Two uncompetitive, activated, and transport sites of the Na +/H + exchanger for pH regulation in perfused rat kidney

• Published in: Comparative biochemistry and physiology. Part A, Molecular & integrative physiology Molecular & integrative physiology, 1999-08, Vol.123 (4), p.417-422
• Main Authors: Bikhazi, Anwar B, Ziadeh, Alexander G, Abbud, Rita A, Nabhan, Suad I, Haddad, George E
• Format: Article
• Language: English
• Subjects: Acidosis and chemical hypoxia; Amiloride; Amiloride - pharmacology; Animals; Diuretics - pharmacology; Female; Hydrogen-Ion Concentration; Intracellular pH; Ion Transport; Kidney - drug effects; Kidney - metabolism; Kinetics; Na +/H + exchanger-1; pH-paradox; Rats; Rats, Sprague-Dawley; Reperfusion injury; Sodium-Hydrogen Exchangers - metabolism; Uncompetitive titratable sites
• ISSN: 1095-6433; 1531-4332
• DOI: 10.1016/S1095-6433(99)00087-2

The purpose of this study is to assess the effect of an apparent alteration in intracellular pH and the effect of amiloride on the activity of the Na +/H + antiporter in perfused rat kidney. Rat kidney-Na + retention was determined using tracer 22Na in perfusate composed of HCl–glycine buffer (pH 3.80 to pH 5.92) or NH 4OH–glycine buffer (pH 6.22–7.95) containing Na + to match physiologic concentrations. Plotting renal Na + retention for 10 min versus pH in absence of amiloride showed two classical uncompetitive activator curves for H +, one curve from pH 4.19 to 5.10 and another from pH 6.22 to 7.95. H + acts as an uncompetitive reversible binding substrate with the receptor triggering activation of the exchanger already sequestered with Na +, thus yielding two K a values for the exchanger suggesting non-first order kinetics. Using an equation derived for uncompetitive-activation binding of Na + o and H + i, plotting [mM Na + mg protein −1 10 min −1] −1 versus [H +], two linear plots are observed on Cartesian coordinates with abscissa intersecting at 47±1 μM, p K a=4.32±0.02 (pH 4.19–5.10) and 4.21±0.02 μM, p K a=5.38±0.01 (pH 6.22–7.95), respectively. Perfusing buffer containing 2 mM amiloride, completely inactivated the antiporter showing stronger inhibition between pH 3.80 and 5.92. Results suggest the presence of two uncompetitive binding sites for H + with the Na +/H + exchanger. One is a high affinity binding site at physiological intracellular apparent pH, and another is a low affinity binding site at ischaemic apparent pH, implying the existence of two titration sites for intracellular pH regulation.