RESUMEN
AIMS: Little is known about the physiological regulation of the human intestinal di/tri-peptide transporter, hPepT1. In the present study we evaluated the effects of epidermal growth factor (EGF) and insulin on hPepT1-mediated dipeptide uptake in the intestinal cell line Caco-2. METHODS: Caco-2 cells were grown on filters for 23-27 days. Apical dipeptide uptake was measured using [14C]glycylsarcosine([14C]Gly-Sar). HPepT1 mRNA levels were investigated using RT-PCR, cytosolic pH was determined using the pH-sensitive fluorescent probe BCECF. RESULTS: Basolateral application of EGF increased [14C]Gly-Sar uptake with an ED50 value of 0.77 +/- 0.25 ng mL-1 (n = 3-6) and a maximal stimulation of 33 +/- 2% (n = 3-6). Insulin stimulated [14C]Gly-Sar uptake with an ED50 value of 3.5 +/- 2.0 ng mL-1 (n = 3-6) and a maximal stimulation of approximately 18% (n = 3-6). Gly-Sar uptake followed simple Michaelis-Menten kinetics. Km in control cells was 0.98 +/- 0.11 mM (n = 8) and Vmax was 1.86 +/- 0.07 nmol cm-2 min-1 (n = 8). In monolayers treated with 200 ng mL-1 of EGF, Km was 1.11 +/- 0.05 mM (n = 5) and Vmax was 2.79 +/- 0.05 nmol cm-2 min-1 (n = 5). In monolayers treated with 50 ng mL-1 insulin, Km was 1.03 +/- 0.08 mM and Vmax was 2.19 +/- 0.06 nmol cm-2 min-1 (n = 5). Kinetic data thus indicates an increase in the number of active transporters, following stimulation. The incrased Gly-Sar uptake was not accompanied by changes in hPepT1 mRNA, nor by measurable changes in cytosolic pH. CONCLUSIONS: Short-term stimulation with EGF and insulin caused an increase in hPepT1-mediated uptake of Gly-Sar in Caco-2 cell monolayers, which could not be accounted for by changes in hPepT1 mRNA or proton-motive driving force.
Asunto(s)
Proteínas Portadoras/metabolismo , Dipéptidos/farmacocinética , Factor de Crecimiento Epidérmico/farmacología , Simportadores , Brefeldino A/farmacología , Células CACO-2/efectos de los fármacos , Colchicina/farmacología , Relación Dosis-Respuesta a Droga , Glucosa/farmacocinética , Humanos , Concentración de Iones de Hidrógeno , Insulina/farmacología , Leucina/farmacocinética , Transportador de Péptidos 1 , Reacción en Cadena de la Polimerasa , ARN Mensajero/análisis , ARN Neoplásico/análisisRESUMEN
One of the long-term effects of growth hormone (GH) in adipocytes is to maintain a state of refractoriness to insulin-like effects, a refractoriness which otherwise declines within a few hours of GH starvation. Here, we examined differences in GH signaling and the possible role for the recently identified family of suppressors of cytokine signaling (SOCS) proteins in the transition between the refractory and the responsive states in rat adipocytes. The ability of GH to stimulate lipogenesis and tyrosine phosphorylation of the GH receptor (GHR), Janus kinase 2 (Jak2), insulin receptor substrate-1 (IRS-1) and -2 (IRS-2) was greatly reduced in refractory as compared to responsive primary rat adipocytes. However, phosphorylation of Signal Transducer and Activator of Transcription 5 (Stat5) was not affected. SOCS-3 and CIS mRNA levels were significantly higher in refractory compared to responsive cells and could be induced by GH, whereas the level of SOCS-2 mRNA was unchanged. With overexpression of GHR, Jak2 and IRS-1 along with each of these SOCS proteins in human A293 cells, we could demonstrate that both SOCS-1 and SOCS-3 completely inhibited the GH-stimulated tyrosine phosphorylation of IRS-1, whereas SOCS-2 and CIS did not. Our data suggest that GH induces refractoriness to the insulin-like effects in a negative-feedback manner by inhibiting GH-induced GHR/Jak2/IRS-1/IRS-2 phosphorylation through upregulation of SOCS-3, which almost completely blocks Jak2 activation.
Asunto(s)
Adipocitos/efectos de los fármacos , Hormona del Crecimiento/farmacología , Insulina/farmacología , Péptidos y Proteínas de Señalización Intracelular , Proteínas de la Leche , Fosfoproteínas/antagonistas & inhibidores , Proteínas Tirosina Quinasas/antagonistas & inhibidores , Proteínas/fisiología , Proteínas Proto-Oncogénicas , Proteínas Represoras , Factores de Transcripción , Adipocitos/química , Adipocitos/metabolismo , Animales , Proteínas Portadoras/genética , Proteínas Portadoras/fisiología , Proteínas de Unión al ADN/metabolismo , Embrión de Mamíferos , Retroalimentación Fisiológica , Expresión Génica , Humanos , Proteínas Inmediatas-Precoces/genética , Proteínas Sustrato del Receptor de Insulina , Janus Quinasa 2 , Riñón , Lípidos/biosíntesis , Masculino , Fosfoproteínas/genética , Fosfoproteínas/metabolismo , Fosforilación , Proteínas Tirosina Quinasas/genética , Proteínas Tirosina Quinasas/metabolismo , Proteínas/genética , ARN Mensajero/análisis , Ratas , Ratas Sprague-Dawley , Receptores de Somatotropina/metabolismo , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa , Factor de Transcripción STAT5 , Transducción de Señal , Proteína 1 Supresora de la Señalización de Citocinas , Proteína 3 Supresora de la Señalización de Citocinas , Proteínas Supresoras de la Señalización de Citocinas , Transactivadores/metabolismo , Transfección , Tirosina/metabolismoRESUMEN
While the zebrafish is commonly used for studies of developmental biology and toxicology, very little is known about their osmoregulatory physiology. The present investigation of Na(+) and Cl(-) transport revealed that the zebrafish is able to tolerate extremely low ambient ion concentrations and that this is achieved at least in part by a greatly enhanced apparent uptake capacity and affinity for both ions. Zebrafish maintain plasma and whole body electrolyte concentrations similar to most other freshwater teleosts even in deionized water containing only 35 microM NaCl, i.e soft water. We recorded an extremely low transport affinity constant (K(m)) of 8+/-1 microM for the active uptake of Cl(-) in soft water acclimated fish, while other transport kinetic parameters were in agreement with reports for other freshwater organisms. While both Na(+) and Cl(-) uptake in soft water clearly depends on apical proton pump activity, changes in abundance and possibly localization of this protein did not appear to contribute to soft water acclimation. Active Cl(-) uptake was strongly dependent on branchial carbonic anhydrase (CA) activity regardless of water type, while the response of Na(+) transport to a CA inhibitor was more variable. Differential response of Na(+) uptake to amiloride depending on acclimation medium suggests that different Na(+) transport mechanisms are employed by zebrafish acclimated to soft and hard water.
Asunto(s)
Amilorida/análogos & derivados , Cloruros/metabolismo , Sodio/metabolismo , Pez Cebra/metabolismo , Animales , Transporte Biológico , Western Blotting , Regulación hacia Abajo , Etoxzolamida , Agua Dulce , Branquias/metabolismo , Inmunohistoquímica , Cinética , Macrólidos , Inhibidores de la Bomba de Protones , Bombas de Protones/análisis , Bombas de Protones/metabolismo , Pez Cebra/sangreRESUMEN
The Ca2+ content of pancreatic juice is closely regulated by yet unknown mechanisms. One aim of the present study was to find whether rat pancreatic ducts have a Na+/Ca2+ exchanger, as found in some Ca2+ transporting epithelia. Another aim was to establish whether the exchanger is regulated by hormones/agonists affecting pancreatic secretion. Whole pancreas, pure pancreatic acini and ducts were obtained from rats and used for RT-PCR and Western blot analysis, immunohistochemistry and intracellular Ca2+ measurements using Fura-2. RT-PCR analysis indicated Na+/Ca2+-exchanger isoforms NCX1.3 and NCX1.7 in acini and pancreas. Western blot with NCX1 antibody identified bands of 70, 120 and 150 kDa in isolated ducts, acini and pancreas. Immunofluorescence experiments showed the Na+/Ca2+ exchanger on the basolateral membrane of acini and small intercalated/intralobular ducts, but in larger intralobular/extralobular ducts the exchanger was predominantly on the luminal membrane. Na+/Ca2+ exchange in ducts was monitored by changes in intracellular Ca2+ activity upon reversal of the Na+ gradient. Secretin (1 nM) and carbachol (1 mM) reduced Na+/Ca2+ exchange by 40% and 51%, respectively. Insulin (1 nM) increased Na+/Ca2+ exchange by 230% within 5 min. The present study shows that pancreatic ducts express the Na+/Ca2+ exchanger. Its distinct localization along the ductal tree and regulation by secretin, carbachol and insulin indicate that ducts might be involved in regulation of Ca2+ concentrations in pancreatic juice.
Asunto(s)
Calcio/metabolismo , Conductos Pancreáticos/metabolismo , Intercambiador de Sodio-Calcio/metabolismo , Animales , Carbacol/farmacología , Femenino , Inmunoquímica , Insulina/farmacología , Miocardio/citología , Miocardio/metabolismo , Conductos Pancreáticos/citología , Potasio/farmacología , Ratas , Ratas Wistar , Secretina/farmacología , Intercambiador de Sodio-Calcio/genéticaRESUMEN
The human intestinal cell line Caco-2 was used as a model system to study the effects of epidermal growth factor (EGF) on peptide transport. EGF decreased apical-to-basolateral fluxes of [(14)C]glycylsarcosine ([(14)C]Gly-Sar) up to 50.2 +/- 3.6% (n = 6) of control values. Kinetic analysis of the fluxes showed that maximal flux (V(max)) of transepithelial transport decreased from 3.00 +/- 0.17 nmol x cm(-2) x min(-1) in control cells to 0.50 +/- 0.07 nmol x cm(-2) x min(-1) in cells treated with 5 ng/ml EGF (n = 6, P < 0.01). The apparent Michaelis-Menten constant (K(m)) was 2.71 +/- 0.31 mM (n = 6) in control cells and 1.89 +/- 0.28 mM (n = 6, not significantly different from control) in EGF-treated cells. Similarly, apical uptake of [(14)C]Gly-Sar decreased in cells treated with EGF, with an ED(50) value of 0.36 +/- 0.06 ng/ml (n = 6) EGF and a maximal inhibition of 80 +/- 0.02% (n = 6). V(max) decreased from 2.61 +/- 0.4 to 1.06 +/- 0.1 nmol x cm(-2) x min(-1) (n = 3, P < 0.05), whereas K(m) remained constant. Basolateral Gly-Sar uptake showed no changes in V(max) or K(m) after EGF treatment (n = 3). RT-PCR showed a decrease in hPepT1 mRNA (using glucose-6-phosphate dehydrogenase mRNA as control) in cells treated with EGF. Western blotting indicated a decrease in hPepT1 protein in cell lysates. We conclude that EGF treatment decreases Gly-Sar transport in Caco-2 cells by decreasing the number of peptide transporter molecules in the apical membrane.
Asunto(s)
Proteínas Portadoras/genética , Proteínas Portadoras/metabolismo , Dipéptidos/farmacocinética , Factor de Crecimiento Epidérmico/farmacología , Mucosa Intestinal/metabolismo , Simportadores , Transporte Biológico/efectos de los fármacos , Transporte Biológico/fisiología , Células CACO-2 , Radioisótopos de Carbono , Proteínas Portadoras/análisis , Relación Dosis-Respuesta a Droga , Expresión Génica/fisiología , Humanos , Inmunohistoquímica , Mucosa Intestinal/citología , Microscopía Confocal , Transportador de Péptidos 1 , ARN Mensajero/análisisRESUMEN
Evidence is discussed that apical CFTR Cl- channels of mitochondria-rich (MR) cells of Bufo bufo skin conduct beta-adrenergic receptor-activated Cl- currents. Ussing chambers studies revealed the following selectivity sequence of the receptor activated conductance, Cl- > Br- > NO3- > I-. With ion selective microelectrode-techniques, it was shown that receptor-coupled Cl- channels are not located in principal cells. A small conductance (7-10 pS) CFTR-like Cl- channel is located in the apical plasma membrane of MR cells. Short life times of sealed patches prevented detailed study of its selectivity to other halide ions and its molecular regulation. With monoclonal hCFTR-antibodies, selective expression in MR cells of the targeted antigens could be demonstrated. A transcript of CFTR was amplified in the skin, and a bbCFTR cDNA clone was generated from toad skin mRNA that exhibits 89% amino acid identity with the human homologue. The frequency of obtaining channels in patch clamp studies was too low for accounting quantitatively for the macroscopic conductance. Since MR cells were isolated by trypsin, and a putative extracellular loop of the deduced bbCFTR protein contains a target peptide bond for trypsin, enzyme treatment may have destroyed apical CFTR molecules.
Asunto(s)
Cloruros/metabolismo , Regulador de Conductancia de Transmembrana de Fibrosis Quística/metabolismo , Piel/metabolismo , Secuencia de Aminoácidos , Animales , Bufo bufo , Clonación Molecular , Regulador de Conductancia de Transmembrana de Fibrosis Quística/química , Regulador de Conductancia de Transmembrana de Fibrosis Quística/genética , ADN Complementario , Humanos , Transporte Iónico , Microscopía Electrónica , Datos de Secuencia Molecular , Técnicas de Placa-Clamp , Receptores Adrenérgicos beta/efectos de los fármacos , Receptores Adrenérgicos beta/metabolismo , Homología de Secuencia de Aminoácido , Piel/ultraestructuraRESUMEN
Growth hormone initiates signaling by inducing homodimerization of two GH receptors. Here, we have sought to determine whether constitutively active receptor can be created in the absence of the extracellular domain by substituting it with high affinity leucine zippers to create dimers of the growth hormone receptor (GHR) signaling domain. The entire extracellular domain of the GHR was replaced by the hemagglutinin-tagged zipper sequence of either the c-Fos or c-Jun transcription factor (termed Fos-GHR and Jun-GHR, respectively). Transient transfection of Fos-GHR or Jun-GHR resulted in activation of the serine protease inhibitor 2.1 promoter in Chinese hamster ovary-K1 cells to a level equal to that achieved by fully activated wild type GHR. Furthermore, stable expression of Jun-GHR alone or Fos-GHR and Jun-GHR together in the interleukin 3-dependent BaF-B03 cell line resulted in cell proliferation after interleukin 3 withdrawal at a rate equal to maximally stimulated wild type GHR-expressing cells. Activation of STAT 5b was also observed in Fos-Jun-GHR-expressing cells at a level equal to that in chronically GH-treated GHR-expressing cells. Thus, forced dimerization of the transmembrane and cytoplasmic domains of the GHR in the absence of the extracellular domain can lead to the constitutive activation of known GH signaling end points, supporting the view that proximity of Janus kinase 2 (JAK2) kinases is the essential element in signaling. Such constitutively active GH receptors may have particular utility for transgenic livestock applications.
Asunto(s)
Hormona del Crecimiento/metabolismo , Leucina Zippers , Proteínas de la Leche , Receptores de Somatotropina/metabolismo , Secuencia de Aminoácidos , Animales , Secuencia de Bases , Células CHO , División Celular , Cricetinae , Cartilla de ADN , Proteínas de Unión al ADN/biosíntesis , Dimerización , Humanos , Proteínas Proto-Oncogénicas c-fos/metabolismo , Proteínas Proto-Oncogénicas c-jun/metabolismo , Receptores de Somatotropina/química , Proteínas Recombinantes de Fusión/metabolismo , Factor de Transcripción STAT5 , Transactivadores/biosíntesisRESUMEN
In epithelia, extracellular nucleotides are often associated with regulation of ion transporters, especially Cl(-) channels. In this study, we investigated which purinoceptors are present in native pancreatic ducts and how they regulate ion transport. We applied whole-cell patch-clamp recordings, intracellular Ca(2+) and pH measurements, and reverse transcription-polymerase chain reaction (RT-PCR) analysis. The data show two types of purinoceptors and cellular responses. UTP and ATP produced large Ca(2+) transients, a decrease in intracellular pH, 8-10-mV depolarization of the membrane voltage, and a decrease in the whole-cell conductance. The membrane effects were due to closure of K(+) channels, as confirmed by dependence on extracellular K(+). UTP/ATP effects could be associated with P2Y(2) purinoceptors, and RT-PCR revealed mRNAs for P2Y(2) and P2Y(4) receptors. On the other hand, 2', 3'-O-4-benzoylbenzoyl-ATP induced Ca(2+) influx and approximately 20-mV depolarization of the membrane voltage with a concomitant increase in the whole-cell conductance. These effects were dependent on extracellular Na(+), not Cl(-), indicating opening of cation channels associated with P2X(7) purinoceptors. RT-PCR showed mRNAs for P2X(7) and P2X(4) receptors. In microperfused ducts, luminal (but not basolateral) ATP caused large depolarizations of membrane voltages recorded with microelectrodes, consistent with luminal localization of P2X(7) receptors. Thus, P2Y(2) (and possibly P2Y(4)) purinoceptors inhibit K(+) channels and may not support secretion in native ducts. P2X(7) (and possibly P2X(4)) receptors are associated with cation channels and may contribute to regulation of secretion.
Asunto(s)
Conductos Pancreáticos/fisiología , Bloqueadores de los Canales de Potasio , Receptores Purinérgicos P2/metabolismo , Adenosina Trifosfato/farmacología , Animales , Electrofisiología , Femenino , Neuropéptidos/genética , Neuropéptidos/metabolismo , Conductos Pancreáticos/metabolismo , Reacción en Cadena de la Polimerasa , Canales de Potasio/efectos de los fármacos , ARN Mensajero/metabolismo , Ratas , Ratas Wistar , Receptores Purinérgicos P2/genética , Receptores Purinérgicos P2X4 , Receptores Purinérgicos P2X7 , Receptores Purinérgicos P2Y2 , Uridina Trifosfato/farmacologíaRESUMEN
Duodenal diverticula occur in 0.016 to 5.19% of patients undergoing roentgenographic investigation of the upper gastrointestinal tract. They are classified into extraluminal and intraluminal types and are usually found near the papilla of Vater. The majority of these cases are asymptomatic. These diverticula occasionally result in the obstruction of the biliary and/or pancreatic ducts, haemorrhage or perforation. Symptomatic cases may require endoscopic or surgical intervention. The management should be individualized to the kind of complication, type, and location of the diverticulum. Surgical procedures in this area may be technically difficult and associated with high mortality and morbidity. Elective treatment of uncomplicated duodenal diverticula is usually not justified. The difficulties in identifying these lesions as the source of symptoms are emphasized.
Asunto(s)
Divertículo , Enfermedades Duodenales , Divertículo/diagnóstico , Divertículo/etiología , Enfermedades Duodenales/diagnóstico , Enfermedades Duodenales/etiología , Enfermedades Duodenales/terapia , HumanosAsunto(s)
Conformación Proteica , Proteínas Tirosina Quinasas/metabolismo , Proteínas Proto-Oncogénicas , Receptores de Somatotropina/química , Receptores de Somatotropina/fisiología , Transducción de Señal , Animales , Calcio/metabolismo , Proteínas Quinasas Dependientes de Calcio-Calmodulina/metabolismo , División Celular/efectos de los fármacos , División Celular/fisiología , Endocitosis , Hormona del Crecimiento/farmacología , Humanos , Janus Quinasa 2 , Mitosis , Modelos Biológicos , Receptores de Somatotropina/metabolismoRESUMEN
Sixty-one surgical departments in Denmark were asked how they used the three possible diagnoses: carcinoma in situ vesicae urinariae (D09.0), neoplasma benignum vesicae urinariae (D30.3) and neoplasma malignum vesicae urinariae (C67.9) for bladder tumours with specific reference to different stages and grades of the tumour. The answers from 59 departments demonstrated great variation in the classification of the same bladder tumour. This variation results in a registration of data which is not valid for a statistical outcome of the real incidence of benign and malignant bladder tumours in Denmark. A consensus from the Danish Bladder Cancer Committee, which will be published in 1996, concerning the criteria for the use of the different diagnoses for bladder tumours, should however in the future make it possible for all departments to make a uniform classification.
Asunto(s)
Neoplasias de la Vejiga Urinaria/clasificación , Dinamarca , Humanos , Sistema de Registros , Servicio de Cirugía en Hospital , Encuestas y Cuestionarios , Terminología como Asunto , Neoplasias de la Vejiga Urinaria/diagnóstico , Neoplasias de la Vejiga Urinaria/cirugíaRESUMEN
Fifteen patients with stones in the common bile duct, in whom treatment with endoscopic papillotomy and stone-extraction had been unsuccessful were treated with extracorporeal shockwave lithotripsy. Nine patients were stone-free after one or two sessions, and two patients after further endoscopic treatment. One patient achieved partial clearance and palliation. One patient had a choledochoduodenostomy performed due to ineffectiveness of the shockwave lithotripsy. Two patients, who were thought to have a stone, turned out to have neoplasma in the common bile duct. Complications were frequent but temporary and needed no treatment. We conclude that extracorporeal shockwave lithotripsy is a valuable and safe alternative in those cases where conventional endoscopic treatment has failed, and should be considered before operation, especially to old for high-risk patients.