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Aquaporins (AQPs) are water channel proteins, and the expression of AQPs in carcinoma cells has received much attention over the last 15 years. In the veterinary field, however, little is known about the expression of AQPs. In the present study using immunohistochemistry, we examined the expression of AQP1, AQP3, and AQP5 in canine mammary gland carcinomas. The 27 samples comprised 10 grade I, 12 grade II, and 5 grade III samples (See Materials and Methods section for grade classification method). AQP1 was expressed in only 2 of the grade III carcinomas, and the expression was limited to spindle-shaped cells in the solid structure and on the outside of the solid mass. AQP3-positive cells were observed in 20 of 22 grade I and II samples. On the other hand, among grade III carcinomas, AQP3 was expressed only in spindle-shaped cells in 1 sample. AQP5 was expressed in all grade I and II carcinomas but not in the grade III tumors. In addition, enhanced expression of basolateral AQP3 and apical AQP5 was observed in lobular hyperplastic cells. These results suggest that the expression patterns of AQP3 and AQP5 can be of help for judging the grading of canine mammary tumors and that AQP1 is likely to be involved in metastasis. Moreover, AQP3 and AQP5 might be relevant to lactation in female dogs. [ABSTRACT FROM AUTHOR]

Phosphorylation of serine 256 ($256) plays a critical role in vasopressin (VP)-mediated membrane accumulation of aquaporin-2 (AQP2). Recently, phosphorylation of serine 261 was also reported, raising the possibility that it has a role in AQP2 trafficking. We addressed this issue using transfected LLC-P[K.sub.1] cells that express point mutations of AQP2 S261 and $256, mimicking the phosphorylated (S to D) or dephosphorylated (S to A) states of these residues. Both AQP2 ($261A) and AQP2 ($261D) were located in the perinuclear cytoplasm without stimulation but, like wild-type AQP2, they both accumulated on the plasma membrane after 20-min exposure to VP or forskolin. Following membrane accumulation, $261A, $261D, and wild-type AQP2 reinternalization was complete over a similar time frame, between 30 and 60 min after VP washout. Using various combinations of point mutations, we showed that the phosphorylation state of $256 is dominant with respect to AQP2 behavior; AQP2 membrane accumulation and internalization were not detectably affected by the phosphorylation state of $261. Finally, blocking AQP2 endocytosis by methyl-[beta]-cyclodextrin caused membrane accumulation of AQP2 in cells expressing either a single S-A mutation or double mutations of $256 and $261, although as previously reported, the $256D mutation was always present at the cell surface. This suggests that constitutive recycling of AQP2 was not modified by the phosphorylation state of $261. Together, our data indicate that the phosphorylation state of AQP2 at $261 does not detectably affect regulated or constitutive trafficking of AQP2. The potential role of $261 phosphorylation/ dephosphorylation in vasopressin action remains to be determined. vasopressin; cAMP

Hypertonicity-induced expression of aquaporin 3 in MDCK cells. Am J Physiol Cell Physiol 281: C55-C63, 2001.--Aquaporins (AQPs) are water channel proteins that participate in water transport. In the principal cells of the kidney collecting duct, water reabsorption is mediated by the combined action of AQP2 in the apical membrane and both AQP3 and AQP4 in the basolateral membrane, and the expression of AQP2 and AQP3 is regulated by antidiuretic hormone and water restriction. The effect of hypertonicity on AQP3 expression in Madin-Darby canine kidney (MDCK) epithelial cells was investigated by exposing the cells to hypertonic medium containing raffinose or NaCl. Northern blot and immunoblot analyses revealed that the amounts of AQP3 mRNA and AQP3 protein, respectively, were markedly increased by exposure of cells to hypertonicity. These effects were maximal at 12 and 24 h, respectively. Immunofluorescence and immunoelectron microscopy also demonstrated that the abundance of AQP3 protein was increased in cells incubated in hypertonic medium and that the protein was localized at the basolateral plasma membrane. These results indicate that the expression of AQP3 is upregulated by hypertonicity. immunohistochemistry; water channel; plasma membrane; Madin-Darby canine kidney cells