RAPID HEPARIN-SENSITIVE CA2+ RELEASE FOLLOWING CA2+-ATPASE INHIBITION IN INTACT HL-60 GRANULOCYTES - EVIDENCE FOR INS(1,4,5)P-3-DEPENDENT CA2+ CYCLING ACROSS THE MEMBRANE OF CA2+ STORES

In many cell types, emptying of intracellular Ca2+ stores after application of inhibitors of the intracellular Ca2+-ATPase (e.g. thapsigargin) is astonishingly rapid. It was the aim of this study to elucidate the underlying mechanism. We first compared thapsigargin-induced emptying of intracellular Ca2+ stores in intact and homogenized HL-60 granulocytes. Thapsigargin-induced Ca2+ release was rapid in intact cells (33.9+/-4.9% of store content/min), but it was slow in permeabilized or homogenized cells (7.7+/-3.9 and 12+/-3.8%, of store content/min respectively). To study whether the Ins(1,4,S)P-3 receptor might be involved in thapsigargin-induced Ca2+ release, we tested the effect of heparin, a competitive Ins(1,4,5)P-3 antagonist. In homogenized and permeabilized preparations, heparin did not interfere with thapsigargin-induced Ca2+ release. In contrast, when introduced into intact cells by an endocytosis/osmotic-shock procedure, heparin, but not the inactive de-N-sulphated heparin, decreased the rate of Ca2+ release by approx. 70%. Heparin inhibited Ca2+ release in response to the Ins(1,4,5)P-3-generating receptor agonist N-formylmethionyl-leucylphenylalanine (f-MLP) (50 nM) and to thapsigargin (50 nM) at comparable concentrations. Heparin inhibition was competitive for f-MLP-induced, but not for thapsigargin-induced, Ca2+ release. In permeabilized cells, the addition of low Ins(1,4,5)P-3 concentrations before thapsigargin increased the rate of thapsigargin-induced Ca2+ release 4-fold. Taken together, our results suggest that the rapid Ca2+-ATPase-inhibitor-induced Ca2+ release is due to a partial activation of the Ins(1,4,5)P-3 receptor in resting cells. This implies Ca2+ cycling across the membrane of Ins(1,4,5)P-3-sensitive Ca2+ stores in resting cells.