EFFECT OF CARBACHOL ON PHOSPHOLIPASE C-MEDIATED PHOSPHATIDYLINOSITOL 4,5-BISPHOSPHATE HYDROLYSIS, AND ITS MODULATION BY ISOPROTERENOL IN RABBIT CORNEAL EPITHELIAL-CELLS

The effects of carbachol (CCh) on phospholipase C(PLC)-mediated phosph atidylinositol 4,5-bisphosphate (PIP2) hydrolysis and its modulation b y isoproterenol were investigated in SV40-adenovirus transformed rabbi t corneal epithelial cells (RCEC). When examined under light microscop e, these cells exhibited a cobblestone-like appearance typical of the corneal epithelial cells grown in primary culture. Addition of CCh (0. 1 mM) for 30 min to RCEC, prelabeled with (32)Pi, decreased the radioa ctivity in phosphatidylinositol LC-phosphate and PIP2 by 15 and 27%, r espectively, and concomitantly increased the radioactivity in phosphat idylinositol and phosphatidic acid by 14 and 38%, respectively. When t he concentration of CCh was increased to 1 mM, the changes in radioact ivity were even more pronounced. Addition of CCh (0.1 mM) to the cells , prelabeled with myo[H-3]inositol, increased the accumulation of [H-3 ]inositol 1,4,5-trisphosphate ([H-3]InsP(3)) by 115%, indicating stimu lation of PLC-mediated PIP2 hydrolysis. Similar increases were also ob served in [H-3]InsP(1) and [H-3]InsP(2). The effects of CCh on inosito l phosphate accumulation were time- and dose-dependent, and were inhib ited by atropine (10 mu M), suggesting that the observed effects of CC h were mediated by activation of muscarinic cholinergic receptors. The effects of CCh were antagonized more potently by 4-diphenylacetoxy N- methyl-piperidine than by pirenzepine, indicating that the muscarinic receptors involved in PLC activation are probably of M(3) type BY West ern immunoblotting analysis with various anti-PLC antibodies, the RCEC were shown to contain PLC gamma 1 and PLC delta 1 in the soluble frac tion and PLC beta 1 in the microsomal fraction. Addition of isoprotere nol to RCEC, increased cAMP both in a time- and dose-dependent manner. Pre-treatment of the cells with isoproterenol or other cAMP elevating agents resulted in a significant inhibition of the CCh-induced InsP(3 ) accumulation. It can be concluded from these data that activation of M(3) muscarinic receptors in RCEC results in stimulation of PLC beta 1-mediated PIP2 hydrolysis, and that activation of beta-adrenergic rec eptors causes inhibition of the CCh-stimulated PIP2 hydrolysis.