A novel role for membrane potential in the modulation of intracellular Ca2+ oscillations in rat megakaryocytes

1. The effect of membrane potential (V-m) on ADP-evoked [Ca2+](i) oscillati ons was investigated in rat megakaryocytes, a non-excitable cell type recen tly shown to exhibit depolarisation evoked Ca2+ release from intracellular stores during metabotropic purinoceptor stimulation. 2. Hyperpolarising voltage steps caused a transient fall in [Ca2+](i) and e ither abolished Ca2+ oscillations or reduced the oscillation amplitude. The se effects were observed in both the presence and absence of extracellular Ca2+ and also in Na+-free saline solutions, suggesting that hyperpolarisati on leads to a reduction in the level of ADP-dependent Ca2+ release without a requirement for altered transmembrane Ca2+ fluxes. 3. In the presence of Ca2+ oscillations, depolarising voltage steps transie ntly enhanced the amplitude of Ca2+ oscillations. Following run-down of Ca2 + oscillations, depolarisation briefly restimulated oscillations. 4. Simultaneous [Ca2+](i) and current-clamp recordings showed that Ca2+ and V-m oscillate in synchrony, with an average fluctuation of approximately 3 0-40 mV, due to activation and inactivation of Ca2+-dependent K+ channels. Application of a physiological oscillating V-m waveform to non-oscillating cells under voltage clamp stimulated [Ca2+](i) oscillations. 5. Analysis of the relationship between [Ca2+](i) and V-m showed a threshol d for activation of hyperpolarisation at about 250-300 nM. The implications of this threshold in the interaction between V-m and Ca2+ release during o scillations are discussed. 6. We conclude that the ability of voltage to control release of endosomal Ca2+ in ADP-stimulated megakaryocytes is bipolar in nature. Our data sugges t that V-m changes are active components of the feedback/feedforward mechan isms contributing to the generation of Ca2+ oscillations.