Authors: Alina B Elperin Shruti A Pore Jordanah M Evans Annabel L Naditz Douglas B Light
Publish Date: 2010/12/10
Volume: 240, Issue: 1, Pages: 1-12
Abstract
The American alligator can hibernate during winter which may lead to osmotic imbalance because of reduced kidney function and lack of food consumption during this period Accordingly we hypothesized that their red blood cells would have a welldeveloped regulatory volume decrease RVD to cope with the homeostatic challenges associated with torpor Osmotic fragility was determined optically mean cell volume was measured by electronic sizing and changes in intracellular Ca2+ concentration were visualized using fluorescence microscopy and fluo4AM Osmotic fragility increased and the ability to regulate volume was inhibited when extracellular Na+ was replaced with K+ or when cells were exposed to the K+ channel inhibitor quinine indicating a requirement of K+ efflux for RVD Addition of the ionophore gramicidin to the extracellular medium decreased osmotic fragility and also potentiated volume recovery even in the presence of quinine In addition hypotonic shock 05× Ringer caused an increase in cytosolic Ca2+ which resulted from Ca2+ influx because it was not observed when extracellular Ca2+ was chelated with EGTA ethylene glycolbis2aminoethyletherNNN′N′tetraacetic acid Furthermore cells loaded with BAPTAAM 12bis2aminophenoxymethylethaneNNN′N′tetraacetic acid tetrakisacetoxymethyl ester or exposed to a low Ca2+EGTA hypotonic Ringer had a greater osmotic fragility and also failed to recover from cell swelling indicating that extracellular Ca2+ was needed for RVD Gramicidin reversed the inhibitory effect of low extracellular Ca2+ Finally and surprisingly the Ca2+ ionophore A23187 increased osmotic fragility and inhibited volume recovery Taken together our results show that cell swelling activated a K+ permeable pathway via a Ca2+dependent mechanism and this process mediated K+ loss during RVDResearch support was provided by Lake Forest College and the Laurence R Lee Family Endowment Portions of this study were presented in abstract form at Experimental Biology 2007 Washington DC EB 2008 San Diego CA and EB 2009 New Orleans LA
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