Two thrombin-activated Ca2+ channels in human platelets.
The regulation of extracellular Ca2+ entry into fura-2-loaded human platelets was examined following stimulation with thrombin. In the presence of external Ca2+, stimulation of platelets with thrombin resulted in a rapid increase, followed by a plateau, in intracellular Ca2+ concentration ([Ca2+]i). Pretreatment with wortmannin, a specific inhibitor of myosin light chain kinase, suppressed only the plateau phase and had no effect on the initial rapid increase in [Ca2+]i. In Ca(2+)-free EGTA buffer, thrombin induced a transient and relatively small increase in [Ca2+]i caused by Ca2+ release from internal stores. When Ca2+ was added subsequently to the Ca(2+)-free medium within 10 min after thrombin activation, a marked increase in [Ca2+]i was seen, reflecting thrombin-stimulated external Ca2+ entry. With the Ca(2+)-free medium, wortmannin did not affect either the Ca2+ mobilization from the internal stores or the rapid external Ca2+ entry at early time points (within 5 s) after thrombin stimulation, whereas it significantly inhibited Ca2+ entry when Ca2+ was added later (at 3 min). Wortmannin inhibition of this late Ca2+ entry and that of 20-kDa myosin light chain phosphorylation after thrombin stimulation were dose- and preincubation time-dependent and correlated well with each other. These results suggest that two different channels are responsible for Ca2+ entry in human platelets at the early and late phases of thrombin stimulation and that the channel responsible for the late phase of Ca2+ entry may be activated by a mechanism involving myosin light chain kinase.
Hashimoto
Y
Y
First Department of Internal Medicine, Faculty of Medicine, University of Tokyo, Japan.
Ogihara
A
A
Nakanishi
S
S
Matsuda
Y
Y
Kurokawa
K
K
Nonomura
Y
Y
D000730:Androstadienes / Q000494:pharmacology; D000893:Anti-Inflammatory Agents / Q000494:pharmacology; D001792:Blood Platelets / Q000187:drug effects / Q000502:physiology*; D002118:Calcium / Q000097:blood; D015220:Calcium Channels / Q000187:drug effects / Q000502:physiology*; D016257:Fura-2; D006801:Humans; D066298:In Vitro Techniques; D007700:Kinetics; D009218:Myosins / Q000097:blood; D010766:Phosphorylation; D013917:Thrombin / Q000494:pharmacology*; D000077191:Wortmannin
D000730:Androstadienes; D000893:Anti-Inflammatory Agents; D015220:Calcium Channels; D013917:Thrombin; D009218:Myosins; D002118:Calcium; D016257:Fura-2; D000077191:Wortmannin