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CONTEXT: Cucumber (Cucumis sativus Linn. [Cucurbitaceae]) is widely known for its purgative, antidiabetic, antioxidant, and anticancer therapeutic potential. However, its effect on gastrointestinal (GI) disease is unrecognised. OBJECTIVE: This study investigated the effect of C. sativus fruit extract (CCE) on intestinal chloride secretion, motility, and motor function, and the role of TMEM16A chloride channels. MATERIALS AND METHODS: CCE extracts were obtained from commercially available cucumber. Active fractions were then purified by HPLC and analysed by high resolution mass spectrometry. The effect of CCE on intestinal chloride secretion was investigated in human colonic T84 cells, ex vivo mouse intestinal tissue using an Ussing chamber, and the two-electrode voltage-clamp technique to record calcium sensitive TMEM16A chloride currents in Xenopus laevis oocytes. In vivo, intestinal motility was investigated using the loperamide-induced C57BL/6 constipation mouse model. Ex vivo contractility of mouse colonic smooth muscles was assessed by isometric force measurements. RESULTS: CCE increased the short-circuit current (ΔIsc 34.47 ± µA/cm2) and apical membrane chloride conductance (ΔICl 95 ± 8.1 µA/cm2) in intestinal epithelial cells. The effect was dose-dependent, with an EC50 value of 0.06 µg/mL. CCE stimulated the endogenous TMEM16A-induced Cl- current in Xenopus laevis oocytes. Moreover, CCE increased the contractility of smooth muscle in mouse colonic tissue and enhanced small bowel transit in CCE treated mice compared to loperamide controls. Mass spectrometry suggested a cucurbitacin-like analogue with a mass of 512.07 g/mol underlying the bioactivity of CCE. CONCLUSION: A cucurbitacin-like analog present in CCE activates TMEM16A channels, which may have therapeutic potential in cystic fibrosis and intestinal hypodynamic disorders.
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Anoctamina-1/metabolismo , Cloruros/metabolismo , Cucumis sativus/química , Intestinos/efectos de los fármacos , Canales Iónicos/efectos de los fármacos , Extractos Vegetales/farmacología , Animales , Línea Celular , Estreñimiento/inducido químicamente , Estreñimiento/tratamiento farmacológico , Motilidad Gastrointestinal/efectos de los fármacos , Humanos , Loperamida/farmacología , Masculino , Ratones , Ratones Endogámicos C57BL , Modelos Animales , Músculo Liso/efectos de los fármacos , Técnicas de Placa-Clamp , Xenopus laevisRESUMEN
Formation of biofilm by Vibrio cholerae plays a crucial role in pathogenesis and transmission of cholera. Lower infective dose of the biofilm form of V. cholerae compared to the planktonic counterpart, and its antibiotic resistance, make it challenging to combat cholera. Nanoparticles may serve as an effective alternative to conventional antibiotics for targeting biofilms and virulence factors. We explored the effectiveness of gold nanoparticles (AuNPs) of different size and shape (spherical: AuNS10 and AuNS100, and rod: AuNR10, the number indicating the diameter in nm) on both the inhibition of formation and eradication of biofilm of the two biotypes of V. cholerae, classical (VcO395) and El Tor (VcN16961). Inhibition of biofilm formation by spherical AuNPs was observed for both the biotypes. Considering eradication, the biofilms for both, particularly El Tor, was destroyed using both the AuNSs, AuNS100 showing higher efficacy. AuNR10 did not affect the biofilm of either biotype. Micrographs of small intestinal sections of VcO395-infected mice indicated the inhibition of biofilm formation by both AuNSs. We also studied the effect of these AuNPs on the structure of cholera toxin (CT), the major toxin produced by V. cholerae. Far-UV CD showed both AuNR10 and AuNS100 compromised the structure of CT, which was also validated from the reduction of fluid accumulation in mice ileal loop. Western blot analysis revealed the reduction of CT production upon treatment with AuNPs. AuNS100 seems to be the best suited to inhibit the formation or destruction of biofilm, as well as to disrupt CT production and function.
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Nanopartículas del Metal , Vibrio cholerae , Animales , Biopelículas , Toxina del Cólera , Oro , RatonesRESUMEN
TMEM16A (Transmembrane protein 16A or Anoctamin1) is a calcium-activated chloride channel. (CaCC),that exerts critical roles in epithelial secretion. However, its localization, function, and regulation in intestinal chloride (Cl-) secretion remain obscure. Here, we show that TMEM16A protein abundance correlates with Cl- secretion in different regions of native intestine activated by the Ca2+-elevating muscarinic agonist carbachol (CCH). Basal, as well as both cAMP- and CCH-stimulated Isc, was largely reduced in Ano1 ± mouse intestine. We found CCH was not able to increase Isc in the presence of apical to serosal Cl- gradient, strongly supporting TMEM16A as primarily a luminal Cl- channel. Immunostaining demonstrated apical localization of TMEM16A where it colocalized with NHERF1 in mouse colonic tissue. Cellular depletion of NHERF1 in human colonic T84 cells caused a significant reduction of both cAMP- and CCH-stimulated Isc. Immunoprecipitation experiments revealed that NHERF1 forms a complex with TMEM16A through a PDZ-based interaction. We conclude that TMEM16A is a luminal Cl- channel in the intestine that functionally interacts with CFTR via PDZ-based interaction of NHERF1 for efficient and specific cholinergic stimulation of intestinal Cl- secretion.
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Because of the emergence of multidrug-resistant pathogenic bacteria, there is a growing interest for the development of an efficient alternative to antibiotics. Gold nanoparticles (AuNPs) are promising candidates due to their inherent non-toxicity and can be used as effective carriers of drugs. Cholera caused by Gram-negative Vibrio cholerae is still a potential threat in many developing countries. Virstatin, a small molecule, has been reported to inhibit virulence regulation in V. cholerae. Herein, we report an efficient synthesis of virstatin-conjugated gold nanoparticles (VL-AuNPs) and their antibacterial efficacy against the El Tor biotype of V. cholerae (VcN16961). The spherical-shaped NPs have an average diameter of â¼17 nm. The uniqueness of VL-AuNPs relies in the enhanced antibacterial efficacy compared to virstatin, as evidenced from the inhibitory concentration obtained from growth kinetics, and attributed to the inhibition of ATPase activity and DNA damage. More importantly, the expression of cholera toxin, the most important virulence factor of V. cholera, is reduced to a far greater extent than by any of the component molecules. The effect of VL-AuNPs on VcN16961 was monitored using various assays such as confocal microscopy, FACS, fluorescence spectroscopy, and so on. Overall, VL-AuNPs could be a potential candidate for the use as an effective agent for combating diarrheal diseases caused by V. cholera.
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Antibacterianos/farmacología , Materiales Biocompatibles/farmacología , Butiratos/farmacología , Oro/farmacología , Nanopartículas del Metal/química , Naftalimidas/farmacología , Vibrio cholerae O1/efectos de los fármacos , Antibacterianos/síntesis química , Antibacterianos/química , Materiales Biocompatibles/síntesis química , Materiales Biocompatibles/química , Butiratos/química , Oro/química , Cinética , Ensayo de Materiales , Pruebas de Sensibilidad Microbiana , Estructura Molecular , Naftalimidas/química , Tamaño de la Partícula , Vibrio cholerae O1/crecimiento & desarrolloRESUMEN
Transepithelial K+ absorption requires apical K+ uptake and basolateral K+ exit. In the colon, apical H+-K+-ATPase mediates cellular K+ uptake, and it has been suggested that electroneutral basolateral K+ exit reflects K+-Cl- cotransporter-1 (KCC1) operating in parallel with K+ and Cl- channels. The present study was designed to identify basolateral transporter(s) responsible for K+ exit in rat distal colon. Active K+ absorption was determined by measuring 86Rb+ (K+ surrogate) fluxes across colonic epithelia under voltage-clamp conditions. With zero Cl- in the mucosal solution, net K+ absorption was reduced by 38%, indicating that K+ absorption was partially Cl--dependent. Serosal addition of DIOA (KCC1 inhibitor) or Ba2+ (nonspecific K+ channel blocker) inhibited net K+ absorption by 21% or 61%, respectively, suggesting that both KCC1 and K+ channels contribute to basolateral K+ exit. Clotrimazole and TRAM34 (IK channel blockers) added serosally inhibited net K+ absorption, pointing to the involvement of IK channels in basolateral K+ exit. GaTx2 (CLC2 blocker) added serosally also inhibited net K+ absorption, suggesting that CLC2-mediated Cl- exit accompanies IK channel-mediated K+ exit across the basolateral membrane. Net K+ absorption was not inhibited by serosal addition of either IbTX (BK channel blocker), apamin (SK channel blocker), chromanol 293B (KV7 channel blocker), or CFTRinh172 (CFTR blocker). Immunofluorescence studies confirmed basolateral membrane colocalization of CLC2-like proteins and Na+-K+-ATPase α-subunits. We conclude that active K+ absorption in rat distal colon involves electroneutral basolateral K+ exit, which may reflect IK and CLC2 channels operating in parallel.NEW & NOTEWORTHY This study demonstrates that during active electroneutral K+ absorption in rat distal colon, K+ exit across the basolateral membrane mainly reflects intermediate conductance K+ channels operating in conjunction with chloride channel 2, with a smaller, but significant, contribution from K+-Cl- cotransporter-1 (KCC1) activity.
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Canales de Cloruro/metabolismo , Colon/fisiología , Mucosa Intestinal/metabolismo , Canales de Potasio/metabolismo , Potasio/metabolismo , Animales , Canales de Cloruro CLC-2 , Canales de Cloruro/genética , Cloruros/metabolismo , Femenino , Transporte Iónico , Masculino , Técnicas de Placa-Clamp , Canales de Potasio/genética , Transporte de Proteínas , Ratas , Ratas Sprague-DawleyRESUMEN
The pathophysiological nature of the common ABCG2 gout and hyperuricemia associated variant Q141K (rs2231142) remains undefined. Here, we use a human interventional cohort study (ACTRN12615001302549) to understand the physiological role of ABCG2 and find that participants with the Q141K ABCG2 variant display elevated serum urate, unaltered FEUA, and significant evidence of reduced extra-renal urate excretion. We explore mechanisms by generating a mouse model of the orthologous Q140K Abcg2 variant and find male mice have significant hyperuricemia and metabolic alterations, but only subtle alterations of renal urate excretion and ABCG2 abundance. By contrast, these mice display a severe defect in ABCG2 abundance and function in the intestinal tract. These results suggest a tissue specific pathobiology of the Q141K variant, support an important role for ABCG2 in urate excretion in both the human kidney and intestinal tract, and provide insight into the importance of intestinal urate excretion for serum urate homeostasis.
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Transportador de Casetes de Unión a ATP, Subfamilia G, Miembro 2/metabolismo , Gota/metabolismo , Hiperuricemia/metabolismo , Ácido Úrico/metabolismo , Transportador de Casetes de Unión a ATP, Subfamilia G, Miembro 2/genética , Alelos , Animales , Modelos Animales de Enfermedad , Epitelio/metabolismo , Epitelio/patología , Proteínas Facilitadoras del Transporte de la Glucosa/genética , Proteínas Facilitadoras del Transporte de la Glucosa/metabolismo , Gota/genética , Gota/patología , Homeostasis , Humanos , Intestinos/patología , Riñón/metabolismo , Riñón/patología , Masculino , Ratones , Ratones Endogámicos C57BL , Ratones Noqueados , Proteínas de Neoplasias , Fenotipo , Ácido Úrico/sangreRESUMEN
Cl- secretion by the human and murine intestinal epithelium occurs through the cystic fibrosis transmembrane conductance regulator (cftr). However, the Ca2+ activated Cl- channel Tmem16a was shown to contribute to Cl- secretion, mainly, but not exclusively, as a basolaterally located Cl- channel that controls basolateral Ca2+ signaling, and thus activation of basolateral Ca2+ dependent Sk4 K+ channels. In intestinal goblet cells, Tmem16a was shown to regulated Ca2+ signals required for exocytosis of mucus. Because a recent report denied the existence and functional role of Tmem16a in murine intestine, we reexamined in detail expression of mRNA and protein for Tmem16a in mouse colon. In experiments using short-circuited Ussing chamber and whole cell patch-clamp techniques, we further compared ion transport in wild type (WT) colon with that in mice with intestinal epithelial specific knockout of Tmem16a. As reported earlier we fully confirm expression of Tmem16a in colonic epithelial cells and the role of Tmem16a for both Ca2+-dependent and cAMP-regulated ion secretion.
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Whether zinc (Zn2+) regulates barrier functions by modulating tight-junction (TJ) proteins when pathogens such as Shigella alter epithelial permeability is still unresolved. We investigated the potential benefits of Zn2+ in restoring impaired barrier function in vivo in Shigella-infected mouse tissue and in vitro in T84 cell monolayers. Basolateral Shigella infection triggered a time-dependent decrease in transepithelial resistance followed by an increase in paracellular permeability of FITC-labeled dextran and altered ion selectivity. This led to ion and water loss into the intestinal lumen. Immunofluorescence studies revealed redistribution of claudin-2 and -4 to an intracellular location and accumulation of these proteins in the cytoplasm following infection. Zn2+ ameliorated this perturbed barrier by redistribution of claudin-2 and -4 back to the plasma membrane and by modulating the phosphorylation state of TJ proteins t hough extracellular signal-regulated kinase (ERK)1/2 dependency. Zn2+ prevents elevation of IL-6 and IL-8. Mice challenged with Shigella showed that oral Zn2+supplementation diminished diverse pathophysiological symptoms of shigellosis. Claudin-2 and -4 were susceptible to Shigella infection, resulting in altered barrier function and increased levels of IL-6 and IL-8. Zn2+ supplementation ameliorated this barrier dysfunction, and the inflammatory response involving ERK-mediated change of phosphorylation status for claudin-2 and -4. Thus, Zn2+ may have potential therapeutic value in inflammatory diarrhea and shigellosis. NEW & NOTEWORTHY Our study addresses whether Zn2+ could be an alternative strategy to reduce Shigella-induced inflammatory response and epithelial barrier dysfunction. We have defined a mechanism in terms of intracellular signaling pathways and tight-junction protein expression by Zn2+. Claudin-2 and -4 are susceptible to Shigella infection, whereas in the presence of Zn2+ they are resistant to infection-related barrier dysfunction involving ERK-mediated change of phosphorylation status of claudins.
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Claudina-2/metabolismo , Claudina-4/metabolismo , Permeabilidad/efectos de los fármacos , Zinc/farmacología , Animales , Claudina-2/efectos de los fármacos , Claudina-4/efectos de los fármacos , Células Epiteliales/efectos de los fármacos , Células Epiteliales/metabolismo , Enfermedades Intestinales/tratamiento farmacológico , Enfermedades Intestinales/metabolismo , Masculino , Ratones Endogámicos BALB C , Ratones Endogámicos C57BL , Proteína Quinasa 3 Activada por Mitógenos/efectos de los fármacos , Proteína Quinasa 3 Activada por Mitógenos/metabolismo , Proteínas Quinasas Activadas por Mitógenos/efectos de los fármacos , Proteínas Quinasas Activadas por Mitógenos/metabolismo , Uniones Estrechas/efectos de los fármacos , Uniones Estrechas/metabolismo , Zinc/metabolismoRESUMEN
BACKGROUND: Accessory cholera enterotoxin (Ace) is a classical enterotoxin produced by Vibrio cholerae, the causative agent for cholera. Considering the crucial role of Ace in pathogenesis of cholera, we explored the modulation of structure/function of Ace using gold nanoparticles (AuNPs) of different size and shape - spherical (AuNS10 and AuNS100, the number indicating the diameter in nm) and rod (AuNR10). METHODS: Biophysical techniques have been used to find out structural modulation of Ace by AuNPs. Effect of AuNP on Ace conformation was monitored by far-UV CD; urea-induced unfolding and binding of Ace to various AuNPs were studied by tryptophan fluorescence. In vivo experiments using mouse ileal loop and Ussing chamber were carried out to corroborate biophysical data. RESULTS: Biophysical data revealed degradation of Ace by AuNR10 and AuNS100, not by AuNS10. The feature of AuNR10 having high aspect ratio, but with the same transverse diameter as that of AuNS10 enabled us to explore the importance of morphology on modulation of protein structure/function. The equilibration time for adsorption shows dependence on the radius of curvature, being largest for AuNR10. In vivo experiments revealed the efficacy of AuNR10 and AuNS100 for reduced fluid accumulation, indicative of the loss of activity of Ace. CONCLUSIONS: We show how biophysical studies and in vivo experiments go hand-in-hand in establishing the efficacy and role of size/shape of AuNPs on a toxin structure. GENERAL SIGNIFICANCE: The effect of AuNP on toxin depends on its morphology. The targeted modulation of Ace could be of therapeutic benefit for gastrointestinal disorders.
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Toxina del Cólera/química , Toxina del Cólera/metabolismo , Oro/química , Nanopartículas del Metal/química , Vibrio cholerae/química , Vibrio cholerae/metabolismo , Animales , Masculino , Ratones , Ratones Endogámicos C57BL , Tamaño de la Partícula , Relación Estructura-ActividadRESUMEN
Accessory cholera enterotoxin (Ace) of Vibrio cholerae has been shown to contribute to diarrhea. However, the signaling mechanism and specific type of Cl- channel activated by Ace are still unknown. We have shown here that the recombinant Ace protein induced ICl of apical plasma membrane, which was inhibited by classical CaCC blockers. Surprisingly, an Ace-elicited rise of current was neither affected by ANO1 (TMEM16A)-specific inhibitor T16A(inh)-AO1(TAO1) nor by the cystic fibrosis transmembrane conductance regulator (CFTR) blocker, CFTR inh-172. Ace stimulated whole-cell current in Caco-2 cells. However, the apical ICl was attenuated by knockdown of ANO6 (TMEM16F). This impaired phenotype was restored by re-expression of ANO6 in Caco-2 cells. Whole-cell patch clamp recordings of ANO currents in HEK293 cells transiently expressing mouse ANO1-mCherry or ANO6-GFP confirmed that Ace induced Cl- secretion. Application of Ace produced ANO6 but not the ANO1 currents. Ace was not able to induce a [Ca2+]i rise in Caco-2 cells, but cellular abundance of phosphatidylinositol 4,5-bisphosphate (PIP2) increased. Identification of the PIP2-binding motif at the N-terminal sequence among human and mouse ANO6 variants along with binding of PIP2 directly to ANO6 in HEK293 cells indicate likely PIP2 regulation of ANO6. The biophysical and pharmacological properties of Ace stimulated Cl- current along with intestinal fluid accumulation, and binding of PIP2 to the proximal KR motif of channel proteins, whose mutagenesis correlates with altered binding of PIP2, is comparable with ANO6 stimulation. We conclude that ANO6 is predominantly expressed in intestinal epithelia, where it contributes secretory diarrhea by Ace stimulation in a calcium-independent mechanism of RhoA-ROCK-PIP2 signaling.
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Cloruros/metabolismo , Toxina del Cólera/toxicidad , Cólera/metabolismo , Proteínas de Transferencia de Fosfolípidos/metabolismo , Fosfotransferasas (Aceptor de Grupo Alcohol)/metabolismo , Proteína de Unión al GTP rhoA/metabolismo , Secuencia de Aminoácidos , Animales , Anoctaminas , Secuencia de Bases , Sistemas CRISPR-Cas , Células CACO-2 , Calcio/metabolismo , Cólera/inducido químicamente , Regulador de Conductancia de Transmembrana de Fibrosis Quística/antagonistas & inhibidores , Células HEK293 , Humanos , Mucosa Intestinal/efectos de los fármacos , Mucosa Intestinal/metabolismo , Mucosa Intestinal/virología , Masculino , Ratones , Ratones Endogámicos C57BL , Técnicas de Placa-Clamp , Proteínas de Transferencia de Fosfolípidos/antagonistas & inhibidores , Proteínas de Transferencia de Fosfolípidos/genética , Fosfotransferasas (Aceptor de Grupo Alcohol)/antagonistas & inhibidores , Fosfotransferasas (Aceptor de Grupo Alcohol)/genética , Homología de Secuencia de Aminoácido , Transducción de Señal/efectos de los fármacos , Vibrio cholerae/patogenicidad , Proteína de Unión al GTP rhoA/antagonistas & inhibidores , Proteína de Unión al GTP rhoA/genéticaRESUMEN
The potency of zinc oxide nanoparticles (NPs), with a core size of ~7-10nm, to inhibit cholera disease was investigated by demonstrating the effect on two biotypes (classical and El Tor) of O1 serogroup of Vibrio cholerae-El Tor was more susceptible both in planktonic and in biofilm forms. Interaction with ZnO NP results in deformed cellular architecture. Increased fluidity and depolarization of membrane, and protein leakage further confirmed the damages inflicted on Vibrio by NP. NP was shown to produce reactive oxygen species (ROS) and induce DNA damage. These results suggest that the antibacterial mechanism of ZnO action is most likely due to generation of ROS and disruption of bacterial membrane. The antimicrobial efficacy of NP has been validated in animal model. The synergistic action of NP and antibiotic suggests an alternative for the treatment of cholera.
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Antiinfecciosos/farmacología , Nanopartículas , Vibrio cholerae/efectos de los fármacos , Óxido de Zinc , Animales , Cólera/tratamiento farmacológicoRESUMEN
Cholera pathogenesis occurs due to synergistic pro-secretory effects of several toxins, such as cholera toxin (CTX) and Accessory cholera enterotoxin (Ace) secreted by Vibrio cholerae strains. Ace activates chloride channels stimulating chloride/bicarbonate transport that augments fluid secretion resulting in diarrhea. These channels have been targeted for drug development. However, lesser attention has been paid to the interaction of chloride channel modulators with bacterial toxins. Here we report the modulation of the structure/function of recombinant Ace by small molecule calcium-activated chloride channel (CaCC) inhibitors, namely CaCCinh-A01, digallic acid (DGA) and tannic acid. Biophysical studies indicate that the unfolding (induced by urea) free energy increases upon binding CaCCinh-A01 and DGA, compared to native Ace, whereas binding of tannic acid destabilizes the protein. Far-UV CD experiments revealed that the α-helical content of Ace-CaCCinh-A01 and Ace-DGA complexes increased relative to Ace. In contrast, binding to tannic acid had the opposite effect, indicating the loss of protein secondary structure. The modulation of Ace structure induced by CaCC inhibitors was also analyzed using docking and molecular dynamics (MD) simulation. Functional studies, performed using mouse ileal loops and Ussing chamber experiments, corroborate biophysical data, all pointing to the fact that tannic acid destabilizes Ace, inhibiting its function, whereas DGA stabilizes the toxin with enhanced fluid accumulation in mouse ileal loop. The efficacy of tannic acid in mouse model suggests that the targeted modulation of Ace structure may be of therapeutic benefit for gastrointestinal disorders.
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Canales de Cloruro/antagonistas & inhibidores , Toxina del Cólera/fisiología , Vibrio cholerae/fisiología , Animales , Cólera/fisiopatología , Toxina del Cólera/antagonistas & inhibidores , Dicroismo Circular , Depsidos/farmacología , Diarrea/fisiopatología , Ácido Gálico/análogos & derivados , Ácido Gálico/farmacología , Masculino , Ratones , Ratones Endogámicos C57BL , Simulación del Acoplamiento Molecular , Proteínas Recombinantes , Espectrometría de Fluorescencia , Taninos/farmacología , Tiofenos/farmacologíaRESUMEN
The apical membrane of intestinal epithelia expresses intermediate conductance K(+) channel (KCNN4), which provides the driving force for Cl(-) secretion. However, its role in diarrhea and regulation by Epac1 is unknown. Previously we have established that Epac1 upon binding of cAMP activates a PKA-independent mechanism of Cl(-) secretion via stimulation of Rap2-phospholipase Cε-[Ca(2+)]i signaling. Here we report that Epac1 regulates surface expression of KCNN4c channel through its downstream Rap1A-RhoA-Rho-associated kinase (ROCK) signaling pathway for sustained Cl(-) secretion. Depletion of Epac1 protein and apical addition of TRAM-34, a specific KCNN4 inhibitor, significantly abolished cAMP-stimulated Cl(-) secretion and apical K(+) conductance (IK(ap)) in T84WT cells. The current-voltage relationship of basolaterally permeabilized monolayers treated with Epac1 agonist 8-(4-chlorophenylthio)-2'-O- methyladenosine 3',5'-cyclic monophosphate showed the presence of an inwardly rectifying and TRAM-34-sensitive K(+) channel in T84WT cells that was absent in Epac1KDT84 cells. Reconstructed confocal images in Epac1KDT84 cells revealed redistribution of KCNN4c proteins into subapical intracellular compartment, and a biotinylation assay showed â¼83% lower surface expression of KCNN4c proteins compared with T84WT cells. Further investigation revealed that an Epac1 agonist activates Rap1 to facilitate IK(ap). Both RhoA inhibitor (GGTI298) and ROCK inhibitor (H1152) significantly reduced cAMP agonist-stimulated IK(ap), whereas the latter additionally reduced colocalization of KCNN4c with the apical membrane marker wheat germ agglutinin in T84WT cells. In vivo mouse ileal loop experiments showed reduced fluid accumulation by TRAM-34, GGTI298, or H1152 when injected together with cholera toxin into the loop. We conclude that Rap1A-dependent signaling of Epac1 involving RhoA-ROCK is an important regulator of intestinal fluid transport via modulation of apical KCNN4c channels, a finding with potential therapeutic value in diarrheal diseases.
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Cloruros/metabolismo , Diarrea/metabolismo , Factores de Intercambio de Guanina Nucleótido/metabolismo , Canales de Potasio de Conductancia Intermedia Activados por el Calcio/metabolismo , 1-(5-Isoquinolinesulfonil)-2-Metilpiperazina/análogos & derivados , 1-(5-Isoquinolinesulfonil)-2-Metilpiperazina/farmacología , Animales , Western Blotting , Línea Celular Tumoral , Toxina del Cólera , Colforsina/farmacología , AMP Cíclico/farmacología , Diarrea/inducido químicamente , Células Epiteliales/efectos de los fármacos , Células Epiteliales/metabolismo , Factores de Intercambio de Guanina Nucleótido/antagonistas & inhibidores , Factores de Intercambio de Guanina Nucleótido/genética , Humanos , Canales de Potasio de Conductancia Intermedia Activados por el Calcio/antagonistas & inhibidores , Mucosa Intestinal/efectos de los fármacos , Mucosa Intestinal/metabolismo , Ratones , Microscopía Confocal , Bloqueadores de los Canales de Potasio/farmacología , Pirazoles/farmacología , Interferencia de ARN , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa , Transducción de Señal/efectos de los fármacos , Proteínas de Unión al GTP rap/genética , Proteínas de Unión al GTP rap/metabolismo , Proteínas de Unión al GTP rap1/genética , Proteínas de Unión al GTP rap1/metabolismo , Quinasas Asociadas a rho/antagonistas & inhibidores , Quinasas Asociadas a rho/metabolismo , Proteína de Unión al GTP rhoA/antagonistas & inhibidores , Proteína de Unión al GTP rhoA/metabolismoRESUMEN
Diarrhea remains a continuous threat to human health worldwide. Scaling up the best practices for diarrhea prevention requires improved therapies. Diarrhea results from dysregulation of normal intestinal ion transport functions. Host-microbe contact is a key determinant of this response. Underlying mechanisms in the disease state are regulated by intracellular signals that modulate the activity of individual transport proteins responsible for ion transport and barrier function. Similarly, virulence factors of pathogens and their complex interaction with the host has shed light on the mechanism of enteric infection. Great advances in our understanding of the pathophysiologic mechanisms of epithelial transport, and host-microbe interaction have been made in recent years. Application of these new advances may represent strategies to decrease pathogen attachment, enhance intestinal cation absorption, decrease anion secretion and repair barrier function. This review highlights the new advances and better understanding in the pathophysiology of diarrheal diseases and their impact on therapy.
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Diarrea/fisiopatología , Diarrea/terapia , Intestinos/fisiopatología , Transporte Iónico/fisiología , Uniones Estrechas/fisiología , Diarrea/prevención & control , Células Epiteliales/citología , Humanos , Infecciones/fisiopatología , Infecciones/terapia , Intestinos/citología , Intestinos/fisiologíaRESUMEN
Vibrio cholerae accessory cholera enterotoxin (Ace) is the third toxin, along with cholera toxin (CT) and zonula occludens toxin (Zot), that causes the endemic disease cholera. Structural characterization of Ace has been restricted because of the limited production of this toxic protein by V. cholerae. We have cloned, overexpressed, and purified Ace from V. cholerae strain O395 in Escherichia coli to homogeneity and determined its biological activity. The unfolding of the purified protein was investigated using circular dichroism and intrinsic tryptophan fluorescence. Because Ace is predominantly a hydrophobic protein, the degree of exposure of hydrophobic regions was identified from the spectral changes of the environment-sensitive fluorescent probe 4,4'-dianilino-1,1'-binaphthyl-5,5'-disulfonic acid (bis-ANS) that quenches the fluorescence of tryptophan residues of Ace in a concentration-dependent manner. Results showed that bis-ANS binds one monomeric unit of Ace with a 1:1 stoichiometry and a K' of 0.72 µM. Ace exists as a dimer, with higher oligomeric forms appearing upon glutaraldehyde cross-linking. This study also reports the binding of virstatin, a small molecule that inhibits virulence regulation in V. cholerae, to Ace. The binding constant (K=9×10(4) M(-1)) and the standard free energy change (ΔG°=-12 kcal mol(-1)) of Ace-virstatin interaction have been evaluated by the fluorescence quenching method. The binding does not affect the oligomeric status of Ace. A cell viability assay of the antibacterial activity of Ace has been performed using various microbial strains. A homology model of Ace, consistent with the experimental results, has been constructed.
Asunto(s)
Toxina del Cólera/química , Toxina del Cólera/metabolismo , Vibrio cholerae/metabolismo , Algoritmos , Secuencia de Aminoácidos , Bacterias/efectos de los fármacos , Bacterias/crecimiento & desarrollo , Secuencia de Bases , Butiratos/química , Butiratos/metabolismo , Toxina del Cólera/genética , Dicroismo Circular , Relación Dosis-Respuesta a Droga , Glutaral/química , Cinética , Viabilidad Microbiana/efectos de los fármacos , Modelos Moleculares , Datos de Secuencia Molecular , Naftalimidas/química , Naftalimidas/metabolismo , Unión Proteica , Multimerización de Proteína , Estructura Secundaria de Proteína , Estructura Terciaria de Proteína , Desplegamiento Proteico , Proteínas Recombinantes/química , Proteínas Recombinantes/metabolismo , Proteínas Recombinantes/toxicidad , Homología de Secuencia de Aminoácido , Homología de Secuencia de Ácido Nucleico , Espectrometría de Fluorescencia , Vibrio cholerae/genéticaRESUMEN
Intestinal Cl- secretion is stimulated by cyclic AMP (cAMP) and intracellular calcium ([Ca2+]i). Recent studies show that protein kinase A (PKA) and the exchange protein directly activated by cAMP (Epac) are downstream targets of cAMP. Therefore, we tested whether both PKA and Epac are involved in forskolin (FSK)/cAMP-stimulated Cl- secretion. Human intestinal T84 cells and mouse small intestine were used for short circuit current (I(sc)) measurement in response to agonist-stimulated Cl- secretion. FSK-stimulated Cl- secretion was completely inhibited by the additive effects of the PKA inhibitor, H89 (1 microM), and the [Ca2+]i chelator, 1,2-bis-(o-aminophenoxy)-ethane-N,N,N',N'-tetraacetic acid, tetraacetoxymethyl ester (BAPTA-AM; 25 microM). Both FSK and the Epac activator 8-pCPT-2'-O-Me-cAMP (50 microM) elevated [Ca2+]i, activated Ras-related protein 2, and induced Cl- secretion in intact or basolateral membrane-permeabilized T84 cells and mouse ileal sheets. The effects of 8-pCPT-2'-O-Me-cAMP were completely abolished by BAPTA-AM, but not by H89. In contrast, T84 cells with silenced Epac1 had a reduced I(sc) response to FSK, and this response was completely inhibited by H89, but not by the phospholipase C inhibitor U73122 or BAPTA-AM. The stimulatory effect of 8-pCPT-2'-O-Me-cAMP on Cl- secretion was not abolished by cystic fibrosis transmembrane conductance (CFTR) inhibitor 172 or glibenclamide, suggesting that CFTR channels are not involved. This was confirmed by lack of effect of 8-pCPT-2'-O-Me-cAMP on whole cell patch clamp recordings of CFTR currents in Chinese hamster ovary cells transiently expressing the human CFTR channel. Furthermore, biophysical characterization of the Epac1-dependent Cl- conductance of T84 cells mounted in Ussing chambers suggested that this conductance was hyperpolarization activated, inwardly rectifying, and displayed a Cl->Br->I- permeability sequence. These results led us to conclude that the Epac-Rap-PLC-[Ca2+]i signaling pathway is involved in cAMP-stimulated Cl- secretion, which is carried by a novel, previously undescribed Cl- channel.
Asunto(s)
Cloro/metabolismo , Colforsina/administración & dosificación , Proteínas Quinasas Dependientes de AMP Cíclico/metabolismo , AMP Cíclico/administración & dosificación , Factores de Intercambio de Guanina Nucleótido/metabolismo , Mucosa Intestinal/metabolismo , Animales , Células Cultivadas , Humanos , Mucosa Intestinal/efectos de los fármacos , RatonesRESUMEN
An increasing amount of data showing the beneficial use of zinc (Zn) in treating diarrhea continues to emerge from epidemiological and clinical trials. However, without a thorough understanding of physiological mechanisms of Zn, it does not support policy recommendation to advocate the therapeutic use of Zn. Our data demonstrate that Zn is a potential antidiarrheal agent that provides substantial benefit by stimulating sodium absorption and inhibiting chloride secretion in intestinal epithelial cells. Thus, inclusion of Zn in oral rehydration solution (ORS) has the potential to markedly augment the effectiveness of ORS in the treatment of diarrhea.
Asunto(s)
Antidiarreicos/uso terapéutico , Diarrea/tratamiento farmacológico , Diarrea/etiología , Zinc/uso terapéutico , Células CACO-2 , AMP Cíclico/metabolismo , Fluidoterapia , Humanos , Absorción Intestinal , Mucosa Intestinal/metabolismo , Intercambiadores de Sodio-Hidrógeno/metabolismo , Zinc/administración & dosificación , Compuestos de Zinc/uso terapéuticoRESUMEN
Nucleoside and nucleobase transporters are important for salvage of purines and pyrimidines and for transport of their analog drugs into cells. However, the pathways for nucleobase translocation in mammalian cells are not well characterized. We identified an Na-independent purine-selective nucleobase/nucleoside transport system in the nucleoside transporter-deficient PK15NTD cells. This transport system has 1,000-fold higher affinity for nucleobases than nucleosides with K(m) values of 2.5 +/- 0.7 microM for [(3)H]adenine, 6.4 +/- 0.5 microM for [(3)H]guanine, 1.1 +/- 0.1 mM for [(3)H]guanosine, and 4.2 +/- 0.5 mM [(3)H]adenosine. The uptake of [(3)H]guanine (0.05 microM) was inhibited by other nucleobases and nucleobase analog drugs (at 0.5-1 mM in the order of potency): 6-mercaptopurine = thioguanine = guanine > adenine >>> thymine = fluorouracil = uracil. Cytosine and methylcytosine had no effect. Nucleoside analog drugs with modification at 2' and/or 5 positions (all at 1 mM) were more potent than adenosine in competing the uptake of [(3)H]guanine: 2-chloro-2'-deoxyadenosine > 2-chloroadenosine > 2'3'-dideoxyadenosine = 2'-deoxyadenosine > 5-deoxyadenosine > adenosine. 2-Chloro-2'-deoxyadenosine and 2-chloroadenosine inhibited [(3)H]guanine uptake with IC(50) values of 68 +/- 5 and 99 +/- 10 microM, respectively. The nucleobase/nucleoside transporter was resistant to nitrobenzylthioinosine {6-[(4-nitrobenzyl) thiol]-9-beta-D-ribofuranosylpurine}, dipyridamole, and dilazep, but was inhibited by papaverine, the organic cation transporter inhibitor decynium-22 (IC(50) of approximately 1 microM), and by acidic pH (pH = 5.5). In conclusion, we have identified a mammalian purine-selective nucleobase/nucleoside transporter with high affinity for purine nucleobases. This transporter is potentially important for transporting naturally occurring purines and purine analog drugs into cells.
Asunto(s)
Riñón/metabolismo , Proteínas de Transporte de Nucleobases/metabolismo , Purinas/metabolismo , Adenosina/metabolismo , Animales , Línea Celular , Guanosina/metabolismo , Riñón/citología , Riñón/efectos de los fármacos , Papaverina/farmacología , Quinolinas/farmacología , Porcinos , TritioRESUMEN
The improved treatment of acute diarrhea in children during the past 35 years has reduced its morbidity and mortality substantially. However, better therapy still is required. This article reviews the role of oral rehydration solution in the treatment of acute diarrhea with particular attention to recent efforts to develop improved oral rehydration solution formulations. One promising approach is the administration of Zinc (Zn). Based on its beneficial effects in infections, including pneumonia, Zn has been shown to be effective in the treatment of acute diarrhea in several randomized controlled trials including subsequent meta-analyses. Thus, an emerging body of clinical data indicates that Zn can be useful for treating acute diarrhea. However, only limited information is known about the mechanism(s) by which Zn reduces diarrhea. Recent studies have indicated that Zn acts as a K channel blocker of adenosine 3',5'-cyclic monophosphate-mediated chlorine secretion, but may not affect either Ca2+- or guanosine 3',5'-cyclic monophosphate-mediated chlorine secretion. These data provide a strong rationale for further trials testing its efficacy in specific clinical settings and for more detailed physiologic studies examining how Zn exerts its antidiarrheal effect.
Asunto(s)
Diarrea/diagnóstico , Diarrea/tratamiento farmacológico , Compuestos de Zinc/uso terapéutico , Enfermedad Aguda , Preescolar , Ensayos Clínicos como Asunto , Diarrea Infantil/diagnóstico , Diarrea Infantil/tratamiento farmacológico , Relación Dosis-Respuesta a Droga , Esquema de Medicación , Femenino , Humanos , Lactante , Masculino , Pronóstico , Índice de Severidad de la Enfermedad , Resultado del TratamientoRESUMEN
Zn, an essential micronutrient and second most abundant trace element in cell and tissues, reduces stool output when administered to children with acute diarrhea. The mechanism by which Zn improves diarrhea is not known but could result from stimulating Na absorption and/or inhibiting anion secretion. The aim of this study was to investigate the direct effect of Zn on intestinal epithelial ion absorption and secretion. Rat ileum was partially stripped of serosal and muscle layers, and the mucosa was mounted in lucite chambers. Potential difference and short-circuit current were measured by conventional current-voltage clamp method. 86Rb efflux and uptake were assessed for serosal K channel and Na-K-2Cl cotransport activity, respectively. Efflux experiments were performed in isolated cells preloaded with 86Rb in the presence of ouabain and bumetanide, whereas uptake experiments were performed in low-Cl isotonic buffer containing Ba and ouabain. Neither mucosal nor serosal Zn affected glucose-stimulated Na absorption. In contrast, forskolin-induced Cl secretion was markedly reduced by serosal but not mucosal addition of Zn. Zn also substantially reversed the increase in Cl secretion induced by 8-bromoadenosine 3',5'-cyclic monophosphate (8-BrcAMP) with half-maximal inhibitory concentration of 0.43 mM. In contrast, serosal Zn did not alter Cl secretion stimulated by carbachol, a Ca-dependent agonist. Zn inhibited 8-BrcAMP-stimulated 86Rb efflux but not carbachol-stimulated 86Rb efflux. Zn had no effect on bumetanide-sensitive 86Rb uptake, Na-K-ATPase, or CFTR. We conclude from these studies that Zn inhibits cAMP-induced Cl secretion by blocking basolateral membrane K channels.