Vasopressin regulates water reabsorption in renal collecting duct principal cells by a cAMP-dependent translocation of the water channel aquaporin-2 (AQP2) from intracellular vesicles into the cell membrane. In the present work primary cultured inner medullary collecting duct cells were used to study the role of the proteins of the Rho family in the translocation of AQP2. Clostridium difficile toxin B, which inhibits all members of the Rho family, Clostridium limosum C3 toxin, which inactivates only Rho, and the Rho kinase inhibitor, Y-27632, induced both depolymerization of actin stress fibers and AQP2 translocation in the absence of vasopressin. The data suggest an inhibitory role of Rho in this process, whereby constitutive membrane localization is prevented in resting cells. Expression of constitutively active RhoA induced formation of actin stress fibers and abolished AQP2 translocation in response to elevation of intracellular cAMP, confirming the inhibitory role of Rho. Cytochalasin D induced both depolymerization of the F-actin cytoskeleton and AQP2 translocation, indicating that depolymerization of F-actin is sufficient to induce AQP2 translocation. Thus Rho is likely to control the intracellular localization of AQP2 via regulation of the F-actin cytoskeleton.The antidiuretic hormone arginine-vasopressin (AVP) 1 regulates water reabsorption in renal collecting duct principal cells by inducing the translocation of the water channel aquaporin-2 (AQP2) from intracellular vesicles primarily into the apical cell membrane (shuttle hypothesis; Refs. 1 and 2). The molecular targets of AVP on the surface of principal cells are heptahelical vasopressin V2 receptors coupled to the G s /adenylyl cyclase system. Activation of this system by the hormone raises the level of intracellular cAMP and results in the activation of protein kinase A (PKA) which then phosphorylates its substrates, one of which is AQP2.The phosphorylation of AQP2 by PKA and also the anchoring of PKA to subcellular compartments via protein kinase A anchoring proteins are prerequisites for AQP2 translocation to the cell membrane (2-5). In addition, the involvement of a heterotrimeric G protein of the G i family in the AQP2 translocation has been demonstrated in CD8 cells (6).The cytoskeleton consists of various components, including microtubules and F-actin, both of which are involved in AVPmediated changes of osmotic water permeability (2, 7-9). Microtubule-disrupting drugs like colchicine and nocodazole inhibit AVP-mediated increases in osmotic water permeability in renal collecting ducts by 65 and 72%, respectively (10 -13). Disruption of the F-actin cytoskeleton by cytochalasin B or dihydrocytochalasin B inhibits the AVP-induced increase in osmotic water permeability in toad bladder epithelium by 25-50% (13, 14). The F-actin cytoskeleton also undergoes rearrangements after stimulation of cells with cAMP-elevating agents. After stimulation with vasopressin, total F-actin decreases in toad bladders by 20 -30% (15) and apical F-actin in rat coll...

The antidiuretic hormone arginine-vasopressin (AVP) regulates water reabsorption in renal collecting duct principal cells by inducing a cAMP-dependent translocation of water channels (aquaporin-2, AQP-2) from intracellular vesicles into the apical cell membranes. In subcellular fractions from primary cultured rat inner medullary collecting duct (IMCD) cells, enriched for intracellular AQP-2-bearing vesicles, catalytic protein kinase A (PKA) subunits and several protein kinase A anchoring proteins (AKAPs) were detected. In nonstimulated IMCD cells the majority of AQP-2 staining was detected intracellularly but became mainly localized within the cell membrane after stimulation with AVP or forskolin. Quantitative analysis revealed that preincubation of the cells with the synthetic peptide S-Ht31, which prevents the binding between AKAPs and regulatory subunits of PKA, strongly inhibited AQP-2 translocation in response to forskolin. Preincubation of the cells with the PKA inhibitor H89 prior to forskolin stimulation abolished AQP-2 translocation. In contrast to H89, S-Ht31 did not affect the catalytic activity of PKA. These data demonstrate that not only the activity of PKA, but also its tethering to subcellular compartments, are prerequisites for cAMP-dependent AQP-2 translocation.The antidiuretic action of arginine-vasopressin (AVP) 1 is mediated by renal collecting duct principal cells. The water channel aquaporin-2 (AQP-2), which is exclusively expressed in principal cells, belongs to a family of water channel proteins (aquaporins), which greatly increase the osmotic water permeability of biological membranes (1, 2). In resting principal cells AQP-2 is localized on intracellular vesicles. The antidiuretic hormone AVP causes its redistribution from the intracellular compartment to the apical cell membrane and thereby facilitates water reabsorption (3, 4). Mutations in the genes coding for the vasopressin V2 receptor or AQP-2 cause nephrogenic diabetes insipidus, a disease characterized by a massive loss of water through the kidney (5-9).The binding of AVP to vasopressin V2 receptors (10) on principal cells stimulates cAMP synthesis via the G s /adenylyl cyclase system. Cyclic AMP activates protein kinase A (PKA), which in turn phosphorylates AQP-2 at 12). The functional consequence of this modification is not clear: Kuwahara et al. (11) found a minor increase in AQP-2 water permeability in oocytes injected with AQP-2 mRNA, while Lande et al. (13) did not find an increased water permeability in isolated AQP-2-bearing vesicles after phosphorylation. Elimination of the PKA phosphorylation site of AQP-2 or incubation with the PKA inhibitor H89 prevents AQP-2 translocation in LLC-PK1 cells stably transfected with the water channel (14). These data indicate that the phosphorylation of AQP-2 is important for its subcellular localization rather than for its function. The finding that isolated AQP-2-bearing vesicles contain endogenous PKA activity (13) led to the premise that PKA might be anchored to the vesicle membra...

. 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...

Cultured renal epithelial cells rapidly downregulate expression of the vasopressin-regulated water channel aquaporin-2 (AQP-2). Our aim was to define conditions that favor maintenance of AQP-2 expression in vitro without genetic manipulation. We show here that primary cultures of rat inner medullary collecting duct (IMCD) cells retain AQP-2 expression for at least 6 days when grown with dibutyryl cAMP (DBcAMP) supplementation. We also found that coating the culture dishes with type IV collagen, rather than rat-tail collagen, retards AQP-2 downregulation. Immunofluorescence and biochemical studies indicate a shuttling of AQP-2-bearing vesicles after stimulation with vasopressin or forskolin. Rab3 proteins, known to be involved in regulated exocytosis, were detected only in cells grown in the presence of DBcAMP. Using the adenylyl cyclase assay, we confirmed the functional integrity of the vasopressin V2receptor in a broken cell preparation. Our data show that cAMP supplementation is sufficient for the maintenance of AQP-2 expression in primary cultured cells. The model system established here allows the study of the regulation of genes encoding the antidiuretic machinery at the cellular level.

. Osmolality and solute composition are strong regulators of AQP2 expression in renal principal cells. Am J Physiol Renal Physiol 284: F189-F198, 2003. First published August 13, 2002; 10.1152/ajprenal.00245.2002The water permeability of the renal collecting duct is regulated by the insertion of aquaporin-2 (AQP2) into the apical plasma membrane of epithelial (principal) cells. Using primary cultured epithelial cells from the inner medulla of rat kidney (IMCD cells), we show that osmolality and solute composition are potent regulators of AQP2 mRNA and protein synthesis, as well as the classical cAMP-dependent pathway, but do not affect the arginine vasopressin-induced AQP2 shuttle. In the presence of the cAMP analog dibutyryl cAMP (DBcAMP, 500 M), NaCl and sorbitol, but not urea, evoked a robust increase of AQP2 expression in IMCD cells, with NaCl being far more potent than sorbitol. cAMP-responsive elementbinding protein phosphorylation increased with DBcAMP concentrations but was not altered by changes in osmolality. In the rat and human AQP2 promoter, we identified a putative tonicity-responsive element. We conclude that, in addition to the arginine vasopressin/cAMP-signaling cascade, a further pathway activated by elevated effective osmolality (tonicity) is crucial for the expression of AQP2 in IMCD cells, and we suggest that the effect is mediated via the tonicityresponsive element. osmolality; aquaporin-2; kidney; gene regulation; toxicity responsive enhancer THE WATER CHANNEL aquaporin-2 (AQP2), expressed in epithelial (principal) cells of renal collecting ducts, is required for the vasopressin-dependent concentration of urine (7). AQP2 abundance increases from the kidney cortex to the inner region of the kidney medulla (29), as does osmolality. The water permeability of the inner medullary collecting duct (IMCD) is rapidly regulated (within minutes) by the antidiuretic hormone arginine vasopressin (AVP), which binds to heptahelical vasopressin V 2 receptors (V 2 R), located mainly in the basolateral plasma membrane of principal cells. Activation of the V 2 R causes stimulation of adenylyl cyclase via the G protein G S , leading to elevation of cAMP. The subsequent activation of protein kinase A (PKA) initiates the translocation of AQP2-bearing vesicles from the cytosol to the plasma membrane, in which AQP2 is inserted by an exocytosis-like process (short-term regulation) (D. Lorenz, A. Krylov, V. Hagen, J. Zipper, W. Rosenthal, P. Pohl, and K. Maric, unpublished observations; 30). In addition, the signaling cascade described above governs the expression of AQP2 (long-term regulation) by PKA-mediated phosphorylation of the transcription factor cAMP-responsive element (CRE)-binding protein (CREB) (13,16). Given the fact that AQP2 biosynthesis is usually shut off shortly after IMCD cells are established in primary culture (10), most studies on AQP2 long-term regulation have been performed using animal models (29, 37). We recently established primary cultured IMCD cells as a model system (17,18,21,22). Thes...