Evidence for Inhibition by Protein Kinase A of Receptor/Gαq/Phospholipase C (PLC) Coupling by a Mechanism Not Involving PLCβ2*

May 16, 1998Endocrinology

Protein kinase A may block receptor signaling through G alpha(q) and phospholipase C without using the usual PLCbeta2 pathway

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Abstract

Preincubation with chlorophenylthio-cAMP (CPT-cAMP) resulted in a 53% reduction in oxytocin-stimulated inositol 1,3,4-trisphosphate formation in PHM1-41 cells.

CPT-cAMP, forskolin, or relaxin inhibited oxytocin-stimulated phosphatidylinositide turnover in human myometrial cells.
The inhibition of phosphatidylinositide turnover was reversed by H-89, a specific protein kinase A inhibitor.
Transient expression of protein kinase A catalytic subunit also inhibited stimulation by oxytocin and carbachol in COS-M6 cells.
CPT-cAMP inhibited stimulation of phosphatidylinositide turnover by endothelin-1, indicating a broad cAMP-inhibitory mechanism.
PLCbeta1 and PLCbeta3 were detected in the analyzed cells, but PLCbeta2 was not present.
Pertussis toxin reduced oxytocin-stimulated inositol 1,3,4-trisphosphate levels, suggesting an indirect effect of cAMP elevation.

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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.

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Evidence for Inhibition by Protein Kinase A of Receptor/Gαq/Phospholipase C (PLC) Coupling by a Mechanism Not Involving PLCβ2*

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