GASTRIN INDUCES IP3 FORMATION THROUGH PHOSPHOLIPASE C-GAMMA-1 AND PP60(C-SRC) KINASE

We have previously reported that gastrin induces a rapid and transient tyrosine phosphorylation of phospholipase C gamma(1) (PLC gamma(1)) i n association with inositol 1,4,5-trisphosphate (IP3) formation in rat colonic epithelial cells (34). In this study, we demonstrate that gas trin regulates IP3 formation mainly through PLC gamma(1) isozyme, Immu noblotting analysis revealed the expression of PLC beta(3) and -gamma( 1), but not PLC beta(1), -beta(2), or -beta(4) in the rat colonic epit heliums, To explore what PLC isozyme(s) modulates gastrin effect on IP 3, immunoneutralizingr antibody to PLC beta(1), -beta(3), or -gamma(1) was introduced into the colonic cells using a lipid carrier. The gast rin-stimulated increase in IP3 concentration was specifically prevente d by anti-PLC gamma(1) but not by anti-PLC beta(1) or -beta(3) antibod y. Immunoprecipitation assays have also revealed that gastrin promoted an increase in tyrosine phosphorylation and co-precipitation of a 60 kDa are kinase with PLC gamma(1). Adminstration of antibody specific t o pp60(c-src) into the colonic cells prevented the gastrin-stimulated increases in IP3. Tyrosine phosphorylation of PLC gamma(1) may be a ma jor mechanism through which gastrin regulates IP3 level in the colonic cells. Pretreatment of cells with the tyrosine kinase inhibitor genis tein abrogated gastrin's effect on IP3, while extended pretreatment wi th pertussis toxin, a G-protein inhibitor, did not affect the ability of gastrin to stimulate IP3 formation. Colonic cells expressed the Ga- i subunits1-3; however, immunoblotting analysis did not reveal any dif ference in Ga-i proteins' expression between control and gastrin treat ed cells, The results provide direct evidence that gastrin regulates I P3 level by a signaling mechanism that involves PLC gamma(1) and pp60( c-src) kinase. (C) 1998 Elsevier Science Inc.