Honors Theses


Carissa Krane



Publication Date

Spring 4-2014

Document Type

Honors Thesis


Cope’s gray treefrog Hyla chrysoscelis, accumulates and distributes glycerol as a cryoprotectant in anticipation of freezing. Transmembrane glycerol and water flux in H. chysoscelis erythrocytes likely occurs through HC-3, an ortholog of mammalian aquaporin 3. HC-3 protein is in higher abundance and is preferentially localized to the plasma membrane in RBCs from cold-acclimated treefrogs as compared to warmacclimated animals. It is hypothesized that neuroendocrine agonists via receptor mediated second messenger pathways integrate signals derived from fasting, dehydration, diurnal, and/or temperature changes during cold-acclimation to regulate HC-3 expression as part of the mechanism of freeze tolerance. In this study, cultured H. chrysoscelis erythrocytes were exposed to 1 uM epinephrine for 30 and 60 minutes. Native HC-3 expression increased 3 fold at 30 minutes and 5.5-fold at 60 minutes relative to controls, whereas glycosylated HC-3 expression increased by 1.1-fold at 30 minutes and by 2 -fold at 60 minutes relative when exposed to epinephrine. Moreover, epinephrine treatment resulted in membrane localization as compared to cytosolic distribution in control cells. Erythrocytes pre-treated with Calphostin C, a PKC inhibitor, showed no HC-3 membrane localization, and native HC-3 expression was reduced by 5% relative to controls and 3- fold relative to epinephrine-treated cells. Thus, epinephrine begins a PKC-dependent mechanism that results in an increase in HC-3 abundance, HC-3 membrane localization, and enhanced glycosylation in erythrocytes. These regulatory mechanisms are consistent with the in vivo regulation of HC-3 expression observed in erythrocytes from coldacclimated treefrogs.

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