Hepatocyte responses to in vitro freezing and β-adrenergic stimulation: Insights into the extreme freeze tolerance of subarctic Rana sylvatica.

The wood frog, Rana sylvatica LeConte 1825, is a freeze-tolerant amphibian widely distributed in North America. Subarctic populations of this species can survive experimental freezing to temperatures below -16 °C, whereas temperate populations tolerate freezing only at temperatures above -6 °C. We investigated whether hepatocytes isolated from frogs indigenous to Interior Alaska (subarctic) or southern Ohio (temperate) had distinct characteristics that could contribute to this variation in freeze tolerance capacity. Following in vitro freezing, cell damage, as assessed from lactate dehydrogenase leakage, was similar between samples from Alaskan and Ohioan frogs. Preincubation of cells in media containing glucose or urea, the two primary cryoprotectants used by R. sylvatica, markedly reduced freezing damage to hepatocytes; however, results suggested that cells of the northern phenotype were comparatively more amenable to cryoprotection by urea. Stimulation of isolated hepatocytes with β-adrenergic agonists, which simulates the freezing-induced cryoprotectant mobilization response, gave rates of glucose production from endogenous glycogen reserves that were similar between the populations. Our findings suggest that extreme freeze tolerance in subarctic R. sylvatica does not require an enhanced ability of the liver to resist freezing stress or rapidly mobilize cryoprotectant.