A significant body of evidence now suggests that signalling via certain G-protein-coupled receptors (GPCRs) can be directly regulated by the membrane potential. The most extensively studied example is the bipolar voltage control of P2Y receptor-evoked Ca²⁺ release in non-excitable rat megakaryocytes (MKs), a phenomenon that requires functional IP₃ receptors (Mason & Mahaut-Smith, 2001). We have now examined (i) the agonist dependence of this phenomenon and (ii) the role of dihydropyridine receptors (DHPRs).

Adult male Wistar rats were humanely killed by exposure to a rising concentration of CO₂ followed by cervical dislocation. Combined whole-cell patch clamp and fura-2 fluorescence recordings from marrow MKs were conducted as previously described (Mason & Mahaut-Smith, 2001). ADP was superfused at a near-threshold concentration of 0.01 or 0.03 µM, and also at 1 µM and 100 µM. In cells showing an agonist-evoked Ca²⁺ increase at all concentrations, depolarisation (80 mV, 10 s from -75 mV) stimulated a larger Ca²⁺ increase in 0.01/0.03 µM ADP (340 ± 58 nM, mean ± S.E.M., n = 13) than in 1 µM ADP (139 ± 16 nM, n = 23) or in 100 µM ADP (75 ± 26, n = 7). Interestingly 6/10 cells that failed to respond to 0.01/0.03 µM ADP alone, showed a large Ca²⁺ increase following depolarisation (239 ± 35 nM).

In megakaryocytes exposed to 1 µM ADP, the Ca²⁺ increase evoked by depolarisation (80 mV, 10 s from -75 mV) was not significantly different (P > 0.05, Student’s unpaired t test) in the presence of 50 µM nifedipine (196 ± 23 nM, n = 19) compared to controls (161 ± 30, n = 19). Nifedipine caused a substantial block of the voltage-dependent outward K⁺ current and thereby acted as a positive control. This result contrasts with the reported complete block of the slow (IP₃-dependent) phase of the depolarisation-induced Ca²⁺ increase in skeletal muscle by 10 µM nifedipine (Araya et al. 2003).

Our data demonstrate that a large depolarisation-evoked Ca²⁺ release is observed during near-threshold stimulation of P2Y receptors in the megakaryocyte. In addition, in contrast to skeletal muscle, the underlying mechanism does not require DHPRs. The physiological relevance of the voltage control of P2Y receptors is unknown, but could represent a means whereby ionotropic receptors or action potentials (Martinez-Pinna et al. 2003) potentiate cellular activation via this and other GPCRs.

This work was funded by the BHF and MRC. I.S.G. is supported by a Gates Cambridge Scholarship and an ORS Award.