Characterization of phosphoinositide-specific phospholipase C in rat colonocyte membranes

The phosphoinositide signal transduction pathway mediates important processes in intestinal physiology, yet the key enzyme, phosphoinositide-specific phospholipase C (PI-PLC), is not well-characterized in the colon. PI-PLC activity was examined in rat colonic membranes using exogenous [³H]phosphatidylinositol 4,5-bisphosphate (PIP₂) as substrate, and β-glycerophosphate to suppress degradation of substrate or product. The activity of membrane PI-PLC increased 6-fold with the addition of alamethicin, and a further 2-3-fold enhancement was observed with 10 μM guanosine 5'-[γ-thio]triphosphate (GTP[S]), suggesting the involvement of G-protein(s). The effect of GTP[S] appeared to be specific, as up to 100 μM adenosine 5'-[γ-thio] triphosphate failed to stimulate PI-PLC activity, and guanosine 5' (β- thio]diphosphate inhibited activity. The response of membrane PI-PLC to Ca²⁺ was biphasic, while > 0.5 mM Mg²⁺ was inhibitory with or without GTP[S]. Comparable total PI-PLC activities and responses to GTP[S] and Ca²⁺ were observed in purified brush-border and basolateral membranes. Western immunoblots probed with monoclonal antibodies to PLC isoenzymes PLC -β1 and -γ1 and δ1 demonstrated that these antipodal plasma membranes contain predominantly the PLC-δ1 isoform, with small amounts of PLC-γ1 present but no detectable PLC-β1. PLC-γ1 was the major isoform detected in cytosol.