. Rho inhibits cAMP-induced translocation of aquaporin-2 into the apical membrane of renal cells. Am J Physiol Renal Physiol 281: F1092-F1101, 2001. First published August 8, 2001; 10.1152/ajprenal.00091.2001We have recently demonstrated that actin depolymerization is a prerequisite for cAMP-dependent translocation of the water channel aquaporin-2 (AQP2) into the apical membrane in AQP2-transfected renal CD8 cells (29). The Rho family of small GTPases, including Cdc42, Rac, and Rho, regulates the actin cytoskeleton. In AQP2-transfected CD8 cells, inhibition of Rho GTPases with Clostridium difficile toxin B or with C. limosum C3 fusion toxin, as well as incubation with the Rho kinase inhibitor, Y-27632, caused actin depolymerization and translocation of AQP2 in the absence of the cAMP-elevating agent forskolin. Both forskolin and C3 fusion toxin-induced AQP2 translocation were associated with a similar increase in the osmotic water permeability coefficient. Expression of constitutively active RhoA induced formation of stress fibers and abolished AQP2 translocation in response to forskolin. Cytochalasin D induced both depolymerization of F-actin and AQP2 translocation, suggesting that depolymerization of F-actin is sufficient to induce AQP2 translocation. Together, these data indicate that Rho inhibits cAMP-dependent translocation of AQP2 into the apical membrane of renal principal cells by controlling the organization of the actin cytoskeleton.aquaporin; C3 toxin; toxin B; actin cytoskeleton; G proteins; adenosine 3Ј,5Ј-cyclic monophosphate IN RENAL PRINCIPAL CELLS, the antidiuretic hormone arginine-vasopressin (AVP) regulates the shuttling of the water channel AQP2 from intracellular vesicles into the plasma membrane (8,14,34). AVP binds vasopressin V 2 receptors coupled to the G s -adenylyl cyclase system. Activation of this system results in the activation of protein kinase A (PKA), which induces phosphorylation of AQP2 at Ser 256 (10, 12). The phosphorylation of AQP2 by PKA and also the anchoring of PKA to subcellular compartments via protein kinase A anchoring proteins (AKAPs) are prerequisites for AQP2 translocation to the cell membrane (11, 12).F-actin has been demonstrated to be involved in AVP-mediated changes of osmotic water permeability (3,4,12,28). Depolymerization of cortical F-actin has been considered an important prerequisite for exocytosis. Calcium/ATP causes cortical F-actin disassembly (calmodulin dependent) and secretion (calmodulin independent) in permeabilized mast cells (25). Thus the cortical actin network may represent a cage that blocks the exocytotic process, and its disassembly constitutes an early stage of this reaction. In toad bladder, a renal-like epithelium, as well as in rat collecting duct epithelium, the total F-actin content decreases by 20-30 and 26%, respectively, after stimulation with vasopressin (5, 24). In AQP2-transfected renal collecting duct cells (CD8 cells), okadaic acid, an inhibitor of 1 and 2A phosphatases, induces actin depolymerization, leading to AQP2 transl...
Nephrogenic syndrome of inappropriate antidiuresis (NSIAD) is a recently identified chromosome X-linked disease associated with gain-of-function mutations of the V2 vasopressin receptor (V2R), a G-protein-coupled receptor. It is characterized by inability to excrete a free water load, hyponatremia, and undetectable vasopressin-circulating levels. Hyponatremia can be quite severe in affected male children. To gain a deeper insight into the functional properties of the V2R active mutants and how they might translate into the pathological outcome of NSIAD, in this study, we have expressed the wild-type V2R and three constitutively active V2R mutants associated with NSIAD (R137L, R137C, and the F229V) in MCD4 cells, a cell line derived from renal mouse collecting duct, stably expressing the vasopressin-sensitive water channel aquaporin-2 (AQP2). Our findings indicate that in cells expressing each active mutant, AQP2 was constitutively localized to the apical plasma membrane in the absence of vasopressin stimulation. In line with these observations, under basal conditions, osmotic water permeability in cells expressing the constitutively active mutants was significantly higher compared to that of cells expressing the wild-type V2R. Our findings demonstrate a direct link between activating mutations of the V2R and the perturbation of water balance in NSIAD. In addition, this study provides a useful cell-based assay system to assess the functional consequences of newly discovered activating mutations of the V2R on water permeability in kidney cells and to screen the effect of drugs on the mutated receptors.
Tolvaptan, a selective vasopressin V2 receptor antagonist, is a new generation diuretic. Its clinical efficacy is in principle due to impaired vasopressin‐regulated water reabsorption via aquaporin‐2 (AQP2). Nevertheless, no direct in vitro evidence that tolvaptan prevents AQP2‐mediated water transport, nor that this pathway is targeted in vivo in patients with syndrome of inappropriate antidiuresis (SIAD) has been provided. The effects of tolvaptan on the vasopressin–cAMP/PKA signalling cascade were investigated in MDCK cells expressing endogenous V2R and in mouse kidney. In MDCK, tolvaptan prevented dDAVP‐induced increase in ser256‐AQP2 and osmotic water permeability. A similar effect on ser256‐AQP2 was found in V1aR −/− mice, thus confirming the V2R selectively. Of note, calcium calibration in MDCK showed that tolvaptan per se caused calcium mobilization from the endoplasmic reticulum resulting in a significant increase in basal intracellular calcium. This effect was only observed in cells expressing the V2R, indicating that it requires the tolvaptan–V2R interaction. Consistent with this finding, tolvaptan partially reduced the increase in ser256‐AQP2 and the water permeability in response to forskolin, a direct activator of adenylyl cyclase (AC), suggesting that the increase in intracellular calcium is associated with an inhibition of the calcium‐inhibitable AC type VI. Furthermore, tolvaptan treatment reduced AQP2 excretion in two SIAD patients and normalized plasma sodium concentration. These data represent the first detailed demonstration of the central role of AQP2 blockade in the aquaretic effect of tolvaptan and underscore a novel effect in raising intracellular calcium that can be of significant clinical relevance.
Healthful dietary patterns and bioactive compounds supplementation can be adopted as simple and easy intervention to prevent, attenuate or cure clinical disorders, especially when it comes degenerative and chronic diseases. In the recent years, a growing body of evidence indicates Aquaporins (AQPs), a family of membrane channel proteins widely expressed in the human body, among the targets underlying the beneficial action played by some food nutrients and phytochemical compounds. Here, we provide an overview of what is known regarding the AQP modulation exerted by healthful dietary patterns and plant polyphenols.
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