Cytosolic acidification stimulates a calcium influx that activates Na(+)-H+ exchange in LLC-PK1.
We have previously shown that cytosolic acidification-stimulated Na(+)-H+ exchange in LLC-PK1 cells is inhibited by calmodulin antagonists. To investigate further the role of Ca(2+)-calmodulin-dependent processes in intracellular pH (pHi) regulation in these cells, we studied the effects of pHi changes on cytosolic Ca2+ concentration ([Ca2+]i). In fura-2/acetoxymethylester (fura-2/AM)-loaded cells maintained in isotonic Na(+)-free buffer containing 1.8 mM CaCl2, [Ca2+]i was 168 +/- 59 nM (n = 5). After NH4Cl-induced alkalinization, [Ca2+]i decreased to 83 +/- 28 nM and partially recovered to 126 +/- 42 nM. Cytosolic acidification, after NH4Cl washout, caused an increase in [Ca2+]i to 481 +/- 166 nM (P less than 0.05; n = 5) that was dependent on extracellular Ca2+. An increase in [Ca2+]i was also observed in cells acidified with KCl-nigericin, with a return of [Ca2+]i to baseline with cell alkalinization. No increase in 45Ca2+ efflux occurred in association with initial NH4Cl-induced [Ca2+]i decrease, suggesting Ca2+ flux into an intracellular store during alkalinization. Membrane depolarization did not alter [Ca2+]i. The acidification-induced [Ca2+]i increase was inhibited by preincubation with verapamil or the calmodulin antagonist N-(6-aminohexyl)-5-chloro-1-naphthalenesulfonamide (W-7). Na(+)-dependent pHi recovery in 2,'7'-bis(carboxyethyl)-5(6)-carboxyfluorescein acetoxymethylester (BCECF/AM)-loaded LLC-PK1 cells and cytosolic acidification-stimulated basolateral Na(+)-H+ exchange activity in LLC-PK1/CL4 cells were both attenuated in the absence of extracellular Ca2+. The results indicate that cytosolic acidification activates an influx of extracellular Ca2+ in LLC-PK1 cells. Furthermore, in the absence of Ca2+ influx, Na(+)-H+ exchange is inhibited.(ABSTRACT TRUNCATED AT 250 WORDS)
Burns
K D
KD
Department of Medicine, Department of Veterans Affairs Medical Center, Nashville, Tennessee.
Homma
T
T
Breyer
M D
MD
Harris
R C
RC
D000643:Ammonium Chloride / Q000494:pharmacology; D000818:Animals; D002118:Calcium / Q000378:metabolism*; D002352:Carrier Proteins / Q000378:metabolism*; D003600:Cytosol / Q000378:metabolism*; D004847:Epithelial Cells; D004848:Epithelium / Q000378:metabolism; D006863:Hydrogen-Ion Concentration; D007668:Kidney / Q000166:cytology / Q000378:metabolism*; D009994:Osmolar Concentration; D017923:Sodium-Hydrogen Exchangers
D002352:Carrier Proteins; D017923:Sodium-Hydrogen Exchangers; D000643:Ammonium Chloride; D002118:Calcium