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

Danny Jans *, Paul De Weer †, S. P. Srinivas ‡, Els Larivière *, Jeannine Simaels * and Willy Van Driessche *

Polarized renal A6 epithelia respond to hyposmotic shock with an increase in transepithelial capacitance (C_T) that is inhibited by extracellular Mg²⁺. Elevation of free cytosolic [Ca²⁺] ([Ca²⁺]_i) is known to increase C_T. 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 (K_i) 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.

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