Fluorescence digital image analysis of thrombin and ADP induced rise in intracellular Ca2+ concentration of single blood platelets.
Cytoplasmic free Ca2+ concentration, [Ca2+]i, was estimated in single rabbit blood platelets by digital imaging microscopy with the use of the specific Ca(2+)-indicator dye Fura-2. Uneven distribution and low level of [Ca2+]i was found in the resting platelet even in the presence of extracellular 1 mM Ca2+. Thrombin at 1 unit/ml immediately caused a transient increase in [Ca2+]i, which was followed by a secondary and sustained increase in [Ca2+]i. The distribution of increased levels of [Ca2+]i was also shown to be uneven within the cell. The presence of 1 mM EGTA in the medium only slightly decreased the initial rise in [Ca2+]i, but completely inhibited the latter phase, a sustained rise in [Ca2+]i. This result shows that the initial rise of [Ca2+]i might not be caused by Ca2+ influx, but might be induced by mobilization of Ca2+ from intracellular Ca2+ storage sites. This speculation is further supported by the fact that the elevated [Ca2+]i induced by thrombin immediately decreased to the base line value when 3 mM EGTA was applied. Thus, thrombin induced elevation of [Ca2+]i is suggested to consist of two different processes, namely the mobilization of Ca2+ from the intracellular storage sites and the successive Ca2+ influx through the receptor activated Ca2+ channels. Stimulation with ADP also caused a rapid elevation of platelet [Ca2+]i, but this effect of ADP was different form that of thrombin. Thus, the ADP induced rise in [Ca2+]i was accompanied by oscillation and was inhibited by extracellular EGTA. Our present experiment is the first report that clearly and directly reveals the differences between the effects of thrombin and ADP on [Ca2+]i of platelets.
Nishio
H
H
Department of Pharmacology, Hiroshima University School of Medicine, Japan.
Ikegami
Y
Y
Nakata
Y
Y
Segawa
T
T
D000244:Adenosine Diphosphate / Q000494:pharmacology*; D000818:Animals; D001792:Blood Platelets / Q000187:drug effects / Q000378:metabolism*; D002118:Calcium / Q000097:blood*; D003593:Cytoplasm / Q000378:metabolism; D004533:Egtazic Acid / Q000494:pharmacology; D016257:Fura-2; D066298:In Vitro Techniques; D007700:Kinetics; D008856:Microscopy, Fluorescence / Q000379:methods; D011817:Rabbits; D013917:Thrombin / Q000494:pharmacology*; D013997:Time Factors
D004533:Egtazic Acid; D000244:Adenosine Diphosphate; D013917:Thrombin; D002118:Calcium; D016257:Fura-2