[Significance of multiple analysis of platelet Ca2+ mobilization by using some available methods measuring cytosolic Ca2+ concentration, membrane-bound Ca2+ and Ca2+ flux across the plasma membrane].
Intracellular Ca2+ mobilization in the resting platelets undergoing Ca2+ influx and in the activated platelets stimulated by thrombin and phorbol ester (TPA) was investigated by measuring cytosolic Ca2+ concentration: [Ca2+]cyt (fura-2, aequorin), membrane-bound Ca2+ (chlortetracycline) and Ca2+ flux across the plasma membrane (45Ca2+ tracer). Some modified methods employed in this study concerning 45Ca2+ flux measurement and aequorin loading into platelets were very useful to elucidate the details of platelet Ca2+ mobilization in the combination with other Ca2+ assay methods. These combined and integrated assays on platelet Ca2+ showed some new interesting findings as follows; (1) The difference of the obtained values of [Ca2+]cyt and of the reaction pattern between fura-2 and aequorin method was responsible to the difference of the distribution of probe molecules in platelet. This was confirmed by the higher sensitivity of aequorin molecules to [Ca2+]cyt elevation induced by Ca2+ influx than by that of fura-2 molecules in thrombin- and TPA-activated platelets. (2) The membrane-bound Ca2+ release in activated platelets was found to be another intracellular Ca2+ movement distinct from organelle Ca2+ release and CTC molecules not to bind to the organelle membranes. (3) The plasma membrane Ca2+ pump was found to exist in both unstimulated platelets and stimulated platelets suggested by its specific time course of 45Ca2+ binding and [Ca2+]cyt increase in platelets. (4) There existed biphasic increase of [Ca2+]cyt in thrombin-stimulated platelets consisted of biphasic Ca2+ influx and biphasic intracellular Ca2+ release with alternating point at 10 seconds after activation. The initial changes seemed to be correlated with receptor operated Ca2+ channel and receptor-linked phospholipase C activation respectively, while both second phase increase seemed to be correlated with the plasma membrane phospholipid metabolisms evoked by phospholipase A2 activation.
Katabami
F
F
Department of Laboratory Medicine, Hokkaido University School of Medicine, Sapporo.
D000331:Aequorin; D001572:Benzofurans; D001792:Blood Platelets / Q000032:analysis*; D002118:Calcium / Q000097:blood*; D002462:Cell Membrane / Q000378:metabolism; D002751:Chlortetracycline; D003600:Cytosol / Q000378:metabolism*; D016257:Fura-2; D006801:Humans; D010974:Platelet Aggregation; D013050:Spectrometry, Fluorescence / Q000379:methods
D001572:Benzofurans; D000331:Aequorin; D002118:Calcium; D016257:Fura-2; D002751:Chlortetracycline