Immunolocalization of aquaporin water channels in the domestic cat male genital tract.

Four different aquaporins (AQP1, 2, 5 and 9), integral membrane water channels that facilitate rapid passive movement of water, were immuno-localized in the excurrent ducts collected from sexually mature cats during orchiectomy. Aquaporins 1, 2 and 9, were immuno-localized at distinct levels, whereas AQP5 was undetectable all along the feline genital tract. No immunoreactivity was present at the level of the rete testis with any of the antibodies tested. In the efferent ducts, AQP1-immunoreactivity was strongly evidenced at the apical surface of the non-ciliated cells, and AQP9-immunoreactivity was shown at the periphery of both ciliated and non-ciliated cells. Aquaporins 2 was absent in the caput epididymidis, either in the efferent ducts or in the epididymal duct. Otherwise, AQP2 was increasingly localized at the adluminal surface of principal cells from the corpus to the cauda epididymidis and more weakly in the vas deferens epithelium. The supranuclear zone of the epididymal principal cells was AQP9-immunoreactive throughout the duct, with the exclusion of the vacuolated sub-region of the caput and with higher reaction intensity in the cauda region. AQP1 was present in the blood vessels all along the genital tract. AQP1 was expressed also in the smooth muscle layer of the vas deferens. The tested AQP molecules showed a different expression pattern in comparison with laboratory mammals, primates and the dog, unique other carnivore species studied to date. The present information is possibly useful in regard to the regional morphology of the feline epididymis and correlated functions, which are still a matter of debate.

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

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.

Functional characterization of three aquaglyceroporins from Trypanosoma brucei in osmoregulation and glycerol transport.

Previous studies using bloodstream form Trypanosoma brucei have shown that glycerol transport in this parasite occurs via specific membrane proteins, namely a glycerol transporter and glycerol channels [1]. Later, we cloned, expressed and characterized the transport properties of all three aquaglyceroporins (AQP1-3) [2], which were found permeable for water, glycerol and other small uncharged solutes like dihydroxyacetone [3]. Here, we report on the cellular localization of TbAQP1 and TbAQP3 in bloodstream form trypanosomes. Indirect immunofluorescence analysis showed that TbAQP1 is exclusively localized in the flagellar membrane, whereas TbAQP3 was found in the plasma membrane.In addition, we analyzed the functions of all 3 AQPs, using an inducible inheritable double-stranded RNA interference methodology. All AQP knockdown cell lines were still able to survive hypo-osmotic stress conditions, except AQP2 knockdown parasites. Depleted TbAQP2 negatively impacted cell growth and the regulatory volume recovery, whereas AQP1 und 3 knockdown trypanosomes displayed phenotypes consistent with their localization in external membranes. A simultaneous knockdown of all 3 AQPs showed that the cells were able to substitute the missing glycerol uptake capability through a putative glycerol transporter.