Regulation of Na(+)-H+ exchange by ATP depletion and calmodulin antagonism in renal epithelial cells.
The present studies examined effects of ATP depletion and calmodulin antagonism on stimulation of Na(+)-H+ exchange by cytosolic acidification in renal epithelial cells (LLC-PK1). ATP depletion significantly inhibited both amiloride-sensitive 22Na+ uptake (P less than 0.001; n = 12) and Na(+)-dependent intracellular pH (pHi) recovery in 2',7'-bis (carboxyethyl)-5(6)-carboxyfluorescein acetoxymethylester (BCECF/AM)-loaded cells. Calmodulin antagonists, N-(6-aminohexyl)-5-chloro-1-naphthalene-sulfonamide (W-7) and calmidazolium, both caused a concentration-dependent inhibition of Na(+)-H+ exchange activity. The W-7-induced inhibition of Na(+)-H+ exchange occurred in cells incubated for 24 h with phorbol 12-myristate 13-acetate, indicating that the effect of W-7 was not mediated by protein kinase C inhibition. Both W-7 and ATP depletion shifted the pHi dependence of the antiporter, and ATP depletion also reduced the maximal activity. In LLC-PK1/CL4 cells grown on permeable filters, W-7 inhibited the cytosolic acidification-stimulated basolateral exchanger by 54 +/- 5% (P less than 0.005; n = 7) and, in contrast, stimulated the apical exchanger by 28 +/- 13% (P less than 0.05; n = 6). ATP depletion significantly inhibited apical Na(+)-H+ exchange. These results suggest that an ATP-Ca(2+)-calmodulin-dependent process is involved in regulation of Na(+)-H+ exchange in LLC-PK1 cells. A Ca(2+)-calmodulin-dependent process activated the amiloride-sensitive basolateral Na(+)-H+ exchanger and inhibited the amiloride-resistant apical antiporter. Phosphorylation of these two Na(+)-H+ exchangers or regulatory proteins by a Ca(2+)-calmodulin-dependent protein kinase may mediate this differential regulation.
Burns
K D
KD
Department of Medicine, Department of Veterans Affairs Medical Center, Nashville, Tennessee.
Homma
T
T
Harris
R C
RC
D000255:Adenosine Triphosphate / Q000172:deficiency*; D000584:Amiloride / Q000031:analogs & derivatives / Q000494:pharmacology; D000818:Animals; D002147:Calmodulin / Q000037:antagonists & inhibitors*; D002352:Carrier Proteins / Q000037:antagonists & inhibitors / Q000378:metabolism*; D002462:Cell Membrane / Q000378:metabolism; D002478:Cells, Cultured; D003600:Cytosol / Q000378:metabolism; D004847:Epithelial Cells; D004848:Epithelium / Q000378:metabolism; D006863:Hydrogen-Ion Concentration; D007668:Kidney / Q000166:cytology / Q000378:metabolism*; D011493:Protein Kinase C / Q000502:physiology; D017923:Sodium-Hydrogen Exchangers; D013449:Sulfonamides / Q000494:pharmacology
D002147:Calmodulin; D002352:Carrier Proteins; D017923:Sodium-Hydrogen Exchangers; D013449:Sulfonamides; C039465:5-dimethylamiloride; C017967:W 7; D000584:Amiloride; D000255:Adenosine Triphosphate; D011493:Protein Kinase C