Intracellular Ca2+ release and Ca2+ influx during regulatory volume decrease in IMCD cells.
Volume changes and cytosolic Ca2+ concentration ([Ca2+]i) of inner medullary collecting duct (IMCD) cells under hypotonic stress were monitored by means of confocal laser scanning microscopy and fura 2 fluorescence, respectively. Reduction of extracellular osmolality from 600 to 300 mosmol/kgH2O by omission of sucrose led to an increase in cell volume within 1 min to 135 +/- 3% (n = 9), followed by a partial regulatory volume decrease (RVD) to 109 +/- 2% (n = 9) within the ensuring 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 reduction of osmolality evoked only a transient increase of [Ca2+]i by 182 +/- 22 nmol/l (n = 6). Preincubation with 100 mumol/l 8-(N,N-diethylamino)octyl-3,4,5-trimethoxy-benzoate hydrochloride (TMB-8) or 20 mmol/l caffeine, both effective inhibitors of Ca2+ release from intracellular stores, in low Ca2+ as well as in high Ca2+, inhibited the Ca2+ response and abolished RVD. The temporal relationship between Ca2+ release from intracellular stores and Ca2+ entry was analyzed by determining fura 2 quenching, using Mn2+ as a substitute for external Ca2+. Intracellular Ca2+ release preceded Mn2+ influx by 17 +/- 3 s (n = 10). Mn2+ influx persisted during the whole period of exposure to hypotonicity, indicating that there is no time-dependent Ca2+ channel inactivation. Preincubation with TMB-8 or caffeine reduced Mn2+ influx to the control level, indicating that activation of Ca2+ channels in the plasma membrane occurs via intracellular Ca2+ release.(ABSTRACT TRUNCATED AT 250 WORDS)
Tinel
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H
Max Planck Institute for Molecular Physiology, Dortmund, Germany.
Wehner
F
F
Sauer
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H
D000818:Animals; D002110:Caffeine / Q000494:pharmacology; D002118:Calcium / Q000378:metabolism*; D002460:Cell Line; D005707:Gallic Acid / Q000031:analogs & derivatives / Q000494:pharmacology; D007038:Hypotonic Solutions / Q000494:pharmacology; D007425:Intracellular Membranes / Q000378:metabolism*; D007679:Kidney Medulla; D007685:Kidney Tubules, Collecting / Q000166:cytology / Q000187:drug effects / Q000378:metabolism*; D008345:Manganese / Q000378:metabolism; D009994:Osmolar Concentration; D051381:Rats; D013997:Time Factors
D007038:Hypotonic Solutions; D002110:Caffeine; D008345:Manganese; C006014:8-(N,N-diethylamino)octyl-3,4,5-trimethoxybenzoate; D005707:Gallic Acid; D002118:Calcium