title: Platelet-derived growth factor-mediated Ca2+ entry is blocked by antibodies to phosphatidylinositol 4,5-bisphosphate but does not involve heparin-sensitive inositol 1,4,5-trisphosphate receptors.
abstract: Elevation of intracellular Ca2+ by platelet-derived growth factor (PDGF) and other growth factors involves both release of Ca2+ from intracellular Ca2+ stores and Ca2+ entry from the extracellular medium. Release from intracellular stores is believed to be mediated by inositol 1,4,5-trisphosphate (IP3) and the heparin-sensitive IP3 receptor. We studied the mechanism by which entry of extracellular Ca2+ is induced by PDGF. Intracellular free Ca2+ (Ca2+i) was measured in single cultured rat vascular smooth muscle cells using fura 2 microspectrofluorometry. In nominally Ca2(+)-free medium, PDGF (recombinant BB, 10 ng/ml) raised intracellular Ca2+ transiently (less than 5 min); addition of 2 mM Ca2+ to the bathing medium after 5 min caused a second, prolonged increase in intracellular Ca2+. Repeated changes in extracellular Ca2+ from 0 to 2 mM over 90 min caused rapid, parallel changes in Ca2+i of approximately 200 nM. This change in Ca2+i in response to changes in extracellular Ca2+ was virtually undetectable in control or thrombin-treated cells. The intracellular response to changes in medium Ca2+ after PDGF was completely blocked by 10 mM CoCl2, but not by 10(-7) M nicardipine. Microinjection of monoclonal antibodies to phosphatidylinositol 4,5-bisphosphate (PIP2) (kt 10, 2 mg/ml) totally abolished both mobilization of intracellular Ca2+ stores and entry of extracellular Ca2+. Consistent with this finding, maintenance of Ca2+ entry required ongoing receptor occupancy, since displacement of PDGF from its receptor with suramin (1 mM) eradicated extracellular Ca2+ entry in less than 5 min. To determine whether extracellular Ca2+ entry involves the heparin-sensitive IP3 receptor, cells were microinjected with heparin (4 mg/ml) prior to addition of PDGF. Heparin, but not chondroitin sulfate, prevented mobilization of intracellular Ca2+ stores but did not affect extracellular Ca2+ entry. We PDGF requires ongoing receptor occupancy and involves PIP2 or PIP2 metabolism. However, the signal which mediates PDGF-induced Ca2+ entry does not require the heparin-sensitive IP3 receptor.
authors.affiliation: Division of Nephrology, University of California, San Francisco 94143.
authors.forename: C L
authors.initials: CL
authors.lastname: Huang
authors.forename: T
authors.initials: T
authors.lastname: Takenawa
authors.forename: H E
authors.initials: HE
authors.lastname: Ives
mesh_terms: D000818:Animals; D001692:Biological Transport; D002118:Calcium / Q000378:metabolism*; D015220:Calcium Channels; D002478:Cells, Cultured; D002809:Chondroitin Sulfates / Q000494:pharmacology; D003593:Cytoplasm / Q000378:metabolism; D006493:Heparin / Q000494:pharmacology; D066298:In Vitro Techniques; D053496:Inositol 1,4,5-Trisphosphate Receptors; D009131:Muscle, Smooth, Vascular; D019269:Phosphatidylinositol 4,5-Diphosphate; D010716:Phosphatidylinositols / Q000502:physiology*; D010982:Platelet-Derived Growth Factor / Q000494:pharmacology*; D051381:Rats; D011956:Receptors, Cell Surface / Q000502:physiology*; D018160:Receptors, Cytoplasmic and Nuclear; D013498:Suramin / Q000494:pharmacology
chemical_list: D015220:Calcium Channels; D053496:Inositol 1,4,5-Trisphosphate Receptors; D019269:Phosphatidylinositol 4,5-Diphosphate; D010716:Phosphatidylinositols; D010982:Platelet-Derived Growth Factor; D011956:Receptors, Cell Surface; D018160:Receptors, Cytoplasmic and Nuclear; D013498:Suramin; D006493:Heparin; D002809:Chondroitin Sulfates; D002118:Calcium