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

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 sensitivityto 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.