In freshly isolated hypoxic rat proximal tubules, Ca2+ uptake rate increases promptly, within 1 min, and remains significantly elevated throughout a 20-min period of hypoxia. Lactate dehydrogenase (LDH) release, a sign of membrane injury, increases only after 5 min of hypoxia and thereafter rises progressively. The potential effect of increased Ca2+ uptake rate to activate phospholipases, which would then initiate membrane injury, was evaluated by treating hypoxic tubules with three dissimilar phospholipase inhibitors, i.e., mepacrine, dibucaine, or p-bromophenacyl bromide (PBPB). LDH release averaged 11.9 and 13.8% after 10 and 20 min of normoxia, respectively. With 10 or 20 min of hypoxia LDH release increased to 46.0 and 65.2%, respectively (P < 0.01), and Ca2+ uptake rate increased from 2.56 in normoxia to 4.71 nmol mg(-1) min(-1) at 10 min of hypoxia (P < 0.01) and from 2.82 in normoxia to 3.76 nmol/mg at 20 min of hypoxia (P < 0.05). In a separate series of tubules, after 10 min of hypoxia LDH release was reduced by pretreatment with 50 mu M mepacrine (66.1 to 47.3%, P < 0.01) or 50 mu M dibucaine (53.1 to 38.5%, P < 0.02). The increase in Ca2+ uptake rate also was significantly reduced. After 20 min of hypoxia neither mepacrine nor dibucaine reduced Ca2+ uptake rate; LDH release was modestly reduced by dibucaine but not mepacrine. Higher doses of mepacrine (500 mu M) and dibucaine (250 mu M) also reduced cell injury at 10 min of hypoxia as assessed by LDH release. With 10 min of hypoxia, 50 mu M mepacrine did not reduce free fatty acids (FFA); 500 mu M mepacrine reduced levels of two of the five species of fatty acids determined. Dibucaine at 50 mu M reduced the levels of three of the five fatty acids, but higher concentrations (250 mu M) failed to lower FFA. No protective effects of PBPB (100 mu M) were observed. Thus mepacrine and dibucaine, but not PBPB, reduce the early cellular damage, as assessed by LDH release, that occurs with hypoxia and attenuate the hypoxia-associated increases in Ca2+ uptake rate. This cellular protection does not correlate consistently with the ability of these agents to inhibit fatty acid release during hypoxia. The protective effects of mepacrine and dibucaine are not demonstrable by 20 min of hypoxia, thus suggesting that continued phospholipase activity or other noxious events such as cell swelling, O-2-free radicals, and/or proteases may predominate at this time.
