James Frisbie

Expression and immunolocalization of aquaporins HC-1, -2, and -3 in Cope's gray treefrog, Hyla chrysoscelis

We have previously identified two aquaporins (HC-1, HC-2) and a glyceroporin (HC-3), homologs, respectively, of mammalian AQP1, AQP2, and AQP3, from the freeze-tolerant treefrog Hyla chrysoscelis. The objective of the present study was to investigate by Western blotting and immunohistofluorescence the expression and localization of these proteins in warm-acclimated, hydrated treefrogs. We hypothesized that patterns of protein expression would reflect unique osmoregulatory roles for the three aquaporins. Western blots revealed a spectrum of protein bands from 28 kDa to 65+ kDa; treatment with N-glycosidase suggested that this reflected variable glycosidation of the aquaporins. HC-1 was expressed in all organs, including dermis of skin, sinusoids and septa of liver, Bowmans capsule of kidney, intestinal lacteal vessels, and perimysium and vasculature of muscle. HC-3 expression was also widespread, but with different localization, including epidermis and dermis of skin, renal collecting ducts, and colonic villous epithelium. HC-2 expression was limited to osmoregulatory organs (renal collecting ducts and epidermis). In many ways, the expression of these proteins paralleled their mammalian homologs. For example, HC-2 and HC-3 expression in collecting ducts appeared similar to the mammalian pattern (the former more apical, the latter more basal). However, some aspects of localization (e.g. HC-1 in Bowmans capsule) were unique, and the ubiquity of HC-3 expression may relate to its facilitation of glycerol transport in this animal that possesses glycerol-dependent freeze tolerance.

Dynamic regulation of aquaglyceroporin expression in erythrocyte cultures from cold- and warm-acclimated cope's gray treefrog, Hyla chrysoscelis

Copes gray treefrog, Hyla chrysoscelis,is a freeze-tolerant anuran which accumulates and distributes glycerol as a cryoprotectant before freezing. We hypothesize that HC-3, an aquaglyceroporin member of the MIP family of water pores, may play an important role in the process of freeze tolerance by mediating transmembrane passage of glycerol and water during cold-acclimation. The objectives of this study were two-fold: to examine HC-3 protein abundance and cellular localization in erythrocytes from cold- and warm-acclimated frogs and to develop and characterize an erythrocyte cell culture system for examining HC-3 gene regulation. Compared with warm-acclimated frogs, erythrocytes from cold-acclimated frogs had higher HC-3 protein expression and enhanced plasma membrane localization. Furthermore, erythrocytes from cold- and warm-acclimated frogs maintained in culture at 4 and 20°C exhibited time- and temperature-dependent regulation of HC-3 expression and an increase in the abundance of high molecular weight immunoreactive species within 24 hr of culture at 20°C. Deglycosylation of erythrocyte proteins resulted in the disappearance of the high molecular weight species, indicating that HC-3 is post-translationally modified by N-linked glycosylation. Erythrocytes cultured in media containing glycerol also showed an increased abundance of the high molecular weight bands and enhanced plasma membrane localization of HC-3, suggesting a role for glycerol in regulating HC-3 subcellular trafficking. Thus, the development of this erythrocyte cell culture system from H. chrysoscelis opened an opportunity to study the properties of cells with changing expression of an aquaglyceroporin, HC-3, and to explore the factors regulating that expression.

Endo-Porter–mediated delivery of phosphorodiamidate morpholino oligos (PMOs) in erythrocyte suspension cultures from Cope's gray treefrog Hyla chrysoscelis

Copes gray treefrog, Hyla chrysoscelis, is a freeze-tolerant anuran that accumulates cryoprotective glycerol during cold acclimation. H. chrysoscelis erythrocytes express the aquaglyceroporin HC-3, which facilitates transmembrane glycerol and water movement. Aquaglyceroporins have no pharmacological inhibitors, and no genetic knockout tools currently exist for H. chrysoscelis. A phosphorodiamidate morpholino oligo (PMO)-mediated expression knockdown approach was therefore pursued to provide a model for testing the role of HC-3. We describe a novel procedure optimized for specific, efficient knockdown of HC-3 expression in amphibian erythrocyte suspensions cultured at nonmammalian physiological temperatures using Endo-Porter. Our protocol includes three critical components: pre-incubation at 37°C, two rounds of Endo-Porter and HC-3 PMO administration at ~23°C, and continuous shaking at 190 rpm. This combination of steps resulted in 94% reduction in HC-3 protein expression (Western blot), substantial decrease in HC-3 expression in >65% of erythrocytes, and no detectable expression in an additional 30% of cells (immunocytochemistry).

Expression of the aquaglyceroporin HC‐9 in a freeze‐tolerant amphibian that accumulates glycerol seasonally

As ambient temperatures fall in the autumn, freeze‐tolerant Copes gray treefrogs, Dryophytes chrysoscelis (formerly Hyla chrysoscelis), accumulate glycerol as a cryoprotective agent. We hypothesized that these treefrogs express an ortholog of the mammalian aquaglyceroporin AQP9 and that AQP9 expression is upregulated in the cold to facilitate glycerol transport. We sequenced 1790 bp from cloned cDNA that codes for a 315 amino acid protein, HC‐9, containing the predicted six transmembrane spanning domains, two Asn‐Pro‐Ala (NPA) motifs, and five amino acid residues characteristic of aquaglyceroporins. Functional characterization after heterologous expression of HC‐9 cRNA in Xenopus laevis oocytes indicated that HC‐9 facilitates glycerol and water permeability and is partially inhibited by 0.5 mmol/L phloretin or 0.3 mmol/L HgCl2. HC‐9 mRNA (qPCR) and protein (immunoblot) were expressed in most treefrog tissues analyzed (muscle, liver, bladder, stomach, kidney, dorsal skin, and ventral skin) except the protein fraction of red blood cells. Contrary to our prediction, both mRNA and protein expression were either unchanged or downregulated in most tissues in response to cold, freezing, and thawing. A notable exception to that pattern occurred in liver, where protein expression was significantly elevated in frozen (~4‐fold over warm) and thawed (~6‐fold over warm) conditions. Immunofluorescence labeling of HC‐9 protein revealed a signal that appeared to be localized to the plasma membrane of hepatocytes. Our results indicate that gray treefrogs express an AQP9‐like protein that facilitates glycerol permeability. Both the transcriptional and translational levels of HC‐9 change in response to thermal challenges, with a unique increase in liver during freezing and thawing.