ENDOTHELIN-1 STIMULATES CYTOSOLIC PHOSPHOLIPASE A(2) ACTIVITY AND GENE-EXPRESSION IN RAT GLOMERULAR MESANGIAL CELLS

Endothelin-1 (ET-1) stimulates vascular smooth muscle and mesangial ce lls to release prostaglandin E(2) (PGE(2)), which can attenuate the va soconstrictor and mitogenic effects of this peptide. Phospholipase A(2 ) (PLA(2))-mediated release of arachidonic acid from the sn-2 position of membrane phospholipids is thought to be one of the rate-limiting s teps in prostaglandin (PG) synthesis. We evaluated the role of ET-1 to regulate gene expression, protein synthesis and enzymatic activity of cytosolic PLA(2) (cPLA(2)), an intracellular form of the PLA(2) enzym e family, in cultured rat mesangial cells using both acute and chronic incubation protocols. Acute ET-1-induced stimulation of cPLA(2) activ ity was maximal after 10 minutes (181.1 +/- 6.84% of control), persist ed for 40 minutes and did not require new protein synthesis. Heparin, a potent inhibitor of intracellular Ca2+ increase as well as mitogen-a ctivated protein (MAP) kinase activation and cell proliferation, did n ot affect the rapid cPLA, stimulation by ET-1. Chronic incubation of g lomerular mesangial cells with ET-1 (1 to 24 hr) led to time- and dose -dependent increases in cPLA(2) mRNA expression which was maximal afte r six hours, persisted up to 24 hours and which was accompanied by bot h cPLA(2) protein formation, as assessed by Western analysis, as well as by stimulation of enzymatic activity. Inhibition of protein synthes is by cycloheximide increased basal cPLA(2) mRNA accumulation in quies cent mesangial cells, and the combination of ET-1 and cycloheximide re sulted in a greater induction of cPLA(2) gene expression when compared to ET-1 alone. Actinomycin D treatment blocked the effect of ET-1 on cPLA, mRNA accumulation. Herbimycin A, an inhibitor of cellular protei n tyrosine kinases, abrogated the ET-1-induced cPLA, gene expression, while protein kinase C (PKC) inhibitors (Calphostin, Sangivamycin) onl y slightly reduced cPLA(2) mRNA. We thus conclude that ET-1 is able to stimulate cPLA(2) in a biphasic manner: Acute stimulation starts afte r five minutes and does not require de novo protein synthesis, while l ong-term activation after three hours is most likely linked to cPLA(2) gene expression via a cellular tyrosine kinase pathway. ET-1-mediated acute and chronic stimulation of cPLA, could serve as an autocrine an d/or paracrine negative regulator for vasoconstrictive and mitogenic e ffects of ET-1.