Evidence for inhibition by protein kinase A of receptor/G alpha(q)/phospholipase C (PLC) coupling by a mechanism not involving PLCbeta2.
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.
Dodge
K L
KL
Department of Biochemistry and Molecular Biology, University of Texas Medical School at Houston, 77030, USA.
Sanborn
B M
BM
D000818:Animals; D015153:Blotting, Western; D019556:COS Cells; D002217:Carbachol / Q000494:pharmacology; D002460:Cell Line; D000242:Cyclic AMP / Q000494:pharmacology; D017868:Cyclic AMP-Dependent Protein Kinases / Q000494:pharmacology*; D005260:Female; D019204:GTP-Binding Proteins / Q000378:metabolism*; D006801:Humans; D007295:Inositol Phosphates / Q000378:metabolism; D009215:Myometrium / Q000378:metabolism; D010121:Oxytocin / Q000494:pharmacology; D037342:Pertussis Toxin; D010766:Phosphorylation; D011247:Pregnancy; D043643:Receptor, Muscarinic M1; D011976:Receptors, Muscarinic / Q000235:genetics / Q000378:metabolism*; D018045:Receptors, Oxytocin / Q000235:genetics / Q000378:metabolism*; D014162:Transfection; D010738:Type C Phospholipases / Q000378:metabolism*; D010566:Virulence Factors, Bordetella / Q000494:pharmacology
D007295:Inositol Phosphates; D043643:Receptor, Muscarinic M1; D011976:Receptors, Muscarinic; D018045:Receptors, Oxytocin; D010566:Virulence Factors, Bordetella; D010121:Oxytocin; D002217:Carbachol; C058097:inositol 1,3,4-trisphosphate; D000242:Cyclic AMP; D037342:Pertussis Toxin; D017868:Cyclic AMP-Dependent Protein Kinases; D010738:Type C Phospholipases; D019204:GTP-Binding Proteins