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 against anoxic injury. We have demonstrated previously that proximal tubule (PT) cells in primary culture were more resistant to anoxia-induced cell injury than were freshly isolated cells. Therefore, we asked the question of whether a difference in cellular acidification during anoxia could explain this difference in susceptibility to anoxia. To answer this question, intracellular pH (pHi) was measured during anoxic incubation of PT cells in culture and those that were freshly isolated. PT cells were incubated in an anoxic chamber at 37 degrees C after loading with 2',7'-bis-(2-carboxyethyl)-5,6-carboxyfluorescein acetoxymethyl ester (BCECF-AM) or fura-2 acetoxymethyl ester (fura-2-AM). pHi and cytosolic free Ca2+ ([Ca2+]i) were measured by digital imaging fluorescence microscopy. During anoxia, pHi in cultured PT cells decreased from 7.3 +/- 0.1 to 6.8 +/- 0.1, whereas pHi in freshly isolated cells did not decrease significantly. In addition, the intrinsic buffering capacities (beta i) in cultured and freshly isolated PT cells were determined and turned out to be the same at a pHi greater than or equal to 7.3. Below pHi 7.3, beta i increased several fold in freshly isolated PT cells, and rose to significantly higher levels than in cultured PT cells. During 1 h of anoxia, cell viability of freshly isolated PT cells decreased significantly to 54% +/- 2% (P < 0.05), while no loss in viability was observed in cultured PT cells.(ABSTRACT TRUNCATED AT 250 WORDS)