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
Atherosclerosis results from multiple factors and involves several mechanisms, including endothelial monocyte and smooth muscle cell (SMC) changes, cholesterol accumulation, plaque rupture and thromboembolism. Calcium ions play a role in the initial and chronic development of atherosclerotic lesions. Several studies in experimental animal models have demonstrated the potential direct antiatherosclerotic effects of calcium antagonists. In this study the antiatherogenic activity of lercanidipine, a new lipophilic, second-generation calcium antagonist, was investigated. Lercanidipine and its enantiomers inhibited the replication and migration of arterial myocytes in concentrations ranging from 10 to 50 microM. The antiproliferative effect of lercanidipine was dose dependent, with a potency similar to that of lacidipine and nifedipine, and was unrelated to the stereoselectivity of enantiomers to bind L-type calcium channels. Lercanidipine and its enantiomers (25 microM) decreased the serum-induced elevation of [Ca2+]i in SMC, with the (S)-enantiomer (69% inhibition) being 2.4-fold more active than the (R)-counterpart (29% inhibition). The studies performed with enantiomers of lercanidipine suggest that the observed effects are not related to the blockade of voltage-dependent Ca2+ channels and confirm, at least in vitro, the pharmacological potential of the compound to influence negatively the process of atherogenesis.
Asunto(s)
Antihipertensivos/farmacología , Arteriosclerosis/prevención & control , Bloqueadores de los Canales de Calcio/farmacología , Dihidropiridinas/farmacología , Animales , Antihipertensivos/química , Arteriosclerosis/metabolismo , Encéfalo/efectos de los fármacos , Encéfalo/metabolismo , Bloqueadores de los Canales de Calcio/química , División Celular/efectos de los fármacos , Dihidropiridinas/química , Técnicas In Vitro , Masculino , Membranas/efectos de los fármacos , Membranas/metabolismo , Músculo Liso Vascular/efectos de los fármacos , Músculo Liso Vascular/patología , Nitrendipino/metabolismo , Ratas , Ratas Sprague-Dawley , EstereoisomerismoRESUMEN
The internalization of [3H]iloprost, a prostacyclin analogue, was studied in human platelets by binding studies. After incubation with [3H]iloprost at 37 degrees C, addition of unlabelled ligand at either 37 degrees C or 4 degrees C caused dissociation of 74% and 52% of the bound ligand respectively, suggesting that a portion had been internalized. The percentage of [3H]iloprost bound at equilibrium to the surface (evaluated by acid treatment) at either 37 degrees C or 4 degrees C was markedly different (80% versus 25%). Internalization was dependent on time and on the ligand nature and concentration. Energy-depleting agents (dinitrophenol and 2-deoxyglucose) completely inhibited internalization, whereas probenecid (inhibitor of organic anion transporters) did not affect it significantly. Subcellular fractionation indicated that, at 4 degrees C or in the absence of ligand, most of the receptor was present in membrane fractions (pellet at 27000 or 105000 g), whereas, when platelets were preincubated at 37 degrees C with iloprost, the receptor was found mainly in the cytosolic fraction. In platelets preincubated with iloprost at 4 degrees C, two classes of binding sites were present, whereas after preincubation at 37 degrees C only the lower-affinity sites were detected. After exposure to the agonist, iloprost-induced inhibition of platelet aggregation and activation of adenylate cyclase and cAMP production were significantly lower. Taken together, these data demonstrate that human platelets can internalize a high-affinity binding site for iloprost, presumably the prostacyclin receptor.
Asunto(s)
Plaquetas/metabolismo , Receptores de Prostaglandina/metabolismo , Adenilil Ciclasas/sangre , Unión Competitiva , Membrana Celular/metabolismo , Regulación hacia Abajo , Epoprostenol/sangre , Humanos , Iloprost/sangre , Iloprost/farmacología , Técnicas In Vitro , Recién Nacido , Cinética , Agregación Plaquetaria , Receptores de Epoprostenol , Receptores de Prostaglandina/biosíntesis , Fracciones Subcelulares/metabolismoRESUMEN
The in vitro effects were investigated of the new dihydropyridine calcium antagonist (CA) lercanidipine and its enantiomers on arterial myocyte (smooth muscle cell; SMC) migration and proliferation as related to L-type calcium channel inhibition. Lercanidipine and its enantiomers inhibited the replication and migration of arterial myocytes in concentration ranging from 10 to 50 microM. The antiproliferative effect of lercanidipine, evaluated as cell number, was dose dependent, with a potency similar to that of lacidipine and nifedipine, and was unrelated to the stereoselectivity of enantiomers to bind L-type calcium channels. The cell doubling time increased with drug concentration < or = 122 versus 38 h for controls. The cell growth inhibition induced by lercanidipine and its enantiomers was reversible. Lercanidipine dose dependently decreased [3H]thymidine incorporation into DNA; the (R)-enantiomer, displaying the lowest CA activity, was the most potent in this respect. The tested compounds were able to inhibit fibrinogen-induced myocyte migration in a dose-dependent manner, with the (R)-enantiomer showing the more pronounced effect. To directly rule out the role of calcium channels in the antiatherosclerotic properties of lercanidipine, we examined the effect of the compounds on serum-stimulated calcium influx in SMC. Fluorimetry of Fluo 3 was used to measure changes in free cytosolic Ca2+ concentration ([Ca2+]i) in SMC after long-term preincubation (24 h) with the tested CA. Lercanidipine and its enantiomers (25 microM) decreased the serum-induced elevation of [Ca2+]i in SMC with the (S)-enantiomer (69% inhibition) 2.4-fold more active than the counterpart and the racemate (29% inhibition). In conclusion, our in vitro results suggest that lercanidipine may directly interfere with events involved in atherogenesis. The studies performed with enantiomers of lercanidipine suggest that the observed effects are not related to the blockade of voltage-dependent Ca2+ channels and confirm at least in vitro a pharmacologic potential of the compound to negatively influence the process of atherogenesis.
Asunto(s)
Bloqueadores de los Canales de Calcio/farmacología , Dihidropiridinas/farmacología , Músculo Liso Vascular/efectos de los fármacos , Animales , Aorta/efectos de los fármacos , Encéfalo/metabolismo , Calcio/metabolismo , Bloqueadores de los Canales de Calcio/metabolismo , Canales de Calcio/metabolismo , División Celular/efectos de los fármacos , Movimiento Celular/efectos de los fármacos , Células Cultivadas , Dihidropiridinas/metabolismo , Relación Dosis-Respuesta a Droga , Humanos , Masculino , Nitrendipino/metabolismo , Ratas , Ratas Sprague-Dawley , EstereoisomerismoRESUMEN
The role of Ca++ as an intracellular messenger in leukotriene (LT)D4-induced muscle contraction was investigated by measuring force development and elevation in cytosolic Ca++ concentration simultaneously in strips of guinea pig ileal longitudinal muscle loaded with the fluorescent calcium indicator Fura 2. Upon addition of LTD4, a simultaneous increase in tension and cytosolic calcium concentration, [Ca++]i, was observed. Cumulative applications of LTD4 induced concentration-dependent increases in both muscle tension and [Ca++]i, being the half-maximal effect reached at approximately 6 to 9 nM. A statistically significant positive correlation (r = 0.993, P < .001) exists between the two parameters examined. Removal of calcium in the bathing solution, accompanied by addition of 7.5 mM EGTA, completely prevented any increase in either calcium levels or force development, thus indicating a role for Ca++ influx, rather than a release from intracellular stores. All of the LTD4 antagonists tested were able to counteract the effect of the leukotriene on both [Ca++]i and tension increase. However, although LY171883 shifted both of the LTD4 curves to the right in a parallel fashion, FPL 55712 and ICI 198,615 behaved as non-competitive antagonists in reversing the effect of LTD4 on [Ca++]i and tension. Thus, these results strongly suggest that changes in muscle tension induced by LTD4 are attributable to changes in cytosolic free Ca++ concentrations in guinea pig ileum.
Asunto(s)
Calcio/metabolismo , Leucotrieno D4/farmacología , Contracción Muscular , Músculo Liso/efectos de los fármacos , Animales , Citosol/efectos de los fármacos , Citosol/metabolismo , Cobayas , Histamina/farmacología , Íleon , Técnicas In Vitro , Masculino , Contracción Muscular/efectos de los fármacos , Músculo Liso/fisiología , Sistemas de Mensajero SecundarioRESUMEN
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
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/metabolismoRESUMEN
Platelets from type IIa hypercholesterolemic subjects have been previously shown to be less sensitive than normal platelets to the antiaggregatory effect of PGI2. We demonstrate here that these platelets display a reduced response to iloprost, a chemically stable analogue of PGI2, as well. In fact, the concentration of iloprost yielding 50% inhibition of PRP aggregation was higher in type IIa patients (0.77 +/- 0.08 nM) than in controls (0.51 +/- 0.06 nM, P less than 0.01). In addition, an inverse relationship existed between the threshold aggregatory concentration for collagen and the concentration of iloprost yielding 50% inhibition of PRP aggregation, both in type IIa and normal individuals. In order to elucidate the mechanism of the different sensitivity of platelets to prostacyclin and its analogue, we characterized the binding of 3H-iloprost to platelet rich plasma from single individuals. The binding was rapid, reversible, inhibited by iloprost, PGI2 and PGE1 (Kd = 50.7; 346.2 and 7500 nM, respectively); no heterogeneity of sites could be demonstrated in the PRP from a single individual. When binding studies were carried out in PRP of type IIa patients and controls, it appeared that the amount of 3H-iloprost bound at a fixed (300 nM) concentration was significantly lower in platelets from type IIa individuals (0.94 +/- 0.17 vs. 1.77 +/- 0.27 fmol/10(6) platelets, for patients and controls, respectively). It is concluded that such difference in binding might represent the mechanism underlying the reduced response to PGI2 and iloprost observed in platelets from type IIa patients.