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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 beta-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 microM guanosine 5'-[gamma-thio]triphosphate (GTP[S]), suggesting the involvement of G-protein(s). The effect of GTP[S] appeared to be specific, as up to 100 microM adenosine 5'-[gamma-thio]-triphosphate failed to stimulate PI-PLC activity, and guanosine 5'-[beta-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-beta 1, -gamma 1 and -delta 1 demonstrated that these antipodal plasma membranes contain predominantly the PLC-delta 1 isoform, with small amounts of PLC-gamma 1 present but no detectable PLC-beta 1. PLC-gamma 1 was the major isoform detected in cytosol.

Diabetes was induced in rats by administration of a single intraperitoneal injection of streptozotocin (50 mg/kg body wt). After 7 days, one group of diabetic animals was treated with insulin for an additional 5 days. Control, diabetic and diabetic + insulin rats were then killed, their distal small intestines were removed and the epithelial cells were examined and compared with respect to polyphosphoinositide turnover, total protein kinase C activity and cellular distribution, and 1,2-diacylglycerol mass and production. The results of these experiments demonstrated that, compared with their control counterparts, the intestines from diabetic rats had a decreased turnover of polyphosphoinositides, but an increase in 1,2-diacylglycerol mass which was a result, at least in part, of an increase in the synthesis of this lipid de novo. Total protein kinase C activity was decreased in the diabetic rats due to a decrease in cytosolic activity, with no significant change in particulate activity. Moreover, insulin administration for 5 days to diabetic animals did not affect their lowered intestinal polyphosphoinositide turnover, but did further accentuate their increased 1,2-diacylglycerol mass and synthesis de novo; this treatment also corrected total protein kinase C activity by increasing the cytosolic activity of this enzyme. These results indicate that signalling mechanisms involving polyphosphoinositides, 1,2-diacylglycerol and protein kinase C are abnormal in the intestines of diabetic rats and that some of these biochemical parameters can be modulated by insulin administration in vivo.