Osmotic swelling-induced changes in cytosolic calcium do not affect regulatory volume decrease in rat cultured suspended cerebellar astrocytes

Abstract

Hyposmotic swelling-induced changes in intracellular Ca2+ concentration ([Ca2+](i)) and their influence on regulatory volume decrease (RVD) were examined in rat cultured suspended cerebellar astrocytes. Hyposmotic media (50 or 30%) evoked an immediate rise in [Ca2+](i) from 117 nM to a mean peak increase of 386 (50%) and 220 nM (30%), followed by a maintained plateau phase. Ca2+ influx through the plasmalemma as well as release from internal stores contributed to this osmosensitive [Ca2+](i) elevation. Omission of external Ca2+ or addition of Cd2+, MR2+, or Gd3+ did not reduce RVD, although it was decreased by La3+ (0.1-1 mM). Verapamil did not affect either the swelling-evoked [Ca2+](i) or RVD. Maneuvers that deplete endoplasmic reticulum (ER) Ca2+ stores, such as treatment (in Ca2+-free medium) with 0.2 μM thapsigargin (Tg), 10 μM 2,5- di-tert-butylhy-droquinone, 1 μM ionomycin, or 100 μM ATP abolished the increase in [Ca2+](i) but did not affect RVD. However, prolonged exposure to 1 μM Tg blocked RVD regardless of ER Ca2+ content or cytosolic Ca2+ levels. Ryanodine (up to 100 μM) and caffeine (10 mM) did not modify [Ca2+](i) or RVD. BAPTA-acetoxymethyl ester (20 μM) abolished [Ca2+](i) elevation without affecting RVD, but at higher concentrations BAPTA prevented cell swelling and blocked RVD. We conclude that the osmosensitive [Ca2+](i) rise occurs as a consequence of increased Ca2+ permeability of plasma and organelle membranes, but it appears not relevant as a transduction signal for RVD in rat cultured cerebellar astrocytes.