Carissa M. Krane

Cope's 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. J. Exp. Zool. 315:424–437, 2011.

Energy-based surgical scalpels are designed to efficiently transect and seal blood vessels using thermal energy to promote protein denaturation and coagulation. Assessment and design improvement of ultrasonic scalpel performance relies on both in vivo and ex vivo testing. The objective of this work was to design and implement a robust, experimental test matrix with randomization restrictions and predictive statistical power, which allowed for identification of those experimental variables that may affect the quality of the seal obtained ex vivo.

Maintenance of fluid homeostasis is critical to establishing and maintaining normal physiology. The landmark discovery of membrane water channels (aquaporins; AQPs) ushered in a new area in osmoregulatory biology that has drawn from and contributed to diverse branches of biology, from molecular biology and genomics to systems biology and evolution, and from microbial and plant biology to animal and translational physiology. As a result, the study of AQPs provides a unique and integrated backdrop for exploring the relationships between genes and genome systems, the regulation of gene expression, and the physiologic consequences of genetic variation. The wide species distribution of AQP family members and the evolutionary conservation of the family indicate that the control of membrane water flux is a critical biological process. AQP function and regulation is proving to be central to many of the pathways involved in individual physiologic systems in both mammals and anurans. In mammals, AQPs are essential to normal secretory and absorptive functions of the eye, lung, salivary gland, sweat glands, gastrointestinal tract, and kidney. In urinary, respiratory, and gastrointestinal systems, AQPs are required for proper urine concentration, fluid reabsorption, and glandular secretions. In anurans, AQPs are important in mediating physiologic responses to changes in the external environment, including those that occur during metamorphosis and adaptation from an aquatic to terrestrial environment and thermal acclimation in anticipation of freezing. Therefore, an understanding of AQP function and regulation is an important aspect of an integrated approach to basic biological research.

Cope's gray tree frog Hyla chrysoscelis accumulates glycerol during cold acclimation. We hypothesized that, during this process, gray tree frogs adjust renal filtration and/or reabsorption rates to retain accumulated glycerol. During cold acclimation, plasma concentrations of glycerol rose >200-fold, to 51 mmol/l. Although fractional water reabsorption decreased, glomerular filtration rate (GFR) and, consequently, urine flow were Xenopus oocyte expression system. HC-1, an AQP1-like water channel conferring low glycerol permeability, is expressed ubiquitously in warm- and cold-acclimated tissues. HC-2, a water channel most similar to AQP2, is primarily expressed in organs of osmoregulation. HC-3, which is most similar to AQP3, is functionally characterized as a GLP, with low permeability to water but high permeability to glycerol. Aspects of expression levels and functional characteristics varied between cold and warm conditions for each of the three AQPs, suggesting a complex pattern of involvement in osmoregulation related to thermal acclimation.