Rat megakaryocytes are prototypical non-excitable cells devoid of both voltage-gated Ca²⁺ channels and ryanodine-sensitive intracellular Ca²⁺ stores. Under whole-cell voltage-clamp, application of ADP or the thromboxane A₂ analogue U46619 evokes transient Ca²⁺ oscillations that subside to an elevated Ca²⁺ plateau. Depolarisation during this plateau phase results in promotion of intracellular Ca²⁺ release whilst hyperpolarisation results in its inhibition via a mechanism requiring the presence of functional InsP₃Rs (Mahaut-Smith et al. 1999; Mason et al. 2000; Mason & Mahaut-Smith, 2001). The ability of membrane potential to modulate Ca²⁺ release from intracellular stores has important influences upon the pattern of Ca²⁺ oscillations. Depolarisation can stimulate oscillations in non-oscillating cells whilst hyperpolarisation inhibits oscillations. Thus in the megakaryocyte, changes in membrane potential are potent stimuli controlling both release of Ca²⁺ from intracellular stores and the pattern of Ca²⁺ signals generated by inositol lipid turnover.

This study has combined confocal fluorescence measurements of Ca²⁺ and whole-cell patch-clamp to investigate the spatiotemporal pattern of agonist- and depolarisation-evoked Ca²⁺ signals in single rat megakaryocytes. Rats were killed by cervical dislocation following exposure to rising CO₂ and bone marrow prepared as described elsewhere (Mahaut-Smith et al. 1999). ADP (1 µM) resulted in a clear increase in Ca²⁺ that in 82 % of cells (45 of 55) was visualised as a wave of Ca²⁺ sweeping across the cell. Depolarisations (-75 to 0 mV) during ADP stimulation resulted in an elevation in Ca²⁺ that also took the form of a wave in 75 % of cells (41 of 55). Ca²⁺ waves generated by either ADP or depolarisation traversed the periphery of the cell significantly faster than across the radius (Fig. 1), leading to the development of a curvilinear wavefront. In 69 % of cells (38 of 55) the large polyploidic nucleus was contained within a single hemisphere of the cell, and both ADP- and depolarisation-induced Ca²⁺ waves predominantly originated within this hemisphere, although not necessarily from the same place. Immunochemistry showed the endoplasmic reticulum marker GRP78/BiP, the Ca²⁺ storage protein calreticulin, and the type III InsP₃R to be preferentially located at the cell periphery, suggesting that the restricted localisation of endosomal Ca²⁺ signalling machinery may underlie formation of curvilinear Ca²⁺ waves.Taken together, these data demonstrate that ADP- and depolarization-stimulated release of intracellular Ca²⁺ is initiated from a peripherally located point source close to the nucleus and leads to the propagation of a Ca²⁺ wave around the periphery of the cell. Given that ADP- and depolarisation-evoked Ca²⁺ waves can originate from different locations, some compartments of the endoplasmic reticulum may show preferential sensitivity to ADP and depolarisation stimulation.M.P.M.-S. acknowledges the support of the BHF, and D.T. is supported by MRC Co-operative Funds.

Figure 1. Ca2+ waves evoked by ADP alone, or depolarisation in the presence of ADP, traverse the periphery of the rat megakaryocytes significantly faster than across the cell radius. Data are means ± S.D. (n = 11) compared using Students’ two-tailed paired t test.

Mason, M.J. & Mahaut-Smith, M.P. (2001). J. Physiol. 533, 175-183. abstract

Mason, M.J., Hussain, J.F. & Mahaut-Smith, M.P. (2000). J. Physiol. 524, 437-446. abstract

Mahaut-Smith, M.P., Hussain, J.F. & Mason, M.J. (1999). J. Physiol. 515, 385-390. abstract