RESUMEN
BACKGROUND: In vitro hemolysis is probably the most common pre-analytic problem in laboratory medicine. However, it introduces variation into results in unknown ways. Therefore, the purpose of this study was to assess the quantitative effects of hemolysis on 80 different, routine laboratory tests. METHODS: We examined the ratio of hemolysis in our hospital from January 1 to March 31, 2015. Next, to study the effect of in vitro hemolysis of whole blood, we added lysed erythrocytes to pooled specimens of serum or plasma to give hemoglobin concentrations of 0.9 to 8.1 g/L and 2.8 to 14 g/L, respectively, and a rating by colorimetry of 0 to 4+ hemolyzed. Then, 80 different laboratory tests were determined with a Hitachi 7700 autoanalyzer for biochemical tests and with AIA 2000, Cobas 6000, and Lumipulus G1200 machines for other tests. RESULTS: Hemolysis occurred in a total in 8.6% of the specimens in our hospital. Significant correlations with the hemolysis ratio were observed in 43 of 80 laboratory tests. At apparent hemolysis, 11 test levels increased and 7 test levels decreased due to hemolysis. Among the 11 tests, potassium, aspartate aminotransferase, lactate dehydrogenase, thymol turbidity test (TTT), zinc sulfate turbidity test (ZTT), and hyaluronic acid tests showed proportional increases due to hemolysis. CONCLUSIONS: Hemolysis is a common problem for accuracy in many routine laboratory tests. Although the quantitative effects of hemolysis can only be roughly estimated in this report, the approximate extent of change in specific laboratory tests is useful for establishing a baseline for future hemolytic studies.
Asunto(s)
Análisis Químico de la Sangre , Recolección de Muestras de Sangre , Técnicas de Laboratorio Clínico , Hemólisis , Biomarcadores/sangre , Errores Diagnósticos , Humanos , Japón , Valor Predictivo de las Pruebas , Reproducibilidad de los ResultadosRESUMEN
Measuring the levels of the plasma free metanephrines (PFMs) represents a recently developed and promising test for the diagnosis of pheochromocytoma in the United States and Europe. As this test has not yet been evaluated in Japan, it is necessary to evaluate the diagnostic efficacy of measuring the levels of PFMs compared with the standard measurement of the urinary excretion of metanephrines (uMNs) whose reliability is well established to detect of pheochromocytoma. A total of 101 Japanese subjects clinically suspected of having pheochromocytoma in were included in this study. Subsequently, we prospectively measured the PFMs levels in all patients, compared with those of biochemical markers of the catecholamine secretion and metabolisms in the plasma and urine. All subjects with adrenal tumors underwent tumor excision. Data were available for 84 of the 101 patients, 47 of whom had histopathologically proven pheochromocytoma and 37 were finally diagnosed with non-pheochromocytoma. The results of comparisons in the accuracy of measurement for diagnosis of pheochromocytoma between PFMs and the urinary excretion of metanephrines (uMNs) were 0.980 VS 0.951 for AUC of receiver operatorating characteristic (ROC) curve, 0.957 VS 0.894 for sensitivity, and 0.973 VS 0.946 for specificity, respectively. Although the differences were small, the results of our study definitely demonstrated that measurement of PFMs was not inferior to standard urinary metanephrines (uMNs) measurement, which is established to be the most reliable biochemical method to detect pheochromocytoma. This study clearly shows measuring the PFMs levels to be a reliable and efficient method for diagnosing pheochromocytoma in Japanese patients, as demonstrated in previous reports.
Asunto(s)
Neoplasias de las Glándulas Suprarrenales/diagnóstico , Metanefrina/sangre , Normetanefrina/sangre , Feocromocitoma/diagnóstico , Neoplasias de las Glándulas Suprarrenales/sangre , Neoplasias de las Glándulas Suprarrenales/patología , Neoplasias de las Glándulas Suprarrenales/orina , Glándulas Suprarrenales/patología , Adulto , Biomarcadores/sangre , Biomarcadores/química , Biomarcadores/orina , Estudios de Cohortes , Femenino , Hospitales Municipales , Hospitales Universitarios , Humanos , Técnicas para Inmunoenzimas , Japón , Masculino , Metanefrina/química , Metanefrina/orina , Persona de Mediana Edad , Normetanefrina/química , Normetanefrina/orina , Feocromocitoma/sangre , Feocromocitoma/patología , Feocromocitoma/orina , Estudios Prospectivos , Sensibilidad y Especificidad , Solubilidad , Ácido Vanilmandélico/orinaRESUMEN
Sunitinib is an oral multitargeted receptor tyrosine kinase inhibitor with antiangiogenic and antitumor activity that mainly targets vascular endothelial growth factor receptors (VEGFRs). Very recently, sunitinib has been shown to be an active agent for the treatment of malignant pheochromocytomas. However, it is unclear whether sunitinib acts only through an antiangiogenic mechanism or whether it may also directly target tumor cells. Sunitinib markedly induced apoptosis of PC12 cells in a dose-dependent and time-dependent manner. Furthermore, in support of these findings, we found that sunitinib induced a reduction in the expression of the antiapoptotic molecule Bcl-2 as well as dephosphorylation of the proapoptotic molecule BAD, which results in the activation of BAD in these cells. Consistent with these apoptotic effects, our results showed that sunitinib inhibited phosphorylation of Akt and mTOR and was followed by a reduction of S6K1, which is a well-known target of mTOR. Knockdown of VEGFR-2 attenuated the sunitinib-induced effects, including apoptosis and inhibition of signaling pathways such as the phosphorylation of Akt as well as mTOR, and Bcl-2, which confirmed that these effects could be mediated by VEGFR-2. In addition, silencing of S6K1 induced apoptosis accompanied by a decrease in the phosphorylation of BAD and Bcl-2, similar to that observed with sunitinib treatment. Thus, these results together suggest that sunitinib initially exerts its apoptotic effect through the inhibition of VEGFR-2, which, when followed by reduction of its downstream effectors, including Akt/mTOR/S6K1, may lead to inhibition of the antiapoptotic molecule Bcl-2 and activation of the proapoptotic molecule BAD in PC12 cells. However, PC12 cells do not precisely reflect the pathogenesis of malignant cells. Therefore, we confirmed the key findings by replicating these experiments in human neuroblastoma SK-N-SH cells.
Asunto(s)
Inhibidores de la Angiogénesis/farmacología , Apoptosis/efectos de los fármacos , Indoles/farmacología , Proteína Oncogénica v-akt/antagonistas & inhibidores , Proteínas Proto-Oncogénicas c-bcl-2/metabolismo , Pirroles/farmacología , Proteínas Quinasas S6 Ribosómicas/antagonistas & inhibidores , Transducción de Señal/efectos de los fármacos , Serina-Treonina Quinasas TOR/antagonistas & inhibidores , Receptor 2 de Factores de Crecimiento Endotelial Vascular/antagonistas & inhibidores , Proteína Letal Asociada a bcl/metabolismo , Animales , Antimetabolitos Antineoplásicos , Western Blotting , Bromodesoxiuridina , Línea Celular Tumoral , Proliferación Celular/efectos de los fármacos , Colorantes , Etiquetado Corte-Fin in Situ , Indicadores y Reactivos , Análisis por Micromatrices , Células PC12 , Proteínas Proto-Oncogénicas c-bcl-2/efectos de los fármacos , ARN Interferente Pequeño/genética , Ratas , Sunitinib , Sales de Tetrazolio , Tiazoles , Proteína Letal Asociada a bcl/efectos de los fármacosRESUMEN
Recently, mutations in nuclear genes encoding two mitochondrial complex II subunit proteins, Succinate dehydrogenase D (SDHD) and SDHB, have been found to be associated with the development of familial pheochromocytomas and paragangliomas (hereditary pheochromocytoma/paraganglioma syndrome: HPPS). Growing evidence suggests that the mutation of SDHB is highly associated with abdominal paraganglioma and the following distant metastasis (malignant paraganglioma). In the present study, we used multiplex ligation dependent probe amplification (MLPA) analysis to identify a large heterozygous SDHB gene deletion encompassing sequences corresponding to the promoter region, in addition to exon 1 and exon 2 malignant paraganglioma patient in whom previously characterized SDHB mutations were undetectable. This is the first Japanese case report of malignant paraganglioma, with a large SDHB deletions. Our present findings strongly support the notion that large deletions in the SDHB gene should be considered in patients lacking characterized SDHB mutations.
Asunto(s)
Eliminación de Gen , Paraganglioma Extraadrenal/genética , Succinato Deshidrogenasa/genética , Adulto , Catecolaminas/sangre , ADN/sangre , ADN/química , Exones/genética , Femenino , Heterocigoto , Humanos , Hipertensión , Neoplasias Hepáticas/secundario , Metástasis de la Neoplasia , Recurrencia Local de Neoplasia/diagnóstico , Norepinefrina/sangre , Normetanefrina/orina , Cuerpos Paraaórticos , Paraganglioma Extraadrenal/diagnóstico , Paraganglioma Extraadrenal/patología , Reacción en Cadena de la Polimerasa , Regiones Promotoras Genéticas/genética , Análisis de Secuencia de ADN , Tomografía Computarizada por Rayos XRESUMEN
Recently, nuclear genes encoding two mitochondrial complex II subunit proteins, SDHD and SDHB, have been found to be associated with the development of familial pheochromocytomas and paragangliomas (hereditary pheochromocytoma/paraganglioma syndrome: HPPS). Growing evidence suggests that the mutation of SDHB is highly associated with abdominal paraganglioma and the following distant metastasis (malignant paraganglioma). In the present study, we report the case of a novel SDHB mutation (L157X) in a Japanese patient with abdominal paraganglioma following malignant lung metastasis. In addition, we identified an asymptomatic carrier of the SDHB mutation in this family.
Asunto(s)
Paraganglioma/genética , Neoplasias Retroperitoneales/genética , Succinato Deshidrogenasa/genética , Adulto , Pueblo Asiatico/genética , Femenino , Mutación de Línea Germinal , Humanos , Neoplasias Pulmonares/patología , Neoplasias Pulmonares/secundario , Masculino , Persona de Mediana Edad , Linaje , Neoplasias Retroperitoneales/patologíaRESUMEN
We investigated the effects of beta(3)-adrenoceptor agonist, 5-[(2R)-2-[[(2R)-2-(3-chlorophenyl)-2-hydroxyethyl]amino]propyl]-1,3-benzodioxole-2,2-dicarboxylate (CL-316,243) in obese diabetic KKAy mice. Two weeks' subcutaneous administration of CL-316,243 reduced serum levels of glucose, insulin, triglyceride, free fatty acid and tumor necrosis factor-alpha (TNF-alpha), and increased adiponectin. Adiponectin, adiponectin receptors and beta(3)-adrenoceptor mRNA expressions were reduced in epididymal white adipose tissue in KKAy mice, and CL-316,243 recovered these mRNA expressions. Meanwhile, CL-316,243 suppressed the overexpressed mRNA level of TNF-alpha in both epididymal white adipose tissue and brown adipose tissue. These data suggest that the normalization of adiponectin, adiponectin receptors and TNF-alpha may result in the amelioration of obesity-induced insulin resistance.
Asunto(s)
Tejido Adiposo/efectos de los fármacos , Agonistas de Receptores Adrenérgicos beta 3 , Agonistas Adrenérgicos beta/farmacología , Diabetes Mellitus/tratamiento farmacológico , Dioxoles/farmacología , Hipoglucemiantes/farmacología , Obesidad/tratamiento farmacológico , Receptores de Adiponectina/metabolismo , Factor de Necrosis Tumoral alfa/metabolismo , Adiponectina/sangre , Adiponectina/genética , Adiponectina/metabolismo , Tejido Adiposo/metabolismo , Agonistas Adrenérgicos beta/administración & dosificación , Animales , Glucemia/efectos de los fármacos , Peso Corporal , Diabetes Mellitus/etiología , Diabetes Mellitus/metabolismo , Diabetes Mellitus/fisiopatología , Grasas de la Dieta/administración & dosificación , Dioxoles/administración & dosificación , Modelos Animales de Enfermedad , Ingestión de Alimentos , Hipoglucemiantes/administración & dosificación , Inyecciones Subcutáneas , Insulina/sangre , Resistencia a la Insulina , Lípidos/sangre , Masculino , Ratones , Obesidad/complicaciones , Obesidad/metabolismo , Obesidad/fisiopatología , Receptores de Adiponectina/sangre , Receptores de Adiponectina/genética , Receptores Adrenérgicos beta 3/genética , Receptores Adrenérgicos beta 3/metabolismo , Factor de Necrosis Tumoral alfa/sangreRESUMEN
Recently, nuclear genes encoding two mitochondrial complex II subunit proteins, SDHD and SDHB, have been found to be associated with the development of familial pheochromocytomas and paragangliomas (hereditary pheochromocytoma/paraganglioma syndrome: HPPS). Growing evidence suggests that a mutation of SDHB is highly associated with abdominal (or thoracic) paraganglioma and the following distant metastasis (malignant paraganglioma). Previously, we identified a novel heterozygous G to A point mutation at the first base of intron 3 of the SDHB gene (IVS3+1G>A) in a malignant abdominal paraganglioma from a Japanese patient. In the present study, we report another case of SDHB mutation (R46Q) in a Japanese patient with both abdominal and thoracic paraganglioma following malignant metastasis. In addition, we identified an asymptomatic carrier of SDHB mutation in this family. Our report highlights the pathogenic role of the SDHB mutation (R46Q) in malignant paraganglioma. We also discuss the desired protocol that should be adopted to follow up an asymptomatic carrier of this mutation.
Asunto(s)
Neoplasias Abdominales/genética , Paraganglioma/genética , Mutación Puntual/genética , Succinato Deshidrogenasa/genética , Neoplasias Torácicas/genética , Neoplasias Abdominales/diagnóstico , Neoplasias Abdominales/secundario , Secuencia de Bases , Predisposición Genética a la Enfermedad , Humanos , Intrones/genética , Masculino , Persona de Mediana Edad , Metástasis de la Neoplasia , Paraganglioma/diagnóstico , Paraganglioma/secundario , Linaje , Neoplasias Torácicas/diagnóstico , Neoplasias Torácicas/secundarioRESUMEN
AIM: Adiponectin reportedly reduces insulin resistance. Exercise has also been shown to lessen insulin resistance, although it is not well known whether exercise increases levels of adiponectin and/or its receptors nor whether it effects are dependent on exercise intensity and/or period. We previously reported that blood adiponectin levels increased by 150% in animals that exercised at a rate of 30 m/min for 60 minutes, 2 days per week, and adiponectin receptor 1 (AdipoR1) mRNA levels in muscle increased up to 4 times in response to exercise at a rate of 25 m/min for 30 min, 5 days per week for 12 weeks. METHODS: In light of this information, we examined the effects of short-term exercise on adiponectin, and adiponectin receptor levels in rats, using ELISA and real-time PCR. RESULTS: Our data showed that adiponectin mRNA levels in adipose tissue increased by 280% in rats exercised at a rate of 30 m/min for 60 minutes for 2 weeks and correlated with the exercise time periods. No effects of short-term exercise on adiponectin receptor 1 mRNA in muscle were observed. CONCLUSION: Thus, long-term exercise may be required to regulate adiponectin receptor 1 mRNA expression in muscle and adiponectin mRNA expression in adipose tissue.
Asunto(s)
Adiponectina/biosíntesis , Condicionamiento Físico Animal/fisiología , Receptores de Adiponectina/biosíntesis , Tejido Adiposo/metabolismo , Animales , Glucemia/fisiología , Ensayo de Inmunoadsorción Enzimática , Epinefrina/orina , Expresión Génica , Lípidos/sangre , ARN Mensajero/análisis , Ratas , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa , TiempoRESUMEN
Recently, the insulin-sensitizing adipokine adiponectin and the insulin resistance-inducing adipokine tumor necrosis factor-alpha (TNF-alpha) were reported to inhibit each other's production in adipocytes. We investigated the effects of two beta(3)-adrenoceptor agonists, 5-[(2R)-2-[[(2R)-2-(3-chlorophenyl)-2-hydroxyethyl]amino]propyl]-1,3-benzodioxole-2,2-dicarboxylate (CL-316,243) and (+/-)-(R(*),R(*))-[4-[2-[[2-(3-chlorophenyl)-2-hydroxyethyl]amino]propyl]phenoxy]acetic acid (BRL37344), on the gene expression of adiponectin, two adiponectin receptors, and TNF-alpha in adipose tissues of C57BL/6J mice. CL-316,243 and BRL37344 downregulated adiponectin, but upregulated adiponectin receptor 2 (not receptor 1) in epididymal or/and subcutaneous white adipose tissues and in brown adipose tissue. TNF-alpha expression was upregulated only in epididymal adipose tissue. To further explore these effects, we treated differentiated 3T3-L1 adipocytes with the non-selective beta-adrenoceptor agonist isoproterenol. As a result, adiponectin receptor 2 (but not receptor 1) gene expression and TNF-alpha protein expression increased, but gene expression and secretion of adiponectin decreased. The upregulation of adiponectin receptor 2 by isoproterenol is most likely via beta(2),beta(3)-adrenoceptors, adenylyl cyclases, and protein kinase A (PKA). However, the accompanying activation of AMP-activated protein kinase (AMPK) may inhibit this upregulation. Our results suggest that upregulation of TNF-alpha and downregulation of adiponectin by beta-adrenoceptor activation may contribute to the pathogenesis of catecholamine-induced insulin resistance, and that upregulation of adiponectin receptor 2 may be a feedback result of reduced adiponectin.
Asunto(s)
Adipocitos/efectos de los fármacos , Adiponectina/metabolismo , Agonistas Adrenérgicos beta/farmacología , Receptores de Superficie Celular/metabolismo , Factor de Necrosis Tumoral alfa/metabolismo , Células 3T3-L1 , Proteínas Quinasas Activadas por AMP , Adenilil Ciclasas/metabolismo , Adipocitos/citología , Adipocitos/metabolismo , Adiponectina/genética , Animales , Western Blotting , Colforsina/farmacología , Proteínas Quinasas Dependientes de AMP Cíclico/metabolismo , Regulación hacia Abajo/efectos de los fármacos , Activación Enzimática/efectos de los fármacos , Etanolaminas/farmacología , Fenoterol/farmacología , Regulación de la Expresión Génica/efectos de los fármacos , Isoproterenol/farmacología , Isoquinolinas/farmacología , Masculino , Ratones , Ratones Endogámicos C57BL , Complejos Multienzimáticos/metabolismo , Proteínas Serina-Treonina Quinasas/metabolismo , ARN Mensajero/genética , ARN Mensajero/metabolismo , Receptores de Adiponectina , Receptores de Superficie Celular/genética , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa , Sulfonamidas/farmacología , Factor de Necrosis Tumoral alfa/genética , Regulación hacia Arriba/efectos de los fármacosRESUMEN
The SDHA, SDHB, SDHC, and SDHD genes code for subunits of succinate dehydrogenase (SDH), which forms part of the mitochondrial respiratory chain. Germline mutations in the genes encoding SDHB and SDHD have been reported in familial paragangliomas/pheochromocytomas and in apparently sporadic pheochromocytomas. SDHB and SDHD mutations are widely distributed along the genes with no apparent hot spots. SDHB mutations are often detected in malignant and extra-adrenal pheochromocytomas. SDHD mutations are also detected frequently in head and neck paragangliomas. We sequenced the entire coding regions of the SDHB and SDHD genes in 17 pheochromocytomas. We identified novel heterozygous G to A point mutations at the first base of intron 3 of the SDHB gene in a malignant extra-adrenal abdominal pheochromocytoma patient, and at the first base of codon 111 of the SDHD gene in an adrenal pheochromocytoma patient. Further, we confirmed the SDHD mutation by DHPLC. The prevalence of SDHB and SDHD mutations in pheochromocytomas we examined was 12% (2/17). Thus, we identified two novel SDH mutations in Japanese pheochromocytomas. Further studies will investigate the oncogenic potential of these mutations.
Asunto(s)
Neoplasias de las Glándulas Suprarrenales/genética , Mutación de Línea Germinal , Proteínas Hierro-Azufre/genética , Feocromocitoma/genética , Succinato Deshidrogenasa/genética , Adulto , Secuencia de Bases , Análisis Mutacional de ADN , Femenino , Frecuencia de los Genes , Pruebas Genéticas , Humanos , Japón , Masculino , Persona de Mediana EdadRESUMEN
The small GTPase, RhoA, and its downstream effecter Rho-kinase (ROK) are reported to be involved in various cellular functions, such as myosin light chain phosphorylation during smooth muscle contraction and exocytosis. Indeed, growing evidence suggests that the RhoA/Rho-kinase pathway plays an important role in regulating exocytosis in these cells. However, it is not known whether the RhoA/Rho-kinase pathway has an effect on catecholamine synthesis. Using the rat pheochromocytoma cell line, PC12, we examined the effects of either Rho-kinase inhibitor (Y27632) or RhoA inhibitor (C3 toxin) on nicotine-induced catecholamine biosynthesis. We show that nicotine (10 microM) induces a significant, though transient, increase in RhoA activation in these cells. Treatment with either Y27632 (1 microM) or C3 toxin (10 microg/ml) significantly inhibited the nicotine-induced increase of tyrosine hydroxylase (TH) mRNA and the corresponding enzyme activity. TH catalyzes the rate-limiting step in the biosynthesis of catecholamine. Y27632 significantly inhibited nicotine-induced phosphorylation of TH at Ser40 as well as Ser19, which are known to be phosphorylated by Ca(2+)/calmodulin kinase II. Furthermore, Y27632 (10 microM) as well as C3 toxin (10 microg/ml) significantly inhibited the nicotine-induced increase of TH at the protein level. Thus, we propose that activation of RhoA, and its downstream effecter Rho-kinase, is a prerequisite for catecholamine biosynthesis in PC12 cells. At the concentrations used in our experiments, Y27632 does not affect cAMP/PKA activity or PKC activity, indicating that the inhibitory effect of Y27632 can be attributed to the inhibition of Rho-kinase activity as observed in chromaffin cells. In contrast, neither Y27632 (10 microM) nor C3 toxin (10 microg/ml) significantly altered catecholamine secretion in PC12 cells. In conclusion, we have demonstrated that inhibition of the Rho/Rho-kinase pathway in chromaffin cells lowers TH activity, probably through CaMKII inhibition. By contrast, neither Y27632 nor C3 toxin affect the secretion of catecholamine.
Asunto(s)
Catecolaminas/biosíntesis , Regulación Enzimológica de la Expresión Génica/efectos de los fármacos , Proteínas Serina-Treonina Quinasas/antagonistas & inhibidores , Transducción de Señal/efectos de los fármacos , Proteína de Unión al GTP rhoA/antagonistas & inhibidores , Amidas/farmacología , Análisis de Varianza , Animales , Western Blotting , Toxinas Botulínicas/farmacología , AMP Cíclico/metabolismo , Proteínas Quinasas Dependientes de AMP Cíclico/metabolismo , Cartilla de ADN , Glutatión Transferasa , Péptidos y Proteínas de Señalización Intracelular , Nicotina/metabolismo , Células PC12 , Fosforilación , Reacción en Cadena de la Polimerasa , Proteína Quinasa C/metabolismo , Proteínas Serina-Treonina Quinasas/metabolismo , Piridinas/farmacología , Ratas , Tirosina 3-Monooxigenasa/metabolismo , Quinasas Asociadas a rho , Proteína de Unión al GTP rhoA/metabolismoRESUMEN
Mutations in the genes encoding succinate dehydrogenase (SDH) have been associated with susceptibility to pheochromocytoma. However, few reports have examined the level of SDH mRNAs expression. In this study, we examined the level of expression of mRNAs encoding SDHB, SDHC, and SDHD in pheochromocytoma, pheochromocytoma subgroups, and normal adrenal gland, and compared the expression of these genes to the level of expression of related genes in the same tissues. The mean relative level of expression of SDHB, SDHC, SDHD and VHL mRNA was 28.7+/-6.2%, 16.6+/-4.8%, 214+/-47.5% and 25.9+/-8.2%, respectively, in pheochromocytoma tissues compared to normal adrenal gland. Furthermore, the mean relative level of the RET proto-oncogene mRNA was 707+/-149% in pheochromocytoma compared to normal adrenal gland. The level of expression of the SDH genes was highly correlated in each individual sample (P<0.0001). The level of expression of the SDH mRNAs correlated with the level of VHL mRNA (P<0.0001), but not with the level of RET mRNA. The level of SDH mRNAs expression also correlated with the expression of phenylethanolamine N-methyl transferase (PNMT), an adrenaline synthesizing enzyme (P<0.01), which may explain the correlation between SDH expression and adrenaline content (P<0.05). The level of SDH mRNAs expression correlated strongly with the expression of VEGF mRNA (P<0.0001). In multiple endocrine neoplasia (MEN) 2a, the expression of the SDH genes and VHL mRNA was significantly higher than that observed in adrenal or extra-adrenal pheochromocytoma. The expression of the corticotropin-releasing hormone (CRH) mRNA was significantly higher in extra-adrenal pheochromocytoma than in adrenal pheochromocytoma or MEN2a. Thus, tumor-specific gene expression exists in pheochromocytoma, which may explain the characteristics of the tumor.
Asunto(s)
Neoplasias de las Glándulas Suprarrenales/genética , Feocromocitoma/genética , ARN Mensajero/genética , Succinato Deshidrogenasa/genética , Adolescente , Adulto , Anciano , Femenino , Humanos , Masculino , Persona de Mediana Edad , Reacción en Cadena de la Polimerasa , Proto-Oncogenes MasRESUMEN
Adenosine monophosphate-activated protein kinase (AMPK) is activated in response to adenosine triphosphate depletion caused by the metabolic and nutritional state. Mammalian AMPK is a heterotrimeric enzyme composed of a catalytic alpha subunit and 2 regulatory subunits (beta and gamma). Although much attention has been focused on exercise-induced AMPK activation in skeletal muscle, little information is available on the role of AMPK in adipose tissue and liver. Acetyl-coenzyme A carboxylase (ACC) is a well-known downstream target of AMPK. The ACC contains serine residues that are phosphorylated by AMPK. The present study was undertaken to determine whether long-term exercise of medium intensity (60% of Vo2max for 12 weeks) may influence AMPK enzyme activity, gene/protein expression, and subsequent ACC phosphorylation in rat adipose tissue (visceral and subcutaneous) and liver. We initially demonstrated that long-term exercise induced a significant increase in phosphorylation of Thr172 in the AMPK alpha1 subunit and of Ser79 in ACC in visceral adipose tissue rather than subcutaneous tissue. We also demonstrated that the AMPK alpha1-,alpha2-subunit messenger RNA (mRNA) level as well as the corresponding protein levels were increased in response to long-term exercise, whereas the other subunits were not altered significantly. In contrast to that of visceral adipose tissue, long-term exercise did not induce any significant effect on any of the AMPK subunit mRNA levels or alpha1-,alpha2-subunit protein levels in subcutaneous adipose tissue. In addition to adipose tissue, we demonstrated that long-term exercise induced an increase in both AMPK/ACC phosphorylation and alpha1-,alpha2-subunit mRNA/protein expression in the liver. Although the precise physiologic relevance of AMPK activation in these tissues remains unknown, it is possible that it might play an important role in long-term exercise-induced adaptation mechanisms and may lead to an improvement in certain metabolic abnormalities in metabolic diseases.
Asunto(s)
Tejido Adiposo/enzimología , Hígado/enzimología , Complejos Multienzimáticos/metabolismo , Condicionamiento Físico Animal/fisiología , Proteínas Serina-Treonina Quinasas/metabolismo , Proteínas Quinasas Activadas por AMP , Animales , Glucemia/metabolismo , Western Blotting , Peso Corporal/fisiología , Colesterol/sangre , HDL-Colesterol/sangre , Masculino , Complejos Multienzimáticos/biosíntesis , Consumo de Oxígeno/fisiología , Proteínas Serina-Treonina Quinasas/biosíntesis , ARN Mensajero/biosíntesis , ARN Mensajero/genética , Ratas , Ratas Wistar , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa , Triglicéridos/sangreRESUMEN
Norepinephrine is a well known major vasoconstricting factor. Recent reports suggest that norepinephrine, in addition to acting as a vasoconstricting factor, may also play several additional roles in endothelial cells. These include: 1] induction of NO release. It has been demonstrated that a small GTP-binding protein, Rho, and its downstream effecter, Rho kinase (ROCK), negatively regulate endothelial nitric oxide synthase (eNOS) production. However, it is not known whether ROCK is directly involved in norepinephrine-induced NO release. 2] Norepinephrine is reported to induce a mitogenic effect, but whether MAPKs are involved in this process is unknown. 3] Recently, we demonstrated an increase in vascular endothelial growth factor (VEGF) mRNA/protein expression in human pheochromocytoma tissue in comparison to normal adrenomedullary tissue. Thus, it is reasonable to speculate that norepinephrine may stimulate the level of VEGF mRNA. The aim of the present study was to clarify the role of norepinephrine and related endothelial adrenoceptor systems in various pathophysiological conditions, such as hypertension and in particular pheochromocytoma, using human umbilical vein endothelial cells (HUVEC). Norepinephrine-induced RhoA attenuation, through cAMP/protein kinase A (PKA) activation coupled with beta-adrenoceptors, may lead to eNOS activation in acute conditions. Norepinephrine stimulates the production of VEGF mRNA through cAMP/PKA activation coupled with beta-adrenoceptors. Norepinephrine stimulates a mitogenic effect through ERK activation coupled with the alpha(1)-adrenoceptor. In conclusion, norepinephrine stimulates eNOS activity via RhoA attenuation, VEGF mRNA synthesis and mitogenic activity in endothelial cells. We propose that an excess of norepinephrine can lead to endothelial dysfunction due to these aforementioned processes.
Asunto(s)
Células Endoteliales/metabolismo , Proteínas Quinasas Activadas por Mitógenos/biosíntesis , Norepinefrina/farmacología , Simpatomiméticos/farmacología , Factor A de Crecimiento Endotelial Vascular/biosíntesis , Proteína de Unión al GTP rhoA/biosíntesis , Western Blotting , Recuento de Células , Proliferación Celular/efectos de los fármacos , Células Endoteliales/efectos de los fármacos , Humanos , Óxido Nítrico Sintasa de Tipo III/biosíntesis , Fosforilación , ARN Mensajero/biosíntesis , Receptores Adrenérgicos/efectos de los fármacos , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa , Venas Umbilicales/citología , Venas Umbilicales/efectos de los fármacos , Venas Umbilicales/metabolismoRESUMEN
Plasma non-HDL cholesterol (HDL-C) concentration that is simply estimated from plasma total cholesterol and HDL-C concentrations, without the influence of plasma triglyceride concentration, has been included as a therapeutic target for hypertriglyceridemic patients in the most recent National Cholesterol Education Program (NCEP) recommendations. In the present study, we estimated plasma non-HDL-C concentration in Japanese subjects to clarify the correlation of plasma non-HDL-C to other plasma lipid concentrations, and to evaluate the NCEP recommendation. Plasma non-HDL-C concentration has a positive correlation with low-density lipoprotein cholesterol (LDL-C) and triglyceride concentrations. From our analysis, 140 mg/dl of plasma LDL-C concentration, which is the level for the diagnosis of hyper-LDL cholesterolemia, corresponds to 169 mg/dl of non HDL-C concentration. The relationship between plasma non-HDL-C and LDL-C concentrations in Japanese subjects is quite similar to that described in the NCEP guideline. Thus, we suggest that non-HDL-C is a useful risk marker in Japan, as recommended by the NCEP.
Asunto(s)
Arteriosclerosis/sangre , HDL-Colesterol/sangre , LDL-Colesterol/sangre , Triglicéridos/sangre , Arteriosclerosis/epidemiología , Biomarcadores/sangre , Femenino , Humanos , Japón/epidemiología , Masculino , Persona de Mediana Edad , Prevalencia , Estudios Retrospectivos , Factores de RiesgoRESUMEN
Urocortin is a novel mammalian member of the corticotrophin releasing factor (CRF)-related peptides. We have investigated the expression, mechanism of action and second messenger for urocortin in rat pheochromocytoma PC12 cells. We initially confirmed the expression of urocortin and CRF-R2beta, which is thought to be an endogenous receptor for urocortin, in PC12 cells. We also demonstrate that urocortin (> or = 1 nM) significantly elevates the level of cAMP in these cells. Moreover, alpha-helical CRF-(9-41), a more specific antagonist of CRF-R2 than CRF-R1 and the adenylate cyclase inhibitor SQ22536, inhibited the urocortin-induced increase in the level of cAMP. Thus, urocortin may exert its physiological role in chromaffin cells via CRF-R2beta coupling to adenylate cyclase. Urocortin (> or = 1 nM) significantly increased the mRNA level and activity of tyrosine hydroxylase (TH), a rate-limiting enzyme in the biosynthesis of catecholamine. Furthermore, urocortin-induced changes in TH-mRNA and activity were inhibited by H89 (a PKA inhibitor) and SQ22536 as well as alpha-helical CRF-(9-41). However, urocortin did not affect DNA synthesis or catecholamine secretion in these cells. In conclusion, we have demonstrated that urocortin stimulates catecholamine biosynthesis via the cAMP/protein kinase A pathway in PC12 cells, where both urocortin and its receptor, CRF-R2, are expressed.
Asunto(s)
Adenina/análogos & derivados , Hormona Liberadora de Corticotropina/fisiología , Proteínas Quinasas Dependientes de AMP Cíclico/fisiología , AMP Cíclico/fisiología , Tirosina 3-Monooxigenasa/metabolismo , Adenina/farmacología , Inhibidores de Adenilato Ciclasa , Animales , Catecolaminas/biosíntesis , Catecolaminas/metabolismo , Hormona Liberadora de Corticotropina/biosíntesis , Hormona Liberadora de Corticotropina/metabolismo , Hormona Liberadora de Corticotropina/farmacología , Proteínas Quinasas Dependientes de AMP Cíclico/antagonistas & inhibidores , ADN/biosíntesis , Inhibidores Enzimáticos/farmacología , Isoquinolinas/farmacología , Células PC12 , Ratas , Sistemas de Mensajero Secundario/fisiología , Sulfonamidas/farmacología , UrocortinasAsunto(s)
Dopamina beta-Hidroxilasa/sangre , Adolescente , Neoplasias de las Glándulas Suprarrenales/enzimología , Adulto , Niño , Preescolar , Dopamina beta-Hidroxilasa/deficiencia , Femenino , Humanos , Lactante , Masculino , Persona de Mediana Edad , Neuroblastoma/enzimología , Feocromocitoma/enzimologíaRESUMEN
It has been reported that endothelins (ETs) stimulate catecholamine release from chromaffin cells. However, it is not known whether ETs also affect catecholamine biosynthesis. Thus, using a rat pheochromocytoma cell line, PC12, we examined the effects of ETs on catecholamine biosynthesis. The mRNA level and activity of tyrosine hydroxylase (TH), a rate-limiting enzyme in catecholamine biosynthesis, were increased significantly by endothelin-1 (ET-1) (100nM). These stimulatory effects were inhibited completely by a blocker for the A-type endothelin receptor, BQ-123 [cyclo(D-alpha-aspartyl-L-prolyl-D-valyl-L-leucyl-D-tryptophyl)] (1 microM), but not by a blocker for the B-type endothelin receptor, BQ-788 (N-cis 2,6-dimethylpiperidinocarbonyl-L-gamma-methylleucyl-D-1-methoxycarbonyltryptophanyl-D-norleucine (1 microM). Also, Ro-32-0432 (3-[8-[(dimethylamino)methyl]-6,7,8,9-tetrahydropyrido-[1,2-a]indol-10-yl]-4-(1-methyl-3-indolyl)-H-pyrrole-2,5-dione hydrochloride) (100nM), a protein kinase C inhibitor, completely inhibited ET-1-induced increases in TH activity and mRNA level. Furthermore, ET-1 (100nM) significantly stimulated protein kinase C activity, as well as inositol 1,4,5-triphosphate production; these stimulatory effects were abolished by BQ-123 but not by BQ-788. Moreover, ET-1 (100nM) significantly increased both the TH-protein level and the intracellular catecholamine content. By contrast to ET-1, endothelin-3 did not affect catecholamine synthesis. These results indicate that ET-1, but not ET-3, stimulates catecholamine synthesis through the PKC pathway in PC12 cells. Also, the use of selective ET receptor antagonists suggests that the effects of ET-1 on catecholamine biosynthesis are mediated through ET(A).