Ca²⁺ spike initiation from sensitized inositol 1,4,5-trisphosphate-sensitive Ca²⁺ stores in megakaryocytes

Abstract

Ca²⁺-mediated Ca²⁺ spikes were analysed in fura-2-loaded megakaryocytes. Direct Ca²⁺ loading using whole-cell dialysis induced an all-or-none Ca²⁺ spike on top of a tonic increase in cellular Ca²⁺ concentration ([Ca²⁺]_i) with a latency of 3–7 s. The latency decreased with increasingly higher concentrations of Ca²⁺ in the dialysing solution. Spike size and its initiation did not correlate with the tonic level of [Ca²⁺]_i. Thapsigargin completely abolished the Ca²⁺-induced spike initiation, suggesting that Ca²⁺ spikes originate from thapsigargin-sensitive Ca²⁺ pools. An inhibitor of phosphatidylinositide-specific phospholipase C (PLC), 2-nitro-4-carboxyphenyl-N,N-diphenyl-carbamate prolonged the latency without changes of spike size in most cases (6/9 cells), but abolished the spike initiation in the other cells (3/9). The results suggest that an increase in [Ca²⁺]_i charges up the inositol-1,4,5-trisphosphate(InsP ₃)- and thapsigargin-sensitive Ca²⁺ pools which progressively sensitize to low or slightly elevated levels of InsP₃ by the action of Ca²⁺-dependent PLC until a critical Ca²⁺ content is reached, and then the Ca²⁺ spike is triggered. Thus, the limiting step of Ca²⁺ spike triggering is the initial filling process and the level of InsP₃ in megakaryocytes.