Actions of thapsigargin on the Ca(2+)-handling systems of the human platelet. Incomplete inhibition of the dense tubular Ca2+ uptake, partial inhibition of the Ca2+ extrusion pump, increase in plasma membrane Ca2+ permeability, and consequent elevation of resting cytoplasmic Ca2+.
Thapsigargin (Tg) effects on Ca2+ handling in the intact human platelet were studied using Quin2 and chlorotetracycline to measure free cytoplasmic and dense tubular (DT) Ca2+ concentrations ([Ca2+]cyt and [Ca2+]dt, respectively). Tg inhibits Ca2+ uptake by the DT Ca(2+)-ATPase pumps, but incompletely, lowering the Vm to 32% of control (IC50,Tg = 0.18 +/- 0.10 microM). The kinetics of loss of DT Ca2+, transient increases in [Ca2+]cyt, and lowered steady-state [Ca2+]dt after Tg addition are all explained by pump inhibition, with no effect on the rate constant of Ca2+ leakage across the DT membrane (kleak,DT = 1.14 min-1). Tg lowers by 30% the Vm of the Ca2+ extrusion pump located in the plasma membrane (PM), as shown by a Quin2-based method measuring active Ca2+ extrusion (Johansson, J. S., and Haynes, D. H. (1988) J. Membr. Biol. 104, 147-163). This effect (IC50,Tg = 0.45 +/- 0.06 microM), together with a 24 +/- 16% increase in kleak,PM,Ca (to 3 x 10(-4) min-1), accounts for a Tg-dependent sustained elevation [Ca2+]cyt (to 708 +/- 78 nM) which is independent of DT Ca2+ status or history. Thrombin and Tg release 30 and 70% (respectively) of the DT Ca2+ available at any instant, independent of order of challenge, consistent with a single class of DT with respect to these agents.
Tao
J
J
Department of Molecular and Cellular Pharmacology, University of Miami School of Medicine, Florida 33101.
Haynes
D H
DH
D001692:Biological Transport; D001792:Blood Platelets / Q000187:drug effects / Q000378:metabolism*; D002118:Calcium / Q000097:blood*; D000252:Calcium-Transporting ATPases / Q000037:antagonists & inhibitors* / Q000097:blood* / Q000187:drug effects*; D002463:Cell Membrane Permeability / Q000187:drug effects*; D003600:Cytosol / Q000187:drug effects / Q000378:metabolism; D006801:Humans; D066298:In Vitro Techniques; D015759:Ionomycin / Q000494:pharmacology; D007700:Kinetics; D008345:Manganese / Q000097:blood; D008433:Mathematics; D008954:Models, Biological; D013050:Spectrometry, Fluorescence; D013729:Terpenes / Q000494:pharmacology*; D019284:Thapsigargin
D013729:Terpenes; D008345:Manganese; D015759:Ionomycin; D019284:Thapsigargin; D000252:Calcium-Transporting ATPases; D002118:Calcium