Intracellular acidosis: can it delay the inevitable?

The relationships between extracellular pH (pH,), intracellular pH (pH,), and loss of cell viability were evaluated in cultured rat hepatocytes after ATP depletion by metabolic inhibition with KCN and iodoacetate (chemical hypoxia). pHi was measured in single cells by ratio imaging of 2’,7’-biscarbox~-ethyl5,6-carboxyfluorescein (BCECF) fluorescence using multiparameter digitized video microscopy. During chemical hypoxia at pH, of 7.4, pHi decreased from 7.36 to 6.33 within 10 min. pHi remained at 6.1-6.5 for 30-40 min. (plateau phase). Thereafter, pHi began to rise and cell death ensued within minutes, as evidenced by nuclear staining with propidium iodide and coincident leakage of BCECF from the cytoplasm. An acidic pH, produced a slightly greater drop in pH,, prolonged the plateau phase of intracellular acidosis, and delayed the onset of cell death. Inhibition of Na+/H+ exchange also prolonged the plateau phase and delayed cell death. In contrast, monensin or substitution of gluconate for Clin buffer containing HC0,abolished the pH gradient across the plasma membrane and shortened cell survival. The results indicate that intracellular acidosis after ATP depletion delays the onset of cell death, whereas reduction of the degree of acidosis accelerates cell killing. We conclude that intracellular acidosis protects against hepatocellular death from ATP depletion, a phenomenon that may represent a protective adaptation against hypoxic and ischemic stress. COMMENTS Hepatocytes possess membrane transport processes, such as Na+-H’ exchange (1) and Na+-HC0,cotransport (21, that maintain intracellular pH (pH,) at levels well above that predicted by the Nernst equation. Numerous studies in other epithelia have demonstrated a relationship between pH, and cellular function. Acidic cytoplasmic conditions are typically associated with a quiescent or dormant cellular state and intracellular alkalinization frequently accompanies cellular growth and activation (3, 4). In contrast, the relationship between pH, and the structural and functional abnormalities that occur during ischemic and hypoxic hepatocellular injury are less clear. Previous studies that examined the relationship between extracellular pH (pH,) and hepatocyte injury suggested that acidosis may exert a protective effect (5, 6). The article under discussion extends these findings with the technological benefit (via multiparameter digitized video microscopy) of serial measurements of pHi in single cells. The advantages of this technique are readily apparent in the figures demonstrating the cellular localization of the probe used, 2‘,7’-biscarboxyethy1-5, 6-carboxyfluorescein (BCECF), to the cytosol. This fluorescent probe, introduced into the cytosol in the form of the lipid-soluble acetoxymethyl ester where it is then cleaved by cytoplasmic esterases was assumed, in previous work, to be trapped in the cytosol. The cellular localization of BCECF is now confirmed by this elegant study using additional probes specific for lysosomes, endosomes and mitochondria and sequential dissolution of fluorescence by increasing concentrations of detergents. Hypoxia was induced by the administration of KCN and iodoacetate to inhibit oxidative phosphorylation and glycolysis, respectively, and thereby deplete cultured hepatocytes of ATP. Maneuvers that prolonged and/or increased intracellular acidosis protected against cell death during ATP depletion, including inhibition of Na’-H’ exchange by amiloride and by Na’ replacement and exposure of hepatocytes to acidic buffer. Conversely, cell death was accelerated by maneuvers that prevented a decrease in pHi. However, there are two potential disadvantages to this form of “chemical hypoxia.” The rapid decline in intracellular ATP levels (95% decrease within 5 min) that is observed may not duplicate the decrease that occurs in vivo during ischemic liver injury. Furthermore, the effect of iodoacetate-induced protein thiol alkylation is not addressed. The authors speculate that the effect of intracellular acidosis is a suppression of autolytic degradative processes. In support of this hypothesis they discuss unpublished observations of a similar protective effect of acidosis against cystamine-induced hepatotoxicity, an injury thought to involve activation of neutral proteases (7). Alterations in cellular Caz+ homeostasis have also been proposed to play a major role in ischemic cell injury, as recently reviewed (8), and cystamine hepatotoxicity has been demonstated to be mediated not only by activation of a nonlysosomal proteolytic system but 708 HEPATOLOGY Elsewhere HEPATOLOGY also by an inhibition of Ca2+ efflux (7). Although not discussed, these findings may provide a unifying theory of hepatocellular injury that incorporates both pertubations in intracellular Ca2’ levels and pHi. Loss of plasma membrane integrity is an important feature in most schema of cell injury and several studies have correlated plasma membrane blebbing with hepatocellular injury (8). It is, therefore, somewhat disconcerting that the authors observed that acidic pH, does not prevent cell surface blebbing, scored with time-lapse video recordings of cultured hepatocytes. Finally, these findings, as well as those of previous work (6), may have important clinical implications for tissue preservation before orthotopic liver transplantation. If the conclusions of this work are accepted, then storage of donor livers in acidic media should enhance organ viability. However, it should be noted that the cold storage solution in current use, the “UW” solution, is of pH 7.4 and has been reported to extend appreciably the preservation of donor livers for transplantation (9). Would a lower pH be better?