein kinase A inhibitor. Both CPT- cAMP and transiently expressed protein kinase A catalytic subunit inhibited stimulation by oxytocin and carbachol of [3H]inositol 1,3,4-trisphosphate formation in COS-M6 cells expressing oxytocin or muscarinic M1 receptors, respectively. CPT-cAMP also inhibited phosphatidylinositide turnover stimulation by endothelin-1 in PHM1-41 cells, further demonstrating the generality of the cAMP-inhibitory mechanism. Since Gβγ activation of phospholipase C(β2) (PLC(β2)) is a suggested target of protein kinase A, the possibility that the oxytocin receptor couples to PLC(β2) via Gα(i)Gβγ activation was explored. Western blot analysis of PHM1-41 cells and COS-M6 cells detected PLC(β1) and PLC(β3), but not PLC(β2). In PHM1- 41 cells, pertussis toxin reduced the oxytocin-stimulated increase in [3H]inositol 1,3,44-trisphosphate by 53%, and this was reversed completely by H-89. Thus, the inhibitory effect of pertussis toxin may result from an indirect effect of cAMP elevation. These data suggest that receptor/Gα(q)- coupled stimulation of PLC(β1) or PLC(β3) can be inhibited by cAMP through a phosphorylation mechanism involving protein kinase A that does not involve PLC(β2) smooth muscle, this mechanism could constitute potentially important cross-talk between pathways regulating contraction and relaxation.