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First published online August 3, 2006
Journal of Experimental Biology 209, 3199-3208 (2006)
Published by The Company of Biologists 2006
doi: 10.1242/jeb.02351

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Molecular and cellular characterization of a new aquaporin, AQP-x5, specifically expressed in the small granular glands of Xenopus skin

Makoto Kubota, Takahiro Hasegawa, Takashi Nakakura, Haruna Tanii, Masakazu Suzuki and Shigeyasu Tanaka^*

Department of Biology, Faculty of Science, Shizuoka University, Ohya 836, Shizuoka 422-8529, Japan

View larger version (42K): [in a new window]   Fig. 1. (A) Nucleotide and deduced amino acid sequence of Xenopus AQP-x5 cDNA. The predicted amino acid is shown below the nucleotide sequence. The asterisk indicates the terminal codon. NPA motifs and polyadenylation signal regions are outlined. The square, diamonds, and open triangles indicate phosphorylation sites for protein kinase A, protein kinase C and mercurial-inhibition sites, respectively. (B) Kyte-Doolittle hydropathy profile (window 11) of the deduced AQP-x5 amino acid sequence.

View larger version (14K): [in a new window]   Fig. 2. RT-PCR of Xenopus tissue extracts. RT-PCR products using primers as described in the Materials and methods were separated on a 2% agarose gel and stained with ethidium bromide. +, mRNA present; -, mRNA absent.

View larger version (49K): [in a new window]   Fig. 3. Expression of AQP-x5 in Xenopus oocytes. (A) Osmotic water permeability (Pf) was calculated from the initial rate of oocyte swelling. Oocytes were microinjected with water or cRNA encoding AQP-x5. A portion of the AQP-x5-injected oocytes was incubated with 0.3 mmol l-1 HgCl2. All data shown are given as the mean ± s.e.m. of measurements from 8-11 oocytes (as indicated above the bars) in each experimental group. *P<0.001 vs water, **P<0.001 vs AQP-x5. (B) Immunofluorescence images of AQP-x5 protein in AQP-x5-injected oocytes. (1) After completion of the swelling experiments, immunoreactive AQP-x5 substances are visible, predominantly in the plasma membrane. (2) The corresponding Nomarski differential interference image. (3) In the absorption test, positive immunoreactive substances obtained with anti-AQP-x5 are nearly eliminated at background levels in AQP-x5-injected oocyte. (4) As in 3, only background levels are seen in the water-injected oocyte with anti-AQP-x5. Arrowheads indicate plasma membrane. Scale bar, 50 µm.

View larger version (29K): [in a new window]   Fig. 4. Characterization of anti-AQP-x5 serum by western blot analysis. (A) Immunoreactive bands are seen at 29.0 kDa in an extract of AQP-x5 cRNA-injected oocytes. (B) The membrane was immunostained with the antiserum preabsorbed with the antigen peptide (10 µg ml-1).

View larger version (114K): [in a new window]   Fig. 5. Mallory's triple staining of Xenopus skin. In sections treated with Mallory's triple stain the skin glands can be classified into three types: the granular gland (g), the mucous gland (m), and the small granular gland (sg). Scale bar, 50 µm.

View larger version (97K): [in a new window]   Fig. 6. Immunofluorescence localization of AQP-x5 in the skin glands. (A,C,D) Fluorescence images of AQP-x5 and (B) the corresponding Nomarski differential interference contrast image. (A,B) The labels (green; arrows) are clearly visible in the apical plasma membrane of granular cells in the small granular glands (sg). Weak positive reaction is visible in the apical plasma membrane of glandular cells located in upper sides of the mucous glands (arrowheads). No labeling is seen in the mucous (m) and granular (g) glands. The asterisk in B indicates red blood cells, displaying a nonspecific label for AQP-x5. (C) No labeling is detected in any of the cells of the skins when anti-AQP-x5 is preabsorbed with the corresponding antigen peptide. (D) Double-labeling for AQP-x5 (green) and V-ATPase E-subunit (red); V-ATPase-expressing cells are observed among the glandular cells (arrowheads). Nuclei are counterstained with DAPI (blue); l, lumen. Scale bars, 50 µm (A-C); 10 µm (D).

View larger version (123K): [in a new window]   Fig. 7. Localization of AQP-x5 by immunofluorescent staining in Hyla japonica (A,B), Rana japonica (C,D) and Bufo marinus (E,F). Fluorescence images (A,C,E) for AQP-x5 and the corresponding Nomarski differential interference contrast images (B,D,F) are shown. AQP-x5 (green; arrows); V-ATPase E-subunit (red; arrowheads) are clearly visible. l, lumen. Scale bars, 10 µm (A-D); 50 µm (E,F).

View larger version (117K): [in a new window]   Fig. 8. A conventional electron micrograph showing secretory cells (sc) and a mitochondria-rich cell (mrc) in the small granular gland. The secretory cell has many secretory granules (sg), whereas the cytoplasm in the mitochondria-rich cell is filled with mitochondria, and microvilli-like structures (arrows) develop throughout the region of the plasma membrane. Scale bar, 1 µm.

View larger version (109K): [in a new window]   Fig. 9. (A) An immunoelectron micrograph showing the small granular gland immunolabeled for AQP-x5. Labels are visible in the apical plasma membrane of the secretory cells (sc) in the small granular gland. (B) An immunoelectron micrograph showing secretory and mitochondria-rich cells labeled with the antibody preabsorbed with the antigen. Immunogold particles were seen at background levels, especially in mitochondria-rich cell (arrowheads). l, lumen; mrc, mitochondria-rich cell; m, mitochondria; asterisks, secretory granule. Scale bar, 1 µm.