Estimation of the number and turnover rate of Na+/H+ exchangers in lymphocytes. Effect of phorbol ester and osmotic shrinking.
Rat thymic lymphocytes possess an amiloride-sensitive Na+/H+ exchanger in their plasma membrane. Kinetic studies revealed that 5-(N-methyl-N-isobutyl)amiloride (MIA) was a more potent inhibitor of the antiport than amiloride (cf. apparent Ki of 174 nM and 6 microM, respectively). Inhibition by MIA was rapid (less than 5 s) and readily reversible. [3H]MIA binding to whole cells was assayed by rapid centrifugation following short (5 s) incubations to minimize nonspecific binding. A saturable binding component (Kd approximately equal to 170 nM) which was displaced by amiloride was detected. In contrast, there was no significant amiloride-displaceable binding to human erythrocytes, which have comparatively little Na+/H+ exchange activity. In lymphocytes, the ability of amiloride and several of its analogs to displace [3H]MIA correlated with their potency as inhibitors of the antiport. Both kinetic and binding studies revealed that extracellular H+, but not Na+, inhibited the interaction of MIA with its receptor(s). Taken together, these data suggest that [3H]MIA binds to the Na+/H+ exchanger. Scatchard analysis revealed that [3H]MIA bound to a maximum of 8000 high affinity sites/cell. Activation of Na+/H+ exchange by osmotic shrinking or by the phorbol ester 12-O-tetradecanoylphorbol 13-acetate was not accompanied by a significant change in [3H]MIA binding. Given an upper limit of 8000 functional sites/thymocyte, we estimate that the turnover number of each maximally activated exchanger is at least 2000 cycles/s.
D000584:Amiloride / Q000031:analogs & derivatives / Q000378:metabolism / Q000494:pharmacology; D000818:Animals; D002352:Carrier Proteins / Q000378:metabolism*; D002462:Cell Membrane / Q000187:drug effects / Q000378:metabolism; D004912:Erythrocytes / Q000378:metabolism; D006801:Humans; D007700:Kinetics; D008297:Male; D051381:Rats; D011919:Rats, Inbred Strains; D017923:Sodium-Hydrogen Exchangers; D013601:T-Lymphocytes / Q000166:cytology / Q000378:metabolism*; D013755:Tetradecanoylphorbol Acetate / Q000494:pharmacology*
D002352:Carrier Proteins; D017923:Sodium-Hydrogen Exchangers; D000584:Amiloride; D013755:Tetradecanoylphorbol Acetate