COLLAGEN TYPE-IV STIMULATES AN INCREASE IN INTRACELLULAR CA2+ IN PANCREATIC ACINAR-CELLS VIA ACTIVATION OF PHOSPHOLIPASE-C

Intracellular Ca2+ responses to extracellular matrix molecules were studied in suspensions of pancreatic acinar cells loaded with Fura-2. Collagen type I, laminin, fibrinogen and fibronectin were unable to raise cytosolic free Ca2+ concentration ([Ca2+](i)), whereas collagen type IV, at concentrations from 5 to 50 mu g/ml, significantly increased it. The effect of collagen type IV was not due to possible contamination with type-I transforming growth factor beta or plasminogen, as neither of these agents was able to increase [Ca2+](i). Using highly specific mass assays, concentrations of inositol lipids, 1,2-diacylglycerol (DAG) and Ins(1,4,5)P-3 were measured in pancreatic acinar cells stimulated with collagen type IV. A decrease in the concentrations of PtdIns(4,5)P-2 and PtdIns4P with a concomitant increase in the concentrations of DAG and InsP(3) mass were observed, showing that collagen type IV increases [Ca2+](i) by activation of phospholipase C. The observed [Ca2+](i) signals had two components, the first resulting from Ca2+ release from the intracellular stores, and the second resulting from Ca2+ flux from the extracellular medium through the verapamil-insensitive channels. A tyrosine kinase inhibitor (tyrphostine) was able to block inositol lipid signalling caused by collagen type IV, which together with the insensitivity of this pathway to cholera toxin and pertussis toxin or to preactivation of protein kinase C, the longer duration of the increase in [Ca2+](i) and a longer lag period needed for observation of increases in DAG and InsP(3) concentration with collagen type IV than with carbachol (50 mM) suggest that activation of phospholipase C by collagen type IV is caused by tyrosine kinase activation. Inositol lipid signalling and increases in [Ca2+](i) were also observed with Arg-Gly-Asp (RGD)-containing peptide but not with Arg-Asp-Gly (RDG)-containing peptide. Collagen type IV and RGD-containing peptide, but not carbachol, competed in increasing [Ca2+](i) and DAG concentration, suggesting that the binding site of collagen type IV responsible for phospholipase C activation contains the RGD sequence. Together the present results suggest that, in pancreatic acinar cells, RGD sequence(s) within collagen type IV molecules cause activation of tyrosine kinase, probably through one of the integrin receptors, which then stimulates phospholipase C and increases [Ca2+](i).