Activation of Na+/H+ exchange and Ca2+ mobilization start simultaneously in thrombin-stimulated platelets. Evidence that platelet shape change disturbs early rises of BCECF fluorescence which causes an underestimation of actual cytosolic alkalinization

Abstract

Although an increase in cytosolic pH (pHi) caused by Na+/H+ exchange enhances Ca2+ mobilization in platelets stimulated by low concentrations of thrombin [Siffert & Akkerman (1987) Nature (London) 325, 456-458], studies using fluorescent indicators for pHi (BCECF) and [Ca2+]i (fura2) suggest that Ca2+ is mobilized while the cytosolic pH decreases. Several lines of evidence indicate that the initial fall in BCECF fluorescence is not due to cytosolic acidification but is caused by a platelet shape change. (1) Pulse stimulation of platelets by successive addition of hirudin (4 unit/ml) and thrombin (0.2 unit/ml) induced a shape change of 43 +/- 8% and a fall in BCECF fluorescence, which both remained unchanged when Na+/H+ exchange was inhibited by ethylisopropylamiloride (EIPA, 100 microM). (2) Increasing the thrombin concentration to 0.4 unit/ml doubled the shape change and the fall in BCECF fluorescence, but again EIPA had no effect on these responses. (3) Treating platelets with 2 microM-ADP induced shape change and a decline in BCECF fluorescence that was unaffected by EIPA. (4) A second addition of thrombin to platelets that had already undergone shape change induced an immediate increase in BCECF fluorescence without a prior decrease. (5) Activation of protein kinase C by 1,2-dioctanoyl-sn-glycerol (DiC8) neither induced shape change nor a decline in BCECF fluorescence; in contrast BCECF fluorescence rapidly increased indicating an immediate cytosolic alkalinization. Concurrent analysis of [Ca2+]i under conditions in which shape change did not interfere with BCECF fluorescence showed that cytosolic alkalinization and Ca2+ mobilization started almost simultaneously. These observations suggest that cytosolic alkalinization is not preceded by a fall in pHi and can support Ca2+ mobilization induced by weak agonists.