Influence of Ca2+ on K+ efflux during regulatory volume decrease in cultured astrocytes

The calcium (Ca2+) dependence of potassium (K+) efflux activated by hyposmolarity in cultured cerebellar astrocytes was investigated, measuring in parallel experiments Rb-86 release and changes in cytosolic Ca2+ ([Ca2+](i)). Hyposmotic (50%) medium increased [Ca2+](i) from 117 to 386 nM, with contributions of extracellular Ca2+ and Ca2+ from the endoplasmic reticulum. Hyposmotic medium increased Rb-86 efflux rate from 0.015 min(-1) to a maximal of 0.049 min(-1) and a net release of 30%. This osmosensitive efflux was inhibited by Ba2+ (0.028 min(-1)), quinidine (0.024 min(-1)), and charybdotoxin (0.040 min(-1)), but was unaffected by TEA, 4-AP, or apamin. Removal of external Ca2+ from the hyposmotic medium increased Rb-86 efflux to a maximal rate constant of 0.056 min(-1) and a net release of 38% and caused a delay of inactivation. These changes were due to the overlaping of an efflux activated by Ca2+ removal in isosmotic medium. This isosmotic Rb-86 efflux was unaffected by TEA or 4-AP, reduced by verapamil, and abolished by Ba2+, nitrendipine, and Mg2+. With the swelling-induced [Ca2+](i) rise suppressed by ethyleneglycoltetraacetic acid-acetoxy-methyl ester (EGTA-AM), hyposmotic Rb-86 was 30 % reduced. The Ca2+ entry blockers Cd2+, Ni2+, La3+, and Gd3+ did not affect Rb-86 efflux. A 40% decrease observed with verapamil and nitrendipine was found unrelated to Ca2+, because these agents did not affect the [Ca2+](i) rise and the inhibition persisted in the absence of external Ca2+. The phospholipase C blocker U-73122 did not affect [Ca2+](i) nor Rb-86 efflux. Blockers of Ca2+/calmodulin W7 and KN-93 decreased Rb-86 efflux to the same extent as EGTA-AM. Ionomycin markedly potentiated Rb-86 release in hyposmotic conditions only when [Ca2+](i) was raised to about 1 mu M, suggesting the implication of maxi-K+ channels at this [Ca2+](i) threshold, which nonetheless, was not attained during hyposmotic swelling. It is concluded that Rb-86 efflux in cerebellar astrocytes is largely (70%) Ca2+-independent and the Ca2+-dependent fraction is sustained essentially by Ca2+ released from the endoplasmic reticulum and mediated by a mechanism involving Ca2+/calmodulin. (C) 1999 Wiley-Liss, Inc.