Non-capacitative calcium entry in Chinese hamster ovary cells expressing the platelet-derived growth factor receptor.
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.
Mathias
R S
RS
Department of Pediatrics, University of California, San Francisco, California 94143, USA. rmathias@peds.ucsf.edu
Zhang
S J
SJ
Wilson
E
E
Gardner
P
P
Ives
H E
HE
D000818:Animals; D016466:CHO Cells; D002118:Calcium / Q000378:metabolism*; D006224:Cricetinae; D006493:Heparin / Q000008:administration & dosage / Q000378:metabolism; D015544:Inositol 1,4,5-Trisphosphate / Q000378:metabolism; D008845:Microinjections; D009154:Mutation; D017479:Receptors, Platelet-Derived Growth Factor / Q000235:genetics / Q000378:metabolism*
D015544:Inositol 1,4,5-Trisphosphate; D006493:Heparin; D017479:Receptors, Platelet-Derived Growth Factor; D002118:Calcium