ROLE OF INTRACELLULAR PH DURING CYTOPROTECTION OF PROXIMAL TUBULE CELLS BY GLYCINE OR ACIDOSIS

Lowering extracellular pH to less than 7.0 strongly protects isolated proximal tubules against ATP depletion and Ca2+-induced injury, but th ere is little information about alterations of intracellular pH (pH(i) ) in renal tubules during either injury or its modification by decreas ing medium pH or other potent protective factors such as glycine. pH(i ) was assessed with 2',7'-bis-(2-carboxyethyl)-5-carboxyfluorescein du ring proximal tubule injury produced by simple ATP depletion with the electron transport inhibitor antimycin or by large increases of cytoso lic free Ca2+ induced by treatment with the calcium ionophore iono myc in, alone and in combination with antimycin. Freshly isolated rabbit p roximal tubules studied under superfusion conditions in the presence o f probenecid were suitable for monitoring pH(i) during relatively prol onged and severe injury states. Probenecid, used to promote the retent ion of intracellular fluorophores, only minimally modified the injury response by transiently delaying lactate dehydrogenase release during antimycin treatment. The tubules did not exhibit spontaneous decreases of pH, during simple ATP depletion, but pH(i) fully equilibrated with cytoprotective decreases of medium pH. Irrespective of the presence o f antimycin, ionomycin induced intracellular alkalinization in Ca2+-re plete medium, which may have further enhanced the severity of injury. When medium Ca2+ was buffered to 100 nM, ionomycin induced intracellul ar acidification, which likely resulted from a combination of Ca2+/Hexchange activity of the ionophore and H+ uptake during Ca2+-ATPase-me diated extrusion of Ca2+ released by ionomycin from intracellular pool s. Alterations of pH(i) did not contribute to glycine cytoprotection b ecause glycine did not affect the behavior of pH(i) during treatment w ith antimycin, ionomycin, or both agents in combination. The lack of e ffect of glycine on pH(i) makes it likely that complementary cytoprote ctive actions of glycine and reduced pH will be relevant to understand ing both in vitro models of injury and in vivo ischemic states.