CELLULAR ACIDIFICATION OCCURS DURING ANOXIA IN CULTURED, BUT NOT IN FRESHLY ISOLATED, RABBIT PROXIMAL TUBULAR CELLS

In a variety of cells it has been shown that acidosis is protective ag ainst anoxic injury. We have demonstrated previously that proximal tub ule (PT) cells in primary culture were more resistant to anoxia-induce d cell injury than were freshly isolated cells. Therefore, we asked th e question of whether a difference in cellular acidification during an oxia could explain this difference in susceptibility to anoxia. To ans wer this question, intracellular pH (pH(i)) was measured during anoxic incubation of PT cells in culture and those that were freshly isolate d. PT cells were incubated in an anoxic chamber at 37 degrees C after loading with 2',7'-bis-(2-carboxyethyl)-5,6-carboxyfluo-rescein acetox ymethyl ester (BCECF-AM) or fura-2 acetoxymethyl ester (fura-2-AM). pH (i) and cytosolic free Ca2+([Ca2+](i)) were measured by digital imagin g fluorescence microscopy. During anoxia, pH(i) in cultured PT cells d ecreased from 7.3 +/- 0.1 to 6.8 +/- 0.1, whereas pH(i) in freshly iso lated cells did not decrease significantly. In addition, the intrinsic buffering capacities (beta(i)) in cultured and freshly isolated PT ce lls were determined and turned out to be the same at a pH(i) greater t han or equal to 7.3. Below pH(i) 7.3, beta(i) increased several fold i n freshly isolated PT cells, and rose to significantly higher levels t han in cultured PT cells. During 1 h of anoxia, cell viability of fres hly isolated PT cells decreased significantly to 54% +/- 2% (P < 0.05) , while no loss in viability was observed in cultured PT cells. Clampi ng the pH(i) during anoxia at 6.7 and 6.1 significantly increased cell viability in freshly isolated PT cells to 76% +/- 5% and 72% +/- 4%, respectively (P < 0.05). In contrast, prevention of acidification in c ultured PT cells during anoxia did not lead to increased cell death. T herefore, the differences in susceptibility to anoxic injury between c ultured and freshly isolated PT cells cannot be explained by cellular acidification in cultured cells, but must be sought elsewhere.