title: Swelling-induced Ca²+ influx and K+ efflux in American alligator erythrocytes.
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 well-developed 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 fluo-4-AM. 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 (0.5 x 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 glycol-bis(2-aminoethylether)-N,N,N',N'-tetraacetic acid). Furthermore, cells loaded with BAPTA-AM (1,2-bis(2-aminophenoxymethyl)ethane-N,N,N',N'-tetraacetic acid tetrakis(acetoxymethyl) 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 RVD.
authors.affiliation: Department of Biology, Lake Forest College, 555 N. Sheridan Road, Lake Forest, IL 60045, USA.
authors.forename: Alina B
authors.initials: AB
authors.lastname: Elperin
authors.forename: Shruti A
authors.initials: SA
authors.lastname: Pore
authors.forename: Jordanah M
authors.initials: JM
authors.lastname: Evans
authors.forename: Annabel L
authors.initials: AL
authors.lastname: Naditz
authors.forename: Douglas B
authors.initials: DB
authors.lastname: Light
mesh_terms: D000489:Alligators and Crocodiles; D000814:Aniline Compounds / Q000378:metabolism; D000818:Animals; D000001:Calcimycin / Q000494:pharmacology; D002118:Calcium / Q000378:metabolism*; D048429:Cell Size; D004533:Egtazic Acid / Q000031:analogs & derivatives / Q000378:metabolism; D004906:Erythrocyte Count; D004912:Erythrocytes / Q000166:cytology / Q000378:metabolism*; D006096:Gramicidin / Q000494:pharmacology; D006706:Homeostasis; D017136:Ion Transport / Q000502:physiology; D007476:Ionophores / Q000494:pharmacology; D009996:Osmotic Fragility / Q000502:physiology; D011188:Potassium / Q000378:metabolism*; D015221:Potassium Channels / Q000378:metabolism; D011803:Quinine / Q000494:pharmacology; D014966:Xanthenes / Q000378:metabolism
chemical_list: D000814:Aniline Compounds; C409648:Fluo 4; D007476:Ionophores; D015221:Potassium Channels; D014966:Xanthenes; C070379:1,2-bis(2-aminophenoxy)ethane N,N,N',N'-tetraacetic acid acetoxymethyl ester; D006096:Gramicidin; D000001:Calcimycin; D004533:Egtazic Acid; D011803:Quinine; D011188:Potassium; D002118:Calcium