RESUMEN
Statins are effective drugs in the prevention of cardiovascular disease. Recent studies suggested that statins have additional beneficial effects on the vascular wall independent of their cholesterol-lowering effects. We investigated whether atorvastatin influences angiotensin-converting enzyme (ACE) production in differentiating human macrophages. Human peripheral blood monocytes (PBM) were isolated from fresh buffy coats. The cells were allowed to differentiate for 0-8 days in macrophage serum-free medium with 5 ng/ml granulocyte-macrophage colony-stimulating factor. Atorvastatin (0.005-0.5 microM), mevalonate (200-400 microM), geranylgeranyl pyrophosphate (1.25-2.5 microM), and/or farnesylpyrophosphate (FPP; 1.25-2.5 microM) was added on the second day of differentiation and then every other day. After incubation time, the ACE amount in intact macrophages was measured. ACE amount in PBM was low. A marked time-dependent ACE induction was noticed during differentiation of monocytes to macrophages. Atorvastatin treatment inhibited ACE induction during differentiation. In the presence of mevalonate, atorvastatin failed to downregulate ACE production. Cotreatment of the cells with atorvastatin and FPP reversed the suppressive effect of atorvastatin on ACE. In conclusion, atorvastatin inhibited ACE upregulation, normally occurring in differentiating human macrophages. This effect was mediated via the mevalonate pathway, and inhibition of FPP was probably involved. The finding that atorvastatin inhibited ACE upregulation may represent a novel pleiotropic action and an additional beneficial effect of statins in treatment of cardiovascular disease.
Asunto(s)
Ácidos Heptanoicos/farmacología , Inhibidores de Hidroximetilglutaril-CoA Reductasas/farmacología , Macrófagos/efectos de los fármacos , Macrófagos/enzimología , Peptidil-Dipeptidasa A/metabolismo , Pirroles/farmacología , Adolescente , Adulto , Anciano , Atorvastatina , Diferenciación Celular/efectos de los fármacos , Diferenciación Celular/inmunología , Inducción Enzimática/efectos de los fármacos , Inducción Enzimática/inmunología , Regulación Enzimológica de la Expresión Génica/efectos de los fármacos , Regulación Enzimológica de la Expresión Génica/inmunología , Humanos , Técnicas In Vitro , Macrófagos/citología , Ácido Mevalónico/metabolismo , Ácido Mevalónico/farmacología , Persona de Mediana Edad , Peptidil-Dipeptidasa A/genética , Fosforilación/efectos de los fármacos , Fosfatos de Poliisoprenilo/metabolismo , Proteína Quinasa C/metabolismo , ARN Mensajero/metabolismo , Sesquiterpenos/metabolismo , Terpenos/metabolismo , Regulación hacia Arriba/efectos de los fármacos , Regulación hacia Arriba/inmunologíaRESUMEN
Nicotine, a component of cigarette smoke, has been implicated in the pathogenesis of cardiovascular disease. We examined whether nicotine regulates angiotensin-converting enzyme (ACE), an enzyme that plays an important role in the pathophysiology of atherosclerosis and hypertension. Human umbilical cord vein endothelial cells were treated with nicotine (0.1-1 microM) alone or in combination with vascular endothelial growth factor (VEGF; 0.5 nM) or GF-109203X (GFX; 2.5 microM). The amount of ACE in intact endothelial cells was measured by an inhibitor-binding assay method, and ACE mRNA levels were quantified using LightCycler technology. Phosphorylated PKC levels were measured by Western immunoblotting. Nicotine did not modulate basal ACE production but significantly potentiated VEGF-induced ACE upregulation. Treatment of endothelial cells with the PKC inhibitor GFX totally blocked VEGF- and nicotine-induced ACE upregulation. VEGF induced PKC phosphorylation, which was potentiated by cotreatment with nicotine. We conclude that nicotine significantly potentiated VEGF-induced ACE upregulation. This effect was probably mediated by PKC phosphorylation. The interaction of nicotine with VEGF in ACE induction may contribute to the pathogenesis of smoking-related cardiovascular disease.
Asunto(s)
Células Endoteliales/enzimología , Nicotina/farmacología , Agonistas Nicotínicos/farmacología , Peptidil-Dipeptidasa A/biosíntesis , Inhibidores de la Enzima Convertidora de Angiotensina/metabolismo , Supervivencia Celular/efectos de los fármacos , Células Cultivadas , Células Endoteliales/efectos de los fármacos , Inhibidores Enzimáticos/farmacología , Humanos , Immunoblotting , Indoles/farmacología , Maleimidas/farmacología , Fosforilación , Proteína Quinasa C/antagonistas & inhibidores , Proteína Quinasa C/metabolismo , ARN Mensajero/biosíntesis , Regulación hacia Arriba/efectos de los fármacos , Factor A de Crecimiento Endotelial Vascular/farmacologíaRESUMEN
BACKGROUND: Calcium salts are used as phosphate binders in renal failure, while high calcium diet also improves vasorelaxation and enhances natriuresis. The influences of calcium intake on renal renin-angiotensin system (RAS) are largely unknown. METHODS: Four weeks after NTX, rats were put on 3.0% or 0.3% calcium diet for 8 weeks (12-week study). In additional experiments, 15 weeks after NTX, rats were put on similar diets for 12 weeks (27-week study). Appropriate blood, urine, and kidney samples were taken. Renal angiotensin-converting enzyme (ACE) and angiotensin II receptors (AT1, AT2) were examined using autoradiography, ACE also using Western blotting, and connective tissue growth factor (CTGF) using immunohistochemistry. RESULTS: In the 12-week study, albuminuria increased 5-fold in NTX rats, but only 2-fold in calcium NTX rats on 3.0% calcium. In the 27-week study, high calcium intake decreased blood pressure, retarded progression of renal failure, reduced glomerulosclerosis, interstitial damage, and aortic calcifications, and improved survival from 50% to 92% in NTX rats. In both experiments plasma parathyroid hormone and phosphate were elevated after NTX, and suppressed by high calcium diet, while kidney ACE was down-regulated by 40% or more after increased calcium intake. In the 27-week study renal CTGF was decreased and cortical AT1 receptor density reduced after high calcium diet. CONCLUSION: High calcium diet down-regulated kidney ACE, reduced albuminuria and blood pressure, and favorably influenced kidney morphology in experimental renal failure. These findings suggest a link between calcium metabolism and kidney ACE expression, which may play a role in the progression of renal damage.
Asunto(s)
Albuminuria/tratamiento farmacológico , Calcio de la Dieta/farmacología , Riñón/enzimología , Peptidil-Dipeptidasa A/metabolismo , Insuficiencia Renal/tratamiento farmacológico , Albuminuria/metabolismo , Albuminuria/patología , Animales , Aorta/patología , Factor de Crecimiento del Tejido Conjuntivo , Regulación hacia Abajo/efectos de los fármacos , Proteínas Inmediatas-Precoces/metabolismo , Péptidos y Proteínas de Señalización Intercelular/metabolismo , Riñón/patología , Masculino , Hormona Paratiroidea/sangre , Fosfatos/sangre , Ratas , Ratas Sprague-Dawley , Insuficiencia Renal/metabolismo , Insuficiencia Renal/patología , Sistema Renina-Angiotensina/efectos de los fármacos , Sistema Renina-Angiotensina/fisiologíaRESUMEN
Both rosiglitazone and metformin increase hepatic insulin sensitivity, but their mechanism of action has not been compared in humans. The objective of this study was to compare the effects of rosiglitazone and metformin treatment on liver fat content, hepatic insulin sensitivity, insulin clearance, and gene expression in adipose tissue and serum adiponectin concentrations in type 2 diabetes. A total of 20 drug-naive patients with type 2 diabetes (age 48 +/- 3 years, fasting plasma glucose 152 +/- 9 mg/dl, BMI 30.6 +/- 0.8 kg/m2) were treated in a double-blind randomized fashion with either 8 mg rosiglitazone or 2 g metformin for 16 weeks. Both drugs similarly decreased HbA1c, insulin, and free fatty acid concentrations. Body weight decreased in the metformin (84 +/- 4 vs. 82 +/- 4 kg, P < 0.05) but not the rosiglitazone group. Liver fat (proton spectroscopy) was decreased with rosiglitazone by 51% (15 +/- 3 vs. 7 +/- 1%, 0 vs. 16 weeks, P = 0.003) but not by metformin (13 +/- 3 to 14 +/- 3%, NS). Rosiglitazone (16 +/- 2 vs. 20 +/- 1 ml.kg(-1).min(-1), P = 0.02) but not metformin increased insulin clearance by 20%. Hepatic insulin sensitivity in the basal state increased similarly in both groups. Insulin-stimulated glucose uptake increased significantly with rosiglitazone but not with metformin. Serum adiponectin concentrations increased by 123% with rosiglitazone but remained unchanged during metformin treatment. The decrease of serum adiponectin concentrations correlated with the decrease in liver fat (r = -0.74, P < 0.001). Rosiglitazone but not metformin significantly increased expression of peroxisome proliferator-activated receptor-gamma, adiponectin, and lipoprotein lipase in adipose tissue. In conclusion, rosiglitazone but not metformin decreases liver fat and increases insulin clearance. The decrease in liver fat by rosiglitazone is associated with an increase in serum adiponectin concentrations. Both agents increase hepatic insulin sensitivity, but only rosiglitazone increases peripheral glucose uptake.
Asunto(s)
Tejido Adiposo/metabolismo , Diabetes Mellitus Tipo 2/metabolismo , Regulación de la Expresión Génica/genética , Metabolismo de los Lípidos , Hígado/metabolismo , Metformina/farmacología , Tiazolidinedionas/farmacología , Tejido Adiposo/efectos de los fármacos , Colesterol/sangre , Diabetes Mellitus Tipo 2/sangre , Femenino , Regulación de la Expresión Génica/efectos de los fármacos , Humanos , Hipoglucemiantes/farmacología , Insulina/sangre , Resistencia a la Insulina/fisiología , Hígado/efectos de los fármacos , Masculino , Persona de Mediana Edad , Rosiglitazona , Triglicéridos/sangreRESUMEN
Angiotensin-converting enzyme (ACE) plays an important role in the pathophysiology of cardiovascular disease. We examined the effect of carvedilol, a cardiovascular drug, on basal and stimulated ACE production in human endothelial cells. Carvedilol (0.625-5 microM), in a concentration-dependent manner, inhibited basal and vascular endothelial growth factor (VEGF, 0.5 nM) or phorbol 12-myristate 13-acetate (PMA, 10 nM) induced ACE up-regulation. Carvedilol has non-selective beta-adrenoceptor and selective alpha1-adrenoceptor blocking activity, calcium channel blocking, and anti-oxidant activity. To study whether these activities were related to ACE down-regulation, endothelial cells were treated with metoprolol (1-10 microM), propranolol (1-10 microM), prazosin (1-5 microM), nicardipine (1-10 microM), probucol (1-100 microM), or ascorbic acid (1-100 microM). None of these compounds modified ACE. VEGF (0.5 nM) and PMA (10 nM) induced PKC phosphorylation, which was inhibited by co-treatment of cell cultures with carvedilol (5 microM). In conclusion, carvedilol inhibited basal and VEGF or PMA induced ACE up-regulation. Inhibition of PKC phosphorylation was probably involved in carvedilol action.
Asunto(s)
Carbazoles/farmacología , Fármacos Cardiovasculares/farmacología , Endotelio Vascular/efectos de los fármacos , Peptidil-Dipeptidasa A/biosíntesis , Propanolaminas/farmacología , Acetato de Tetradecanoilforbol/análogos & derivados , Vasodilatadores/farmacología , Carvedilol , Células Cultivadas , Endotelio Vascular/citología , Endotelio Vascular/enzimología , Humanos , Immunoblotting , Peptidil-Dipeptidasa A/genética , Fosforilación , Proteína Quinasa C/metabolismo , ARN Mensajero/biosíntesis , Acetato de Tetradecanoilforbol/farmacología , Venas Umbilicales/citología , Venas Umbilicales/efectos de los fármacos , Venas Umbilicales/enzimología , Factor A de Crecimiento Endotelial Vascular/farmacologíaRESUMEN
Highly active antiretroviral therapy (HAART) has improved the prognosis of human immunodeficiency virus (HIV)-infected patients but is associated with severe adverse events, such as lipodystrophy and insulin resistance. Rosiglitazone did not increase subcutaneous fat in patients with HAART-associated lipodystrophy (HAL) in a randomized, double-blind, placebo-controlled trial, although it attenuated insulin resistance and decreased liver fat content. The aim of this study was to examine effects of rosiglitazone on gene expression in subcutaneous adipose tissue in 30 patients with HAL. The mRNA concentrations in subcutaneous adipose tissue were measured using real-time PCR. Twenty-four-week treatment with rosiglitazone (8 mg/day) compared with placebo significantly increased the expression of adiponectin, peroxisome proliferator-activated receptor-gamma (PPARgamma), and PPARgamma coactivator 1 and decreased IL-6 expression. Expression of other genes involved in lipogenesis, fatty acid metabolism, or glucose transport, such as acyl-CoA synthase, adipocyte lipid-binding protein, CD45, fatty acid transport protein-1 and -4, GLUT1, GLUT4, keratinocyte lipid-binding protein, lipoprotein lipase, PPARdelta, and sterol regulatory element-binding protein-1c, remained unchanged. Rosiglitazone also significantly increased serum adiponectin concentration. The change in serum adiponectin concentration was inversely correlated with the change in fasting serum insulin concentration and liver fat content. In conclusion, rosiglitazone induced significant changes in gene expression in subcutaneous adipose tissue and ameliorated insulin resistance in patients with HAL. Increased expression of adiponectin might have mediated most of the favorable insulin-sensitizing effects of rosiglitazone in these patients.
Asunto(s)
Tejido Adiposo/efectos de los fármacos , Terapia Antirretroviral Altamente Activa/efectos adversos , Síndrome de Lipodistrofia Asociada a VIH/tratamiento farmacológico , Hipoglucemiantes/administración & dosificación , Péptidos y Proteínas de Señalización Intercelular , Tejido Subcutáneo/efectos de los fármacos , Tiazolidinedionas/administración & dosificación , Adiponectina , Tejido Adiposo/fisiología , Composición Corporal , Ácidos Grasos/metabolismo , Expresión Génica/efectos de los fármacos , Síndrome de Lipodistrofia Asociada a VIH/fisiopatología , Humanos , Resistencia a la Insulina , Hígado/metabolismo , Proteínas/genética , Rosiglitazona , Tejido Subcutáneo/fisiologíaRESUMEN
Angiotensin-converting enzyme (ACE) plays an important role in the pathophysiology of cardiovascular disease. We investigated whether atorvastatin, a powerful agent for the prevention and treatment of cardiovascular disease, influences ACE production in endothelial cells. Human umbilical cord vein endothelial cells were treated with VEGF (476 pM), which induced ACE upregulation. Cotreatment with atorvastatin (0.1-10 microM) dose dependently inhibited VEGF-induced ACE upregulation. In the presence of mevalonate (100 microM), atorvastatin failed to downregulate VEGF-induced ACE production. Cotreatment of the cells with either farnesylpyrophosphate (FPP; 5 microM) or geranylgeranylpyrophosphate (GGPP; 5 microM) partially inhibited the suppressive effect of atorvastatin. Pretreatment of the cells with Rho-associated protein kinase inhibitor, Y-27632 (10 microM), partially inhibited VEGF-induced ACE upregulation. VEGF (476 pM) caused PKC phosphorylation, which was inhibited by cotreatment of the cells with atorvastatin. Atorvastatin inhibited VEGF-induced ACE upregulation probably by inhibiting PKC phosphorylation. This effect was mediated via inhibition of the mevalonate pathway. ACE downregulation may be an additional beneficial effect of statins in the treatment of cardiovascular disease.
Asunto(s)
Endotelio Vascular/efectos de los fármacos , Ácidos Heptanoicos/farmacología , Inhibidores de Hidroximetilglutaril-CoA Reductasas/farmacología , Peptidil-Dipeptidasa A/metabolismo , Pirroles/farmacología , Factor A de Crecimiento Endotelial Vascular/farmacología , Amidas/farmacología , Atorvastatina , División Celular/efectos de los fármacos , Células Cultivadas , Interacciones Farmacológicas , Endotelio Vascular/citología , Endotelio Vascular/enzimología , Inhibidores Enzimáticos/farmacología , Regulación Enzimológica de la Expresión Génica/efectos de los fármacos , Humanos , Péptidos y Proteínas de Señalización Intracelular , Ácido Mevalónico/farmacología , Peptidil-Dipeptidasa A/genética , Fosforilación/efectos de los fármacos , Fosfatos de Poliisoprenilo/farmacología , Proteínas Serina-Treonina Quinasas/antagonistas & inhibidores , Piridinas/farmacología , ARN Mensajero/análisis , Sesquiterpenos , Venas Umbilicales/citología , Regulación hacia Arriba/efectos de los fármacos , Quinasas Asociadas a rhoRESUMEN
OBJECTIVE: To determine the expressions of multiple genes in the subcutaneous adipose tissue of HIV-positive, highly active antiretroviral therapy (HAART)-treated patients with and without lipodystrophy. DESIGN AND METHODS: Real-time polymerase chain reaction was used to measure gene expressions in this cross-sectional study. RESULTS: The messenger RNA concentrations of adipose transcription factors (peroxisome proliferator-activated receptor (PPAR) gamma and delta and sterol regulatory element binding protein 1c) were all significantly lower in the lipodystrophic than the non-lipodystrophic group. The mRNA concentration of PPAR-gamma co-activator 1 (PGC-1), which regulates mitochondrial biogenesis, was lower in the lipodystrophic than the non-lipodystrophic group. The mRNA expression of lipoprotein lipase, acyl coenzyme A synthase and glucose transport protein 4 were significantly lower in the lipodystrophic than the non-lipodystrophic group, but the mRNA concentrations of fatty acid transport and binding proteins were similar in both groups. The mRNA concentrations of IL-6 and CD45 (a common leukocyte marker) were significantly higher in the lipodystrophic than the non-lipodystrophic group. CONCLUSION: Multiple alterations characterize gene expression in the subcutaneous adipose tissue of patients with HAART-associated lipodystrophy compared with HIV-positive, HAART-treated patients without lipodystrophy. The low expression of transcription factors inhibits adipocyte differentiation. The low expression of PGC-1 may contribute to mitochondrial defects. In addition, IL-6 and CD45 expressions are increased, the latter implying an excessive number of cells of leukocyte origin in lipodystrophic adipose tissue. Mitochondrial injury and an excess of proinflammatory cytokines may lead to increased apoptosis. All these changes may contribute to the loss of subcutaneous fat in HAART-associated lipodystrophy.
Asunto(s)
Tejido Adiposo/metabolismo , Síndrome de Lipodistrofia Asociada a VIH/genética , Interleucina-6/genética , Antígenos Comunes de Leucocito/genética , Proteínas de Transporte de Membrana , Proteínas Musculares , Proteínas de Neoplasias , Factores de Transcripción/genética , Proteínas Supresoras de Tumor , Actinas/genética , Tejido Adiposo/inmunología , Adulto , Terapia Antirretroviral Altamente Activa , Antivirales/uso terapéutico , Proteínas Potenciadoras de Unión a CCAAT/genética , Proteínas Portadoras/genética , Estudios de Casos y Controles , Coenzima A Ligasas/genética , Estudios Transversales , Proteínas de Unión al ADN/genética , Proteínas de Transporte de Ácidos Grasos , Proteína de Unión a los Ácidos Grasos 7 , Proteínas de Unión a Ácidos Grasos , Femenino , Expresión Génica , Transportador de Glucosa de Tipo 1 , Transportador de Glucosa de Tipo 4 , Infecciones por VIH/tratamiento farmacológico , Infecciones por VIH/metabolismo , Síndrome de Lipodistrofia Asociada a VIH/tratamiento farmacológico , Humanos , Lipoproteína Lipasa/genética , Masculino , Proteínas de la Membrana/genética , Proteínas de Transporte de Monosacáridos/genética , Proteína Tirosina Fosfatasa no Receptora Tipo 1 , ARN Mensajero/análisis , Receptores Citoplasmáticos y Nucleares/genética , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa , Proteína 1 de Unión a los Elementos Reguladores de Esteroles , Microglobulina beta-2/genéticaRESUMEN
The regulation of both angiotensin receptors and bradykinin receptors during sodium intake is poorly understood. We hypothesized that an altered balance between renal angiotensin type 1 (AT1) receptors and bradykinin type 2 (B2) receptors might contribute to an increase in blood pressure during periods of high-sodium intake. We studied the effects of high-sodium intake on renal AT1 receptors and B2 receptors in 5-6-week-old spontaneously hypertensive rats (SHR) receiving high-sodium chloride (6% NaCl) or mineral salts (10.5%, composition: 57% NaCl, 28% KCl, 12% MgSO4) compared to those receiving a low-sodium (NaCl 0.125%) diet for 10 weeks. Mineral salt intake was included due to its beneficial effects on blood pressure and cardiac hypertrophy. Receptor densities were measured by quantitative autoradiography. AT1 receptors were quantified using incubation with 125I-Sar1-Ile8-angiotensin II and displacement was measured with PD123319 (10 micromol/l), whereas B2 receptors were quantified using 125I-HPP-icatibant and displacement was measured with icatibant (3 micromol/l). Compared to the SHR controls, a further increase in blood pressure occurred after 2 weeks in the 6% NaCl group and after 6 weeks in the mineral salt group. AT1 receptor density increased in the renal cortex by 41% (p<0.01) in the 6% NaCl group and by 26% (p<0.05) in the mineral salt group. B2 receptor density decreased in the renal medulla by 26% (p<0.01) in the 6% NaCl group, and decreased even more i.e., by 45% (p<0.001), in the mineral salt group. It was shown that a 6% NaCl or a 10.5% mineral salt loading was capable of increasing renal AT1 receptor density and decreasing renal B2 receptor density. An altered balance between these receptors might be associated with hypertension under conditions of sodium loading.
Asunto(s)
Riñón/metabolismo , Receptor de Angiotensina Tipo 1/metabolismo , Receptor de Bradiquinina B2/metabolismo , Cloruro de Sodio Dietético/farmacología , Animales , Autorradiografía , Riñón/efectos de los fármacos , Ratas , Ratas Endogámicas SHR , Receptor de Angiotensina Tipo 1/efectos de los fármacos , Receptor de Bradiquinina B2/efectos de los fármacos , Cloruro de Sodio Dietético/administración & dosificaciónRESUMEN
Highly active antiretroviral therapy (HAART) has dramatically reduced HIV-related mortality, but is associated with severe metabolic adverse events, such as lipodystrophy and insulin resistance, the mechanisms of which are unknown. Adiponectin is a adipocytokine that is decreased in insulin resistant conditions. In mice, adiponectin decreases liver and muscle fat content and enhances insulin sensitivity. We determined serum adiponenctin and adiponectin mRNA concentrations in subcutaneous adipose tissue in HIV-positive HAART-treated patients with (HAART+LD+, n = 30) and without lipodystrophy (HAART+LD-, n = 13). The HAART+ LD+ group had significantly less subcutaneous and more intra-abdominal fat than the HAART+LD- group. Liver fat content (spectroscopy), serum insulin, C-peptide and triglyceride concentrations were significantly higher, and HDL cholesterol concentration lower in the HAART+LD+ than the HAART+LD- group. Serum adiponectin (3.4 +/- 0.4 vs 8.5 +/- 1.0 micro g/mL, p < 0.001) and adiponectin mRNA concentration in subcutaneous adipose tissue (7 +/- 1 x 10(-4) vs 24 +/- 6 x 10(-4), p < 0.001) were significantly lower in the HAART+LD+ than the HAART+LD- group. Both serum adiponectin and mRNA concentrations correlated closely with features of insulin resistance, including liver fat content. These data suggest that the decreased production of adiponectin in lipoatrophic adipose tissue may contribute to hepatic insulin resistance in these patients.
Asunto(s)
Tejido Adiposo/química , Terapia Antirretroviral Altamente Activa/efectos adversos , Síndrome de Lipodistrofia Asociada a VIH/sangre , Síndrome de Lipodistrofia Asociada a VIH/inducido químicamente , Péptidos y Proteínas de Señalización Intercelular , Proteínas/análisis , Proteínas/genética , Abdomen , Actinas/genética , Adiponectina , Tejido Adiposo/patología , Adulto , Composición Corporal , Péptido C/sangre , HDL-Colesterol/sangre , Femenino , Expresión Génica , Humanos , Insulina/sangre , Resistencia a la Insulina , Hígado/efectos de los fármacos , Hígado/patología , Imagen por Resonancia Magnética , Masculino , Persona de Mediana Edad , ARN Mensajero/análisis , Triglicéridos/sangreRESUMEN
OBJECTIVE: To investigate regulation of angiotensin converting enzyme (ACE) by tumour necrosis factor alpha (TNF-alpha) in differentiating human peripheral blood monocytes (PBM). METHODS: Human PBM were allowed to differentiate to macrophages for 0-7 days and ACE amount was measured during differentiation. Experiments with TNF-alpha were performed after 2 days of differentiation. Cell cultures were incubated with TNF-alpha (0.5-10ng/ml) without or with SB 202190 (5microM), or PD 98059 (40microM). ACE amounts were measured by an inhibitor binding assay (IBA) and ACE mRNA levels by RNase protection assay (RPA). Activated p44/42 and p38 MAP kinases were measured by Western Blot analysis using phospho-p44/42 and -p38 MAPK antibodies. RESULTS: ACE amount increased by 40-fold along with macrophage differentiation. TNF-alpha caused dose dependent suppression of the amount of ACE and decreased levels of ACE mRNA. TNF-alpha activated p44/42 and p38 MAP kinases, which was inhibited by the specific inhibitors of these kinases, PD98059 or SB202190, respectively. Pretreatment of the cells with SB 202190, or PD 98059 both partly reversed TNF-alpha induced ACE suppression. CONCLUSIONS: TNF-alpha downregulated ACE, which effect was probably mediated by both p44/42 and p38 MAPK pathways. Local downregulation of ACE by TNF-alpha may be a counterbalancing mechanism in inflammatory processes.
Asunto(s)
Regulación hacia Abajo , Inhibidores Enzimáticos/farmacología , Macrófagos/citología , Macrófagos/metabolismo , Peptidil-Dipeptidasa A/metabolismo , Factor de Necrosis Tumoral alfa/metabolismo , Western Blotting , Diferenciación Celular , Movimiento Celular , Células Cultivadas , Humanos , Immunoblotting , Proteína Quinasa 1 Activada por Mitógenos/metabolismo , Proteína Quinasa 3 Activada por Mitógenos , Proteínas Quinasas Activadas por Mitógenos/metabolismo , Fosforilación , ARN Mensajero/metabolismo , Factores de Tiempo , Proteínas Quinasas p38 Activadas por MitógenosRESUMEN
Subendothelial mast cells have been implicated in the pathogenesis of allergic inflammation, in atherosclerosis, and in the regulation of vascular tone. Because endothelin-1 (ET-1) is an important regulator of vascular tone and has also been implicated in the pathogenesis of atherosclerosis, we studied the role of mast cells in the metabolism of endothelial cell-derived ET-1. In mast cell-endothelial cell cocultures, activation of the mast cells with ensuing degranulation was accompanied by the increased expression of ET-1 mRNA in the endothelial cells, yet the immunoreactive ET-1 protein in the coculture medium disappeared almost completely during the 24-hour coculture. Activation of the mast cells with the ensuing degranulation resulted in proteolytic degradation of ET-1 by the 2 neutral proteases, chymase and carboxypeptidase A, of the exocytosed mast cell granules. With synthetic ET-1 and purified mast cell granule enzymes, efficient degradation of ET-1 by chymase and carboxypeptidase A was verified. These in vitro results imply a novel role for mast cell-derived neutral proteases in ET-1 metabolism and suggest that activated subendothelial mast cells are important local regulators of ET-1 metabolism.