Gastrin Induces IP 3 Formation Through Phospholipase Cγ 1 and pp60 c-src Kinase

Abstract

Yassin, R. R. and J. T. Abrams. Gastrin induces IP 3 formation through phospholipase Cγ 1 and pp60 c-src kinase. Peptides 19(1) 47–55, 1998.—We have previously reported that gastrin induces a rapid and transient tyrosine phosphorylation of phospholipase Cγ 1 (PLCγ 1) in association with inositol 1,4,5-trisphosphate (IP 3) formation in rat colonic epithelial cells [34]. In this study, we demonstrate that gastrin regulates IP 3 formation mainly through PLCγ 1 isozyme. Immunoblotting analysis revealed the expression of PLCβ 3 and −γ 1, but not PLCβ 1, −β 2, or −β 4 in the rat colonic epitheliums. To explore what PLC isozyme(s) modulates gastrin effect on IP 3, immunoneutralizing antibody to PLCβ 1, −β 3, or −γ 1 was introduced into the colonic cells using a lipid carrier. The gastrin-stimulated increase in IP 3 concentration was specifically prevented by anti-PLCγ 1 but not by anti-PLCβ 1 or −β 3 antibody. Immunoprecipitation assays have also revealed that gastrin promoted an increase in tyrosine phosphorylation and co-precipitation of a 60 kDa src kinase with PLCγ 1. Adminstration of antibody specific to pp60 c-src into the colonic cells prevented the gastrin-stimulated increases in IP 3. Tyrosine phosphorylation of PLCγ 1 may be a major mechanism through which gastrin regulates IP 3 level in the colonic cells. Pretreatment of cells with the tyrosine kinase inhibitor genistein abrogated gastrin’s effect on IP 3, while extended pretreatment with pertussis toxin, a G-protein inhibitor, did not affect the ability of gastrin to stimulate IP 3 formation. Colonic cells expressed the Gα i subunits1-3; however, immunoblotting analysis did not reveal any difference in Gα i proteins’ expression between control and gastrin treated cells. The results provide direct evidence that gastrin regulates IP 3 level by a signaling mechanism that involves PLCγ 1 and pp60 c-src kinase.