Protein kinase-A inhibits phospholipase-C activity and alters protein phosphorylation in rat myometrial plasma membranes

Abstract

Our previous studies implicated the involvement of protein kinase-A in the inhibitory effects of isoproterenol and relaxin on oxytocin-stimulated phosphoinositide turnover in rat myometrium. To understand the possible mechanisms involved, the properties and regulation of phospholipase-C (PLC) in purified myometrial plasma membranes from estrogen-primed rats were studied. The PLC activity measured with exogenous [3H]phosphatidylinositol 4,5-bisphosphate as substrate was Ca2+ dependent. The nonhydrolyzable GTP analog guanosine 5'-(3-O-thio)triphosphate stimulated PLC activity with a ED50 of 1.6 microM and shifted the calcium dependence curve to the left. Guanosine 5'-(3-O-thio)triphosphate-stimulated phosphatidylinositol 4,5-bisphosphate hydrolysis was inhibited by activation of endogenous and exogenous cAMP-dependent protein kinase (PKA). The effects of endogenous and exogenous PKA were significantly reversed by IP20, a potent synthetic peptide inhibitor of PKA. In the presence of [gamma-32Pi]ATP and exogenous PKA, 32Pi was incorporated in an IP20-sensitive manner into major bands at approximately 17,000, 20,000-24,000, 33,000, 38,000, 40,000-44,000, and other higher mol wt. These data indicate that one or more GTP-binding proteins mediate activation of membrane-bound PLC in rat myometrium. Phosphorylation of one or more membrane-associated proteins by PKA may regulate myometrial PLC activity and play a role in the inhibitory effects of isoproterenol and relaxin.

Multiple Signals Regulate Phospholipase CBeta3 in Human Myometrial Cells1

“…Ku, unpublished observations). On the basis of data presented here and previously [14,18], it appears that a significant proportion of OXTR/Ga q is coupled to PLCB3 in PHM1 cells, primary human cells, and rat myometrium. PLCB1 is not a substrate of PRKA, and may account for the PRKA-resistant PLC component of the activity elicited by OXT.…”

“…Inhibition by cAMP of OXT-induced PI turnover has been observed in pregnant rat uterus, but not at term, coincident with a partial loss of membrane-and AKAP-associated PRKA [16,17,31,32]. PRKA inhibited GTPcS-stimulated PI turnover in rat myometrial plasma membrane, indicating that this effect was distal to possible effects on the OXTR itself [18]. These data support the presence of negative crosstalk from the receptor/Ga s / adenylyl cyclase/PRKA pathway that contributes to the inhibition of the OXTR/Ga q /PLCB3 pathway via phosphorylation of PLCB3-S 1105 (Fig.…”

“…The inhibition of OXT-induced PI turnover by increasing intracellular cAMP in myometrial cells is both PRKA and A-kinase anchoring protein (AKAP) dependent [16,17]. Activation of PRKA by 8- [4-chlorophenylthio] (CPT)-cAMP inhibits GTPcS-stimulated PI turnover in rat myometrial plasma membrane, indicating that the effect of PRKA is distal to OXT receptor (OXTR)/G protein coupling [18].…”

Correlation between Connexin43 Expression, Cell-Cell Communication, and Oxytocin-lnduced Ca2+ Responses in an Immortalized Human Myometrial Cell Line1

“…The antagonistic effect of 8-bromo-cAMP on the oxytocin-induced [Ca 2 +]i response is presumably attributable, in part, to antagonism of oxytocin-stimulated phospholipase C activation [45]. The results obtained in the present study using fluo-3 complement the observed inhibition of oxytocin-stimulated increase in [Ca 2 +]i measured with fura-2 and the inhibition of phosphatidyl inositol turnover in PHM1-41 cells by acute exposure to the cAMP analog CPT-cAMP described by Monga et al [24].…”

“…The 3 2 -adrenoreceptor agonists are thought to stimulate adenylyl cyclase to elevate intracellular cAMP levels in myometrial cells [16]. Possible mechanisms by which cAMP has been postulated to bring about smooth muscle relaxation include lowering of [Ca 2 +]i by intracellular sequestration or efflux from the cytoplasm [31], interference with agonist-stimulated increases in inositol trisphosphate [45], and regulation of Ca 2 +-activated K + channel activity [31,46]. Current tocolytic treatment is usually initiated after the initial events of preterm labor (including contraction and cervical dilation) are underway.…”