RESUMEN
Cysteine-containing leukotrienes (cysteinyl-LTs) are potent bronchoconstrictors and play a key role in asthma. We found that histamine and LTD4 markedly constrict strips of human bronchi (HB) with similar efficacy. However, in human airway smooth-muscle (HASM) cells, LTD4, at variance with histamine, elicited only a small, transient change in intracellular calcium ion concentration. HASM cells express both Ca2+-dependent and -independent isoforms of protein kinase C (PKC) (i.e., PKC-alpha and PKC-alpha ). Western blot analysis showed that PKC-alpha is activated by histamine and, to a lesser extent, by LTD4, whereas only LTD4 translocates PKC-alpha. This translocation was specifically inhibited by the LTD4 antagonist pobilukast. Phorbol-dibutyrate ester (PDBu) (a PKC activator) contracted HB strips to the same extent in the presence as in the absence of extra- and intracellular Ca2+. In the absence of Ca2+, LTD4 contracted HB strips to the same extent as did PDBu, suggesting the involvement of a Ca2+-independent PKC in LTD4-mediated signal transduction. PDBu-induced desensitization and the PKC inhibitor H7 abolished the slow and sustained LTD4-triggered contraction of HB strips in the absence of Ca2+, although H7 did not greatly affect the response in the presence of the ion. Thus, in human airways, we identified a novel LTD4 transduction mechanism linked to bronchial smooth-muscle contraction, which is partly independent of Ca2+ and involves the activation of PKC-alpha.
Asunto(s)
Bronquios/fisiología , Calcio/fisiología , Leucotrieno D4/fisiología , Músculo Liso/citología , Músculo Liso/fisiología , Humanos , Contracción Muscular , Músculo Liso/química , Proteína Quinasa C/análisisRESUMEN
We studied the effect of endogenous and exogenous prostaglandin E(2) (PGE(2)), a metabolite of arachidonic acid through the cyclooxygenase (COX) pathway, on interleukin (IL)-1 beta-induced COX-2 expression, using primary cultures of human bronchial smooth-muscle cells (HBSMC). Treatment with exogenous PGE(2) resulted in enhanced expression of IL-1 beta-induced COX-2 protein and messenger RNA (mRNA) as compared with the effect of the cytokine per se. Inhibition of PGE(2) production with a nonselective COX inhibitor (flurbiprofen, 10 microM) resulted in a significant reduction in IL-1 beta- induced COX-2 expression, supporting a role of endogenous COX metabolites in the modulation of COX-2 expression. None of the experimental conditions used in the study affected the expression of constitutive cyclooxygenase (COX-1). Treatment with cycloheximide to inhibit translation, and with dexamethasone or actinomycin D to inhibit transcription, linked the effect of PGE(2) to the transcriptional level of COX-2 mRNA rather than to a potential effect on protein and/or mRNA stabilization. PGE(2) increased adenylate cyclase activity in a concentration dependent manner, and forskolin, a direct activator of adenylate cyclase, caused a marked increase in IL-1 beta-dependent COX-2, suggesting the existence of a causal relationship between the two events. The same results were observed with salbutamol, a bronchodilator that acts by increasing cyclic adenosine monophosphate. The effect of PGE(2) on COX-2 expression may contribute to the hypothesized antiinflammatory role of PGE(2) in human airways, providing a self-amplifying loop leading to increased biosynthesis of PGE(2) during an inflammatory event.
Asunto(s)
Dinoprostona/farmacología , Interleucina-1/farmacología , Músculo Liso/efectos de los fármacos , Peroxidasas/efectos de los fármacos , Prostaglandina-Endoperóxido Sintasas/efectos de los fármacos , Prostaglandinas E Sintéticas/farmacología , Adenilil Ciclasas/análisis , Análisis de Varianza , Northern Blotting/métodos , Northern Blotting/estadística & datos numéricos , Western Blotting/métodos , Western Blotting/estadística & datos numéricos , Bronquios/citología , Bronquios/efectos de los fármacos , Bronquios/enzimología , Células Cultivadas , Ciclooxigenasa 2 , Inducción Enzimática/efectos de los fármacos , Humanos , Isoenzimas/análisis , Isoenzimas/biosíntesis , Isoenzimas/efectos de los fármacos , Proteínas de la Membrana , Músculo Liso/citología , Músculo Liso/enzimología , Peroxidasas/análisis , Peroxidasas/biosíntesis , Prostaglandina-Endoperóxido Sintasas/análisis , Prostaglandina-Endoperóxido Sintasas/biosíntesisRESUMEN
The purpose of this study was to determine the mechanism of enhanced prostaglandin synthesis in cultured human bronchial smooth-muscle cells challenged with interleukin-1 beta (IL-1 beta). Cells were incubated with IL-1 beta (10 to 50 U/ml) for 0 to 24 h. Prostaglandin E2 (PGE2) production was evaluated through the conversion of exogenous (14C)-arachidonic acid and specific enzyme immunoassay of endogenous products. IL-1 beta enhanced PGE2 formation in a concentration- and time-dependent manner, reaching its peak at 6 to 8 h and fading at 18 to 24 h. Immunoblot analysis showed that the inducible cyclooxygenase enzyme (COX-2) was expressed only in IL-1 beta treated cells, whereas the constitutive isoform of cyclooxygenase (COX-1) remained unaltered. COX-2 expression and PGE2 formation were inhibited by dexamethasone (2 microM), cycloheximide (10 microM), and IL-1-receptor antagonist (IL-1 ra) (250 ng/ml), independently. PGE2 synthesis was significantly reduced by compound SC-58125, a specific COX-2 inhibitor. The close parallelism between the kinetics of COX-2 protein expression and PGE2 accumulation, as well as the constitutive nature of COX-1 isoform, indicate that IL-1 beta-driven PGE2 formation in human bronchial smooth-muscle cells is mediated by de novo expression of COX-2 enzyme.
Asunto(s)
Bronquios/metabolismo , Dinoprostona/biosíntesis , Isoenzimas/biosíntesis , Músculo Liso/metabolismo , Peroxidasas/metabolismo , Prostaglandina-Endoperóxido Sintasas/biosíntesis , Ácido Araquidónico/farmacología , Western Blotting , Bronquios/citología , Células Cultivadas , Cicloheximida/farmacología , Ciclooxigenasa 1 , Ciclooxigenasa 2 , Inhibidores de la Ciclooxigenasa 2 , Inhibidores de la Ciclooxigenasa/farmacología , Dexametasona/farmacología , Relación Dosis-Respuesta a Droga , Humanos , Immunoblotting , Proteína Antagonista del Receptor de Interleucina 1 , Interleucina-1/farmacología , Proteínas de la Membrana , Músculo Liso/efectos de los fármacos , Pirazoles/farmacología , Receptores de Interleucina-1/antagonistas & inhibidores , Sialoglicoproteínas/farmacologíaRESUMEN
The role of mevalonate and geranylgeraniol in the control of cellular proliferation of cultured human bronchial myocytes was examined by investigating the effect of simvastatin, an inhibitor of 3-hydroxy-3-methylglutaryl coenzyme A reductase, the rate-limiting enzyme in mevalonate synthesis. Simvastatin inhibited the rate of growth of human bronchial smooth muscle cells in a concentration-dependent manner, with an IC50 value of 0.97 +/- 0.1 microM. Mevalonate (100 microM), as well as geranylgeraniol (5 microM), at their highest non-toxic concentrations, restored cell proliferation to control levels.
Asunto(s)
Bronquios/efectos de los fármacos , Diterpenos/farmacología , Lovastatina/análogos & derivados , Pulmón/efectos de los fármacos , Ácido Mevalónico/farmacología , División Celular/efectos de los fármacos , Inhibidores Enzimáticos/farmacología , Humanos , Lovastatina/farmacología , Músculo Liso/efectos de los fármacos , SimvastatinaRESUMEN
The (+/-)-3'-hydroxyfarrerol (IdB 1031) is a new drug endowed with an interesting mucokinetic activity. In this study the effectiveness of IdB 1031 has been verified in a model of airway hyperreactivity and lung inflammation induced in anaesthetized guinea pig by active cigarette smoke exposure. IdB 1031 (500 mg/kg per os) completely inhibited the capacity of cigarette smoke to induce airway hyperreactivity. IdB 1031 also inhibited the recruitment of proinflammatory cells within the airway lumen as showed in bronchoalveolar lavage fluids. In line with these experiments IdB 1031 inhibited 5-lipoxygenase with an IC50 of 7.36 x 10(-6) M in human leukocytes challenged by A-23187 (2 microM). A significant reduction of the above parameters was observed also in animals exposed to smoke after repeated treatment with IdB 1031 at 200 mg/kg per os for 15 days. These results show that IdB 1031 is a promising drug with a favourable spectrum of activities on the respiratory tract.
Asunto(s)
Hiperreactividad Bronquial/prevención & control , Flavonoides/farmacología , Fumar/efectos adversos , Anestesia , Animales , Líquido del Lavado Bronquioalveolar/citología , Relación Dosis-Respuesta a Droga , Eosinófilos/efectos de los fármacos , Cobayas , Humanos , Técnicas para Inmunoenzimas , Técnicas In Vitro , Leucotrieno B4/biosíntesis , Inhibidores de la Lipooxigenasa/farmacología , Macrófagos/efectos de los fármacos , Masculino , Neutrófilos/efectos de los fármacos , Neutrófilos/inmunologíaRESUMEN
SCH 37224, 1-(1,2-dihydro-4-hydroxy-2-oxo-1-phenyl-1,8-naphthyridin-3-yl) pyrrolidinium, is a structurally novel compound that had been shown to inhibit leukotriene D4 formation in guinea pig lung in vitro. We tested whether SCH 37224 is able to inhibit both the formation of eicosanoids from human lung parenchyma in vitro and the binding of sulfidopeptide leukotrienes to membranes of lung parenchyma and bronchi. SCH 37224, at a concentration of 30 and 100 microM, was able to inhibit antigen-induced formation of sulfidopeptide leukotrienes, measured as leukotriene E4, while it did not significantly affect the formation of prostaglandin D2. At concentrations up to 100 microM, it did not affect either the binding of [3H]leukotriene C4 to membranes of human lung parenchyma or human bronchi, or the binding of [3H]leukotriene D4 to the parenchyma. In conclusion, SCH 37224 is a selective inhibitor of leukotriene formation in human lung in vitro, which might be of potential therapeutic interest in the treatment of asthma.
Asunto(s)
Inmunoglobulina E/inmunología , Leucotrienos/biosíntesis , Pulmón/metabolismo , Naftiridinas/farmacología , Eicosanoides/biosíntesis , Humanos , Técnicas In Vitro , Pulmón/efectos de los fármacos , SRS-A/metabolismoRESUMEN
The ability of arachidonic acid (AA) and bradykinin to release calcitonin gene-related peptide-like immunoreactivity (CGRP-LI) from capsaicin-sensitive primary afferents was studied in guinea pig isolated and perfused heart. Infusion of AA (50 microM to 5 mM, 0.5 ml/min over 2 min) produced a remarkable and dose-dependent CGRP-LI release that was abolished by in vitro capsaicin (10 microM) pretreatment or in the presence of indomethacin (10 microM). The capsaicin antagonist ruthenium red (10 microM) did not affect the CGRP-LI release produced by AA, whereas it blocked that produced by capsaicin (10 microM). In vitro capsaicin pretreatment reduced the increase in heart rate evoked by AA, whereas it did not affect the increase in coronary flow and decrease in contractility. Bradykinin (10 microM, 0.5 ml/min over 2 min) induced CGRP-LI release in a similar manner to that produced by AA, with the only difference being that in the presence of indomethacin, a residual increase in CGRP-LI outflow was still observed. AA increased the outflow of 6-keto-prostaglandin (PG) F1 alpha, PGE2 and leukotriene B4, whereas bradykinin enhanced only the release of 6-keto-PGF1 alpha. Infusion of either PGI2 or PGE2 (1-100 microM) released CGRP-LI in a dose-dependent manner and with a similar potency. PGI2 (100 microM)- or PGE2 (100 microM, 0.5 ml/min over 2 min)-evoked release was abolished by previous exposure to capsaicin and not affected by indomethacin.(ABSTRACT TRUNCATED AT 250 WORDS)
Asunto(s)
Ácido Araquidónico/farmacología , Bradiquinina/farmacología , Péptido Relacionado con Gen de Calcitonina/metabolismo , Capsaicina/farmacología , Corazón/inervación , Fibras Nerviosas/metabolismo , Neuronas Aferentes/metabolismo , Animales , Ácido Araquidónico/metabolismo , Péptido Relacionado con Gen de Calcitonina/inmunología , Dinoprostona/farmacología , Eicosanoides/metabolismo , Epoprostenol/farmacología , Cobayas , Corazón/efectos de los fármacos , Corazón/fisiología , Técnicas In Vitro , Fibras Nerviosas/efectos de los fármacos , Neuronas Aferentes/efectos de los fármacos , PerfusiónRESUMEN
Unlike other classic NSAIDs, some fenamates given at therapeutic concentrations, have been shown to inhibit, both in vitro and in vivo, the 5-lipoxygenase pathway of arachidonic acid cascade as well as the synthesis of cyclooxygenase products. This dual inhibitory property might represent an improvement in anti-inflammatory therapy. The aim of this work was to characterize the effect of morniflumate, administered at therapeutic dosages to normal human volunteers, on leukotriene B4 (LTB4) and thromboxane (TXB2) synthesis, both in purified PMNs and in whole blood. PMNs, isolated two hours after a single oral administration of morniflumate and at steady-state condition, fully retain their capacity to release LTB4 and TXB2. Since intracellular concentrations of the drug were undetectable, in spite of its elevated concentrations in platelet poor plasma, the results obtained using PMNs suggest a drug loss during the cells purification procedure. In whole blood experiments, morniflumate reduced blood LTB4 synthesis induced by Ca-ionophore A23187 Bx approximately 50%, both after single dose and at steady state; the degree of inhibition showed a pattern similar to the plasma levels of the bioactive metabolite of morniflumate (M1). The inhibition of serum TXB2 levels was higher than 85%. Hence, morniflumate is capable of reducing arachidonic acid metabolism acting both on cyclooxygenase and 5-lipoxygenase. This characteristic might provide a better approach in anti-inflammatory therapy.
Asunto(s)
Antiinflamatorios no Esteroideos/farmacología , Ácido Araquidónico/metabolismo , Ácido Niflúmico/análogos & derivados , Administración Oral , Adulto , Calcimicina/farmacología , Femenino , Humanos , Técnicas para Inmunoenzimas , Técnicas In Vitro , Leucotrieno B4/biosíntesis , Leucotrienos/biosíntesis , Masculino , Persona de Mediana Edad , Neutrófilos/efectos de los fármacos , Neutrófilos/metabolismo , Ácido Niflúmico/farmacología , Tromboxano B2/biosíntesis , Tromboxano B2/sangreRESUMEN
In vitro passive sensitization of human lung parenchyma with hyper-immune serum did not affect the release of prostaglandin D2 (PGD2) or leukotriene (LT)-like activity upon challenge with anti-IgE antibody with respect to control lung, despite a marked difference in IgE levels between control (C) and sensitized (S) tissue. Binding studies with [3H]LTC4, [3H]LTD4 and [3H]mepyramine (a histamine H1 antagonist) showed a statistically significant increase in the amount bound in sensitized vs control lung for [3H]mepyramine only. Contractile response to 5 x 10(-5) M histamine (H) in C and S lung parenchymal strips did not correlate with binding data. It is concluded that in vitro elevated IgE levels do not affect the interaction of sulfidopeptide leukotrienes with their putative receptors. As for the observed increase in [3H]mepyramine binding, this might not represent a true increase in histamine receptors on lung smooth muscle cells.
Asunto(s)
Eicosanoides/metabolismo , Pulmón/metabolismo , Pirilamina/metabolismo , SRS-A/metabolismo , Animales , Cobayas , Histamina/farmacología , Humanos , Inmunización Pasiva , Inmunoglobulina E/farmacología , Técnicas In Vitro , Pulmón/inmunología , Músculo Liso/efectos de los fármacos , Prostaglandina D2/análisis , Receptores Histamínicos H1/metabolismoAsunto(s)
Ácidos Araquidónicos/metabolismo , Macrófagos/metabolismo , Alveolos Pulmonares/patología , Sarcoidosis/metabolismo , Adulto , Ácido Araquidónico , Líquido del Lavado Bronquioalveolar/patología , Células Cultivadas , Dinoprostona/metabolismo , Humanos , Leucotrieno B4/metabolismo , Activación de Macrófagos/efectos de los fármacos , Macrófagos/efectos de los fármacos , Persona de Mediana Edad , Fagocitosis/efectos de los fármacos , Sarcoidosis/patología , Zimosan/farmacologíaRESUMEN
Esaprazole, N-cyclohexyl-1-piperazineacetamide monohydrochloride, was studied for its activity to prevent gastric mucosal damage induced by several necrotizing agents in the rat. Its effects on acid gastric secretion and the role of gastric mucosal prostaglandin generation were also investigated. Esaprazole, given orally, dose dependently prevented the formation of mucosal damage induced by absolute ethanol, 0.2 N NaOH or 0.6 N HCl. This activity occurred at doses lower than the antisecretory doses. Esaprazole was also found to increase the gastric mucosal prostaglandin content but at doses that exceeded the cytoprotective doses. The failure of indomethacin to impair the gastric mucosal protection provided by esaprazole suggests that mechanisms other than mobilization of endogenous prostaglandins may be involved.
Asunto(s)
Antiulcerosos/farmacología , Mucosa Gástrica/metabolismo , Piperazinas/farmacología , Prostaglandinas/biosíntesis , 6-Cetoprostaglandina F1 alfa/farmacología , Animales , Dinoprostona/metabolismo , Relación Dosis-Respuesta a Droga , Epoprostenol/biosíntesis , Femenino , Ácido Gástrico/metabolismo , Mucosa Gástrica/efectos de los fármacos , Técnicas In Vitro , Indometacina/farmacología , Perfusión , Píloro/fisiología , Ratas , Ratas EndogámicasRESUMEN
We studied the ex vivo production of prostaglandin D2, prostaglandin E2, 6-ketoprostaglandin F1 alpha, and leukotriene C4 in the brain tissue of rats subjected to experimental subarachnoid hemorrhage. The ex vivo method allows the study of arachidonic acid metabolites released from brain slices at different times after subarachnoid hemorrhage induction and reflects the residual capacity for arachidonic acid metabolism after the pathologic event. The rats were sacrificed 30 minutes, 1 and 6 hours, and 2 days after subarachnoid hemorrhage was induced by the injection of 0.30 ml autologous arterial blood into the cisterna magna. Concentration of prostaglandin D2 and 6-ketoprostaglandin F1 alpha was increased significantly relative to control 2 days after induction. The concentration of prostaglandin E2 was increased significantly 6 hours after induction, while ex vivo production of leukotriene C4 was increased significantly at 1 and 6 hours and 2 days. The correlation between these results and the occurrence of vasospasm after subarachnoid hemorrhage is discussed. The results obtained from the ex vivo incubation of brain tissue slices after experimental subarachnoid hemorrhage suggest that after the hemorrhage there is a significant modification of brain eicosanoid metabolism, which could be of great importance in interpreting the pathogenesis of subarachnoid hemorrhage-related neuronal impairment.
Asunto(s)
Ácidos Araquidónicos/metabolismo , Hemorragia Subaracnoidea/metabolismo , Animales , Ácido Araquidónico , Masculino , Concentración Osmolar , Prostaglandinas/metabolismo , Ratas , Ratas Endogámicas , SRS-A/metabolismo , Hemorragia Subaracnoidea/fisiopatología , Factores de TiempoRESUMEN
Immunological challenge of human lung parenchyma causes formation of arachidonate metabolites: prostaglandin D2 (PGD2) (70% of the formed mediators), leukotrienes E4 (LTE4) (15%) and D4 (LTD4) (10%). Leukotriene B4 (LTB4) was barely detectable (2%). Inhibition of PGD2 formation by indomethacin (15 microM) was approximately 90%, but was not accompanied by redistribution of arachidonate metabolism towards sulphidopeptide leukotrienes, as postulated for aspirin-sensitive asthma. Specific binding sites for leukotrienes C4 (LTC4) have been identified in membrane preparations of human bronchi. Binding of 3H-LTC4 is rapid (1 min) and quickly reversible following addition of excess. The sites are specific for LTC4 and competition curves fitted a two-site model. Moreover, clinical studies on specific endobronchial challenge of patients allergic to Dermatophagoides pteronyssinus, revealed narrowing of bronchial diameter and oedema of the bronchial mucosa; these symptoms were accompanied by an increase of immunoreactive-LTC4 and PGD2 present in the bronchial lavage fluids.
Asunto(s)
Asma/inmunología , Leucotrienos/inmunología , Sistema Respiratorio/inmunología , Asma/metabolismo , Asma/fisiopatología , Pruebas de Provocación Bronquial , Femenino , Humanos , Leucotrienos/biosíntesis , Leucotrienos/farmacología , Masculino , Sistema Respiratorio/efectos de los fármacos , Sistema Respiratorio/metabolismoRESUMEN
Normal human lung parenchyma, passively sensitized with an hyperimmune serum, releases eicosanoids from the cyclo-oxygenase pathway and from different lipoxygenases (5-, 12-, 15-HETE) upon anti-IgE challenge. Prostaglandin D2 represents almost 50% of the total arachidonate metabolites whereas, among sulphidopeptide leukotrienes, LTE4 is the one present in larger amounts. Pretreatment of the passively sensitized tissue with indomethacin (1.5 X 10(-5) M) inhibits cyclo-oxygenase by more than 90% without a concomitant shift of arachidonate metabolism towards leukotriene formation.
Asunto(s)
Inhibidores de la Ciclooxigenasa , Lipooxigenasa/metabolismo , Pulmón/metabolismo , Bioensayo , Cromatografía Líquida de Alta Presión , Humanos , Sueros Inmunes , Técnicas para Inmunoenzimas , Indometacina/farmacología , Leucotrienos/metabolismo , Pulmón/enzimología , Pulmón/inmunología , Prostaglandina D2/análisis , Valores de Referencia , SRS-A/análisisRESUMEN
Gross and microscopic examination of rat gastric mucosa demonstrated that intragastric administration to rats of tripotassium dicitrate bismuthate (TDB), a colloidal bismuth compound, protects against gastric lesions induced by 85% ethanol. Indomethacin, a prostaglandin synthetase inhibitor, significantly blocked the gastric mucosal protective effect of TDB. The release of gastric mucosal prostaglandins was greater in animals treated with TDB than in control animals, both time- and dose-dependently. These results seem to indicate involvement of prostaglandins in the action of TDB.
Asunto(s)
Epoprostenol/biosíntesis , Etanol/farmacología , Mucosa Gástrica/efectos de los fármacos , Compuestos Organometálicos/farmacología , Prostaglandinas E/biosíntesis , Animales , Dinoprostona , Relación Dosis-Respuesta a Droga , Mucosa Gástrica/metabolismo , Mucosa Gástrica/patología , Masculino , Ratas , Ratas EndogámicasRESUMEN
Defibrotide, a polydeoxyribonucleotide obtained from mammalian lungs, reduced in a dose-dependent fashion the ischemic contracture due to low perfusion (0.2 ml/min) of isovolumic left heart of rabbit and abolished the irregular rhythm of the heart, thereby restoring the cardiomechanical activity upon reperfusion (20 ml/min). Defibrotide stimulated the release of PG-like material from the heart in a dose-dependent manner without modifying the basal contractility. Both PGE2 and PGI2 (10 ng/ml) have an antiischemic activity on this preparation as shown by the partial reduction of the ischemic contracture and by the improvement of heart contractility upon reperfusion. Indomethacin infusion (1 microgram/ml) completely removed both the antiischemic activity of Defibrotide (400 micrograms/ml) and its ability to increase the generation of prostaglandins in the rabbit heart. These results suggest that Defibrotide has a beneficial influence on ischemic rabbit heart through an increase in prostaglandin synthesis. However other mechanisms not necessarily related to prostaglandin generation, such as a direct effect on membrane function deactivation and mitochondrial Ca2+ overload, should be considered in explaining the antiischemic activity of Defibrotide in the rabbit heart.