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 (pH(i)) regulation and Na+/H+ exchange. Cyanide (CN-; 5 mM) plus fructose (20 mM) were used as a model of hypo;dc acidosis. pH(i) was measured with quantitative fluorescence microscopy of cells loaded with the pH indicator, 2',7'-bis-(2-carboxyethyl)-5,6-carboxyfluorescein. In control cells, pH(i) was 7.09 +/- 0,01 SE (n = 106). After 60 min in CN--fructose, pH(i) 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. To test whether Na+/H+ exchange inhibition was partially due to a change in the Na+ gradient, we Na+ depleted both control and CN--treated cells with tetramethylammonium and then suddenly exposed them to 140 mM Na+. In control cells an amiloride-inhibitable allialinization occurred. In CN--treated cells this Na+/-induced alkalinization was inhibited 95%, thereby confirming that Na+/H+ exchange activity was minimal. In the presence of 25 mM: HCOB-buffered Ringer solution, the control rate of pH(i) recovery was increased but CN--fructose still inhibited pH(i) recovery. In conclusion, Na+/H+ exchange contributes to pH(i) regulation in control conditions but is reversibly inhibited after blockage of mitochondrial respiration with CN--fructose. This inhibition of Na+/H+ exchange helps to maintain intracellular acidification, a condition that prolongs cell survival during hypoxia.