Cell swelling induced by water and glycerol gradients in hepatocytes from warm‐and cold‐acclimated Hyla chrysoscelis , a freeze‐tolerant amphibian

Gray tree frogs, Hyla chrysoscelis , survive freezing in part by accumulating cryoprotective glycerol, which is likely synthesized by hepatocytes. During cold acclimation, freezing, and thawing, glycerol and water must be transported into and out of those cells, likely via aqua/glyceroporins. We therefore hypothesized that hepatocytes of cold‐acclimated frogs have enhanced permeability to glycerol and water. We tested this hypothesis by assessing cell volume change in cells subjected to an osmotic or glycerol gradient. We perfused liver with media containing collagenase, dissociated hepatocytes in isosmotic media, and tracked cell swelling upon exposure to hypotonic challenge. In response to a glycerol gradient, cells from cold‐acclimated animals swelled and lysed more quickly than those from warm‐acclimated animals. This response was slower for cells tested at ~4°C and was inhibited by pre‐incubation with 0.3 mM HgCl 2 , which blocks aqua/glyceroporin‐mediated permeability. Both HgCl 2 and incubation temperature had a lesser effect on the response to an osmotic gradient than to a glycerol gradient. These findings imply that cold‐acclimated hepatocytes have enhanced glycerol permeability conferred by aqua/glyceroporins, but that water permeability may occur via a separate pathway. Supported by NSF IOS‐1121457 to D. Goldstein and C. M. Krane.