Direct characterization of the Na+/H+ exchanger in human platelets.
The kinetic properties of the Na+/H+ exchanger in human platelets were investigated by direct measurements of pHi as detected with the fluorescent dye, BCECF. In acid-loaded cells, the antiporter displayed a hyperbolic dependence regarding external Na+ with an apparent Km of 38 +/- 4 mM (pHo 7.2 at 25 degrees C) whereas its pHi-dependent activation between 7.3 to 6.4 did not obey a Michaelian model. External acidification from 7.7 to 6.5 decreased significantly the initial rate of Na(+)-dependent H+ efflux. The amiloride derivative, ethylisopropylamiloride blocked this exchanger and exerted a non-competitive inhibition with respect to Na+o (Ki = 17 nM). The cation selectivity of the external site of the antiporter was Na+ greater than Li+ greater than K+ and choline. These results indicate that the BCECF technique allows to evaluate the main features of the Na+/H+ exchanger in human platelets, which possesses kinetic properties similar to those reported in other cell types.
Astarie
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Department of Pharmacology, CNRS SDI 61670, Necker Medical School, Paris, France.
David-Dufilho
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Devynck
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MA
D000584:Amiloride / Q000031:analogs & derivatives / Q000494:pharmacology; D001792:Blood Platelets / Q000378:metabolism*; D002352:Carrier Proteins / Q000378:metabolism*; D005452:Fluoresceins / Q000737:chemistry; D006801:Humans; D006863:Hydrogen-Ion Concentration; D066298:In Vitro Techniques; D007700:Kinetics; D009550:Nigericin / Q000737:chemistry; D012964:Sodium / Q000378:metabolism*; D017923:Sodium-Hydrogen Exchangers
D002352:Carrier Proteins; D005452:Fluoresceins; D017923:Sodium-Hydrogen Exchangers; C039465:5-dimethylamiloride; D000584:Amiloride; C043829:2',7'-bis(carboxyethyl)-5(6)-carboxyfluorescein; D012964:Sodium; D009550:Nigericin