Mg2+‐sensitive non‐capacitative basolateral Ca2+ entry secondary to cell swelling in the polarized renal A6 epithelium

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Abstract
Polarized renal A6 epithelia respond to hyposmotic shock with an increase in transepithelial capacitance (CT) that is inhibited by extracellular Mg2+. Elevation of free cytosolic [Ca2+] ([Ca2+]i) is known to increase CT. Therefore, we examined [Ca2+]i dynamics and their sensitivity to extracellular Mg2+ during hyposmotic conditions. Fura-2-loaded A6 monolayers, cultured on permeable supports were subjected to a sudden reduction in osmolality at both the basolateral and apical membranes from 260 to 140 mosmol (kg H2O)−1. Reduction of apical osmolality alone did not affect [Ca2+]i. In the absence of extracellular Mg2+, the hyposmotic shock induced a biphasic rise in [Ca2+]i. The first phase peaked within 40 s and [Ca2+]i increased from 245 ± 12 to 606 ± 24 nm. This phase was unaffected by removal of extracellular Ca2+, but was abolished by activating P2Y receptors with basolateral ATP or by exposing the cells to the phospholipase C (PLC) inhibitor U73122 prior to the osmotic shock. Suramin also severely attenuated this first phase, suggesting that the first phase of the [Ca2+]i rise followed swelling-induced ATP release. The PLC inhibitor, the ATP treatment or suramin did not affect a second rise of [Ca2+]i to a maximum of 628 ± 31 nm. The second phase depended on Ca2+ in the basolateral perfusate and was largely suppressed by 2 mm basolateral Mg2+. Acute exposure of the basolateral membrane to Mg2+ during the upstroke of the second phase caused a rapid decline in [Ca2+]i. Basolateral Mg2+ inhibited Ca2+ entry in a dose-dependent manner with an inhibition constant (Ki) of 0.60 mm. These results show that polarized A6 epithelia respond to hyposmotic shock by Ca2+ release from inositol trisphosphate-sensitive stores, followed by basolateral Ca2+ influx through a Mg2+-sensitive pathway. The second phase of the [Ca2+]i response is independent of the initial intracellular Ca2+ release and therefore constitutes non-capacitative Ca2+ entry.