Thrombin-induced changes of intracellular [Ca2+] and pH in human platelets. Cytoplasmic alkalinization is not a prerequisite for calcium mobilization.
We have studied the effects of thrombin (0.1 U/ml) on intracellular Ca2+ ([Ca2+]i) and pH (pHi) in human platelets loaded with fluorescent indicators. Thrombin produced a transient decrease of pHi which reached its maximum within 15-25 seconds (s) and was followed by a sustained alkalinization which brought pHi above the resting value. [Ca2+]i increased transiently peaking at 5-10 s. The late alkalinization induced by thrombin was antagonized by ethylisopropylamiloride, an inhibitor of Na+-H+ exchange, and by sphingosine, an inhibitor of protein kinase C, with little effect on the [Ca2+]i transient. The early acidification was not inhibited by these treatments. We conclude tha the thrombin-induced changes of [Ca2+]i and pHi are mediated by different mechanisms. The late alkalinization is due to activation of Na+/H+ exchange mediated by protein kinase C and, contrarily to previous proposals (Siffert, W. and Akkerman, J.W.N. (1987) Nature 325, 456-458), it is not necessary for calcium mobilization from intracellular stores.
Sanchez
A
A
Departamento de Bioquímica y Biología Molecular y Fisiología, Facultad de Medicina, Universidad de Valladolid, Valladolid, Spain.
Alonso
M T
MT
Collazos
J M
JM
D000584:Amiloride / Q000031:analogs & derivatives / Q000494:pharmacology; D001792:Blood Platelets / Q000187:drug effects / Q000378:metabolism*; D002118:Calcium / Q000097:blood*; D002352:Carrier Proteins / Q000037:antagonists & inhibitors / Q000378:metabolism; D003593:Cytoplasm / Q000378:metabolism; D004987:Ethers / Q000494:pharmacology; D006801:Humans; D006863:Hydrogen-Ion Concentration; D015759:Ionomycin; D011493:Protein Kinase C / Q000037:antagonists & inhibitors / Q000378:metabolism; D017923:Sodium-Hydrogen Exchangers; D013110:Sphingosine / Q000494:pharmacology; D013917:Thrombin / Q000494:pharmacology*
D002352:Carrier Proteins; D004987:Ethers; D017923:Sodium-Hydrogen Exchangers; D015759:Ionomycin; D000584:Amiloride; D011493:Protein Kinase C; D013917:Thrombin; D013110:Sphingosine; D002118:Calcium; C039614:ethylisopropylamiloride