title: Two uncompetitive, activated, and transport sites of the Na+/H+ exchanger for pH regulation in perfused rat kidney.
abstract: 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 NH4OH-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 Ka values for the exchanger suggesting non-first order kinetics. Using an equation derived for uncompetitive-activation binding of Nao+ and Hi+, 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 microM, pKa = 4.32 +/- 0.02 (pH 4.19-5.10) and 4.21 +/- 0.02 microM, pKa = 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.
authors.affiliation: Department of Physiology, Faculty of Medicine, American University of Beirut, Lebanon. ab04@aub.edu.lb
authors.forename: A B
authors.initials: AB
authors.lastname: Bikhazi
authors.forename: A G
authors.initials: AG
authors.lastname: Ziadeh
authors.forename: R A
authors.initials: RA
authors.lastname: Abbud
authors.forename: S I
authors.initials: SI
authors.lastname: Nabhan
authors.forename: G E
authors.initials: GE
authors.lastname: Haddad
mesh_terms: D000584:Amiloride / Q000494:pharmacology; D000818:Animals; D004232:Diuretics / Q000494:pharmacology; D005260:Female; D006863:Hydrogen-Ion Concentration; D017136:Ion Transport; D007668:Kidney / Q000187:drug effects / Q000378:metabolism*; D007700:Kinetics; D051381:Rats; D017207:Rats, Sprague-Dawley; D017923:Sodium-Hydrogen Exchangers / Q000378:metabolism*
chemical_list: D004232:Diuretics; D017923:Sodium-Hydrogen Exchangers; D000584:Amiloride