RESUMEN
Fatty acids (including lubiprostone and cobiprostone) are human ClC-2 (hClC-2) Cl- channel activators. Molecular and cellular mechanisms underlying this activation were examined. Role of a four-amino acid PKA activation site, RGET691, of hClC-2 was investigated using wild-type (WT) and mutant (AGET, RGEA, and AGAA) hClC-2 expressed in 293EBNA cells as well as involvement of PKA, intracellular cAMP concentration ([cAMP]i), EP2, or EP4 receptor agonist activity. All fatty acids [lubiprostone, cobiprostone, eicosatetraynoic acid (ETYA), oleic acid, and elaidic acid] caused significant rightward shifts in concentration-dependent Cl- current activation (increasing EC50s) with mutant compared with WT hClC-2 channels, without changing time and voltage dependence, current-voltage rectification, or methadone inhibition of the channel. As with lubiprostone, cobiprostone activation of hClC-2 occurred with PKA inhibitor (myristoylated protein kinase inhibitor) present or when using double PKA activation site (RRAA655/RGEA691) mutant. Cobiprostone did not activate human CFTR. Fatty acids did not increase [cAMP]i in hClC-2/293EBNA or T84 cells. Using T84 CFTR knockdown cells, cobiprostone increased hClC-2 Cl- currents without increasing [cAMP]i, while PGE2 and forskolin-IBMX increased both. Fatty acids were not agonists of EP2 or EP4 receptors. L-161,982, a supposed EP4-selective inhibitor, had no effect on lubiprostone-activated hClC-2 Cl- currents but significantly decreased T84 cell barrier function measured by transepithelial resistance and fluorescent dextran transepithelial movement. The present findings show that RGET691 of hClC-2 (possible binding site) plays an important functional role in fatty acid activation of hClC-2. PKA, [cAMP]i, and EP2 or EP4 receptors are not involved. These studies provide the molecular basis for fatty acid regulation of hClC-2.
Asunto(s)
Canales de Cloruro/metabolismo , Lubiprostona/farmacología , Prostaglandinas/farmacología , Tiofenos/farmacología , Triazoles/farmacología , 1-Metil-3-Isobutilxantina/farmacología , Secuencias de Aminoácidos , Ácidos Araquidónicos/metabolismo , Sitios de Unión , Canales de Cloruro CLC-2 , Canales de Cloruro/química , Cloruros/metabolismo , Colforsina/farmacología , AMP Cíclico/metabolismo , Proteínas Quinasas Dependientes de AMP Cíclico/metabolismo , Regulador de Conductancia de Transmembrana de Fibrosis Quística/deficiencia , Regulador de Conductancia de Transmembrana de Fibrosis Quística/metabolismo , Células HEK293 , Humanos , Transporte Iónico , Cinética , Lubiprostona/química , Metadona/farmacología , Ácido Oléico/metabolismo , Ácidos Oléicos , Prostaglandinas/química , Unión Proteica , Inhibidores de Proteínas Quinasas/farmacología , Subtipo EP2 de Receptores de Prostaglandina E/metabolismo , Subtipo EP4 de Receptores de Prostaglandina E/metabolismo , Tiofenos/química , Triazoles/químicaAsunto(s)
Ácido Clorhídrico/metabolismo , Modelos Biológicos , Células Parietales Gástricas/fisiología , Potasio/fisiología , Estómago/fisiología , Animales , Antiulcerosos/farmacología , Transporte Biológico Activo , Cloruros , Simulación por Computador , ATPasa Intercambiadora de Hidrógeno-Potásio/metabolismo , Omeprazol/farmacología , ATPasa Intercambiadora de Sodio-Potasio/metabolismo , Programas InformáticosRESUMEN
The present studies were designed to examine the effects of ClC-2 ablation on cellular morphology, parietal cell abundance, H/K ATPase expression, parietal cell ultrastructure and acid secretion using WT and ClC-2-/- mouse stomachs. Cellular histology, morphology and proteins were examined using imaging techniques, electron microscopy and western blot. The effect of histamine on the pH of gastric contents was measured. Acid secretion was also measured using methods and secretagogues previously established to give maximal acid secretion and morphological change. Compared to WT, ClC-2-/- gastric mucosal histological organization appeared disrupted, including dilation of gastric glands, shortening of the gastric gland region and disorganization of all cell layers. Parietal cell numbers and H/K ATPase expression were significantly reduced by 34% (P<0.05) and 53% (P<0.001) respectively and cytoplasmic tubulovesicles appeared markedly reduced on electron microscopic evaluation without evidence of canalicular expansion. In WT parietal cells, ClC-2 was apparent in a similar cellular location as the H/K ATPase by immunofluorescence and appeared associated with tubulovesicles by immunogold electron microscopy. Histamine-stimulated [H+] of the gastric contents was significantly (P<0.025) lower by 9.4 fold (89%) in the ClC-2-/- mouse compared to WT. Histamine/carbachol stimulated gastric acid secretion was significantly reduced (range 84-95%, P<0.005) in ClC-2-/- compared to WT, while pepsinogen secretion was unaffected. Genetic ablation of ClC-2 resulted in reduced gastric gland region, reduced parietal cell number, reduced H/K ATPase, reduced tubulovesicles and reduced stimulated acid secretion.
Asunto(s)
Canales de Cloruro/genética , Digestión/fisiología , Ácido Gástrico/metabolismo , ATPasa Intercambiadora de Hidrógeno-Potásio/biosíntesis , Células Parietales Gástricas/metabolismo , Animales , Transporte Biológico , Canales de Cloruro CLC-2 , Recuento de Células , Vesículas Citoplasmáticas/metabolismo , Digestión/genética , Técnica del Anticuerpo Fluorescente , ATPasa Intercambiadora de Hidrógeno-Potásio/metabolismo , Ratones , Ratones Noqueados , Microscopía Electrónica , Células Parietales Gástricas/ultraestructura , Pepsinógeno A/metabolismoRESUMEN
This investigation was conducted to study the relationship between intracellular Ca(2+) and activation of large conductance Ca(2+)-activated K(+) (BK) currents by unoprostone, the first synthetic docosanoid. We used HEK293 cells stably transfected with two BK channel splice variants, one sensitive to unoprostone and the other insensitive. We examined the effects of unoprostone on channel activity in excised inside-out patches and cell-attached patches. The half-maximal stimulation of the sensitive BK channels by Ca(2+) was shifted from 3.4±0.017 nM to 0.81±.0058 nM in the presence of 10 nM unoprostone. There was no effect on insensitive channels even at unoprostone concentrations as high as 1000 nM. There was no effect of unoprostone on the voltage dependence of the BK channels. Changes in open probability and effects of Ca(2+) and unoprostone were best described by a synergistic binding model. These data would suggest that Ca(2+) and unoprostone were binding to sites close to one another on the channel protein and that unoprostone binding causes the affinity of the calcium binding site to increase. This idea is consistent with three dimensional models of the Ca(2+) binding site and a putative unoprostone binding domain. Our results have important implications for the clinical use of unoprostone to activate BK channels. Channel activation will be limited if intracellular Ca(2+) is not elevated.
Asunto(s)
Calcio/metabolismo , Dinoprost/análogos & derivados , Activación del Canal Iónico/efectos de los fármacos , Subunidades alfa de los Canales de Potasio de Gran Conductancia Activados por Calcio/fisiología , Empalme Alternativo , Secuencia de Aminoácidos , Animales , Dinoprost/farmacología , Relación Dosis-Respuesta a Droga , Células HEK293 , Humanos , Espacio Intracelular/efectos de los fármacos , Espacio Intracelular/metabolismo , Subunidades alfa de los Canales de Potasio de Gran Conductancia Activados por Calcio/genética , Potenciales de la Membrana/efectos de los fármacos , Datos de Secuencia Molecular , Técnicas de Placa-Clamp , Isoformas de Proteínas/genética , Isoformas de Proteínas/fisiología , Ratas , Homología de Secuencia de Aminoácido , TransfecciónRESUMEN
It has been difficult to separate/identify the roles of ClC-2 and CFTR in Cl(-) transport studies. Using pharmacological agents, we aimed to differentiate functionally between ClC-2 and CFTR Cl(-) channel currents. Effects of CFTR inhibitor 172 (CFTRinh172), N-(4-methylphenylsulfonyl)-N'-(4-trifluoromethylphenyl)urea (DASU-02), and methadone were examined by whole cell patch clamp on Cl(-) currents in recombinant human ClC-2/human embryonic kidney 293 (ClC-2/HEK293) cells stably transformed with Epstein-Barr nuclear antigen 1 (hClC-2/293EBNA) and human CFTR/HEK293 (hCFTR/HEK293) cells and by short-circuit current (Isc) measurements in T84 cells. Lubiprostone and forskolin-IBMX were used as activators. CFTRinh172 inhibited forskolin-IBMX-stimulated recombinant human CFTR (hCFTR) and lubiprostone-stimulated recombinant human ClC-2 (hClC-2) Cl(-) currents in a concentration-dependent manner equipotently. DASU-02 inhibited forskolin-IBMX-stimulated Cl(-) currents in hCFTR/HEK293 cells, but not lubiprostone-stimulated Cl(-) currents in hClC-2/293EBNA cells. In T84 cells with basolateral nystatin or 1-ethyl-2-benzimidazolinone (1-EBIO), lubiprostone-stimulated and forskolin-IBMX-cyclosporin A (FICA)-stimulated Isc components were observed. CFTRinh172 inhibited major portions of both components. DASU-02 had no effect on lubiprostone-stimulated Isc but partially inhibited FICA-stimulated Isc. T84 cells in which ClC-2 or CFTR was knocked down using siRNAs were constructed. T84 ClC-2 knockdown cells did not respond to lubiprostone but did respond to forskolin-IBMX in a methadone-insensitive, DASU-02-sensitive manner, indicating CFTR function. T84 CFTR knockdown cells responded separately to lubiprostone and forskolin-IBMX in a methadone-sensitive and DASU-02-insensitive manner, indicating ClC-2 function. Low lubiprostone concentrations activated ClC-2, but not CFTR, and both channels were activated by forskolin-IBMX but have different inhibitor sensitivities. Methadone, but not DASU-02, inhibited ClC-2. DASU-02, but not methadone, inhibited CFTR. In T84 cells, both ClC-2 and CFTR are present and likely play roles in Cl(-) secretion.
Asunto(s)
Canales de Cloruro/antagonistas & inhibidores , Canales de Cloruro/fisiología , Regulador de Conductancia de Transmembrana de Fibrosis Quística/antagonistas & inhibidores , Regulador de Conductancia de Transmembrana de Fibrosis Quística/fisiología , 1-Metil-3-Isobutilxantina/farmacología , Canales de Cloruro CLC-2 , Línea Celular , Relación Dosis-Respuesta a Droga , Células Epiteliales/efectos de los fármacos , Células HEK293 , Humanos , Metadona/farmacología , Urea/análogos & derivados , Urea/farmacologíaRESUMEN
In clinical trials, methadone, but not morphine, appeared to prevent beneficial effects of lubiprostone, a ClC-2 Cl(-) channel activator, on opioid-induced constipation. Effects of methadone and morphine on lubiprostone-stimulated Cl(-) currents were measured by short circuit current (Isc) across T84 cells. Whole cell patch clamp of human ClC-2 (hClC-2) stably expressed in HEK293 cells and in a high expression cell line (HEK293EBNA) as well as human CFTR (hCFTR) stably expressed in HEK293 cells was used to study methadone and morphine effects on recombinant hClC-2 and hCFTR Cl(-) currents. Methadone but not morphine inhibited lubiprostone-stimulated Isc in T84 cells with half-maximal inhibition at 100 nM. Naloxone did not affect lubiprostone stimulation or methadone inhibition of Isc. Lubiprostone-stimulated Cl(-) currents in hClC-2/HEK293 cells, but not forskolin/IBMX-stimulated Cl(-) currents in hCFTR/HEK293 cells, were inhibited by methadone, but not morphine. HEK293EBNA cells expressing hClC-2 showed time-dependent, voltage-activated, CdCl2-inhibited Cl(-) currents in the absence (control) and the presence of lubiprostone. Methadone, but not morphine, inhibited control and lubiprostone-stimulated hClC-2 Cl(-) currents with half-maximal inhibition at 100 and 200-230 nM, respectively. Forskolin/IBMX-stimulated hClC-2 Cl(-) currents were also inhibited by methadone. Myristoylated protein kinase inhibitor (a specific PKA inhibitor) inhibited forskolin/IBMX- but not lubiprostone-stimulated hClC-2 Cl(-) currents. Methadone caused greater inhibition of lubiprostone-stimulated currents added before patching (66.1 %) compared with after patching (28.7 %). Methadone caused inhibition of lubiprostone-stimulated Cl(-) currents in T84 cells and control; lubiprostone- and forskolin/IBMX-stimulated recombinant hClC-2 Cl(-) currents may be the basis for reduced efficacy of lubiprostone in methadone-treated patients.
Asunto(s)
Alprostadil/análogos & derivados , Canales de Cloruro/efectos de los fármacos , Regulador de Conductancia de Transmembrana de Fibrosis Quística/metabolismo , Metadona/farmacología , Morfina/farmacología , Alprostadil/farmacología , Transporte Biológico , Cloruro de Cadmio/farmacología , Canales de Cloruro/antagonistas & inhibidores , Colforsina/farmacología , Regulador de Conductancia de Transmembrana de Fibrosis Quística/genética , Evaluación Preclínica de Medicamentos , Células HEK293 , Humanos , Lubiprostona , Naloxona/farmacología , Técnicas de Placa-Clamp , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Factores de Tiempo , TransfecciónRESUMEN
BACKGROUND: CFTR function is tightly regulated by many interacting proteins. RESULTS: Intermediate filament protein keratin 18 increases the cell surface expression of CFTR by interacting with the C-terminal hydrophobic patch of CFTR. CONCLUSION: K18 controls the function of CFTR. SIGNIFICANCE: These findings offer novel insights into the regulation of CFTR and suggest that K18 and its dimerization partner, K8, may be modifier genes in cystic fibrosis. Malfunction of the cystic fibrosis transmembrane conductance regulator (CFTR) leads to cystic fibrosis, but the regulation of CFTR is not fully understood. Here, we identified the intermediate filament protein keratin K18 (K18) as a CFTR-binding protein by various approaches. We mapped a highly conserved "hydrophobic patch" ((1413)FLVI(1416)) in the CFTR C-terminus, known to determine plasmalemmal CFTR stability, as the K18-binding site. On the other hand, the C-terminal tail of K18 was found to be a critical determinant for binding CFTR. Overexpression of K18 in cells robustly increased the surface expression of wild-type CFTR, whereas depletion of K18 through RNA interference specifically diminished it. K18 binding increased the surface expression of CFTR by accelerating its apical recycling rate without altering CFTR biosynthesis, maturation, or internalization. Importantly, CFTR surface expression was markedly reduced in duodenal and gallbladder epithelia of K18(-/-) mice. Taken together, our results suggest that K18 increases the cell surface expression of CFTR by interacting with the CFTR C-terminal hydrophobic patch. These findings offer novel insights into the regulation of CFTR and suggest that K18 and its dimerization partner, K8, may be modifier genes in cystic fibrosis.
Asunto(s)
Regulador de Conductancia de Transmembrana de Fibrosis Quística/química , Regulador de Conductancia de Transmembrana de Fibrosis Quística/metabolismo , Fibrosis Quística/metabolismo , Queratina-18/metabolismo , Animales , Línea Celular , Fibrosis Quística/genética , Regulador de Conductancia de Transmembrana de Fibrosis Quística/genética , Humanos , Interacciones Hidrofóbicas e Hidrofílicas , Queratina-18/química , Queratina-18/genética , Ratones , Ratones Noqueados , Unión Proteica , Estructura Terciaria de Proteína , Técnicas del Sistema de Dos HíbridosRESUMEN
PURPOSE: Effects of cis-unoprostone isopropyl, its primary metabolite M1, trans-unoprostone isopropyl, latanoprost free acid, and fluprostenol were studied on Ca(2+)-activated K(+) (BK) channels, plasma membrane potential, [cAMP](i), [cGMP](i), and steady state [Ca(2+)](i), and protection against endothelin-1 (ET-1)-induced steady state [Ca(2+)](i) increases in human cortical neuronal (HCN-1A), trabecular meshwork (HTMC), and pulmonary artery smooth muscle (PASMC) cells. Effects on recombinant human prostaglandin (PG) receptors were determined. METHODS: BK channel currents were measured using whole-cell patch clamp; [cAMP](i), [cGMP](i) with ELISAs; [Ca(2+)](i) with indo-1; plasma membrane potential using diBAC(4)(3); and PG receptor effects with PG receptor-expressing cells and FLIPR fluo-4 Ca(2+) assays. RESULTS: Unoprostone isopropyl and M1 activated sustained iberiotoxin (IbTX)-sensitive, AL-8810 (FP receptor antagonist)-insensitive BK channel currents with EC(50)s of 0.51 ± 0.03 nM (n = 5) and 0.52 ± 0.03 nM (n = 6) in HTMCs; 0.61 ± 0.06 nM (n = 8) and 0.46 ± 0.04 nM (n = 5) for M1 in HCN-1A cells and PASMC, respectively. They caused AL-8810-insensitive, IbTX-sensitive membrane hyperpolarization at 10 nM; up to 100 nM had no effect on or decreased [cAMP](i), [cGMP](i), and [Ca(2+)](i); and prevented ET-1-induced [Ca(2+)](i) increases. In contrast, 10 nM latanoprost free acid and fluprostenol caused membrane depolarization; increased [cAMP](i), [cGMP](i), and [Ca(2+)](i); and did not prevent ET-1-induced [Ca(2+)](i) increases. Trans-unoprostone isopropyl had no effects. Unoprostone isopropyl (1.25 µM) had no effect on PG receptors, and neither did M1, except for activating the FP receptor with EC(50) = 557.9 ± 55.2 nM (n = 4). CONCLUSIONS: Prostones, unoprostone isopropyl and M1, are potent AL-8810-insensitive, stereospecific BK channel activators, without [cAMP](i), [cGMP](i), or [Ca(2+)](i) involvement, and prevent ET-1-induced steady state Ca(2+) increases in HTMCs.
Asunto(s)
Agonistas de los Canales de Calcio/farmacología , Dinoprost/análogos & derivados , Canales de Potasio de Gran Conductancia Activados por el Calcio/efectos de los fármacos , Músculo Liso/efectos de los fármacos , Malla Trabecular/efectos de los fármacos , Calcio/metabolismo , Membrana Celular/efectos de los fármacos , AMP Cíclico , GMP Cíclico/metabolismo , Dinoprost/farmacología , Relación Dosis-Respuesta a Droga , Endotelina-1/farmacología , Humanos , Potenciales de la Membrana/efectos de los fármacos , Músculo Liso/metabolismo , Técnicas de Placa-Clamp , Péptidos/farmacología , Malla Trabecular/metabolismoRESUMEN
BACKGROUND: Linaclotide has been proposed as a treatment for the same gastrointestinal indications for which lubiprostone has been approved, chronic idiopathic constipation and irritable bowel syndrome with constipation. Stressors damage the epithelial cell barrier and cellular homeostasis leading to loss of these functions. Effects of active linaclotide on repair of barrier and cell function in pig jejunum after ischemia and in T84 cells after treatment with proinflammatory cytokines, interferon-γ and tumor necrosis factor-α were examined. Comparison with effects of lubiprostone, known to promote repair of barrier function was carried out. RESULTS: In ischemia-damaged pig jejunum, using measurements of transepithelial resistance, (3)H-mannitol fluxes, short-circuit current (Cl(-) secretion) and occludin localization, active linaclotide failed to effectively promote repair of the epithelial barrier or recovery of short-circuit current, whereas lubiprostone promoted barrier repair and increased short-circuit current. In control pig jejunum, 1 µM linaclotide and 1 µM lubiprostone both caused similar increases in short-circuit current (Cl(-) secretion). In T84 cells, using measurements of transepithelial resistance, fluxes of fluorescent macromolecules, occludin and mitochondrial membrane potential, active linaclotide was virtually ineffective against damage caused by interferon-γ and tumor necrosis factor-α, while lubiprostone protected or promoted repair of epithelial barrier and cell function. Barrier protection/repair by lubiprostone was inhibited by methadone, a ClC-2 inhibitor. Linaclotide, but not lubiprostone increased [cGMP]i as expected and [Ca(2+)]i and linaclotide depolarized while lubiprostone hyperpolarized the T84 plasma membrane potential suggesting that lubiprostone may lead to greater cellular stability compared to linaclotide. In T84 cells, as found with linaclotide but not with lubiprostone, transepithelial resistance was slightly but significantly decreased by guanylin, STa and 8-bromo cGMP and fluorescent dextran fluxes were increased by guanylin. However the physiological implications of these small but statistically significant changes remain unclear. CONCLUSIONS: Considering the physiological importance of epithelial barrier function and cell integrity and the known impact of stressors, the finding that lubiprostone, but not active linaclotide, exhibits the additional distinct property of effective protection or repair of the epithelial barrier and cell function after stress suggests potential clinical importance for patients with impaired or compromised barrier function such as might occur in IBS.
Asunto(s)
Alprostadil/análogos & derivados , Células Epiteliales/efectos de los fármacos , Fármacos Gastrointestinales/farmacología , Péptidos/farmacología , Alprostadil/farmacología , Animales , Calcio/fisiología , Línea Celular Tumoral , Permeabilidad de la Membrana Celular/efectos de los fármacos , Células Epiteliales/fisiología , Femenino , Homeostasis/efectos de los fármacos , Humanos , Técnicas In Vitro , Interferón gamma/farmacología , Isquemia/fisiopatología , Yeyuno/efectos de los fármacos , Yeyuno/fisiología , Lubiprostona , Masculino , Potenciales de la Membrana/efectos de los fármacos , Ocludina/metabolismo , Porcinos , Factor de Necrosis Tumoral alfa/farmacologíaRESUMEN
Lubiprostone, a bicyclic fatty acid derivative and member of a new class of compounds called prostones, locally activates ClC-2 Cl(-) channels without activation of prostaglandin receptors. The present study was specifically designed to test and compare lubiprostone and prostaglandin effects at the cellular level using human uterine smooth muscle cells. Effects on [Ca(2+)](i), membrane potential and [cAMP](i) in human uterine smooth muscle cells were measured. 10 nM lubiprostone significantly decreased [Ca(2+)](i) from 188 to 27 nM, which was unaffected by 100 nM SC-51322, a prostaglandin EP receptor antagonist. In contrast 10nM PGE(2) and PGE(1) both increased [Ca(2+)](i) 3-5-fold which was blocked by SC-51322. Similarly, lubiprostone and prostaglandins had opposite/different effects on membrane potential and [cAMP](i). Lubiprostone caused SC-51322-insensitive membrane hyperpolarization and no effect on [cAMP](i). PGE(2) and PGE(1) both caused SC-51322-sensitive membrane depolarization and increased [cAMP](i). Lubiprostone has fundamentally different cellular effects from prostaglandins that are not mediated by EP receptors.
Asunto(s)
Alprostadil/análogos & derivados , Miocitos del Músculo Liso/efectos de los fármacos , Útero/citología , Alprostadil/farmacología , Calcio/metabolismo , Línea Celular , AMP Cíclico/metabolismo , Dinoprostona/farmacología , Femenino , Humanos , Lubiprostona , Potenciales de la Membrana/efectos de los fármacos , Miocitos del Músculo Liso/metabolismo , Miocitos del Músculo Liso/fisiología , Receptores de Prostaglandina E/antagonistas & inhibidores , Receptores de Prostaglandina E/fisiologíaRESUMEN
Effects of unoprostone isopropyl (unoprostone), a prostaglandin metabolite analog; latanoprost, a PGF(2alpha) analog; and PGF(2alpha) were examined in HCN-1A cells, a model system for studies of large conductance Ca(2+) activated K(+)(BK) channel activator-based neuroprotective agents. Unoprostone and latanoprost, both used as anti-glaucoma agents, have been suggested to act through FP receptors and have neuroprotective effects. Ion channel activation, plasma membrane polarization, [Ca(2+)](i) changes and protection against long-term irreversible glutamate-induced [Ca(2+)](i) increases were studied. Unoprostone activated iberiotoxin (IbTX)-sensitive BK channels in HCN-1A cells with an EC(50) of 0.6+/-0.2 nM and had no effect on Cl(-) currents. Unoprostone caused IbTX-sensitive plasma membrane hyperpolarization that was insensitive to AL8810, an FP receptor antagonist. In contrast, latanoprost and PGF(2alpha) activated a Cl(-) current sensitive to [Ca(2+)](i) chelation, tamoxifen and AL8810, and caused IbTX-insensitive, AL8810-sensitive membrane depolarization consistent with FP receptor-mediated Ca(2+) signaling Cl(-) current activation. Latanoprost and PGF(2alpha), but not unoprostone, increased [Ca(2+)](i). Unoprostone, PGF(2alpha) only partially, but not latanoprost protected HCN-1A cells against glutamate-induced Ca(2+) deregulation. These findings show that unoprostone has a distinctly different mechanism of action from latanoprost and PGF(2alpha). Whether unoprostone affects the BK channel directly or an unidentified signaling mechanism has not been determined.
Asunto(s)
Dinoprost/análogos & derivados , Activación del Canal Iónico/efectos de los fármacos , Canales de Potasio Calcio-Activados/metabolismo , Antihipertensivos/farmacología , Calcio/metabolismo , Calibración , Línea Celular , Quelantes/farmacología , Canales de Cloruro/metabolismo , Dinoprost/farmacología , Ácido Glutámico/farmacología , Humanos , Latanoprost , Potenciales de la Membrana/efectos de los fármacos , Péptidos/farmacología , Prostaglandinas F Sintéticas/farmacología , Receptores de Prostaglandina/antagonistas & inhibidores , Tamoxifeno/farmacologíaRESUMEN
The use of solid supported membranes (SSM) was investigated for reconstitution of ion channels and for potential application to screen pharmacological reagents affecting ion channel function. The voltage-gated Kv1.5 K+ channel was reconstituted on an SSM and a current was measured. This current was dependent on the presence of K+, but not Na+, indicating that the Kv1.5 K+ channel maintained cation specificity when reconstituted on SSM. Two pharmacological reagents applied to Kv1.5 K+ channels reconstituted on SSM had similar inhibitory effects as those measured using Kv1.5 in biological membranes. SSM-mounted ion channels were stable enough to be washed with buffer solution and reused many times, allowing solution exchange essential for pharmacological drug screening.
Asunto(s)
Evaluación Preclínica de Medicamentos/métodos , Canales Iónicos/efectos de los fármacos , Membranas Artificiales , Animales , Línea Celular , Equipo Reutilizado , Canal de Potasio Kv1.5 , Ratones , Técnicas de Placa-Clamp , Canales de Potasio con Entrada de Voltaje/efectos de los fármacosRESUMEN
The purpose of this study was to determine the mechanism of action of SPI-0211 (lubiprostone), a novel bicyclic fatty acid in development for the treatment of bowel dysfunction. Adult rabbit intestine was shown to contain mRNA for ClC-2 using RT-PCR, Northern blot analysis, and in situ hybridization. T84 cells grown to confluence on permeable supports were shown to express ClC-2 channel protein in the apical membrane. SPI-0211 increased electrogenic Cl- transport across the apical membrane of T84 cells, with an EC50 of approximately 18 nM measured by short-circuit current (Isc) after permeabilization of the basolateral membrane with nystatin. SPI-0211 effects on Cl- currents were also measured by whole cell patch clamp using the human embryonic kidney (HEK)-293 cell line stably transfected with either recombinant human ClC-2 or recombinant human cystic fibrosis transmembrane regulator (CFTR). In these studies, SPI-0211 activated ClC-2 Cl- currents in a concentration-dependent manner, with an EC50 of approximately 17 nM, and had no effect in nontransfected HEK-293 cells. In contrast, SPI-0211 had no effect on CFTR Cl- channel currents measured in CFTR-transfected HEK-293 cells. Activation of ClC-2 by SPI-0211 was independent of PKA. Together, these studies demonstrate that SPI-0211 is a potent activator of ClC-2 Cl- channels and suggest a physiologically relevant role for ClC-2 Cl- channels in intestinal Cl- transport after SPI-0211 administration.
Asunto(s)
Canales de Cloruro/metabolismo , Cloruros/metabolismo , Ácidos Grasos/farmacología , Mucosa Intestinal/metabolismo , Animales , Northern Blotting , Canales de Cloruro CLC-2 , Canales de Cloruro/efectos de los fármacos , Regulador de Conductancia de Transmembrana de Fibrosis Quística , Relación Dosis-Respuesta a Droga , Humanos , Hibridación in Situ , Intestinos/efectos de los fármacos , Transporte Iónico/efectos de los fármacos , Potenciales de la Membrana/efectos de los fármacos , Potenciales de la Membrana/fisiología , Microscopía Confocal , Técnicas de Placa-Clamp , ARN Mensajero/análisis , Conejos , Reacción en Cadena de la Polimerasa de Transcriptasa InversaRESUMEN
Human ClC-2 Cl(-) (hClC-2) channels are activated by protein kinase A (PKA) and low extracellular pH(o). Both of these effects are prevented by the PKA inhibitor, myristoylated PKI. The aims of the present study were to identify the PKA phosphorylation site(s) important for PKA activation of hClC-2 at neutral and low pH(o) and to examine the relationship between PKA and low pH(o) activation. Recombinant hClC-2 with point mutations of consensus phosphorylation sites was prepared and stably expressed in HEK-293 cells. The responses to forskolin plus isobutylmethylxanthine at neutral and acidic pH(o) were studied by whole cell patch clamp in the presence and absence of phosphatase inhibitors. The double phosphorylation site (RRAT655(A) plus RGET691(A)) mutant hClC-2 lost PKA activation and low pH(o) activation. Either RRAT or RGET was sufficient for PKA activation of hClC-2 at pH(o) 7.4, as long as phosphatase inhibitors (cyclosporin A or endothal) were present. At pH(o) 6 only RGET was needed for PKA activation of hClC-2. Low pH(o) activation of hClC-2 Cl(-) channel activity was PKA-dependent, retained in RGET(A) mutant hClC-2, but lost in RRAT(A) mutant hClC-2. RRAT655(D) mutant hClC-2 was constitutively active and was further activated by PKA at pH(o) 7.4 and 6.0, consistent with the above findings. These results show that activation of hClC-2 is differentially regulated by PKA at two sites, RRAT655 and RGET691. Either RRAT655 or RGET691 was sufficient for activation at pH(o) 7.4. RGET, but not RRAT, was sufficient for activation at pH(o) 6.0. However, in the RGET691(D) mutant, there was PKA activation at pH(o) 6.0.
Asunto(s)
Canales de Cloruro/química , Proteínas Quinasas Dependientes de AMP Cíclico/química , Ácido Araquidónico/farmacología , Sitios de Unión , Canales de Cloruro CLC-2 , Línea Celular , Cloruros/química , Proteínas Quinasas Dependientes de AMP Cíclico/metabolismo , Ciclosporina/farmacología , ADN Complementario/metabolismo , Ácidos Dicarboxílicos/farmacología , Activación Enzimática , Inhibidores Enzimáticos/farmacología , Humanos , Concentración de Iones de Hidrógeno , Mutagénesis Sitio-Dirigida , Mutación , Ácidos Mirísticos/metabolismo , Técnicas de Placa-Clamp , Fosforilación , Mutación Puntual , Proteínas Recombinantes/química , TransfecciónRESUMEN
Our objective was to identify and localize a K+ channel involved in gastric HCl secretion at the parietal cell secretory membrane and to characterize and compare the functional properties of native and recombinant gastric K+ channels. RT-PCR showed that mRNA for Kir2.1 was abundant in rabbit gastric mucosa with lesser amounts of Kir4.1 and Kir7.1, relative to beta-actin. Kir2.1 mRNA was localized to parietal cells of rabbit gastric glands by in situ RT-PCR. Resting and stimulated gastric vesicles contained Kir2.1 by Western blot analysis at approximately 50 kDa as observed with in vitro translation. Immunoconfocal microscopy showed that Kir2.1 was present in parietal cells, where it colocalized with H+ -K+ -ATPase and ClC-2 Cl- channels. Function of native K+ channels in rabbit resting and stimulated gastric mucosal vesicles was studied by reconstitution into planar lipid bilayers. Native gastric K+ channels exhibited a linear current-voltage relationship and a single-channel slope conductance of approximately 11 pS in 400 mM K2SO4. Channel open probability (Po) in stimulated vesicles was high, and that of resting vesicles was low. Reduction of extracellular pH plus PKA treatment increased resting channel Po to approximately 0.5 as measured in stimulated vesicles. Full-length rabbit Kir2.1 was cloned. When stably expressed in Chinese hamster ovary (CHO) cells, it was activated by reduced extracellular pH and forskolin/IBMX with no effects observed in nontransfected CHO cells. Cation selectivity was K+ = Rb+ >> Na+ = Cs+ = Li+ = NMDG+. These findings strongly suggest that the Kir2.1 K+ channel may be involved in regulated gastric acid secretion at the parietal cell secretory membrane.
Asunto(s)
Proteínas Quinasas Dependientes de AMP Cíclico/metabolismo , Ácido Gástrico/metabolismo , Células Parietales Gástricas/metabolismo , Canales de Potasio de Rectificación Interna/metabolismo , Animales , Células CHO , Clonación Molecular , Cricetinae , Células Epiteliales/metabolismo , Mucosa Gástrica/citología , Mucosa Gástrica/metabolismo , ATPasa Intercambiadora de Hidrógeno-Potásio/metabolismo , Concentración de Iones de Hidrógeno , Activación del Canal Iónico/fisiología , Potenciales de la Membrana/fisiología , Proteínas de la Membrana/metabolismo , Oocitos/fisiología , Técnicas de Placa-Clamp , Canales de Potasio de Rectificación Interna/genética , ARN Mensajero/análisis , Conejos , Xenopus laevisRESUMEN
Tumor necrosis factor (TNF) increases epithelial permeability in many model systems. Protein kinase C (PKC) isozymes regulate epithelial barrier function and alter ligand-receptor interactions. We sought to define the impact of PKC on TNF-induced barrier dysfunction in T84 intestinal epithelia. TNF induced a dose- and time-dependent fall in transepithelial electrical resistance (TER) and an increase in [(3)H]mannitol flux. The TNF-induced fall in TER was not PKC mediated but was prevented by pretreatment with bryostatin-1, a PKC agonist. As demonstrated by a pattern of sensitivity to pharmacological inhibitors of PKC, this epithelial barrier preservation was mediated by novel PKC isozymes. Bryostatin-1 reduced TNF receptor (TNF-R1) surface availability, as demonstrated by radiolabeled TNF binding and cell surface biotinylation assays, and increased TNF-R1 receptor shedding. The pattern of sensitivity to isozyme-selective PKC inhibitors suggested that these effects were mediated by activation of PKC-epsilon. In addition, after bryostatin-1 treatment, PKC-delta and TNF-R1 became associated, as determined by mutual coimmunoprecipitation assay, which has been shown to lead to receptor desensitization in neutrophils. TNF-induced barrier dysfunction occurs independently of PKC, but selective modulation of novel PKC isozymes may regulate TNF-R1 signaling.
Asunto(s)
Antineoplásicos/farmacología , Mucosa Intestinal/metabolismo , Lactonas/farmacología , Proteína Quinasa C/metabolismo , Factor de Necrosis Tumoral alfa/farmacología , Antígenos CD/metabolismo , Transporte Biológico/efectos de los fármacos , Transporte Biológico/fisiología , Brioestatinas , Células Cultivadas , Relación Dosis-Respuesta a Droga , Células Epiteliales/efectos de los fármacos , Células Epiteliales/metabolismo , Humanos , Mucosa Intestinal/citología , Radioisótopos de Yodo , Macrólidos , Manitol/farmacocinética , Proteína Quinasa C-delta , Proteína Quinasa C-epsilon , Receptores del Factor de Necrosis Tumoral/metabolismo , Receptores Tipo I de Factores de Necrosis Tumoral , Transducción de Señal/efectos de los fármacos , Transducción de Señal/fisiología , Tritio , Factor de Necrosis Tumoral alfa/metabolismoRESUMEN
Recent evidence indicates that Pseudomonas aeruginosa residing as biofilms in airway mucus of cystic fibrosis (CF) patients is undergoing anaerobic metabolism, a form of growth requiring gene products that are not utilized during aerobic growth. The outer membrane protein, OprF, and the rhl quorum sensing circuit are two previously unrecognized cellular factors that are required for optimal anaerobic biofilm viability. Without OprF, bacteria grow extremely poorly because they lack nitrite reductase activity while lacking rhlR or rhlI forces bacteria to undergo metabolic suicide by overproduction of nitric oxide. Furthermore, anaerobic growth favors maintenance of the mucoid, alginate-overproducing phenotype. Thus, with increasing age of CF patients, mucoid populations predominate, indicating that anaerobic bacteria reside in the inspissated airway mucus. Because many frontline antibiotics used in the treatment of CF airway disease are either ineffective or show reduced efficacy during anaerobic conditions, we propose development of new drugs to combat anaerobic metabolism by P. aeruginosa for more effective treatment of chronic CF lung infections.