Rapid heparin-sensitive Ca2+ release following Ca(2+)-ATPase inhibition in intact HL-60 granulocytes. Evidence for Ins(1,4,5)P3-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 Ca(2+)-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(1,4,5)P3 receptor might be involved in thapsigargin-induced Ca2+ release, we tested the effect of heparin, a competitive Ins(1,4,5)P3 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)P3-generating receptor agonist N-formylmethionyl-leucyl-phenylalanine (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)P3 concentrations before thapsigargin increased the rate of thapsigargin-induced Ca2+ release 4-fold. Taken together, our results suggest that the rapid Ca(2+)-ATPase-inhibitor-induced Ca2+ release is due to a partial activation of the Ins(1,4,5)P3 receptor in resting cells. This implies Ca2+ cycling across the membrane of Ins(1,4,5)P3-sensitive Ca2+ stores in resting cells.
Favre
C J
CJ
Division of Infectious Diseases, University Hospital, Genève, Switzerland.
Lew
D P
DP
Krause
K H
KH
D002118:Calcium / Q000378:metabolism*; D000252:Calcium-Transporting ATPases / Q000037:antagonists & inhibitors*; D002463:Cell Membrane Permeability; D002478:Cells, Cultured; D006098:Granulocytes / Q000187:drug effects / Q000201:enzymology / Q000378:metabolism*; D006493:Heparin / Q000494:pharmacology*; D006801:Humans; D015544:Inositol 1,4,5-Trisphosphate / Q000378:metabolism*; D009240:N-Formylmethionine Leucyl-Phenylalanine / Q000494:pharmacology; D013729:Terpenes / Q000494:pharmacology; D019284:Thapsigargin
D013729:Terpenes; D009240:N-Formylmethionine Leucyl-Phenylalanine; D019284:Thapsigargin; D015544:Inositol 1,4,5-Trisphosphate; D006493:Heparin; D000252:Calcium-Transporting ATPases; D002118:Calcium