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

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 (<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 <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 conclude that entry of extracellular Ca²⁺ induced by 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.