THE EFFECT OF L-TYPE CA2-INDUCED INCREASES IN INTRACELLULAR CA2+ CONCENTRATION IN RABBIT PROXIMAL TUBULE CELLS IN PRIMARY CULTURE( CHANNEL BLOCKERS ON ANOXIA)

Ca2+ channel blockers (CCB) have been shown to be protective against i schaemic damage of the kidney, suggesting an important role for intrac ellular Ca2+ ([Ca2+]i) in generating cell damage. To delineate the mec hanism behind this protective effect, we studied [Ca2+]i in cultured p roximal tubule (PT) cells during anoxia in the absence of glycolysis a nd the effect of methoxyverapamil (D600) and felodipine on [Ca2+]i dur ing anoxia. A method was developed whereby [Ca2+]i in cultured PT cell s could be measured continuously with a fura-2 imaging technique durin g anoxic periods up to 60 min. Complete absence of O2 was realised by inclusion of a mixture of oxygenases in an anoxic chamber. [Ca2+]i in PT cells started to rise after 10 min of anoxia and reached maximal le vels at 30 min, which remained stable up to 60 min. The onset of this increase and the maximal levels reached varied markedly among individu al cells. The mean values for normoxic and anoxic [Ca2+]i were 118 +/- 2 (n = 98) and 662 +/- 22 (n = 160) nM, respectively. D600 (1 muM), b ut not felodipine (10 muM), significantly reduced basal [Ca2+]i in nor moxic incubations. During anoxia 1 muM and 100 muM D 600 significantly decreased anoxic [Ca2+]i levels by 22 and 63% respectively. Felodipin e at 10 muM was as effective as 1 muM D600. Removal of extracellular C a2+ and addition of 0.1 mM La3+ completely abolished anoxia-induced in creases in [Ca2+]i. We conclude that anoxia induces increases in [Ca2]i in rabbit PT cells in primary culture, which results from Ca2+ infl ux. Since this Ca2+ influx is partially inhibited by low doses of CCBs , L-type Ca2+ channels may be involved.