RESUMEN
Propionate, a metabolite from the microbial fermentation of carbohydrates, evokes a release of epithelial acetylcholine in rat caecum resulting in an increase of short-circuit current (Isc) in Ussing chamber experiments. The present experiments were performed in order to characterize the ionic mechanisms underlying this response which has been thought to be due to Cl- secretion. As there are regional differences within the caecal epithelium, the experiments were conducted at oral and aboral rat corpus caeci. In both caecal segments, the propionate-induced Isc (IProp) was inhibited by > 85%, when the experiments were performed either in nominally Cl-- or nominally HCO3--free buffer. In the case of Cl-, the dependency was restricted to the presence of Cl- in the serosal bath. Bumetanide, a blocker of the Na+-K+-2Cl--cotransporter, only numerically reduced IProp suggesting that a large part of this current must be carried by an ion other than Cl-. In the aboral caecum, IProp was significantly inhibited by mucosally administered stilbene derivatives (SITS, DIDS, DNDS), which block anion exchangers. Serosal Na+-free buffer reduced IProp significantly in the oral (and numerically also in aboral) corpus caeci. RT-PCR experiments revealed the expression of several forms of Na+-dependent HCO3--cotransporters in caecum, which might underlie the observed Na+ dependency. These results suggest that propionate sensing in caecum is coupled to HCO3- secretion, which functionally would stabilize luminal pH when the microbial fermentation leads to an increase in the concentration of short-chain fatty acids in the caecal lumen.
Asunto(s)
Bicarbonatos/metabolismo , Ciego/metabolismo , Cloruros/metabolismo , Propionatos/farmacología , Ácido 4,4'-Diisotiocianostilbeno-2,2'-Disulfónico/farmacología , Ácido 4-Acetamido-4'-isotiocianatostilbeno-2,2'-disulfónico/farmacología , Acetilcolina/metabolismo , Animales , Bumetanida/farmacología , Ciego/efectos de los fármacos , Masculino , Ratas , Ratas Wistar , Inhibidores del Simportador de Cloruro Sódico y Cloruro Potásico/farmacología , Simportadores de Sodio-Bicarbonato/antagonistas & inhibidores , Simportadores de Sodio-Bicarbonato/metabolismo , Simportadores de Cloruro de Sodio-Potasio/metabolismoRESUMEN
RAD51 is the central protein in DNA repair by homologous recombination (HR), involved in several steps of this process. It is shown that overexpression of the RAD51 protein is correlated with increased survival of cancer cells to cancer treatments. For the past decade, RAD51 overexpression-mediated resistance has justified the development of targeted inhibitors. One of the first molecules described to inhibit RAD51 was the 4,4'-diisothiocyanato-stilbene-2,2'-disulfonic acid (DIDS) molecule. This small molecule is effective in inhibiting different functions of RAD51, however its mode of action and the chemical functions involved in this inhibition have not been identified. In this work, we used several commercial molecules derived from DIDS to characterize the structural determinants involved in modulating the activity of RAD51. By combining biochemical and biophysical approaches, we have shown that DIDS and two analogs were able to inhibit the binding of RAD51 to ssDNA and prevent the formation of D-loop by RAD51. Both isothiocyanate substituents of DIDS appear to be essential in the inhibition of RAD51. These results open the way to the synthesis of new molecules derived from DIDS that should be greater modulators of RAD51 and more efficient for HR inhibition.
Asunto(s)
Ácido 4,4'-Diisotiocianostilbeno-2,2'-Disulfónico/análogos & derivados , Recombinasa Rad51/química , Recombinasa Rad51/metabolismo , Ácido 4,4'-Diisotiocianostilbeno-2,2'-Disulfónico/administración & dosificación , Ácido 4,4'-Diisotiocianostilbeno-2,2'-Disulfónico/farmacología , Ácido 4-Acetamido-4'-isotiocianatostilbeno-2,2'-disulfónico/administración & dosificación , Ácido 4-Acetamido-4'-isotiocianatostilbeno-2,2'-disulfónico/farmacología , ADN de Cadena Simple/metabolismo , Relación Dosis-Respuesta a Droga , Recombinasa Rad51/antagonistas & inhibidoresRESUMEN
We have studied inorganic phosphate (Pi) handling in rat aortic vascular smooth muscle cells (VSMC) using 32P-radiotracer assays. Our results have revealed a complex set of mechanisms consisting of 1) well-known PiT1/PiT2-mediated sodium-dependent Pi transport; 2) Slc20-unrelated sodium-dependent Pi transport that is sensitive to the stilbene derivatives 4,4'-diisothiocyanatostilbene-2,2'-disulphonic acid (DIDS) and 4-acetamido-4-isothiocyanostilbene-2,2-disulfonate (SITS); 3) a sodium-independent Pi uptake system that is competitively inhibited by sulfate, bicarbonate, and arsenate and is weakly inhibited by DIDS, SITS, and phosphonoformate; and 4) an exit pathway from the cell that is partially chloride dependent and unrelated to the known anion-exchangers expressed in VSMC. The inhibitions of sodium-independent Pi transport by sulfate and of sodium-dependent transport by SITS were studied in greater detail. The maximal inhibition by sulfate was similar to that of Pi itself, with a very high inhibition constant (212 mM). SITS only partially inhibited sodium-dependent Pi transport, but the Ki was very low (14 µM). Nevertheless, SITS and DIDS did not inhibit Pi transport in Xenopus laevis oocytes expressing PiT1 or PiT2. Both the sodium-dependent and sodium-independent transport systems were highly dependent on VSMC confluence and on the differentiation state, but they were not modified by incubating VSMC for 7 days with 2 mM Pi under nonprecipitating conditions. This work not only shows that the Pi handling by cells is highly complex but also that the transport systems are shared with other ions such as bicarbonate or sulfate.NEW & NOTEWORTHY In addition to the inorganic phosphate (Pi) transporters PiT1 and PiT2, rat vascular smooth muscle cells show a sodium-dependent Pi transport system that is inhibited by DIDS and SITS. A sodium-independent Pi uptake system of high affinity is also expressed, which is inhibited by sulfate, bicarbonate, and arsenate. The exit of excess Pi is through an exchange with extracellular chloride. Whereas the metabolic effects of the inhibitors, if any, cannot be discarded, kinetic analysis during initial velocity suggests competitive inhibition.
Asunto(s)
Transporte Biológico/fisiología , Músculo Liso Vascular/metabolismo , Músculo Liso Vascular/fisiología , Miocitos del Músculo Liso/metabolismo , Fosfatos/metabolismo , Ácido 4,4'-Diisotiocianostilbeno-2,2'-Disulfónico/farmacología , Ácido 4-Acetamido-4'-isotiocianatostilbeno-2,2'-disulfónico/farmacología , Animales , Cloruros/metabolismo , Cinética , Oocitos/efectos de los fármacos , Oocitos/metabolismo , Ratas , Sodio/metabolismo , Proteínas Cotransportadoras de Sodio-Fosfato de Tipo III/metabolismo , Estilbenos/farmacología , Xenopus laevisRESUMEN
The SLC4A11 gene mutations cause a variety of genetic corneal diseases, including congenital hereditary endothelial dystrophy 2 (CHED2), Harboyan syndrome, some cases of Fuchs' endothelial dystrophy (FECD), and possibly familial keratoconus. Three NH2-terminal variants of the human SLC4A11 gene, named SLC4A11-A, -B, and -C are known. The SLC4A11-B variant has been the focus of previous studies. Both the expression of the SLC4A11-C variant in the cornea and its functional properties have not been characterized, and therefore its potential pathophysiological role in corneal diseases remains to be explored. In the present study, we demonstrate that SLC4A11-C is the predominant SLC4A11 variant expressed in human corneal endothelial mRNA and that the transporter functions as an electrogenic H(+)(OH(-)) permeation pathway. Disulfonic stilbenes, including 4,4'-diisothiocyano-2,2'-stilbenedisulfonate (DIDS), 4,4'-diisothiocyanatodihydrostilbene-2,2'-disulfonate (H2DIDS), and 4-acetamido-4'-isothiocyanato-stilbene-2,2'-disulfonate (SITS), which are known to bind covalently, increased SLC4A11-C-mediated H(+)(OH(-)) flux by 150-200% without having a significant effect in mock-transfected cells. Noncovalently interacting 4,4'-diaminostilbene-2,2'-disulfonate (DADS) was without effect. We tested the efficacy of DIDS on the functionally impaired R109H mutant (SLC4A11-C numbering) that causes CHED2. DIDS (1 mM) increased H(+)(OH(-)) flux through the mutant transporter by â¼40-90%. These studies provide a basis for future testing of more specific chemically modified dilsulfonic stilbenes as potential therapeutic agents to improve the functional impairment of specific SLC4A11 mutant transporters.
Asunto(s)
Ácido 4,4'-Diisotiocianostilbeno-2,2'-Disulfónico/farmacología , Proteínas de Transporte de Anión/metabolismo , Antiportadores/metabolismo , Hidróxidos/metabolismo , Permeabilidad/efectos de los fármacos , Transducción de Señal/efectos de los fármacos , Ácido 4,4'-Diisotiocianostilbeno-2,2'-Disulfónico/análogos & derivados , Ácido 4-Acetamido-4'-isotiocianatostilbeno-2,2'-disulfónico/análogos & derivados , Ácido 4-Acetamido-4'-isotiocianatostilbeno-2,2'-disulfónico/farmacología , Proteínas de Transporte de Anión/genética , Antiportadores/genética , Transporte Biológico/fisiología , Línea Celular , Córnea/efectos de los fármacos , Córnea/metabolismo , Células Endoteliales/efectos de los fármacos , Células Endoteliales/metabolismo , Células HEK293 , Humanos , Mutación/genética , ARN Mensajero/genéticaRESUMEN
BACKGROUND: Bicarbonate transport has crucial roles in regulating intracellular pH (pHi) in a variety of cells. The purpose of this study was to evaluate its participation in the regulation of pHi in resting and stimulated human neutrophils. METHODS: Freshly isolated human neutrophils acidified by an ammonium prepulse were used in this study. RESULTS: We demonstrated that resting neutrophils have a bicarbonate transport mechanism that prevents acidification when the Na(+)/H(+) exchanger is blocked by EIPA. Neutrophils acidified by an ammonium prepulse showed an EIPA-resistant recovery of pHi that was inhibited by the blocker of the anionic transporters SITS or the Na(+)/HCO3(-) cotransporter (NBC) selective inhibitor S0859, and abolished when sodium was removed from the extracellular medium. In western blot and RT-PCR analysis the expression of NBCe2 but not NBCe1 or NBCn1 was detected in neutrophils Acidified neutrophils increased the EIPA-insensitive pHi recovery rate when its activity was stimulated with fMLF/ cytochalasin B. This increase in the removal of acid equivalents was insensitive to the blockade of the NADPH oxidase with DPI. CONCLUSION: It is concluded that neutrophils have an NBC that regulates basal pHi and is modulated by chemotactic agents.
Asunto(s)
Neutrófilos/metabolismo , Simportadores de Sodio-Bicarbonato/metabolismo , Ácido 4-Acetamido-4'-isotiocianatostilbeno-2,2'-disulfónico/farmacología , Amilorida/análogos & derivados , Amilorida/farmacología , Cloruro de Amonio/farmacología , Benzamidas/farmacología , Bicarbonatos/farmacología , Citocalasina B/farmacología , Células HEK293 , Humanos , Concentración de Iones de Hidrógeno , Transporte Iónico/efectos de los fármacos , N-Formilmetionina Leucil-Fenilalanina/farmacología , NADPH Oxidasas/antagonistas & inhibidores , NADPH Oxidasas/metabolismo , Neutrófilos/efectos de los fármacos , Isoformas de Proteínas/genética , Isoformas de Proteínas/metabolismo , ARN Mensajero/metabolismo , Simportadores de Sodio-Bicarbonato/genética , Intercambiadores de Sodio-Hidrógeno/metabolismo , Sulfonamidas/farmacologíaRESUMEN
Bicarbonate transporter (BCT) plays a crucial role in maintaining pH homeostasis of tumor cells by import of HCO3(-). This helps the tumor cells in manifesting extracellular tumor acidosis, accompanied by a relative intracellular alkalinization, which in turn promotes tumor progression. Therefore, blocking BCT-mediated HCO3(-) transport is envisaged as a promising anticancer therapeutic approach. Thus, using a murine model of a T cell lymphoma, designated as Dalton's lymphoma (DL), in the present in vitro investigation the antitumor consequences of blocking BCT function by its inhibitor 4-acetamido-4-isothiocyanostilbene-2,2-disulfonate (SITS) were explored. Treatment of DL cells with SITS resulted in an increase in the extracellular pH, associated with a decline in DL cell survival and augmented induction of apoptosis. BCT inhibition also elevated the expression of cytochrome c, caspase-9, caspase-3, Bax, reactive oxygen species, and nitric oxide along with inhibition of HSP-70 and Bcl2, which regulate tumor cell survival and apoptosis. SITS-treated DL cells displayed upregulated production of IFN-γ and IL-6 along with a decline of IL-10. Treatment of DL cells with SITS also inhibited the expression of fatty acid synthase, which is crucial for membrane biogenesis in neoplastic cells. The expression of lactate transporter MCT-1 and multidrug resistance regulating protein MRP-1 got inhibited along with hampered uptake of glucose and lactate production in SITS-treated DL cells. Thus, the declined tumor cell survival following inhibition of BCT could be the consequence of interplay of several inter-connected regulatory molecular events. The outcome of this study indicates the potential of BCT inhibition as a novel therapeutic approach for treatment of hematological malignancies.
Asunto(s)
Ácido 4-Acetamido-4'-isotiocianatostilbeno-2,2'-disulfónico/farmacología , Proteínas de Transporte de Anión/antagonistas & inhibidores , Apoptosis/efectos de los fármacos , Homeostasis/efectos de los fármacos , Linfoma de Células T/tratamiento farmacológico , Proteínas de Neoplasias/metabolismo , Neoplasias Experimentales/tratamiento farmacológico , Simportadores/antagonistas & inhibidores , Animales , Proteínas de Transporte de Anión/genética , Proteínas de Transporte de Anión/metabolismo , Apoptosis/genética , Proteínas Reguladoras de la Apoptosis/genética , Proteínas Reguladoras de la Apoptosis/metabolismo , Bicarbonatos/metabolismo , Citocinas/genética , Citocinas/metabolismo , Homeostasis/genética , Concentración de Iones de Hidrógeno , Linfoma de Células T/genética , Linfoma de Células T/metabolismo , Linfoma de Células T/patología , Ratones , Ratones Endogámicos BALB C , Proteínas de Neoplasias/antagonistas & inhibidores , Proteínas de Neoplasias/genética , Neoplasias Experimentales/genética , Neoplasias Experimentales/metabolismo , Neoplasias Experimentales/patología , Simportadores/genética , Simportadores/metabolismoRESUMEN
We identified 11 human pedigrees with dominantly inherited hemolytic anemias in both the hereditary stomatocytosis and spherocytosis classes. Affected individuals in these families had an increase in membrane permeability to Na and K that is particularly marked at 0 degrees C. We found that disease in these pedigrees was associated with a series of single amino-acid substitutions in the intramembrane domain of the erythrocyte band 3 anion exchanger, AE1. Anion movements were reduced in the abnormal red cells. The 'leak' cation fluxes were inhibited by SITS, dipyridamole and NS1652, chemically diverse inhibitors of band 3. Expression of the mutated genes in Xenopus laevis oocytes induced abnormal Na and K fluxes in the oocytes, and the induced Cl transport was low. These data are consistent with the suggestion that the substitutions convert the protein from an anion exchanger into an unregulated cation channel.
Asunto(s)
Proteína 1 de Intercambio de Anión de Eritrocito/genética , Cationes/metabolismo , Cloruros/metabolismo , Eritrocitos/metabolismo , Potasio/metabolismo , Sodio/metabolismo , Ácido 4-Acetamido-4'-isotiocianatostilbeno-2,2'-disulfónico/farmacología , Sustitución de Aminoácidos , Anemia Hemolítica/genética , Anemia Hemolítica/metabolismo , Animales , Proteína 1 de Intercambio de Anión de Eritrocito/metabolismo , Benzoatos/farmacología , Transporte Biológico , Permeabilidad de la Membrana Celular , Dipiridamol/farmacología , Humanos , Datos de Secuencia Molecular , Oocitos/citología , Oocitos/metabolismo , Linaje , Compuestos de Fenilurea/farmacología , Inhibidores de Fosfodiesterasa/farmacología , Estructura Terciaria de Proteína , ARN/metabolismo , Esferocitosis Hereditaria/genética , Xenopus laevisRESUMEN
OBJECTIVE: To explore the impact of chloride ion channel and its blockers 4, 4'-diisothiocyanostilbene-2, 2'-disulfonic acid (DIDS), cyanato-stilbene-2, 2'-disulfonic acid (SITS) and 5-nitro-2-(3-phenyl-propylamino) benzoic acid (NPPB) on arrhythmias caused by myocardial ischemia reperfusion. METHODS: A total of 40 rabbits were divided into control, ischemia reperfusion, DIDS low-dose, DIDS high-dose, SITS low-dose, SITS high-dose, NPPB low-dose and NPPB high-dose groups. Myocardial ischemia reperfusion model was established by ligation of anterior descending coronary artery. And standard limb lead II of electrocardiogram (ECG) was continuously monitored during the experimental process. Then comparisons of heart rate, ECG P wave, R wave, T wave, ST segment changes and arrhythmias score were made between the above groups. RESULTS: During 30-minute ischemia, compared with the control group, all other groups showed significantly decreased heart rate ((199.8 ± 4.0) - (253.6 ± 2.1) vs (267.0 ± 3.4), all P < 0.01), elevated ECG P wave ((0.216 ± 0.019) - (0.356 ± 0.024) vs (0.186 ± 0.019), all P < 0.01), R wave ((0.564 ± 0.017) - (1.138 ± 0.048) vs (0.506 ± 0.018), all P < 0.01), T wave ((0.542 ± 0.013) - (0.856 ± 0.045) vs (0.278 ± 0.015), all P < 0.01) and ST segment ((0.326 ± 0.027) - (0.668 ± 0.054) vs (0.024 ± 0.023), all P < 0.01) and increased arrhythmia score ((1.4 ± 0.5) - (4.6 ± 0.5) vs (0.4 ± 0.5), all P < 0.01). Compared with the ischemia reperfusion group, the above indices significantly improved in the intervention groups (heart rate: (214.8 ± 3.4) - (246.8 ± 4.0) vs (199.8 ± 4.0), all P < 0.01; P wave: (0.216 ± 0.019) - (0.316 ± 0.011) vs (0.356 ± 0.024), all P < 0.01; R wave: (0.564 ± 0.017) - (0.980 ± 0.035) vs (1.138 ± 0.048), all P < 0.01; T wave: (0.542 ± 0.013) - (0.792 ± 0.026) vs (0.856 ± 0.045), all P < 0.01; ST segment: (0.326 ± 0.027) - (0.596 ± 0.018) vs (0.668 ± 0.054), all P < 0.01; arrhythmia score: (1.4 ± 0.5) - (3.8 ± 0.4) vs (4.6 ± 0.5), all P < 0.01). Among which, the DIDS group was the best, followed by the SITS group and then the NPPB group. And the high-dose subgroups were better than those of the low-dose subgroups. During 60-minute reperfusion, the decreased heart rate upswing significantly in each group and the elevated P wave, R wave, T wave and ST segment fell back gradually. The DIDS group showed the most obvious amplitude change, followed by the SITS group and then the NPPB group. And the high-dose subgroups were better than those of the low-dose subgroups. The arrhythmia score during reperfusion showed the same trend. CONCLUSION: Chloride ion channel is involved in the generation of myocardial ischemia reperfusion arrhythmia.Early application of chloride ion channel blockers DIDS, SITS and NPPB may improve the ECG manifestations and reduce the degree of reperfusion arrhythmia.
Asunto(s)
Arritmias Cardíacas/fisiopatología , Canales de Cloruro , Isquemia Miocárdica/fisiopatología , Daño por Reperfusión Miocárdica/fisiopatología , Ácido 4,4'-Diisotiocianostilbeno-2,2'-Disulfónico/farmacología , Ácido 4-Acetamido-4'-isotiocianatostilbeno-2,2'-disulfónico/farmacología , Animales , Antiarrítmicos/farmacología , Canales de Cloruro/antagonistas & inhibidores , Nitrobenzoatos/farmacología , ConejosRESUMEN
Transient receptor potential vanilloid type 1 (TRPV1) is a nonselective cation channel activated by capsaicin, low pH, and noxious heat and plays a key role in nociception. Understanding mechanisms for functional modulation of TRPV1 has important implications. One characteristic of TRPV1 is that channel activity induced by either capsaicin or other activators can be sensitized or modulated by factors involving different cell signaling mechanisms. In this study, we describe a novel mechanism for the modulation of TRPV1 function: TRPV1 function is modulated by 4,4'-diisothiocyanatostilbene-2,2'-disulfonic acid (DIDS) and its analogs. We found that, in rat dorsal root ganglion neurons, although DIDS did not induce the activation of TRPV1 per se but drastically increased the TRPV1 currents induced by either capsaicin or low pH. DIDS also blocked the tachyphylaxis of the low pH-induced TRPV1 currents. 4-Acetamido-4'-isothiocyanatostilbene-2,2'-disulfonic acid (SITS), a DIDS analog, failed to enhance the capsaicin-evoked TRPV1 current but increased the low pH-evoked TRPV1 currents, with an effect comparable with that of DIDS. SITS also blocked the low pH-induced tachyphylaxis. DIDS also potentiated the currents of TRPV1 channels expressed in human embryonic kidney 293 cells, with an effect of left-shifting the concentration-response curve of the capsaicin-induced TRPV1 currents. This study demonstrates that DIDS and SITS, traditionally used chloride channel blockers, can modify TRPV1 channel function in an agonist-dependent manner. The results provide new input for understanding TRPV1 modulation and developing new modulators of TRPV1 function.
Asunto(s)
Estilbenos/farmacología , Canales Catiónicos TRPV/efectos de los fármacos , Ácido 4,4'-Diisotiocianostilbeno-2,2'-Disulfónico/farmacología , Ácido 4-Acetamido-4'-isotiocianatostilbeno-2,2'-disulfónico/farmacología , Animales , Ácidos Araquidónicos/farmacología , Calcio/metabolismo , Capsaicina/farmacología , Células Cultivadas , Endocannabinoides , Humanos , Concentración de Iones de Hidrógeno , Alcamidas Poliinsaturadas/farmacología , Ratas , Ratas Sprague-Dawley , Canales Catiónicos TRPV/agonistas , Canales Catiónicos TRPV/fisiologíaRESUMEN
Mutations in the SLC4A11 protein, reported as a sodium-coup-led borate transporter of the human plasma membrane, are responsible for three corneal dystrophies (CD): congenital hereditary endothelial dystrophy type 2, Harboyan syndrome, and late-onset Fuch's CD. To develop a rational basis to understand these diseases, whose point mutations are found throughout the SLC4A11 sequence, we analyzed the protein biochemically. Hydropathy analysis and an existing topology model for SLC4A1 (AE1), a bicarbonate transporter with the lowest evolutionary sequence divergence from SLC4A11, formed the basis to propose an SLC4A11 topology model. Immunofluorescence studies revealed the cytosolic orientation of N- and C-termini of SLC4A11. Limited trypsinolysis of SLC4A11 partially mapped the folding of the membrane and cytoplasmic domains of the protein. The binding of SLC4A11 to a stilbenedisulfonate inhibitor resin (SITS-Affi-Gel) was prevented by preincubation with H(2)DIDS, with a significantly higher half-maximal effective concentration than AE1. We conclude that stilbenedisulfonates interact with SLC4A11 but with a lower affinity than other SLC4 proteins. Disease-causing mutants divided into two classes on the basis of the half-maximal [H(2)DIDS] required for resin displacement and the fraction of protein binding H(2)DIDS, likely representing mildly misfolded and grossly misfolded proteins. Disease-causing SLC4A11 mutants are retained in the endoplasmic reticulum of HEK 293 cells. This phenotype could be partially rescued in some cases by growing the cells at 30 °C.
Asunto(s)
Proteínas de Transporte de Anión/química , Proteínas de Transporte de Anión/metabolismo , Antiportadores/química , Antiportadores/metabolismo , Distrofias Hereditarias de la Córnea/metabolismo , Ácido 4-Acetamido-4'-isotiocianatostilbeno-2,2'-disulfónico/metabolismo , Ácido 4-Acetamido-4'-isotiocianatostilbeno-2,2'-disulfónico/farmacología , Alelos , Secuencia de Aminoácidos , Proteínas de Transporte de Anión/antagonistas & inhibidores , Proteínas de Transporte de Anión/genética , Antiportadores/antagonistas & inhibidores , Antiportadores/genética , Extractos Celulares , Membrana Celular/metabolismo , Distrofias Hereditarias de la Córnea/genética , Retículo Endoplásmico/metabolismo , Epítopos/metabolismo , Células HEK293 , Humanos , Datos de Secuencia Molecular , Mutación , Pliegue de Proteína , Estructura Terciaria de Proteína , Análisis de Secuencia de ADN , Temperatura , Tripsina/metabolismoRESUMEN
During anoxia/reoxygenation (A/R) injury, intracellular chloride ion concentration ([Cl(-)](i)) homeostasis may play a role in maintaining the normal physiological function of cardiomyocytes. Various chloride transport systems could have influenced the concentration of chloride ion, but what kinds of chloride transport systems could play an important role in cardiomyocytes subjected to A/R injury and its mechanism are unknown. The aim of our study was to clarify the contributions of various chloride transport systems to anoxia/reoxygenation in rat neonatal cardiac myocytes and further to investigate the involved mechanisms. Oxidative stress and redox-sensitive transcription factor (NF-kappaB) activation are believed to play an important role in the A/R injury. To assess whether oxidative stress and NF-kappaB involve [Cl(-)](i) changes resulting in cardiomyocytes injury, the anoxia-reoxygenation (A/R) injury model was successfully established and administered with inhibitors of various chloride transport systems. Administration with Cl(-)-substitution and Cl(-)/HCO(3) (-) exchange inhibitor(SITS) has been shown to produce a protective effect against A/R injury by decreasing [Cl(-)](i) concentration, lipid peroxidation (malondialdehyde (MDA)) levels, and NF-kappaB activity, and by increasing antioxidant enzyme (glutathione peroxidase (GSHPx), superoxide dismutase (SOD), and catalase(CAT)) activity. However, inhibitors for the Cl(-)-channel (9-AC) and Na(+)-K(+)-2Cl(-) co-transporter (bumetanide) had no effects. Our results indicate that Cl(-)/HCO(3) (-) exchange system plays an important role in the cardiocyte A/R injury by influencing [Cl(-)](i) concentration. The protective effects of SITS and Cl(-)-substitution on cardiomyocytes may be due to the attenuation of oxidative stress and inhibition of NF-kappaB activation.
Asunto(s)
Cloruros/metabolismo , Daño por Reperfusión Miocárdica/metabolismo , Miocitos Cardíacos/metabolismo , FN-kappa B/metabolismo , Estrés Oxidativo , Ácido 4-Acetamido-4'-isotiocianatostilbeno-2,2'-disulfónico/farmacología , Animales , Antracenos/farmacología , Apoptosis , Bumetanida/farmacología , Catalasa/metabolismo , Hipoxia de la Célula , Supervivencia Celular , Canales de Cloruro/antagonistas & inhibidores , Canales de Cloruro/metabolismo , Antiportadores de Cloruro-Bicarbonato/metabolismo , Productos Lácteos Cultivados , Pruebas de Enzimas , Glutatión Peroxidasa/metabolismo , L-Lactato Deshidrogenasa/metabolismo , Peroxidación de Lípido , Masculino , Daño por Reperfusión Miocárdica/patología , Miocitos Cardíacos/patología , Oxígeno/metabolismo , Cultivo Primario de Células , Transporte de Proteínas , Ratas , Ratas Sprague-Dawley , Especies Reactivas de Oxígeno/metabolismo , Inhibidores del Simportador de Cloruro Sódico y Cloruro Potásico/farmacología , Simportadores de Cloruro de Sodio-Potasio/metabolismo , Superóxido Dismutasa/metabolismoRESUMEN
Anion secretion by colonic epithelium is dependent on apical CFTR-mediated anion conductance and basolateral ion transport. In many tissues, the NKCC1 Na(+)-K(+)-2Cl(-) cotransporter mediates basolateral Cl(-) uptake. However, additional evidence suggests that the AE2 Cl(-)/HCO(3)(-) exchanger, when coupled with the NHE1 Na(+)/H(+) exchanger or a Na(+)-HCO(3)(-) cotransporter (NBC), contributes to HCO(3)(-) and/or Cl(-) uptake. To analyze the secretory functions of AE2 in proximal colon, short-circuit current (I(sc)) responses to cAMP and inhibitors of basolateral anion transporters were measured in muscle-stripped wild-type (WT) and AE2-null (AE2(-/-)) proximal colon. In physiological Ringer, the magnitude of cAMP-stimulated I(sc) was the same in WT and AE2(-/-) colon. However, the I(sc) response in AE2(-/-) colon exhibited increased sensitivity to the NKCC1 inhibitor bumetanide and decreased sensitivity to the distilbene derivative SITS (which inhibits AE2 and some NBCs), indicating that loss of AE2 results in a switch to increased NKCC1-supported anion secretion. Removal of HCO(3)(-) resulted in robust cAMP-stimulated I(sc) in both AE2(-/-) and WT colon that was largely mediated by NKCC1, whereas removal of Cl(-) resulted in sharply decreased cAMP-stimulated I(sc) in AE2(-/-) colon relative to WT controls. Inhibition of NHE1 had no effect on cAMP-stimulated I(sc) in AE2(-/-) colon but caused a switch to NKCC1-supported secretion in WT colon. Thus, in AE2(-/-) colon, Cl(-) secretion supported by basolateral NKCC1 is enhanced, whereas HCO(3)(-) secretion is diminished. These results show that AE2 is a component of the basolateral ion transport mechanisms that support anion secretion in the proximal colon.
Asunto(s)
Proteínas de Transporte de Anión/metabolismo , Aniones/metabolismo , Antiportadores/metabolismo , Colon/metabolismo , AMP Cíclico/fisiología , 1-Metil-3-Isobutilxantina/farmacología , Ácido 4-Acetamido-4'-isotiocianatostilbeno-2,2'-disulfónico/farmacología , Acetazolamida/farmacología , Amilorida/análogos & derivados , Amilorida/farmacología , Animales , Animales Recién Nacidos , Proteínas de Transporte de Anión/antagonistas & inhibidores , Proteínas de Transporte de Anión/genética , Antiportadores/antagonistas & inhibidores , Antiportadores/genética , Bicarbonatos/metabolismo , Bumetanida/farmacología , Anhidrasa Carbónica II/genética , Anhidrasa Carbónica II/metabolismo , Anhidrasa Carbónica III/genética , Anhidrasa Carbónica III/metabolismo , Inhibidores de Anhidrasa Carbónica/farmacología , Anhidrasas Carbónicas/genética , Anhidrasas Carbónicas/metabolismo , Proteínas de Transporte de Catión/antagonistas & inhibidores , Proteínas de Transporte de Catión/metabolismo , Ciego/patología , Cloruros/metabolismo , Colforsina/farmacología , Colon/efectos de los fármacos , Colon/patología , Fenómenos Electrofisiológicos , Expresión Génica/genética , Canales Iónicos/genética , Bombas Iónicas/genética , Ratones , Ratones Endogámicos , Ratones Noqueados , Proteínas SLC4A , Intercambiador 1 de Sodio-Hidrógeno , Intercambiadores de Sodio-Hidrógeno/antagonistas & inhibidores , Intercambiadores de Sodio-Hidrógeno/metabolismo , Simportadores de Cloruro de Sodio-Potasio/efectos de los fármacos , Simportadores de Cloruro de Sodio-Potasio/metabolismo , Miembro 2 de la Familia de Transportadores de Soluto 12RESUMEN
In earlier studies, we have established that l-arginine enhances motility and metabolic rate in spermatozoa of goat, bull and mouse. In the present study this work was extended to human sperm cells obtained from the semen samples of asthenospermic patients, which are characterised by low motility. The metabolic rate was followed by monitoring the glucose consumption (1-(13)C glucose as substrate) and the production of lactate in sperm cells, using (13)C NMR. The stimulatory effect of l-arginine was neutralised on adding an NO-synthase inhibitor like N(omega)-nitro-L-arginine methyl ester. On the other hand, the inactive d-enantiomorph did not affect the stimulatory effect of l-arginine. This strongly suggests that L-arginine acts through the NO signal pathway. We also demonstrated that the stimulatory effect of L-arginine was inhibited in the presence of anion channel inhibitors like 4-acetamido-4'-isothiocyanostilbene-2,2'-disulphonic acid, 2,4-dinitrophenol and carbonyl cyanide m-chlorophenylhydrazone. Furthermore, bicarbonate supplementation was found to be essential for the action of L-arginine. These observations indicate that L-arginine induces NO synthesis and stimulates motility and metabolism only when an active anion transport system is present.
Asunto(s)
Arginina/farmacología , Astenozoospermia/metabolismo , Transportadores de Anión Orgánico/antagonistas & inhibidores , Motilidad Espermática/efectos de los fármacos , 2,4-Dinitrofenol/farmacología , Ácido 4-Acetamido-4'-isotiocianatostilbeno-2,2'-disulfónico/farmacología , Bicarbonatos/farmacología , Carbonil Cianuro m-Clorofenil Hidrazona/farmacología , Humanos , Ácido Láctico/metabolismo , Masculino , NG-Nitroarginina Metil Éster/farmacología , Óxido Nítrico/fisiología , Espermatozoides/efectos de los fármacos , Espermatozoides/metabolismo , Estereoisomerismo , Desacopladores/farmacologíaRESUMEN
Proteins of the CLCA gene family including the human ClCa1 (hClCa1) have been suggested to constitute a new family of chloride channels mediating Ca(2+)-dependent Cl- currents. The present study examines the relationship between the hClCa1 protein and Ca(2+)-dependent Cl- currents using heterologous expression of hClCa1 in HEK293 and NCIH522 cell lines and whole cell recordings. By contrast to previous reports claiming the absence of Cl- currents in HEK293 cells, we find that HEK293 and NCIH522 cell lines express constitutive Ca(2+)-dependent Cl- currents and show that hClCa1 increases the amplitude of Ca(2+)-dependent Cl- currents in those cells. We further show that hClCa1 does not modify the permeability sequence but increases the Cl- conductance while decreasing the G(SCN-)/G(Cl-) conductance ratio from approximately 2-3 to approximately 1. We use an Eyring rate theory (two barriers, one site channel) model and show that the effect of hClCa1 on the anionic channel can be simulated by its action on lowering the first and the second energy barriers. We conclude that hClCa1 does not form Ca(2+)-dependent Cl- channels per se or enhance the trafficking/insertion of constitutive channels in the HEK293 and NCIH522 expression systems. Rather, hClCa1 elevates the single channel conductance of endogenous Ca(2+)-dependent Cl- channels by lowering the energy barriers for ion translocation through the pore.
Asunto(s)
Canales de Cloruro/fisiología , Fenómenos Electrofisiológicos/fisiología , Ácido 4,4'-Diisotiocianostilbeno-2,2'-Disulfónico/farmacología , Ácido 4-Acetamido-4'-isotiocianatostilbeno-2,2'-disulfónico/farmacología , Calcio/farmacología , Línea Celular , Línea Celular Tumoral , Membrana Celular/metabolismo , Canales de Cloruro/antagonistas & inhibidores , Cloruros/metabolismo , Estimulación Eléctrica , Fenómenos Electrofisiológicos/efectos de los fármacos , Expresión Génica/genética , Gluconatos/farmacología , Humanos , Potenciales de la Membrana/fisiología , Modelos Moleculares , Ácido Niflúmico/farmacología , Permeabilidad , Termodinámica , Tiocianatos/farmacología , TransfecciónRESUMEN
In our attempt to assess the topology of glucosylceramide biosynthesis, we have employed a truncated ceramide analogue that permeates cell membranes and is converted into water soluble sphingolipid analogues both in living and in fractionated cells. Truncated sphingomyelin is synthesized in the lumen of the Golgi, whereas glucosylceramide is synthesized at the cytosolic surface of the Golgi as shown by (a) the insensitivity of truncated sphingomyelin synthesis and the sensitivity of truncated glucosylceramide synthesis in intact Golgi membranes from rabbit liver to treatment with protease or the chemical reagent DIDS; and (b) sensitivity of truncated sphingomyelin export and insensitivity of truncated glucosylceramide export to decreased temperature and the presence of GTP-gamma-S in semiintact CHO cells. Moreover, subfractionation of rat liver Golgi demonstrated that the sphingomyelin synthase activity was restricted to fractions containing marker enzymes for the proximal Golgi, whereas the capacity to synthesize truncated glucosylceramide was also found in fractions containing distal Golgi markers. A similar distribution of glucosylceramide synthesizing activity was observed in the Golgi of the human liver derived HepG2 cells. The cytosolic orientation of the reaction in HepG2 cells was confirmed by complete extractability of newly formed NBD-glucosylceramide from isolated Golgi membranes or semiintact cells by serum albumin, whereas NBD-sphingomyelin remained protected against such extraction.
Asunto(s)
Glucosilceramidas/biosíntesis , Glucosiltransferasas/metabolismo , Aparato de Golgi/metabolismo , Ácido 4,4'-Diisotiocianostilbeno-2,2'-Disulfónico , Ácido 4-Acetamido-4'-isotiocianatostilbeno-2,2'-disulfónico/análogos & derivados , Ácido 4-Acetamido-4'-isotiocianatostilbeno-2,2'-disulfónico/farmacología , Fraccionamiento Celular , Línea Celular , Citosol/metabolismo , Endopeptidasas/metabolismo , Glucosiltransferasas/antagonistas & inhibidores , Aparato de Golgi/enzimología , Guanosina 5'-O-(3-Tiotrifosfato)/farmacología , Humanos , Hígado/enzimología , Hígado/metabolismo , Esfingomielinas/biosíntesisRESUMEN
We have explored the possibilities that cell volume is regulated by the status of microtubule assembly and cyclic AMP metabolism and may be coordinated with shape change. Treatment of J774.2 mouse macrophages with colchicine caused rapid microtubule disassembly and was associated with a striking increase (from 15-20 to more than 90 percent) in the proportion of cells with a large protuberance at one pole. This provided a simple experimental system in which shape changes occurred in virtually an entire cell population in suspension. Parallel changes in cell volume could then be quantified by isotope dilution techniques. We found that the shape change caused by colchicine was accompanied by a decrease in cell volume of approximately 20 percent. Nocodozole, but not lumicolchicine, caused identical changes in both cell shape and cell volume. The volume loss was not due to cell lysis nor to inhibition of pinocytosis. The mechanism of volume loss was also examined. Colchicine induced a small but reproducible increase in activity of the ouabain-sensitive Na(+), K(+)-dependent ATPase. However, inhibition of this enzyme/transport system by ouabain did not change cell volume nor did it block the colchicines-induced decrease in volume. One the other hand, SITS (4'acetamido, 4-isothiocyano 2,2' disulfonic acid stilbene), an inhibitor of anion transport, inhibited the effects of colchicines, thus suggesting a role for an anion transport system in cell volume regulation. Because colchicine is known to activate adenylate cyclase in several systems and because cell shape changes are often induced by hormones that elevate cyclic AMP, we also examined the effects of cyclic AMP on cell volume. Agents that act to increase syclic AMP (cholera toxin, which activates adenylate cyclase; IBMX, and inhibitor of phosphodiesterase; and dibutyryl cyclic AMP) all caused a volume decrease comparable to that of colchicine. To define the effective metabolic pathway, we studied two mutants of J774.2, one deficient in adenylate cyclase and the other exhibiting markedly reduced activity of cyclic AMP-dependent protein kinase. Cholera toxin did not produce a volume change in either mutant. Cyclic AMP produced a decrease in the cyclase-deficient line comparable to that in wild type, but did not cause a volume change in the kinase- deficient line. This analysis established separate roles for cyclic AMP and colchicine. The volume decrease induced by cyclic AMP requires the action of a cyclic AMP-dependent protein kinase. Colchicine, on the other hand, induced a comparable volume change in both mutants and wild type, and thus does not require the kinase.
Asunto(s)
AMP Cíclico/fisiología , Macrófagos/citología , Microtúbulos/metabolismo , Ácido 4-Acetamido-4'-isotiocianatostilbeno-2,2'-disulfónico/farmacología , Animales , Calcio/farmacología , Línea Celular , Colchicina/farmacología , Activación Enzimática , Iones/metabolismo , Macrófagos/fisiología , Ratones , Pinocitosis , ATPasa Intercambiadora de Sodio-Potasio/metabolismoRESUMEN
Upon engagement of chemoattractant receptors, neutrophils generate inositol trisphosphate and diacylglycerol (DG) by means of a phosphatidylinositol-specific phospholipase C (PI-PLC) which is regulated by a GTP-binding protein(s). We have previously reported (Reibman, J., H. M. Korchak, L. B. Vosshall, K. A. Haines, A. M. Rich, and G. Weissmann. 1988. J. Biol. Chem. 263:6322-6328) a biphasic rise in DG after exposure of neutrophils to the chemoattractant FMLP: a rapid (less than or equal to 15 s) phase ("triggering") and a slow (greater than or equal to 30 s) phase ("activation"). These derive from distinct intracellular lipid pools. To study the source of rapid and slow DG, we have used a unique probe, protein I, a porin that is the major outer membrane protein of Neisseria gonorrhoeae. Treatment of neutrophils with protein I inhibits exocytosis and homotypic cell adhesion provoked by FMLP without inhibiting assembly of the NADPH oxidase responsible for O2-. generation. DG turnover in PMN labeled with [3H]arachidonate and [14C]glycerol was profoundly altered by protein I. Whereas the rapid peak of DG was only modestly diminished (FMLP vs. FMLP plus protein I = DG labeled with [3H]arachidonic acid (3H-a.a.-DG): 142 +/- 14% SEM vs. 125 +/- 22%; DG labeled with the glycerol backbone with [14C]glycerol (D-14C-G): 125 +/- 10% SEM vs. 107 +/- 8.5% SEM), the slow rise in both 3H-a.a.-DG and D-14C-G was essentially abolished. Moreover, treatment of neutrophils with 4-4'-diisothiocyanatostilbene-2,2'-disulfonic acid (DIDS), which, like protein I, inhibits exocytosis without affecting O2-. generation also inhibited slow DG. However, protein phosphorylation and dephosphorylation (47phox, 66phox) were unaffected in the absence of slow DG. To determine the source of the slow DG, we have analyzed radiolabeled phospholipid (PL) turnover after FMLP +/- protein I (P.I.). Treatment of PMN with FMLP (0.1 microM) resulted in breakdown of phosphatidylcholine (PC), beginning at 30 s, and reaching a nadir at 60 s (3H-PC = 59 +/- 10.2% SEM of resting, 14C-PC = 57 +/- 6.4%). Protein I (0.25 microM) significantly inhibited PC turnover after FMLP ([3H]PC = 95 +/- 5.6% and [14C]PC = 86 +/- 8.4% of resting at 60 s), but failed to alter the metabolism of 3H- or 14C-phosphatidylinositol after FMLP (91 +/- 19.6 and 88 +/- 16.5% vs. 92 +/- 9.2 and 91 +/- 16% at 60 s).(ABSTRACT TRUNCATED AT 400 WORDS)
Asunto(s)
Proteínas de la Membrana Bacteriana Externa/inmunología , Diglicéridos/metabolismo , Activación de Linfocitos , Neisseria gonorrhoeae/inmunología , Neutrófilos/metabolismo , Fosfatidilcolinas/metabolismo , Porinas , Fosfolipasas de Tipo C/metabolismo , Ácido 4,4'-Diisotiocianostilbeno-2,2'-Disulfónico , Ácido 4-Acetamido-4'-isotiocianatostilbeno-2,2'-disulfónico/análogos & derivados , Ácido 4-Acetamido-4'-isotiocianatostilbeno-2,2'-disulfónico/farmacología , Adulto , Células Cultivadas , Activación Enzimática , Etanol/farmacología , Exocitosis , Humanos , Cinética , N-Formilmetionina Leucil-Fenilalanina , Neutrófilos/inmunología , Fosfolípidos/metabolismo , Propranolol/farmacología , Proteína Quinasa C/metabolismoRESUMEN
Chick myoblast fusion in culture was investigated using prostanoid synthesis inhibitors to delay spontaneous fusion. During this delay myoblast fusion could be induced by prostaglandin E1 (PGE1), by raising extracellular potassium and by addition of carbachol. Carbachol-induced fusion, but not PGE-induced fusion, was prevented by the acetylcholine receptor blocker alpha-bungarotoxin. Fusion induced by any of these agents was prevented by the Ca channel blockers lanthanum and D600. The threshold for potassium-induced fusion was 7-8 mM; maximal fusion occurred at 16-20 mM. Low extracellular potassium inhibited spontaneous fusion. Intracellular potassium in fusion competent myoblasts was 101 m-moles/l cell. Calcium flux measurements demonstrated that high potassium increased calcium permeability in fusion-competent myoblasts. A 30-s exposure to high potassium or PGE1 was sufficient to initiate myoblast fusion. Anion-exchange inhibitors (SITS and DIDS) delayed spontaneous myoblast fusion and blocked fusion induced by PGE1 but not carbachol. Blocking the acetylcholine receptor shifted the dose-response relation for PGE-induced fusion to higher concentrations. PGE1-induced fusion required chloride ions; carbachol-induced fusion required sodium ions. Provided calcium channels were available, potassium always induced fusion. We conclude that myoblasts possess at least three, independent pathways, each of which can initiate myoblast fusion and that the PGE-activated pathway and the acetylcholine receptor-activated pathway act synergistically. We suggest that fusion competent myoblasts have a high resting membrane potential and that fusion is controlled by depolarization initiated directly (potassium), by an increase in permeability to chloride ions (PGE), or by activation of the acetylcholine receptor (carbachol); depolarization triggers a rise in calcium permeability. The consequent increase in intracellular calcium initiates myoblast fusion.
Asunto(s)
Calcio/metabolismo , Canales Iónicos/fisiología , Músculos/embriología , Prostaglandinas E/farmacología , Receptores Colinérgicos/metabolismo , Ácido 4,4'-Diisotiocianostilbeno-2,2'-Disulfónico , Ácido 4-Acetamido-4'-isotiocianatostilbeno-2,2'-disulfónico/análogos & derivados , Ácido 4-Acetamido-4'-isotiocianatostilbeno-2,2'-disulfónico/farmacología , Animales , Bungarotoxinas/farmacología , Bloqueadores de los Canales de Calcio/farmacología , Carbacol/farmacología , Fusión Celular/efectos de los fármacos , Permeabilidad de la Membrana Celular , Embrión de Pollo , Cloroquina/farmacología , Indometacina/farmacología , Canales Iónicos/efectos de los fármacos , Lantano/farmacología , Potenciales de la Membrana , Músculos/citología , Potasio/metabolismo , Sodio/metabolismoRESUMEN
A chloride (Cl-)-dependent, nonselective cation conductance was activated during cellular shrinkage and inhibited during cellular swelling or by extracellular gadolinium. The shrinking-induced, nonselective cation conductance and the swelling-induced anion conductance appear to function in the regulation of cell volume in airway epithelia. The shrinking-induced cation conductance had an unusual dependence on Cl-: partial replacement of extracellular Cl- with aspartate reduced the magnitude of the shrinking-enhanced current without accompanying changes in the reversal potential. The Cl- dependence of the nonselective cation conductance could provide a mechanism that tightly regulates Cl- secretion and sodium reabsorption in cells under osmotic stress.
Asunto(s)
Cationes , Membrana Celular/fisiología , Cloruros/farmacología , Canales Iónicos/fisiología , Ácido 4,4'-Diisotiocianostilbeno-2,2'-Disulfónico , Ácido 4-Acetamido-4'-isotiocianatostilbeno-2,2'-disulfónico/análogos & derivados , Ácido 4-Acetamido-4'-isotiocianatostilbeno-2,2'-disulfónico/farmacología , Amilorida/farmacología , Calcio/farmacología , Permeabilidad de la Membrana Celular , Cesio/metabolismo , Conductividad Eléctrica , Epitelio/fisiología , Epitelio/ultraestructura , Humanos , Litio/metabolismo , Pulmón/fisiología , Pulmón/ultraestructura , Potenciales de la Membrana , Concentración Osmolar , Potasio/metabolismo , Sodio/metabolismoRESUMEN
Barrett's specialized columnar epithelium (SCE) replaces reflux-damaged squamous epithelium. The benefits of SCE lie in its superior protection of the esophagus against further reflux damage. It was shown that this protection is dependent on ion transport and barrier function of SCE. The risks of SCE lie in its higher predisposition to malignant transformation. An understanding of underlying mechanisms of both processes would benefit considerably from greater knowledge of the structure and function of native SCE - the latter recently advanced by the availability of a telomerase-immortalized, nonneoplastic, human Barrett's cell line (BAR-T). Some of BAR-T characteristics for growth and differentiation have been described recently, but not its capacity to serve as a model for ion transport and barrier function of SCE. To determine the latter, BAR-T cells were grown in enriched media, seeded on permeable supports, and subjected to electrical, biochemical, and morphologic study. HET-1A (esophageal epithelial cell line), a nonneoplastic, human esophageal squamous cell line, was also studied for comparison. BAR-T, but not HET-1A cells in HEPES Ringer solution behaved as polarized monolayers with the capacity for ion transport and barrier function. This was evident electrically with a volt-ohm meter (EVOM),which recorded in BAR-T a resting potential difference of 2.0 +/- 0.2 mV, Isc of 17.4 +/- 3.3 microAmps/cm2 and resistance of 103 +/- 12 ohms x cm2. Further, Isc in BAR-T was inhibitable by exposure to Na-free solution, serosal ouabain, and luminal 4-acetamido4'-isothiocyano-2,2'-stilbenedisulfonic acid. Expression of tight junction genes were determined in BAR-T and HET-1A cells using quantitative reverse transcriptase-polymerase chain reaction, with expression of zonula occludens-1 (ZO-1) set at 1 as reference. Claudins 1, 4, and 12 were prominently expressed in BAR-T (0.2-0.6 of ZO-1), while claudins 1, 11, and 12 were prominently expressed in HET-1A(0.1-0.8 of ZO-1). BAR-T, but not HET-1A, expressed claudins 4, 8, 16, 18, and 23, and HET-1A, but not BAR-T, expressed claudins 11, 15, and 20. Protein expression of prominently expressed claudins in BAR-T correlated with mRNA expression. Immunofluorescence and confocal microscopy localized claudins 1 and 4 in BAR-T to cell membranes and claudin 18, specifically to the tight junction. Membrane polarization was also documented in BAR-T by immunolocalization of NaK,ATPase to the basolateral membrane. BAR-T, but not HET-1A cells grown on permeable supports form a polarized monolayer with both ion transport and barrier function. Since a number of features of BAR-T are similar to Barrett's SCE and distinct from HET-1A, the BAR-T cell line represents a valuable resource for the study of ion transport and barrier function of nondysplastic SCE.