22Na+ fluxes in thymic lymphocytes. II. Amiloride-sensitive Na+/H+ exchange pathway; reversibility of transport and asymmetry of the modifier site.
22Na+ flux and cytoplasmic pH (pHi) determinations were used to study the reversibility, symmetry, and mechanism of activation of the Na+/H+ exchange system in rat thymic lymphocytes. In acid-loaded cells, the antiport can be detected as an Na+-induced, amiloride-sensitive alkalinization. At pHi greater than or equal to 7.0, amiloride-sensitive net H+ fluxes are not detectable. To investigate whether at this pHi the transporter is operative in a different mode, e.g., Na+/Na+ exchange, 22Na+ uptake was measured as a function of pHi. The results indicate that the antiport is relatively inactive at pHi greater than or equal to 7.0. Comparison of the rates of H+ efflux (or equivalent OH- uptake) and Na+ uptake indicate that Na+/Na+ countertransport through this system is negligible at all values of pHi and that the Na+:H+ stoichiometry is 1:1. Measurements of pHi in Na+-loaded cells suspended in Na+-free medium revealed an amiloride-sensitive cytoplasmic acidification, which is indicative of exchange of internal Na+ for external H+. The symmetry of the system was analyzed by measuring the effect of extracellular pH (pHo) on Na+ efflux. Unlike cytoplasmic acidification, lowering pHo failed to activate the antiport. The results indicate that the amiloride-sensitive Na+/H+ exchanger is reversible but asymmetric. The system is virtually inactive at pHi greater than or equal to 7.0 but can be activated by protonation of a modifier site on the cytoplasmic surface. Activation can also occur by depletion of cellular Na+. It is proposed that Na+ may also interact with the modifier site, stabilizing the unprotonated (inactive) form.
D000584:Amiloride / Q000494:pharmacology*; D000818:Animals; D001692:Biological Transport; D006859:Hydrogen / Q000378:metabolism; D006863:Hydrogen-Ion Concentration; D008094:Lithium / Q000494:pharmacology; D008297:Male; D009994:Osmolar Concentration; D011719:Pyrazines / Q000494:pharmacology*; D051381:Rats; D011919:Rats, Inbred Strains; D012964:Sodium / Q000378:metabolism*; D012979:Sodium Radioisotopes; D013601:T-Lymphocytes / Q000378:metabolism*; D013696:Temperature; D013950:Thymus Gland / Q000166:cytology
D011719:Pyrazines; D012979:Sodium Radioisotopes; D000584:Amiloride; D006859:Hydrogen; D008094:Lithium; D012964:Sodium