Multiple Signals Regulate Phospholipase CBeta3 in Human Myometrial Cells1

Abstract

Phospholipase CB3 (PLCB3) serine(1105) (S(1105)), a substrate for multiple protein kinases, represents a potential point of convergence of several signaling pathways in the myometrium. To explore this hypothesis, the regulation of PLCB3-S(1105) phosphorylation (P-S(1105)) was studied in immortalized and primary human myometrial cells. 8-[4-chlorophenylthio] (CPT)-cAMP and calcitonin gene-related peptide (CALCA) transiently increased P-S(1105). Relaxin also stimulated P-S(1105); this effect was partially blocked by the protein kinase A (PRKA) inhibitor, Rp-8-CPT-cAMPS. Oxytocin, which stimulates Galphaq-mediated pathways, also rapidly increased P-S(1105), as did prostaglandin F2alpha and ATP. Oxytocin-stimulated phosphorylation was blocked by protein kinase C (PRKC) inhibitor Go6976 and by pretreatment overnight with a phorbol ester. Cypermethrin, a PP2B phosphatase inhibitor, but not okadaic acid, a PP1/PP2A inhibitor, prolonged the effect of CALCA on P-S(1105), whereas the reverse was the case for the oxytocin-stimulated increase in P-S(1105). PLCB3 was the predominant PLC isoform expressed in the myometrial cells and PLCB3 short hairpin RNA constructs significantly attenuated oxytocin-stimulated increases in intracellular calcium. oxytocin-induced phosphatidylinositol (PI) turnover was inhibited by CPT-cAMP and okadaic acid, but was enhanced by pretreatment with Go6976. CPT-cAMP inhibited oxytocin-stimulated PI turnover in the presence of overexpressed PLCB3, but not overexpressed PLCB3-S(1105)A. These data demonstrate that both negative crosstalk from the cAMP/PRKA pathway and a negative feedback loop in the oxytocin/G protein/PLCB pathway involving PRKC operate in myometrial cells and suggest that different protein phosphatases predominate in mediating P-S(1105) dephosphorylation in these pathways. The integration of multiple signal components at the level of PLCB3 may be important to its function in the myometrium.

Quantitative determination of α2B-adrenoceptor-evoked myosin light chain phosphorylation in vascular smooth muscle cells

“…The tested protein kinase C inhibitors very markedly potentiated the dexmedetomidine-induced pMLC 20 response, which might be due to increased responsiveness of the inositol-lipid signalling pathways, as reported for other smooth muscle cells (Pfeilschifter, Ochsner, Whitebread, & De Gasparo, 1989;Zhong, Murtazina, Phillips, Ku, & Sanborn, 2008). Blockade of protein kinase A also augmented the pMLC 20 response, although the interpretation of this effect is complicated by a reduction of basal pMLC 20 levels (by 33%) by the PKA inhibitor H-89.…”

Phenylephrine, a common cold remedy active ingredient, suppresses uterine contractions through cAMP signalling

“…Acting via the β2-adrenoceptor, phenylephrine activates adenylyl cyclase, thereby increasing the intracellular concentration of cAMP (Figs 4 and 7 ). cAMP is known to relax uterine smooth muscles; and it has been proposed that the cAMP-induced relaxation involves the activation of cAMP-dependent protein kinase A (PKA), which phosphorylates myosin light chain kinase (MYLK) 22 and phospholipase C (PLC) 23 , inhibiting their activity. In the present study, we indeed found that phenylephrine not only increased the level of cAMP, but also inhibited the oxytocin-induced Ca 2+ signalling in primary cultured mouse uterine smooth muscle cells, likely due to the cAMP-dependent inhibition of PLC.…”

Decreased Expression of the Rat Myometrial Relaxin Receptor (RXFP1) in Late Pregnancy Is Partially Mediated by the Presence of the Conceptus1

“…Various mechanisms of relaxin action have been proposed to account for the uterine quiescence. Relaxin increases calcium efflux from smooth muscle cells [31], decreases myosin light-chain kinase phosphorylation [32], stimulates calcium-activated potassium channels via a protein kinase A-mediated mechanism [33,34], and has been shown to induce phosphorylation of phospholipase Cb3 [35]. In addition, relaxin decreases oxytocinstimulated myometrial contractions by inhibiting phosphatidyl-inositol phosphate turnover and suppressing phospholipase C [36].…”