Kinetics and regulation of three cloned mammalian Na+/H+ exchangers stably expressed in a fibroblast cell line.
The kinetics and second messenger regulation of three cloned mammalian intestinal Na+/H+ exchangers were studied using fluorometric techniques. These exchangers, NHE1, NHE2, and NHE3, were stably expressed in PS120 fibroblasts, which lack an endogenous Na+/H+ exchanger. H+ kinetic data indicated cooperativity by internal protons, with Hill coefficients of approximately 2 for all three isoforms. In contrast, Na+ kinetic data fit Michaelis-Menten kinetics, with Km (Na+) 15-18 mM and a Hill coefficient of approximately 1. The exchangers were all activated by growth factors and thrombin; in NHE1 these agonists increased the apparent affinity for intracellular H+, but did not change Vmax, while for NHE2 and NHE3 the effect was on Vmax alone. Phorbol ester stimulated NHE1 and NHE2, but inhibited NHE3 with a decrease in Vmax. ATP-depletion decreased Vmax and the apparent affinity for H+ for all three isoforms, and reduced the Hill coefficient to approximately 1, suggesting that a basal level of phosphorylation was required for the cooperativity. The differences in kinetics and second messenger regulation suggest that the NHE isoforms may serve different cellular functions. The up- and down-regulation of NHE3 by kinases indicates that this isoform may be involved in a specialized function such as Na+ absorption.
Levine
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SA
Department of Medicine, Johns Hopkins University School of Medicine, Baltimore, Maryland 21205-2195.
Montrose
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MH
Tse
C M
CM
Donowitz
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D015124:8-Bromo Cyclic Adenosine Monophosphate / Q000494:pharmacology; D000255:Adenosine Triphosphate / Q000378:metabolism; D000818:Animals; D002352:Carrier Proteins / Q000378:metabolism; D002460:Cell Line; D002999:Clone Cells; D003001:Cloning, Molecular; D006152:Cyclic GMP / Q000031:analogs & derivatives / Q000494:pharmacology; D005346:Fibroblast Growth Factors / Q000494:pharmacology; D005347:Fibroblasts / Q000378:metabolism; D015870:Gene Expression; D006863:Hydrogen-Ion Concentration; D007700:Kinetics; D011994:Recombinant Proteins / Q000096:biosynthesis / Q000378:metabolism; D012964:Sodium / Q000378:metabolism*; D017923:Sodium-Hydrogen Exchangers / Q000096:biosynthesis / Q000378:metabolism*; D013755:Tetradecanoylphorbol Acetate / Q000494:pharmacology; D013917:Thrombin / Q000494:pharmacology; D014162:Transfection
D002352:Carrier Proteins; D011994:Recombinant Proteins; D017923:Sodium-Hydrogen Exchangers; C069139:growth factor-activatable Na-H exchanger NHE-1; D015124:8-Bromo Cyclic Adenosine Monophosphate; C016276:8-bromocyclic GMP; D005346:Fibroblast Growth Factors; D000255:Adenosine Triphosphate; D012964:Sodium; D013917:Thrombin; D006152:Cyclic GMP; D013755:Tetradecanoylphorbol Acetate