Regulation of water absorption in the frog skins by two vasotocin-dependent water-channel aquaporins, AQP-h2 and AQP-h3.

A new frog aquaporin (AQP) cDNA was cloned from a cDNA library constructed from the ventral skin of the tree frog Hyla japonica. This AQP (Hyla AQP-h2) consisted of 268 amino acid residues with a high homology to mammalian AQP2. The predicted amino acid sequence contained the two conserved Asn-Pro-Ala motifs found in all the major intrinsic protein family members and the putative six transmembrane domains. The sequence also contained a mercurial compound: cysteine, one potential N-glycosylation site at Asn-124, and a putative phosphorylation site recognized by protein kinase A at Ser-262. In a swelling assay using Xenopus oocytes, AQP-h2 facilitated water permeability, especially in response to cAMP. Expression of AQP-h2 mRNA was restricted to several tissues including the ventral skin, kidney, and urinary bladder; but with immunofluorescence staining using an antipeptide antibody (ST-140) against the AQP-h2 protein, immunopositive cells were found only in the ventral skin and urinary bladder. In the ventral pelvic skin, the label for AQP-h2 was localized in the entire plasma membrane of the granular cells beneath the outmost layer of the skin and in the basolateral membrane of the granular cells in this layer. In response to vasotocin, however, the label for AQP-h2 became more intense in the apical membrane in the granular cells of the outermost layer, similar to the case for the earlier studied AQP-h3, which was specifically expressed in the ventral skin. Taken together, these findings suggest that not only AQP-h3, but also AQP-h2 acts as a regulator of the water balance in this frog.

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

A new toad aquaporin (AQP) cDNA was cloned from a cDNA library constructed from the ventral skin of Xenopus laevis. This AQP (Xenopus AQP-x5) consisted of 273 amino acid residues with a high sequence homology to mammalian AQP5. The predicted amino acid sequence contained the two conserved Asn-Pro-Ala motifs found in all major intrinsic protein (MIP) family members and six putative transmembrane domains. The sequence also contained a mercurial-sensitive cysteine and a putative phosphorylation motif site for protein kinase A at Ser-257. The swelling assay using Xenopus oocytes revealed that AQP-x5 facilitated water permeability. Expression of AQP-x5 mRNA was restricted to the skin, brain, lungs and testes. Immunofluorescence and immunoelectron microscopical studies using an anti-peptide antibody (ST-156) against the C-terminal region of the AQP-x5 protein revealed the presence of immunopositive cells in the skin, with the label predominately localized in the apical plasma membrane of the secretory cells of the small granular glands. These glands are unique both in being close to the epidermal layer of the skin and in containing mitochondria-rich cells with vacuolar H+-ATPase dispersed among its secretory cells. Results from immunohistochemical experiments on the mucous or seromucous glands of several other anurans verified this result. We conclude that the presence of AQP-x5 in the apical plasma membrane of the small granular glands suggests its involvement in water secretion from the skins. The physiological roles of the AQP-x5 protein in the small or mucous glands are discussed.