Maintenance of cellular acidification in cyanide-treated hepatocytes results from inhibition of Na+/H+ exchange.
Inhibition of respiration by metabolic inhibitors or hypoxia is accompanied by intracellular acidification. Although this acidification is known to promote cell survival during hypoxia, little is known about its mechanism. Given that the Na+/H+ exchanger is known to be a major component of pH regulation in normal hepatocytes, the aim of this study was to determine the effects of inhibition of mitochondrial respiration on intracellular pH (pHi) regulation and Na+/H+ exchange. Cyanide (CN-; 5 mM) plus fructose (20 mM) were used as a model of hypoxic acidosis. pHi was measured with quantitative fluorescence microscopy of cells loaded with the pH indicator, 2',7'-bis-(2-carboxyethyl)-5,6-carboxyfluorescein. In control cells, pHi was 7.09 +/- 0.01 SE (n = 106). After 60 min in CN(-)-fructose, pHi fell to 6.74 +/- 0.01 (n = 129, P < 0.001). The pHi recovery rate (expressed as mmol H+.l-1.min-1) was determined under both conditions after acid loading by transient exposure and removal of 20 mM NH4Cl. Control and CN(-)-treated cells recovered at 3.59 +/- 0.25 (n = 42) and 0.69 +/- 0.09 (n = 38, P < 0.001), respectively. Amiloride treatment (1 mM) in the absence of CN- reduced pHi recovery similarly to that caused by CN- (0.34 +/- 0.07, n = 14). CN(-)-treated cells exposed to amiloride demonstrated no additional inhibition (efflux rate 0.65 +/- 0.11, n = 27), suggesting that the inhibition is directed at Na+/H+ exchange. Twenty minutes after CN- removal, CN(-)-treated cells regained their ability to recover from an acid load, thus demonstrating the reversibility of this effect.(ABSTRACT TRUNCATED AT 250 WORDS)
Schoenecker
J A
JA
Department of Internal Medicine, University of Texas Medical Branch, Galveston 77555.
Weinman
S A
SA
D000643:Ammonium Chloride / Q000494:pharmacology; D000818:Animals; D002478:Cells, Cultured; D003486:Cyanides / Q000494:pharmacology*; D006863:Hydrogen-Ion Concentration; D007700:Kinetics; D007773:Lactates / Q000032:analysis; D008099:Liver / Q000187:drug effects / Q000378:metabolism*; D008297:Male; D008856:Microscopy, Fluorescence; D051381:Rats; D017207:Rats, Sprague-Dawley; D012044:Regression Analysis; D017923:Sodium-Hydrogen Exchangers / Q000037:antagonists & inhibitors*; D013997:Time Factors
D003486:Cyanides; D007773:Lactates; D017923:Sodium-Hydrogen Exchangers; D000643:Ammonium Chloride