Role of cytoplasmic domain of the Na+/H+ exchanger in hormonal activation.
The growth factor-activated Na+/H+ exchanger is regulated by numerous stimuli, including polypeptide hormones, phorbol esters, cell acidity, and cell shrinkage. To determine whether this regulation occurs at a common site on the cytoplasmic domain of the Na+/H+ exchanger, we microinjected polyclonal antibodies (RP1-c28) to the C-terminal 157 amino acids of the molecule and measured cell pH changes after application of a variety of stimuli known to activate the Na+/H+ exchanger. Microinjection of approximately 10 fg of RP1-c28 antibody, but not control IgG, into single cultured fibroblasts blocked subsequent activation of the exchanger by both endothelin and alpha-thrombin. In contrast, microinjected RP1-c28 did not prevent activation of Na+/H+ exchange by phorbol esters, consistent with the observation that both endothelin-1 and alpha-thrombin retained the ability to activate exchange activity in protein kinase C-depleted cells. Finally, activation of Na+/H+ exchange by both cell acidity and osmotic shrinkage was also unaffected by microinjected RP1-c28 antibody. These data indicate that activation of Na+/H+ exchange by endothelin-1 and alpha-thrombin is mechanistically distinct both from activation by protein kinase C and activation by physical factors and probably occurs at a separate site on the exchanger molecule.
Nephrology Division, University of California, San Francisco 94143.
G K
GK
Winkel
C
C
Sardet
J
J
Pouyssegur
H E
HE
Ives
D000584:Amiloride / Q000494:pharmacology; D000643:Ammonium Chloride / Q000494:pharmacology; D000818:Animals; D016466:CHO Cells; D002352:Carrier Proteins / Q000276:immunology / Q000378:metabolism*; D006224:Cricetinae; D003593:Cytoplasm / Q000378:metabolism; D016232:Endothelins / Q000494:pharmacology*; D006863:Hydrogen-Ion Concentration; D007074:Immunoglobulin G; D011493:Protein Kinase C / Q000378:metabolism; D017466:Receptors, Endothelin / Q000235:genetics / Q000378:metabolism; D017923:Sodium-Hydrogen Exchangers; D013050:Spectrometry, Fluorescence; D013395:Sucrose / Q000494:pharmacology; D013755:Tetradecanoylphorbol Acetate / Q000494:pharmacology; D013917:Thrombin / Q000494:pharmacology; D014162:Transfection
D002352:Carrier Proteins; D016232:Endothelins; D007074:Immunoglobulin G; D017466:Receptors, Endothelin; D017923:Sodium-Hydrogen Exchangers; D000643:Ammonium Chloride; D013395:Sucrose; D000584:Amiloride; D011493:Protein Kinase C; D013917:Thrombin; D013755:Tetradecanoylphorbol Acetate