RESUMEN
Protein kinase C (PKC) plays a key role in a variety of signal transduction processes. The promoter region of the endothelial constitutive nitric oxide synthase (ecNOS) gene contains a transcriptional factor AP-1 binding element. In the present study, we sought to determine the effect of PKC inhibition on the expression of ecNOS in cultured bovine aortic endothelial cells (BAEC). The PKC inhibitor staurosporine (10 to 100 nmol/L) increased the expression of ecNOS mRNA, assessed by Northern analysis, in a dose-dependent manner. A newly developed, more specific PKC inhibitor, chelerythrine (1 to 3 mumol/L), also increased the level of ecNOS mRNA. Incubation of BAEC with phorbol 12-myristate 13-acetate (100 nmol/L) for 24 hours, which downregulates PKC, increased ecNOS mRNA expression. The protein content of ecNOS, assessed by Western analysis, was also increased in staurosporine-treated or chelerythrine-treated BAEC. The release of nitrogen oxides from staurosporine-treated or chelerythrine-treated cells both under basal conditions and in response to calcium ionophore A23187 was significantly increased (P < .05). In conclusion, the present study suggests that regulation of ecNOS is mediated by PKC. The increased release of nitric oxide induced by PKC inhibition may play a protective role against atherogenic process.
Asunto(s)
Aminoácido Oxidorreductasas/metabolismo , Endotelio Vascular/enzimología , Proteína Quinasa C/fisiología , Alcaloides/farmacología , Aminoácido Oxidorreductasas/genética , Animales , Benzofenantridinas , Bovinos , Células Cultivadas , Endotelio Vascular/citología , Óxido Nítrico Sintasa , Fenantridinas/farmacología , Proteína Quinasa C/antagonistas & inhibidores , ARN Mensajero/metabolismo , Estaurosporina , Acetato de Tetradecanoilforbol/farmacologíaRESUMEN
Endothelial nitric oxide synthase (eNOS) is a calmodulin (CaM)-dependent, membrane-associated, myristoylated enzyme, which has an important role in regulation of vascular tone and platelet aggregation. In this study, wild-type and mutant forms of bovine eNOS were overexpressed in a baculovirus/Sf9 insect cell system and examined for interactions with membrane phospholipids. Purified wild-type eNOS binds to pure anionic phospholipid vesicles but not to neutral phospholipid vesicles, demonstrating that eNOS attachment to lipid bilayers requires electrostatic as well as hydrophobic interactions. Moreover, catalytic activity of the enzyme is potently inhibited by anionic phospholipids, notably phosphatidylserine (PS), but not by neutral phospholipids. eNOS activity is also significantly inhibited upon enzyme binding to biological membranes isolated from cultured cells. Binding of eNOS to PS vesicles prevents subsequent binding of the enzyme to CaM-Sepharose. Interactions of eNOS with PS are not affected by site-specific mutation of the myristic acid acceptor site in the enzyme. Deletional mutation of the eNOS CaM-binding domain, however, results in loss of binding capacity of the enzyme not only for CaM-Sepharose but also for PS vesicles. Furthermore, removal of the CaM-binding domain converts eNOS from a membrane to a cytosolic protein when the enzyme is expressed in Sf9 cells. These data demonstrate that electrostatic interactions between anionic membrane phospholipids and basic residues in the eNOS CaM-binding domain are important for enzyme membrane association. Membrane association can thus function to inhibit eNOS catalytic activity by interfering with the interaction of the enzyme with calmodulin.
Asunto(s)
Aminoácido Oxidorreductasas/antagonistas & inhibidores , Calmodulina/metabolismo , Endotelio Vascular/enzimología , Fosfatidilserinas/farmacología , Aminoácido Oxidorreductasas/genética , Aminoácido Oxidorreductasas/aislamiento & purificación , Secuencia de Aminoácidos , Animales , Baculoviridae/genética , Bovinos , Línea Celular , Membrana Celular/metabolismo , Clonación Molecular , Datos de Secuencia Molecular , Óxido Nítrico Sintasa , Fosfatidilserinas/metabolismo , Unión Proteica , SpodopteraRESUMEN
The calmodulin (CaM)-binding regions in bovine endothelial nitric oxide synthase (eNOS) and murine inducible nitric oxide synthase (iNOS) are identified in this study as eNOS residues 493-512 and iNOS residues 501-532. Peptides corresponding to eNOS 493-512 and NOS 501-532 produce a (Ca2+)-dependent, electrophoretic mobility shift of CaM on 4 M urea gels. The two peptides are also potent inhibitors of the CaM-mediated activation of neuronal nitric oxide synthase and have dissociation constants for CaM binding of 4.0 and 1.5 nM respectively. Substitution of eNOS and iNOS CaM-binding domains in eNOS/iNOS chimeric proteins produces major alterations in the Ca2+ and CaM dependence of the intact enzymes expressed and purified from a baculovirus/Sf9 insect cell system. Replacement of aligned NOS sequence with eNOS 493-512 creates a functional, chimeric iNOS that is both (Ca2+)- and CaM-dependent. Replacement of aligned eNOS sequence with NOS 501-532 creates a functional, chimeric eNOS that is CaM-independent but that remains (Ca2+)-dependent. Specific amino acid residues critical for CaM binding by eNOS are also identified in this study as Phe-498, Lys-499, and Leu-511 in the bovine eNOS sequence.
Asunto(s)
Calmodulina/metabolismo , Óxido Nítrico Sintasa/metabolismo , Secuencia de Aminoácidos , Animales , Baculoviridae/genética , Sitios de Unión/genética , Calcio/metabolismo , Bovinos , Línea Celular , Pollos , Endotelio/enzimología , Inducción Enzimática , Ratones , Datos de Secuencia Molecular , Óxido Nítrico Sintasa/química , Óxido Nítrico Sintasa/genética , Conejos , Ratas , Proteínas Recombinantes de Fusión/química , Proteínas Recombinantes de Fusión/genética , Proteínas Recombinantes de Fusión/metabolismo , Homología de Secuencia de Aminoácido , SpodopteraRESUMEN
Previous studies have shown the effect of nitroglycerin on coronary microvessels < 100 microns in diameter is markedly enhanced by L-cysteine. These studies were performed to examine the mechanisms responsible for this effect. Under control conditions, nitroglycerin caused potent dilations of large (> 200 microns diam) coronary microvessels while having minimal effects on small (< 100 microns diam) coronary microvessels [peak relaxations 85 +/- 4 vs. 23 +/- 3% (mean +/- SE) of endothelin-1-constricted vessels, respectively]. L-Cysteine (100 microM) and N-acetylcysteine (100 microM) markedly enhanced nitroglycerin-induced relaxations of small coronary microvessels (peak relaxation 84 +/- 6 and 87 +/- 12%, respectively) while having no effect on relaxations of vessels > 100 microns. In contrast, neither L-methionine (100 microM) nor glutathione (100 microM) enhanced nitroglycerin's vasodilation of small coronary microvessels. The effects of L-cysteine and N-acetylcysteine on the augmentation of nitroglycerin vasodilatation in smaller coronary microvessels was abolished in the presence of buthionine sulfoximine (100 microM), a potent inhibitor of intracellular glutathione synthesis. Buthionine sulfoximine had no effect on the vasodilatation produced by nitroprusside. These data demonstrate that, in smaller coronary microvessels, L-cysteine and N-acetylcysteine enhance nitroglycerin-induced vasodilatation by increasing intracellular glutathione concentrations. Intracellular glutathione, formed from either L-cysteine or N-acetylcysteine, may participate in the formation of an intermediate of nitroglycerin biotransformation or may maintain a redox potential within coronary microvessels that favors enzymatic bioconversion of nitroglycerin.
Asunto(s)
Circulación Coronaria/efectos de los fármacos , Nitroglicerina/farmacología , Compuestos de Sulfhidrilo/farmacología , Animales , Vasos Sanguíneos/metabolismo , Tampones (Química) , Butionina Sulfoximina , Cisteína/metabolismo , Perros , Interacciones Farmacológicas , Femenino , Glutatión/antagonistas & inhibidores , Glutatión/biosíntesis , Masculino , Metionina Sulfoximina/análogos & derivados , Metionina Sulfoximina/farmacología , Microcirculación/efectos de los fármacos , Óxido Nítrico/metabolismo , Nitroglicerina/metabolismo , Oxígeno/farmacología , Vasodilatación/efectos de los fármacosRESUMEN
The promoter region of the endothelial cell nitric oxide synthase (ecNOS) gene contains potential response elements for transforming growth factor-beta 1 (TGF beta 1). TGF beta 1 plays an important role in the pathogenesis of atherosclerosis, vascular hypertrophy, and angiogenesis. We therefore sought to determine whether TGF beta 1 might modulate ecNOS expression in bovine aortic endothelial cells (BAEC). TGF beta 1 increased ecNOS mRNA in a dose-dependent manner. TGF beta 1 also increased ecNOS protein content. The production of nitrogen oxides (NOx), assessed by chemiluminescence, and nitric oxide synthase activity, assessed by arginine/citrulline conversion were increased in TGF beta 1-treated cells. Transcriptional activity of the 5'-flanking promoter region of the ecNOS gene was increased by TGF beta 1, as assessed by transfection with promoter/luciferase constructs. Deletion analysis suggested that the TGF beta 1-response element was present between nucleotides -1269 and -935 from the first transcription start site, in which a putative nuclear factor-1 (NF-1) binding site existed. Gel shift assays showed that nuclear protein(s), immunologically similar to CCAAT transcription factor/NF-1, bound to the putative NF-1 binding site in a sequence-specific manner. Mutation of the putative NF-1 binding site in the promoter/luciferase construct significantly decreased the responsiveness to TGF beta 1. In conclusion, TGF beta 1 increases ecNOS expression associated with an increase in production of NO in BAEC. This response is probably mediated by transcriptional activation of the ecNOS gene promoter.
Asunto(s)
Aminoácido Oxidorreductasas/genética , Endotelio Vascular/enzimología , Óxido Nítrico/metabolismo , Factor de Crecimiento Transformador beta/farmacología , Animales , Secuencia de Bases , Sitios de Unión , Bovinos , División Celular/efectos de los fármacos , Núcleo Celular/metabolismo , Células Cultivadas , Proteínas de Unión al ADN/metabolismo , Técnicas In Vitro , Datos de Secuencia Molecular , Mutagénesis Sitio-Dirigida , Factores de Transcripción NFI , Óxido Nítrico Sintasa , Oligodesoxirribonucleótidos/química , Regiones Promotoras Genéticas , Relación Estructura-Actividad , Factores de Transcripción/metabolismo , Transcripción Genética/efectos de los fármacosRESUMEN
BACKGROUND: We have shown that hypercholesterolemia increases vascular superoxide anion (O2-) production, which could be responsible for augmented inactivation of endothelium-derived vascular relaxing factor. We sought to determine whether this increased vascular O2- production is due to infiltration of macrophages into the intima and whether dietary treatment of hypercholesterolemia normalizes O2- production. METHODS AND RESULTS: A specific and sensitive assay for O2- based on chemiluminescence of lucigenin was used; the amount of O2- produced by vascular ring segments was quantified based on known quantities of O2- produced by xanthine-xanthine oxidase standards. O2- production of aortic segments from normal rabbits (n = 9), cholesterol-fed rabbits (1% cholesterol diet for 1 month, n = 7), and rabbits fed a 1% cholesterol diet for 1 month followed by a normal diet for 1 month (regression rabbits, n = 5) was measured. At the end of these diets, serum cholesterol levels were 1.5 +/- 0.2, 26.0 +/- 3.9, and 1.8 +/- 0.5 mmol/L (58 +/- 6, 1000 +/- 150, and 71 +/- 19 mg/dL) in the normal, cholesterol-fed, and regression animals, respectively. Vessels from normal rabbits with endothelium produced 0.32 +/- 0.06 nmol O2-/mg dry wt per minute, whereas those without endothelium produced approximately twice as much O2- (0.66 +/- 0.12 nmol O2- mg dry wt per minute. Vessels with endothelium from cholesterol-fed rabbits produced 4.5-fold more O2- than vessels from normal animals. This increased production of O2- was normalized by endothelial removal. This increased production of O2- was not due to infiltration of macrophages in the intima, because there was no correlation between vascular O2- production and macrophage infiltration assessed by immunohistochemistry with use of a specific antibody against rabbit macrophage. O2- production by vessels from regression rabbits was similar to that observed in normal animals, and as in the normal rabbits, endothelial removal increased O2- production. Aortic rings from these animals also were studied in organ chambers. Dietary lowering of cholesterol dramatically improved vasodilator responses to acetylcholine and A23187 (P < .05 versus cholesterol-fed rabbits). CONCLUSIONS: Dietary lowering of cholesterol not only improves endothelium-dependent vascular relaxation but also normalizes endothelial O2- production. Decreases of O2- production by dietary lowering of cholesterol not only may improve vasomotor control but also may improve other aspects of vascular integrity in atherosclerosis.