Effects of Ca2+ channel blockers, low Ca2+ medium and glycine on cell Ca2+ and injury in anoxic rabbit proximal tubules

Effects of Ca2+ channel blockers, low Ca2+ medium and glycine on cell Ca2+ and injury in anoxic rabbit proximal tubules. L-type Ca2+ channel blockers (CCBs) have been shown to be protective against ischemiainduced injury of the kidney, suggesting that increased intracellular Ca2+ levels ([Ca2+]i) play an important role in the pathogenesis of ischemic cell injury. To assess the role of [Ca2+]i in anoxic injury of the proximal tubule (PT) and the protective effect of CCBs, digital imaging fluorescence microscopy was used to monitor [Ca2+]i in individual PT cells during 60 minutes of anoxia. [Ca2+]i started to rise within 10 minutes and reached maximal levels between 30 to 45 minutes of anoxia. The onset of this increase and the maximal levels reached varied markedly among individual cells. The mean values for initial and maximal anoxic [Ca2+]i were 109 ± 2(N = 209) and 422 ± 14 (N = 240) nM, respectively. Methoxyverapamil (D600; 1 µM) significantly reduced anoxic [Ca2+]i to 122 ± 5nM (P < 0.05; N = 79). Removal of extracellular Ca2+ completely abolished anoxia-induced increases in [Ca2+]i, confirming that these increases in [Ca2+]i result from Ca2+ influx. During 60 minutes of anoxia, PT cells showed a gradual decrease in cell viability to 54 ± 2%. D600 (1 µM) significantly increased cell viability to 64 ± 3% (P < 0.05). Glycine (5mM), however, increased cell viability to 77 ± 4% without a significant reduction in anoxic [Ca2+]i levels. Low Ca2+ medium only protected when 0.1mM La3+ was included, a condition which increased cell viability to 82 ± 5%. La3+ did not enter PT cells and probably protected via a membranestabilizing effect. The combination of glycine and La3+ did not further increase protection. In conclusion, D600 almost completely prevented anoxia-induced increases in [Ca2+]i by blocking Ca2+ influx via L-type Ca2+ channels. Since D600 only partly protected PT cells against anoxic injury, [Ca2+]i unrelated cell injury, which is attenuated by glycine, is a more prominent factor in anoxia-induced cell injury in rabbit PT cells