Non-capacitative calcium entry in Chinese hamster ovary cells expressing the platelet-derived growth factor receptor.
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abstract
Platelet-derived growth factor (PDGF) is believed to produce intracellular calcium (Ca2+i) transients by inositol trisphosphate (InsP3)-mediated release of intracellular Ca2+ stores followed by "capacitative" Ca2+ entry due to emptying of these stores. We examined the roles for the phospholipase Cgamma-InsP3 pathway and the emptying of InsP3-dependent intracellular Ca2+ stores in PDGF-mediated Ca2+ entry. Intracellular Ca2+ release and Ca2+ entry were measured with fluorometric methods in Chinese hamster ovary cells expressing wild type or mutant PDGF receptors. Activation of the wild type PDGF receptor caused both intracellular "Ca2+ release, " measured in nominally 0 Ca2+ extracellular medium, and "Ca2+ entry, " measured upon addition of 2 mM Ca2+ medium. Both phases were absent in Chinese hamster ovary cells expressing a PDGF receptor mutant (Y977F,Y989F) that fails to bind phospholipase Cgamma. Blockade of the InsP3 receptor, by microinjection of single cells with low molecular weight heparin (5-50 mg/ml), blocked only Ca2+i release (following PDGF or flash photolysis of caged InsP3) and had no effect on PDGF-induced Ca2+ entry. In whole cell patch-clamp experiments, intracellular heparin also failed to block PDGF-evoked ion currents. Release of InsP3-dependent intracellular Ca2+ stores, by flash photolysis of caged InsP3, was apparently not sufficient to maximally activate Ca2+ entry. Intracellular InsP3 caused significantly less Ca2+ entry than PDGF alone. These data suggest that InsP3 alone is not sufficient to maximally activate Ca2+ entry by the capacitative pathway and that products of phosphatidylinositol 4,5-bisphosphate breakdown other than InsP3 probably play a role in PDGF-mediated Ca2+ entry.
