Anoxia-induced increases in intracellular calcium concentration in primary cultures of rabbit thick ascending limb of Henle's loop

Abstract

The effect of anoxia on intracellular Ca 2+ concentration ([Ca 2+] i) in primary cultures of medullary (mTAL) and cortical (cTAL) thick ascending limb of Henle's loop was investigated. Previously, we reported a method to monitor [Ca 2+] i continuously in cultured proximal tubule cells during 1 h of anoxic incubation in the absence of glycolytic substrates [1]. Complete absence of O 2 was realised by inclusion of a mixture of oxygenases in an anoxic chamber. As a result of substrate-free anoxia, [Ca 2+] i started to rise in individual cells of mTAL and cTAL monolayers and reached maximal levels within 60 min after starting the anoxic incubation. Anoxia induced significant increases in [Ca 2+] i from 76 ± 1 ( n = 176) to 469 ± 18 nM ( n = 203) in mTAL monolayers and from 58 ± 1 ( n = 91) to 442 ± 27 nM ( n = 106) in cTAL monolayers ( P < 0.05). At the re-introduction of oxygen and glucose, elevated [Ca 2+] i rapidly declined to 110 ± 4 ( n =167) and 105 ± 5 nM ( n = 87) in mTAL and cTAL, respectively ( P < 0.05). Removal of extracellular Ca 2+ and addition of 0.1 mM La 3+ partially prevented anoxia-induced increase in [Ca 2+] i in both cell types. The L-type Ca 2+ channel blocker D600 (1 μM) was as effective as Ca 2+ removal and La 3+ addition. Comparing mTAL and cTAL cells, only one difference was consistently observed. Prevention of Ca 2+ influx by exposure to La 3+ combined with Ca 2+ removal or addition of 1 μM D600 had a greater inhibitory effect on anoxic [Ca 2+] i values in mTAL than in cTAL monolayers, indicative of a larger role of Ca 2+ influx through L-type Ca 2+ channels in anoxia-induced increases in [Ca 2+] i in the former cell type. In conclusion, substrate-free anoxia reversibly increases [Ca 2+] i in primary cultures of cTAL and mTAL, which results from Ca 2+ release from stores as well as from Ca 2+ influx via D600-sensitive Ca 2+ channels.