Aquaglyceroporin expression and erythrocyte omoregulation in cultures from the freeze tolerant anuran, Cope's gray tree frog, Hyla chrysoscelis

Abstract

Cope's Gray Tree Frog, Hyla chrysoscelis is a freeze tolerant anuran that accumulates glycerol during cold acclimation. We hypothesize that the osmoregulatory capabilities of erythrocytes‐‐ to tolerate changes in cell volume and to affect rapid water and solute fluxes‐‐are likely to be most important during the events of freezing and thawing, and are dependent upon the expression and function of HC‐3, an ortholog of the aquaglyceroporin AQP3. Erythrocytes of H. chrysoscelis cultured for 48 hrs in media made hyperosmotic (400 mOsM) through the addition of 150 mM glycerol or urea showed enhanced HC‐3 glycosylation and membrane localization compared to those cultured in control or hypertonic media (250 mOsM culture media + 150 mM sorbitol or 75 mM NaCl). Cultured erythrocytes transferred from hyperosmotic culture media (glycerol or urea) to a hypotonic solution containing 250 mM glycerol or urea underwent a series of shape changes as they swelled, initially elongating, then becoming swollen and round. Cells cultured in hypertonic media (sorbitol or NaCl) responded with significantly less osmotically‐induced swelling (i.e. cell shape change). When aquaporins were blocked by HgCl2, cells retained normal dimensions, indicating the importance of aquaporins in conferring water and solute permeability in these cells. This research was supported by NSF Research Grant IOS‐1121457.