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

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....

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Published in:	Comparative biochemistry and physiology. Part A, Molecular & integrative physiology Vol. 123; no. 4; pp. 417 - 422
Main Authors:	Bikhazi, Anwar B, Ziadeh, Alexander G, Abbud, Rita A, Nabhan, Suad I, Haddad, George E
Format:	Journal Article
Language:	English
Published:	United States Elsevier Inc 01-08-1999
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 Na +/H + exchanger-1 Intracellular pH pH-paradox Amiloride Uncompetitive titratable sites Reperfusion injury Acidosis and chemical hypoxia
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Summary:	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.
Bibliography:	ObjectType-Article-1 SourceType-Scholarly Journals-1 ObjectType-Feature-2 content type line 23
ISSN:	1095-6433 1531-4332
DOI:	10.1016/S1095-6433(99)00087-2