RESUMEN
BACKGROUND AND PURPOSE: Since endothelin (ET) may act as pro-fibrotic mediator, expression and release of ET isoforms, their receptors and potential pro-fibrotic ET effects were studied in human lung fibroblasts. EXPERIMENTAL APPROACH: MRC-5 and primary human lung fibroblasts (phLFb) were cultured. Expression of prepro-ET isoforms was determined by qPCR and release of ET-1 by elisa. ET receptor function was analysed by real-time measurement of dynamic mass redistribution (DMR). Incorporation of [(3) H]-thymidine was determined as measure of proliferation and that of [(3) H]-proline for collagen synthesis. Phospho-p42/44 MAP kinase was determined by Western blot. KEY RESULTS: ET-1 is the predominant ET in human lung fibroblasts (hLF), and TGF-ß caused a further, selective and sustained up-regulation of ET-1 resulting in increased extracellular ET-1 accumulation. hLFb express mRNA encoding ET-A and ET-B receptors. Expression of both receptors was confirmed at protein level. ET-1 induced marked DMR signals, an effect that involved ET-A and ET-B receptors. Stimulatory effects of ET-1 on hLFb proliferation and collagen synthesis were mediated exclusively via ET-A receptors. ET-1, again via ET-A receptors, induced rapid activation of ERK MAPK, shown to be a crucial cellular signal in ET-1-induced collagen synthesis. ET-1-induced activation of ERK and collagen synthesis was, in contrast to corresponding effect of a muscarinic agonist, largely insensitive to pertussis toxin. CONCLUSIONS AND IMPLICATIONS: hLFb are endowed with all elements necessary to build a functional autocrine/paracrine endothelinergic system, which appears to drive pro-fibrotic airway and lung remodelling processes, effects for which only ET-A, but not ET-B receptors appear to be of significance.
Asunto(s)
Endotelinas/metabolismo , Fibroblastos/metabolismo , Fibrosis/metabolismo , Línea Celular , Células Cultivadas , Colágeno/metabolismo , Antagonistas de los Receptores de Endotelina , Fibroblastos/efectos de los fármacos , Humanos , Pulmón/citología , Masculino , Prolina/metabolismo , Isoformas de Proteínas/metabolismo , Receptores de Endotelina/metabolismo , Timidina/metabolismo , Factor de Crecimiento Transformador beta/farmacologíaRESUMEN
BACKGROUND AND PURPOSE: Artificial agonists may have higher efficacy for receptor activation than the physiological agonist. Until now, such 'superagonism' has rarely been reported for GPCRs. Iperoxo is an extremely potent muscarinic receptor agonist. We hypothesized that iperoxo is a 'superagonist'. EXPERIMENTAL APPROACH: Signalling of iperoxo and newly synthesized structural analogues was compared with that of ACh at label-free M2 muscarinic receptors applying whole cell dynamic mass redistribution, measurement of G-protein activation, evaluation of cell surface agonist binding and computation of operational efficacies. KEY RESULTS: In CHO-hM2 cells, iperoxo significantly exceeds ACh in Gi /Gs signalling competence. In the orthosteric loss-of-function mutant M2 -Y104(3.33) A, the maximum effect of iperoxo is hardly compromised in contrast to ACh. 'Superagonism' is preserved in the physiological cellular context of MRC-5 human lung fibroblasts. Structure-signalling relationships including iperoxo derivatives with either modified positively charged head group or altered tail suggest that 'superagonism' of iperoxo is mechanistically based on parallel activation of the receptor protein via two orthosteric interaction points. CONCLUSION AND IMPLICATIONS: Supraphysiological agonist efficacy at muscarinic M2 ACh receptors is demonstrated for the first time. In addition, a possible underlying molecular mechanism of GPCR 'superagonism' is provided. We suggest that iperoxo-like orthosteric GPCR activation is a new avenue towards a novel class of receptor activators.
Asunto(s)
Fibroblastos/efectos de los fármacos , Proteínas de Unión al GTP/metabolismo , Isoxazoles/farmacología , Agonistas Muscarínicos/farmacología , Compuestos de Amonio Cuaternario/farmacología , Receptor Muscarínico M2/agonistas , Acetilcolina/farmacología , Animales , Células CHO , Línea Celular , Cricetulus , Fibroblastos/metabolismo , Humanos , Pulmón/citología , Pulmón/efectos de los fármacos , Pulmón/metabolismo , Receptor Muscarínico M2/metabolismo , Receptores Acoplados a Proteínas G/metabolismo , Transducción de Señal/efectos de los fármacosRESUMEN
Insulin has been approved for inhaled application, but safety concerns remain, because of un-physiologically high insulin concentrations in the lung. Since insulin may act as growth factor, possible proliferative effects of insulin, insulin analogues and insulin-like growth factor-1 (IGF-1) on human lung fibroblasts were studied. As measure of proliferation [(3)H]-thymidine incorporation was studied in HEL-299, MRC-5, IMR-90 and primary human lung fibroblasts. In all cells, mRNA encoding IGF-1 receptors and two variants of insulin receptors was detected. Insulin and IGF-1 stimulated [(3)H]-thymidine incorporation in all cells. Comparison of the concentration-dependent effects in HEL-299 cells showed that IGF-1 and insulin glargine were more potent (EC(50), 3 and 6 nM) and more effective (maximum increase, by 135-150%) than insulin and insulin detemir (EC(50), 22 and 110 nM; maximum increase: by 80%). Proliferative effects of IGF-1 and insulin were inhibited to the same extent by an antibody (1H7) directed against the IGF-1 receptor α-subunit. Insulin-induced stimulation of [(3)H]-thymidine incorporation was reduced by 83% after siRNA-mediated down-regulation of IGF-1 receptor by about 75%, but not affected by a similar down-regulation of the insulin receptor. Insulin and IGF-1 caused rapid up-regulation of the early genes FOS, EGR-1 and EGR-2 as well as of the gene coding for IGF-1. In conclusion, in human lung fibroblasts insulin exerts marked proliferative effects and the pharmacological profile of this response as well as specific receptor knock-down experiments suggest mediation via IGF-1 receptors. The risk of unwanted structural lung alterations by long-term inhalative application of insulin should be considered.
Asunto(s)
Fibroblastos/efectos de los fármacos , Hipoglucemiantes/farmacología , Factor I del Crecimiento Similar a la Insulina/farmacología , Insulina/análogos & derivados , Pulmón/efectos de los fármacos , Proliferación Celular , Células Cultivadas , Fibroblastos/metabolismo , Humanos , Insulina/farmacología , Insulina Detemir , Insulina Glargina , Insulina de Acción Prolongada , Pulmón/citología , ARN Mensajero/biosíntesis , Receptor IGF Tipo 1/biosíntesis , Receptor IGF Tipo 1/genética , Receptor de Insulina/biosíntesis , Receptor de Insulina/genética , Transducción de Señal , Timidina/metabolismoRESUMEN
Arginase was shown to be up-regulated in different animal models of inflammatory and fibrotic airway diseases. Since arginase provides L-ornithine, one precursor for L-proline, an essential substrate for collagen synthesis, it has been suggested that arginase might be a key enzyme in airway remodelling. The present study aimed to characterize expression of arginase isoenzymes in rat and human pulmonary fibroblasts, and to test whether arginase inhibition affects collagen synthesis. In primary rat tracheal and lung fibroblasts, mRNA for arginase I and II could be detected, with arginase I as predominant isoenzyme. In contrast, in human lung fibroblasts (primary cells and different cells lines) mRNA levels for arginase I were at or below detection limit whereas arginase II mRNA was markedly higher than in rat pulmonary fibroblasts. Arginase activity in rat tracheal and lung fibroblasts was between 20 and 30 mU/mg protein, but was below detection limit (2.5 mU/mg) in human lung fibroblasts. In rat tracheal and lung fibroblasts cultured in proline-free medium, arginase inhibition by N(omega)-hydroxy-nor-L-arginine caused a reduction by about one-third of basal collagen I accumulation (determined by western blot analysis) and largely attenuated transforming growth factor beta 1 (TGF-beta(1))-induced increase in collagen accumulation, whereas basal and TGF-beta(1)-induced collagen accumulation by human lung fibroblasts was not affected by arginase inhibition. In conclusion, arginase isoenzymes reveal a species specific expression pattern. Arginase contributes significantly to L-proline supply for collagen synthesis in rat fibroblasts, in which arginase I is the predominant isoenzyme, but not in human fibroblasts, in which arginase II is the only isoenzyme expressed.
Asunto(s)
Arginasa/metabolismo , Colágeno/biosíntesis , Fibroblastos/metabolismo , Regulación Enzimológica de la Expresión Génica , Animales , Arginasa/antagonistas & inhibidores , Arginasa/genética , Arginina/análogos & derivados , Arginina/farmacología , Femenino , Fibroblastos/enzimología , Humanos , Isoenzimas , Pulmón/citología , Pulmón/enzimología , Masculino , Prolina/metabolismo , ARN Mensajero/metabolismo , Ratas , Ratas Sprague-Dawley , Especificidad de la Especie , Tráquea/citología , Tráquea/enzimología , Factor de Crecimiento Transformador beta1/farmacologíaRESUMEN
Tiotropium is commonly used in the treatment of chronic obstructive pulmonary disease. Although largely considered to be a long-acting bronchodilator, its demonstrated efficacy in reducing the frequency of exacerbations and preliminary evidence from early studies indicating that it might slow the rate of decline in lung function suggested mechanisms of action in addition to simple bronchodilation. This hypothesis was examined in the recently published UPLIFT study and, although spirometric and other clinical benefits of tiotropium treatment extended to four years, the rate of decline in lung function did not appear to be reduced by the addition of tiotropium in this study. This article summarizes data from a variety of investigations that provide insights into possible mechanisms to account for the effects of tiotropium. The report summarizes the discussion on basic and clinical research in this field.
Asunto(s)
Broncodilatadores/farmacología , Antagonistas Colinérgicos/farmacología , Derivados de Escopolamina/farmacología , Acetilcolina/fisiología , Animales , Broncodilatadores/uso terapéutico , Antagonistas Colinérgicos/uso terapéutico , Tos/tratamiento farmacológico , Tos/fisiopatología , Humanos , Inflamación/patología , Pulmón/inervación , Pulmón/fisiología , Moco/metabolismo , Sistema Nervioso Parasimpático/efectos de los fármacos , Sistema Respiratorio/efectos de los fármacos , Sistema Respiratorio/patología , Derivados de Escopolamina/uso terapéutico , Bromuro de TiotropioRESUMEN
Lysosulfatide is a derivative of the glycosphingolipid sulfatide. It is a major component of high density lipoproteins and was detected in the human brain. Here, we show that lysosulfatide acts as an extracellular signal regulating the migration of a neural precursor cell line (B35 neuroblastoma cells) by rapidly promoting process retraction and cell rounding. These cells express the lysosulfatide receptor S1P3 according to RT-PCR, western blotting and immunocytochemistry, but S1P3 does not mediate the effect since preincubation with three different compounds known to inhibit S1P3 did not block lysosulfatide-induced cell rounding. The signal transduction after stimulation with 3 microM lysosulfatide involves a rapid increase of [Ca2+]i which causes process retraction. This mechanism may be relevant under conditions where neural cells encounter elevated lysosulfatide levels as for example under pathological conditions after breakdown of the blood brain barrier or possibly in the lysosomal sulfatide storage disorder metachromatic leukodystrophy.
Asunto(s)
Calcio/fisiología , Neuronas/fisiología , Psicosina/análogos & derivados , Células Madre/fisiología , Animales , Adhesión Celular , Línea Celular Tumoral , Movimiento Celular/efectos de los fármacos , Forma de la Célula , Neuronas/citología , Neuronas/efectos de los fármacos , Psicosina/farmacología , Psicosina/fisiología , Ratas , Receptores de Lisoesfingolípidos/biosíntesis , Transducción de Señal , Células Madre/citología , Células Madre/efectos de los fármacosRESUMEN
In lung fibroblasts, proliferation is inhibited by activation of EP(2) prostanoid receptors which are known to couple to adenylyl cyclase. Beside the classic target of cAMP, protein kinase A (PKA), alternative cAMP effectors have been identified, among them Epac (exchange protein activated by cAMP). The present study aimed to illuminate transduction pathways mediating the anti-proliferative effects of EP(2) receptors in lung fibroblasts. Proliferative activity of human lung fibroblasts was determined by measuring [(3)H]-thymidine incorporation. The selective EP(2) receptor agonist butaprost inhibited [(3)H]-thymidine incorporation by 75%, an effect mimicked by forskolin, the phosphodiesterase inhibitor IBMX, the stable cAMP analogues dibutyryl-cAMP and bromo-cAMP, as well as by the Epac selective cAMP analogues 8-pCPT-2'-O-Me-cAMP and Sp-8-pCPT-2'-O-Me-cAMPS, whereas the PKA selective agonist 6-Bnz-cAMP was inactive. The PKA inhibitor Rp-8-Br-cAMPS inhibited butaprost-induced phosphorylation of CREB (cAMP response element-binding protein), but did not affect butaprost-induced inhibition of [(3)H]-thymidine incorporation. Partial knockdown of Epac1 by specific siRNA transfection resulted in a marked attenuation of the inhibitory potency of butaprost, whereas transfection of Epac2 siRNA or non-silencing siRNA did not affect the effectiveness of butaprost to inhibit [(3)H]-thymidine incorporation. In conclusion, Epac1 rather than the classic cAMP effector PKA is a crucial element in the signal transduction pathway mediating anti-proliferative effects of EP(2) receptor activation.
Asunto(s)
Fibroblastos/metabolismo , Factores de Intercambio de Guanina Nucleótido/fisiología , Pulmón/citología , Receptores de Prostaglandina E/metabolismo , 8-Bromo Monofosfato de Adenosina Cíclica/análogos & derivados , 8-Bromo Monofosfato de Adenosina Cíclica/farmacología , Alprostadil/análogos & derivados , Alprostadil/farmacología , Proliferación Celular/efectos de los fármacos , Células Cultivadas , Colágeno/metabolismo , AMP Cíclico/fisiología , Proteínas Quinasas Dependientes de AMP Cíclico/antagonistas & inhibidores , Proteínas Quinasas Dependientes de AMP Cíclico/metabolismo , Activación Enzimática , Fibroblastos/citología , Fibroblastos/efectos de los fármacos , Técnicas de Silenciamiento del Gen , Factores de Intercambio de Guanina Nucleótido/agonistas , Factores de Intercambio de Guanina Nucleótido/metabolismo , Humanos , Fosforilación , ARN Interferente Pequeño/genética , Receptores de Prostaglandina E/genética , Transducción de Señal , Tionucleótidos/farmacologíaRESUMEN
Clinical observations indicate that in chronic obstructive pulmonary disease patients, the long-acting muscarinic antagonist tiotropium delays decline in airway function, suggesting that cholinergic mechanisms contribute to long-term structural changes. Human lung fibroblasts express muscarinic receptors and the present study aimed to explore their role in controlling collagen synthesis. MRC-5, HEL-299 and primary human lung fibroblasts (phLFb) were cultured. Incorporation of [(3)H]-proline into cellular proteins was determined as measure of collagen synthesis. In MRC-5 cells, the muscarinic agonist carbachol enhanced [(3)H]-proline incorporation in a concentration-dependent manner (effective concentration of 50%: 220 nM, increase at 10 microM by 40-55%, in a different series of experiments). Likewise, 10 microM oxotremorine caused an increase of approximately 65%. For comparison, transforming growth factor-beta1 (5 ng x mL(-1)) caused an increase of approximately 80%. Effects of carbachol on total [(3)H]-proline incorporation and collagenase-sensitive [(3)H]-proline fraction were similar. The effect of 10 microM carbachol was inhibited by tiotropium (inhibitory concentration of 50%: 110 pM), prevented by pertussis toxin and the mitogen-activated protein kinase inhibitor, PD 98059. Muscarinic agonists also enhanced [(3)H]-proline incorporation in a tiotropium-sensitive manner in HEL-299 cells and phLFb. In human lung fibroblasts, muscarinic receptors exert stimulatory effects on collagen synthesis. Prolonged blockade of muscarinic-induced collagen synthesis may contribute to reported beneficial long-term effects of anticholinergics in chronic obstructive pulmonary disease.
Asunto(s)
Colágeno/metabolismo , Fibroblastos/metabolismo , Pulmón/metabolismo , Receptores Muscarínicos/fisiología , Línea Celular , Antagonistas Colinérgicos/farmacología , Humanos , Pulmón/citologíaAsunto(s)
Broncodilatadores , Parasimpatolíticos , Enfermedad Pulmonar Obstructiva Crónica/tratamiento farmacológico , Derivados de Escopolamina , Animales , Broncodilatadores/efectos adversos , Broncodilatadores/farmacocinética , Broncodilatadores/farmacología , Broncodilatadores/uso terapéutico , Humanos , Pulmón/metabolismo , Parasimpatolíticos/efectos adversos , Parasimpatolíticos/farmacocinética , Parasimpatolíticos/farmacología , Parasimpatolíticos/uso terapéutico , Calidad de Vida , Receptores Muscarínicos/clasificación , Receptores Muscarínicos/efectos de los fármacos , Derivados de Escopolamina/efectos adversos , Derivados de Escopolamina/farmacocinética , Derivados de Escopolamina/farmacología , Derivados de Escopolamina/uso terapéutico , Bromuro de TiotropioRESUMEN
The present review summarizes the current knowledge of the cholinergic systems in the airways with special emphasis on the role of acetylcholine both as neurotransmitter in ganglia and postganglionic parasympathetic nerves and as non-neuronal paracrine mediator. The different cholinoceptors, various nicotinic and muscarinic receptors, as well as their signalling mechanisms are presented. The complex ganglionic and prejunctional mechanisms controlling the release of acetylcholine are explained, and it is discussed whether changes in transmitter release could be involved in airway dysfunctions. The effects of acetylcholine on different target cells, smooth muscles, nerves, surface epithelial and secretory cells as well as mast cells are described in detail, including the receptor subtypes involved in signal transmission.
Asunto(s)
Acetilcolina/metabolismo , Receptores Colinérgicos/fisiología , Fenómenos Fisiológicos Respiratorios/efectos de los fármacos , Sistema Respiratorio/metabolismo , Animales , Ganglios Parasimpáticos/fisiología , Humanos , Mastocitos/metabolismo , Músculo Liso/inervación , Músculo Liso/fisiología , Comunicación Paracrina/fisiología , Mucosa Respiratoria/citología , Mucosa Respiratoria/metabolismo , Sistema Respiratorio/inervación , Transducción de Señal/fisiologíaRESUMEN
Endothelial differentiation gene (EDG) receptors are a new family of eight G protein-coupled receptors for the lysophospholipids lysophosphatitic acid and sphingosine-1-phosphate. In the present experiments, the expression of EDG receptors in rat and human alveolar macrophages was studied by reverse transcription-polymerase chain reaction (RT-PCR). In alveolar macrophages of both species, mRNA for multiple EDG receptors could be detected, but the pattern of expression was different in both species. In human alveolar macrophages, mRNA for EDG1, EDG2, EDG4, EDG7 receptors and, to a lesser extent, for the EDG7 receptor was detected, whereas in rat macrophages, mRNA for EDG2, EDG5 receptors and, to a lesser extent, for the EDG6 receptor was found. In functional experiments, it was observed that lysophosphatitic acid and sphingosine-1-phosphate can stimulate O(2)(-) generation in rat and human alveolar macrophages suggesting that lysophosphatitic acid and sphingosine-1-phosphate possibly acting via EDG receptors may play a role in controlling the activation of macrophages.
Asunto(s)
Endotelio/citología , Endotelio/metabolismo , Proteínas Inmediatas-Precoces/biosíntesis , Macrófagos Alveolares/metabolismo , Proteínas Nucleares/biosíntesis , Receptores de Superficie Celular/biosíntesis , Receptores Acoplados a Proteínas G , Esfingosina/análogos & derivados , Factores de Transcripción/biosíntesis , Animales , Diferenciación Celular , Femenino , Humanos , Proteínas Inmediatas-Precoces/genética , Lisofosfolípidos/farmacología , Lisofosfolípidos/fisiología , Macrófagos Alveolares/efectos de los fármacos , Masculino , Proteínas Nucleares/genética , ARN Mensajero/biosíntesis , Ratas , Ratas Sprague-Dawley , Receptores de Superficie Celular/genética , Receptores del Ácido Lisofosfatídico , Receptores Lisofosfolípidos , Estallido Respiratorio/efectos de los fármacos , Estallido Respiratorio/inmunología , Esfingosina/farmacología , Esfingosina/fisiología , Factores de Transcripción/genéticaRESUMEN
By screening nucleotide databases, sequences containing the complete genes of the human cationic amino acid transporters (hCATs) 1, 2 and 4 were identified. Analysis of the genomic organization revealed that hCAT-2 consists of 12 translated exons and most likely of 2 untranslated exons. The splice variants hCAT-2A and hCAT-2B use exon 7 and 6, respectively. The hCAT-2 gene structure is closely related to the structure of hCAT-1, suggesting that they belong to a common gene family. hCAT-4 consists of only 4 translated exons and 3 short introns. Exons of identical size and highly homologous to exon 3 of hCAT-4 are present in hCAT-1 and hCAT-2.
Asunto(s)
Sistema de Transporte de Aminoácidos y+/genética , Transportador de Aminoácidos Catiónicos 1/genética , Transportador de Aminoácidos Catiônicos 2/genética , Sistema de Transporte de Aminoácidos y+/química , Sistemas de Transporte de Aminoácidos Básicos , Transportador de Aminoácidos Catiónicos 1/química , Transportador de Aminoácidos Catiônicos 2/química , Bases de Datos de Ácidos Nucleicos , Exones , Genes , Humanos , IntronesRESUMEN
Polyamines can inhibit NO synthesis in activated macrophages (Mphi). Since NO synthesis in Mphi depends on cellular uptake of L-arginine, effects of polyamines on L-arginine uptake were studied. Rat alveolar Mphi (AMphi) were cultured in absence or presence of lipopolysaccharides (LPS) and/or different polyamines for up to 20 h. LPS increased nitrite accumulation about 10-fold and [(3)H]-L-arginine uptake about 2.5-fold, effects almost abolished by spermidine. Spermine had much weaker and putrescine no effects. The effects of spermine depended largely on the presence of serum in the culture medium, suggesting that spermine aldehyde might be involved. Spermine suppressed the mRNA for inducible nitric oxide synthase (iNOS) and that for a specific cationic amino acid transporter (CAT), CAT-2B. In conclusion, in Mphi spermine concomitantly down-regulates NO synthesis and cellular L-arginine uptake by suppressing the expression of iNOS and CAT-2B. By inhibiting specific functions of activated Mphi the polyamine oxidase-polyamine system may play a role as immuno-suppressive modulator.
Asunto(s)
Arginina/farmacocinética , Macrófagos Alveolares/fisiología , Óxido Nítrico/biosíntesis , Espermina/farmacología , Animales , Transporte Biológico , Cationes , Regulación hacia Abajo , Femenino , Regulación de la Expresión Génica , Lipopolisacáridos , Macrófagos Alveolares/efectos de los fármacos , Masculino , Óxido Nítrico Sintasa/biosíntesis , Óxido Nítrico Sintasa/metabolismo , Ratas , Ratas Sprague-DawleyRESUMEN
It was tested whether the inducible nitric oxide synthase (iNOS) pathway might be involved in lipopolysaccharides-(LPS)-induced up-regulation of L-arginine transport in rat alveolar macrophages (AM). AM were cultured in absence or presence of LPS. Nitrite accumulation was determined in culture media and cells were used to study [3H]-L-arginine uptake or to isolate RNA for RT - PCR. Culture in presence of LPS (1 microg ml(-1), 20 h) caused 11 fold increase of nitrite accumulation and 2.5 fold increase of [3H]-L-arginine uptake. The inducible NO synthase (iNOS) inhibitor 2-amino-5,6-dihydro-6-methyl-4H-1,3-thiazine (AMT) present alone during culture had only marginal effects on [3H]-L-arginine uptake. However, AMT present during culture additionally to LPS, suppressed LPS-induced nitrite accumulation and LPS-stimulated [3H]-L-arginine uptake in the same concentration-dependent manner. AMT present only for the last 30 min of the culture period had similar effects on [3H]-L-arginine uptake. AMT present only during the uptake period also inhibited LPS-stimulated [3H]-L-arginine uptake, but with lower potency. The inhibitory effect of AMT could not be opposed by the NO releasing compound DETA NONOate. LPS caused an up-regulation of the mRNA for the cationic amino acid transporter CAT-2B, and this effect was not affected by AMT. AMT (100 microM) did not affect L-arginine transport studied by electrophysiological techniques in Xenopus laevis oocytes expressing either the human cationic amino acid transporter hCAT-1 or hCAT-2B. In conclusion, iNOS inhibition in rat AM abolished LPS-activated L-arginine uptake. This effect appears to be caused by reduced flow of L-arginine through the iNOS pathway.
Asunto(s)
Arginina/metabolismo , Inhibidores Enzimáticos/farmacología , Lipopolisacáridos/antagonistas & inhibidores , Lipopolisacáridos/farmacología , Macrófagos Alveolares/metabolismo , Óxido Nítrico Sintasa/antagonistas & inhibidores , Sistemas de Transporte de Aminoácidos Básicos , Animales , Arginina/farmacología , Transporte Biológico/efectos de los fármacos , Proteínas Portadoras/genética , Proteínas Portadoras/metabolismo , Femenino , Humanos , Macrófagos Alveolares/efectos de los fármacos , Macrófagos Alveolares/enzimología , Masculino , Potenciales de la Membrana/efectos de los fármacos , Proteínas de la Membrana/genética , Proteínas de la Membrana/metabolismo , Óxido Nítrico Sintasa/metabolismo , Óxido Nítrico Sintasa de Tipo II , Nitritos/metabolismo , Oocitos/efectos de los fármacos , Oocitos/metabolismo , ARN Mensajero/genética , ARN Mensajero/metabolismo , Ratas , Ratas Sprague-Dawley , Transducción de Señal/efectos de los fármacos , Tiazinas/farmacología , Xenopus laevisRESUMEN
1. As arginase by limiting nitric oxide (NO) synthesis may play a role in airway hyperresponsiveness and glucocorticoids are known to induce the expression of arginase I in hepatic cells, glucocorticoid effects on arginase in alveolar macrophages (AM Phi) were studied. 2. Rat AM Phi were cultured in absence or presence of test substances. Thereafter, nitrite accumulation, arginase activity, and the expression pattern of inducible NO synthase, arginase I and II mRNA (RT - PCR) and proteins (immunoblotting) were determined. 3. Lipopolyssacharides (LPS, 20 h) caused an about 2 fold increase in arginase activity, whereas interferon-gamma (IFN-gamma), like LPS a strong inducer of NO synthesis, had no effect. 4. Dexamethasone decreased arginase activity by about 25% and prevented the LPS-induced increase. Mifepristone (RU-486) as partial glucocorticoid receptor agonist inhibited LPS-induced increase by 45% and antagonized the inhibitory effect of dexamethasone. 5. Two different inhibitors of the NF-kappa B-pathway also prevented LPS-induced increase in arginase activity. 6. Rat AM Phi expressed mRNA and protein of arginase I and II, but arginase I expression was stronger. Arginase I mRNA and protein was not affected by IFN-gamma, but increased by LPS and this effect was prevented by dexamethasone. Both, LPS and IFN-gamma enhanced the levels of arginase II mRNA and protein, effects also inhibited by dexamethasone. As IFN-gamma did not affect total arginase activity, arginase II may represent only a minor fraction of total arginase activity. 7. In rat AM Phi glucocorticoids inhibit LPS-induced up-regulation of arginase activity, an effect which may contribute to the beneficial effects of glucocorticoids in the treatment of inflammatory airway diseases.
Asunto(s)
Arginasa/metabolismo , Dexametasona/farmacología , Glucocorticoides/farmacología , Lipopolisacáridos/farmacología , Macrófagos Alveolares/efectos de los fármacos , Animales , Células Cultivadas , Interacciones Farmacológicas , Femenino , Interferón gamma/farmacología , Macrófagos Alveolares/enzimología , Masculino , FN-kappa B/antagonistas & inhibidores , Ratas , Ratas Sprague-Dawley , Regulación hacia Arriba/efectos de los fármacosRESUMEN
The connection between the regulation of L-arginine transport and nitric oxide (NO) synthesis was studied in rat alveolar macrophages. Lipopolysaccharides (LPSs) and interferon-gamma stimulated in the same concentration- and time-dependent manner NO synthesis (measured by nitrite accumulation) and L-[(3)H]arginine uptake. This correlated with an increased mRNA expression for iNOS and the cationic amino acid transporter CAT-2B (analyzed by reverse transcription-polymerase chain reaction), with the same kinetics observed for the up-regulation of both mRNAs. Because nuclear factor-kappaB (NF-kappaB) is essential for induction of iNOS its role for the regulation of CAT-2B expression and L-arginine transport was investigated. The NF-kappaB inhibitors pyrrolidine dithiocarbamate and N(alpha)-p-tosyl-L-lysine chloromethyl ketone abrogated LPS- and interferon-gamma-induced increase of nitrite accumulation and L-[(3)H]arginine uptake as well as up-regulation of iNOS and CAT-2B mRNA. LPS-induced increase in iNOS and CAT-2B mRNA was also suppressed by specific NF-kappaB decoy oligodesoxynucleotides, confirming the essential role of NF-kappaB for iNOS and CAT-2B expression. Dexamethasone did not affect the initial (5 h) LPS-induced increase of iNOS and CAT-2B mRNA, but down-regulated both mRNAs after prolonged (20 h) exposure and this was accompanied by partial inhibition of LPS-stimulated nitrite accumulation and L-[(3)H]arginine uptake. These findings demonstrate parallel regulation of the expression of iNOS and CAT-2B, and of NO synthesis and L-arginine uptake in rat alveolar macrophages. NF-kappaB is an essential transcription factor not only for the induction of iNOS, but also for the up-regulation of CAT-2B. The simultaneous up-regulation of CAT-2B with iNOS is considered as a mechanism to ensure a high substrate supply for iNOS.
Asunto(s)
Proteínas Portadoras/genética , Macrófagos Alveolares/fisiología , Proteínas de la Membrana/genética , FN-kappa B/fisiología , Óxido Nítrico Sintasa/biosíntesis , Sistemas de Transporte de Aminoácidos Básicos , Animales , Arginina/metabolismo , Células Cultivadas , Cicloheximida/farmacología , Dexametasona/farmacología , Interacciones Farmacológicas , Inducción Enzimática , Femenino , Proteínas Ligadas a GPI , Regulación de la Expresión Génica , Interferón gamma/farmacología , Lipopolisacáridos/farmacología , Macrófagos Alveolares/enzimología , Masculino , FN-kappa B/antagonistas & inhibidores , Óxido Nítrico/metabolismo , Óxido Nítrico Sintasa/genética , Óxido Nítrico Sintasa de Tipo II , Inhibidores de la Síntesis de la Proteína/farmacología , ARN Mensajero/biosíntesis , ARN Mensajero/efectos de los fármacos , Ratas , Ratas Sprague-Dawley , Receptores del Factor de Necrosis Tumoral/metabolismo , Miembro 10c de Receptores del Factor de Necrosis Tumoral , Transducción de Señal/efectos de los fármacos , Receptores Señuelo del Factor de Necrosis Tumoral , Regulación hacia ArribaRESUMEN
In rat alveolar macrophages (AMphi) it was tested whether induction of iNOS by lipopolysaccharides (LPS) is accompanied by changes in L-arginine transport and whether L-ornithine, the product of arginase released from AMphi, could, via inhibition of L-arginine uptake, act as a paracrine inhibitor of NO synthesis. Rat AMphi (cultured for 20 h in the absence or presence of 1 microg/ml LPS) were incubated in Krebs-HEPES solution containing [3H]-L-arginine (0.1 microM for 2 min or 100 microM for 5 min) and the cellular radioactivity was determined as a measure of L-arginine uptake. In parallel, cells were incubated for 6 h in Krebs-HEPES solution containing 0-1 mM L-arginine and nitrite accumulation was determined. [3H]-L-arginine uptake (0.1 microM or 100 microM) occurred independently of sodium ions and was inhibited by L-ornithine (EC50: 117 and 562 microM, respectively) and with similar potencies by L-lysine. In LPS-treated AMphi the concentration inhibition curve of L-ornithine was shifted to the right by about a factor of 4, whereas that of L-lysine was only marginally shifted to the right. L-Leucine (0.1 and 1 mM) inhibited [3H]-L-arginine (0.1 microM) by 43 and 58%, respectively, and the effect of 0.1 mM L-leucine was partially sodium dependent. In LPS-treated AMphi, 0.1 mM L-leucine no longer inhibited [3H]-L-arginine and the effect of 1 mM L-leucine was attenuated. Kinetic analysis of the transport of [3H]-L-arginine and [14C]-L-ornithine revealed two components for each amino acid with Km values of 21 and 114 microM (L-arginine) and 39 and 1050 microM (L-ornithine), respectively. After LPS treatment Km2 of L-arginine transport was reduced to 63 microM and Vmax of both components was increased, whereas Km2 of L-ornithine transport was enhanced to 1392 microM and Vmax1 reduced. LPS-stimulated AMphi, incubated in amino acid-free Krebs-HEPES solution, produced about 4 nmol nitrite/10(6) cells per 6 h, and L-arginine enhanced nitrite accumulation maximally about threefold (EC50: 30 microM). L-ornithine, up to 3 mM, failed to affect significantly nitrite accumulation observed in the presence of 30 or 100 microM L-arginine. Rat AMphi express mRNA for two cationic amino acid transporters (CAT-1 and CAT-2B), and LPS markedly up-regulated mRNA for CAT-2B in parallel with mRNA for iNOS, but had no effect on that for CAT-1. In conclusion, in rat AMphi LPS up-regulates L-arginine transport and induces changes in the characteristics of the cationic amino acid transport resulting in preferential transport of L-arginine. These effects may be regarded as cellular measures to ensure a high L-arginine supply for iNOS.
Asunto(s)
Arginina/metabolismo , Lipopolisacáridos/farmacología , Lisina/metabolismo , Macrófagos Alveolares/metabolismo , Ornitina/metabolismo , Animales , Transporte Biológico/efectos de los fármacos , Líquido del Lavado Bronquioalveolar , Células Cultivadas , Leucina/metabolismo , Macrófagos Alveolares/enzimología , Óxido Nítrico Sintasa/genética , Óxido Nítrico Sintasa/metabolismo , Óxido Nítrico Sintasa de Tipo II , ARN Mensajero/aislamiento & purificación , Ensayo de Unión Radioligante , Ratas , Ratas Sprague-Dawley , Sodio/metabolismoRESUMEN
The role of lipid mediators derived from membrane glycerophospholipids and sphingolipids as intracellular messenger has been studied intensively during the last two decades, but with the recent discovery of high affinity G-protein coupled receptors for the lysophospholipids lysophosphatidic acid (LPA), sphingosine-1-phosphate (S1P) and sphingosylphosphorylcholine (SPC), increasing attention has been paid to the role of these lipid mediators as extracellular mediators. This review will summarize the biosynthesis and metabolism of lysophospholipids and describe the family of endothelial differentiation gene (EDG) receptors as high affinity receptors for lysophospholipids. Furthermore, an overview of the numerous biological effects of lysophospholipids which might be mediated by EDG receptors will be given together with an outlook on the potential role of such mechanisms in pulmonary physiology and pathophysiology.
Asunto(s)
Pulmón/fisiología , Lisofosfolípidos/metabolismo , Receptores de Superficie Celular/fisiología , Receptores Acoplados a Proteínas G , Animales , Plaquetas/efectos de los fármacos , Fibroblastos/efectos de los fármacos , Humanos , Pulmón/efectos de los fármacos , Lisofosfolípidos/farmacología , Músculo Liso/efectos de los fármacos , Receptores del Ácido Lisofosfatídico , Transducción de Señal , Esfingosina/análogos & derivados , Esfingosina/farmacologíaRESUMEN
Alveolar macrophages (AMsmall ef, Cyrillic) express considerable arginase activity which can be modulated by various mediators. As inhibitors of phosphodiesterase (PDE) play an increasing role in the treatment of chronic inflammatory and obstructive airway disease, we tested whether PDE inhibitors affect arginase activity in AMsmall ef, Cyrillic. Isolated rabbit AMsmall ef, Cyrillic were cultured for 20 h in the absence or presence of bacterial lipopolysaccharides (LPS) and/or different test substances. Thereafter arginase activity was determined by measuring the formation of [(3)H]-L-ornithine during 1 h incubation with [(3)H]-L-arginine. Lipopolysaccharide-enhanced (0. 01-5 microg/ml) maximal arginase activity by about 2.5-fold. The non-selective PDE inhibitor IBMX and the PDE4 selective inhibitor rolipram (each up to 30 microM) caused a 2.4-fold increase in arginase activity, and these effects were additive to those of LPS. The PDE3-selective inhibitor siguazodan had only marginal effects. Forskolin (10 microM) also enhanced arginase activity in the absence and presence of LPS. The effect of forskolin was almost prevented by cycloheximide (30 microM) and largely attenuated by the protein kinase A inhibitor KT 5720 (300 nM). In conclusion, inhibition of the cAMP-specific PDE4, like direct activation of adenylyl cyclase, causes an up-regulation of arginase activity in rabbit AMsmall ef, Cyrillic.
Asunto(s)
Arginasa/biosíntesis , Colforsina/farmacología , Macrófagos Alveolares/enzimología , Inhibidores de Fosfodiesterasa/farmacología , 1-Metil-3-Isobutilxantina/farmacología , Animales , Femenino , Isoenzimas/metabolismo , Lipopolisacáridos/farmacología , Masculino , Ornitina/biosíntesis , Hidrolasas Diéster Fosfóricas/metabolismo , Conejos , Regulación hacia ArribaRESUMEN
Nearly 30% of patients treated with metformin experience gastrointestinal side effects. Since release of 5-hydroxytryptamine (5-HT) from the intestine is associated with nausea, vomiting, and diarrhea, we examined whether metformin induces 5-HT release from the intestinal mucosa. In 40% of tissue biopsy specimens of human duodenal mucosa, metformin (1, 10, and 30 microM) caused an increase in 5-HT outflow by 35, 70, and 98%, respectively. Peak increases in 5-HT outflow were observed after 10-15 min exposure to metformin, returning to baseline levels after 25 min. Tetrodotoxin (1 microM) reduced by about 50% the metformin-evoked increase in 5-HT outflow (P<0.05). Metformin-evoked release was not affected by scopolamine + hexamethonium, propranolol, the 5-HT3 receptor antagonist dolasetron, naloxone, or the NK1 receptor antagonist L703606. In the presence of tetrodotoxin (1 microM), somatostatin (1 microM) further reduced metformin-induced 5-HT release by 15-20%. In view of the 5-HT releasing effects of selective 5-HT3 receptor agonists to which metformin (N-N-dimethylbiguanide) is structurally related, we investigated whether metformin directly interacts with 5-HT3 receptors. Receptor binding (inhibition of [3H]-GR65630 binding) and agonist effects (stimulation of [14C]-guanidinium influx) at 5-HT3 receptors were studied in murine neuroblastoma N1E-115 cells, which express functional 5-HT3 receptors. Metformin up to 0.3 mM failed to inhibit [3H]-GR65630 binding and to modify displacement of [3H]-GR65630 binding induced by 5-HT. 5-HT (3 microM) stimulated the influx of [14C]-guanidinium in intact N1E-115 cells. Metformin up to 1 mM failed to modify basal influx, 5-HT-induced influx, and 5-HT+ substance P-induced influx of [14C]-guanidinium. Our results indicate that metformin induces 5-HT3 receptor-independent release of 5-HT from human duodenal mucosa via neuronal and non-neuronal mechanisms. Part of the gastrointestinal side effects observed during treatment with metformin could, thus, be produced by the release of 5-HT and other neurotransmitter substances within the duodenal mucosa.