Evidence for inhibition by protein kinase A of receptor/G alpha(q)/phospholipase C (PLC) coupling by a mechanism not involving PLCbeta2

• Published in: Endocrinology (Philadelphia) 1998-05, Vol.139 (5), p.2265-2271
• Main Authors: Dodge, K L, Sanborn, B M
• Format: Article
• Language: English
• Subjects: Animals; Blotting, Western; Carbachol - pharmacology; Cell Line; COS Cells; Cyclic AMP - pharmacology; Cyclic AMP-Dependent Protein Kinases - pharmacology; Female; GTP-Binding Proteins - metabolism; Humans; Inositol Phosphates - metabolism; Myometrium - metabolism; Oxytocin - pharmacology; Pertussis Toxin; Phosphorylation; Pregnancy; Receptor, Muscarinic M1; Receptors, Muscarinic - genetics; Receptors, Muscarinic - metabolism; Receptors, Oxytocin - genetics; Receptors, Oxytocin - metabolism; Transfection; Type C Phospholipases - metabolism; Virulence Factors, Bordetella - pharmacology
• ISSN: 0013-7227

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 G betagamma activation of phospholipase Cbeta2 (PLCbeta2) is a suggested target of protein kinase A, the possibility that the oxytocin receptor couples to PLCbeta2 via G alpha(i)G betagamma activation was explored. Western blot analysis of PHM1-41 cells and COS-M6 cells detected PLCbeta1 and PLCbeta3, but not PLCbeta2. In PHM1-41 cells, pertussis toxin reduced the oxytocin-stimulated increase in [3H]inositol 1,3,4-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/G alpha(q)-coupled stimulation of PLCbeta1 or PLCbeta3 can be inhibited by cAMP through a phosphorylation mechanism involving protein kinase A that does not involve PLCbeta2. In smooth muscle, this mechanism could constitute potentially important cross-talk between pathways regulating contraction and relaxation.