title: 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.
authors.affiliation: Institut für Physiologie, Ruhr-Universität, Bochum, Federal Republic of Germany.
authors.forename: W
authors.initials: W
authors.lastname: Siffert
authors.forename: G
authors.initials: G
authors.lastname: Siffert
authors.forename: P
authors.initials: P
authors.lastname: Scheid
authors.forename: J W
authors.initials: JW
authors.lastname: Akkerman
mesh_terms: D001792:Blood Platelets / Q000187:drug effects*; D002118:Calcium / Q000378:metabolism*; D003600:Cytosol / Q000378:metabolism; D005452:Fluoresceins / Q000378:metabolism*; D005456:Fluorescent Dyes / Q000378:metabolism*; D006801:Humans; D006863:Hydrogen-Ion Concentration; D011522:Protons; D012964:Sodium / Q000378:metabolism; D013917:Thrombin / Q000494:pharmacology*
chemical_list: D005452:Fluoresceins; D005456:Fluorescent Dyes; D011522:Protons; C043829:2',7'-bis(carboxyethyl)-5(6)-carboxyfluorescein; D012964:Sodium; D013917:Thrombin; D002118:Calcium