Ligand-affected shift of Na+/H+ exchange pHi set point in human blood platelets, rapidly revealed by a novel approach.
The Na+/H+ exchange time-course of BCECF-loaded human platelets, suspended in isotonic media containing NaCl and sodium propionate and activated by intracellular acidification, was measured spectrofluorimetrically. Sequential alkalinization rates decline exponentially as a function of the changing intracellular pH (pHi) and its linear expression (log rate vs. pHi) extrapolates reproducibly to the pHi set point for the Na+/H+ exchange activation. The set point of control platelets (7.28 +/- 0.01) is shifted rapidly (discernibly less than or equal to 30 s) and markedly to alkaline pHi (7.62 +/- 0.03) by PMA, that activates protein kinase C and is shifted to acidic pHi (7.05 +/- 0.01) by staurosporine, which inhibits protein kinases. The addition of 5-N-(3-aminophenyl)amiloride reveals that the alkalinization measured is predominantly Na+/H+ exchange with only a minute contribution (delta pHi = 0.012 +/- 0.002 in 1 min) of an acid loading component, at pHi greater than 7.2. The results support recent studies concluding that the set point indeed reflects the phosphorylation state of the Na+/H+ exchanger.
Livne
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AA
Department of Life Sciences, Ben-Gurion University of the Negev, Beer-Sheva, Israel.
Aharonovitz
O
O
D001792:Blood Platelets / Q000187:drug effects / Q000378:metabolism*; D005452:Fluoresceins; D005456:Fluorescent Dyes; D006801:Humans; D006863:Hydrogen-Ion Concentration; D066298:In Vitro Techniques; D007700:Kinetics; D012964:Sodium / Q000097:blood*; D013050:Spectrometry, Fluorescence; D013755:Tetradecanoylphorbol Acetate / Q000494:pharmacology
D005452:Fluoresceins; D005456:Fluorescent Dyes; C043829:2',7'-bis(carboxyethyl)-5(6)-carboxyfluorescein; D012964:Sodium; D013755:Tetradecanoylphorbol Acetate