RESUMO
Transepithelial sodium transport is a process that involves active Na(+) transport at the basolateral membrane of the epithelial cell. This process is mediated by the Na(+)/K(+) pump, which exchanges 3 internal Na(+) by 2 external K(+) inducing a net charge movement and the second Na(+) pump, which transports Na(+) accompanied by Cl(-) and water. It has been suggested that this pump could also be electrogenic. Herein, we evaluated, in MDCK cells, the short-circuit current (Isc) generated by these Na(+) pumps at the basolateral membrane of the epithelial cells, using amphotericin B as an apical permeabilizing agent. In Cl(-)-containing media, Isc induced by amphotericin B is totally inhibited by ouabain, indicating that only the electrogenic Na(+)/K(+) pump is detectable in the presence of Cl(-). Electrogenicity of the second Na(+) pump can be demonstrated in Cl(-)-free media. The existence of a furosemide-sensitive component of Isc, in addition to an ouabain-sensitive one, was identified in absence of chloride. Passive Cl(-) movement associated with the function of the second Na(+) pump seems to be regulated by the pump itself. These results demonstrate that the second Na(+) pump is an electroneutral mechanism result from the stoichiometric movement of Na(+) and Cl(-) across the basolateral plasma membrane of the epithelial cell.
Assuntos
Células Epiteliais/metabolismo , ATPase Trocadora de Sódio-Potássio/metabolismo , Animais , Cloretos/química , Cloretos/metabolismo , Cães , Células Epiteliais/química , Transporte de Íons/fisiologia , Células Madin Darby de Rim Canino , Potássio/química , Potássio/metabolismo , Sódio/química , Sódio/metabolismo , ATPase Trocadora de Sódio-Potássio/químicaRESUMO
Secretory antibodies of the immunoglobulin A (sIgA) class constitute the first line of antigen-specific immune protection against pathogens and other antigens at mucosal surfaces. Although initially perceived as potentially deleterious, catalytic antibodies have been proposed to participate in the removal of metabolic wastes and in protection against infection. Here we show that the presence of sIgA endowed with serine protease-like hydrolytic activity in milk strongly correlates with PAR-2 activation in human intestinal epithelial cells. F(ab')(2) fragments of sIgA activated the epithelial cells in culture to produce beta-defensin-2 (hBD2). Intracellular Ca(2+) mobilization was induced by treatment with (1) sIgA-F(ab')(2) fragments; (2) trypsin, a recognized PAR-2 agonist; or (3) a synthetic PAR-2 agonist peptide (SLIGKV). The co-treatment with a synthetic PAR-2 antagonist peptide (FSLLRY) and sIgA-F(ab')(2) fragments eliminates the latter's effect; nevertheless, cells were not refractory to subsequent stimulation with sIgA-F(ab')(2) fragments. Both the induction of hBD-2 expression in epithelial cells and the increase in intracellular [Ca(2+)] stimulated by sIgA-F(ab')(2) fragments were inhibited by treatment with serine protease inhibitors or pertussis toxin (PTX). These findings suggest that catalytic antibodies can activate intestinal epithelial cells through G-protein-coupled PAR-2, and could actively participate in the immune system of breastfed babies inducing the production of peptides related to innate defense, such as defensins.
Assuntos
Anticorpos Catalíticos/imunologia , Imunoglobulina A Secretora/imunologia , Mucosa Intestinal/imunologia , Leite Humano/imunologia , Peptídeo Hidrolases/imunologia , Receptor PAR-2/agonistas , beta-Defensinas/biossíntese , Adolescente , Adulto , Anticorpos Catalíticos/isolamento & purificação , Anticorpos Catalíticos/metabolismo , Linhagem Celular Tumoral , Feminino , Células HT29 , Humanos , Imunidade Inata , Imunoglobulina A Secretora/isolamento & purificação , Imunoglobulina A Secretora/metabolismo , Fragmentos Fab das Imunoglobulinas/imunologia , Fragmentos Fab das Imunoglobulinas/isolamento & purificação , Fragmentos Fab das Imunoglobulinas/metabolismo , Leite Humano/enzimologia , Peptídeo Hidrolases/isolamento & purificação , Peptídeo Hidrolases/metabolismo , Receptor PAR-2/biossíntese , Adulto JovemRESUMO
K+ -conductive pathways were evaluated in isolated surface and crypt colonic cells, by measuring (86)Rb efflux. In crypt cells, basal K+ efflux (rate constant: 0.24 +/- 0.044 min(-1), span: 24 +/- 1.3%) was inhibited by 30 mM TEA and 5 mM Ba2+ in an additive way, suggesting the existence of two different conductive pathways. Basal efflux was insensitive to apamin, iberiotoxin, charybdotoxin and clotrimazole. Ionomycin (5 microM) stimulated K+ efflux, increasing the rate constant to 0.65 +/- 0.007 min(-1) and the span to 83 +/- 3.2%. Ionomycin-induced K+ efflux was inhibited by clotrimazole (IC(50) of 25 +/- 0.4 microM) and charybdotoxin (IC(50) of 65 +/- 5.0 nM) and was insensitive to TEA, Ba2+, apamin and iberiotoxin, suggesting that this conductive pathway is related to the Ca2+-activated intermediate-conductance K+ channels (IK(ca)). Absence of extracellular Ca2+ did neither affect basal nor ionomycin-induced K+ efflux. However, intracellular Ca2+ depletion totally inhibited the ionomycin-induced K+ efflux, indicating that the activation of these K+ channels mainly depends on intracellular calcium liberation. K+ efflux was stimulated by intracellular Ca(2+) with an EC(50) of 1.1 +/- 0.04 microM. In surface cells, K+ efflux (rate constant: 0.17 +/- 0.027 min(-1); span: 25 +/- 3.4%) was insensitive to TEA and Ba2+. However, ionomycin induced K+ efflux with characteristics identical to that observed in crypt cells. In conclusion, both surface and crypt cells present IK(Ca) channels but only crypt cells have TEA- and Ba2+-sensitive conductive pathways, which would determine their participation in colonic K+ secretion.
Assuntos
Cálcio/farmacologia , Colo/fisiologia , Canais de Potássio Cálcio-Ativados/metabolismo , Potássio/metabolismo , Animais , Cálcio/metabolismo , Células Cultivadas , Colo/citologia , Inibidores Enzimáticos/farmacologia , Cobaias , Transporte de Íons/efeitos dos fármacos , Transporte de Íons/fisiologia , MasculinoRESUMO
The mechanisms involved in D-glucose and amino acid transport in the intestine of birds are still not clear. In chickens, D-glucose and amino acid absorption occurs via carrier-mediated transport, but in wild birds a passive paracellular mechanism seems to be the predominant pathway. The purpose of this work was to determine the existence of carrier-mediated sodium cotransport of D-glucose and L-alanine in the small intestine of Japanese quail (Coturnix coturnix), a granivorous bird. Intestinal transport was determined by changes in the short-circuit current (Isc), proportional to ion transmembrane flux, in the middle segment of the intestine of Japanese quail with a Ussing chamber. D-Glucose produced an increase of the Isc, and this effect was reverted by phloridzin, indicating the presence of a D-glucose transport mediated by the sodium/glucose cotranspoter 1. Addition of L-alanine also produced an increase of the Isc. We concluded that there is carrier-mediated cotransport of D-glucose and L-alanine with sodium in the small intestine of the Japanese quail.
Assuntos
Alanina/metabolismo , Coturnix/metabolismo , Glucose/metabolismo , Animais , Transporte Biológico/efeitos dos fármacos , Condutividade Elétrica , Impedância Elétrica , Glucose/farmacologia , Absorção Intestinal/efeitos dos fármacos , Absorção Intestinal/fisiologia , Intestino Delgado/fisiologia , Masculino , Proteínas de Transporte de Monossacarídeos/efeitos dos fármacos , Proteínas de Transporte de Monossacarídeos/fisiologia , Florizina/farmacologiaRESUMO
This paper describes a rapid and sensitive method to determine inorganic phosphate, even in the presence of labile organic phosphate compounds and large quantities of proteins. The method eliminates the use of sodium arsenite, a highly toxic compound, substituting bismuth citrate for it to stabilize the phosphomolybdic acid complex formed during the interaction of inorganic phosphate and molybdate reduced by ascorbic acid. This method has also been adapted to microplates and has been used to determine the activities of Na/K ATPase and alkaline phosphatase of intestinal basolateral and luminal plasma membranes.
Assuntos
Membrana Celular/enzimologia , Enterócitos/enzimologia , Compostos Organometálicos/química , Fosfatos/análise , Monoéster Fosfórico Hidrolases/análise , Fosfatase Alcalina/análise , Animais , Arsenitos/química , Arsenitos/toxicidade , Ácido Ascórbico/química , Soluções Tampão , Mucosa Intestinal/enzimologia , Modelos Lineares , Molibdênio/química , Compostos Organofosforados/análise , Fosfatos/normas , Ácidos Fosfóricos/química , Compostos de Sódio/química , Compostos de Sódio/toxicidade , ATPase Trocadora de Sódio-Potássio/análise , EspectrofotometriaRESUMO
Epinephrine and beta-adrenergic agonists (beta1 and beta2 for isoproterenol, beta1 for dobutamine, beta2 for salbutamol) stimulated K+ (or 86Rb) influx mediated by the Na+-K+-2Cl- cotransporter and the Na+-K+ pump in isolated colonic crypt cells. Preincubation with bumetanide abolished the epinephrine effect on the Na+-K+ pump, suggesting that the primary effect is on the cotransporter. Maximal effect was obtained with 1 microM epinephrine with an EC50 of 91.6 +/- 9.98 nM. Epinephrine-induced K+ transport was blocked by propranolol with an IC50 of 134 +/- 28.2 nM. alpha-Adrenergic drugs did not modify K+ transport mechanisms. Neither Ba2+ nor tetraethylammonium nor DIDS modified the adrenergic enhancement on the cotransporter. In addition, epinephrine did not affect K+ efflux. Dibutyryl cAMP did not alter K+ transport. Reduction of extracellular Ca2+ to 30 nM did not influence the response to epinephrine. However, 1,2-bis(2-aminophenoxy)ethane-N,N,N',N'-tetraacetic acid-AM abolished epinephrine-induced K+ transport. Ionomycin increased Na+-K+-2Cl- cotransport activity. Moreover, epinephrine increased intracellular Ca2+ concentration in a process inhibited by propranolol. In conclusion, epinephrine stimulated the Na+-K+-2Cl- cotransporter in a process mediated by beta1- and beta2-receptors and modulated by intracellular Ca2+ liberation.
Assuntos
Agonistas Adrenérgicos beta/farmacologia , Cálcio/metabolismo , Proteínas de Transporte/metabolismo , Colo/metabolismo , Membranas Intracelulares/metabolismo , Adrenérgicos/farmacologia , Animais , Transporte Biológico/efeitos dos fármacos , Bumetanida/farmacologia , Colo/citologia , Epinefrina/farmacologia , Cobaias , Masculino , Potássio/metabolismo , Sistemas do Segundo Mensageiro/fisiologia , Simportadores de Cloreto de Sódio-PotássioRESUMO
We present three cases of Leiomioblastomas of stomach, diagnosed at the unit of Pathology of the Gastrointestinal Cancer Center "Dr Luis E. Anderson" -San Cristóbal-Táchira State Venezuela. The lesions are analyzed from the clinical, morphological and immunohistological points of view, utilizing Queratine and Vimentin techniques. This neoplasm is not frequent but it is interesting because its biologic behavior not always correspond to its histology-pattern.
Assuntos
Leiomioma Epitelioide/patologia , Neoplasias Gástricas/patologia , Humanos , Imuno-Histoquímica , Leiomioma Epitelioide/diagnóstico por imagem , Leiomioma Epitelioide/cirurgia , Masculino , Pessoa de Meia-Idade , Radiografia , Neoplasias Gástricas/diagnóstico por imagem , Neoplasias Gástricas/cirurgiaRESUMO
Neutral amino acid transport was examined by using isolated enterocytes. Cells transport L-alanine by at least three different mechanisms: two Na(+)-dependent systems (A and ASC) and one Na(+)-independent mechanism (system L), in addition to passive entry. System A was characterized acterized by measuring the Na(+)-dependent alpha-(methylamino)isobutyric acid (MeAIB) uptake. Na(+)-dependent MeAIB uptake was concentrative and saturable. Vmax was obtained at 80mM Na+ in the incubation medium and Kt app for Na+ was 21.5 mM. Kt app for MeAIB was 6.75 +/- 0.37 mM and the Vmax was 14.2 +/- 0.3 nmol.mg-1.min-1. System ASC was studied by evaluating the Na(+)-dependent L-alanine uptake, insensitive to MeAIB and inhibitable by L-serine and L-cysteine. Uptake by this mechanism was also concentrative and saturable. Maximal uptake was obtained with 80 mM Na+ in the incubation medium and Kt app for Na+ was 29.7 mM. Kt app for L-alanine was 7.02 +/- 0.61 mM and Vmax was 5.44 +/- 0.19 nmol.mg-1.min-1. The Na(+)-independent system L was studied by measuring cycloleucine uptake in Na(+)-free medium. It had a saturable and a nonsaturable component. Only the saturable component was concentrative; it was inhibited by 2-amino-2-norbornanecarboxylic acid and was capable of mediating exchange diffusion. Kt app for cycloleucine was 4.05 +/- 0.72 mM and the Vmax was 31.9 +/- 1.3 nmol.mg-1.min-1. These results confirm the existence of Na(+)-dependent systems A and ASC and Na(+)-independent system L in isolated enterocytes.
Assuntos
Aminoácidos/metabolismo , Intestino Delgado/metabolismo , Alanina/metabolismo , Animais , Transporte Biológico , Separação Celular , Cobaias , Técnicas In Vitro , Intestino Delgado/citologia , Cinética , Masculino , Sódio/metabolismoRESUMO
A fluorimetric assay using ethidium bromide (EB) was employed to quantify cell death in monolayer cell cultures (MA-104 cells) in situ and isolated cell suspensions (isolated colonic cells and Leishmania). Fluorescence of EB stained cells was measured with a photometer coupled to an inverted microscope for cell monolayers or in a spectrofluorometer for cell suspensions. Dead cells stained with trypan blue were fluorescent with EB in all preparations studied, but the latter gave an unequivocal signal. Staining with EB and fluorescein diacetate was mutually exclusive. The relationship between the number of EB fluorescent cells and the intensity of fluorescence measured in the microphotometer was linear for a large range of cell numbers (1-14000) from different types of preparations. Applicability of the method for measuring living and dead cells in two different time scales (minutes and hours) is shown using MA-104 cell monolayers infected with rotavirus and Leishmania suspensions treated with amphotericin B. The method is fast, simple, sensitive and reliable, enabling quantification of living and dead cells in monolayers and suspensions.
Assuntos
Morte Celular/fisiologia , Fluorometria , Animais , Sobrevivência Celular/fisiologia , Células Cultivadas , Chlorocebus aethiops , Colo/citologia , Etídio , Técnicas In Vitro , Leishmania braziliensis/citologia , Microscopia de Fluorescência , Ratos , Reprodutibilidade dos Testes , Infecções por Rotavirus/patologia , Sensibilidade e EspecificidadeRESUMO
Infection of MA-104 cells with the OSU strain of rotavirus induced an increase in Na+ and a decrease in K+ intracellular concentrations, starting at 4 h post-infection. These changes were not related to an inhibition of the Na+/K+ pump since ouabain-sensitive 86Rb uptake was augmented in rotavirus-infected cells compared to control cells, whereas the [3H]ouabain binding and Na+/K+ ATPase activity in the cell homogenate were unaffected. Furosemide-sensitive 86Rb uptake (Na+/K+/2Cl- cotransport) was not modified by the infection. Passive 86Rb efflux and 22Na influx were augmented in infected cells suggesting an increase in the plasma membrane permeability. The increase in intracellular Na+ concentration might be responsible for the observed stimulation of the Na+/K+ pump. This effect was dependent upon the synthesis of viral proteins because it was abolished by addition of cycloheximide up to 4 h post-infection. Prevention of the increase in intracellular Na+ by the use of low Na(+)-containing media did not modify the pattern of protein synthesis. This suggests that changes in intracellular Na+ and K+ concentrations were not related to shutoff of cellular protein synthesis. Alterations of ion contents in the rotavirus-infected enterocytes might impair intestinal absorptive capacity before the appearance of histopathological lesions.
Assuntos
Potássio/metabolismo , Rotavirus/fisiologia , Sódio/metabolismo , Animais , Transporte Biológico Ativo/efeitos dos fármacos , Linhagem Celular , Permeabilidade da Membrana Celular , Cicloeximida/farmacologia , Furosemida/farmacologia , Homeostase , Ouabaína/farmacologia , ATPase Trocadora de Sódio-Potássio/metabolismo , Proteínas Virais/biossínteseRESUMO
We describe a method to isolate surface cells from guinea pig distal colon that obtains a good yield and high viability, as demonstrated by a 99% exclusion of trypan blue, only a 10% liberation of lactate dehydrogenase after 30-min incubation at 37 degrees C, and the inability of succinate to stimulate oxygen consumption before plasma membrane permeabilization. Oxygen consumption (QO2) measured after the sequential addition of the following drugs showed that oligomycin inhibited QO2 by 67%, carbonyl cyanide p-trifluoromethoxyphenylhydrazone increased QO2 by approximately 70% of the basal consumption, and rotenone inhibited QO2 by 90%. Cells at 37 degrees C for 30 min maintained intracellular Na+ and K+ concentrations of 25 and 120 mM, respectively. The ATP consumed by the Na(+)-K+ pump was derived from oxidative phosphorylation (79%) and from glycolysis (21%). Initial rates for Na+ and K+ transported by the pump were 105 +/- 10 and 65 +/- 5 nmol.mg protein-1.min-1, respectively. The rates of Na+ and K+ transported per ATP consumed were estimated to be 2.5 Na+/ATP and 1.6 K+/ATP.