RESUMEN

We report the first observation of the charmless hyperonic B decay, B0-->pLambda(pi)(-), using a 78 fb(-1) data sample recorded on the Upsilon(4S) resonance with the Belle detector at KEKB. The measured branching fraction is B(B0-->pLambda(pi)(-))=(3.97(+1.00)(-0.80)+/-0.56)x10(-6). Searches for B0-->pLambda(K)- and pSigma(0)pi(-) yield no significant signals and we set 90% confidence-level upper limits of B(B0-->pLambda(K)-)<8.2x10(-7) and B(B0-->pSigma(0)pi(-))<3.8x10(-6).

RESUMEN

Two experiments were conducted to investigate the relation between resistance to change and preference. Four pigeons responded in concurrent chained schedules in which variable-interval (VI) 60-s schedules were arranged in the initial link. In Experiment 1, VI and fixed-interval (FI) schedules of equal mean reinforcement rates were arranged in the terminal link. Response rates were higher in the initial link leading to VI terminal link. Under the prefeeding test, the initial-link response rates leading to VI terminal link were more resistant to change than were those leading to FI terminal link, but under the extinction test there were no consistent differences between the two initial-link response rates. In Experiment 2, FI value of the terminal link was manipulated so that pigeons maintained approximately equal responding in the initial link. The two initial-link response rates showed equal resistance to change under the prefeeding and extinction tests. Thus, the data suggest that although the use of extinction as a manipulation to study resistance to change is questioned, resistance to change and preference are different measures of a single object.

Asunto(s)

Conducta Animal , Columbidae , Esquema de Refuerzo , Animales , Condicionamiento Operante/fisiología , Tiempo de Reacción

RESUMEN

The Bcl-2/CED-9 family of proteins, which includes both antiapoptotic and proapoptotic members, plays key regulating roles in programmed cell death. We report here the identification and characterization of Drob-1, the first Drosophila member of the Bcl-2/CED-9 family to be isolated. Drob-1 contains four conserved Bcl-2 homology domains (BH1, BH2, BH3, and BH4) and a C-terminal hydrophobic domain. Ectopic expression of Drob-1 in the developing Drosophila eye resulted in a rough-eye phenotype. Furthermore, when overexpressed in Drosophila S2 cells, Drob-1 induced apoptosis accompanied by elevated caspase activity. This Drob-1-induced cell death, however, could not be antagonized by baculovirus p35, a broad-spectrum caspase inhibitor. Drob-1 was localized to the intracytoplasmic membranes, predominantly to the mitochondrial membranes, and a mutant Drob-1 lacking the hydrophobic C terminus lost both its mitochondrial localization and its proapoptotic activity. These results suggest that Drob-1 promotes cell death by inducing both caspase-dependent and -independent pathways at the mitochondria. Our identification of Drob-1 and further genetic analysis should provide increased understanding of the universal mechanisms by which the Bcl-2/CED-9 family members and other related proteins regulate apoptosis.

Asunto(s)

Proteínas de Caenorhabditis elegans , Proteínas de Drosophila , Drosophila/genética , Proteínas de Insectos/genética , Proteínas de la Membrana/genética , Proteínas Proto-Oncogénicas/genética , Secuencia de Aminoácidos , Animales , Animales Modificados Genéticamente , Proteínas Reguladoras de la Apoptosis , Northern Blotting , Células COS , Caspasas/metabolismo , Caspasas/fisiología , Muerte Celular/fisiología , ADN Complementario/química , ADN Complementario/genética , Drosophila/embriología , Drosophila/crecimiento & desarrollo , Activación Enzimática , Ojo/embriología , Ojo/crecimiento & desarrollo , Ojo/metabolismo , Regulación del Desarrollo de la Expresión Génica , Proteínas del Helminto/genética , Hibridación in Situ , Proteínas de Insectos/fisiología , Membranas Intracelulares/química , Proteínas de la Membrana/fisiología , Microscopía Confocal , Mitocondrias/química , Datos de Secuencia Molecular , Fenotipo , Proteínas Proto-Oncogénicas/fisiología , Proteínas Proto-Oncogénicas c-bcl-2/genética , ARN Mensajero/genética , ARN Mensajero/metabolismo , Alineación de Secuencia , Análisis de Secuencia de ADN , Homología de Secuencia de Aminoácido

RESUMEN

BACKGROUND: Vascular endothelial growth factor (VEGF) is an important angiogenic factor reported to induce migration and proliferation of endothelial cells, enhance vascular permeability, and modulate thrombogenicity. VEGF expression in cultured cells (smooth muscle cells, macrophages, endothelial cells) is controlled by growth factors and cytokines. Hence, the question arises of whether VEGF could play a role in atherogenesis. METHODS AND RESULTS: Frozen sections from 38 coronary artery segments were studied. The specimens were characterized as normal with diffuse intimal thickening, early atherosclerosis with hypercellularity, and advanced atherosclerosis (atheromatous plaques, fibrous plaques, and totally occlusive lesions). VEGF expression as well as the expression of 2 VEGF receptors, flt-1 and Flk-1, were studied with immunohistochemical techniques in these samples at the different stages of human coronary atherosclerosis progression. The expression of VEGF mRNA was also studied with reverse transcription-polymerase chain reaction. Normal arterial segments showed no substantial VEGF expression. Hypercellular and atheromatous lesions showed distinct VEGF positivity of activated endothelial cells, macrophages, and partially differentiated smooth muscle cells. VEGF positivity was also detected in endothelial cells of intraplaque microvessels within advanced lesions. In totally occlusive lesions with extensive neovascularization, intense immunostaining for VEGF was observed in accumulated macrophages and endothelial cells of the microvessels. Furthermore, VEGF mRNA expression was detected in atherosclerotic coronary segments but not in normal coronary segments. The immunostainings for flt-1 and Flk-1 were detected in aggregating macrophages in atherosclerotic lesions and also in endothelial cells of the microvessels in totally occlusive lesions. CONCLUSIONS: These results demonstrate distinct expression of VEGF and its receptors (flt-1 and Flk-1) in atherosclerotic lesions in human coronary arteries. Considering the multipotent actions of VEGF documented experimentally in vivo and in vitro, our findings suggest that VEGF may have some role in the progression of human coronary atherosclerosis, as well as in recanalization processes in obstructive coronary diseases.

Asunto(s)

Enfermedad de la Arteria Coronaria/metabolismo , Vasos Coronarios/química , Factores de Crecimiento Endotelial/análisis , Linfocinas/análisis , Adulto , Anciano , Southern Blotting , Enfermedad de la Arteria Coronaria/etiología , Factores de Crecimiento Endotelial/genética , Factores de Crecimiento Endotelial/fisiología , Terapia Genética , Humanos , Inmunohistoquímica , Linfocinas/genética , Linfocinas/fisiología , Persona de Mediana Edad , Neovascularización Fisiológica , ARN Mensajero/análisis , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa , Factor A de Crecimiento Endotelial Vascular , Factores de Crecimiento Endotelial Vascular

RESUMEN

The purpose of the study is to determine if intravitreal injection of c-type natriuretic peptide (CNP) affects intraocular pressure (IOP), aqueous humor dynamics and guanosine 3',5'-cyclic monophosphate (cGMP) concentration in the aqueous humor of the rabbit eye. Also we investigated whether CNP-like immunoreactivities (CNP-LI) were present in porcine aqueous humor and whether CNP-LI were detected in rabbit and porcine ciliary body. The IOP was measured after intravitreal injection of 2 pmol approximately 20 nmol CNP into rabbit eyes. Aqueous humor dynamics (aqueous humor flow, outflow facility, and uveoscleral outflow) and cGMP concentration in the aqueous humor were determined at approximately 6 hr after CNP injection. The CNP concentration in aqueous was measured by radioimmunoassay in porcine eye, and CNP-LI were detected with a monoclonal antibody in porcine and rabbit eyes. Intravitreally injected CNP caused IOP reduction in a dose-dependent manner (P<0.0001) and the maximum effect was observed at 4 approximately 6 hr. CNP increased total outflow facility by approximately 35%, but did not affect aqueous humor flow or uveoscleral outflow. The cGMP concentration in the aqueous of CNP-treated eyes was about 4- to 14-fold higher than that in the contralateral untreated eyes. CNP concentration in aqueous was about 2-fold higher than that in plasma, and CNP-LI were found in non-pigmented epithelium of the ciliary body of both rabbit and porcine eyes. CNP may play an important role in the regulation of IOP.

Asunto(s)

Hipotensión Ocular/inducido químicamente , Proteínas/efectos adversos , Animales , Humor Acuoso/química , Humor Acuoso/fisiología , GMP Cíclico/metabolismo , Inmunohistoquímica , Presión Intraocular/efectos de los fármacos , Péptido Natriurético Tipo-C , Conejos , Porcinos , Factores de Tiempo

RESUMEN

To address endothelial function on vascular walls exposed to oxidative stress, we investigated the effect of oxidative stress on the secretion of endothelin-1 (ET-1) from cultured bovine carotid artery endothelial cells (BAECs). Concentrations of ET-1 in the media were measured by a specific radioimmunoassay and ET-1 mRNA expression was estimated by Northern blot analysis. Treatment of BAECs with 0.5-2.0 mM H2O2 for 3 h suppressed both ET-1 secretion and ET-1 mRNA expression in a dose-dependent manner compared to control. Attenuation of ET-1 mRNA expression by H2O2 was revealed to take place at the transcriptional level. The addition of NG-nitro-L-arginine-methyl ester (L-NAME) 10 microns, a specific nitric oxide synthase inhibitor, had no effect on H2O2-induced suppression of ET-1 mRNA expression. Suppression of ET secretion under oxidative stress observed in the present study is proposed to be a compensatory mechanism of endothelial cells to inhibit vasoconstriction and proliferation during oxidative stress.

Asunto(s)

Endotelinas/metabolismo , Endotelio Vascular/metabolismo , Estrés Oxidativo/fisiología , Animales , Northern Blotting , Arterias Carótidas/citología , Arterias Carótidas/efectos de los fármacos , Arterias Carótidas/metabolismo , Bovinos , Células Cultivadas , Medios de Cultivo Condicionados , Endotelinas/biosíntesis , Endotelio Vascular/efectos de los fármacos , Peróxido de Hidrógeno/farmacología , Oxidantes/farmacología , Estrés Oxidativo/efectos de los fármacos , ARN Mensajero/biosíntesis , Radioinmunoensayo , Especies Reactivas de Oxígeno/metabolismo

RESUMEN

We have previously reported that C-type natriuretic peptide (CNP), the third member of the natriuretic peptide family, is produced in vascular endothelial cells and suggested that CNP might be a local regulator of vascular tone and growth. To evaluate the biological actions of CNP as compared with human atrial natriuretic peptide (hANP), we intravenously administered synthetic CNP (0.43 nmol/kg) and alpha-hANP (0.43 and 0.043 nmol/kg) to healthy humans. The experiments were done on different days in the same five healthy volunteers (31+/-1 yr old). CNP injection caused a transient but significant decrease in both systolic and diastolic blood pressure (-4.3+/-1.3, -4.1+/-1.0 mmHg) with a significant increase in heart rate (+7.6+/-2.6 bpm), and exerted significant diuretic and natriuretic activities (+130+/-80%, +160+/-100% over the basal level). These effects of CNP (0.43 nmol/kg) were comparable to, or less than, those of alpha-hANP (0.043 nmol/kg). CNP injection also significantly suppressed aldosterone secretion (22% reduction as compared with the basal level). Our results demonstrate that intravenously-administered CNP acts as a natriuretic peptide with less potency than ANP.

Asunto(s)

Factor Natriurético Atrial/administración & dosificación , Presión Sanguínea/efectos de los fármacos , Diuresis/efectos de los fármacos , Proteínas/administración & dosificación , Adulto , Aldosterona/sangre , Aldosterona/metabolismo , Factor Natriurético Atrial/sangre , GMP Cíclico/sangre , GMP Cíclico/orina , Frecuencia Cardíaca/efectos de los fármacos , Humanos , Inyecciones Intravenosas , Masculino , Péptido Natriurético Tipo-C , Proteínas/metabolismo

RESUMEN

We demonstrated endothelial production of C-type natriuretic peptide (CNP), the third member of the natriuretic peptide family, and its regulation by cytokines, including tumor necrosis factor-alpha (TNF alpha). We thus proposed that CNP can control vascular tone and growth as an endothelium-derived relaxing peptide. We also revealed the marked elevation of plasma CNP concentration in patients with septic shock, in which TNF alpha plays a significant part. As the interaction between endothelial cells (EC) and monocytes-macrophages plays a pivotal role in the pathogenesis of atherosclerosis, we investigated the effect of coculture of EC and macrophages on endothelial production of CNP. We used a human monocytic leukemia cell line, THP-1, which differentiates into macrophages when treated with phorbol 12-myristate 13-acetate. The coculture of EC and THP-1-derived macrophages enhanced CNP secretion by more than 10-fold compared with the single culture of EC or the coculture of EC and THP-1 without phorbol 12-myristate 13-acetate treatment. Prevention of direct contact between EC and THP-1-derived macrophages did not attenuate the increase in CNP secretion. Northern blotting revealed the augmentation of CNP messenger RNA expression in EC in the coculture. We detected TNF alpha in the conditioned medium from the coculture of EC and THP-1-derived macrophages. Furthermore, anti-TNF alpha antibody inhibited the stimulation of CNP secretion in the coculture. CNP at a concentration of 1 nM did not stimulate cGMP production in EC or THP-1-derived macrophages, but it elevated cGMP production significantly in vascular smooth muscle cells. These results indicate that endothelial production of CNP is stimulated mainly by TNF alpha released from THP-1-derived macrophages in the coculture. Endothelial CNP at the enhanced level may be one of the vascular mediators to regulate local vascular tone and growth through cGMP production by vascular smooth muscle cells, suggesting the potential significance of endothelial CNP in atherosclerosis.

Asunto(s)

Técnicas de Cocultivo , Endotelio Vascular/citología , Endotelio Vascular/metabolismo , Macrófagos/fisiología , Biosíntesis de Proteínas , Animales , Factor Natriurético Atrial/farmacología , Arterias Carótidas , Bovinos , Comunicación Celular , Línea Celular , Células Cultivadas , Medios de Cultivo Condicionados , GMP Cíclico/biosíntesis , Expresión Génica , Humanos , Sueros Inmunes/farmacología , Péptido Natriurético Tipo-C , Proteínas/genética , Proteínas/metabolismo , ARN Mensajero/metabolismo , Factor de Necrosis Tumoral alfa/antagonistas & inhibidores , Factor de Necrosis Tumoral alfa/metabolismo

RESUMEN

It has been reported that the Trp 64 Arg mutation of the human beta3-adrenergic receptor (beta3-AR) gene is related to an earlier age of onset of non-insulin-dependent diabetes mellitus (NIDDM) and features of insulin resistance and weight gain in morbidly obese patients. However, such findings have not been consistent in varying ethnic populations. In the present study, we investigated the frequency of the Trp 64 Arg mutation of the human beta3-AR gene in Japanese control subjects (n = 253) and in NIDDM (n = 314) and impaired glucose tolerance (IGT) patients (n = 100). We compared the frequency of the mutation with the body-mass index (BMI) in these groups and with the metabolic clearance rate (MCR) of glucose in the NIDDM patients. A Trp 64 Arg mutation was observed in 36.7%, 31.6%, and 37.0% of the control, NIDDM, and IGT subjects, respectively. The frequency of the homozygotes for the mutation was 4.3%, 4.8%, and 3.0%, respectively. Neither the genotype frequency (Trp/Arg, Arg/Arg) nor the frequency of the mutated allele was significantly different among the three groups. The BMI of the subjects with the mutation was not significantly higher than that of the subjects without the mutation in each group. Furthermore, the allele frequency (A) was not different among the subjects with different BMIs (BMI < 22.0, 22.0 < or = BMI < or = 26.4, BMI > 26.4) in each group. In a separate group of NIDDM patients, the MCR of the subjects with intermediate BMIs (22.0 < or = BMI < or = 26.4) with the mutation tended to be lower than that of those without the mutation. In addition, the MCR of the subjects with the mutation in this group was significantly lower compared with that of those with a BMI less than 22. These results indicate that the Trp 64 Arg mutation of the beta3-AR gene may not contribute to the development of NIDDM or be a determinant of obesity in the Japanese population. However, the mutation may contribute to insulin resistance in NIDDM patients with an intermediate BMI.

Asunto(s)

Arginina , Diabetes Mellitus Tipo 2/fisiopatología , Intolerancia a la Glucosa/genética , Mutación Puntual , Receptores Adrenérgicos beta/genética , Triptófano , Adulto , Sustitución de Aminoácidos , Presión Sanguínea/fisiología , Índice de Masa Corporal , Estudios de Casos y Controles , Diabetes Mellitus Tipo 2/sangre , Diabetes Mellitus Tipo 2/genética , Femenino , Genotipo , Humanos , Resistencia a la Insulina/genética , Japón , Lípidos/sangre , Masculino , Persona de Mediana Edad

RESUMEN

BACKGROUND: In cardiac hypertrophy, both excessive enlargement of cardiac myocytes and progressive interstitial fibrosis are well known to occur simultaneously. In the present study, to investigate the interaction between ventricular myocytes (MCs) and cardiac nonmyocytes (NMCs), mostly fibroblasts, during cardiocytes hypertrophy, we examined the change in cell size and gene expression of atrial natriuretic peptide (ANP) and brain natriuretic peptide (BNP) in cultured MCs as markers for hypertrophy in the neonatal rat ventricular cardiac cell culture system. METHODS AND RESULTS: The size of cultured MCs significantly increased in the MC-NMC coculture. Concomitantly, secretions of ANP and BNP into culture media were significantly increased in the MC-NMC coculture compared with in the MC culture (with the possible contamination of NMC <1% of MC). Moreover, in the MC culture, enlargement of MC and an increase in ANP and BNP secretions were induced by treatment with conditioned media of the NMC culture. A considerable amount of endothelin (ET)-1 production was detected in the NMC-conditioned media. BQ-123, an ET-A receptor antagonist, and bosentan, a nonselective ET receptor antagonist, significantly blocked the hypertrophic response of MCs induced by treatment with NMC-conditioned media. Angiotensin II (Ang II) (10(-10) to 10(-6) mol/L) and transforming growth factor-beta1 (TGF-beta1) (10(-13) to 10(-9) mol/L), both of which are known to be cardiac hypertrophic factors, did not induce hypertrophy in MC culture, but both Ang II and TGF-beta1 increased the size of MCs and augmented ANP and BNP productions in the MC-NMC coculture. This hypertrophic activity of Ang II and TGF-beta1 was associated with the potentiation of ET-1 production in the MC-NMC coculture, and the effect of Ang II or TGF-beta1 on the secretions of ANP and BNP in the coculture was significantly suppressed by pretreatment with BQ-123. CONCLUSIONS: These results demonstrate that NMCs regulate MC hypertrophy at least partially via ET-1 secretion and that the interaction between MCs and NMCs plays a critical role during the process of Ang II- or TGF-beta1-induced cardiocyte hypertrophy.

Asunto(s)

Cardiomegalia/patología , Cardiomegalia/fisiopatología , Endotelina-1/fisiología , Miocardio/patología , Comunicación Paracrina/fisiología , Función Ventricular/fisiología , Angiotensina II/farmacología , Animales , Factor Natriurético Atrial/metabolismo , Bovinos , Técnicas de Cocultivo , Medios de Cultivo Condicionados/farmacología , Antagonistas de los Receptores de Endotelina , Hipertrofia , Lipoproteínas LDL/metabolismo , Péptido Natriurético Encefálico , Proteínas del Tejido Nervioso/metabolismo , ARN Mensajero/metabolismo , Ratas , Ratas Wistar , Receptores de Angiotensina/genética , Receptores de Endotelina/genética , Factor de Crecimiento Transformador beta/farmacología

RESUMEN

We have proposed the "vascular natriuretic peptide system", in which C-type natriuretic peptide (CNP), the third member of the natriuretic peptide family, can control vascular tone and growth as an endothelium-derived relaxing peptide. We aimed at overexpression of CNP gene in vascular smooth muscle cells (SMCs) by adenovirus-mediated gene transfer to examine the growth characteristics of SMCs via the augmentation of cGMP production. Rat aortic SMCs infected with Ad.CNP, a replication-deficient adenovirus driving rat CNP cDNA, produced 162 +/- 55 fmol/mL of CNP, which was 4,000 times higher than that produced by endothelial cells. cGMP production was also augmented in Ad.CNP-infected SMCs (2200 +/- 270 fmol/10(4) cells). Accordingly, significant growth inhibition was observed in SMCs infected with Ad.CNP. The flow cytometry analysis revealed that the population of the S and G2 + M phases was reduced by 60% of the control in Ad.CNP-infected SMCs. The gene expression of ANP-B receptor, which is expressed abundantly in SMCs with the synthetic phenotype, was suppressed in Ad.CNP-infected SMCs, while the gene expression of ANP-A receptor, which is expressed predominantly in SMCs with the contractile phenotype, became detectable in Ad.CNP-infected SMCs. In addition, the gene expression of smooth muscle myosin heavy chain-2 (SM-2), which is the molecular marker of highly-differentiated SMCs, was also induced in Ad.CNP-treated SMCs. These results suggest that cGMP cascade activation induces re-differentiation of SMCs. The present study demonstrated that overexpression of CNP induced growth inhibition of SMCs at the G1 phase with possible alteration of the phenotype.

Asunto(s)

Adenoviridae/genética , Factor Natriurético Atrial/genética , Fase G1/genética , Técnicas de Transferencia de Gen , Inhibidores de Crecimiento/genética , Músculo Liso Vascular/metabolismo , Proteínas/genética , Animales , Aorta Torácica , Factor Natriurético Atrial/biosíntesis , Células Cultivadas , GMP Cíclico/metabolismo , Ciclina A/biosíntesis , Ciclina A/genética , Regulación de la Expresión Génica , Masculino , Músculo Liso Vascular/citología , Músculo Liso Vascular/virología , Péptido Natriurético Tipo-C , Biosíntesis de Proteínas , ARN Mensajero/biosíntesis , Ratas

RESUMEN

Shear stress is known to dilate blood vessels and exert antiproliferative effects on vascular walls: these effects have been ascribed to shear stress-induced upregulation of endothelium-derived vasoactive substances, mainly nitric oxide and prostacyclin. We have demonstrated the significance of C-type natriuretic peptide (CNP) as a novel endothelium-derived relaxing peptide (EDRP) that shares a cGMP pathway with nitric oxide. Adrenomedullin is a recently isolated EDRP that elevates intracellular cAMP as prostacyclin does. To elucidate the possible role of these EDRPs under shear stress, we examined the effect of physiological shear stress on CNP mRNA expression in endothelial cells derived from the human umbilical vein (HUVECs), bovine aorta (BAECs), and murine lymph nodes (MLECs) as well as adrenomedullin mRNA expression in HUVECs. CNP mRNA was stimulated prominently in HUVECs under shear stress of 15 dyne/cm2 in a time-dependent manner (4 hours, sixfold increase compared with that in the static condition; 24 hours, 30-fold increase). Similar results were obtained in BAECs (4 hours, twofold increase; 24 hours, threefold increase) and MLECs (4 hours, threefold increase; 24 hours, 10-fold increase). Augmentation of CNP mRNA expression that was dependent on shear stress intensity was also observed (5 dyne/cm2, 2.5-fold increase of static; 15 dyne/cm2, 4.5-fold increase). Increased CNP secretion was also confirmed by the specific radioimmunoassay for CNP. Adrenomedullin mRNA expression in HUVECs increased under shear stress of 15 dyne/cm2 in a time-dependent manner (4 hours, 1.2-fold increase of static: 24 hours, threefold increase) and shear stress intensity-dependent manner (15 dyne/cm2, threefold increase compared with that at 5 dyne/cm2). These results suggest that the coordinated augmentation of mRNA expression of these novel EDRPs may constitute shear stress-dependent vasodilator and antiproliferative effects.

Asunto(s)

Endotelio Vascular/fisiología , Péptidos/metabolismo , Proteínas/metabolismo , Vasodilatadores/metabolismo , Adrenomedulina , Animales , Bovinos , Células Cultivadas , Endotelio Vascular/metabolismo , Hemorreología , Humanos , Ratones , Péptido Natriurético Tipo-C , Reacción en Cadena de la Polimerasa , Proteínas/análisis , Especificidad de la Especie , Estrés Mecánico , Regulación hacia Arriba/efectos de los fármacos

Asunto(s)

Factor Natriurético Atrial/fisiología , Vasos Sanguíneos/patología , Enfermedades Vasculares/patología , Animales , Vasos Sanguíneos/metabolismo , Terapia Genética , Humanos , Ratas , Enfermedades Vasculares/metabolismo , Enfermedades Vasculares/terapia

RESUMEN

The natriuretic peptide family consists of atrial natriuretic peptide (ANP), brain natriuretic peptide (BNP), and C-type natriuretic peptide (CNP). We have elucidated that CNP is synthesized by endothelial cells. We have also shown that CNP secretion is potently suppressed by vascular endothelial growth factor (VEGF). In the present study, we examined the developmental gene expression of the natriuretic peptide system with the expression of VEGF and endothelial cell-specific receptor tyrosine kinases (RTKs), which expression is necessary for vasculogenesis, using embryoid bodies (EB) as an in vitro model for vascular development. When mouse embryonic stem (ES) cells were cultured in suspension culture, ES cells spontaneously differentiated into EB on day 4 and then into cystic EB (day 10). The VEGF gene transcript was detected early, on day 4. The expression of Flk-1, and flt-1 (the two VEGF receptors) and also of tie-2, which is crucial for blood island formation, was detected as early as day 4, and also on days 8 and 21. In contrast, the expression of flt-4, the receptor for VEGF-C, and tie-1, was first detected on day 21. Along with the developmental expression of these markers of differentiation for endothelial cells, the gene expression of CNP and its specific receptor, ANP-B receptor, was detected on days 4, 8, and 21. In contrast, the gene expression of BNP, which acts as a cardiac hormone, and the gene expression of the ANP-A receptor, which is specific to ANP and BNP, was first detected on days 8 and 21, respectively. These results indicate the distinct role of CNP in the natriuretic peptide family and the close linkage of CNP expression and endothelial cell differentiation, suggesting a possible role of CNP in vasculogenesis.

Asunto(s)

Factor Natriurético Atrial/metabolismo , Desarrollo Embrionario y Fetal/fisiología , Regulación del Desarrollo de la Expresión Génica , Neovascularización Fisiológica/fisiología , Proteínas del Tejido Nervioso/metabolismo , Proteínas/metabolismo , Células Madre , Animales , Factor Natriurético Atrial/genética , Diferenciación Celular , División Celular , Desarrollo Embrionario y Fetal/genética , Factores de Crecimiento Endotelial/genética , Endotelio Vascular/citología , Técnicas In Vitro , Linfocinas/genética , Ratones , Músculo Liso Vascular/citología , Péptido Natriurético Encefálico , Péptido Natriurético Tipo-C , Neovascularización Fisiológica/genética , Proteínas del Tejido Nervioso/genética , Reacción en Cadena de la Polimerasa , Proteínas/genética , Receptores del Factor Natriurético Atrial/metabolismo , Transcripción Genética , Factor A de Crecimiento Endotelial Vascular , Factores de Crecimiento Endotelial Vascular

RESUMEN

Growth arrest-specific homeobox (Gax) gene was isolated from rat aorta cDNA library and its expression was largely confined to the cardiovascular tissues. Gax gene was rapidly downregulated by platelet-derived growth factor in vascular smooth muscle cells (VSMCs) and overexpressed Gax was reported to reduce the neointimal thickening after balloon injury in vivo. We have demonstrated that angiotensin II (Ang II) stimulates vascular growth. In contrast, we also reported that C-type natriuretic peptide (CNP) is secreted from vascular endothelial cells to act as a novel endothelium-derived relaxing peptide and inhibits vascular growth via cGMP cascade. In the present study, we examined the effects of Ang II and CNP on Gax gene expression in VSMCs. In quiescent rat aortic VSMCs. Gax mRNA (2 3 kb) level became negligible 6 hours after the addition of Ang II (10(-6) mol/L). The inhibitory action of Ang II on Gax mRNA expression (ED50: 10(-11) mol/L) was almost completely blocked by an AT1R antagonist, CV11974. In contrast, CNP 10(-6) mol/L augmented Gax mRNA expression to exhibit 1.8-fold increase of the control 12 hours after the stimulation. This effect of CNP was mimicked by the addition of 8-bromoadenosine 3'-5'-cyclic monophosphate. The addition of C-ANF[4-23], an atrial natriuretic peptide-C receptor-specific agonist and devoid of stimulating cGMP production, exhibited no effect on Gax mRNA expression. Simultaneous administration of Ang II and CNP revealed that CNP (10(-6) mol/L) significantly attenuated the inhibitory action of Ang II (10(-10) mol/L) on Gax mRNA expression. These results suggest that Gax is a common transcription factor involved in the signaling pathway of vascular growth for Ang II and CNP and regulates the cell cycle and/or phenotype of VSMCs for vascular remodeling in hypertension and atherosclerosis.

Asunto(s)

Angiotensina II/farmacología , Regulación de la Expresión Génica/efectos de los fármacos , Proteínas de Homeodominio/metabolismo , Proteínas Musculares/metabolismo , Músculo Liso Vascular/metabolismo , Proteínas/farmacología , ARN Mensajero/metabolismo , Animales , GMP Cíclico/análogos & derivados , GMP Cíclico/farmacología , Regulación hacia Abajo , Interacciones Farmacológicas , Proteínas de Homeodominio/genética , Masculino , Proteínas Musculares/genética , Péptido Natriurético Tipo-C , Ratas , Ratas Wistar

RESUMEN

We have previously reported that C-type natriuretic peptide (CNP), the third member of the natriuretic peptide family, is produced in vascular endothelial cells (ECs) and acts as an endothelium-derived relaxing peptide. We further demonstrated the detection of the gene transcripts of CNP and atrial natriuretic peptide (ANP) B receptor, a specific receptor for CNP, in human blood vessels. We thus propose the existence of a vascular natriuretic peptide system (NPS). CNP secretion was also demonstrated to be stimulated by various growth factors and cytokines. To clarify the significance of vascular NPS in proliferative vascular complications associated with diabetes, hypertension, or atherosclerosis, in the present study we examined the effect of insulin on CNP secretion from cultured ECs. Insulin at a concentration in the physiological range (10(-10)-10(-7) mol/l) potently suppressed CNP secretion, whereas insulin at the same concentration did not suppress endothelin (ET) secretion from EC. IGF-I had no significant effect on CNP secretion. Insulin, therefore, can be a potent inhibitor of CNP secretion through the activation of insulin receptor. Since CNP has been shown to be a potent inhibitor of vascular smooth muscle cell proliferation, the present study suggests the possibility that attenuated activity of vascular NPS is associated with hyperinsulinemia, which might result in proliferative vascular lesions.

Asunto(s)

Endotelio Vascular/metabolismo , Insulina/farmacología , Proteínas/metabolismo , Animales , Bovinos , Células Cultivadas , Endotelinas/metabolismo , Factor I del Crecimiento Similar a la Insulina/farmacología , Péptido Natriurético Tipo-C , Tasa de Secreción/efectos de los fármacos

RESUMEN

We have previously reported that C-type natriuretic peptide (CNP), the third member of the natriuretic peptide family, is produced in vascular endothelial cells and acts as an endothelium-derived relaxing peptide. To clarify the clinical significance of CNP in renal disorders, we examined the plasma level of CNP in patients with various cardiovascular diseases, including chronic renal failure (CRF) patients who were under hemodialysis therapy. We also investigated biological effects of intravenously-administered CNP (0.43 nmol/kg) by bolus injection from the peripheral vein in healthy volunteers and measured systemic hemodynamic variables, plasma levels of CNP, atrial natriuretic peptide (ANP), brain natriuretic peptide (BNP), cGMP, aldosterone and also urine volume, urinary excretions of sodium, potassium, chloride and cGMP. The plasma CNP levels in healthy humans (N = 13) was 1.4 +/- 0.6 fmol/ml. In CRF patients, the plasma CNP significantly increased up to 3.0 +/- 1.1 fmol/ml. The administration of CNP elicited significant increase of plasma cGMP level (from 4.77 +/- 1.25 to 8.33 +/- 1.59 pmol/ml 15 min after the administration) and of urinary cGMP excretion (from 30.7 +/- 4.3 to 74.9 +/- 13.4 nmol/30 min). Intravenously-administered CNP exerted significant diuretic (% increase: +117 +/- 85.0), natriuretic, kalliuretic and chloriuretic actions with the increase of endogenous creatinine clearance. CNP also elicited significant hypotensive actions (delta BPs/delta BPd: -4.3 +/- 1.3/-4.1 +/- 1.0 mm Hg) with the concomitant increase of heart rate (+7.6 +/- 2.6 bpm). Plasma aldosterone concentration significantly decreased from 45.4 +/- 2.3 to 35.4 +/- 4.9 pg/ml 30 minutes after the administration. Taken together, these results suggest a role for CNP in human renal function.

Asunto(s)

Factor Natriurético Atrial/metabolismo , Factor Natriurético Atrial/farmacología , Fallo Renal Crónico/metabolismo , Proteínas/metabolismo , Proteínas/farmacología , Adulto , Anciano , Aldosterona/sangre , Factor Natriurético Atrial/administración & dosificación , Presión Sanguínea/efectos de los fármacos , Diuresis/efectos de los fármacos , Humanos , Inyecciones Intravenosas , Masculino , Persona de Mediana Edad , Péptido Natriurético Encefálico , Péptido Natriurético Tipo-C , Proteínas del Tejido Nervioso/sangre , Proteínas/administración & dosificación

RESUMEN

OBJECTIVE: To clarify the significance of C-type natriuretic peptide in the interaction between endothelial cells and vascular smooth muscle cells by investigating the endothelial production of C-type natriuretic peptide and the clearance mechanism of C-type natriuretic peptide using the endothelial cells-smooth muscle cells co-culture system. RESULTS: Secretion of C-type natriuretic peptide in the direct co-culture of endothelial cells with smooth muscle cells elicited as much as a 60-fold increase compared with endothelial cells alone. The accumulation of intracellular cyclic GMP in the co-culture was consequently increased and the elevation of cyclic GMP level in the co-culture was abolished by the anti-C-type natriuretic peptide monoclonal antibody. The elevated cyclic GMP production in the co-culture was abolished by the anti-transforming growth factor-beta neutralizing antibody. Candoxatrilat (10(-6)-10(-4) mol/l), a neutral endopeptidase inhibitor, dose-dependently increased the concentrations of C-type natriuretic peptide in the culture medium with endothelial cells alone, but not in the endothelial cells-smooth muscle cells co-culture. The transcript of neutral endopeptidase messenger RNA was detected in endothelial cells but not in smooth muscle cells by reverse transcriptase polymerase chain reaction. Treatment with C-atrial natriuretic factor4-23 (10(-9)-10(-6) mol/l), the specific ligand for the clearance receptor of the natriuretic peptides, resulted in dose-dependent augmentation of C-type natriuretic peptide concentration and concomitant intracellular cyclic GMP production in the endothelial cells-smooth muscle cells co-culture but not in endothelial cells alone. CONCLUSION: The present study demonstrated that direct interaction between endothelial cells and smooth muscle cells augments C-type natriuretic peptide secretion from endothelial cells through transforming growth factor-beta activation, and revealed that the enzymatic degradation is responsible for the steady state level of C-type natriuretic peptide in endothelial cells alone and that the receptor-mediated clearance mainly determines the augmented level of C-type natriuretic peptide in the interaction between endothelial cells and smooth muscle cells. The results taken together raise the possibility that endothelial C-type natriuretic peptide might play a role in regulation of vascular tone and remodelling.

Asunto(s)

Factor Natriurético Atrial/metabolismo , Endotelio Vascular/metabolismo , Músculo Liso Vascular/metabolismo , Proteínas/metabolismo , Animales , Secuencia de Bases , Bovinos , Células Cultivadas , Técnicas de Cocultivo , GMP Cíclico/biosíntesis , Endotelio Vascular/citología , Guanilato Ciclasa/fisiología , Tasa de Depuración Metabólica , Datos de Secuencia Molecular , Músculo Liso Vascular/citología , Péptido Natriurético Tipo-C , Neprilisina/genética , ARN Mensajero/análisis , Receptores del Factor Natriurético Atrial/fisiología , Factor de Crecimiento Transformador beta/fisiología

RESUMEN

Recently, we have demonstrated that C-type natriuretic peptide (CNP) is produced in vascular endothelial cells (ECs). In the present study, we investigated the interaction of ECs and vascular smooth muscle cells (SMCs) for endothelial production of CNP and its action on vascular growth, using the EC/SMC coculture system. The concentration of CNP-like immunoreactivity in the medium was increased 60-fold within 48 hours in the EC/SMC coculture with direct contact compared with that in EC alone. Northern blot analysis revealed the augmented expression of CNP mRNA in the EC/SMC coculture. The accumulation of intracellular cGMP in the coculture was concomitantly increased, and this response was blocked by anti-CNP monoclonal antibody and HS-142-1, a nonpeptide atrial natriuretic peptide receptor antagonist. The concentration of biologically active transforming growth factor-beta (TGF-beta) in the culture medium of the coculture with direct contact of ECs and SMCs was elevated to the level to stimulate endothelial production of CNP. Actually, the neutralizing antibody against TGF-beta abrogated the cGMP accumulation in the coculture. These results show that endothelial production of CNP in the EC/SMC coculture is at least in part regulated by TGF-beta. Furthermore, the conditioned medium from ECs stimulated by TGF-beta was demonstrated to have a growth-inhibitory effect on SMCs, which was abolished by anti-CNP monoclonal antibody and HS-142-1. The treatment with anti-CNP monoclonal antibody and HS-142-1 also significantly increased the cell number of the EC/SMC coculture. The present study reveals the pathophysiological significance of endothelial CNP as a paracrine/autocrine vascular regulator for vascular growth in the interaction of ECs and SMCs.

Asunto(s)

Endotelio Vascular/metabolismo , Músculo Liso Vascular/metabolismo , Biosíntesis de Proteínas , Animales , Anticuerpos Monoclonales , Factor Natriurético Atrial/metabolismo , Vasos Sanguíneos/crecimiento & desarrollo , Vasos Sanguíneos/fisiología , Bovinos , División Celular/efectos de los fármacos , Técnicas de Cocultivo , GMP Cíclico/biosíntesis , Endotelio Vascular/química , Endotelio Vascular/citología , Músculo Liso Vascular/química , Músculo Liso Vascular/citología , Natriuréticos/fisiología , Péptido Natriurético Tipo-C , Polisacáridos/farmacología , Proteínas/genética , Proteínas/farmacología , ARN Mensajero/metabolismo , Factor de Crecimiento Transformador beta/metabolismo

RESUMEN

Angiogenesis plays a pivotal role not only in wound healing and tumor progression but also in diabetic angiopathy, arteriosclerosis, and collateral formation of obstructive vascular diseases. Vascular endothelial growth factor (VEGF) is now thought to be an endothelium-specific and potent angiogenic factor. We previously demonstrated that C-type natriuretic peptide (CNP), originally isolated from porcine brain, is produced by endothelial cells and proposed that CNP can exert control over vascular tone and growth as a local vascular regulator. In the present study, we examined the effect of VEGF on CNP secretion from endothelial cells using the specific radioimmunoassay for CNP we developed. VEGF (1 to 100 ng/mL) dose-dependently suppressed CNP secretion from cultured bovine endothelial cells, and 100 ng/mL VEGF suppressed endothelial CNP secretion to 28% of control levels (31.7 +/- 5.5 versus 8.9 +/- 0.8 fmol/mL, vehicle versus VEGF). VEGF also suppressed CNP mRNA expression in endothelial cells 9 hours after administration. In contrast, basic fibroblast growth factor (20 ng/mL), an endothelium-nonspecific angiogenic factor, significantly stimulated CNP secretion by 290%. These results indicate that VEGF can regulate vascular tone and growth in the process of angiogenesis through suppression of endothelial secretion of CNP, which is an endothelium-derived vasorelaxing and growth-inhibitory peptide.

Asunto(s)

Factores de Crecimiento Endotelial/farmacología , Endotelio Vascular/metabolismo , Linfocinas/farmacología , Proteínas/metabolismo , Animales , Bovinos , Células Cultivadas , Relación Dosis-Respuesta a Droga , Factor 2 de Crecimiento de Fibroblastos/farmacología , Péptido Natriurético Tipo-C , Radioinmunoensayo/métodos , Factor A de Crecimiento Endotelial Vascular , Factores de Crecimiento Endotelial Vascular