title: Irregular spiking in free calcium concentration in single, human platelets. Regulation by modulation of the inositol trisphosphate receptors.
abstract: Fluorescence ratio imaging indicates that immobilized, aspirin-treated platelets, loaded with Fura-2, respond to inositol 1,4,5-trisphosphate- (InsP3)-generating agonists such as thrombin by high-frequency, irregular rises in cytosolic [Ca2+]i with spikes that vary in peak level and peak-to-peak interval. This differs from the regular [Ca2+]i oscillations observed in other, larger cells. We found that the thiol-reactive compounds thimerosal (10 microm) and U73122 (10 microm) evoked similar irregular Ca2+ responses in platelets, but in this case in the absence of InsP3 generation. Thrombin-induced spiking was acutely abolished by inhibiting phospholipase C or elevating intracellular cAMP levels, while spiking with sulfhydryl reagents was only partially blocked by cAMP elevation. Confocal laser scanning microscopy using fluo-3-loaded platelets indicated that, with all agonists or conditions, the irregular spikes were almost instantaneously raised in various regions within a single platelet. When using saponin-permeabilized platelets, we found that InsP3-induced Ca2+ release from stores was stimulated by modest Ca2+ concentrations, pointing to a mechanism of InsP3-dependent Ca2+-induced Ca2+ release (CICR). This process was completely inhibitable by heparin. The Ca2+ release by InsP3, but not the CICR sensor, was negatively regulated by cAMP elevation. Thimerosal treatment did not release Ca2+ from intracellular stores, but markedly potentiated the stimulatory effect of InsP3. In contrast, U73122 caused a heparin/cAMP-insensitive Ca2+ leak from stores that differed from those used by InsP3. Taken together, these results demonstrate that InsP3 receptor channels play a crucial role in the irregular, spiking Ca2+ signal of intact platelets, even when induced by agents such as thimerosal or U73122 which do not stimulate InsP3 formation. The irregular Ca2+ release events appear to be subjected to extensive regulation by: (a) InsP3 level, (b) the potentiating effect of elevated Ca2+ on InsP3 action via CICR, (c) InsP3 channel sensitization by sulfhydryl (thimerosal) modification, (d) InsP3 channel-independent Ca2+ leak with U73122, and (e) down-regulation via cAMP elevation. The observation that individual Ca2+ peaks were generated in various parts of a platelet at similar intervals and amplitudes points to effective cooperation of the various stores in the Ca2+-release process.
authors.affiliation: Department of Biochemistry, University of Maastricht, the Netherlands.
authors.forename: Roosje M A
authors.initials: RM
authors.lastname: van Gorp
authors.forename: Marion A H
authors.initials: MA
authors.lastname: Feijge
authors.forename: Wim M J
authors.initials: WM
authors.lastname: Vuist
authors.forename: Martin B
authors.initials: MB
authors.lastname: Rook
authors.forename: Johan W M
authors.initials: JW
authors.lastname: Heemskerk
mesh_terms: D000527:Alprostadil / Q000494:pharmacology; D001792:Blood Platelets / Q000378:metabolism*; D002118:Calcium / Q000378:metabolism*; D015220:Calcium Channels / Q000502:physiology*; D000242:Cyclic AMP / Q000096:biosynthesis; D006801:Humans; D015544:Inositol 1,4,5-Trisphosphate / Q000096:biosynthesis; D053496:Inositol 1,4,5-Trisphosphate Receptors; D018160:Receptors, Cytoplasmic and Nuclear / Q000502:physiology*; D013849:Thimerosal / Q000494:pharmacology
chemical_list: D015220:Calcium Channels; C506926:ITPR1 protein, human; D053496:Inositol 1,4,5-Trisphosphate Receptors; D018160:Receptors, Cytoplasmic and Nuclear; D013849:Thimerosal; D015544:Inositol 1,4,5-Trisphosphate; D000242:Cyclic AMP; D000527:Alprostadil; D002118:Calcium