[Lentivirus-mediated siRNA targeting sae1 induces cell cycle arrest and apop- tosis in colon cancer cell RKO].

Deregulated expression of proteins involved in the SUMOylation pathway has been detected in several tumors. SUMO1-activating enzyme subunit 1 (SAE1) plays an important role in this process. We found that SAE1 was highly expressed in the colon cancer cell line RKO and used lentivirus-mediated siRNA to suppress SAE1 expressionin RKO cells. RNA-interference efficiently and specifically downregulated the target gene expression in RKO on both mRNA and protein levels. Silencing of SAE1 inhibited proliferation and reduced colony formation of RKO cells. Furthermore, as it has been shown by flow cytometry analysis, specific knockdown of SAE1 slowed down the cell population at G0/G1 phase and induced apoptosis of RKO cells. On the base of results obtained, one can suppose the biological significance of SAE1 in colon tumorigenesis and a use of this protein as a novel molecular target for colon cancer therapy.

[Epidemiological characteristics of adenovirus infection in hospitalized children with acute respiratory tract infection in Kunming during 2019].

Objective: To investigate the epidemiology and clinical characteristics of adenovirus (ADV)-caused acute respiratory tract infection among hospitalized children in Kunming, China. Methods: Clinical and laboratory data were collected from 467 children with adenovirus infection who were hospitalized from January 1, 2019 to December 31, 2019 in 6 grade A class Ⅲ hospitals in Kunming area. The basic characteristics, epidemiology, mixed infection and adenovirus genotypes of the patients were retrospectively analyzed. The patients diagnosed with adenovirus pneumonia (AP) were divided into two groups, severe AP (SAP) group and general AP(GAP) group according to the severity of illness. Mann-Whitney U test or χ2 test was used for comparison between groups, while multivariate regression was applied to analyze the risk factors of SAP. Results: Among 15 635 hospitalized children with respiratory tract infection, 467 cases were adenovirus positive, with a detection rate of 2.99%. Of the 467 patients with adenovirus infection, 284 were male and 183 female, the age was 2.4 (1.1,3.9) years, including 44 cases (9.4%) < 0.5 years, 59 cases (12.6%) of 0.5 to<1.0 years, 176 cases (37.7%) of 1.0 to <3.0 years, 150 cases (32.1%) of 3.0 to <7.0 years, and 38 cases (8.1%) of 7.0 to 14.0 years. Adenovirus infection was common in autumn and winter, and the high incidence months were October to December, which accounted for 51.6% (241/467) of the whole year cases. Co-infection was detected in 226 cases (48.4%) out of 467 patients, in which one pathogen co-infection was the most frequent form (172 cases, 76.1%). Of the 262 pathogen detected 108 (41.2%) were Mycoplasma pneumoniae. In 144 of ADV-positve cases (30.8%) were taken geno-typing was done by PCR amplification, the results showed that 74 cases (51.4%) were ADV 3, 7 subtypes and 65 cases (45.1%) of ADV 1, 2,6 subtypes. Of the 467 cases of ADV infection, 320 (68.5%) were diagnosed with pneumonia, 82 (17.6%) with upper respiratory tract infection and pharyngeal tonsillitis, and 65 (13.9%) with bronchitis, laryngeal bronchitis, and asthmatic bronchitis. Among the 320 patients with AP, 56 cases were severe and 264 cases were general. Two cases (3.6%) in severe group died. Compared with the GAP group, the age was young [17 (11,42) months vs. 24 (14,44) months, Z=2.222, P=0.026], the fever duration was long [8 (5,14) days vs. 6 (3,9) days, Z=3.380, P<0.01], and the proportions of preterm birth and having underlying diseases were high [respectively 19.6% (11/56) vs. 6.1% (16/264), 26.8% (15/56) vs. 10.2% (27/264), χ2=8.965,11.109, P<0.05] in SAP group. Referring to laboratory markers, white blood cell count, C-reactive protein, creatine kinase-MB and lactate dehydrogenase were significantly increased in SAP group as compared to GAP group(all P<0.05). Multivariate Logistic regression analysis showed that preterm birth (OR=3.284, 95%CI 1.079-9.993, P=0.036), underlying disease (OR=3.284, 95%CI 1.079-9.993, P=0.036), fever duration ≥10 d (OR=2.523,95%CI 1.195-5.328, P=0.015) and C-reactive protein ≥50 mg/L (OR=3.156, 95%CI 1.324-7.524, P=0.010) were positively correlated with the risk of SAP. Conclusions: The incidence of adenovirus infection among hospitalized children in Kunming was lower than the national level, and no outbreak occurred in 2019. Subtype 3 and 7 of ADV are the predominant strains for infection, which usually occurs in autumn and winter and mainly causes pneumonia. Premature birth, underlining diseases, long fever duration and markedly increased C-reactive protein are the risk factors for developing into severe pneumonia. This paper presents the prevalence and clinical characteristics of adenovirus infection in children at high altitude area.

Apical endosomes isolated from kidney collecting duct principal cells lack subunits of the proton pumping ATPase.

Endocytic vesicles that are involved in the vasopressin-stimulated recycling of water channels to and from the apical membrane of kidney collecting duct principal cells were isolated from rat renal papilla by differential and Percoll density gradient centrifugation. Fluorescence quenching measurements showed that the isolated vesicles maintained a high, HgCl2-sensitive water permeability, consistent with the presence of vasopressin-sensitive water channels. They did not, however, exhibit ATP-dependent luminal acidification, nor any N-ethylmaleimide-sensitive ATPase activity, properties that are characteristic of most acidic endosomal compartments. Western blotting with specific antibodies showed that the 31- and 70-kD cytoplasmically oriented subunits of the vacuolar proton pump were not detectable in these apical endosomes from the papilla, whereas they were present in endosomes prepared in parallel from the cortex. In contrast, the 56-kD subunit of the proton pump was abundant in papillary endosomes, and was localized at the apical pole of principal cells by immunocytochemistry. Finally, an antibody that recognizes the 16-kD transmembrane subunit of oat tonoplast ATPase cross-reacted with a distinct 16-kD band in cortical endosomes, but no 16-kD band was detectable in endosomes from the papilla. This antibody also recognized a 16-kD band in affinity-purified H+ ATPase preparations from bovine kidney medulla. Therefore, early endosomes derived from the apical plasma membrane of collecting duct principal cells fail to acidify because they lack functionally important subunits of a vacuolar-type proton pumping ATPase, including the 16-kD transmembrane domain that serves as the proton-conducting channel, and the 70-kD cytoplasmic subunit that contains the ATPase catalytic site. This specialized, non-acidic early endosomal compartment appears to be involved primarily in the hormonally induced recycling of water channels to and from the apical plasma membrane of vasopressin-sensitive cells in the kidney collecting duct.

Biogenesis and transmembrane topology of the CHIP28 water channel at the endoplasmic reticulum.

CHIP28 is a 28-kD hydrophobic integral membrane protein that functions as a water channel in erythrocytes and renal tubule epithelial cell membranes. We examined the transmembrane topology of CHIP28 in the ER by engineering a reporter of translocation (derived from bovine prolactin) into nine sequential sites in the CHIP28 coding region. The resulting chimeras were expressed in Xenopus oocytes, and the topology of the reporter with respect to the ER membrane was determined by protease sensitivity. We found that although hydropathy analysis predicted up to seven potential transmembrane regions, CHIP28 spanned the membrane only four times. Two putative transmembrane helices, residues 52-68 and 143-157, reside on the lumenal and cytosolic surfaces of the ER membrane, respectively. Topology derived from these chimeric proteins was supported by cell-free translation of five truncated CHIP28 cDNAs, by N-linked glycosylation at an engineered consensus site in native CHIP28 (residue His69), and by epitope tagging of the CHIP28 amino terminus. Defined protein chimeras were used to identify internal sequences that direct events of CHIP28 topogenesis. A signal sequence located within the first 52 residues initiated nascent chain translocation into the ER lumen. A stop transfer sequence located in the hydrophobic region from residues 90-120 terminated ongoing translocation. A second internal signal sequence, residues 155-186, reinitiated translocation of a COOH-terminal domain (residues 186-210) into the ER lumen. Integration of the nascent chain into the ER membrane occurred after synthesis of 107 residues and required the presence of two membrane-spanning regions. From this data, we propose a structural model for CHIP28 at the ER membrane in which four membrane-spanning alpha-helices form a central aqueous channel through the lipid bilayer and create a pathway for water transport.

Transcellular water flow modulates water channel exocytosis and endocytosis in kidney collecting tubule.

The regulation of osmotic water permeability (Pf) by vasopressin (VP) in kidney collecting tubule involves the exocytic-endocytic trafficking of vesicles containing water channels between an intracellular compartment and apical plasma membrane. To examine effects of transcellular water flow on vesicle movement, Pf was measured with 1-s time resolution in the isolated perfused rabbit cortical collecting tubule in response to addition and removal of VP (250 microU/ml) in the presence of bath greater than lumen (B greater than L), lumen greater than bath (L greater than B), and lumen = bath (L = B) osmolalities. With VP addition, Pf increased from 12 to 240-270 x 10(-4) cm/s (37 degrees C) in 10 min. At 1 min, Pf was approximately 70 x 10(-4) cm/s for B greater than L, L greater than B, and L = B conditions. At later times, Pf increased fastest for L greater than B and slowest for B greater than L osmolalities; at 5 min, Pf was 250 x 10(-4) cm/s (L greater than B) and 158 x 10(-4) cm/s (B greater than L). With VP removal, Pf returned to pre-VP levels at the fastest rate for B greater than L and the slowest rate for L greater than B osmolalities; at 30 min, Pf was 65 x 10(-4) cm/s (B greater than L) and 183 x 10(-4) cm/s (L greater than B). For a series of osmotic gradients of different magnitudes and directions, the rates of Pf increase and decrease were dependent upon the magnitude of transcellular volume flow; control studies showed that paracellular water flux, asymmetric transcellular water pathways, or changes in cell volume could not account for the data. VP-dependent endocytosis was measured by apical uptake of rhodamine-dextran; in paired studies where the same tubule was used for + and - gradients, B greater than L and L greater than B osmolalities gave 168% and 82% of uptake measured with no gradient. In contrast, endocytosis in proximal tubule was not dependent on gradient direction. These data provide evidence that transcellular volume flow modulates the vasopressin-dependent cycling of vesicles containing water channels, suggesting a novel driving mechanism to aid or oppose the targeted, hormonally directed movement of subcellular membranes.

Proteinases inhibit H(+)-ATPase and Na+/H+ exchange but not water transport in apical and endosomal membranes from rat proximal tubule.

A marked increase in water permeability can be induced in Xenopus oocytes by injection of mRNA from tissues that express water channels, suggesting that the water channel is a protein. In view of this and previous reports which showed that proteinases may interfere with mercurial inhibition of water transport in red blood cells (RBC), we examined the influence of trypsin, chymotrypsin, papain, pronase, subtilisin and thermolysin on water permeability as well as on ATPase activity, H(+)-pump, passive H+ conductance, and Na+/H+ exchange in apical brush-border vesicles (BBMV) and endosomal (EV) vesicles from rat renal cortex. H+ transport was measured by Acridine orange fluorescence quenching and water transport by stopped-flow light scattering. As measured by potential-driven H+ accumulation in BBMV and EV, proteinase treatment had little effect on vesicle integrity. In BBMV, ecto-ATPase activity was inhibited by 15-30%, Na+/H+ exchange by 20-55%, and H+ conductance was unchanged. Osmotic water permeability (Pf) was 570 microns/s and was inhibited 85-90% by 0.6 mM HgCl2; proteinase treatment did not affect Pf or the HgCl2 inhibition. In EV, NEM-sensitive H+ accumulation and ATPase activity were inhibited by greater than 95%. Pf (140 microns/s) and HgCl2 inhibition (75-85%) were not influenced by proteinase treatment. SDS-PAGE showed selective digestion of multiple polypeptides by proteinases. These results confirm the presence of water channels in BBMV and EV and demonstrate selective inhibition of ATPase function and Na+/H+ exchange by proteinase digestion. The lack of effect of proteinases on water transport by mercurials. We conclude that the water channel may be a small integral membrane protein which, unlike the H(+)-ATPase and Na+/H+ exchanger, has no functionally important membrane domains that are sensitive to proteolysis.

Very high water permeability in vasopressin-induced endocytic vesicles from toad urinary bladder.

The regulation of transepithelial water permeability in toad urinary bladder is believed to involve a cycling of endocytic vesicles containing water transporters between an intracellular compartment and the cell luminal membrane. Endocytic vesicles arising from luminal membrane were labeled selectively in the intact toad bladder with the impermeant fluid-phase markers 6-carboxyfluorescein (6CF) or fluorescein-dextran. A microsomal preparation containing labeled endocytic vesicles was prepared by cell scraping, homogenization, and differential centrifugation. Osmotic water permeability was measured by a stopped-flow fluorescence technique in which microsomes containing 50 mM mannitol, 5 mM K phosphate, pH 8.5 were subject to a 60-mM inwardly directed gradient of sucrose; the time course of endosome volume, representing osmotic water transport, was inferred from the time course of fluorescence self-quenching. Endocytic vesicles were prepared from toad bladders with hypoosmotic lumen solution treated with (group A) or without (group B) serosal vasopressin at 23 degrees C, and bladders in which endocytosis was inhibited by treatment with vasopressin at 0-2 degrees C (group C), or with vasopressin plus sodium azide at 23 degrees C (group D). Stopped-flow results in all four groups showed a slow rate of 6CF fluorescence decrease (time constants 1.0-1.7 s for exponential fit) indicating a component of nonendocytic 6CF entrapment into sealed vesicles. However, in vesicles from group A only, there was a very rapid 6CF fluorescence decrease (time constant 9.6 +/- 0.2 ms, SEM, 18 separate preparations) with an osmotic water permeability coefficient (Pf) of greater than 0.1 cm/s (18 degrees C) and activation energy of 3.9 +/- 0.8 kcal/mol (16 kJ/mol). Pf was inhibited reversibly by greater than 60% by 1 mM HgCl2. The rapid fluorescence decrease was absent in vesicles in groups B, C, and D. These results demonstrate the presence of functional water transporters in vasopressin-induced endocytic vesicles from toad bladder, supporting the hypothesis that water channels are cycled to and from the luminal membrane and providing a functional marker for the vasopressin-sensitive water channel. The calculated Pf in the vasopressin-induced endocytic vesicles is the highest Pf reported for any biological or artificial membrane.

Solvent drag measurement of transcellular and basolateral membrane NaCl reflection coefficient in kidney proximal tubule.

The NaCl reflection coefficient in proximal tubule has important implications for the mechanisms of near isosmotic volume reabsorption. A new fluorescence method was developed and applied to measure the transepithelial (sigma NaClTE) and basolateral membrane (sigma NaClcl) NaCl reflection coefficients in the isolated proximal straight tubule from rabbit kidney. For sigma NaClTE measurement, tubules were perfused with buffers containing 0 Cl, the Cl-sensitive fluorescent indicator 6-methoxy-N-[3-sulfopropyl] quinolinium and a Cl-insensitive indicator fluorescein sulfonate, and bathed in buffers of differing cryoscopic osmolalities containing NaCl. The transepithelial Cl gradient along the length of the tubule was measured in the steady state by a quantitative ratio imaging technique. A mathematical model based on the Kedem-Katchalsky equations was developed to calculate the axial profile of [Cl] from tubule geometry, lumen flow, water (Pf) and NaCl (PNaCl) permeabilities, and sigma NaClTE. A fit of experimental results to the model gave PNaCl = (2.25 +/- 0.2) x 10(-5) cm/s and sigma NaClTE = 0.98 +/- 0.03 at 23 degrees C. For measurement of sigma NaClbl, tubule cells were loaded with SPQ in the absence of Cl. NaCl solvent drag was measured from the time course of NaCl influx in response to rapid (less than 1 s) Cl addition to the bath solution. With bath-to-cell cryoscopic osmotic gradients of 0, -60, and +30 mosmol, initial Cl influx was 1.23, 1.10, and 1.25 mM/s; a fit to a mathematical model gave sigma NaClbl = 0.97 +/- 0.04. These results indicate absence of NaCl solvent drag in rabbit proximal tubule. The implications of these findings for water and NaCl movement in proximal tubule are evaluated.

Water, proton, and urea transport in toad bladder endosomes that contain the vasopressin-sensitive water channel.

Vasopressin (VP) increases the water permeability of the toad urinary bladder epithelium by inducing the cycling of vesicles containing water channels to and from the apical membrane of granular cells. In this study, we have measured several functional characteristics of the endosomal vesicles that participate in this biological response to hormonal stimulation. The water, proton, and urea permeabilities of endosomes labeled in the intact bladder with fluorescent fluid-phase markers were measured. The diameter of isolated endosomes labeled with horse-radish peroxidase was 90-120 nm. Osmotic water permeability (Pf) was measured by a stopped-flow fluorescence quenching assay (Shi, L.-B., and A. S. Verkman. 1989. J. Gen. Physiol. 94:1101-1115). The number of endosomes formed when bladders were labeled in the absence of a transepithelial osmotic gradient increased with serosal [VP] (0-50 mU/ml), and endosome Pf was very high and constant (0.08-0.10 cm/s, 18 degrees C). When bladders were labeled in the presence of serosal-to-mucosal osmotic gradient, the number of functional water channels per endosome decreased (at [VP] = 0.5 mU/ml, Pf = 0.09 cm/s, 0 osmotic gradient; Pf = 0.02 cm/s, 180 mosmol gradient). Passive proton permeability was measured from the rate of pH decrease in voltage-clamped endosomes in response to a 1 pH unit gradient (pHin = 7.5, pHout = 6.5). The proton permeability coefficient (PH) was 0.051 cm/s at 18 degrees C in endosomes containing the VP-sensitive water channel; PH was not different from that measured in vesicles not containing water channels. Measurement of urea transport by the fluorescence quenching assay gave a urea reflection coefficient of 0.97 and a permeability coefficient of less than 10(-6) cm/s. These results demonstrate: (a) VP-induced endosomes from toad urinary bladder have extremely high Pf. (b) In states of submaximal bladder Pf, the density of functional water channels in endosomes in constant in the absence of an osmotic gradient, but decreases in the presence of a serosal-to-mucosal gradient, suggesting that the gradient has a direct effect on the efficiency of packaging of water channels into endosomes. (c) The VP-sensitive water channel does not have a high proton permeability. (d) Endosomes that cycle the water channel do not contain urea transporters. These results establish a labeling procedure in which greater than 85% of labeled vesicles from toad urinary bladder are endosomes that contain the VP-sensitive water channel in a functional form.

Functional colocalization of water channels and proton pumps in endosomes from kidney proximal tubule.

The apical membrane of mammalian proximal tubule undergoes rapid membrane cycling by exocytosis and endocytosis. Osmotic water and ATP-driven proton transport were measured in endocytic vesicles from rabbit and rat proximal tubule apical membrane labeled in vivo with the fluid phase marker fluorescein-dextran. Osmotic water permeability (Pf) was determined from the time course of fluorescein-dextran fluorescence after exposure of endosomes to an inward osmotic gradient in a stopped-flow apparatus. Pf was 0.009 (rabbit) and 0.029 cm/s (rat) (23 degrees C) and independent of osmotic gradient size. Pf in rabbit endosomes was inhibited reversibly by HgCl2 (KI = 0.2 mM) and had an activation energy of 6.4 +/- 0.5 kcal/mol (15-35 degrees C). Endosomal proton ATPase activity was measured from the time course of internal pH, measured by fluorescein-dextran fluorescence, after the addition of external ATP. Endosomes contained an ATP-driven proton pump that was sensitive to N-ethylmaleimide and insensitive to vanadate and oligomycin. In response to saturating [ATP] the pump acidified the endosomal compartment at a rate of 0.17 (rat) and 0.029 pH unit/s (rabbit); at an external pH of 7.4, the steady-state pH was 6.4 (rat) and 6.5 (rabbit). To examine whether water channels and the proton ATPase were present in the same endosome, the time course of fluorescein-dextran fluorescence was measured in response to an osmotic gradient in the presence and absence of ATP. ATP did not alter endosome Pf, but decreased the amplitude of the fluorescence signal by 43 +/- 3% (rabbit) and 47 +/- 4% (rat).(ABSTRACT TRUNCATED AT 250 WORDS)

Regulation of the formation and water permeability of endosomes from toad bladder granular cells.

Osmotic water permeability (Pf) in toad bladder is regulated by the vasopressin (VP)-dependent movement of vesicles containing water channels between the cytoplasm and apical membrane of granular cells. Apical endosomes formed in the presence of serosal VP have the highest Pf of any biological or artificial membrane (Shi and Verkman. 1989. J. Gen. Physiol. 94:1101-1115). We examine here: (a) the influence of protein kinase A and C effectors on transepithelial Pf (Pfte) in intact bladders and on the number and Pf of labeled endosomes, and (b) whether endosome Pf can be modified physically or biochemically. In paired hemibladder studies, Pfte induced by maximal serosal VP (50 mU/ml, 0.03 cm/s) was not different than that induced by 8-Br-cAMP (1 mM), forskolin (50 microM), VP + 8-Br-cAMP, or VP + forskolin. Pf was measured in endosomes labeled in intact bladders with carboxyfluorescein by a stopped-flow, fluorescence-quenching assay using an isolated microsomal suspension; the number and Pf (0.08-0.11 cm/s, 18 degrees C) of labeled endosomes was not different in bladders treated with VP, forskolin, and 8-Br-cAMP. Protein kinase C activation by 1 microM mucosal phorbol myristate acetate (PMA) induced submaximal bladder Pfte (0.015 cm/s) and endosome Pf (0.022 cm/s) in the absence of VP, but had little effect on maximal Pfte and endosome Pf induced by VP. However, PMA increased by threefold the number of apical endosomes with high Pf formed in response to serosal VP. Pf of endosomes containing the VP-sensitive water channel decreased fourfold by increasing membrane fluidity with hexanol or chloroform (0-75 mM); Pf of phosphatidylcholine liposomes (0.002 cm/s) increased 2.5-fold under the same conditions. Endosome Pf was mildly pH dependent, strongly inhibited by HgCl2, but not significantly altered by GTP gamma S, Ca, ATP + protein kinase A, and phosphatase action. We conclude that: (a) water channels cycled in endocytic vesicles are functional and not subject to physiological regulation, (b) VP and forskolin do not have cAMP-independent cellular actions, (c) activation of protein kinase C stimulates trafficking of water channels, but does not increase the number of apical membrane water channels induced by maximal VP, and (d) water channel function is sensitive to membrane fluidity. By using VP and PMA together, large quantities of endosomes containing the VP-sensitive water channel are labeled with fluid-phase endocytic markers.

Diagnosis and treatment of congenital choledochal cyst: 20 years' experience in China.

AIM: To summarize the experience of diagnosis and treatment of congenital choledochal cyst in the past 20 years (1980-2000). METHODS: The clinical data of 108 patients admitted from 1980 to 2000 were analyzed retrospectively. RESULTS: Abdominal pain,jaundice and abdominal mass were presented in most child cases. Clinical symptoms in adult cases were non-specific, resulting in delayed diagnosis frequently. Fifty-seven patients (52.7%) had coexistent pancreatiobiliary disease. Carcinoma of the biliary duct occurred in 18 patients (16.6%). Ultrasonic examination was undertaken in 94 cases, ERCP performed in 46 cases and CT in 71 cases. All of the cases were correctly diagnosed before operation. Abnormal pancreatobiliary duct junction was found in 39 patients. Before 1985 the diagnosis and classification of congenital choledochal cyst were established by ultrasonography preoperatively and confirmed during operation, the main procedures were internal drainage by cyst enterostomy. After 1985, the diagnosis was established by ERCP and CT, and cystectomy with Roux-en-Y hepaticojejunostomy was the conventional procedures.In 1994, we reported a new and simplified operative procedure in order to reduce the risk of choledochal cyst malignancy. Postoperative complication was mainly retrograde infection of biliary tract, which could be controlled by the administration of antibiotics, there was no perioperative mortality. CONCLUSION: The concept in diagnosis and treatment of congenital choledochal cyst has obviously been changed greatly.CT and ERCP were of great help in the classification of the disease.Currently, cystectomy with Roux-en-Y hepaticojejunostomy is strongly recommended as the choice for patients with type I and type IV cysts. Piggyback orthotopic liver transplantation is indicated in type V cysts (Caroli's disease) with frequently recurrent cholangitis.

[Characterization of proton pump and osmotic water transport in endocytic vesicles from rat kidney proximal tubule].

The characteristics of ATP-driven proton and osmotic water transport were studied in endocytic vesicles isolated from rat kidney proximal tubule labelled in vivo with fluorescein isothiocyanate-dextran (FITC-dextran). ATP-driven proton transport was measured from the time course of endosome pH following addition of external ATP. The rate of endosome acidification and the minimum pH were dependent on the ATP concentration. At an initial endosome pH of 7.4, the final pH values were 7.30, 6.99, 6.68, 6.38 and 6.39 at [ATP] = 0.005, 0.05, 0.5, 5 and 10 mmol/L, respectively. The acidification was inhibited by 97% at 0.5 mmol/L N-ethylmaleimide but was not affected by vanadate and oligomycin. Osmotic water permeability was determined in the same endosomes from the rapid kinetics of FITC-dextran fluorescence following an inward sucrose gradient. The osmotic water permeability coefficient was 0.03 cm/s at 23 degrees C. Water permeability was inhibited by 70% with addition of 0.5 mmol/L mercuric chloride. The inhibition was reversed completely by adding 5 mmol/L mercaptoethanol. These data demonstrate that proximal tubule endosomes contain a proton ATPase and water channel. The endocytic process may be important for regulation of acidification and fluid resorption in the proximal tubule.

Selected cysteine point mutations confer mercurial sensitivity to the mercurial-insensitive water channel MIWC/AQP-4.

The mercurial-insensitive water channel (MIWC or AQP-4) is a 30-32 kDA integral membrane protein expressed widely in fluid-transporting epithelia [Hasegawa et al. (1994) J. Biol. Chem. 269, 5497-5500]. To investigate the mercurial insensitivity and key residues involved in MIWC-mediated water transport, amino acids just proximal to the conserved NPA motifs (residues 69-74 and 187-190) were mutated individually to cysteine. Complementary RNAs were expressed in Xenopus oocytes for assay of osmotic water permeability (Pf) and HgCl2 inhibition dose-response. Oocytes expressing the cysteine mutants were highly water permeable, with Pf values (24-33 x 10(-3) cm/s) not different from that of wild-type (WT) MIWC. Pf was reversibly inhibited by HgCl2 in mutants S70C, G71C, G72C, H73C, and S189C but insensitive to HgCl2 in the other mutants. K1/2 values for 50% inhibition of Pf by HgCl2 were as follows (in millimolar): 0.40 (S70C), 0.36 (G71C), 0.14 (G72C), 0.45 (H73C), 0.24 (S189C), and > 1 for WT MIWC and the other mutants. To test the hypothesis that these residues are near the MIWC aqueous pore, residues 72 and 188 were mutated individually to the larger amino acid tryptophan. Pf in oocytes expressing mutants G72W or A188W (1.3-1.4 x 10(-3) cm/s) was not greater than that in water-injected oocytes even though these proteins were expressed at the oocyte plasma membrane as shown by quantitative immunofluorescence. Coinjection of cRNAs encoding WT MIWC and G72W or A188W indicated a dominant negative effect; Pf (x 10(-3) cm/s) was 22 (0.25 ng of WT), 10 (0.25 ng of WT + 0.25 ng of G72W), and 12 (0.25 ng of WT + 0.25 ng of A188W). Taken together, these results suggest the MIWC is mercurial-insensitive because of absence of a cysteine residue near the NPA motifs and that residues 70-73 and 189 are located at or near the MIWC aqueous pore. In contrast to previous data for the channel-forming integral protein of 28kDa (CHIP28), the finding of a dominant negative phenotype for mutants G72W and A188W indicates that MIWC monomers interact at a functional level.

Functional water channels and proton pumps are in separate populations of endocytic vesicles in toad bladder granular cells.

Functional water channels are retrieved by endocytosis from the apical membrane of toad bladder granular cells in response to vasopressin [Shi, L.-B., & Verkman, A.S. (1989) J. Gen. Physiol. 94, 1101-1115]. To examine whether endocytic vesicles which contain the vasopressin-sensitive water channel fuse with acidic vesicles for entry into a lysosomal pathway, ATP-dependent acidification and osmotic water permeability were measured in endosomes from control bladders and bladders treated with vasopressin (VP) and/or phorbol myristate acetate (PMA). Endosomes were labeled with the fluid-phase markers 6-carboxyfluorescein or fluorescein-dextran. Osmotic water permeability (Pf) was measured by stopped-flow fluorescence quenching and proton ATPase activity by ATP-dependent, N-ethylmaleimide-inhibitable acidification. In a microsomal pellet, Pf was low (less than 0.002 cm/s, 20 degrees C) in labeled endocytic vesicles from control bladders but high (0.05-0.1 cm/s) in a subpopulation (50-70%) of vesicles from VP- and PMA-treated bladders. Following ATP addition, the average drop in pH was 0.1 (control), 0.3 (VP), and 0.2 (PMA) unit. Measurement of pH in individual endocytic vesicles by quantitative image analysis showed that less than 20% of vesicles from VP-treated bladders acidified by greater than 0.5 pH unit. To examine whether water channels and proton pumps were present in the same endocytic vesicles, the pH of endosomes with high and low water permeability was measured from the effect of ATP on the amplitude of the fluorescence quenching signal in response to an osmotic gradient.(ABSTRACT TRUNCATED AT 250 WORDS)

Functional independence of monomeric CHIP28 water channels revealed by expression of wild-type mutant heterodimers.

CHIP28 is a major water transporting protein in erythrocytes and kidney which forms tetramers in membranes (Verbavatz, J. M., Brown, D., Sabolic, I., Valenti, G., Ausiello, D. A., Van Hoek, A. N., Ma, T., and Verkman, A. S. (1993) J. Cell Biol. 123, 605-618). To determine whether CHIP28 monomers function independently, chimeric cDNA dimers were constructed which contained wild-type CHIP28 in series with either wild-type CHIP28, a non-water transporting CHIP28 mutant (C189W), or a functional but mercurial-insensitive CHIP28 mutant (C189S). Transcribed cRNAs were injected in Xenopus oocytes and plasma membrane expression was assayed by quantitative immunofluorescence. Water channel function was measured by osmotically induced swelling. CHIP28 homo- and heterodimers were targeted to the oocyte plasma membrane and functioned as water channels. Relative osmotic water permeability (Pf) values (normalized for plasma membrane expression of monomeric subunits) were: 1.0 (CHIP28 monomer), 0.0 (C189W), 1.07 (C189S), 1.10 (CHIP28-CHIP28 dimer) and 0.52 (CHIP28-C189W). The increase in oocyte Pf was linearly related to plasma membrane expression of wild-type CHIP28 and C189S subunits. HgCl2 (0.3 mM) inhibited channel-mediated Pf in oocytes expressing wild-type CHIP28 monomers and dimers by 85-90%, but did not inhibit Pf in oocytes expressing C189S. HgCl2 inhibited Pf in oocytes expressing CHIP28-C189S dimers by 44 +/- 7%, consistent with one mercurial-sensitive and one insensitive subunit in the heterodimer. These results indicate that despite their assembly in tetramers, monomeric CHIP28 subunits function independently as water channels.

Distinct biogenesis mechanisms for the water channels MIWC and CHIP28 at the endoplasmic reticulum.

MIWC is a 32 kDa mercurial-insensitive water channel [Hasegawa et al. (1994) J. Biol. Chem. 269, 5497-5500] expressed in kidney collecting duct, brain ependymal cells, airways, and other tissues. We showed recently that the homologous water channel CHIP28 spanned the endoplasmic reticulum (ER) membrane 4 times with N- and C-termini in the cytoplasm [Skach et al., (1994) J. Cell Biol. 125, 803-815]. Hydropathy analysis of MIWC indicated up to eight hydrophobic regions (HRs) comprising potential membrane-spanning domains. To determine MIWC transmembrane topology at the ER, 10 cDNA chimeras were constructed which encoded increasing lengths of MIWC upstream from a reporter epitope (prolactin P-domain) at residues 13, 46, 73, 92, 120, 140, 164, 209, 276, and 2097, corresponding to putative polar extramembrane loops in the MIWC sequence. The chimeras were translated cell-free (rabbit reticulocyte lysate+ER-derived microsomes) and in Xenopus oocytes. Peptide chains were labeled with [35S]methionine and immunoprecipitated with a P-domain antibody. Transmembrane topology as determined by protease accessibility of the P-reporter indicated six membrane-spanning domains with N- and C-termini in the cytoplasm. The predicted topology was confirmed by demonstrating N-linked glycosylation at native residue N131 and an engineered consensus site at residue 197. Membrane integration of the nascent chain, as assayed by extractability at pH 11.5, occurred after synthesis of the first HR (residues 1-46). Translocation was terminated by a stop transfer sequence in the second HR (residues 32-73) as demonstrated by translation of the heterologous construct, [prolactin signal sequence]-[globin]-[HR2]-P.(ABSTRACT TRUNCATED AT 250 WORDS)

Nonpolar environment of tryptophans in erythrocyte water channel CHIP28 determined by fluorescence quenching.

CHIP28 is an abundant water-transporting protein in erythrocytes, kidney proximal tubule, and other fluid-transporting tissues. To determine the environment of the four tryptophans in CHIP28, fluorescence spectra and quenching by polar and nonpolar compounds were measured in stripped human erythrocyte membranes containing CHIP28 and in proteoliposomes reconstituted with purified CHIP28; comparative studies were performed in membranes containing MIP26. Functional analysis showed that CHIP28 water permeability was not affected by the polar quenchers iodide and acrylamide nor the nonpolar n-anthroyloxy fatty acids (n-AF). The emission maximum of CHIP28 tryptophan fluorescence was at 324 +/- 2 nm and did not change with the addition of quenchers; the maximum for MIP26 was at 335 +/- 5 nm. There was weak quenching of CHIP28 tryptophan fluorescence by the polar compounds iodide and acrylamide, with Stern-Volmer constants of 0.13 and 0.71 M-1, respectively. HgCl2 inhibited water permeability by > 95% at 50 microM and quenched CHIP28 fluorescence reversibly by up to 70% with a biphasic concentration dependence; quenching by HgCl2 and acrylamide was not additive. The membrane-associated n-AF probes quenched CHIP28 fluorescence by up to 80% with the greatest quenching for n = 2 and 12; addition of HgCl2 or acrylamide after n-AF caused a small, anthroyloxy-position-dependent increase in quenching which was greatest at n = 6. These studies indicate that the tryptophans in CHIP28 are in a nonpolar, membrane-associated environment. Mathematical modeling of the n-AF results suggests that the tryptophans are clustered near the surface and center of the bilayer.(ABSTRACT TRUNCATED AT 250 WORDS)

Heterogeneity in ATP-dependent acidification in endocytic vesicles from kidney proximal tubule. Measurement of pH in individual endocytic vesicles in a cell-free system.

Measurement of membrane transport in suspensions of isolated membrane vesicles provides averaged information over a potentially very heterogeneous vesicle population. To examine the regulatory mechanisms for ATP-dependent acidification, methodology was developed to measure pH in individual endocytic vesicles. Endocytic vesicles from proximal tubule apical membrane of rat kidney were labeled in vivo by intravenous infusion of FITC-dextran (9 kD); a microsomal fraction was obtained from dissected renal cortex by homogenization and differential centrifugation. Vesicles were immobilized on a polylysine coated coverglass and imaged at high magnification by a silicon intensified target camera. ATP-dependent acidification was not influenced by endosome immobilization. Endosome pH was determined from the integrated fluorescence intensity of individual labeled vesicles after background subtraction. Calibration studies with high K and nigericin showed nearly identical fluorescence vs. pH curves for different endosomes with a standard deviation for a single pH measurement in a single endosome of approximately 0.2 pH units. In response to addition of 1 mM MgATP in the presence of K and valinomycin, endosome pH decreased from 7.2 to a mean of 6.4 with a unimodal distribution with width at half-maximum of approximately 1 pH unit. The drop in endosome pH increased and the shape of the distribution changed when the time between FITC-dextran infusion and kidney removal was increased from 5 to 20 min. Differences in ATP-dependent acidification could not be attributed to heterogeneity in passive proton conductance. These results establish a direct method to measure pH in single endocytic vesicles and demonstrate remarkable heterogeneity in ATP-dependent acidification which was interpreted in terms of heterogeneity in the number and/or activity of proton pumps at serial stages of endocytosis.