Concentrating urine is mandatory for most mammals to prevent water loss from the body. Concentrated urine is produced in response to vasopressin by the transepithelial recovery of water from the lumen of the kidney collecting tubule through highly water-permeable membranes. In this nephron segment, vasopressin regulates water permeability by endo- and exocytosis of water channels from or to the apical membrane. CHIP28 is a water channel in red blood cells and the kidney proximal tubule, but it is not expressed in the collecting tubule. Here we report the cloning of the complementary DNA for WCH-CD, a water channel of the apical membrane of the kidney collecting tubule. WCH-CD is 42% identical in amino-acid sequence to CHIP28. WCH-CD transcripts are detected only in the collecting tubule of the kidney. Immunohistochemically, WCH-CD is localized to the apical region of the kidney collecting tubule cells. Expression of WCH-CD in Xenopus oocytes markedly increases osmotic water permeability. The functional expression and the limited localization of WCH-CD to the apical region of the kidney collecting tubule suggest that WCH-CD is the vasopressin-regulated water channel.

Water transport in highly water-permeable membranes is conducted by water-selective pores-namely, water channels. The recent cloning of water channels revealed the water-selective characteristics of these proteins when expressed in Xenopus oocytes or reconstituted in liposomes. Currently, It is as d that the function of water ch ls is to transport only water. We now report the cloning of a member of the water channel that also transports nonionic small molecules such as urea and glycerol. We named this channel aquaporin 3 (AQP3) for its predominant water permeabilit. AQP3 has amino acid sequence Identity with major intrinsic protein (MIP) family proteins including AQPchannel-forming integral membrane protein, AQP-colecting duct, MIP, AQP-ytonoplast intrinsic protein, nodulin 26, and glycerol facilitator (33-42%). Thus, AQP3 is an additional member of the MEP family. Osmotic water permeability of Xenopus oocytes measured by videomicroscopy was 10-fold higher in oocytes injected with AQP3 transcript than with water-injected oocytes. The increase in osmotic water permeability was inhibited by HgC12, and this effect was reversed by a reducing agent, 2-mercaptoethanol. Although to a smaller degree, AQP3 also facilitated the transport of nonionic small solutes such as urea and glycerol, while the previously cloned water channels are permeable only to water when expressed in Xenopus oocytes. AQP3 mRNA was expressed abundantly in kidney medulla and colon. In kidney, it was exclusively immunokoalzed at the baolateral membrane of collecting duct cells. AQP3 may functon as a water and urea exit hanism in antidlure in ollecting duct cells.Water channels have been postulated for the pathway of selective water permeation in highly water-permeable membranes. Recent

The aquaporin-2 (AQP2) vasopressin water channel is translocated to the apical membrane upon vasopressin stimulation. Phosphorylation of serine 256 of AQP2 by cAMP-dependent protein kinase has been shown, but its relation to vasopressin-regulated translocation has not been elucidated. To address this question, wild type (WT) AQP2 and a mutant with alanine in place of serine 256 of AQP2 (S256A) were expressed in LLC-PK1 cells by electroporation. Measurements by a stopped-flow lightscattering method revealed that the osmotic water permeability (P f ) of LLC-PK1 cells transfected with WT was 69.6 ؎ 6.5 m/s (24.8 ؎ 2.2 m/s for mock-transfected), and stimulation by 500 M 8-(4-chlorophenylthio)-cAMP increased the P f by 85 ؎ 12%. When S256A AQP2 was transfected, the cAMP-dependent increase in the P f was only 8 ؎ 5%. After cAMP stimulation, the increase in surface expression of AQP2 determined by surface biotin labeling was 4 ؎ 10%, significantly less than that for WT (88 ؎ 5%). In addition, an in vivo [ 32 P]orthophosphate labeling assay demonstrated significant phosphorylation of WT AQP2 and only minimal phosphorylation of S256A AQP2 in LLC-PK1 cells. Our results indicated that serine 256 of AQP2 is necessary for regulatory exocytosis and that cAMP-responsive redistribution of AQP2 may be regulated by phosphorylation of AQP2.The urine concentration system in the kidney is regulated mainly by vasopressin, an antidiuretic hormone that increases the osmotic water permeability of the collecting duct cells, resulting in bulk reabsorption of free water (1-3). The cellular actions of vasopressin in the collecting duct cell have been partially resolved since the identification of the vasopressin water channel aquaporin-2 (AQP2) 1 (4, 5). Vasopressin has been shown to induce regulatory redistribution of AQP2 from endosomal compartments to the apical membrane (6 -8). These observations have directly proved the shuttle hypothesis that exo-and endocytosis of water channel-containing vesicles account for the regulatory effects of vasopressin on water permeability of the collecting duct cells (9, 10).Despite elucidation of the regulatory translocation of AQP2, the potential interactions between vasopressin-induced intracellular cAMP accumulation and AQP2 trafficking remain unknown. It has been shown that cAMP-dependent phosphorylation of AQP2 expressed in the Xenopus oocyte slightly increased osmotic water permeability without changing AQP2 surface expression (11), but the increase was far too small to account for the dramatic increase in water permeability of collecting duct cells. Many channels have been shown to be functionally regulated by their phosphorylation (12), but the direct regulation of AQP2 channel functions through phosphorylation is minimal at best and is still controversial (13). On the other hand, the physiological significance of regulatory effects on AQP2 trafficking has been emphasized. Regulatory delivery and sorting of membrane proteins have been observed in many cell types (14, 15). Many vesicle-associated...

Aquaporin-2 is detectable in the urine, and changes in the urinary excretion of this protein can be used as an index of the action of vasopressin on the kidney.

Abstract. Information about the rheological characteristics of the aqueous cytoplasm can be provided by analysis of the rotational motion of small polar molecules introduced into the cell. To determine fluidphase cytoplasmic viscosity in intact cells, a polarization microscope was constructed for measurement of picosecond anisotropy decay of fluorescent probes in the cell cytoplasm. We found that the rotational correlation time (to) of the probes, 2,7-bis-(2-carboxyethyl)-5-(and-6-)carboxyfluorescein (BCECF), 6-carboxyfluorescein, and 8-hydroxypyrene-l,3,6-trisulfonic acid (HPTS) provided a direct measure of fluid-phase cytoplasmic viscosity that was independent of probe binding. In quiescent Swiss 3T3 fibroblasts, tc values were 20-40% longer than those in water, indicating that the fluid-phase cytoplasm is only 1.2-1.4 times as viscous as water. The activation energy of fluid-phase cytoplasmic viscosity was 4 kcal/mol, which is similar to that of water. Fluid-phase cytoplasmic viscosity was altered by <10% upon addition of sucrose to decrease cell volume, cytochalasin B to disrupt cell cytoskeleton, and vasopressin to activate phospholipase C. Nucleoplasmic and peripheral cytoplasmic viscosities were not different. Our results establish a novel method to measure fluid-phase cytoplasmic viscosity, and indicate that fluid-phase cytoplasmic viscosity in fibroblasts is similar to that of free water.

Our analyses show that all of the ICD-10 versions of the Charlson algorithm performed satisfactorily (c-statistics 0.70-0.86), with the Quan version showing a trend toward outperforming the other versions in all data sets.

The effect of vasopressin on subcellular localization of AQP-CD and AQP3 water channels was examined in thirsted Brattleboro rats by immunohistochemistry and immunoelectron microscopy. AQP-CD was mainly present in the cytoplasm of the collecting duct cells in association with cytoplasmic vesicles but was sparse in the apical membrane in control vehicle-injected rats. In rats given vasopressin 15 min before death, the number of immunogold particles for AQP-CD in the apical membrane increased significantly (P < 0.002) from 1.8 +/- 0.2 to 10.0 +/- 0.4/microns with a significant decrease (P < 0.05) of cytoplasmic labeling from 32.6 +/- 6.4 to 24.6 +/- 5.6/microns 2, indicating that AQP-CD is the vasopressin-regulated water channel predicted by the "shuttle" hypothesis. In contrast, AQP3 was restricted to the basolateral membrane of the collecting duct cells, and the labeling density of AQP3 was unchanged by vasopressin treatment, indicating that AQP3 is constitutively expressed and may maintain high water permeability of the basolateral membrane.

Among water channel proteins (aquaporins), aquaporin-collecting duct (AQP-CD) is the vasopressin-regulated water channel. Vasopressin causes cAMP production in the renal collecting duct cells, and this is believed to lead to exocytic insertion of water channel into the apical membrane (shuttle hypothesis). AQP-CD contains a consensus sequence for cAMP-dependent protein kinase, residues at positions 253-256 (Arg-Arg-Gln-Ser). To determine the role of this site, Ser-256 was substituted for Ala, Leu, Thr, Asp, or Glu by site-directed mutagenesis. In Xenopus oocytes injected with wild-type or mutated AQP-CD cRNAs, osmotic water permeability (Pf) was 4.8-7.7 times higher than Pf of water-injected oocytes. Incubation with cAMP plus forskolin or direct cAMP injection into the oocytes increased Pf of wild-type, but not mutated, AQP-CD-expressing oocytes, whereas the amounts of AQP-CD expression were similar in wild and mutated types as identified by Western blot analysis. In vitro phosphorylation studies of AQP-CD proteins expressed in oocyte showed that cAMP-dependent protein kinase phosphorylated wild-type, but not mutated, AQP-CD proteins. Phosphoamino acid analysis revealed that this phosphorylation occurred at the serine residue. Moreover, phosphorylation of AQP-CD protein in intact rat kidney medulla tissues was stimulated by incubation with cAMP. Our data suggest that cAMP stimulates water permeability of AQP-CD by phosphorylation. This process may contribute to the vasopressin-regulated water permeability of collecting duct in addition to the apical insertion of AQP-CD by exocytosis.