RESUMO
Airway mucociliary clearance (MCC) is the main mechanism of lung defense keeping airways free of infection and mucus obstruction. Airway surface liquid volume, ciliary beating, and mucus are central for proper MCC and critically regulated by sodium absorption and anion secretion. Impaired MCC is a key feature of muco-obstructive diseases. The calcium-activated potassium channel KCa.3.1, encoded by Kcnn4, participates in ion secretion, and studies showed that its activation increases Na+ absorption in airway epithelia, suggesting that KCa3.1-induced hyperpolarization was sufficient to drive Na+ absorption. However, its role in airway epithelium is not fully understood. We aimed to elucidate the role of KCa3.1 in MCC using a genetically engineered mouse. KCa3.1 inhibition reduced Na+ absorption in mouse and human airway epithelium. Furthermore, the genetic deletion of Kcnn4 enhanced cilia beating frequency and MCC ex vivo and in vivo. Kcnn4 silencing in the Scnn1b-transgenic mouse (Scnn1btg/+), a model of muco-obstructive lung disease triggered by increased epithelial Na+ absorption, improved MCC, reduced Na+ absorption, and did not change the amount of mucus but did reduce mucus adhesion, neutrophil infiltration, and emphysema. Our data support that KCa3.1 inhibition attenuated muco-obstructive disease in the Scnn1btg/+ mice. K+ channel modulation may be a therapeutic strategy to treat muco-obstructive lung diseases.
Assuntos
Canais de Potássio Ativados por Cálcio de Condutância Intermediária/genética , Pneumopatias Obstrutivas/etiologia , Depuração Mucociliar/fisiologia , Animais , Cálcio/metabolismo , Células Cultivadas , Cílios/efeitos dos fármacos , Cílios/metabolismo , Modelos Animais de Doenças , Epitélio/metabolismo , Feminino , Humanos , Canais de Potássio Ativados por Cálcio de Condutância Intermediária/antagonistas & inibidores , Canais de Potássio Ativados por Cálcio de Condutância Intermediária/metabolismo , Pulmão/fisiopatologia , Pneumopatias Obstrutivas/genética , Masculino , Camundongos Endogâmicos C57BL , Camundongos Mutantes , Camundongos Transgênicos , Depuração Mucociliar/efeitos dos fármacos , Sódio/metabolismoRESUMO
BACKGROUND: Ketotifen is a second-generation noncompetitive H1-antihistamine and mast-cell stabilizer. It is commonly used to treat or prevent allergic conjunctivitis, asthma, chronic urticaria, anaphylaxis, mast-cell, and other allergic-type disorders. However, it has never been studied in aspirin-exacerbated respiratory disease (AERD), an aggressive phenotype of chronic rhinosinusitis with nasal polyps, where the mast cell plays a prominent role its pathogenesis. METHODS: Human sinonasal epithelial cells were grown at an air-liquid interface (ALI). Ketotifen powder was dissolved in saline to make 4 test solutions at 1.04, 2.08, 10.4, and 20.8 µg/mL. Control (saline) or ketotifen solution was added apically to ALI cultures from tissue of 5 unique patients, and ciliary beat frequency (CBF) changes were recorded. Lactate dehydrogenase was measured at 24 and 48 hours to estimate long-term cellular toxicity. RESULTS: Apical application of ketotifen at all concentrations was neither ciliotoxic nor ciliostimulatory, with no change in CBF over a period of 15 minutes after application. Cellular toxicity for all concentrations at 24 and 48 hours after application was <3% and <7%, respectively, that of lysed cultures. CONCLUSION: Topical application of ketotifen to an in vitro model of sinonasal epithelium is safe, as evaluated by CBF and lactate dehydrogenase. Ketotifen is neither ciliotoxic nor ciliostimulatory, and no long-term cellular toxicity was observed. Ketotifen may have promise as a topical nasal rinse in the treatment of AERD.
Assuntos
Antialérgicos/farmacologia , Antagonistas dos Receptores Histamínicos H1/farmacologia , Cetotifeno/farmacologia , Administração Tópica , Células Cultivadas , Cílios/efeitos dos fármacos , Cílios/fisiologia , Células Epiteliais/efeitos dos fármacos , Células Epiteliais/fisiologia , Humanos , L-Lactato Desidrogenase/metabolismo , Irrigação TerapêuticaRESUMO
BACKGROUND: Broncho-Vaxom® (OM-85 BV) is an extract of infectious respiratory bacteria that is used as an immunostimulant outside of the United States for the prevention and treatment of bronchitis and rhinosinusitis. Prior studies have shown that use of OM-85 BV is associated with reduction in frequency of respiratory infection and decreased duration of antibiotic usage. However, the effects of OM-85 BV on respiratory mucosal innate immunity are unknown. METHODS: Human sinonasal epithelial cells were grown at an air-liquid interface (ALI). Ciliary beat frequency (CBF) and nitric oxide (NO) production in response to stimulation with OM-85 BV was measured in vitro. Pharmacologic inhibitors of bitter taste receptor (T2R) signaling were used to determine if this pathway was taste-receptor-mediated. RESULTS: Apical application of OM-85 BV resulted in an NO-mediated increase in CBF (p < 0.05) and increased NO production (p < 0.0001) when compared to saline-stimulated control cultures. ALI pretreatment with taste receptor pathway inhibitors blocked OM-85 BV-induced increases in NO. CONCLUSION: OM-85 BV has ciliostimulatory and immunogenic properties that may be partially responsible for its observed efficacy as a respiratory therapeutic. These responses were NO-dependent and consistent with T2R activation. Further work is necessary to elucidate specific component-receptor signaling relationships.
Assuntos
Adjuvantes Imunológicos/farmacologia , Extratos Celulares/farmacologia , Imunidade Inata/efeitos dos fármacos , Mucosa Nasal/efeitos dos fármacos , Células Cultivadas , Cílios/efeitos dos fármacos , Cílios/fisiologia , Células Epiteliais/efeitos dos fármacos , Células Epiteliais/imunologia , Células Epiteliais/fisiologia , Humanos , Mucosa Nasal/imunologia , Mucosa Nasal/fisiologia , Óxido Nítrico/imunologiaRESUMO
Ciliary beat frequency (CBF) regulates the oviductal transport of oocytes and embryos, which are important components of the reproductive process. Local release of ATP transiently increases CBF by increasing [Ca2+]i. Ovarian hormones also regulate ciliary activity and oviductal transport. Progesterone (P4) induces nitric oxide (NO) production and high P4 concentrations induce ciliary dysfunction. However, the mechanism by which P4 affects CBF has not been elucidated. To evaluate the role of P4 in NO production and its effect on ATP-induced increases in CBF, we measured CBF, NO concentrations and [Ca2+]i in cultures of oviductal ciliated cells treated with P4 or NO signalling-related molecules. ATP induced a [Ca2+]i peak, followed by an increase in NO concentrations that were temporally correlated with the decreased phase of the transiently increased CBF. Furthermore, P4 increased the expression of nitric oxide synthases (iNOS and nNOS) and reduced the ATP-induced increase in CBF via a mechanism that involves the NO signalling pathway. These results have improved our knowledge about intracellular messengers controlling CBF and showed that NO attenuates oviduct cell functions. Furthermore, we showed that P4 regulates neurotransmitter (ATP) actions on CBF via the NO pathway, which could explain pathologies where oviductal transport is altered and fertility decreased.
Assuntos
Trifosfato de Adenosina/farmacologia , Cílios/efeitos dos fármacos , Óxido Nítrico/metabolismo , Oviductos/efeitos dos fármacos , Progesterona/farmacologia , Animais , Cálcio/metabolismo , Cílios/metabolismo , Feminino , Oviductos/metabolismo , Transdução de Sinais/fisiologiaRESUMO
The toxic actions of acute exposition to different diesel exhaust particles (DEPA) fractions on the mucociliary epithelium are not yet fully understood due to different concentrations of organic and inorganic elements. These chemicals elements produce damage to the respiratory epithelium and exacerbate pre-existent diseases. In our study we showed these differences in two experimental studies. Study I (dose-response curve - DRCS): Forty frog-palates were exposed to the following dilutions: frog ringer, intact DEPA diluted in frog-ringer at 3mg/L, 6mg/L and 12mg/L. Study II (DEPF) (DEPA fractions diluted at 12mg/L): Fifty palates - Frog ringer, intact DEPA, DEPA treated with hexane, nitric acid and methanol. Variables analyzed: relative time of mucociliary transport (MCT), ciliary beating frequency (CBF) and morphometric analysis for mucin profile (neutral/acid) and vacuolization. The Results of DRCS: Group DEPA-12mg/L presented a significant increase in the MCT (p<0.05), proportional volume of acid mucus (p<0.05) and decreased proportional volume of neutral mucus and vacuoles (p<0.05). In relation of DEPF: A significant increase in the MCT associated to a decrease in the proportional volume of neutral mucus was founded in nitric acid group. In addition, a significant increase in the proportional volume of acid mucus was found in methanol group. We concluded that: 1) Increasing concentrations of intact DEPA can progressively increase MCT and promote an acidification of intra-epithelial mucins associated to a depletion of neutral mucus. 2) Intact DEPA seem to act as secretagogue substance, promoting mucus extrusion and consequently reducing epithelial thickness. 3) Organic fraction of low polarity seems to play a pivotal role on the acute toxicity to the mucociliary epithelium, by promoting a significant increase in the MCT associated to changes in the chemical profile of the intracellular mucins.
Assuntos
Epitélio/efeitos dos fármacos , Depuração Mucociliar/efeitos dos fármacos , Mucosa/efeitos dos fármacos , Muco/metabolismo , Sistema Respiratório/efeitos dos fármacos , Emissões de Veículos/toxicidade , Poluição do Ar , Animais , Anuros , Cílios/efeitos dos fármacos , Mucinas/metabolismo , Mucosa/metabolismo , Palato , Ranidae , Sistema Respiratório/metabolismoRESUMO
BACKGROUND: Off-label use of topical ophthalmologic formulations for treatment of rhinologic disease is cited in recent literature and is anecdotally prevalent among practicing otolaryngologists. Steroids, antibiotics, and other drugs designed for ocular use have subjective clinical efficacy in the nose and sinuses, but their specific effects on the ciliated epithelium are less well defined. This study examines 9 commercially available ophthalmologic drug formulations for effects on ciliary motility in sinonasal cultures, in an effort to characterize their utility as topical therapies for sinonasal diseases. METHODS: Ophthalmologic solutions were tested on human sinonasal cultures grown at an air-liquid interface. Baseline ciliary beat frequency (CBF) was recorded and compared with CBF changes in the 20 minutes after drug addition. Substances were categorized by degree of ciliostimulation or cilioinhibition. RESULTS: All ophthalmologic solutions tested had only moderate effects on CBF during the 20-minute experimental period, with no solutions causing overt ciliostasis. Azithromycin, neomycin, and olopatadine were slightly ciliostimulatory, whereas levofloxacin, tobramycin, dexamethasone, azelastine, and prednisolone acetate were cilioinhibitory. Ciprofloxacin elicited moderate cilioinhibition that progressed to ciliostimulation. CONCLUSION: All solutions tested appear to have moderate effects on ciliated cell surfaces for a period of time typical of mucociliary clearance (10 to 20 minutes). Both active drugs and excipients can play a role in ciliary modulation, and specific formulations can show unique or unexpected properties. Any other individual ophthalmologic solutions to be used in a nasal drug delivery system should be tested in this manner to evaluate potential ciliary effects before clinical use.
Assuntos
Cílios/efeitos dos fármacos , Mucosa Nasal/efeitos dos fármacos , Soluções Oftálmicas/farmacologia , Cílios/fisiologia , Humanos , Mucosa Nasal/fisiologia , Uso Off-Label , Seios Paranasais/fisiologiaRESUMO
The sensory neurons in the olfactory epithelium (OSNs) are equipped with a large repertoire of olfactory receptors and the associated signal transduction machinery. In addition to the canonical OSNs, which express odorant receptors (ORs), the epithelium contains specialized subpopulations of sensory neurons that can detect specific information from environmental cues and relay it to relevant neuronal circuitries. Here we describe a subpopulation of mature OSNs in the main olfactory epithelium (MOE) which expresses CD36, a multifunctional receptor involved in a series of biological processes, including sensory perception of lipid ligands. The Cd36 expressing neurons coexpress markers of mature OSNs and are dispersed throughout the MOE. Unlike several ORs analyzed in our study, we found frequent coexpression of the OR Olfr287 in these neurons, suggesting that only a specific set of ORs may be coexpressed with CD36 in OSNs. We also show that CD36 is expressed in the cilia of OSNs, indicating a possible role in odorant detection. CD36-deficient mice display no signs of gross changes in the organization of the olfactory epithelium, but show impaired preference for a lipid mixture odor. Our results show that CD36-expressing neurons represent a distinct population of OSNs, which may have specific functions in olfaction.
Assuntos
Antígenos CD36/genética , Subunidades alfa de Proteínas de Ligação ao GTP/genética , Proteína de Marcador Olfatório/genética , Mucosa Olfatória/metabolismo , Neurônios Receptores Olfatórios/metabolismo , Receptores Odorantes/genética , Animais , Antígenos CD36/deficiência , Cílios/efeitos dos fármacos , Cílios/metabolismo , Subunidades alfa de Proteínas de Ligação ao GTP/metabolismo , Regulação da Expressão Gênica , Lipídeos/farmacologia , Masculino , Camundongos , Camundongos Knockout , Odorantes/análise , Proteína de Marcador Olfatório/metabolismo , Mucosa Olfatória/citologia , Neurônios Receptores Olfatórios/citologia , Neurônios Receptores Olfatórios/efeitos dos fármacos , Feromônios/farmacologia , Isoformas de Proteínas/genética , Isoformas de Proteínas/metabolismo , Receptores Odorantes/metabolismo , Olfato/fisiologiaRESUMO
Primary cilia are specialized organelles that extend from the cell surface and concentrate signal transduction components. In the nervous system, primary cilia-associated signals, such as sonic hedgehog (Shh), regulate cell proliferation and neuronal fate. Primary cilia assembly and maintenance require a multi-subunit intraflagellar transport (IFT) protein complex. Defects in primary cilia and IFT proteins are associated to severe pathological phenotypes. In the retina, the study of primary cilia has been mainly restricted to the specialized photoreceptor outer segment. The presence and physiological role of primary cilia in other retinal cells have not been clearly elucidated. Müller cells are the main glia of the retina where they exert distinct functions to maintain homeostasis. In pathological conditions, Müller cells mount a unique regenerative response through the processes of dedifferentiation, proliferation, and differentiation into neuronal lineages. The involvement of IFT proteins or a primary cilium in these processes has not been explored. In this study, we used mature Müller glia primary cultures to reveal the presence of the primary cilia by immunoreactivity to acetylated α-tubulin and γ-tubulin, which localize to the axoneme and ciliar basal body, respectively. We demonstrate that si-RNA-mediated downregulation of IFT20 gene expression, a main component of the IFT machinery, blocks Shh-induced Müller cell proliferation. We present evidence that IFT20 ablation impairs the dedifferentiation capacity of Müller cells induced by Shh and by glutamate. Our demonstration that Müller glia expresses IFT20 and harbors primary cilia, and opens new venues of research on the role of primary cilia in the retina.
Assuntos
Proteínas de Transporte/genética , Desdiferenciação Celular , Cílios/metabolismo , Regulação para Baixo , Células Ependimogliais/metabolismo , Proteínas Hedgehog/metabolismo , Animais , Animais Recém-Nascidos , Biomarcadores/metabolismo , Proteínas de Transporte/metabolismo , Desdiferenciação Celular/efeitos dos fármacos , Desdiferenciação Celular/genética , Proliferação de Células/efeitos dos fármacos , Células Cultivadas , Cílios/efeitos dos fármacos , Regulação para Baixo/efeitos dos fármacos , Células Ependimogliais/efeitos dos fármacos , Imunofluorescência , Ácido Glutâmico/farmacologia , Nestina/metabolismo , Ratos Long-Evans , Células-Tronco/efeitos dos fármacos , Células-Tronco/metabolismoRESUMO
Patients undergoing continuous ambulatory peritoneal dialysis are classified according to their peritoneal permeability as low transporter (low solute permeability) or High transporter (high solute permeability). Factors that determine the differences in permeability between them have not been fully disclosed. We investigated morphological features of cultured human peritoneal mesothelial cells from low or high transporter patients and its response to All trans retinoic Acid (ATRA, vitamin A active metabolite), as compared to non-uremic human peritoneal mesothelial cells. Control cells were isolated from human omentum. High or low transporter cells were obtained from dialysis effluents. Cells were cultured in media containing ATRA (0, 50, 100 or 200 nM). We studied length and distribution of microvilli and cilia (scanning electron microscopy), epithelial (cytokeratin, claudin-1, ZO-1 and occludin) and mesenchymal (vimentin and α-smooth muscle actin) transition markers by immunofluorescence and Western blot, and transforming growth factor ß1 expression by Western blot. Low and high transporter exhibited hypertrophic cells, reduction in claudin-1, occludin and ZO-1 expression, cytokeratin and vimentin disorganization and positive α-smooth muscle actin label. Vimentin, α-smooth muscle actin and transforming growth factor-ß1 were overexpressed in low transporter. Ciliated cells were diminished in low and high transporters. Microvilli number and length were severely reduced in high transporter. ATRA reduced hypertrophic cells number in low transporter. It also improved cytokeratin and vimentin organization, decreased vimentin and α-smooth muscle actin expression, and increased claudin 1, occludin and ZO-1 expression, in low and high transporter. In low transporter, ATRA reduced transforming growth factor-ß1 expression. ATRA augmented percentage of ciliated cells in low and high transporter. It also augmented cilia length in high transporter. Alterations in structure, epithelial mesenchymal markers and transforming growth factor-ß1 expression were differential between low and high transporter. Beneficial effects of ATRA were improved human peritoneal mesothelial cells morphology tending to normalize structures.
Assuntos
Cavidade Peritoneal/patologia , Diálise Peritoneal Ambulatorial Contínua , Tretinoína/farmacologia , Actinas/metabolismo , Adolescente , Adulto , Idoso , Transporte Biológico/efeitos dos fármacos , Contagem de Células , Células Cultivadas , Cílios/efeitos dos fármacos , Cílios/metabolismo , Epitélio/efeitos dos fármacos , Epitélio/metabolismo , Epitélio/patologia , Feminino , Regulação da Expressão Gênica/efeitos dos fármacos , Humanos , Masculino , Microvilosidades/efeitos dos fármacos , Microvilosidades/metabolismo , Pessoa de Meia-Idade , Fator de Crescimento Transformador beta1/genética , Vimentina/metabolismo , Adulto JovemRESUMO
Glycosphingolipids (GSLs), which are highly concentrated at the apical membrane of polarized epithelial cells, are key components of cell membranes and are involved in a large number of processes. Here, we investigated the ability of hypertonicity (high salt medium) to induce Madin-Darby Canine Kidney (MDCK) cell differentiation and found an increase in GSL synthesis under hypertonic conditions. Then, we investigated the role of GSLs in MDCK cell differentiation induced by hypertonicity by using two approaches. First, cultured cells were depleted of GSLs by exposure to D-threo-1-phenyl-2-decanoylamino-3-morpholino-1-propanol (D-PDMP). Second, cells were transfected with an siRNA specific to glucosylceramide synthase, the key enzyme in GSL synthesis. Exposure of cells to both treatments resulted in the impairment of the development of the apical membrane domain and the formation of the primary cilium. Enzymatic inhibitions of the de novo and the salvage pathway of GSL synthesis were used to determine the source of ceramide responsible of the GSL increase involved in the development of the apical membrane domain induced by hypertonicity. The results from this study show that extracellular hypertonicity induces the development of a differentiated apical membrane in MDCK cells by performing a sphingolipid metabolic program that includes the formation of a specific pool of GSLs. The results suggest as precursor a specific pool of ceramides formed by activation of a Fumonisin B1-resistant ceramide synthase as a component of the salvage pathway.