Voltage-dependent Ca2+ release in rat megakaryocytes requires functional IP3 receptors

1. Using simultaneous whole-cell patch-clamp and fluorescence measurements of [Ca2+](i) in rat megakaryocytes we have investigated the requirement for functional inositol 1,4,5-trisphosphate (IP,) receptors in Ca2+ release in duced by membrane depolarization during agonist stimulation. 2. Voltage-dependent Ca2+ release was observed during application of the IP 3-generating agonists U46619 (a thromboxane A(2) analogue) and ADP. Further more, voltage-dependent Ca2+ release was observed in the absence of exogeno us agonist following sensitization of IP3 receptors with thimerosal. 3. Depolarization-induced Ca2+ release was not detected during depletion of intracellular Ca2+ stores by thapsigargin. Thus, depletion of stores alone is not sufficient to confer voltage dependence upon the Ca2+ release mecha nism. 4. Block of IP3 receptors by carbacyclin-stimulated elevations in cAMP, unc aging of cAMP or exposure to a high concentration of caffeine reversibly ab olished Ca2+ increases stimulated by both ADP and depolarization. 5. The cAMP-dependent block was prevented by a peptide inhibitor of protein kinase A, indicating that an alteration of adenylate cyclase activity lead ing to modulation of protein kinase A activity does not underlie the contro l of Ca2+ release by voltage. 6. These results are consistent with the requirement for functional IP3 rec eptors for voltage control of Ca2+ release from intracellular stores during inositol lipid signalling. The data also indicate the involvement of a vol tage sensor downstream of surface membrane receptors in the depolarization- evoked Ca2+ response.