Estimation of carrier density and turnover rate of the Na+/H+ exchanger in human platelets using 5-(N-methyl-N-[3H]isobutyl)- amiloride.

We used the radiolabelled inhibitor of Na+/H+ exchange 5-(N-methyl-N-[3H]isobutyl)amiloride ([3H]-MIA) for assessment of the amount of Na+/H+ exchanger in intact human blood platelets. The inhibition constant, KI, of unlabelled MIA toward the antiport was determined at 100 nM. Washed platelets were incubated for 5 s with different concentrations of [3H]-MIA in the presence or absence of an excess concentration of unlabelled amiloride (400 microM). The platelets were rapidly centrifuged and the radioactivity in the pellet was determined. Scatchard analysis revealed one single class of specific binding sites (KD = 63 nM) and a maximum binding capacity of 500 sites/cell. The turnover rate of the Na+/H(+)-exchanger in unstimulated platelets was estimated at 800/s at 25 degrees C.