Signal transduction of arginine vasopressin-induced arachidonic acid release in H9c2 cardiac myoblasts: role of Ca2+ and the protein kinase C-dependent activation of p42 mitogen-activated protein kinase.

Abstract

The mechanism of arginine vasopressin (AVP)-induced arachidonic acid (AA) release was examined in the cardiac myoblast cell line, H9c2. Stimulation of cells with AVP induced dose-dependent AA release, and this effect was completely inhibited by the V1 receptor antagonist, d(CH)5[Tyr(Me)2]AVP. AVP also produced dose-dependent stimulation of inositol phosphate formation; this was not affected by pertussis toxin, indicating the presence of the V1 receptor/Gq protein/PLCbeta pathway in H9c2 cells. The concentration-response curves for these two effects of AVP overlapped. AVP induced a rapid increase in [Ca2+]i, followed by a sustained increase. The Ca2+ ionophore, A23187 or ionomycin, mimicked the effect of AVP, whereas the protein kinase C (PKC) activator, TPA, only induced a slight increase in AA release. Both the AVP- or A23187-stimulated AA release and the AVP-induced sustained [Ca2+]i increase were completely blocked in the absence of external Ca2+. The receptor-operated Ca2+ channel blocker, SKF 96365, and the inorganic Ca2+ channel blockers, Ca2+ and Ni2+, also inhibited the AVP-induced AA release. Western blots demonstrated expression of PKCalpha, betaI, epsilon, delta, and zeta in H9c2 cells; PKC inhibitors (staurosporine or Ro 31-8220) or down-regulation of PKCalpha, betaI, epsilon, and delta by long-term (24 h) TPA treatment caused a partial blockade of the AVP-induced response, whereas the A23187-induced AA release was unaffected by down-regulation of these isoforms. AVP-induced, but not A23187-induced, AA release was partially blocked by the p42 MAPK cascade inhibitor, PD 98059. AVP and TPA, but not A23187, induced an increase in activity and tyrosine phosphorylation of p42 MAPK, together with a molecular weight shift, consistent with phosphorylation, of cytosolic PLA2. AVP- or TPA-induced activation and tyrosine phosphorylation of p42 MAPK were completely blocked by down-regulation of PKCalpha, betaI, epsilon, and delta, but still occurred, together with the cytosolic PLA2 mobility shift, in the absence of external Ca2+. These results show that AVP-induced AA release in H9c2 cells is secondary to activation of the V1 receptor/Gq protein/PLCP pathway, leading to an influx of extracellular Ca2+ and activation of PKCalpha, betaI, epsilon, and delta. The influx of extracellular Ca2- and DAG act, respectively, through PKC-/MAPK-independent or PKC-dependent MAPK pathways to mediate AA release.