RESUMEN
Chinese hamster ovary (CHO) cells are by far the most important mammalian cell lines used for producing antibodies and other therapeutic proteins. It is critical to fully understand their physiological conditions during a bioprocess in order to achieve the highest productivity and the desired product quality. Flow cytometry technology possesses unique advantages for measuring multiple cellular attributes for a given cell and examining changes in cell culture heterogeneity over time that can be used as metrics for enhanced process understanding and control strategy. Flow cytometry-based assays were utilized to examine the progression of cell cycle and apoptosis in three case studies using different antibody-producing CHO cell lines in both fed-batch and perfusion bioprocesses. In our case studies, we found that G0/G1 phase distribution and early apoptosis accumulation responded to subtle changes in culture conditions, such as pH shifting or momentary glucose depletion. In a perfusion process, flow cytometry provided an insightful understanding of the cell physiological status under a hypothermic condition. More importantly, these changes in cell cycle and apoptosis were not detected by a routine trypan blue exclusion-based cell counting and viability measurement. In summary, integration of flow cytometry into bioprocesses as a process analytical technology tool can be beneficial for establishing optimum process conditions and process control.
Asunto(s)
Apoptosis , Técnicas de Cultivo de Célula , Animales , Técnicas de Cultivo Celular por Lotes , Reactores Biológicos , Células CHO , Ciclo Celular , Cricetinae , Cricetulus , Citometría de FlujoRESUMEN
NADPH oxidase isoform-2 (NOX2) generates reactive oxygen species (ROS) that contribute to neurodegenerative and cardiovascular pathologies. However, validation of NOX2 as a pharmacotherapeutic target has been hampered by a lack of mechanistically-defined inhibitors. Using cellular and biochemical assays, we explored previously reported inhibitors of ROS production (perhexiline, suramin, VAS2870 and two Shionogi patent compounds) as direct NOX2 inhibitors. All but suramin, which presumably lacks cell penetrance, inhibit cellular ROS production. However, only perhexiline and suramin inhibit biochemical NOX2 activity. Indeed, our data suggest that NOX2 inhibition by perhexiline may contribute significantly to its demonstrated cardioprotective effects. Inhibition of protein kinase CßII explains the cellular activity of the Shionogi compounds, whereas VAS2870 inhibits by an as-yet unidentified mechanism unrelated to direct NOX2 function or subunit assembly. These data delineate the mechanisms of action of these compounds and highlight their strengths and limitations for use in future target validation studies.
Asunto(s)
Inhibidores Enzimáticos/farmacología , Glicoproteínas de Membrana/antagonistas & inhibidores , NADPH Oxidasas/antagonistas & inhibidores , Especies Reactivas de Oxígeno/metabolismo , Secuencia de Bases , Benzoxazoles/farmacología , Fármacos Cardiovasculares/farmacología , Células Cultivadas , Humanos , Glicoproteínas de Membrana/genética , Glicoproteínas de Membrana/metabolismo , Datos de Secuencia Molecular , NADPH Oxidasa 2 , NADPH Oxidasas/genética , NADPH Oxidasas/metabolismo , Neutrófilos/efectos de los fármacos , Neutrófilos/enzimología , Neutrófilos/metabolismo , Perhexilina/farmacología , Proteína Quinasa C/antagonistas & inhibidores , Proteína Quinasa C beta , Especies Reactivas de Oxígeno/antagonistas & inhibidores , Suramina/farmacología , Triazoles/farmacologíaRESUMEN
Asymmetric dimethylarginine (ADMA) has been implicated in the progression of cardiovascular disease as an endogenous inhibitor of nitric oxide synthase. The regulation of dimethylarginine dimethylaminohydrolase (DDAH), the enzyme responsible for metabolizing ADMA, is poorly understood. The transcription factor sterol response element binding protein (SREBP) is activated by statins via a reduction of membrane cholesterol content. Because the promoters of both DDAH1 and DDAH2 isoforms contain sterol response elements, we tested the hypothesis that simvastatin regulates DDAH1 and DDAH2 transcription via SREBP. In cultured endothelial cells, simvastatin increased DDAH1 mRNA expression compared with vehicle. In an ADMA loading experiment, simvastatin treatment resulted in a decrease in ADMA content, an indication of increased DDAH activity. The knockdown of SREBP1c protein led to an increase in DDAH1 mRNA expression and activity, whereas the knockdown of SREBP2 led to a decrease in DDAH1 mRNA expression. The role of SREBP2 in the activation of the DDAH1 was supported by chromatin immunoprecipitation studies demonstrating increased binding of SREBP2 to the DDAH1 promoter upon simvastatin stimulation. These data indicate that SREBP1c might act as a repressor and SREBP2 as an activator of DDAH transcription and activity. This study describes a novel mechanism of reciprocal regulation by the SREBP family members of the DDAH-ADMA system, which represents a potential link between cellular cholesterol content and endothelial dysfunction observed in cardiovascular disease.
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Amidohidrolasas/fisiología , Arginina/análogos & derivados , Células Endoteliales/fisiología , Proteína 1 de Unión a los Elementos Reguladores de Esteroles/fisiología , Proteína 2 de Unión a Elementos Reguladores de Esteroles/fisiología , Amidohidrolasas/genética , Arginina/fisiología , Western Blotting , Células Cultivadas , Cromatina/metabolismo , Cromatografía Líquida de Alta Presión , Cartilla de ADN , Células Endoteliales/enzimología , Humanos , Inhibidores de Hidroximetilglutaril-CoA Reductasas/farmacología , Inmunoprecipitación , Lentivirus/genética , Nitritos/metabolismo , ARN Mensajero/biosíntesis , ARN Mensajero/genética , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa , Simvastatina/farmacología , Proteína 1 de Unión a los Elementos Reguladores de Esteroles/genética , Proteína 2 de Unión a Elementos Reguladores de Esteroles/genética , TransfecciónRESUMEN
Heme oxygenase-1 (HO) metabolizes heme to form the vasodilator carbon monoxide and antioxidant biliverdin. Upregulation of HO-1 by hemin, which is also a substrate attenuates thrombosis in rodent models, however, whether protection is due to HO-1 upregulation or to increased substrate availability is unknown. This study tested the hypothesis that treatment of mice with cobalt protoporphyrin (CoPP), a non-substrate HO-1 inducer, would protect the endothelium from laser injury. C57Bl/J6 mice were treated with vehicle, CoPP (20 mg/kg), CoPP plus the HO-1 inhibitor tin protoporphyrin (SnPP; 20 mg/kg) or SnPP alone for 18 h. Intravital microscopy was used to quantitate thrombus formation in cremaster arterioles in response to laser ablation of the endothelium. CoPP treatment inhibited thrombosis by 43% compared to vehicle (P<0.05). SnPP co-treatment negated the inhibitory effect of CoPP while SnPP alone potentiated thrombosis compared to vehicle. In CoPP-treated animals, cremaster HO-1 mRNA expression was increased 59+/-17-fold over vehicle (P<0.001). Co-treatment with CoPP+SnPP attenuated this effect by 36%, however the increase in HO-1 protein induced by CoPP was unaffected by SnPP. Induction of HO-1 by the non-substrate inducer CoPP protects against laser induced endothelial injury without the need for increased substrate. Small molecule, substrate-independent upregulation of HO-1 expression represents a feasible approach to ameliorate endothelial dysfunction in cardiovascular disease.
Asunto(s)
Arteriolas/efectos de los fármacos , Arteriolas/patología , Hemo-Oxigenasa 1/biosíntesis , Protoporfirinas/farmacología , Trombosis/enzimología , Animales , Arteriolas/metabolismo , Inducción Enzimática/efectos de los fármacos , Hemo-Oxigenasa 1/genética , Hemo-Oxigenasa 1/metabolismo , Hemodinámica/efectos de los fármacos , Rayos Láser/efectos adversos , Masculino , Ratones , Ratones Endogámicos C57BL , Trombosis/etiología , Trombosis/genética , Trombosis/fisiopatología , Regulación hacia Arriba/efectos de los fármacosRESUMEN
Angiotensin II (Ang II) activates p38 mitogen-activated protein kinase (p38 MAPK) and increases reactive oxygen species (ROS), but the nature of the relationship in vivo is not fully understood. We assess the effect of SB239063AN, a highly selective, orally active, p38 MAPK inhibitor, on Ang II-dependent hypertension, target-organ damage and ROS production. Sprague-Dawley rats and MAPKAP kinase-2 knockout mice were infused with Ang II. Ang II infusion increased the levels of phosphorylated p38 MAPK in the heart and aorta. Production of superoxide anion and expression of NAD(P)H oxidase subunit gp91 in the aorta were increased 4- and 5-fold, respectively. In addition, Ang II infusion led to endothelial dysfunction, progressive and sustained hypertension, and cardiac hypertrophy. Treatment with SB239063AN (800 ppm in the diet) significantly attenuated the levels of phosphorylated p38 MAPK in the heart and aorta, reduced superoxide anion generation by 57% (P < 0.01), markedly suppressed gp91 mRNA expression, prevented endothelial dysfunction, and blunted both the hypertension and cardiac hypertrophy. Ang II-dependent hypertension was also significantly attenuated in MAPKAP kinase-2 knockout mice. The results suggest that Ang II induced hypertension, organ damage, and ROS production are possibly mediated by p38 MAPK and inhibition of p38 MAPK may offer a therapeutic approach for cardiovascular disease.
Asunto(s)
Angiotensina II/efectos adversos , Inhibidores Enzimáticos , Hipertensión/tratamiento farmacológico , Imidazoles , Pirimidinas , Superóxidos/metabolismo , Remodelación Ventricular/efectos de los fármacos , Proteínas Quinasas p38 Activadas por Mitógenos/antagonistas & inhibidores , Animales , Aorta Abdominal/efectos de los fármacos , Aorta Abdominal/enzimología , Aorta Abdominal/metabolismo , Presión Sanguínea/efectos de los fármacos , Arterias Carótidas/efectos de los fármacos , Arterias Carótidas/enzimología , Arterias Carótidas/metabolismo , Ecocardiografía , Endotelio Vascular/efectos de los fármacos , Inhibidores Enzimáticos/administración & dosificación , Inhibidores Enzimáticos/farmacología , Inhibidores Enzimáticos/uso terapéutico , Hipertensión/inducido químicamente , Hipertensión/enzimología , Hipertensión/metabolismo , Imidazoles/administración & dosificación , Imidazoles/farmacología , Imidazoles/uso terapéutico , Péptidos y Proteínas de Señalización Intracelular , Masculino , Glicoproteínas de Membrana/metabolismo , Ratones , Ratones Noqueados , Miocardio/enzimología , Miocardio/metabolismo , NADPH Oxidasa 2 , NADPH Oxidasas/metabolismo , Proteínas Quinasas/genética , Proteínas Serina-Treonina Quinasas , Pirimidinas/administración & dosificación , Pirimidinas/farmacología , Pirimidinas/uso terapéutico , Ratas , Ratas Sprague-Dawley , Vasodilatación/efectos de los fármacos , Proteínas Quinasas p38 Activadas por Mitógenos/biosíntesisRESUMEN
Dendroaspis natriuretic peptide (DNP) is a newly-described natriuretic peptide which lowers blood pressure via vasodilation. The natriuretic peptide clearance receptor (NPR-C) removes natriuretic peptides from the circulation, but whether DNP interacts with human NPR-C directly is unknown. The purpose of this study was to test the hypothesis that DNP binds to NPR-C. ANP, BNP, CNP, and the NPR-C ligands AP-811 and cANP(4-23) displaced [(125)I]-ANP from NPR-C with pM-to-nM K(i) values. DNP displaced [(125)I]-ANP from NPR-C with nM potency, which represents the first direct demonstration of binding of DNP to human NPR-C. DNP showed high pM affinity for the GC-A receptor and no affinity for GC-B (K(i)>1000 nM). DNP was nearly 10-fold more potent than ANP at stimulating cGMP production in GC-A expressing cells. Blockade of NPR-C might represent a novel therapeutic approach in augmenting the known beneficial actions of DNP in cardiovascular diseases such as hypertension and heart failure.
Asunto(s)
Venenos Elapídicos/metabolismo , Péptidos Natriuréticos/metabolismo , Péptidos/metabolismo , Receptores del Factor Natriurético Atrial/metabolismo , Animales , Humanos , Péptidos y Proteínas de Señalización Intercelular , Unión Proteica , Ensayo de Unión Radioligante , Proteínas Recombinantes/metabolismoRESUMEN
Lysophosphatidylcholine (LPC) is the major bioactive lipid component of oxidized LDL, thought to be responsible for many of the inflammatory effects of oxidized LDL described in both inflammatory and endothelial cells. Inflammation-induced transformation of vascular smooth muscle cells from a contractile phenotype to a proliferative/secretory phenotype is a hallmark of the vascular remodeling that is characteristic of atherogenesis; however, the role of LPC in this process has not been fully described. The present study tested the hypothesis that LPC is an inflammatory stimulus in coronary artery smooth muscle cells (CASMCs). In cultured human CASMCs, LPC stimulated time- and concentration-dependent release of arachidonic acid that was sensitive to phospholipase A2 and C inhibition. LPC stimulated the release of arachidonic acid metabolites leukotriene-B4 and 6-keto-prostaglandin F1alpha, within the same time course. LPC was also found to stimulate basic fibroblast growth factor release as well as stimulating the release of the cytokines GM-CSF, IL-6, and IL-8. Optimal stimulation of these signals was obtained via palmitic acid-substituted LPC species. Stimulation of arachidonic acid, inflammatory cytokines and growth factor release, implies that LPC might play a multifactorial role in the progression of atherosclerosis, by affecting inflammatory processes.
Asunto(s)
Vasos Coronarios/citología , Vasos Coronarios/efectos de los fármacos , Inflamación/patología , Lisofosfatidilcolinas/farmacología , Miocitos del Músculo Liso/efectos de los fármacos , 6-Cetoprostaglandina F1 alfa/biosíntesis , Ácido Araquidónico/metabolismo , Ácidos Araquidónicos/farmacología , Células Cultivadas , Citocinas/metabolismo , Factor 2 de Crecimiento de Fibroblastos/biosíntesis , Humanos , Leucotrieno B4/biosíntesis , Lípidos/farmacología , Miocitos del Músculo Liso/metabolismo , Fosfolipasas/antagonistas & inhibidores , TritioRESUMEN
Increased susceptibility to atherosclerosis increases the risk of mortality in type 2 diabetic patients. Leukocyte adhesion to the endothelium is a critical step in atherogenesis. In addition to its insulin-sensitizing effects, rosiglitazone (RSG) possesses anti-inflammatory properties. However, the effects of RSG on the initial phase of leukocyte recruitment (rolling, adhesion) have not been studied in vivo. This study tested the hypothesis that RSG treatment of Zucker diabetic fatty (ZDF) rats inhibits ischemia/reperfusion-induced leukocyte adhesion to the endothelium. Male ZDF rats (16 weeks) were treated with RSG (3 mg/kg/day, p.o.) 7 days before experimentation. Leukocyte-endothelial interactions in cremaster venules were recorded using intravital microscopy prior to 30 min of ischemia and during a 90-min reperfusion period. Although blood pressure, plasma glucose, and insulin were not different between treatment groups, RSG treatment was associated with reduced leukocyte rolling and inhibition of leukocyte adhesion throughout the reperfusion period (P < 0.01). Cremaster mRNA expression of vascular cell adhesion molecule-1 (VCAM-1) was reduced by 35% in RSG-treated animals (P < 0.01), whereas P- and E-selectin and intercellular adhesion molecule-1 (ICAM-1) were unchanged. Immunostaining for P-selectin, E-selectin, and VCAM-1 was reduced by 21, 61, and 50%, respectively (for all, P < 0.05), in RSG-treated animals. Inhibition of ischemia/reperfusion-induced leukocyte adhesion might contribute to the utility of RSG as a therapy for vascular disease.
Asunto(s)
Hipoglucemiantes/farmacología , Leucocitos/fisiología , Infarto del Miocardio/prevención & control , Daño por Reperfusión Miocárdica/prevención & control , Tiazolidinedionas/farmacología , Adiponectina/sangre , Animales , Glucemia/análisis , Presión Sanguínea/efectos de los fármacos , Adhesión Celular/efectos de los fármacos , Ácidos Grasos no Esterificados/sangre , Inmunohistoquímica , Insulina/sangre , Leucocitos/efectos de los fármacos , Masculino , Infarto del Miocardio/patología , Ratas , Ratas Zucker , Rosiglitazona , Molécula 1 de Adhesión Celular Vascular/metabolismoRESUMEN
Urotensin-II is purported to influence pulmonary function by modulating smooth muscle tone/growth. In the present study, Northern blot and reverse transcription polymerase chain reaction (RT-PCR) analysis indicated the presence of UT receptor mRNA in cat trachea, bronchi and lung parenchyma. Urotensin-II contracted cat isolated trachea and bronchi with similar potencies (pEC(50)s 8.61+/-0.07-8.81+/-0.10). Contractile efficacies ranged from 19+/-9% to 63+/-11% KCl in the primary and secondary bronchi. The peptidic UT receptor antagonists BIM-23127, SB-710411 and GSK248451 (7.18+/-0.12, 7.52+/-0.08 and 9.05+/-0.16 cat recombinant UT pK(i)s) inhibited urotensin-II-induced contraction of cat isolated trachea with pK(b)s 6.36+/-0.11, 6.74+/-0.07 and 9.27+/-0.12, respectively. As such, feline lung contains significant amounts of UT mRNA and this receptor appears to be functionally coupled to bronchoconstriction (the peptidic tool compound GSK248451 representing a sub-nanomolar inhibitor of such effects). These findings suggest that the cat represents a suitable species for future studies designed to assess the effects of the urotensin-II receptor on pulmonary (patho)physiology.
Asunto(s)
Contracción Muscular/efectos de los fármacos , Músculo Liso/efectos de los fármacos , Péptidos Cíclicos/farmacología , Receptores Acoplados a Proteínas G/antagonistas & inhibidores , Urotensinas/farmacología , Animales , Unión Competitiva , Northern Blotting , Bronquios/efectos de los fármacos , Bronquios/metabolismo , Bronquios/fisiología , Gatos , Línea Celular , Membrana Celular/efectos de los fármacos , Membrana Celular/metabolismo , Relación Dosis-Respuesta a Droga , Perfilación de la Expresión Génica , Humanos , Técnicas In Vitro , Radioisótopos de Yodo , Pulmón/efectos de los fármacos , Pulmón/metabolismo , Pulmón/fisiología , Masculino , Músculo Liso/fisiología , Péptidos Cíclicos/metabolismo , Cloruro de Potasio/farmacología , ARN Mensajero/genética , ARN Mensajero/metabolismo , Ensayo de Unión Radioligante , Receptores Acoplados a Proteínas G/genética , Receptores Acoplados a Proteínas G/metabolismo , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa , Tráquea/efectos de los fármacos , Tráquea/metabolismo , Tráquea/fisiología , Urotensinas/metabolismoAsunto(s)
Proteínas de Ciclo Celular/metabolismo , Reacción en Cadena de la Polimerasa/métodos , Receptores Acoplados a Proteínas G/metabolismo , Animales , Células Sanguíneas/química , Proteínas de Ciclo Celular/genética , Humanos , ARN Mensajero/análisis , ARN Mensajero/metabolismo , Ratas , Receptores Acoplados a Proteínas G/genéticaRESUMEN
Urotensin-II (U-II), acting through its G-protein-coupled receptor, UT, is a possible contributor to hypertension. Variable functional responses to U-II, both within and between species studied to date, complicate the characterization of UT antagonists. In the cat, however, U-II causes systemic hypertension and constricts arterial segments isolated from several vascular beds. The purpose of this study was to clone and pharmacologically characterize cat recombinant UT to determine whether this system represents a model for characterizing UT antagonists. Cloned cat UT displayed 74% identity to primate UT, and 77% identity to rodent UT. [(125)I] hU-II bound in a saturable manner to a single site on recombinant cat UT with high affinity (K(D) 288+/-13pM) and high density (B(max) 747+/-66fmol/mg protein). U-II isopeptides displayed equipotent, high affinity binding to cat UT (K(i) 1.8-5.3nM). Cat UT was coupled to intracellular [Ca(2+)] release (EC(50) 0.6+/-0.2nM) and total inositol phosphate (IP) formation (EC(50) 0.4+/-0.1nM). Protein kinase C activation desensitized cat, but not human, UT-mediated IP formation. UT mRNA expression was detected in cat blood vessels, trachea, lung, and kidney, where the medulla (K(D) 815+/-34) and cortex and (K(D) 316+/-39pM) displayed high affinity binding for human U-II (hU-II). The cat urotensin-II receptor represents a suitable in vitro model to examine the role of the U-II/UT system in the etiology of hypertension, assisting in the evaluation of the UT antagonists to help treat cardiovascular disease.
Asunto(s)
Clonación Molecular , Receptores Acoplados a Proteínas G/genética , Secuencia de Aminoácidos , Animales , Señalización del Calcio/fisiología , Gatos , Línea Celular , Membrana Celular/fisiología , Secuencia Conservada , Cartilla de ADN , Humanos , Fosfatos de Inositol/metabolismo , Ratones , Datos de Secuencia Molecular , Especificidad de Órganos , Reacción en Cadena de la Polimerasa , ARN Mensajero/genética , Ratas , Receptores Acoplados a Proteínas G/efectos de los fármacos , Receptores Acoplados a Proteínas G/metabolismo , Proteínas Recombinantes/efectos de los fármacos , Proteínas Recombinantes/metabolismo , Alineación de Secuencia , Homología de Secuencia de Aminoácido , TransfecciónRESUMEN
Inflammation and leukocyte activation/infiltration play a major role in the initiation and progression of cardiovascular diseases including atherosclerosis and heart failure. Acute p38 mitogen-activated protein kinase (MAPK) pathway inhibition attenuates tissue damage and leukocyte accumulation in myocardial ischemia/reperfusion injury, although its effect on the acute phase of leukocyte recruitment has not been elucidated. The purpose of this study was to test the hypothesis that acute treatment of rats with a selective p38 inhibitor, SB-239063, inhibits ischemia/reperfusion-induced leukocyte-endothelial adhesion in vivo. Male Sprague-Dawley rats were treated with either SB-239063 (10 mgkg(-1)), dexamethasone (3 mgkg(-1)) or vehicle 1h prior to ischemia. Postcapillary venules were observed microscopically in exteriorized, superfused cremaster tissue. Leukocytes were fluorescently labeled in vivo using intravenous rhodamine 6G. Leukocyte adhesion, rolling, and rolling velocities were quantitated prior to 30 min ischemia, and at several time points during a 90 min reperfusion period. Ischemia caused a 3-fold increase in adherent leukocytes 5 min following reperfusion, a response that was maintained throughout the monitoring period (90 min) in vehicle-treated animals. SB-239063, at a dose known to inhibit p38 MAPK activity in vivo (10 mgkg(-1)), had no effect on ischemia/reperfusion-induced leukocyte adhesion, the number of rolling leukocytes, rolling velocities during the reperfusion period or adhesion molecule expression (P-, E-selectin, VCAM-1, ICAM-1). In contrast, dexamethasone completely blocked leukocyte adhesion in response to ischemia/reperfusion, and reduced expression of E-selectin, intercellular adhesion molecule-1 (ICAM-1) and vascular cell adhesion molecule-1 (VCAM-1). We conclude that p38 MAPK may not play a role in initial leukocyte recruitment in response to ischemia/reperfusion injury, but could affect leukocyte emigration, thereby resulting in increased leukocyte accumulation in ischemic-reperfused tissue.
Asunto(s)
Endotelio Vascular/inmunología , Leucocitos/fisiología , Daño por Reperfusión/inmunología , Proteínas Quinasas p38 Activadas por Mitógenos/antagonistas & inhibidores , Animales , Adhesión Celular/efectos de los fármacos , Movimiento Celular/efectos de los fármacos , Dexametasona/farmacología , Selectina E/biosíntesis , Selectina E/genética , Células Endoteliales/fisiología , Endotelio Vascular/patología , Imidazoles/farmacología , Leucocitos/efectos de los fármacos , Masculino , Microscopía Fluorescente , Músculo Esquelético/irrigación sanguínea , Músculo Esquelético/efectos de los fármacos , Músculo Esquelético/metabolismo , Selectina-P/biosíntesis , Selectina-P/genética , Pirimidinas/farmacología , ARN Mensajero/biosíntesis , Ratas , Ratas Sprague-Dawley , Daño por Reperfusión/patología , Molécula 1 de Adhesión Celular Vascular/biosíntesis , Molécula 1 de Adhesión Celular Vascular/genética , Vénulas/inmunologíaRESUMEN
Urotensin-II (U-II), the most potent mammalian vasoconstrictor identified, and its receptor, UT, exhibits increased expression in cardiac tissue and plasma in congestive heart failure (CHF) patients. Cardiomyocyte hypertrophy is primarily responsible for increased myocardial mass associated with cardiac injury. Neurohumoral factors such as angiotensin-II, endothelin-1, catecholamines, and inflammatory cytokines are thought to mediate this response. U-II shares similar biological activities with other hypertrophic G(q)-coupled receptor ligands such as angiotensin-II and endothelin-1, but a role for U-II in cardiomyocyte hypertrophy has not been characterized. The hypothesis of the current study was that U-II, acting through its G(q)-coupled receptor UT plays a hypertrophic role in cardiac hypertrophic remodeling. We report that adenoviral upregulation of the UT receptor "unmasked" U-II-induced hypertrophy in H9c2 cardiomyocytes, with a threshold response of 202+/-8 binding sites/cell. U-II was equally as efficacious as phenylephrine in inducing hypertrophy, measured by a reporter assay (EC(50) 0.7+/-0.2 nM) and [(3)H]-leucine incorporation (EC(50) 150+/-40 nM). A competitive peptidic UT receptor antagonist, BIM-23127, inhibited U-II-induced hypertrophy ( K(B) 34+/-6 nM). U-II did not affect cell proliferation or apoptosis, indicating that U-II is more hypertrophic than apoptotic or hyperplastic in cardiomyocytes. U-II (10 nM) stimulated interleukin-6 release in UT-expressing cardiomyocytes (4.6-fold at 6 h). Finally, in a rat heart failure model, cardiac ventricular mRNA expression of U-II, UT receptor, interleukin-6, and interleukin-1-beta is increased time-dependently following myocardial injury. These results indicate that U-II might play a role in cardiac remodeling associated with CHF by stimulation of cardiomyocyte hypertrophy via UT, and through upregulation of inflammatory cytokines. As such, UT antagonism may represent a novel therapeutic target for the clinical management of heart failure.
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Cardiomegalia/metabolismo , Cardiomegalia/patología , Mediadores de Inflamación/fisiología , Miocitos Cardíacos/efectos de los fármacos , Miocitos Cardíacos/patología , Receptores Acoplados a Proteínas G/antagonistas & inhibidores , Urotensinas/farmacología , Animales , Línea Celular , Citocinas/biosíntesis , Citocinas/fisiología , Relación Dosis-Respuesta a Droga , Insuficiencia Cardíaca/metabolismo , Insuficiencia Cardíaca/patología , Masculino , Miocitos Cardíacos/metabolismo , Ratas , Ratas Endogámicas Lew , Receptores Acoplados a Proteínas G/agonistas , Receptores Acoplados a Proteínas G/biosíntesisRESUMEN
Plasma and urinary levels of "urotensin(U)-II-like" substances determined in healthy human volunteers were 12.4 +/- 0.6 ng/ml and 2.2 +/- 0.3 ng/ml by RIA, an order of magnitude lower than that seen by RRA, 167.5 +/- 9.5 ng/ml and 65.2 +/- 4.3 ng/ml. HPLC demonstrated the existence of at least three prominent activity peaks in plasma and urine, the more hydrophobic of which did not co-elute with U-II, degradation products or URP. RRA and RIA recognized these peaks with contrasting efficacy. As such, published levels of "U-II-like" activity should be interpreted with caution until a better understanding is obtained regarding what species specific RIA and RRA assay reagents interact with.
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Ensayo de Unión Radioligante/métodos , Urotensinas , Adulto , Femenino , Humanos , Masculino , Persona de Mediana Edad , Radioinmunoensayo/métodos , Valores de Referencia , Reproducibilidad de los Resultados , Urotensinas/análisis , Urotensinas/sangre , Urotensinas/orinaRESUMEN
1 In an effort to identify endogenous, native mammalian urotensin-II (U-II) receptors (UT), a diverse range of human, primate and rodent cell lines (49 in total) were screened for the presence of detectable [125I]hU-II binding sites. 2 UT mRNA (Northern blot, PCR) and protein (immunocytochemistry) were evident in human skeletal muscle tissue and cells. 3 [(125)I]hU-II bound to a homogenous population of high-affinity, saturable (Kd 67.0+/-11.8 pm, Bmax 9687+/-843 sites cell(-1)) receptors in the skeletal muscle (rhabdomyosarcoma) cell line SJRH30. Radiolabel was characteristically slow to dissociate (< or =15% dissociation 90 min). A lower density of high-affinity U-II binding sites was also evident in the rhabdomyosarcoma cell line TE671 (1667+/-165 sites cell(-1), Kd 74+/-8 pm). 4 Consistent with the profile recorded in human recombinant UT-HEK293 cells, [125I]hU-II binding to SJRH30 cells was selectively displaced by both mammalian and fish U-II isopeptides (Kis 0.5+/-0.1-1.2+/-0.3 nm) and related analogues (hU-II[4-11]>[Cys(5,10)]Acm hU-II; Kis 0.4+/-0.1 and 864+/-193 nm, respectively). 5 U-II receptor activation was functionally coupled to phospholipase C-mediated [Ca2+]i mobilization (EC50 6.9+/-2.2 nm) in SJRH30 cells. 6 The present study is the first to identify the presence of 'endogenous' U-II receptors in SJRH30 and TE671 cells. SJRH30 cells, in particular, might prove to be of utility for (a) investigating the pharmacological properties of hU-II and related small molecule antagonists at native human UT and (b) delineating the role of this neuropeptide in the (patho)physiological regulation of mammalian neuromuscular function.
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Receptores Acoplados a Proteínas G/metabolismo , Rabdomiosarcoma/metabolismo , Animales , Sitios de Unión , Unión Competitiva/efectos de los fármacos , Northern Blotting , Péptido Relacionado con Gen de Calcitonina/farmacología , Calcio/metabolismo , Línea Celular , Línea Celular Tumoral , Relación Dosis-Respuesta a Droga , Femenino , Perfilación de la Expresión Génica , Células HeLa , Humanos , Hormonas Hipotalámicas/farmacología , Inmunohistoquímica , Espacio Intracelular/efectos de los fármacos , Espacio Intracelular/metabolismo , Radioisótopos de Yodo , Cinética , Masculino , Melaninas/farmacología , Neuropéptido Y/farmacología , Neurofisinas/farmacología , Toxina del Pertussis/farmacología , Hormonas Hipofisarias/farmacología , Precursores de Proteínas/farmacología , Ensayo de Unión Radioligante , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa , Rabdomiosarcoma/patología , Tapsigargina/farmacología , Urotensinas/genética , Urotensinas/metabolismo , Urotensinas/farmacología , Vasopresinas/farmacologíaRESUMEN
We report the production and characterization of four monoclonal antibodies (MAbs) against human urotensin-II (hU-II). The antibodies were raised against human hU-II, which contains the C-terminus cyclic ring (CFWKYC) that is conserved across species. Multiple selection assays were applied to ensure antibody potency and reactivity against the ring structure. The MAbs reacted via ELISA with hU-II bound to plastic, immunoprecipitated [(125)I-Y(9)] hU-II, bound to biotinylated hU-II in BIAcore analysis and, by Western analysis, recognized the full-length human preprourotensin-II expressed in transfected HEK293 cells. All four MAbs cross-reacted with porcine A, porcine B, rat, mouse, and goby U-II in ELISA. By competitive RIA, hU-II(5-11) (identical to the C-terminus of goby U-II) reacted equivalently to hU-II and goby U-II. The IC(50)s were 0.8 nM for one MAb and 1.6 nM for the others. All four MAbs reacted 15-fold less potently with hU-II(5-10) and 50-fold less potently with hU-II(5-10) amide. Thus, the ring structure and terminal Val/Ile comprise the binding site for this group of MAbs. This panel of antibodies could be useful tools to help delineate the biology and pharmacology of U-II. They may also be of diagnostic value in monitoring hU-II in body fluids.
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Anticuerpos Monoclonales/química , Urotensinas/química , Urotensinas/inmunología , Animales , Sitios de Unión , Unión Competitiva , Bioensayo , Biotinilación , Western Blotting , Línea Celular , Ensayo de Inmunoadsorción Enzimática , Humanos , Hibridomas/metabolismo , Concentración 50 Inhibidora , Cinética , Ratones , Péptidos/química , Pruebas de Precipitina , Unión Proteica , Estructura Terciaria de Proteína , Radioinmunoensayo , Ratas , Porcinos , Transfección , Vasoconstrictores/químicaRESUMEN
1 Urotensin-II (U-II) is among the most potent mammalian vasoconstrictors identified and may play a role in the aetiology of essential hypertension. Currently, only one mouse U-II receptor (UT) gene has been cloned. It is postulated that this protein is solely responsible for mediating U-II-induced vasoconstriction. 2 This hypothesis has been investigated in the present study, which assessed basal haemodynamics and vascular reactivity to hU-II in wild-type (UT((+/+))) and UT receptor knockout (UT((-/-))) mice. 3 Basal left ventricular end-diastolic and end-systolic volumes/pressures, stroke volumes, mean arterial blood pressures, heart rates, cardiac outputs and ejection fractions in UT((+/+)) mice and in UT((-/-)) mice were similar. 4 Relative to UT((+/+)) mouse isolated thoracic aorta, where hU-II was a potent spasmogen (pEC(50)=8.26+/-0.08) that evoked relatively little vasoconstriction (17+/-2% 60 mM KCl), vessels isolated from UT((-/-)) mice did not respond to hU-II. However, in contrast, the superior mesenteric artery isolated from both the genotypes did not contract in the presence of hU-II. Reactivity to unrelated vasoconstrictors (phenylephrine, endothelin-1, KCl) and endothelium-dependent/independent vasodilator agents (carbachol, sodium nitroprusside) was similar in the aorta and superior mesenteric arteries isolated from both the genotypes. 5 The present study is the first to directly link hU-II-induced vasoconstriction with the UT receptor. Deletion of the UT receptor gene results in loss of hU-II contractile action with no 'nonspecific' alterations in vascular reactivity. However, as might be predicted based on the limited contractile efficacy recorded in vitro, the contribution that hU-II and its receptor make to basal systemic haemodynamics appears to be negligible in this species.
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Músculo Liso Vascular/fisiología , Receptores Acoplados a Proteínas G/genética , Urotensinas/metabolismo , Vasoconstricción/fisiología , Animales , Aorta Torácica/efectos de los fármacos , Aorta Torácica/fisiología , Peso Corporal , Marcación de Gen , Genotipo , Hemodinámica , Humanos , Técnicas In Vitro , Masculino , Arteria Mesentérica Superior/efectos de los fármacos , Arteria Mesentérica Superior/fisiología , Ratones , Ratones Noqueados , Músculo Liso Vascular/efectos de los fármacos , Receptores Acoplados a Proteínas G/metabolismo , Urotensinas/farmacología , Urotensinas/fisiología , Vasoconstricción/efectos de los fármacos , Vasoconstrictores/farmacología , Vasodilatadores/farmacologíaRESUMEN
Recent studies have shown that the vasoactive peptide urotensin-II (U-II) exerts a wide range of action on the cardiovascular system of various species. In the present study, we determined the in vivo effects of U-II on basal hemodynamics and cardiac function in the anesthetized intact rat. Intravenous bolus injection of human U-II resulted in a dose-dependent decrease in mean arterial pressure and left ventricular systolic pressure. Cardiac contractility represented by +/-dP/dt was decreased after injection of U-II. However, there was no significant change in heart rate or diastolic pressure. The present study suggests that upregulation of myocardial U-II may contribute to impaired myocardial function in disease conditions such as congestive heart failure.
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Presión Sanguínea/fisiología , Hemodinámica/efectos de los fármacos , Hemodinámica/fisiología , Contracción Miocárdica/efectos de los fármacos , Contracción Miocárdica/fisiología , Urotensinas/administración & dosificación , Urotensinas/farmacología , Función Ventricular Izquierda/efectos de los fármacos , Función Ventricular Izquierda/fisiología , Animales , Relación Dosis-Respuesta a Droga , Humanos , Bombas de Infusión , Inyecciones Intravenosas , RatasRESUMEN
A complementary DNA encoding calcitonin receptor-like receptor (CRLR) was isolated from a bovine aortic endothelial cell library. The bovine CRLR has 462 amino acids and 92% homology with the human CRLR. In a reverse transcriptase-polymerase chain reaction assay, bovine CRLR was found to be widely distributed, including in the heart and lungs. Stable transfection of bovine CRLR in human embryonic kidney cells (HEK-293) resulted in specific high-affinity [125I] rat adrenomedulin (rADM)-binding (dissociation constant=145+/-15 pM). ADM-stimulated adenylyl cyclase activity with an EC50 value of 5.0+/-1.2 nM. The human ADM receptor antagonist hADM(22-52) inhibited [125I]rADM-binding and ADM-stimulated adenylyl cyclase activity. Interactions between bovine CRLR and individual receptor activity modifying proteins (RAMPs) were also investigated. Transient co-transfection of bovine CRLR cDNA with human receptor activity modifying protein 1 (hRAMP1) cDNA in HEK-293 cells resulted in the expression of a CRLR that displayed high-affinity binding to calcitonin gene-related peptide. Co-transfection of bovine CRLR with human RAMP2 or RAMP3 cDNAs in HEK-293 cells displayed high-affinity ADM receptors. These observations suggest that in the absence of exogenous RAMPs heterologous expression of bovine CRLR results in an ADM receptor phenotype.
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Endotelio Vascular/metabolismo , Receptores de Calcitonina/genética , Receptores de Péptidos/metabolismo , Adrenomedulina , Secuencia de Aminoácidos , Animales , Aorta/citología , Proteína Similar al Receptor de Calcitonina , Bovinos , Células Cultivadas , Clonación Molecular , Endotelio Vascular/efectos de los fármacos , Humanos , Péptidos y Proteínas de Señalización Intracelular , Proteínas de la Membrana/metabolismo , Datos de Secuencia Molecular , Péptidos/farmacología , Proteína 2 Modificadora de la Actividad de Receptores , Proteína 3 Modificadora de la Actividad de Receptores , Proteínas Modificadoras de la Actividad de Receptores , Receptores de Adrenomedulina , Receptores de Calcitonina/metabolismo , Receptores de Péptidos/efectos de los fármacos , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa , Homología de Secuencia de AminoácidoRESUMEN
Urotensin-II (U-II) and its receptor (UT) represent novel therapeutic targets for management of a variety of cardiovascular diseases. To test such hypothesis, it will be necessary to develop experimental animal models for the manipulation of U-II/UT receptor system. The goal of this study was to clone mouse and primate preproU-II and UT for pharmacological profiling. Monkey and mouse preproU-II genes were identified to encode 123 and 125 amino acids. Monkey and mouse UT receptors were 389, and 386 amino acids, respectively. Genomic organization of mouse genes showed that the preproU-II has four exons, while the UT receptor has one exon. Although initially viewed by many exclusively as cardiovascular targets, the present study demonstrates expression of mouse and monkey U-II/UT receptor mRNA in extra-vascular tissue including lung, pancreas, skeletal muscle, kidney and liver. Ligand binding studies showed that [125I]h U-II bound to a single sites to the cloned receptors in a saturable/high affinity manner (Kd 654+/-154 and 214+/-65 pM and Bmax of 1011+/-125 and 497+/-68 fmol mg-1 for mouse and monkey UT receptors, respectively). Competition binding analysis demonstrated equipotent, high affinity binding of numerous mammalian, amphibian and piscine U-II isopeptides to these receptors (Ki=0.8 - 3 nM). Fluorescein isothiocyanate (FITC) labelled U-II, bound specifically to HEK-293 cells expressing mouse or monkey UT receptor, confirming cell surface expression of recombinant UT receptor. Exposure of these cells to human U-II resulted in an increase in intracellular [Ca2+] concentrations (EC50 3.2+/-0.8 and 1.1+/-0.3 nM for mouse and monkey UT receptors, respectively) and inositol phosphate (Ip) formation (EC50 7.2+/-1.8 and 0.9+/-0.2 nM for mouse and monkey UT receptors, respectively) consistent with the primary signalling pathway for UT receptor involving phospholipase C activation.