Mg(2+)-sensitive non-capacitative basolateral Ca(2+) entry secondary to cell swelling in the polarized renal A6...

Polarized renal A6 epithelia respond to hyposmotic shock with an increase in transepithelial capacitance (C(T)) that is inhibited by extracellular Mg(2+). Elevation of free cytosolic [Ca(2+)] ([Ca(2+)](i)) is known to increase C(T). Therefore, we examined [Ca(2+)](i) dynamics and their sensitivity to extracellular Mg(2+) 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 H(2)O)(-1). Reduction of apical osmolality alone did not affect [Ca(2+)](i). In the absence of extracellular Mg(2+), the hyposmotic shock induced a biphasic rise in [Ca(2+)](i). The first phase peaked within 40 s and [Ca(2+)](i) increased from 245 +/- 12 to 606 +/- 24 nM. This phase was unaffected by removal of extracellular Ca(2+), 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 [Ca(2+)](i) rise followed swelling-induced ATP release. The PLC inhibitor, the ATP treatment or suramin did not affect a second rise of [Ca(2+)](i) to a maximum of 628 +/- 31 nM. The second phase depended on Ca(2+) in the basolateral perfusate and was largely suppressed by 2 mM basolateral Mg(2+). Acute exposure of the basolateral membrane to Mg(2+) during the upstroke of the second phase caused a rapid decline in [Ca(2+)](i). Basolateral Mg(2+) inhibited Ca(2+) 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 Ca(2+) release from inositol trisphosphate-sensitive stores, followed by basolateral Ca(2+) influx through a Mg(2+)-sensitive pathway. The second phase of the [Ca(2+)](i) response is independent of the initial intracellular Ca(2+) release and therefore constitutes non-capacitative Ca(2+) entry.