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+)) is known to increase C(T). Therefore, we examined Ca(2+) 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+). In the absence of extracellular Mg(2+), the hyposmotic shock induced a biphasic rise in Ca(2+). The first phase peaked within 40 s and Ca(2+) 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+) rise followed swelling-induced ATP release. The PLC inhibitor, the ATP treatment or suramin did not affect a second rise of Ca(2+) 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+). 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+) response is independent of the initial intracellular Ca(2+) release and therefore constitutes non-capacitative Ca(2+) entry.