RESUMO
Aquaporins (AQPs), ordinarily regarded as water channels, have recently been shown to participate in other cellular functions such as cell-to-cell adhesion, cell migration, cell proliferation etc. The current investigation was undertaken to find out whether AQP5 water channel plays a role in corneal epithelial wound healing. Expression of AQP5 in mouse cornea and transfected Madin-Darby canine kidney (MDCK) cells was detected using immunofluorescence or EGFP tag. Cell migration and proliferation, the two major events in wound healing, were studied in vitro using cell culture scratch-wound healing model and cell proliferation assay, in vivo by conducting wound healing experiments on corneas of wild-type and AQP5 knockout mouse model and ex vivo on corneal epithelial cells isolated from wild type and AQP5 knockout mice. MDCK cells stably expressing AQP5 showed significantly higher levels of cell migration and proliferation compared to control cells. Likewise, corneal epithelial cells of wild type mouse with innate AQP5 exhibited faster wound healing than those of AQP5 knockout in vivo and under ex vivo culture conditions. In vitro, in vivo and ex vivo studies showed that presence of AQP5 improved cell migration, proliferation and wound healing. The data collected suggest that AQP5 plays a significant role in corneal epithelial wound healing.
Assuntos
Aquaporina 5/fisiologia , Movimento Celular/fisiologia , Reepitelização/fisiologia , Cicatrização/fisiologia , Animais , Western Blotting , Técnicas de Cultura de Células , Proliferação de Células/fisiologia , Córnea/metabolismo , Cães , Epitélio Corneano/fisiologia , Técnica Indireta de Fluorescência para Anticorpo , Proteínas de Fluorescência Verde/metabolismo , Células Madin Darby de Rim Canino/metabolismo , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , TransfecçãoRESUMO
Maintenance of proper biomechanics of the eye lens is important for its structural integrity and for the process of accommodation to focus near and far objects. Several studies have shown that specialized cytoskeletal systems such as the beaded filament (BF) and spectrin-actin networks contribute to mammalian lens biomechanics; mutations or deletion in these proteins alters lens biomechanics. Aquaporin 0 (AQP0), which constitutes â¼45% of the total membrane proteins of lens fiber cells, has been shown to function as a water channel and a structural cell-to-cell adhesion (CTCA) protein. Our recent ex vivo study on AQP0 knockout (AQP0 KO) mouse lenses showed the CTCA function of AQP0 could be crucial for establishing the refractive index gradient. However, biomechanical studies on the role of AQP0 are lacking. The present investigation used wild type (WT), AQP5 KO (AQP5(-/-)), AQP0 KO (heterozygous KO: AQP0(+/-); homozygous KO: AQP0(-/-); all in C57BL/6J) and WT-FVB/N mouse lenses to learn more about the role of fiber cell AQPs in lens biomechanics. Electron microscopic images exhibited decreases in lens fiber cell compaction and increases in extracellular space due to deletion of even one allele of AQP0. Biomechanical assay revealed that loss of one or both alleles of AQP0 caused a significant reduction in the compressive load-bearing capacity of the lenses compared to WT lenses. Conversely, loss of AQP5 did not alter the lens load-bearing ability. Compressive load-bearing at the suture area of AQP0(+/-) lenses showed easy separation while WT lens suture remained intact. These data from KO mouse lenses in conjunction with previous studies on lens-specific BF proteins (CP49 and filensin) suggest that AQP0 and BF proteins could act co-operatively in establishing normal lens biomechanics. We hypothesize that AQP0, with its prolific expression at the fiber cell membrane, could provide anchorage for cytoskeletal structures like BFs and together they help to confer fiber cell shape, architecture and integrity. To our knowledge, this is the first report identifying the involvement of an aquaporin in lens biomechanics. Since accommodation is required in human lenses for proper focusing, alteration in the adhesion and/or water channel functions of AQP0 could contribute to presbyopia.
Assuntos
Aquaporinas/fisiologia , Proteínas do Olho/fisiologia , Cristalino/fisiologia , Animais , Fenômenos Biomecânicos , Cristalino/ultraestrutura , Camundongos , Camundongos Endogâmicos C57BL , Microscopia Eletrônica de Varredura , Reação em Cadeia da PolimeraseRESUMO
PURPOSE: Aquaporins (AQPs) play a significant role in the movement of water across the plasma membrane. In the eye, the cornea and lens are avascular with unique microcirculatory mechanisms to meet the metabolic demands. We have previously shown that AQP0 and AQP1 water channels participate in maintaining lens transparency and homeostasis. In the present investigation, we explored the expression and spatial distribution of AQP5 in the cornea and lens, and its regulation during membrane localization. METHODS: AQP5 expression and cellular localization were investigated by reverse transcription polymerase chain reaction (RT-PCR) using gene-specific primers, and by western blot and immunocytochemistry analyses using specific antibodies. AQP5 phosphorylation was studied using calf intestinal alkaline phosphatase for dephosphorylation. Effects of phosphokinase A (PKA) agonist cyclic AMP (cAMP), and antagonist H-89 on AQP5 expression and localization were studied in vitro using MDCK (Madin-Darby Canine Kidney) cells, and ex vivo using isolated corneas from wild type mice. RESULTS: RT-PCR revealed the presence of AQP5 transcripts in the cornea, lens epithelial cells and fiber cells. Western blotting identified the presence of both non-phosphorylated and phosphorylated forms of AQP5 protein. Immunostaining showed the distribution of AQP5 in the epithelial layer and stromal keratocytes of the cornea, and epithelial and fiber cells of the lens. In vitro and ex-vivo experiments revealed PKA-induced AQP5 internalization; PKA inhibition prevented such internalization. CONCLUSIONS: This is the first report on the spatial expression of AQP5 in the corneal keratocytes and lens epithelial cells, as well as on the regulation of AQP5 localization by PKA in the corneal epithelial cells. PKA-mediated regulation of AQP5 holds promise for therapeutic intervention to control corneal and lens diseases.
Assuntos
Aquaporina 5/genética , Córnea/metabolismo , Expressão Gênica/genética , Cristalino/metabolismo , Fosfotransferases (Aceptor do Grupo Álcool)/metabolismo , Fosfatase Alcalina/metabolismo , Animais , Aquaporina 5/metabolismo , Transporte Biológico , Bovinos , Linhagem Celular , Córnea/efeitos dos fármacos , Ceratócitos da Córnea/efeitos dos fármacos , Ceratócitos da Córnea/metabolismo , AMP Cíclico/farmacologia , Cães , Células Epiteliais/efeitos dos fármacos , Células Epiteliais/metabolismo , Expressão Gênica/efeitos dos fármacos , Isoquinolinas/farmacologia , Cristalino/efeitos dos fármacos , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Fosforilação , Fosfotransferases (Aceptor do Grupo Álcool)/antagonistas & inibidores , Inibidores de Proteínas Quinases/farmacologia , Sulfonamidas/farmacologia , Água/metabolismoRESUMO
The mechanisms for provisioning maternal resources to offspring in placental mammals involve complex interactions between maternally regulated and fetally regulated gene networks in the placenta, a tissue that is derived from the zygote and therefore of fetal origin. Here we describe a novel use of an embryo transfer system in mice to identify gene networks in the placenta that are regulated by the mother. Mouse embryos from the same strain of inbred mice were transferred into a surrogate mother either of the same strain or from a different strain, allowing maternal and fetal effects on the placenta to be separated. After correction for sex and litter size, maternal strain overrode fetal strain as the key determinant of fetal weight (P < 0.0001). Computational filtering of the placental transcriptome revealed a group of 81 genes whose expression was solely dependent on the maternal strain [P < 0.05, false discovery rate (FDR) < 0.10]. Network analysis of this group of genes yielded highest statistical significance for pathways involved in the regulation of cell growth (such as insulin-like growth factors) as well as those involved in regulating lipid metabolism [such as the low-density lipoprotein receptor-related protein 1 (LRP1), LDL, and HDL], both of which are known to play a role in fetal development. This novel technique may be generally applied to identify regulatory networks involved in maternal-fetal interaction and eventually help identify molecular targets in disorders of fetal growth.
Assuntos
Transferência Embrionária/métodos , Redes Reguladoras de Genes/fisiologia , Placenta/metabolismo , Animais , Feminino , Peso Fetal/genética , Peso Fetal/fisiologia , Redes Reguladoras de Genes/genética , Genótipo , Masculino , Camundongos , GravidezRESUMO
Hg(2+) is commonly used as an inhibitor of many aquaporins during measurements of transcellular water transport. To investigate whether it could also act on the paracellular water transport pathway, we asked whether addition of Hg(2+) affected transport of radiolabeled probes through tight junctions of a salivary epithelial cell monolayer. Inclusion of 1 mM Hg(2+) decreased transepithelial electrical resistance by 8-fold and augmented mannitol and raffinose flux by 13-fold, which translated into an estimated 44% increase in pore radius at the tight junction. These Hg(2+)-induced effects could be partially blocked by the protein kinase A (PKA) inhibitor N-[2-((p-bromocinnamyl) amino) ethyl]-5-isoquinolinesulfonamide, 2HCl (H89), suggesting that both-PKA dependent and PKA-independent mechanisms contribute to tight junction regulation. Western blot analyses showed a 2-fold decrease in tight junction-associated occludin after Hg(2+) treatment and the presence of a novel hyperphosphorylated form of occludin in the cytoplasmic fraction. These findings were corroborated by confocal imaging. The results from this study reveal a novel contribution of the PKA pathway in Hg(2+)-induced regulation of tight junction permeability in the salivary epithelial barrier. Therapeutically, this could be explored for pharmacological intervention in the treatment of dry mouth, Sjögren's syndrome, and possibly other disorders of fluid transport.
Assuntos
Permeabilidade da Membrana Celular/fisiologia , Proteínas Quinases Dependentes de AMP Cíclico/metabolismo , Células Epiteliais/metabolismo , Proteínas de Membrana/metabolismo , Mercúrio/toxicidade , Junções Íntimas/metabolismo , Animais , Permeabilidade da Membrana Celular/efeitos dos fármacos , Células Cultivadas , Células Epiteliais/citologia , Células Epiteliais/efeitos dos fármacos , Ocludina , Fosforilação/efeitos dos fármacos , Ratos , Glândulas Salivares/citologia , Glândulas Salivares/efeitos dos fármacos , Glândulas Salivares/metabolismo , Transdução de Sinais/efeitos dos fármacos , Transdução de Sinais/fisiologia , Glândula Submandibular/citologia , Glândula Submandibular/efeitos dos fármacos , Glândula Submandibular/metabolismo , Junções Íntimas/efeitos dos fármacosRESUMO
PURPOSE: This study was designed to clarify the physiological function and tissue distribution of aquaporin 5 (AQP5) in the lacrimal and parotid glands. METHODS: Saliva and tear volumes were compared in AQP5 knockout (AQP5-/-) mice and wild-type mice. Immunohistochemistry and immunoblot analysis were performed in wild-type and AQP5-/- mice. RESULTS: Immunofluorescence of AQP5 staining showed that AQP5 was localized mainly in the ductal cells rather than in the acinar cells of the lacrimal gland. In contrast, in the parotid gland, AQP5 was observed abundantly in acinar cells with undetectable staining in ductal cells. Tear secretion was not changed in AQP5-/- mouse, although saliva secretion was significantly reduced. CONCLUSIONS: AQP5 distribution in acinar cells and ductal cells was completely opposite in the lacrimal and parotid glands. The physiological role of AQP5 might be dependent on the characteristic tissue distribution of the protein in the lacrimal and parotid glands.
Assuntos
Aquaporina 5/metabolismo , Aparelho Lacrimal/metabolismo , Glândula Parótida/metabolismo , Animais , Aquaporina 5/genética , Técnica Indireta de Fluorescência para Anticorpo , Immunoblotting , Aparelho Lacrimal/citologia , Masculino , Camundongos , Camundongos Knockout , Glândula Parótida/citologia , Saliva/química , Saliva/metabolismo , Lágrimas/química , Lágrimas/metabolismoRESUMO
The high mobility group A2 protein (HMGA2) has been implicated in the pathogenesis of mesenchymal tumors such as leiomyoma, lipoma and hamartoma. HMGA2 was pinpointed by mapping the breakpoints in the chromosomal translocations in 12q15, especially the t(12;14) that is commonly seen in uterine leiomyoma. It is generally assumed that altered expression of HMGA2 is an early event in the pathway to tumor formation. Here, we show evidence that three novel transcripts, A15, B6 and D12 are located within the HMGA2 gene itself and are transcribed from the opposite strand. These embedded transcripts are expressed at 6-20-fold higher levels in tumors compared to matched myometrium from the same patients. We estimate that the domain of increased expression extends 500kb on chromosome 12q15, and encompasses the majority of t(12;14) translocation breakpoints. However, a corresponding domain of consistently altered expression is not seen on chromosome 14 or outside of the chromosome 12 multiple aberration region. These data suggest that t(12;14) breakpoints contribute to the pathogenesis of uterine leiomyoma by interrupting a complex regulation of HMGA2 and other genes embedded within and around it. We also discovered a novel laminin receptor gene, transcribed from the opposite strand, within the promoter region of HMGA2. Although the roles for these embedded transcripts are still unknown, preliminary data suggest that they are members of the family of non-coding RNA and that they may play an important role in the pathology of uterine leiomyoma.
Assuntos
Cromossomos Humanos Par 12 , Cromossomos Humanos Par 14 , Proteína HMGA2/genética , Leiomioma/genética , Translocação Genética , Neoplasias Uterinas/genética , Processamento Alternativo , Sequência de Aminoácidos , Sequência de Bases , Etiquetas de Sequências Expressas , Feminino , Proteína HMGA2/metabolismo , Humanos , Leiomioma/metabolismo , Leiomioma/patologia , Modelos Genéticos , Dados de Sequência Molecular , Miométrio/metabolismo , Regiões Promotoras Genéticas , Receptores de Laminina/genética , Neoplasias Uterinas/metabolismoRESUMO
BACKGROUND AND PURPOSE: Intracerebral hemorrhage (ICH) has a 30-day mortality rate of 40% to 50% and lacks a proven treatment. We report a preplanned, midpoint analysis of the first population-based, case-control study that examines both genetic and environmental risk factors of ICH. METHODS: We prospectively identified cases of hemorrhagic stroke at all 16 hospitals in the Greater Cincinnati/Northern Kentucky region. All cases underwent medical record and neuroimaging review. Cases enrolled in the direct interview and genetic sampling arm of the study were matched to population-based control subjects by age, race, and sex. Multivariable logistic regression was performed to identify significant independent risk factors. RESULTS: We enrolled 188 cases of ICH (67 lobar, 121 nonlobar) and 366 control subjects in the direct interview arm of the study. Significant independent risk factors for lobar ICH included the presence of an apolipoprotein E2 or E4 allele, frequent alcohol use, prior stroke, and first-degree relative with ICH. Significant independent risk factors for nonlobar ICH were hypertension, prior stroke, and first-degree relative with ICH. An increasing level of education was associated with a decreased risk of nonlobar ICH. The attributable risk of apolipoprotein E2 or E4 for lobar ICH was 29%, and the attributable risk of hypertension for nonlobar ICH was 54%. CONCLUSIONS: There is significant epidemiological evidence that the pathophysiology of ICH varies by location. We estimate that a third of all cases of lobar ICH are attributable to possession of an apolipoprotein E4 or E2 allele and that half of all cases of nonlobar ICH are attributable to hypertension.
Assuntos
Hemorragia Cerebral/epidemiologia , Hemorragia Cerebral/genética , Predisposição Genética para Doença/epidemiologia , Distribuição por Idade , Consumo de Bebidas Alcoólicas , Alelos , Apolipoproteína E2 , Apolipoproteína E4 , Apolipoproteínas E/genética , Estudos de Casos e Controles , Hemorragia Cerebral/mortalidade , Comorbidade , Demografia , Escolaridade , Feminino , Humanos , Hipertensão/epidemiologia , Kentucky/epidemiologia , Modelos Logísticos , Masculino , Pessoa de Meia-Idade , Razão de Chances , Ohio/epidemiologia , Estudos Prospectivos , Grupos Raciais , Medição de Risco , Fatores de Risco , Distribuição por SexoRESUMO
To examine the potential roles of aquaporins 1 and 5 (AQP1 and AQP5, respectively) in inner ear development and function, we defined their spatial and temporal expression patterns in the developing mouse inner ear and examined the morphologic and physiologic effects of loss of Aqp5 function. Standard in situ hybridization (ISH) and immunohistochemical (IHC) assays were used for expression studies with routine morphologic, behavioral, and physiologic assessments of hearing and balance in Aqp5 null mutant mice. AQP1 was first detected at embryonic day 10.5 (E10.5) in the otocyst but eventually localized to specific nonsensory portions of the inner ear and connective tissue cells surrounding the membranous labyrinth. AQP5 displayed specific cochlear expression, first detectable at E15.5 in the nonsensory epithelium and later restricted to the lateral wall of the cochlear duct near the spiral prominence. AQP5 expression continued through postnatal periods with a change of expression domain to the stria vascularis between postnatal day 7 (P7) and P14. By in situ hybridization and immunohistochemical techniques, subtle differences between transcript and protein expression patterns were noted for both AQP1 and 5. Although AQP5 is dynamically expressed in the developing mouse inner ear, adult Aqp5 knockout mice show normal hearing when tested and normal inner ear structural development. These results suggest redundant or alternative mechanisms that likely regulate water homeostasis in the developing and mature inner ear.
Assuntos
Envelhecimento/metabolismo , Aquaporinas/metabolismo , Orelha Interna/embriologia , Orelha Interna/metabolismo , Proteínas de Membrana , Animais , Animais Recém-Nascidos/crescimento & desenvolvimento , Animais Recém-Nascidos/metabolismo , Aquaporina 1 , Aquaporina 5 , Aquaporinas/deficiência , Comportamento Animal , Orelha Interna/patologia , Orelha Interna/fisiopatologia , Embrião de Mamíferos/metabolismo , Desenvolvimento Embrionário e Fetal , Potenciais Evocados Auditivos do Tronco Encefálico , Audição , Camundongos , Camundongos Endogâmicos , Camundongos KnockoutRESUMO
Caco-2 cell permeability was evaluated in isotonic media containing high (25 mM) or physiological (5.5 mM) glucose concentrations. Transepithelial electrical resistance (TEER) and membrane fluidity were measured to assess glucose-induced alterations in physical barrier properties. In parallel, distribution of the actin filament (F-actin) and zonula occludens-1 (ZO-1) proteins was assessed by confocal microscopy. Transepithelial fluxes of mannitol, hydrocortisone, digoxin, and glycyl sarcosine (Gly-Sar) that permeate the intestinal mucosa by various pathways were measured to quantify the effect of glucose-induced changes on Caco-2 cell permeability. High glucose decreased maximum TEER of cell monolayers by 47%, whereas membrane fluidity at the hydrophobic core and lipid/polar head interphase was significantly increased. F-actin distribution in high glucose cells appeared more diffuse while ZO-1 was unchanged. Mannitol and hydrocortisone fluxes across Caco-2 cells cultured in high glucose increased by 65% and 24%, respectively. In addition, high glucose decreased the maximum transport capacity (Vmax) of PepT-1. P-glycoprotein activity, however, was unchanged. In conclusion, high extracellular glucose concentration in isotonic media significantly alters physical barrier properties of Caco-2 cell monolayers, which predominantly affects transepithelial transport of solutes permeating the cell barrier by paracellular and transcellular passive diffusion and facilitated transport mediated by the proton-dependent oligopeptide transporter (PepT-1).
Assuntos
Células CACO-2/efeitos dos fármacos , Permeabilidade da Membrana Celular/efeitos dos fármacos , Glucose/administração & dosagem , Transporte Biológico/efeitos dos fármacos , Células CACO-2/fisiologia , Glucose/farmacologia , Humanos , Fluidez de Membrana/efeitos dos fármacosRESUMO
The alveolar epithelium plays a central role in gas exchange and fluid transport, and is therefore critical for normal lung function. Since the bulk of water flux across this epithelium depends on the membrane water channel Aquaporin 5 (AQP5), we asked whether hypoxia had any effect on AQP5 expression. We show that hypoxia causes a significant (70%) decrease in AQP5 expression in the lungs of mice exposed to hypoxia. Hypoxia and the hypoxia mimetic, cobalt, also caused similar decreases in AQP5 mRNA and protein expression in the mouse lung epithelial cell line MLE-12. The action of hypoxia and cobalt on AQP5 transcription was demonstrated by directly quantifying heternonuclear RNA by real-time PCR. Dominant negative mutants of Hypoxia Inducible Factor (HIF-1α) and HIF-1α siRNA blocked the action of cobalt, showing that HIF-1α is a key component in this mechanism. The proteasome inhibitors, lactacystin or proteasome inhibitor-III completely abolished the effect of hypoxia and cobalt both at the protein and mRNA level indicating that the proteasome pathway is probably involved not only for the stability of HIF-1α protein, but for the stability of unidentified transcription factors that regulate AQP5 transcription. These studies reveal a potentially important physiological mechanism linking hypoxic stress and membrane water channels.
Assuntos
Aquaporina 5/genética , Células Epiteliais/metabolismo , Expressão Gênica/efeitos dos fármacos , Subunidade alfa do Fator 1 Induzível por Hipóxia/genética , Hipóxia/metabolismo , Oxigênio/farmacologia , Água/metabolismo , Acetilcisteína/análogos & derivados , Acetilcisteína/farmacologia , Animais , Aquaporina 5/metabolismo , Hipóxia Celular/genética , Linhagem Celular , Cobalto/farmacologia , Células Epiteliais/citologia , Células Epiteliais/efeitos dos fármacos , Hipóxia/genética , Subunidade alfa do Fator 1 Induzível por Hipóxia/antagonistas & inibidores , Subunidade alfa do Fator 1 Induzível por Hipóxia/metabolismo , Pulmão/citologia , Pulmão/efeitos dos fármacos , Pulmão/metabolismo , Camundongos , Oxigênio/metabolismo , Complexo de Endopeptidases do Proteassoma/efeitos dos fármacos , Complexo de Endopeptidases do Proteassoma/metabolismo , Inibidores de Proteassoma/farmacologia , Estabilidade Proteica , RNA Mensageiro/antagonistas & inibidores , RNA Mensageiro/genética , RNA Interferente Pequeno/genética , Mucosa Respiratória/citologia , Mucosa Respiratória/efeitos dos fármacos , Mucosa Respiratória/metabolismo , Transdução de SinaisRESUMO
Chronic obstructive pulmonary disease (COPD) causes significant morbidity and mortality. Cigarette smoke, the most common risk factor for COPD, induces airway and alveolar epithelial barrier permeability and initiates an innate immune response. Changes in abundance of aquaporin 5 (AQP5), a water channel, can affect epithelial permeability and immune response after cigarette smoke exposure. To determine how AQP5-derived epithelial barrier modulation affects epithelial immune response to cigarette smoke and development of emphysema, WT and AQP5(-/-) mice were exposed to cigarette smoke (CS). We measured alveolar cell counts and differentials, and assessed histology, mean-linear intercept (MLI), and surface-to-volume ratio (S/V) to determine severity of emphysema. We quantified epithelial-derived signaling proteins for neutrophil trafficking, and manipulated AQP5 levels in an alveolar epithelial cell line to determine specific effects on neutrophil transmigration after CS exposure. We assessed paracellular permeability and epithelial turnover in response to CS. In contrast to WT mice, AQP5(-/-) mice exposed to 6 months of CS did not demonstrate a significant increase in MLI or a significant decrease in S/V compared with air-exposed mice, conferring protection against emphysema. After sub-acute (4 weeks) and chronic (6 mo) CS exposure, AQP5(-/-) mice had fewer alveolar neutrophil but similar lung neutrophil numbers as WT mice. The presence of AQP5 in A549 cells, an alveolar epithelial cell line, was associated with increase neutrophil migration after CS exposure. Compared with CS-exposed WT mice, neutrophil ligand (CD11b) and epithelial receptor (ICAM-1) expression were reduced in CS-exposed AQP5(-/-) mice, as was secreted LPS-induced chemokine (LIX), an epithelial-derived neutrophil chemoattractant. CS-exposed AQP5(-/-) mice demonstrated decreased type I pneumocytes and increased type II pneumocytes compared with CS-exposed WT mice suggestive of enhanced epithelial repair. Absence of AQP5 protected against CS-induced emphysema with reduced epithelial permeability, neutrophil migration, and altered epithelial cell turnover which may enhance repair.
RESUMO
Salivary gland cells are joined by junctional complexes consisting of a tight junction (TJ), zonula adherens and one or more desmosomes. TJs regulate paracellular permeability, maintain separate apical and basolateral membrane domains, and serve as signaling centers. We examined TJs of mouse submandibular glands (SMG) in thin sections and freeze-fracture replicas. TJs between acinar cells and between intercalated duct cells had 2-6 parallel strands on the protoplasmic fracture face, with occasional branches, interconnections and free ends, and corresponding grooves on the extracellular face. Granular duct cell TJs had 2-30 strands, a depth of Assuntos
Aquaporina 5/fisiologia
, Glândula Submandibular/ultraestrutura
, Junções Íntimas/ultraestrutura
, Animais
, Feminino
, Técnica de Fratura por Congelamento
, Masculino
, Camundongos
, Camundongos Knockout
RESUMO
To investigate potential physiological interactions between the transcellular and paracellular pathways of water transport, we asked whether targeted deletion of Aquaporin 5 (AQP5), the major transcellular water transporter in salivary acinar cells, affected paracellular transport of 4-kDa FITC-labeled dextran (FITC-D), which is transported through the paracellular but not the transcellular route. After i.v. injection of FITC-D into either AQP5 wild-type or AQP5-/- mice and saliva collection for fixed time intervals, we show that the relative amount of FITC-D transported in the saliva of AQP5-/- mice is half that in matched AQP5+/+ mice, indicating a 2-fold decrease in permeability of the paracellular barrier in mice lacking AQP5. We also found a significant difference in the proportion of transcellular vs. paracellular transport between male and female mice. Freeze-fracture electron microscopy revealed an increase in the number of tight junction strands of both AQP5+/+ and AQP5-/- male mice after pilocarpine stimulation but no change in strand number in female mice. Average acinar cell volume was increased by approximately 1.4-fold in glands from AQP5-/- mice, suggesting an alteration in the volume-sensing machinery of the cell. Western blots revealed that expression of Claudin-7, Claudin-3, and Occludin, critical proteins that regulate the permeability of the tight junction barrier, were significantly decreased in AQP5-/- compared with AQP5+/+ salivary glands. These findings reveal the existence of a gender-influenced molecular mechanism involving AQP5 that allows transcellular and paracellular routes of water transport to act in conjunction.
Assuntos
Aquaporina 5/metabolismo , Junções Íntimas/metabolismo , Água/metabolismo , Animais , Aquaporina 5/genética , Transporte Biológico/fisiologia , Western Blotting , Claudina-3 , Claudinas , Dextranos , Feminino , Fluoresceína-5-Isotiocianato/análogos & derivados , Proteínas de Membrana/metabolismo , Camundongos , Camundongos Knockout , Microscopia Eletrônica de Transmissão , Ocludina , Saliva/química , Glândulas Salivares/metabolismo , Junções Íntimas/ultraestruturaRESUMO
Activity of the epithelial Na(+) channel (ENaC) is limiting for Na(+) reabsorption at the distal nephron. Gain-of-function mutations in ENaC cause Liddle's syndrome: a severe form of inheritable hypertension. Several polymorphisms in alpha-hENaC possibly associated with abnormal Na(+) handling by the kidney and the salt-sensitive hypertension prevalent in black populations have been reported. The functional effects of alpha-hENaC polymorphisms on channel activity, however, remain controversial and have not been directly tested in a mammalian background. We ask here whether polymorphisms at positions 334, 618, and 663 in alpha-hENaC influence channel activity. Activity of wild-type (A334, C618, A663) and polymorphic ENaC expressed in Chinese hamster ovary cells was assessed with patch-clamp electrophysiology. While the A334T polymorphism had little effect on macroscopic ENaC currents, the C618F and A663T polymorphisms significantly increased ENaC activity >3.3- and 1.6-fold, respectively. Similarly, polymorphic ENaC had greater activity compared with wild-type channels in excised patches with activity of C618F and A663T channels increased 3.8- and 2.6-fold, respectively. Unitary channel conductances and reversal potentials were not different for polymorphic and wild-type ENaC. Increases in activity resulted primarily from increases in the apparent number of active (polymorphic) channels in the plasma membrane. Moreover, addition of a reducing agent to the cytosol significantly increased activity of wild-type ENaC equal to that of C618F polymorphic channels but had no effect on these latter channels. These results are consistent with the C618F and A663T polymorphisms leading to elevated ENaC activity with the possibility that they facilitate altered Na(+) handling by the kidney.
Assuntos
Hipertensão/genética , Rim/fisiologia , Polimorfismo Genético , Canais de Sódio/genética , Canais de Sódio/fisiologia , Animais , Células CHO , Cricetinae , Cricetulus , Eletrofisiologia , Canais Epiteliais de Sódio , Hipertensão/fisiopatologia , Técnicas de Patch-Clamp , SíndromeRESUMO
The membrane water channel aquaporin 5 (AQP5) plays an important role in transporting water across the apical surface of the alveolar epithelium and the epithelia of submucosal glands in the upper airway and nasopharynx. It is thus a potentially important target for modulating the fluid content of upper airway and nasopharyngeal secretions in disorders such as cystic fibrosis. Here, we have used an in vitro system to identify a molecular mechanism through which transcriptional and post-transcriptional regulation of AQP5 is accomplished. In a murine lung epithelial cell line (MLE-12), the addition of chlorophenylthio-cAMP (cpt-cAMP) causes a 4-fold increase in AQP5 mRNA and protein levels and induces translocation of AQP5 to the apical plasma membrane. Treatment with forskolin and isoproternol also caused similar increases in AQP5 expression both in vitro and in mouse lung tissue slices. The addition of actinomycin D abolished the cAMP-mediated increase in AQP5 mRNA and showed that there was no increase in the half-life of AQP5 mRNA, and inhibition of protein kinase A by H-89 blocked the cpt-cAMP-mediated increase of AQP5. Pretreatment of cells with cycloheximide blocked the cpt-cAMP-mediated increase of AQP5 mRNA, indicating that de novo protein synthesis is essential for increased AQP5 transcription. Immunofluorescent micrographs of cells treated with cpt-cAMP showed a significantly stronger AQP5 signal at the plasma membrane as compared with untreated cells. These results show that cAMP regulates AQP5 at multiple levels, by increasing synthesis of AQP5 mRNA and by triggering translocation of AQP5 to the plasma membrane.
Assuntos
Aquaporinas/genética , Proteínas Quinases Dependentes de AMP Cíclico/metabolismo , AMP Cíclico/fisiologia , Proteínas de Membrana , Processamento Pós-Transcricional do RNA , Transcrição Gênica , Aquaporina 5 , Aquaporinas/metabolismo , Linhagem Celular , Colforsina/farmacologia , Regulação da Expressão Gênica/efeitos dos fármacos , Regulação da Expressão Gênica/fisiologia , Isoproterenol/farmacologia , RNA Mensageiro/genética , RNA Mensageiro/metabolismoRESUMO
The expression of aquaporin-5, the major water channel expressed in alveolar, tracheal, and upper bronchial epithelium, is significantly down-regulated during acute lung injury. In the present study, the expression of aquaporin-5 in two different mouse models of lung fibrosis was evaluated. Lung fibrosis was induced by intratracheal and by subcutaneous infusion of bleomycin. The expression of aquaporin-5 was investigated by immunohistochemical studies and by polymerase chain reaction. There were many cells with loss of aquaporin-5 immunoreactivity in type I alveolar epithelial cells in the mouse models of lung fibrosis. Immunohistochemistry of lung tissue in aquaporin-5 knockout mice revealed a fibrotic phenotype with increased deposition of extracellular collagen type I in thickened alveolar walls. Semiquantitative analysis of aquaporin-5 mRNA expression showed more abundant content of aquaporin-5 in the lung of the normal mouse compared to the mouse with lung fibrosis. The results of this study showed, for the first time, that chronic lung injury and lung fibrosis is associated with decreased protein and mRNA expression of aquaporin-5 in the lung.
Assuntos
Aquaporinas/biossíntese , Regulação para Baixo , Proteínas de Membrana/biossíntese , Fibrose Pulmonar/metabolismo , Animais , Aquaporina 5 , Aquaporinas/genética , Bleomicina/toxicidade , Modelos Animais de Doenças , Feminino , Técnica Indireta de Fluorescência para Anticorpo , Expressão Gênica/efeitos dos fármacos , Técnicas Imunoenzimáticas , Proteínas de Membrana/genética , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Fibrose Pulmonar/induzido quimicamente , Fibrose Pulmonar/patologia , RNA Mensageiro/metabolismo , Reação em Cadeia da Polimerase Via Transcriptase ReversaRESUMO
The distribution and function of aquaporins (AQPs) have not previously been defined in sweat glands. In this study, AQP1, AQP3, and AQP5 mRNA were demonstrated in rat paw by reverse transcription (RT)-PCR, but AQP2 and AQP4 were not. AQP1, AQP3, and AQP5 protein were confirmed in these tissues by immunoblotting. AQP1 was identified in capillary endothelial cells by immunohistochemical labeling, but not in sweat glands or epidermis. Abundant AQP3 expression was seen in basal levels of epidermis, but not in sweat glands. AQP2 and AQP4 were not observed in either skin or sweat glands. Immunohistochemical labeling revealed abundant AQP5 in secretory parts of rat and mouse sweat glands, where immunoelectron microscopy demonstrated abundant AQP5 labeling in the apical plasma membrane. AQP5 immunolabeling of human sweat glands yielded a similar pattern. To establish the role of AQP5 in sweat secretion, we tested the response of adult mice to s.c. injection of pilocarpine, as visualized by reaction of secreted amylase with iodine/starch. The number of active sweat glands was dramatically reduced in AQP5-null (-/-) mice compared with heterozygous (+/-) and wild-type (+/+) mice. We conclude that the presence of AQP5 in plasma membranes of sweat glands is essential for secretion, providing potential insight into mechanisms underlying mammalian thermoregulation, tactile sensitivity, and the pathophysiology of hyperhidrosis.