RESUMEN
Experiments were performed to examine the mechanism by which specific hemostatic proteins regulate the release of nitric oxide (NO) from interleukin-1 beta (IL-1beta) stimulated cultured rate aortic smooth muscle cells. Treatment of smooth muscle cells with IL-Beta stimulated inducible nitric oxide synthase (iNOS) mRNA expression, which preceded the release of NO (as measured by the accumulation of nitrite in the culture media). The cytokine-stimulated production of nitrite was blocked by the protein synthesis inhibitor cycloheximide, the transcriptional inhibitor actinomycin D, and the competitive inhibitor of NOS nitro-L-arginine. However, only actinomycin D inhibited IL-1beta-stimulated iNOS mRNA expression, Treatment of smooth muscle cells with IL-1beta in the presence of platelet derived growth factor or thrombin resulted in the inhibition of cytokine-stimulated expression of iNOS mRNA and NO release. The inhibitory effect of thrombin was reversed by hirudin and was mimicked by a 14 amino acid thrombin receptor activating peptide. In contract, the concomitant exposure of smooth muscle cells to IL-1beta-and plasmin resulted resulted in the potentiation of both IL-1beta-stimulated iNOS expression and NO generation. Finally, treatment of smooth muscle cells with IL-1beta in the presence of the hemostatic proteins did not affect the half-life of iNOS mRNA. These results demonstrate that specific protein components of the hemostatic system regulate IL- 1beta-stimulated iNOS mRNA expression in vascular smooth muscle cells. The capacity of hemostatic proteins to modulate the induction of vascular iNOS activity may play an important role in governing the release of NO and regulating thrombogenesis in vivo.
Asunto(s)
Interleucina-1/farmacología , Proteínas Musculares/fisiología , Músculo Liso Vascular/efectos de los fármacos , Músculo Liso Vascular/enzimología , Óxido Nítrico Sintasa/biosíntesis , Secuencia de Aminoácidos , Animales , Aorta Torácica/efectos de los fármacos , Aorta Torácica/enzimología , Bovinos , Células Cultivadas , Inducción Enzimática/efectos de los fármacos , Fibrinolisina/farmacología , Hemostasis/fisiología , Cinética , Datos de Secuencia Molecular , Nitritos/metabolismo , Fragmentos de Péptidos/farmacología , Factor de Crecimiento Derivado de Plaquetas/farmacología , ARN Mensajero/metabolismo , Ratas , Estimulación QuímicaAsunto(s)
Trasplante de Médula Ósea , Transfusión de Leucocitos , Leucocitos Mononucleares/trasplante , Linfoma de Células B/terapia , Leucemia-Linfoma Linfoblástico de Células Precursoras/terapia , Adulto , Trasplante de Médula Ósea/efectos adversos , Resultado Fatal , Enfermedad Injerto contra Huésped/prevención & control , Infecciones por Herpesviridae , Herpesvirus Humano 4 , Humanos , Leucemia Promielocítica Aguda/terapia , Linfoma de Células B/etiología , Masculino , Leucemia-Linfoma Linfoblástico de Células Precursoras/etiología , Trasplante Homólogo , Infecciones Tumorales por VirusRESUMEN
Macrophages activated by exposure to cytokines and/or to endotoxin produce nitric oxide (NO.), a free radical that is a mediator of the host response to infection. Activation induces the expression of nitric oxide synthase, the enzyme that catalyzes formation of NO. from L-arginine and molecular oxygen. We report the cloning of a cDNA encoding the inducible nitric oxide synthase from a murine macrophage cell line, RAW264.7, exposed to interferon-gamma and lipopolysaccharide. Oocytes injected with mRNA transcribed from this cDNA demonstrate arginine-dependent production of nitrite, a stable metabolite of NO.. Nitric production is blocked by the enzyme inhibitor, NG-monomethylarginine, and is independent of calcium/calmodulin. RAW264.7 cells demonstrate rapid accumulation of the nitric oxide synthase-encoding mRNAs upon activation. Comparison of the deduced amino acid sequence to the calcium/calmodulin-dependent nitric oxide synthase previously purified (Bredt, D. S., and Synder, S.H. (1990) Proc. Natl. Acad. Sci. U. S. A. 87, 682-685) and cloned (Bredt, D. S., Hwang, P. M., Glatt, C. E., Lowenstein, C., Reed, R. R., and Synder, S. H. (1991) nature 351, 714-718) from rat brain identifies shared binding sites for the cofactors NADPH and flavins in the C-terminal half of both proteins and an additional conserved region near the N terminus that may recognize L-arginine and/or contribute to the active site.