RESUMEN
BACKGROUND AND PURPOSE: The recent development of the UT ligand palosuran (1-[2-(4-benzyl-4-hydroxy-piperidin-1-yl)-ethyl]-3-(2-methyl-quinolin-4-yl)-urea sulphate salt) has led to the proposition that urotensin-II (U-II) plays a significant pathological role in acute and chronic renal injury in the rat. EXPERIMENTAL APPROACH: In the present study, the pharmacological properties of palosuran were investigated further using a series of radioligand binding and functional bioassays. KEY RESULTS: Palosuran functioned as a 'primate-selective' UT ligand in recombinant cell membranes (monkey and human UT K(i) values of 4 +/- 1 and 5 +/- 1 nM), lacking appreciable affinity at other mammalian UT isoforms (rodent and feline K(i) values >1 microM). Paradoxically, however, palosuran lost significant (10- to 54-fold) affinity for native and recombinant human UT when radioligand binding was performed in intact cells (K(i) values of 50 +/- 3 and 276 +/- 67 nM). In accordance, palosuran also exhibited diminished activity in hUT (human urotensin-II receptor)-CHO (Chinese hamster ovary) cells (IC50 323 +/- 67 nM) and isolated arteries (K(b)>10 microM in rat aorta; K(b)>8.5 microM in cat arteries; K(b)>1.6 microM in monkey arteries; K(b) 2.2 +/- 0.6 microM in hUT transgenic mouse aorta). Relative to recombinant binding K(i) values, palosuran was subjected to a 392- to 690-fold reduction in functional activity in monkey isolated arteries. Such phenomena were peculiar to palosuran and were not apparent with an alternative chemotype, SB-657510 (2-bromo-N-[4-chloro-3-((R)-1-methyl-pyrrolidin-3-yloxy)-phenyl]-4,5-dimethoxybenzenesulphonamide HCl). CONCLUSIONS AND IMPLICATIONS: Collectively, such findings suggest that caution should be taken when interpreting data generated using palosuran. The loss of UT affinity/activity observed in intact cells and tissues cf. membranes offers a potential explanation for the disappointing clinical efficacy reported with palosuran in diabetic nephropathy patients. As such, the (patho)physiological significance of U-II in diabetic renal dysfunction remains uncertain.
Asunto(s)
Quinolinas/farmacología , Receptores Acoplados a Proteínas G/efectos de los fármacos , Urea/análogos & derivados , Urotensinas/efectos de los fármacos , Animales , Células CHO , Gatos , Línea Celular , Membrana Celular/efectos de los fármacos , Membrana Celular/metabolismo , Cricetinae , Cricetulus , Humanos , Concentración 50 Inhibidora , Macaca fascicularis , Masculino , Ratones , Quinolinas/administración & dosificación , Ensayo de Unión Radioligante , Ratas , Ratas Sprague-Dawley , Ratas Wistar , Receptores Acoplados a Proteínas G/metabolismo , Especificidad de la Especie , Urea/administración & dosificación , Urea/farmacología , Urotensinas/metabolismoRESUMEN
BACKGROUND: Diabetes is associated with increased risk of mortality as a consequence of acute myocardial infarction. This study determined whether rosiglitazone (ROSI) could reduce myocardial infarction after ischemia/reperfusion injury. METHODS AND RESULTS: Male Lewis rats were anesthetized, and the left anterior descending coronary artery was ligated for 30 minutes. After reperfusion for 24 hours, the ischemic and infarct sizes were determined. ROSI at 1 and 3 mg/kg IV reduced infarct size by 30% and 37%, respectively (P<0.01 versus vehicle). Pretreatment with ROSI (3 mg. kg(-1). d(-1) PO) for 7 days also reduced infarct size by 24% (P<0.01). ROSI also improved ischemia/reperfusion-induced myocardial contractile dysfunction. Left ventricular systolic pressure and positive and negative maximal values of the first derivative of left ventricular pressure (dP/dt) were significantly improved in ROSI-treated rats. ROSI reduced the accumulation of neutrophils and macrophages in the ischemic heart by 40% and 43%, respectively (P<0.01). Ischemia/reperfusion induced upregulation of CD11b/CD18 and downregulation of L-selectin on neutrophils and monocytes; these effects were significantly attenuated in ROSI-treated animals. Likewise, intercellular adhesion molecule-1 expression in ischemic hearts was markedly diminished by ROSI, as was the ischemia/reperfusion-stimulated upregulation of monocyte chemoattractant protein-1. CONCLUSIONS: ROSI reduced myocardial infarction and improved contractile dysfunction caused by ischemia/reperfusion injury. The cardioprotective effect of ROSI was most likely due to inhibition of the inflammatory response.
Asunto(s)
Hipoglucemiantes/uso terapéutico , Infarto del Miocardio/tratamiento farmacológico , Daño por Reperfusión Miocárdica/complicaciones , Receptores Citoplasmáticos y Nucleares/agonistas , Tiazoles/uso terapéutico , Tiazolidinedionas , Factores de Transcripción/agonistas , Animales , Antígenos CD18/metabolismo , Moléculas de Adhesión Celular/genética , Moléculas de Adhesión Celular/metabolismo , Quimiocina CCL2/biosíntesis , Quimiocina CCL2/genética , Complicaciones de la Diabetes , Hipoglucemiantes/farmacología , Antígeno de Macrófago-1/metabolismo , Macrófagos/inmunología , Masculino , Monocitos/inmunología , Contracción Miocárdica/efectos de los fármacos , Infarto del Miocardio/etiología , Infarto del Miocardio/inmunología , Infiltración Neutrófila/efectos de los fármacos , Neutrófilos/inmunología , ARN Mensajero/biosíntesis , Ratas , Ratas Endogámicas Lew , Rosiglitazona , Tiazoles/farmacologíaRESUMEN
Evidence suggests that endothelin receptor antagonists may have therapeutic potential for the chronic treatment of heart failure. In the current study, the effects of an orally active mixed endothelin-A/endothelin-B (ETA /ETB ) receptor antagonist (enrasentan) were assessed in a model of cardiac hypertrophy and dysfunction (spontaneously hypertensive stroke prone rats) maintained on a high-salt/high-fat diet. Echocardiography was used to quantify cardiac performance and left ventricular dimensions. Enrasentan (1,200 and 2,400 parts per million in the high-salt/high-fat diet) had no significant effects on body weight and systolic blood pressure. However, increases in heart rate were not observed in the enrasentan-treated groups at 12 weeks (p < 0.05). Enrasentan-treated groups exhibited significantly improved survival (90-95% vs. 30% [control rats] at 18 weeks; p < 0.001). Enrasentan treatments also increased stroke volume (at 8, 12, and 16 weeks) and cardiac index (at 8 and 16 weeks) 33-50% and 45-63%, respectively. Enrasentan treatments reduced the relative wall thickness (14-27% at 8 and 12 weeks), ratio of left ventricular mass to body weight (20% at 12 weeks), and ratio of terminal heart weight to body weight (16-23%, p < 0.05). Finally, circulating aldosterone concentration (54-57%) and proANF fragment (33%) were reduced in enrasentan-treated groups (54-57% and 33%, respectively). Mixed ETA /ETB receptor antagonism improves cardiac performance and attenuates ventricular remodeling and premature mortality in an aggressive hypertension model.
Asunto(s)
Ácidos Carboxílicos/uso terapéutico , Hipertensión/tratamiento farmacológico , Hipertrofia Ventricular Izquierda/tratamiento farmacológico , Indanos/uso terapéutico , Remodelación Ventricular/efectos de los fármacos , Aldosterona/sangre , Animales , Factor Natriurético Atrial/sangre , Presión Sanguínea/efectos de los fármacos , Presión Sanguínea/fisiología , Antagonistas de los Receptores de Endotelina , Frecuencia Cardíaca/efectos de los fármacos , Frecuencia Cardíaca/fisiología , Hipertensión/sangre , Hipertensión/mortalidad , Hipertrofia Ventricular Izquierda/sangre , Hipertrofia Ventricular Izquierda/mortalidad , Masculino , Miocardio/metabolismo , Precursores de Proteínas/sangre , Ratas , Ratas Endogámicas SHR , Tasa de Supervivencia , Remodelación Ventricular/fisiologíaRESUMEN
BACKGROUND: Numerous pathological mediators of cardiac hypertrophy (eg, neurohormones, cytokines, and stretch) have been shown to activate p38 MAPK. The purpose of the present study was to examine p38 MAPK activation and the effects of its long-term inhibition in a model of hypertensive cardiac hypertrophy/dysfunction and end-organ damage. METHODS AND RESULTS: In spontaneously hypertensive stroke-prone (SP) rats receiving a high-salt/high-fat diet (SFD), myocardial p38 MAPK was activated persistently during the development of cardiac hypertrophy and inactivated during decompensation. Long-term oral treatment of SFD-SP rats with a selective p38 MAPK inhibitor (SB239063) significantly enhanced survival over an 18-week period compared with the untreated group (100% versus 50%). Periodic echocardiographic analysis revealed a significant reduction in LV hypertrophy and dysfunction in the SB239063-treatment groups. Little or no difference in blood pressure was noted in the treatment or vehicle groups. Basal and stimulated (lipopolysaccharide) plasma tumor necrosis factor-alpha concentrations were reduced in the SB239063-treatment groups. In vitro vasoreactivity studies demonstrated a significant preservation of endothelium-dependent relaxation in animals treated with the p38 MAPK inhibitor without effects on contraction or NO-mediated vasorelaxation. Proteinuria and the incidence of stroke (53% versus 7%) were also reduced significantly in the SB239063-treated groups. CONCLUSIONS: These results demonstrate a crucial role for p38 MAPK in hypertensive cardiac hypertrophy and end-organ damage. Interrupting its function with a specific p38 MAPK inhibitor halts clinical deterioration.
Asunto(s)
Cardiomegalia/fisiopatología , Hipertensión/fisiopatología , Proteínas Quinasas Activadas por Mitógenos/metabolismo , Animales , Cardiomegalia/enzimología , Cardiomegalia/mortalidad , Modelos Animales de Enfermedad , Relación Dosis-Respuesta a Droga , Ecocardiografía , Endotelio Vascular/efectos de los fármacos , Endotelio Vascular/fisiopatología , Activación Enzimática , Corazón/efectos de los fármacos , Corazón/fisiopatología , Hemodinámica/efectos de los fármacos , Imidazoles/farmacología , Riñón/efectos de los fármacos , Riñón/fisiopatología , Lipopolisacáridos/farmacología , Masculino , Proteínas Quinasas Activadas por Mitógenos/antagonistas & inhibidores , Miocardio/metabolismo , Miocardio/patología , Fosforilación , Proteinuria/prevención & control , Proteinuria/orina , Pirimidinas/farmacología , Ratas , Ratas Endogámicas SHR , Ratas Endogámicas WKY , Accidente Cerebrovascular/patología , Accidente Cerebrovascular/prevención & control , Tasa de Supervivencia , Factores de Tiempo , Factor de Necrosis Tumoral alfa/efectos de los fármacos , Factor de Necrosis Tumoral alfa/metabolismo , Vasodilatación/efectos de los fármacos , Proteínas Quinasas p38 Activadas por MitógenosRESUMEN
BACKGROUND: It is postulated that apoptosis contributes to ischemia-reperfusion graft dysfunction after lung transplantation. The purpose of this study was to determine whether the improvement in lung function that we previously observed with the use of an endothelin-1 (ET-1) receptor antagonist after ischemia-reperfusion injury is associated with a reduction in inducible nitric oxide synthase (NOSII) expression and programmed cell death. METHODS: Left lung canine allotransplantation was performed. Harvested lung blocks were preserved with modified Eurocollins solution and stored at 4 degrees C for 18 to 20 hours. Lung allografts were tested for the expression of NOSII by immunohistochemistry, and extent of apoptosis by terminal dUTP nick end-labeling (TUNEL). Animals blindly received either an intravenous infusion of saline (control) or the ET-1 receptor antagonist (SB209670) (15 microg/kg/min). Infusion began 30 minutes pretransplantation and continued to 6 hours posttransplantation. RESULTS: Immunohistochemical analysis demonstrated significantly stronger NOSII immunostaining in the allografts of the saline control group (36.5%+/-3.6%) compared with native right lungs (6.9%+/-1.3%, p < 0.001) or the ET-receptor antagonist treatment group (9.6%+/-1.4%, p < 0.001). The TUNEL staining revealed a significantly stronger labeling in the allografts of the saline treatment control group (40.7%+/-6.2%) compared with native right lungs (5.0%+/-0.6%, p < 0.005) or the ET receptor antagonist treatment group (14.1%+/-2.8%, p < 0.01). CONCLUSIONS: We conclude that treatment of lung allografts with the ET-1 receptor antagonist SB209670 reduces the area of NOSII expression and the extent of apoptosis, factors known to contribute to the process of prolonged ischemia-reperfusion injury.
Asunto(s)
Apoptosis/efectos de los fármacos , Antagonistas de los Receptores de Endotelina , Indanos/farmacología , Trasplante de Pulmón/patología , Pulmón/irrigación sanguínea , Óxido Nítrico Sintasa/genética , Daño por Reperfusión/patología , Animales , Perros , Regulación Enzimológica de la Expresión Génica/efectos de los fármacos , Técnicas para Inmunoenzimas , Etiquetado Corte-Fin in Situ , Infusiones Intravenosas , Pulmón/patología , Óxido Nítrico Sintasa de Tipo II , Trasplante HomólogoRESUMEN
Matrix metalloproteinases (MMPs) are a family of proteolytic enzymes that degrade the extracellular matrix (ECM). The membrane-type matrix metalloproteinases (MT-MMPs) are a new family of MMPs that differ from other MMPs in that they have a transmembrane domain that anchors them to the cell surface. MT-MMPs have been shown to function as receptors and activators for other MMPs and to localize extracellular matrix proteolysis at the pericellular region. Here we report on mRNA and protein expression of the fifth human MT-MMP (MT5-MMP), a 64-kDa protein that is capable of converting pro-MMP-2 to its active form, in human kidney as well as its upregulation in diabetes. We also demonstrate upregulation of the active form of MMP-2 in kidney samples from patients with diabetes. Through immunohistochemistry, MT5-MMP expression was localized to the epithelial cells of the proximal and distal tubules, the collecting duct, and the loop of Henle. Furthermore, the tubular epithelial cells that expressed MT5-MMP were associated with tubular atrophy. Because renal tubular atrophy is a significant factor in the pathogenesis of diabetic nephropathy and renal failure and the molecular mechanisms regulating this process remain unknown, it is hypothesized that the elevated expression of MT5-MMP contributes to the activation of pro-MMP-2, which participates in the remodeling of the proximal and distal tubules as well as in the collecting duct. These results provide the first evidence of the expression of a MT-MMP in diabetes and suggest a novel role for MT5-MMP in the pathogenesis of renal tubular atrophy and end-stage renal disease.
Asunto(s)
Diabetes Mellitus Tipo 1/enzimología , Riñón/enzimología , Metaloendopeptidasas/metabolismo , Adolescente , Adulto , Anciano , Diabetes Mellitus Tipo 1/patología , Femenino , Humanos , Immunoblotting , Inmunohistoquímica , Riñón/citología , Riñón/metabolismo , Masculino , Metaloproteinasa 2 de la Matriz/metabolismo , Metaloproteinasas de la Matriz Asociadas a la Membrana , Metaloendopeptidasas/genética , Persona de Mediana Edad , Reacción en Cadena de la Polimerasa , ARN Mensajero/genética , ARN Mensajero/metabolismo , Extractos de Tejidos/metabolismo , Regulación hacia ArribaAsunto(s)
Benzodiazepinas , Caspasas/metabolismo , Fibrinolíticos/farmacología , Corazón/efectos de los fármacos , Miocardio/enzimología , Oligopéptidos/farmacología , Piperidinas , Inhibidores de Agregación Plaquetaria/farmacología , Complejo GPIIb-IIIa de Glicoproteína Plaquetaria/antagonistas & inhibidores , Animales , Caspasa 3 , Células Cultivadas/efectos de los fármacos , Cumarinas/metabolismo , Activación Enzimática , Fibrinolíticos/toxicidad , Miocardio/citología , Oligopéptidos/metabolismo , Agregación Plaquetaria/efectos de los fármacos , Inhibidores de Agregación Plaquetaria/toxicidad , Ratas , Ratas Sprague-DawleyRESUMEN
Aberrant gene expression is a fundamental cause of many disease-associated pathophysiologies. The pharmacological modulation of transcription factor activity therefore represents an attractive therapeutic approach to such disorders. With the exception of nuclear receptors, which are the direct targets of pharmaceuticals, other known classes of transcription factors are largely regulated indirectly by drugs that impact upon those signal transduction cascades that alter transcription factor phosphorylation and dephosphorylation and/or nuclear import. However, recent advances in drug discovery technologies now enable high-throughput screens that can identify molecules that act directly at the level of transcription factor complexes.
Asunto(s)
Regulación de la Expresión Génica/efectos de los fármacos , Factores de Transcripción/efectos de los fármacos , Animales , Regulación de la Expresión Génica/genética , Humanos , Factores de Transcripción/química , Factores de Transcripción/genéticaRESUMEN
OBJECTIVE: Eprosartan is a selective angiotensin II type I receptor antagonist approved for the treatment of hypertension. In the present studies, eprosartan's ability to provide end-organ protection was evaluated in a model of cardiomyopathy and renal failure in stroke-prone rats (SP). METHODS: SP were fed a high fat (24.5% in food) and high salt (1% in water) diet (SFD). Eprosartan (60 mg/kg/day) or vehicle (saline control) (n = 25/group) was administered by intraperitoneally-implanted minipumps to these SP on the SFD for 12 weeks. Normal diet fed SP and WKY rats (n = 25/group) were also included for comparison (i.e. served as normal controls). Mortality, hemodynamics, and both renal and cardiac function and histopathology were monitored in all treatment groups. RESULTS: Eprosartan decreased the severely elevated arterial pressure (-12%; P < 0.05) produced by SFD but did not affect heart rate. Vehicle-treated SP-SFD control rats exhibited significant weight loss (-13%; P < 0.05) and marked mortality (50% by week 6 and 95% by week 9; P < 0.01). Eprosartan-treated SP-SFD rats maintained normal weight, and exhibited zero mortality at week 12 and beyond. Eprosartan prevented the increased urinary protein excretion (P < 0.05) that was observed in vehicle-treated SP-SFD rats. Echocardiographic (i.e. 2-D guided M-mode) evaluation indicated that SP-SFD vehicle control rats exhibited increased septal (+22.2%) and posterior left ventricular wall (+30.0%) thickness, and decreased left ventricular chamber diameter (-15.9%), chamber volume (-32.7%), stroke volume (-48.7%) and ejection fraction (-22.3%), and a remarkable decrease in cardiac output (-59.3%) compared to controls (all P < 0.05). These same parameters in eprosartan-treated SP-SFD rats were normal and differed markedly and consistently from vehicle-treated SP-SFD rats (i.e. treatment prevented pathology; all P < 0.05). Cardiac-gated MRI data confirmed the ability of eprosartan to prevent cardiac pathology/remodeling (P < 0.05). Histopathological analysis of hearts and kidneys indicated that eprosartan treatment significantly reduced end-organ damage (P < 0.01) and provided corroborative evidence that eprosartan reduced remodeling of these organs. Vehicle-treated SP-SFD rats exhibited a 40% increase in the plasma level of pro-atrial natiuretic factor that was reduced to normal by eprosartan (P < 0.05). CONCLUSION: These data demonstrate that eprosartan, at a clinically relevant dose, provides significant end-organ protection in the severely hypertensive stroke-prone rat. It preserves cardiac and renal structural integrity, reduces cardiac hypertrophy and indices of heart failure, maintains normal function of the heart and kidneys, and eliminates premature mortality due to hypertension-induced end-organ failure.
Asunto(s)
Acrilatos/uso terapéutico , Antihipertensivos/uso terapéutico , Cardiomegalia/tratamiento farmacológico , Hipertensión/tratamiento farmacológico , Imidazoles/uso terapéutico , Tiofenos , Animales , Factor Natriurético Atrial/sangre , Presión Sanguínea/efectos de los fármacos , Peso Corporal/efectos de los fármacos , Frecuencia Cardíaca/efectos de los fármacos , Riñón/patología , Imagen por Resonancia Magnética , Masculino , Miocardio/patología , Natriuresis/fisiología , Tamaño de los Órganos/efectos de los fármacos , Fragmentos de Péptidos/sangre , Precursores de Proteínas/sangre , Proteinuria/prevención & control , Ratas , Ratas Endogámicas SHR , Accidente Cerebrovascular/prevención & control , Tasa de Supervivencia , Remodelación Ventricular/efectos de los fármacosRESUMEN
This study investigated whether idoxifene, a selective estrogen receptor modulator (SERM), exerted protective effects against ischemia-reperfusion-induced shock. Ovariectomized rats were treated with vehicle, idoxifene, or 17beta-estradiol for 4 days. Rats were subjected to splanchnic artery occlusion (SAO) followed by reperfusion (SOA/R). In vehicle-treated rats, SAO/R resulted in hypotension, hemoconcentration, increased plasma tumor necrosis factor (TNF)-alpha levels, intestinal neutrophil accumulation, and endothelial dysfunction. 17beta-Estradiol treatment increased plasma estradiol concentration and reduced SAO/R-induced tissue injury. Idoxifene treatment had no effect on plasma estradiol concentration but reduced SAO/R-induced hemoconcentration (+8.8 +/- 1.3 vs. +14 +/- 1.3% in the vehicle group, P < 0.01), TNF-alpha production (98 +/- 3.2 vs. 214 +/- 13 pg/ml, P < 0.01), and neutrophil accumulation (0.025 +/- 0.005 vs. 0.047 +/- 0.005 U/g protein, P < 0.01). It also improved endothelial function, prolonged survival time (172 +/- 3.5 vs. 147 +/- 8 min, P < 0.01), and increased survival rate (69 vs. 23%, P < 0.01). Moreover, treatment with 17beta-estradiol or idoxifene in vivo reduced TNF-alpha-induced endothelial dysfunction in vitro. Taken together, these results demonstrated that idoxifene exerted estrogen-like, endothelial-protective, and antishock effects in ovariectomized rats, suggesting that SERMs have therapeutic potential in tissue injury resulting from ischemia-reperfusion.
Asunto(s)
Antagonistas de Estrógenos/farmacología , Receptores de Estrógenos/metabolismo , Daño por Reperfusión/metabolismo , Choque/metabolismo , Tamoxifeno/análogos & derivados , Tamoxifeno/farmacología , Animales , Líquidos Corporales/metabolismo , Endotelio Vascular/efectos de los fármacos , Endotelio Vascular/inmunología , Estradiol/sangre , Estradiol/farmacología , Femenino , Íleon/irrigación sanguínea , Isquemia/inmunología , Isquemia/metabolismo , Neutrófilos/citología , Neutrófilos/inmunología , Ovariectomía , Ratas , Ratas Sprague-Dawley , Daño por Reperfusión/tratamiento farmacológico , Moduladores Selectivos de los Receptores de Estrógeno/farmacología , Choque/tratamiento farmacológico , Circulación Esplácnica/efectos de los fármacos , Circulación Esplácnica/fisiología , Factor de Necrosis Tumoral alfa/inmunología , Factor de Necrosis Tumoral alfa/metabolismo , Vasodilatación/efectos de los fármacos , Vasodilatación/fisiologíaRESUMEN
The peptide human urotensin-II (hUT-II) and its receptor have recently been cloned. The vascular function of this peptide in humans, however, has yet to be determined. Vasoconstrictor and vasodilator responses to hUT-II were investigated in human small muscular pulmonary arteries [approximately 70 microm internal diameter (ID)] and human abdominal resistance arteries (approximately 200 microm ID). Vasodilator responses were investigated in endothelin-1 (3 nM) precontracted vessels and, in the small pulmonary vessels, compared with the known vasodilators adrenomedullin, sodium nitroprusside, and acetylcholine. In human small pulmonary arteries, hUT-II did not induce vasoconstriction but was a potent vasodilator [-log M concentration causing 50% of the maximum vasodilator effect (pIC(50)) 10.4 +/- 0.5; percentage of reduction in tone (E(max)) 81 +/- 8% (vs. 23 +/- 11% in time controls), n = 5]. The order of potency for vasodilation was human urotensin-II = adrenomedullin (pIC(50) 10.1 +/- 0.4, n = 6) > sodium nitroprusside (pIC(50) 7.4 +/- 0.2, n = 6) = acetylcholine (pIC(50) 6.8 +/- 0.3, n = 6). In human abdominal arteries, hUT-II did not induce vasoconstriction but was a potent vasodilator [pIC(50) 10.3 +/- 0.7; E(max) 96 +/- 8% (vs. 43 +/- 16% in time controls), n = 4]. This is the first report that hUT-II is a potent vasodilator but not a vasoconstrictor of human small pulmonary arteries and systemic resistance arteries.
Asunto(s)
Arteria Pulmonar/efectos de los fármacos , Urotensinas/farmacología , Resistencia Vascular/efectos de los fármacos , Vasodilatación/efectos de los fármacos , Abdomen/irrigación sanguínea , Acetilcolina/farmacología , Adrenomedulina , Adulto , Anciano , Endotelina-1/farmacología , Humanos , Técnicas In Vitro , Masculino , Persona de Mediana Edad , Nitroprusiato/farmacología , Péptidos/farmacología , Arteria Pulmonar/fisiología , Resistencia Vascular/fisiología , Vasoconstricción/efectos de los fármacos , Vasodilatadores/farmacologíaRESUMEN
Mitogen-activated protein kinases (MAPKs) are involved in many cellular processes. The stress-activated MAPK, p38, has been linked to inflammatory cytokine production and cell death following cellular stress. Here, we demonstrate focal ischemic stroke-induced p38 enzyme activation (i.e., phosphorylation) in the brain. The second generation p38 MAPK inhibitor SB 239063 was identified to exhibit increased kinase selectivity and improved cellular and in vivo activity profiles, and thus was selected for evaluation in two rat models of permanent focal ischemic stroke. SB 239063 was administered orally pre- and post-stroke and intravenously post-stroke. Plasma concentration levels were achieved in excess of those that effectively inhibit p38 activity. In both moderate and severe stroke, SB 239063 reduced infarct size by 28-41%, and neurological deficits by 25-35%. In addition, neuroprotective plasma concentrations of SB 239063 that reduced p38 activity following stroke also reduced the stroke-induced expression of IL-1beta and TNFalpha (i.e., cytokines known to contribute to stroke-induced brain injury). SB 239063 also provided direct protection of cultured brain tissue to in vitro ischemia. This robust SB 239063-induced neuroprotection emphasizes a significant opportunity for targeting MAPK pathways in ischemic stroke injury, and also suggests that p38 inhibition be evaluated for protective effects in other experimental models of nervous system injury and neurodegeneration.
Asunto(s)
Isquemia Encefálica/tratamiento farmacológico , Imidazoles/uso terapéutico , Proteínas Quinasas Activadas por Mitógenos/antagonistas & inhibidores , Fármacos Neuroprotectores/uso terapéutico , Pirimidinas/uso terapéutico , Animales , Isquemia Encefálica/metabolismo , Células Cultivadas , Corteza Cerebral/enzimología , Corteza Cerebral/metabolismo , Corteza Cerebral/patología , Humanos , Imidazoles/administración & dosificación , Imidazoles/farmacocinética , Interleucina-1/metabolismo , Proteínas Quinasas Activadas por Mitógenos/metabolismo , Fármacos Neuroprotectores/administración & dosificación , Fármacos Neuroprotectores/farmacocinética , Pirimidinas/administración & dosificación , Pirimidinas/farmacocinética , Ratas , Factor de Necrosis Tumoral alfa/metabolismo , Proteínas Quinasas p38 Activadas por MitógenosRESUMEN
Myocardial infarction (MI), leads to cardiac remodeling, thinning of the ventricle wall, ventricular dilation, and heart failure, and is a leading cause of death. Interactions between the contractile elements of the cardiac myocytes and the extracellular matrix (ECM) help maintain myocyte alignment required for the structural and functional integrity of the heart. Following MI, reorganization of the ECM and the myocytes occurs, contributing to loss of heart function. In certain pathological circumstances, the ECM is modulated such that the structure of the tissue becomes damaged. The matrix metalloproteinases (MMPs) are a family of enzymes that degrade molecules of the ECM. The present experiments were performed to define the time-course, isozyme subtypes, and cellular source of increased MMP expression that occurs following MI in an experimental rabbit model. Heart tissue samples from infarcted and sham animals were analyzed over a time-course of 1-14 days. By zymography, it was demonstrated that, unlike the sham controls, MMP-9 expression was induced within 24 hours following MI. MMP-3 expression, also absent in sham controls, was induced 2 days after MI. MMP-2 expression was detected in both the sham and infarcted samples and was modestly up-regulated following MI. Tissue inhibitor of metalloproteinase-1 (TIMP-1) expression was evaluated and shown to be down-regulated following MI, inverse of MMP-9 and MMP-3 expression. Further, MMP-9 and MMP-3 expression was detected by immunohistochemistry in myocytes within the infarct. Additional studies were conducted in which cultured rat cardiac myocytes were exposed to a hypoxic environment (2% O2) for 24 hours and the media analyzed for MMP expression. MMP-9 and MMP-3 were induced following exposure to hypoxia. It is speculated that the net increase in proteolytic activity by myocytes is a contributing factor leading to myocyte misalignment and slippage. Additional studies with a MMP inhibitor would elucidate this hypothesis.
Asunto(s)
Metaloproteinasas de la Matriz/biosíntesis , Infarto del Miocardio/enzimología , Miocardio/metabolismo , Animales , Western Blotting , Hipoxia de la Célula , Electroforesis en Gel de Poliacrilamida , Femenino , Regulación Enzimológica de la Expresión Génica , Inmunohistoquímica , Metaloproteinasa 3 de la Matriz/biosíntesis , Metaloproteinasa 9 de la Matriz/biosíntesis , Metaloproteinasas de la Matriz/genética , Infarto del Miocardio/patología , Isquemia Miocárdica/metabolismo , Miocardio/citología , Proteínas/química , Conejos , Inhibidor Tisular de Metaloproteinasa-1/metabolismoRESUMEN
The stress-activated mitogen-activated protein kinase (MAPK) p38 has been linked to the production of inflammatory cytokines/mediators/inflammation and death/apoptosis following cell stress. In these studies, a second-generation p38 MAPK inhibitor, SB 239063 (IC(50) = 44 nM), was found to exhibit improved kinase selectivity and increased cellular (3-fold) and in vivo (3- to 10-fold) activity over first-generation inhibitors. Oral SB 239063 inhibited lipopolysaccharide-induced plasma tumor necrosis factor production (IC(50) = 2.6 mg/kg) and reduced adjuvant-induced arthritis (51% at 10 mg/kg) in rats. SB 239063 reduced infarct volume (48%) and neurological deficits (42%) when administered orally (15 mg/kg, b.i.d.) before moderate stroke. Intravenous SB 239063 exhibited a clearance of 34 ml/min/kg, a volume of distribution of 3 l/kg, and a plasma half-life of 75 min. An i.v. dosing regimen that provided effective plasma concentrations of 0.38, 0.75, or 1.5 microg/ml (i.e., begun 15 min poststroke and continuing over the initial 6-h p38 activation period) was used. Significant and dose-proportional brain penetration of SB 239063 was demonstrated during these infusion periods. In both moderate and severe stroke, intravenous SB 239063 produced a maximum reduction of infarct size by 41 and 27% and neurological deficits by 35 and 33%, respectively. No effects of the drug were observed on cerebral perfusion, hemodynamics, or body temperature. Direct neuroprotective effects from oxygen and glucose deprivation were also demonstrated in organotypic cultures of rat brain tissue. This robust in vitro and in vivo SB 239063-induced neuroprotection emphasizes the potential role of MAPK pathways in ischemic stroke and also suggests that p38 inhibition warrants further study, including protection in other models of nervous system injury and neurodegeneration.
Asunto(s)
Encéfalo/patología , Inhibidores Enzimáticos/uso terapéutico , Imidazoles/uso terapéutico , Ataque Isquémico Transitorio/tratamiento farmacológico , Ataque Isquémico Transitorio/patología , Proteínas Quinasas Activadas por Mitógenos/antagonistas & inhibidores , Fármacos Neuroprotectores/uso terapéutico , Pirimidinas/uso terapéutico , Animales , Antiinflamatorios no Esteroideos/uso terapéutico , Temperatura Corporal/efectos de los fármacos , Circulación Cerebrovascular/efectos de los fármacos , Hemodinámica/efectos de los fármacos , Hipocampo/patología , Inflamación/patología , Inflamación/prevención & control , Técnicas de Cultivo de Órganos , Piridinas/uso terapéutico , Ratas , Ratas Endogámicas Lew , Ratas Endogámicas SHR , Proteínas Quinasas p38 Activadas por MitógenosRESUMEN
1. Urotensin-II (U-II) and its G-protein-coupled receptor, GPR14, are expressed within mammalian cardiac and peripheral vascular tissue and, as such, may regulate mammalian cardiovascular function. The present study details the vasoconstrictor profile of this cyclic undecapeptide in different vascular tissues isolated from a diverse range of mammalian species (rats, mice, dogs, pigs, marmosets and cynomolgus monkeys). 2. The vasoconstrictor activity of human U-II was dependent upon the anatomical origin of the vessel studied and the species from which it was isolated. In the rat, constrictor responses were most pronounced in thoracic aortae and carotid arteries: -log[EC(50)]s 9.09+/-0.19 and 8.84+/-0.21, R(max)s 143+/-21 and 67+/-26% 60 mM KCl, respectively (compared, for example, to -log[EC(50)] 7.90+/-0.11 and R(max) 142+/-12% 60 mM KCl for endothelin-1 [ET-1] in thoracic aortae). Responses were, however, absent in mice aortae (-log[EC(50)] <6.50). These findings were further contrasted by the observation that U-II was a 'coronary-selective' spasmogen in the dog (-log[EC(50)] 9.46+/-0.11, R(max) 109+/-23% 60 mM KCl in LCX coronary artery), yet exhibited a broad spectrum of vasoconstrictor activity in arterial tissue from Old World monkeys (-log[EC(50)]s range from 8.96+/-0.15 to 9.92+/-0.13, R(max)s from 43+/-16 to 527+/-135% 60 mM KCl). Interestingly, significant differences in reproducibility and vasoconstrictor efficacy were seen in tissue from pigs and New World primates (vessels which responded to noradrenaline, phenylephrine, KCl or ET-1 consistently). 3. Thus, human U-II is a potent, efficacious vasoconstrictor of a variety of mammalian vascular tissues. Although significant species/anatomical variations exist, the data support the hypothesis that U-II influences the physiological regulation of mammalian cardiovascular function.
Asunto(s)
Vasos Sanguíneos/efectos de los fármacos , Urotensinas/farmacología , Vasoconstricción/efectos de los fármacos , Vasoconstrictores/farmacología , Animales , Aorta Abdominal/efectos de los fármacos , Aorta Abdominal/fisiología , Aorta Torácica/efectos de los fármacos , Aorta Torácica/fisiología , Arterias/efectos de los fármacos , Arterias/fisiología , Vasos Sanguíneos/fisiología , Callithrix , Arterias Carótidas/efectos de los fármacos , Arterias Carótidas/fisiología , Vasos Coronarios/efectos de los fármacos , Vasos Coronarios/fisiología , Perros , Relación Dosis-Respuesta a Droga , Arteria Femoral/efectos de los fármacos , Arteria Femoral/fisiología , Humanos , Técnicas In Vitro , Macaca fascicularis , Ratones , Músculo Liso Vascular/efectos de los fármacos , Músculo Liso Vascular/fisiología , Arteria Pulmonar/efectos de los fármacos , Arteria Pulmonar/fisiología , Venas Pulmonares/efectos de los fármacos , Venas Pulmonares/fisiología , Ratas , Porcinos , Tráquea/efectos de los fármacos , Tráquea/fisiología , Venas/efectos de los fármacos , Venas/fisiologíaRESUMEN
BACKGROUND: Idoxifene (ID) is a tissue-selective estrogen receptor modulator (SERM). The pharmacological profile of ID in animal studies suggests that it behaves like an estrogen receptor (ER) agonist in bone and lipid metabolism while having negligible ER activity on the reproductive system. It is unknown whether ID retains the vascular protective effects of estrogen. METHODS AND RESULTS: In cultured vascular smooth muscle cells (VSMCs), ID inhibited platelet-derived growth factor-induced DNA synthesis and mitogenesis with IC(50) values of 20.4 and 27.5 nmol/L, respectively. Treatment with ID resulted in S-phase cell cycle arrest in serum-stimulated VSMCs. ID 1 to 100 nmol/L significantly protected endothelial cells from tumor necrosis factor-alpha (TNF-alpha)-induced apoptosis in vitro. Virgin Sprague-Dawley rats ovariectomized 1 week before the study were treated with ID (1 mg x kg(-1) x d(-1)) or vehicle by gavage for 3 days before balloon denudation in carotid artery. The SMC proliferation in injured vessels was determined by immunostaining for proliferating cell nuclear antigen (PCNA). The number of PCNA-positive SMCs was reduced by 69%, 82%, and 86% in the media at days 1, 3 and 7, respectively, and by 78% in the neointima at day 7 after injury in ID- versus vehicle-treated group (P:<0.01). ID significantly enhanced reendothelialization in the injured carotid arteries as determined by Evans blue stain and immunohistochemical analysis for von Willebrand factor. In the former assay, the reendothelialized area in injured vessels was 43% in ID-treated group versus 24% in the vehicle group (P:<0.05); in the latter assay, the numbers of von Willebrand factor-positive cells per cross section increased from 24. 8 (vehicle) to 60.5 (ID) (P:<0.01) at day 14 after injury. In addition, the production of nitric oxide from excised carotid arteries was significantly higher in ID-treated than the vehicle group (8.5 versus 2.7 nmol/g, P:<0.01). Finally, ID treatment reduced neointimal area and the ratio of intima to media by 45% and 40%, respectively (P:<0.01), at day 14 after balloon angioplasty. CONCLUSIONS: The results indicate that ID beneficially modulates the balloon denudation-induced vascular injury response. Inhibition of VSMC proliferation and acceleration of endothelial recovery likely mediate this protective effect of ID.
Asunto(s)
Endotelio Vascular/lesiones , Endotelio Vascular/patología , Moduladores de los Receptores de Estrógeno/farmacología , Músculo Liso Vascular/efectos de los fármacos , Tamoxifeno/análogos & derivados , Tamoxifeno/farmacología , Heridas no Penetrantes/patología , Adulto , Animales , Arterias Carótidas/efectos de los fármacos , Arterias Carótidas/metabolismo , Arterias Carótidas/cirugía , Estenosis Carotídea/metabolismo , Estenosis Carotídea/patología , Estenosis Carotídea/prevención & control , Recuento de Células , Ciclo Celular/efectos de los fármacos , División Celular/efectos de los fármacos , Células Cultivadas , Modelos Animales de Enfermedad , Endotelio Vascular/efectos de los fármacos , Femenino , Humanos , Inmunohistoquímica , Músculo Liso Vascular/citología , Músculo Liso Vascular/metabolismo , Ovariectomía , Antígeno Nuclear de Célula en Proliferación/metabolismo , Ratas , Ratas Sprague-Dawley , Túnica Íntima/efectos de los fármacos , Túnica Íntima/patología , Factor de von Willebrand/metabolismoRESUMEN
The stimulatory mAb F11 binds two platelet membrane proteins of 32 and 35 kDa and causes activation of platelets when cross-linked with the FcgammaRII receptor. We used bioinformatics to identify expressed sequence tags from libraries of cytokine-stimulated human endothelial cell (EC) cDNAs. The protein sequence deduced from full-length F11 cDNA was identical to partial sequences of peptides derived from affinity-purified platelet F11 antigen. F11 mRNA is expressed in human EC, macrophages, and a variety of non-hematopoietic vascular tissues. Expression of F11 mRNA is modulated by cytokines in EC and is up-regulated by oxidized low-density lipoprotein in human macrophages. The F11 receptor contains two immunoglobulin-like domains in its 236-amino-acid-long extracellular region, and has identity to the recently described junctional adhesion molecule. The data indicate that the F11 antigen is a novel receptor or cell adhesion molecule belonging to the immunoglobulin superfamily.
Asunto(s)
Moléculas de Adhesión Celular/metabolismo , Endotelio Vascular/metabolismo , Macrófagos/metabolismo , Receptores de Superficie Celular/metabolismo , Secuencia de Aminoácidos , Secuencia de Bases , Moléculas de Adhesión Celular/efectos de los fármacos , Moléculas de Adhesión Celular/genética , Células Cultivadas , Citocinas/farmacología , Endotelio Vascular/citología , Endotelio Vascular/efectos de los fármacos , Etiquetas de Secuencia Expresada , Regulación de la Expresión Génica , Humanos , Inmunoglobulinas/genética , Inflamación/metabolismo , Moléculas de Adhesión de Unión , Lipopolisacáridos/farmacología , Lipoproteínas LDL/metabolismo , Lipoproteínas LDL/farmacología , Datos de Secuencia Molecular , Receptores de Superficie Celular/efectos de los fármacos , Receptores de Superficie Celular/genética , Alineación de Secuencia , Homología de Secuencia de AminoácidoRESUMEN
Endothelin-1 (ET-1) has been suggested to have a potential function as an inflammatory mediator. The study reported here assessed the putative inflammatory/nociceptive actions of the ET isopeptides using endothelin-B (ET(B))-receptor knockout (KO) mice and ET(A)- (SB 234551) and ET(B)- (A192621) selective antagonists. Phenylbenzoquinone (PBQ)-induced algesia was evident in the wild-type (WT) ET(B) (+/+) mice, attenuated by 80% in the heterozygous ET(B) (+/-) mice, and absent in the ET(B) (-/-) homozygotes. This was reproduced pharmacologically in WT ET(B) (+/+) mice where the algesic effect of PBQ was inhibited 74% by A192621, but unaffected by SB 234551 (both at 25 mg/kg p.o.). Similar observations were made in a model of cutaneous inflammation: ET(B) (+/+) mice had a marked inflammatory response to topical arachidonic acid, ET(B) (+/-) and ET(B) (-/-) mice had significantly reduced edema responses (37% and 65% inhibition). Neutrophil infiltration was reduced in the ET(B) (+/-) and ET(B) (-/-) mice (51% and 65% reduction, respectively). Topical administration of A192621 (500 microg/ear) inhibited arachidonic acid-induced swelling (39%) in WT ET(B) (+/+) mice. Collectively, these results support a role for the ET(B)-receptor in the mediation of inflammatory pain and cutaneous inflammatory responses. As such, the development of ET(B)-receptor-selective antagonists may be of therapeutic utility in the treatment of inflammatory disorders.
Asunto(s)
Dermatitis/etiología , Dolor/etiología , Receptores de Endotelina/fisiología , Animales , Ácido Araquidónico/farmacología , Benzoquinonas/farmacología , Ratones , Ratones Endogámicos BALB C , Ratones Noqueados , Receptor de Endotelina B , Receptores de Endotelina/genéticaRESUMEN
The observation that the novel G-protein-coupled receptor (GPCR) GPR14 and its cognate ligand, urotensin-II (U-II), are expressed within the mammalian vasculature raises the possibility that they may influence cardiohemodynamic homeostasis. To this end, this study examined the vasoactive properties of U-II in rodents, dogs and primates. In vitro, human U-II was a sustained vasoconstrictor with a potency (pD2s < or = 9) approximately an order of magnitude greater than that seen with endothelin-1 (ET-1), making it one of the most, if not the most, potent mammalian vasoconstrictor identified to date. However, in vitro responses exhibited significant anatomical and/or species-dependency, that is, human U-II was a selective 'aorto-coronary' vasoconstrictor in rats and dogs, inactive in mice and contracted all primate arteries studied. In vivo, this peptide evoked a complex, dose-dependent hemodynamic response in the anesthetized primate, culminating in severe myocardial depression and fatal circulatory collapse. As such, U-II may represent a novel neurohumoral regulator of mammalian cardiovascular physiology and pathology in particular disorders characterized by aberrant vascular smooth muscle and/or myocardial function.
Asunto(s)
Urotensinas/farmacología , Vasoconstricción/efectos de los fármacos , Animales , Perros , Relación Dosis-Respuesta a Droga , Hemodinámica/efectos de los fármacos , Humanos , Técnicas In Vitro , Macaca fascicularis , Masculino , Ratones , Ratones Endogámicos C57BL , Ratas , Ratas Sprague-Dawley , Especificidad de la EspecieRESUMEN
Increased levels of endothelin (ET) have been demonstrated in the ischemic brain, and ET receptor antagonism has been shown to improve outcome in cerebral ischemia. However, no previous work has been carried out evaluating the role of ET and its antagonism in brain trauma as compared to experimental stroke. In this study, we evaluated changes in brain ET levels following closed head injury (CHI) and the effects of SB 234551, an endothelin-A- (ET(A)) selective antagonist, and SB 209670, a mixed endothelin-A- and -B- (ET(A)/ET(B)) antagonist, on outcome in CHI and focal stroke. Male Sabra rats were subjected to CHI (weight drop model). Male Sprague Dawley rats were subjected to focal stroke (intraluminal suture model). Motor function(s) were assessed and immunoreactive ET (irET) and the degree of cerebral edema were measured for 24 h after CHI. Brain swelling (edema), neurological deficits and forebrain infarct volumes were measured 24 h after focal stroke. Antagonists (total doses of 7.5, 15, 30 or 60 mg/kg) were administered intravenously for 6-24 h (beginning 15 min after injury). Control rats were infused with vehicle. CHI resulted in increased ET levels in the directly contused hemisphere at 12 and 24 h. In addition, SB 234551 significantly reduced neurological deficits (decreased 30%) and brain edema (decreased 40%) following CHI (p < 0.05 at 60 mg/kg dose). SB 209670 had no effects on CHI outcome. Focal stroke studies yielded similar results. SB 234551 reduced focal stroke-induced neurological deficits by 50%, brain swelling by 54% and the degree of infarction by 36% (p < 0.05 at 30 mg/kg). SB 209670 did not provide any neuroprotection in focal stroke. These data indicate that ET plays a significant role in the pathophysiology of CHI, and that selectively targeting ET(A)-receptors similarly in both CHI and stroke might be a therapeutic opportunity.