Ca2+ entry pathways activated by the tumor promoter thapsigargin in human platelets.
Thapsigargin-activated Ca2+ entry into platelets was examined in the presence of S-145, a thromboxane A2 receptor antagonist, to inhibit indirect effects by endogenously formed prostaglandin H2/thromboxane A2. With external Ca2+ present, 0.2 microM thapsigargin caused a prompt increase in intracellular Ca2+ concentration ([Ca2+]i) followed by a gradual increase. Pretreatment with 6 microM wortmannin, a specific inhibitor of myosin light chain kinase, partly inhibited the increase in [Ca2+]i. In Ca(2+)-free EGTA buffer, thapsigargin induced a smaller increase in [Ca2+]i, and subsequent addition of Ca2+ to the buffer caused a further prompt increase in [Ca2+]i, demonstrating external Ca2+ entry. Wortmannin only partly inhibited this entry of external Ca2+. The wortmannin-insensitive Ca2+ entry pathway remained open for more than 6 min in Ca(2+)-free buffer. On the other hand, when receptor agonists such as thrombin and U46619 were substituted for thapsigargin, activation of the wortmannin-insensitive Ca2+ entry was transient (Hashimoto et al., J. Biol. Chem (1992) 267, 17078-17081). In the presence of S-145 and wortmannin, thapsigargin stimulated phosphorylation of neither the 20-kDa myosin light chain nor the 47-kDa protein, a substrate of protein kinase C. These results suggest that thapsigargin induces external Ca2+ entry by two mechanisms: (1) a mechanism involving myosin light chain kinase; (2) a mechanism, not activated by receptor agonists, that is independent of the major protein kinases of platelets.
Hashimoto
Y
Y
First Department of Internal Medicine, Faculty of Medicine, University of Tokyo, Japan.
Watanabe
T
T
Kinoshita
M
M
Tsukamoto
K
K
Togo
M
M
Horie
Y
Y
Matsuda
Y
Y
Kurokawa
K
K
D019796:15-Hydroxy-11 alpha,9 alpha-(epoxymethano)prosta-5,13-dienoic Acid; D000730:Androstadienes / Q000494:pharmacology; D001792:Blood Platelets / Q000187:drug effects / Q000378:metabolism*; D002118:Calcium / Q000378:metabolism*; D002273:Carcinogens / Q000494:pharmacology*; D006801:Humans; D066298:In Vitro Techniques; D011450:Prostaglandin Endoperoxides, Synthetic / Q000494:pharmacology; D013729:Terpenes / Q000494:pharmacology*; D019284:Thapsigargin; D013917:Thrombin / Q000502:physiology; D013928:Thromboxane A2 / Q000031:analogs & derivatives / Q000494:pharmacology; D000077191:Wortmannin
D000730:Androstadienes; D002273:Carcinogens; D011450:Prostaglandin Endoperoxides, Synthetic; D013729:Terpenes; D013928:Thromboxane A2; D019284:Thapsigargin; D019796:15-Hydroxy-11 alpha,9 alpha-(epoxymethano)prosta-5,13-dienoic Acid; D013917:Thrombin; D002118:Calcium; D000077191:Wortmannin