Regulatory volume decrease by SV40-transformed rabbit corneal epithelial cells requires ryanodine-sensitive Ca2+-induced Ca2+ release.
The relationship between relative cell volume and time-dependent changes in intracellular Ca2+ concentration ([Ca2+]i) during exposure to hypotonicity was characterized in SV-40 transformed rabbit corneal epithelial cells (tRCE) (i). Light scattering measurements revealed rapid initial swelling with subsequent 97% recovery of relative cell volume (characteristic time (tauvr) was 5.9 min); (ii). Fura2-fluorescence single-cell imaging showed that [Ca2+]i initially rose by 216% in 30 sec with subsequent return to near baseline level after another 100 sec. Both relative cell volume recovery and [Ca2+]i transients were inhibited by either: (a) Ca2+-free medium; (b) 5 mM Ni2+ (inhibitor of plasmalemma Ca2+ influx); (c) 10 microM cyclopiazonic acid, CPA (which causes depletion of intracellular Ca2+ content); or (d) 100 microM ryanodine (inhibitor of Ca2+ release from intracellular stores). To determine the temporal relationship between an increased plasmalemma Ca2+ influx and the emptying of intracellular Ca2+ stores during the [Ca2+]i transients, Mn2+ quenching of fura2-fluorescence was quantified. In the presence of CPA, hypotonic challenge increased plasmalemma Mn2+ permeability 6-fold. However, Mn2+ permeability remained unchanged during exposure to either: 1.100 microM ryanodine; 2.10 microM CPA and 100 microM ryanodine. This report for the first time documents the time dependence of the components of the [Ca2+]i transient required for a regulatory volume decrease (RVD). The results show that ryanodine sensitive Ca2+ release from an intracellular store leads to a subsequent increase in plasmalemma Ca2+ influx, and that both are required for cells to undergo RVD.
Wu
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Department of Biological Sciences, State University of New York, College of Optometry, New York, NY 10010, USA.
Yang
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Iserovich
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Fischbarg
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Reinach
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PS
D000818:Animals; D002118:Calcium / Q000378:metabolism*; D002462:Cell Membrane / Q000187:drug effects / Q000378:metabolism; D048429:Cell Size; D002472:Cell Transformation, Viral; D003315:Cornea / Q000166:cytology* / Q000187:drug effects / Q000378:metabolism*; D004847:Epithelial Cells; D004848:Epithelium / Q000187:drug effects / Q000378:metabolism; D005110:Extracellular Space / Q000378:metabolism; D007038:Hypotonic Solutions; D007211:Indoles / Q000494:pharmacology; D007424:Intracellular Fluid / Q000378:metabolism; D017136:Ion Transport / Q000187:drug effects; D008954:Models, Biological; D009532:Nickel / Q000494:pharmacology; D009997:Osmotic Pressure; D011817:Rabbits; D012433:Ryanodine / Q000494:pharmacology; D013539:Simian virus 40
D007038:Hypotonic Solutions; D007211:Indoles; D012433:Ryanodine; D009532:Nickel; D002118:Calcium; C000543:cyclopiazonic acid