GOLDSTEIN, D. L.; KRANE, C.; FRISBIE, J.; RIVERA, K.; Wright State University, Dayton, OH; University of Dayton, Dayton, OH; Wright State University, Dayton, OH; University of Dayton, Dayton, OH: Aquaporins in an amphibian that accumulates glycerol during cold acclimation.

Cope�s gray treefrog, Hyla chrysoscelis, is a freeze-tolerant amphibian. As part of its strategy of cold acclimation, this species accumulates high concentrations of glycerol, up to 50 � 100 mM/L in extracellular fluid. The glycerol is likely produced in the liver, liberated to the circulation, and then enters cells in other tissues as a cryoprotectant, where it stabilizes cellular structures and prevents excessive cellular dehydration during extracellular freezing. We are initiating study of the mechanisms by which glycerol crosses cell membranes during these processes. Preliminary measures of glycerol entry into erythrocytes of warm-acclimated frogs indicate a low rate of uptake. We hypothesize that this uptake, and associated water movement, is regulated in cold-acclimation and accomplished by a combination of aquaporins and aqua-glycero-porins, the latter of which permit passage of small organic molecules like glycerol in addition to water. At present, we have identified two aquaporins in these animals. One, present in several tissues including kidney and bladder, has high sequence identity (>95%) at the nucleotide level to an aquaporin (AQP-h1) identified in another hylid species, Hyla japonica. The second aquaporin, found so far only in the kidney, has poor nucleotide homology to any previously documented aquaporin but is similar in amino acid sequence to AQP-t4, a water channel identified in the toad Bufo marinus. We are pursuing aquaglyceroporins based on primers designed around conserved regions of one published amphibian aquaqlyceroporin and the bacterial protein GlpF.