Aquaporin 4 is a Ubiquitously Expressed Isoform in the Dogfish (\u3cem\u3eSqualus acanthias\u3c/em\u3e) Shark

The dogfish ortholog of aquaporin 4 (AQP4) was amplified from cDNA using degenerate PCR followed by cloning and sequencing. The complete coding region was then obtained using 5′ and 3′ RACE techniques. Alignment of the sequence with AQP4 amino acid sequences from other species showed that dogfish AQP4 has high levels (up to 65.3%) of homology with higher vertebrate sequences but lower levels of homology to Agnathan (38.2%) or teleost (57.5%) fish sequences. Northern blotting indicated that the dogfish mRNA was approximately 3.2 kb and was highly expressed in the rectal gland (a shark fluid secretory organ). Semi-quantitative PCR further indicates that AQP4 is ubiquitous, being expressed in all tissues measured but at low levels in certain tissues, where the level in liver \u3e gill \u3e intestine. Manipulation of the external environmental salinity of groups of dogfish showed that when fish were acclimated in stages to 120% seawater (SW) or 75% SW, there was no change in AQP4 mRNA expression in either rectal gland, kidney, or esophagus/cardiac stomach. Whereas quantitative PCR experiments using the RNA samples from the same experiment, showed a significant 63.1% lower abundance of gill AQP4 mRNA expression in 120% SW-acclimated dogfish. The function of dogfish AQP4 was also determined by measuring the effect of the AQP4 expression in Xenopus laevis oocytes. Dogfish AQP4 expressing-oocytes, exhibited significantly increased osmotic water permeability (Pf) compared to controls, and this was invariant with pH. Permeability was not significantly reduced by treatment of oocytes with mercury chloride, as is also the case with AQP4 in other species. Similarly AQP4 expressing-oocytes did not exhibit enhanced urea or glycerol permeability, which is also consistent with the water-selective property of AQP4 in other species

Functional Characterization of Four Aquaporins (AQPs) Cloned From the European Eel, \u3cem\u3eAnguilla anguilla\u3c/em\u3e

The eel is a euryhaline teleost which exhibits remarkable adaptation in moving between fresh (FW) and sea water (SW) environments. We are investigating how the eel osmoregulates by functionally characterizing four eel aquaporins (AQP1, AQP1dup [a gene duplication of AQP1], AQP3 and AQPe [possibly an AQP10 homolog]) using the Xenopus oocyte expression system. Water fluxes were measured by exposing oocytes to hypotonic solutions and quantitating cross-sectional area. Urea and glycerol fluxes were measured by isotope uptake. AQP1, expressed in intestine (up in SW), oesophagus and kidney (down in SW) showed high Hg2+-inhibitable water fluxes, but no urea or glycerol transport. AQP1dup found in the same tissues but with high variability in expression, exhibited the same transport specificities but water permeability was ~50% of AQP1. AQP3 in the gill is dramatically downregulated in SW and exhibited high water, urea and glycerol permeabilities. These permeabilities were \u3e90% inhibited at pH6.5. None of the other AQPs were inhibited by pH. AQPe was also an aquaglyceroporin which could transport glycerol and urea as well as water at high rates. It is found in intestine and kidney and exhibits little change in SW or FW. Eel AQPs have similar transport specificities to their human orthologs but are uniquely regulated by environmental salinity

Partial Functional Characterization of an Aquaporin 3 Ortholog From the European Eel, \u3cem\u3eAnguilla anguilla\u3c/em\u3e

Maintenance of body fluid composition and osmoregulation are essential for metazoan animals to survive. Aquatic animals face the particular problem of directly interacting with an aqueous environment that differs markedly in osmolality from their internal physiology. Freshwater (FW) fish experience driving forces for osmotic water uptake across the exposed epithelial surfaces of the gill and the gut. They compensate by drinking little and producing large volumes of dilute urine. Conversely, marine fish experience osmotic water loss to the sea and compensate by drinking seawater (SW) and excreting the excess salts ingested via the gills and the kidney. A number of species are euryhaline, exhibiting remarkable physiological adaptation as they move at some stage of their life cycle between FW and SW environments. One such example is the European eel Anguilla anguilla, a euryhaline teleost. In order for species such as the eel to acclimate to environments of different salinity, water and ion transport pathways in epithelia which interface with the environment must be under tight regulation

Expression and Functional Characterization of Four Aquaporin Water Channels From the European Eel (\u3cem\u3eAnguilla anguilla\u3c/em\u3e)

The eel is a euryhaline teleost. In order to maintain osmotic equilibrium the animal must resist loss of body water and solutes to the sea, and to resist volume expansion from influx of free water in fresh water (FW) environments. The epithelial contact surfaces of the eel are prime sites for water and solute exchange and the gill with its high surface area for gas exchange represents a vulnerable site for osmotically driven uptake or loss of body water1. Likewise, the gastrointestinal tract must employ ion exchange and water transport mechanisms which permit volume homeostasis6

Aquaporin (AQP) Channels in the Spiny Dogfish, \u3cem\u3eSqualus acanthias\u3c/em\u3e I: Characterization of AQP3 and AQP15 Function and Expression, and Localization of the Proteins in Gill and Spiral Valve Intestine

Complementary DNAs (cDNAs) for two aquaporin water channel genes (AQP3 and AQP15) were amplified cloned and sequenced to initiate this study. Northern blot analysis was carried out to confirm the mRNA sizes of these AQP genes with AQP3 mRNA bands exhibiting sizes of 1.2 and 1.6 k bases and AQP15 had a mRNA band of 2.1 k bases. Northern blot analysis was also performed on kidney and esophagus total RNA samples from fish acclimated to 75%, 100% or 120% seawater (SW). The level of AQP15 mRNA expression was shown to significantly decrease following salinity acclimation from 100 to 120% SW. An opposite but non-significantly different trend was observed for AQP3 mRNA levels. Full length cDNAs were then used to generate AQP3 and AQP15 mRNAs for microinjection into Xenopus oocytes. Both AQP3- and AQP15- microinjected oocytes exhibited significantly elevated apparent water permeability compared to control oocytes at neutral pH. The apparent water permeability was mercury-inhibitable, significantly so in the case of AQP3. AQP3 microinjected oocytes showed pH sensitivity in their apparent water permeability, showing a lack of permeability at acidic pH values. The Carboxyl-terminal derived amino acid sequences of AQP3 and AQP15 were used to generate rabbit affinity-purified polyclonal antibodies. Western blots with the antibodies showed a band of 31.3 kDa for AQP3 in the kidney, with minor bands at 26, 24 and 21 kDa. For AQP15 a band of 26 kDa was seen in gill and kidney. Fainter bands at 28 and 24 kDa were also seen in the kidney. There was also some higher molecular weight banding. None of the bands were seen when the antibodies were pre- blocked with their peptide antigens. Immunohistochemical localization studies were also performed in the gill and spiral valve intestine. In the gill, AQP15 antibody staining was seen sporadically in the membranes of surface epithelial cells of the secondary lamellae. Tyramide amplification of signals was employed in the spiral valve intestine. Tyramide-amplified AQP3 antibody staining was observed in the basal membrane of the invaginated epithelial cell layer of secondary intestinal folds in luminal surface of either the side wall of the spiral valve intestine or in internal valve tissue ‘flaps’. For the AQP15 antibody, tyramide-amplified staining was instead found on the apical and to a lesser extent the lateral membranes of the same invaginated epithelial cell layer. The localization of AQP3 and AQP15 in the spiral valve intestine suggests that a trans-cellular water absorption pathway may exist in this tissue