Anoxia-induced increases in intracellular calcium concentration in primary cultures of rabbit thick ascending limb of Henle's loop.
The effect of anoxia on intracellular Ca2+ concentration ([Ca2+]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 [Ca2+]i continuously in cultured proximal tubule cells during 1 h of anoxic incubation in the absence of glycolytic substrates [1]. Complete absence of O2 was realised by inclusion of a mixture of oxygenases in an anoxic chamber. As a result of substrate-free anoxia, [Ca2+]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 [Ca2+]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 [Ca2+]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 Ca2+ and addition of 0.1 mM La3+ partially prevented anoxia-induced increases in [Ca2+]i in both cell types. The L-type Ca2+ channel blocker D600 (1 microM) was as effective as Ca2+ removal and La3+ addition. Comparing mTAL and cTAL cells, only one difference was consistently observed. Prevention of Ca2+ influx by exposure to La3+ combined with Ca2+ removal or addition of 1 microM D600 had a greater inhibitory effect on anoxic [Ca2+]i values in mTAL than in cTAL monolayers, indicative of a larger role of Ca2+ influx through L-type Ca2+ channels in anoxia-induced increases in [Ca2+]i in the former cell type. In conclusion, substrate-free anoxia reversibly increases [Ca2+]i in primary cultures of cTAL and mTAL, which results from Ca2+ release from stores as well as from Ca2+ influx via D600-sensitive Ca2+ channels.
Rose
U M
UM
Department of Cell Physiology, University of Nijmegen, The Netherlands.
Hartog
A
A
Jansen
J W
JW
Van Os
C H
CH
Bindels
R J
RJ
D000818:Animals; D002118:Calcium / Q000378:metabolism*; D015220:Calcium Channels / Q000378:metabolism; D015687:Cell Hypoxia; D002478:Cells, Cultured; D005711:Gallopamil / Q000494:pharmacology; D007672:Kidney Cortex / Q000378:metabolism*; D007679:Kidney Medulla / Q000378:metabolism*; D008138:Loop of Henle / Q000378:metabolism*; D011817:Rabbits
D015220:Calcium Channels; D005711:Gallopamil; D002118:Calcium