INTRACELLULAR CA2+ RELEASE AND CA2+ INFLUX DURING REGULATORY VOLUME DECREASE IN IMCD CELLS

Volume changes and cytosolic Ca2+ concentration ([Ca2+](i)) of inner m edullary collecting duct (IMCD) cells under hypotonic stress were moni tored by means of confocal laser scanning microscopy and fura 2 fluore scence, respectively. Reduction of extracellular osmolality from 600 t o 300 mosmol/kgH(2)O by omission of sucrose led to an increase in cell volume within 1 min to 135 +/- 3% (n = 9), followed by a partial regu latory volume decrease (RVD) to 109 +/- 2% (n = 9) within the ensuing 5 min. In parallel, [Ca2+](i) rose from 145 +/- 9 to 433 +/- 16 nmol/l (n = 9) and thereafter reached a lower steady state of 259 +/- 9 nmol /l. Under low-Ca2+ conditions (10 nmol/l) RVD was not impeded and redu ction of osmolality evoked only a transient increase of [Ca2+](i) by 1 82 +/- 22 nmol/l (n = 6). Preincubation with 100 mu mol/l -(N,N-diethy lamino)octyl-3,4,5-trimethoxy-benzoate hydrochloride (TMB-8) or 20 mmo l/l caffeine, both effective inhibitors of Ca2+ release from intracell ular stores, in low Ca2+ as well as in high Ca2+, inhibited the Ca2+ r esponse and abolished RVD. The temporal relationship between Ca2+ rele ase from intracellular stores and Ca2+ entry was analyzed by determini ng fura 2 quenching, using Mn2+ as a substitute for external Ca2+. Int racellular Ca2+ release preceded Mn2+ influx by 17 +/- 3 s (n = 10). M n2+ influx persisted during the whole period of exposure to hypotonici ty, indicating that there is no time-dependent Ca2+ channel inactivati on. Preincubation with TMB-8 or caffeine reduced Mn2+ influx to the co ntrol level, indicating that activation of Ca2+ channels in the plasma membrane occurs via intracellular Ca2+ release. We conclude that RVD in IMCD cells is associated with a Ca2+ release from intracellular sto res that precedes Ca2+ entry on hypotonic stress.