Cutler CP et al. (2012), Aquaporin 4 is a Ubiquitously Expressed Isoform...

XB-ART-44753

Front Physiol 2012 Jan 10;2:107. doi: 10.3389/fphys.2011.00107.

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Aquaporin 4 is a Ubiquitously Expressed Isoform in the Dogfish (Squalus acanthias) Shark.

Cutler CP , Maciver B , Cramb G , Zeidel M .

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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 > gill > 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 (P(f)) 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.

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Species referenced: Xenopus laevis
Genes referenced: aqp4 cyp26a1

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	Figure 1. Alignment of Dogfish AQP4 [Accession number (Ac. No.) JF944824] amino acid sequence with AQP4 sequences from Human (Homo sapiens; Ac. No. NM_001650.4; 63.0%), Rat (Rattus norvegicus; Ac. No. AF144082; 62.5%), Chicken (Gallus gallus; Ac. No. NM_001004765; 65.3%), African Clawed Toad (Xenopus laevis; Ac. No. NM_001130949.1; 56.7%), Zebrafish (Danio rerio; NM_001003749; 57.5%), and Hagfish (Eptatretus burgeri; Ac. No. AB258403.1; 38.2%). Percentages in parentheses represent amino acid homologies. Numbers indicate position within the alignment. â¢ Symbols indicate positions with identical amino acids. \| Symbols indicate positions with chemically similar amino acids. Bold underline _ indicates the position of the peptide sequences used to raise the polyclonal antibodies. Wavy underline indicates the position of amino acids sequences used to make degenerate primers for initial AQP4 OCR amplifications. Double underline indicates the positions of putative N-glycosylation sites with the dogfish AQP4 sequence.
	Figure 2. Northern blot of 5âÎ¼g of total RNA extracted from various tissues of the dogfish (S. acanthias) and probed with a 32P radioisotope-labeled cDNA fragment of the AQP4 gene. The probe hybridizes to a single band of 3.2âkbp.
	Figure 3. Semi-quantitative PCR amplification of dogfish AQP4 (274âbp) and GAPDH (271âbp) cDNA fragments. The primers used were also used for quantitative PCR and were designed across conserved intronâexon splice junctions of the respective genes to avoid genomic DNA amplification. 0.5âÎ¼l of cDNA template (made with the following microgramâs of total RNA; 1.15 gill; 0.67 rectal gland; 0.93 kidney; 0.88 esophagus/cardiac stomach; 0.99 stomach; 1.56 intestine; 0.41 brain; 0.21 muscle; 0.28 eye; 0.71 liver) was used in all reactions and GAPDH was used as a positive control. The negative (Neg.) control reactions were without cDNA.
	Figure 4. Analysis of a Northern blot experiment measuring the mRNA expression of aquaporin 4 (AQP4) in the tissue of dogfish acclimated to different external environmental salinities. No significant differences were seen in any tissue between any of the salinities.
	Figure 5. Relative dogfish AQP4 mRNA abundance in the gills of fish acclimated to 75, 100, or 120% seawater (SW). mRNA expression was determined using quantitative PCR, with primers designed at conserved exonâintron splice junctions within the gene sequence to avoid amplification of genomic DNA. nâ=â6 Fish per group. *â=âStatistically significant difference between 75 and 100% SW fish, where pâ<â0.05.
	Figure 6. Osmotic water permeability (Pf) of Xenopus laevis oocytes micro-injected with dogfish AQP4 cRNA (-â -) or with H2O (-â-). The data are averages of four experiments where measurements were made at six different pH values with an average of eight oocytes per group, at each pH value, in each experiment.
	Figure 7. The effect of mercury chloride (Hg) on the osmotic water permeability (Pf) of Xenopus oocytes micro-injected with dogfish AQP4 cRNA or in with H2O (Control). Data represent averages of three experiments, with approximately eight oocytes per group in each. There was no statistically significant difference in Pf with or without mercury chloride.
	Figure 8. The uptake of C-labeled urea in Xenopus laevis oocytes micro-injected with dogfish AQP4 cRNA or uninjected. Results are averages from three experiments with approximately eight oocytes used per group.
	Figure 9. The uptake of C-labeled glycerol in Xenopus laevis oocytes micro-injected with dogfish AQP4 cRNA or uninjected. Results are averages from three experiments with approximately eight oocytes used per group.

References [+] :

Amiry-Moghaddam, Aquaporins: multifarious roles in brain. 2010, Pubmed