RESUMEN
BACKGROUND AND AIMS: The progression of nonalcoholic fatty liver disease (NAFLD) into severe histological forms (steatohepatitis - NASH) is paralleled by the occurrence of complex molecular processes. Mitochondrial dysfunction is a hallmark feature of advanced disease. Mitochondrially encoded cytochrome B (cytochrome b, MT-CYB), a member of the oxidative phosphorylation system, is a key component of the respirasome supercomplex. Here, we hypothesized that NAFLD severity is associated with liver tissue cytochrome b mutations and damaged mitochondrial DNA (mtDNA). METHODS: We included 252 liver specimens of NAFLD patients - in whom histological disease ranged from mild to severe - which were linked to clinical and biochemical information. Tissue molecular explorations included MT-CYB sequencing and analysis of differential mtDNA damage. Profiling of circulating Krebs cycle metabolites and global liver transcriptome was performed in a subsample of patients. Tissue levels of 4-hydroxynonenal - a product of lipid peroxidation and 8-hydroxy-2'-deoxyguanosine, a marker of oxidative damage - were measured. RESULTS: Compared to simple steatosis, NASH is associated with a higher level of MT-CYB variance, 12.1 vs. 15.6 substitutions per 103 bp (P = 5.5e-10). The burden of variants was associated with increased levels of 2-hydroxyglutarate, branched-chain amino acids, and glutamate, and changes in the global liver transcriptome. Liver mtDNA damage was associated with advanced disease and inflammation. NAFLD severity was associated with increased tissue levels of DNA oxidative adducts and lipid peroxyl radicals. CONCLUSION: NASH is associated with genetic alterations of the liver cellular respirasome, including high cytochrome b variation and mtDNA damage, which may result in broad cellular effects.
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Citocromos b/genética , Daño del ADN , ADN Mitocondrial , Hígado/metabolismo , Enfermedad del Hígado Graso no Alcohólico/genética , Enfermedad del Hígado Graso no Alcohólico/metabolismo , 8-Hidroxi-2'-Desoxicoguanosina/sangre , Adulto , Anciano , Aldehídos/sangre , Aminoácidos de Cadena Ramificada/sangre , Progresión de la Enfermedad , Ácido Glutámico/sangre , Glutaratos/sangre , Humanos , Peroxidación de Lípido , Persona de Mediana Edad , Mutación , Enfermedad del Hígado Graso no Alcohólico/complicaciones , Obesidad/complicaciones , Obesidad/genética , Obesidad/metabolismo , Fosforilación Oxidativa , Estrés Oxidativo , Índice de Severidad de la Enfermedad , TranscriptomaRESUMEN
Angiotensin II released serotonin from neuron terminals and accelerated synthesis of the serotonin. This increase in synthesis depended on the activation of tryptophan hydroxylase. A biphasic effect was observed: at high doses the stimulatory effect depended on conversion of angiotensin II to angiotensin III. At low doses an inhibitory effect was found, possible dependent on an angiotensin II metabolite. These actions represent a subtle regulation of the open-loop serotonin system.
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Angiotensina III/farmacología , Angiotensina II/análogos & derivados , Angiotensina II/farmacología , Serotonina/metabolismo , Triptófano Hidroxilasa/metabolismo , Animales , Presión Sanguínea/efectos de los fármacos , Tronco Encefálico/metabolismo , Activación Enzimática/efectos de los fármacos , Fenclonina/farmacología , Hipotálamo/metabolismo , Morfina/farmacología , Ratas , Receptores de Serotonina/efectos de los fármacos , Serotonina/farmacologíaRESUMEN
BACKGROUND: Current evidence suggests that lean and obese patients with nonalcoholic fatty liver disease (NAFLD) share an altered metabolic and cardiovascular profile. However, there is an incomplete understanding of the natural history of "lean-NAFLD." Indeed, an unanswered question is whether lean (BMI ≤ 25 Kg/m2 ) NAFLD-patients are protected from severe histological outcomes. AIM: To perform a meta-analysis with the goal of providing a quantitative estimation of the magnitude of fibrosis, as well as histological features associated with the disease severity, in lean versus overweight/obese-NAFLD patients. METHODS: Through a systematic search up to July 2017, we identified eight studies that compared histological outcomes in lean (n = 493) versus overweight/obese (n = 2209) patients. RESULTS: Relative to lean-NAFLD, overweight/obese-NAFLD patients showed significantly (P = .032) higher fibrosis scores; the observed difference in means between the two groups, which is the absolute difference between the mean value of fibrosis score [0-4] ± standard error, was 0.28 ± 0.13. The risk of having nonalcoholic steatohepatitis-NASH (OR 0.58 95% CI 0.34-0.97) was significantly lower in lean-NAFLD (n = 322) than in overweight/obese-NAFLD (n = 1357), P = .04. Relative to lean-NAFLD, overweight/obese-NAFLD patients also have significantly greater NAFLD activity (difference in means ± SE: 0.58 ± 0.16, P = .0004) and steatosis (difference in means ± SE: 0.23 ± 0.07, P = .002) scores. CONCLUSIONS: Lean-NAFLD patients tend to show less severe histological features as compared to overweight/obese-NAFLD patients. Subsequent longitudinal assessment is needed to understand the clinical impact of these findings; however, the significant ~ 25% increment of mean fibrosis score in overweight/obese patients suggests that obesity could predict a worse long-term prognosis.
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Enfermedad del Hígado Graso no Alcohólico/patología , Obesidad/complicaciones , Sobrepeso/complicaciones , Índice de Masa Corporal , Humanos , Hígado/patologíaRESUMEN
Shortened leukocyte telomere length (LTL) is a novel biomarker for age and age-related diseases. Several epidemiological studies have examined the association between telomere length in surrogate tissues (for example, blood cells) and hypertension, and meanwhile the majority of studies reported an association some individual studies do not. We carried out a systematic review and meta-analysis to address the hypothesis that, in humans, telomere length is related with hypertension. Searches were conducted in Pubmed by September 2015 and reference lists of retrieved citations were hand searched. Eligible studies measured telomeres for both hypertensive and normotensive subjects. No restrictions were placed on sample size, publication type, age or gender. We calculated summary estimates using fixed and random effects meta-analysis. Publication bias and heterogeneity among studies were further tested. Meta-analyses from 3097 participants (1415 patients with hypertension and 1682 control subjects) showed a significant standardized mean difference between LTL in hypertensive patients and controls, either in the fixed (P<5 × 10-6) or the random model (P<0.005). Heterogeneity among studies was substantial (Q-statistic P-value <0.001, I2 97.73%). Sensitivity analysis indicated that no single study changed the standardized mean difference qualitatively (0.022> random model P-value >0.002). Egger's test for asymmetry of effect sizes (intercept±s.e.=-7.278±3.574; P=0.072) did not show evidence for strong study publication bias. Leukocyte telomeres may be shorter in hypertensive than in normotensive individuals. Larger studies controlling for confounder effects are needed to confirm these findings and further explore sources of heterogeneity.
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Hipertensión/fisiopatología , Homeostasis del Telómero , Humanos , LeucocitosRESUMEN
BACKGROUND: The pathogenesis of non-alcoholic fatty liver disease (NAFLD) is closely associated with the co-occurrence of multiple pathological conditions characterising the metabolic syndrome (MetS), obesity in particular. However, NAFLD also develops in lean subjects, whose risk factors remain poorly defined. METHODS: We performed a meta-analysis of 15 studies, along with the data pertaining to our own population (n=336 patients). Data from lean (n=1966) and obese (n=5938) patients with NAFLD were analysed; lean (n=9946) and obese (n=6027) subjects without NAFLD served as controls. RESULTS: Relative to the lean non-NAFLD controls, lean patients with NAFLD were older (3.79±0.72 years, P=1.36×10-6 ) and exhibited the entire spectrum of the MetS risk factors. Specifically, they had a significant (P=10-10 ) increase in plasma glucose levels (6.44±1.12 mg/dL) and HOMA-IR (0.52±0.094-unit increment), blood lipids (triglycerides: 48.37±3.6, P=10-10 and total cholesterol: 7.04±3.8, mg/dL, P=4.2×10-7 ), systolic (5.64±0.7) and diastolic (3.37±0.9) blood pressure (mm Hg), P=10-10 , and waist circumference (5.88±0.4 cm, P=10-10 ); values denote difference in means±SE. Nevertheless, the overall alterations in the obese group were much more severe when compared to lean subjects, regardless of the presence of NAFLD. Meta-regression suggested that NAFLD is a modifier of the level of blood lipids. CONCLUSION: Lean and obese patients with NAFLD share a common altered metabolic and cardiovascular profile. The former, while having normal body weight, showed excess of abdominal adipose tissue as well as other MetS features.
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Síndrome Metabólico/epidemiología , Enfermedad del Hígado Graso no Alcohólico/epidemiología , Obesidad/epidemiología , Factores de Edad , Glucemia , Presión Sanguínea , Índice de Masa Corporal , Colesterol/sangre , Humanos , Lípidos/sangre , Factores de Riesgo , Circunferencia de la CinturaRESUMEN
BACKGROUND: Previous epidemiological studies suggest that patients diagnosed with nonalcoholic fatty liver disease (NAFLD) who drink light to moderate amounts of alcohol (up to ~30 g per day) have less severe histological lesions compared with nondrinkers. However, while the cross-sectional nature of current evidence precludes assessment of causality, cumulative lifetime-exposure of moderate alcohol consumption on histological outcomes has never been evaluated. AIM: To overcome these limitations, a Mendelian randomisation study was performed using a validated genetic variant (rs1229984 A;G) in the alcohol dehydrogenase (ADH1B) gene as a proxy of long-term alcohol exposure. METHODS: We first assessed whether the instrumental variant (rs1229984) was associated with the amount of alcohol consumption in our cohort. We further explored the association between the variant and histological outcomes; a sample of 466 individuals, including 266 patients with NAFLD confirmed by liver biopsy, was studied. RESULTS: We found that carriers of the A-allele consumed significantly lower amounts of alcohol compared with noncarriers (2.3 ± 5.3 vs. 8.18 ± 21 g per day, mean ± s.d., P = 0.03). The analysis of association with the disease severity showed that carriers of the A-allele had lower degree of histological steatosis (1.76 ± 0.83 vs. 2.19 ± 0.78, P = 0.03) and lower scores of lobular inflammation (0.54 ± 0.65 vs. 0.95 ± 0.92, P = 0.02) and NAFLD-Activity Score (2.9 ± 1.4 vs. 3.7 ± 1.4, P = 0.015) compared with noncarriers. CONCLUSION: Mendelian randomisation analysis suggests no beneficial effect of moderate alcohol consumption on NAFLD disease severity.
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Alcohol Deshidrogenasa/genética , Consumo de Bebidas Alcohólicas/genética , Enfermedad del Hígado Graso no Alcohólico/genética , Enfermedad del Hígado Graso no Alcohólico/patología , Adulto , Anciano , Alelos , Biopsia , Femenino , Variación Genética , Humanos , Masculino , Análisis de la Aleatorización Mendeliana , Persona de Mediana EdadRESUMEN
Our objective was to search for differences in genotypes of peroxisome proliferator-activated receptor gamma (PPARgamma) (Pro12 Ala) and its coactivator PGC-1alpha (Gly482 Ser) in adolescents harboring features of metabolic syndrome. In a population-based study, we determined medical history, anthropometric variables, biochemical measurements and arterial blood pressures of 934 high-school students of Caucasian origin. We selected 220 adolescents who had systolic or diastolic blood pressures more than the 80th or less than the 20th percentiles based on the previous single set of measurements. One hundred and seventy-five adolescents completed the study and underwent two additional blood pressure measurements on different days, as well as biochemical analysis and genotyping. We found no association between insulin resistance, body mass index (BMI) and leptin levels and PPARgamma and PGC-1alpha genotypes. The 12 Ala PPARgamma allele was associated with increased waist-to-hip ratio (WHR) and carriers seemed to have higher diastolic blood pressure and lower pulse pressure than non-carriers, particularly in the hypertensive and overweight group. Although Ser482 Ser PGC-1alpha homozygotes had lower WHRs than other PGC-1alpha genotypes, they were more frequent in the hypertensive group than in the normotensive (44.4 vs 24.5%, P<0.03), so the 482 Ser PGC-1 allele was in our population a risk factor for hypertension independently of WHR, homeostasis model assessment of insulin resistance, BMI and Pro12 Ala PPARgamma variant (odds ratio=4.0, 95% confidence interval 1.5-10.6, P<0.01). Multiple regression analysis showed that age- and sex-adjusted systolic blood pressure correlated with the 482 Ser PGC-1 allele regardless of those covariates. In conclusion, the Gly482 Ser variant of the PGC-1alpha gene may be an independent genetic risk factor for young-onset hypertension.
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Predisposición Genética a la Enfermedad , Proteínas de Choque Térmico/metabolismo , Hipertensión/fisiopatología , Síndrome Metabólico/metabolismo , PPAR gamma/metabolismo , Factores de Transcripción/metabolismo , Adolescente , Presión Sanguínea/fisiología , Peso Corporal , Femenino , Proteínas de Choque Térmico/genética , Humanos , Masculino , Síndrome Metabólico/fisiopatología , Obesidad/fisiopatología , Coactivador 1-alfa del Receptor Activado por Proliferadores de Peroxisomas gamma , Análisis de Regresión , Factores de Riesgo , Factores de Transcripción/genética , Población BlancaRESUMEN
PTH-related protein (PTHrP) is produced in vascular smooth muscle, where it is believed to act as a local vasorelaxant by activating either the classical PTH or a unique PTHrP receptor. We used a newly cloned complementary DNA encoding the rat PTH/PTHrP receptor to study the expression of its messenger RNA (mRNA) in primary aortic vascular smooth muscle cells (VSMC) and in UMR-106 osteoblast-like cells under basal conditions and in response to treatment with agonists. Both cell types expressed a 2.4-kilobase PTH/PTHrP receptor mRNA transcript and exhibited hormone-induced desensitization of PTHrP-(1-34)NH2-stimulated cAMP. In VSMC, angiotensin-II, which induces PTHrP expression, also rapidly (30 min) desensitized the cAMP response and down-regulated (75-90%) receptor mRNA within 1 h. Treatment of cells with phorbol 12-myristate 13-acetate (0.1 microM) mimicked these effects, whereas neither PTHrP-(1-34)NH2, forskolin, nor (Bu)2cAMP altered receptor mRNA expression. By contrast, in UMR-106 cells, PTHrP-(1-34)NH2 induced time- and dose-dependent decreases in receptor mRNA that were preceded by pronounced desensitization (cAMP and ligand binding) of cell surface receptors. These effects were mimicked by (Bu)2cAMP and forskolin, but not by phorbol 12-myristate 13-acetate, suggesting that both receptor mRNA down-regulation and receptor desensitization in UMR cells were mediated through a protein kinase-A pathway. We suggest that VSMC and UMR cells express a common receptor, which is subject to cell-specific regulation. Such diversity in the PTH/PTHrP receptor regulatory mechanisms provides a means for restricting the length and duration of the cellular response to hormone in a cell/tissue-specific manner.
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Aorta/metabolismo , Músculo Liso Vascular/metabolismo , Osteoblastos/metabolismo , Hormona Paratiroidea/metabolismo , Proteínas/fisiología , ARN Mensajero/metabolismo , Receptores de Hormona Paratiroidea/metabolismo , Angiotensina II/fisiología , Animales , Aorta/citología , Línea Celular , AMP Cíclico/metabolismo , Proteínas Quinasas Dependientes de AMP Cíclico/metabolismo , Activación Enzimática , Músculo Liso Vascular/citología , Hormona Paratiroidea/genética , Proteína Relacionada con la Hormona Paratiroidea , Proteína Quinasa C/metabolismo , Ratas , Receptor de Hormona Paratiroídea Tipo 1 , Receptores de Hormona Paratiroidea/genéticaRESUMEN
N-Terminal analogs of PTH-related protein (PTHrP) and PTH bind to a common receptor and exhibit similar biological properties. However, recent studies suggest that certain midregion and C-terminal PTHrP peptides have activities distinct from those of PTH in the placenta and in osteoclasts, respectively. In this study we determined the biological activities of full-length recombinant PTHrP-(1-141) and several synthetic N-terminal, midregion, and C-terminal PTHrP fragments in two PTHrP-producing cell types. Peptides were tested for their ability to stimulate cAMP production and raise intracellular free calcium ([Ca2+]i) in primary rat aortic smooth muscle cells (VSMC) and UMR-106 rat osteoblast-like (UMR) cells. In UMR cells PTHrP-(1-34)NH2, PTHrP-(1-141), and bovine PTH-(1-34) all increased cAMP (approximately 50 fold) and [Ca2+]i (180 nM). By contrast, in VSMC, these N-terminal peptides increased cAMP (3-fold) but had no detectable effect on [Ca2+]i. PTHrP-(1-34) and PTHrP-(1-141) significantly blunted the angiotensin II-induced rise in cAMP (but not the calcium signal) consistent with the concept that PTHrP opposes angiotensin II activity in VSMC. PTHrP-(67-86)NH2, PTHrP-(107-138)NH2, and PTHrP-(107-111)NH2 had no effect on either cAMP or [Ca2+]i in either cell type. VSMC and UMR-106 cells both expressed a 2.5-kilobase PTH/PTHrP receptor messenger RNA (mRNA) transcript. However, high affinity specific binding of 125I-labeled [Tyr36] PTHrP-(1-36)NH2 was detected in UMR cells but not in VSMC. We conclude that the PTH-like, N terminus of the PTHrP molecule is critical in induction of cAMP and [Ca2+]i pathways in UMR cells, and for cAMP stimulation in VSMC. In addition, PTHrP, like other established vasodilators, signals in VSMC mainly (if not exclusively) by increasing the production of cAMP.
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Calcio/metabolismo , AMP Cíclico/metabolismo , Músculo Liso Vascular/metabolismo , Osteoblastos/metabolismo , Proteínas/farmacología , Animales , Aorta/citología , Aorta/metabolismo , Línea Celular , Citoplasma/metabolismo , Membranas Intracelulares/metabolismo , Músculo Liso Vascular/citología , Concentración Osmolar , Proteína Relacionada con la Hormona Paratiroidea , Fragmentos de Péptidos/farmacología , Ratas , Receptores de Hormona Paratiroidea/metabolismoRESUMEN
PTH-related protein (PTHrP) is induced in aortic vascular smooth muscle cells (VSMC) in association with mitogen-stimulated proliferation. In this study we examined the role of the cell cycle in the control of PTHrP gene expression. In asynchronously cycling cells grown in serum-containing medium, PTHrP-immunoreactive cells were enriched in G2+M, as revealed by fluorescence-activated cell sorting using a specific monoclonal antibody. PTHrP messenger RNA (mRNA) increased transiently in cells after release from chemically induced cell cycle blockade; levels increased by 10-fold at 2 h, coincident with expression of histone-4 mRNA and enrichment of VSMC in the early S phase. However, PTHrP mRNA levels then declined abruptly while the proportion of cells in the S phase and histone-4 mRNA levels remained constant for 8 h. When cell cycle-arrested cells were exposed to fresh serum-containing medium, angiotensin-II, or phorbol ester without removing the cell cycle blocking agents, PTHrP mRNA levels were induced over a time course identical to that observed in cells released from the blockade. This suggests that progression through the cycle per se is not necessary for mitogen-induced PTHrP mRNA expression, and that conventional chemical synchronization is not adequate to examine the cell cycle dependency of PTHrP mRNA abundance in VSMC. By contrast, in two different PTHrP-producing carcinoma cell lines, PTHrP and its mRNA were not altered as a function of cell cycle, demonstrating that different mechanisms control PTHrP expression in these cancer cells. In conclusion, constitutive immunoreactive levels of PTHrP are low in normally cycling VSMC (but not cancer cells) and accumulate during the latter stages of the cell cycle, suggesting a role for this protein in the process of smooth muscle cell division. However, separate mechanisms, which are independent of cell cycle, operate through a protein kinase-C-dependent pathway(s) to mediate the stimulation of PTHrP gene expression by vasoconstrictors such as angiotensin-II.
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Ciclo Celular/fisiología , Expresión Génica , Mitógenos/farmacología , Músculo Liso Vascular/metabolismo , Proteínas/genética , Animales , Aorta , Sangre , Células Cultivadas , Medios de Cultivo , Citometría de Flujo , Masculino , Proteína Relacionada con la Hormona Paratiroidea , ARN Mensajero/metabolismo , Ratas , Ratas Sprague-DawleyRESUMEN
PTH-related peptide (PTHrP), the factor mediating the syndrome of humoral hypercalcemia of malignancy, is also expressed in smooth muscle cells (SMC) of the urinary bladder and uterus in response to mechanical distention and fetal occupancy, respectively. Vascular SMC also produce PTHrP, and its expression is induced by serum and vasoconstrictors, such as angiotensin-II. To determine whether mechanical distension affected vascular PTHrP gene expression, the abdominal aorta of adult male rats was balloon-distended, and aortae were collected at various times after the intervention. PTHrP mRNA was determined by competitive reverse transcriptase-polymerase chain reaction, using sequential dilutions of a cloned internally truncated PTHrP RNA fragment as standard. The molar concentration of PTHrP mRNA was obtained by extrapolating at a standard/wild-type band intensity ratio of 1:1. Aortic PTHrP mRNA was induced from a basal level of 19, to 22, 46, 36, 13, 12, 22, and 20 attamoles/mg total RNA 1, 2, 12, 24, and 48 h and 7 and 48 days after balloon distension, respectively. To determine whether mechanical events directly regulate vascular PTHrP gene expression, primary rat aortic SMC were plated and placed on a rocking device at 20 oscillations/min to create a gentle flowing motion of the culture medium. Rocking induced PTHrP mRNA of SMC exposed to either serum-free medium or 10% serum by 2.5-and 4.0-fold at 4 h, and 2.9- and 3.7-fold at 24 h, respectively. These effects were oscillation rate dependent, potentiated by angiotensin-II, and specific, as similar changes were not observed in alpha-actin mRNA content. Flow motion-induced PTHrP mRNA at 24 h was partially decreased by 10(-6) M colchicine (which inhibits microtubule assembly), but not by cytochalasin-E (which disrupts actin polymerization). As PTHrP is a known vasorelaxant, we propose that mechanical events induce the release of PTHrP by SMC, possibly to serve as a compliance factor or an agent for vascular remodeling.
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Expresión Génica , Músculo Liso Vascular/metabolismo , Proteínas/genética , Alcaloides/farmacología , Angiotensina II/farmacología , Animales , Aorta/metabolismo , Secuencia de Bases , Células Cultivadas , Masculino , Datos de Secuencia Molecular , Proteína Relacionada con la Hormona Paratiroidea , Estimulación Física , Reacción en Cadena de la Polimerasa , Presión , Proteína Quinasa C/antagonistas & inhibidores , Proteína Quinasa C/metabolismo , ARN Mensajero/metabolismo , Ratas , Ratas Sprague-Dawley , EstaurosporinaRESUMEN
Thyrotropin-releasing hormone (TRH) plays an important role in central cardiovascular regulation through the activation of different neurotransmitter systems at distinct extrahypothalamic sites. To study possible alterations in the TRH system in the hypertensive state, we measured TRH concentration in cerebrospinal fluid and TRH content of the preoptic area in spontaneously hypertensive rats (SHR) and Wistar-Kyoto rats (WKY) by radioimmunoassay. In addition, we also measured the density of the TRH receptor in this area by a rapid filtration technique using [3H]methyl-TRH. We found a significant increase in both the TRH content (634 +/- 61 versus 350 +/- 26 pg/mg protein, SHR versus WKY; P < .01, n = 5) and density of TRH receptors without changes in affinity (Bmax, 5.0 +/- 0.1 versus 3.3 +/- 0.1 fmol/mg protein, P < .01, n = 4). An increase in TRH concentration was also found in the cerebrospinal fluid of SHR (30 +/- 3 versus 21 +/- 2 pg/mL, P < .01, n = 5), suggesting increased TRH release in the central nervous system. Northern blot analysis indicated a threefold augmented abundance of TRH precursor mRNA in the preoptic area of SHR. A polyclonal antibody raised against TRH injected peripherally or intracerebroventricularly lowered arterial blood pressure in SHR but not in WKY. In addition, long-term treatment with enalapril (5 mg/kg twice daily), which was effective in inhibiting serum angiotensin-converting enzyme activity by more than 50%, decreased arterial blood pressure and preoptic area TRH content of SHR, whereas another vasodilator, diltiazem (10 mg/kg every 8 hours), failed to produce a similar change.(ABSTRACT TRUNCATED AT 250 WORDS)
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Hipertensión/fisiopatología , Área Preóptica/química , Hormona Liberadora de Tirotropina/fisiología , Análisis de Varianza , Inhibidores de la Enzima Convertidora de Angiotensina/farmacología , Animales , Antihipertensivos/farmacología , Presión Sanguínea/efectos de los fármacos , Northern Blotting , Bloqueadores de los Canales de Calcio/farmacología , Diltiazem/administración & dosificación , Diltiazem/farmacología , Enalapril/administración & dosificación , Enalapril/farmacología , Hipertensión/tratamiento farmacológico , Hipertensión/etiología , Hipertensión/genética , Masculino , ARN Mensajero/análisis , Radioinmunoensayo , Ratas , Ratas Endogámicas SHR , Ratas Endogámicas WKY , Receptores de Hormona Liberadora de Tirotropina/análisis , Receptores de Hormona Liberadora de Tirotropina/genética , Hormona Liberadora de Tirotropina/análisis , Hormona Liberadora de Tirotropina/líquido cefalorraquídeo , Factores de Tiempo , Regulación hacia ArribaRESUMEN
Extrahypothalamic TRH participates in cardiovascular regulation and spontaneous hypertension of the rat. To investigate whether an increase in central TRH activity produces hypertension we studied the effect of the preTRH overproduction induced by I.C.V. transfection with a naked eukaryotic expression plasmid vector which encodes preTRH (pCMV-TRH). Northern blot analysis and RT-PCR showed that pCMV-TRH was transcribed in vitro and in vivo. At 24, 48, and 72 hours, pCMV-TRH (100 microg) in a significant and dose-dependent manner increased 37%, 84%, and 49%, respectively, the diencephalic TRH content and SABP (42+/-3, 50+/-2, and 22+/-2 mm Hg, respectively) with respect to the vector without the preTRH cDNA insert (V[TRH(-)]) as measured by RIA and the plethysmographic method, respectively, in awake animals. In addition, using immunohistochemistry we found that the increase of TRH was produced in circumventricular areas where the tripeptide is normally located. To further analyze the specificity of these effects we studied the actions of 23-mer sense (S), antisense (AS), and 3'self-stabilized sense (Ss) and antisense (ASs) phosphorothioate oligonucleotides against the initiation codon region. Only ASs inhibited the increase of TRH content and SABP induced by pCMV-TRH treatment. In addition, pCMV-TRH-induced hypertension seems not to be mediated by central Ang II or serum TSH. To summarize, central TRH overproduction in periventricular areas induced by I.C.V. transfection produces hypertension in rats which is reversed by specific antisense treatment. This model may help in testing effective antisense oligodeoxynucleotides against other candidate genes.
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Encéfalo/metabolismo , Regulación de la Expresión Génica , Hipertensión/etiología , Oligonucleótidos Antisentido/farmacología , Precursores de Proteínas/genética , Hormona Liberadora de Tirotropina/genética , Animales , Humanos , Masculino , Ratas , Ratas Wistar , Hormona Liberadora de Tirotropina/fisiología , TransfecciónRESUMEN
The infusion of acetylcholine, bradykinin, angiotensin II, norepinephrine and serotonin into the lateral septal area produced a dose-dependent increase of arterial blood pressure and heart rate. A pattern of inhibition of these cardiovascular responses, produced by pretreatment of the lateral septal area with phentolamine, 6-hydroxydopamine, methysergide and 5,7-dihydroxytryptamine was disclosed. These results suggest that the effects of acetylcholine, bradykinin and partially of angiotensin II, depend on the release of norepinephrine and the actions of this neurotransmitter in turn depend on the integrity of the serotonergic system in the lateral septal area.
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Hemodinámica/efectos de los fármacos , Neuropéptidos/farmacología , Neurotransmisores/farmacología , Tabique Pelúcido/fisiología , Serotonina/fisiología , 5,7-Dihidroxitriptamina/farmacología , Acetilcolina/farmacología , Angiotensina II/farmacología , Animales , Bradiquinina/farmacología , Hidroxidopaminas/farmacología , Masculino , Neuropéptidos/antagonistas & inhibidores , Neurotransmisores/antagonistas & inhibidores , Norepinefrina/farmacología , Oxidopamina , Ratas , Ratas Endogámicas , Tabique Pelúcido/efectos de los fármacosRESUMEN
The lateral septal area was used as a model to study the interaction between acetylcholine (Ach) and bradykinin on arterial blood pressure, since both mediators are present in this region. In the lateral septal area, the administration of the peptide or Ach produced a long-lasting, sympathetic-mediated increase of arterial blood pressure which was blocked by atropine. Pretreatment of the lateral septal area with hemicholinium-3, which depletes stores of acetylcholine, partially blocked the pressor effect of bradykinin but not that of Ach. Captopril--an inhibitor of kininase II--enhanced the pressor effects of bradykinin and Ach. Synaptosomal studies showed that bradykinin increased sodium-dependent, high-affinity uptake of choline and the conversion of [3H]choline to [3H]acetylcholine. Competition experiments using the highly specific muscarinic antagonist [3H]quinuclidinyl benzilate, demonstrated that bradykinin displaced the muscarinic antagonist from its receptor-ligand complexes. These results suggest that in the lateral septal area acetylcholine and bradykinin interact in a positive feed-back which amplifies pressor responses.
Asunto(s)
Acetilcolina/farmacología , Bradiquinina/farmacología , Encéfalo/efectos de los fármacos , Hemodinámica/efectos de los fármacos , Receptores Muscarínicos/efectos de los fármacos , Animales , Presión Sanguínea/efectos de los fármacos , Colina/metabolismo , Técnicas In Vitro , Cinética , Masculino , Quinuclidinil Bencilato/metabolismo , Ratas , Ratas Endogámicas , Sinaptosomas/metabolismoRESUMEN
In the lateral septal area of spontaneously hypertensive rats, but not in Wistar-Kyoto rats, the selective M1 antagonist, pirenzepine, and the depletion of acetylcholine storage, by hemicholinium-3 (HC-3), decreased blood pressure. The selective M1 agonist McNeil-A-343, produced a pressor response only after treatment of the lateral septal area with HC-3 in spontaneously hypertensive rats. Carbachol, at doses that mainly affect M2 muscarinic receptors, caused no cardiovascular changes in either strain, pointing to the main intervention of the M1 subtype of muscarinic receptor in the hypertensive condition. In addition, increases in the density of binding sites for [3H]QNB and in Vmax of sodium-dependent, HC-3-inhibitable, high affinity uptake of choline were demonstrated, without significant changes of the activity of choline acetyltransferase in the lateral septal area of spontaneously hypertensive rats. These results suggest that a hyperactivity of the cholinergic system of this area could play a role in the development and/or maintenance of hypertension in spontaneously hypertensive rats.
Asunto(s)
Presión Sanguínea/efectos de los fármacos , Hemicolinio 3/farmacología , Pirenzepina/farmacología , Tabique Pelúcido/fisiología , Cloruro de (4-(m-Clorofenilcarbamoiloxi)-2-butinil)trimetilamonio/farmacología , Animales , Presión Sanguínea/fisiología , Colina/metabolismo , Colina O-Acetiltransferasa/metabolismo , Relación Dosis-Respuesta a Droga , Masculino , Quinuclidinil Bencilato/metabolismo , Ratas , Ratas Endogámicas SHR , Ratas Endogámicas WKY , Receptores Colinérgicos/metabolismo , Sinaptosomas/metabolismoRESUMEN
The infusion of pilocarpine, acetylcholine, bradykinin and the selective M1 muscarinic agonist McNeil-A-343 into the lateral septal area produced a dose-dependent increase of arterial blood pressure and heart rate. The M1 muscarinic agonist carbamylcholine that causes a rise in arterial blood pressure when injected into the anterior lateral ventricles did not produce any cardiovascular effects when infused into the lateral septal area. Chronic treatment with atropine induced supersensitivity to the muscarinic agonists and a significant increase in the number of muscarinic receptors. In this study bradykinin failed to produce any significant change in cardiovascular activity. Pirenzepine, a M1 muscarinic blocking agent, inhibited completely the effect of both muscarinic agonists and bradykinin on cardiovascular activity. In fact, in vitro studies shows that the displacement of the binding of [3H]QNB by pirenzepine is compatible with the presence of the M1 subtype of muscarinic receptor in the lateral septal area, where it may play a major role on cardiovascular regulation.
Asunto(s)
Atropina/farmacología , Presión Sanguínea/efectos de los fármacos , Bradiquinina/farmacología , Ventrículos Cerebrales/fisiología , Frecuencia Cardíaca/efectos de los fármacos , Parasimpaticomiméticos/farmacología , Receptores Muscarínicos/fisiología , Cloruro de (4-(m-Clorofenilcarbamoiloxi)-2-butinil)trimetilamonio/farmacología , Acetilcolina/farmacología , Animales , Carbacol/farmacología , Ventrículos Cerebrales/efectos de los fármacos , Masculino , Pilocarpina/farmacología , Ratas , Ratas Endogámicas , Receptores Muscarínicos/efectos de los fármacosRESUMEN
OBJECTIVE: We studied the expression of parathyroid hormone (PTH)-related protein in vascular smooth muscle cells of spontaneously hypertensive rats (SHR) using Wistar-Kyoto (WKY) and Sprague-Dawley rats as normotensive controls. METHODS: Aortae from 4- and 18-week-old SHR versus age-matched WKY and Sprague-Dawley rats were excised to obtain total RNA or smooth muscle cells. The cells were subcultured in Dulbecco's Modified Eagle's Medium containing 10% fetal calf serum, then serum-deprived for 72 h and stimulated with 0.1 micromol/I angiotensin II. PTH-related protein, c-myc and angiotensin II type qa receptor (AT1aR) messenger (m)RNA levels were measured by Northern blot, using total RNA extracted by phenol/chloroform. The effects of PTH-related protein(1-34)NH2 intravenous injections on arterial blood pressure and the heart rate were studied in anesthetized SHR and WKY rats. RESULTS: The Northern blots showed a significantly higher abundance of PTH-related protein mRNA in aortae of SHR versus WKY rats in the prehypertensive state but no significant difference in adult animals. In cultured aortic smooth muscle cells, angiotensin II induced a four- to sixfold increase in PTH-related protein mRNA levels in smooth muscle cells from normotensive animals, but failed to elicit a significant response in smooth muscle cells derived from SHR in either the prehypertensive or the hypertensive state. This lack of response to angiotensin II in SHR smooth muscle cells was not due to decreased expression or responsiveness of the AT1aR, since SHR smooth muscle cells had more AT1aR mRNA than Sprague-Dawley smooth muscle cells, and angiotensin II-induced activation of c-myc was faster and greater in smooth muscle cells derived from 4- or 18-week-old SHR than in Sprague-Dawley smooth muscle cells. In contrast, PTH-related protein(1-34)NH2 induced a long-lasting dose-dependent hypotensive and tachycardic response in both SHR and WKY rats, indicating that SHR retained responsiveness to the vasodilator. CONCLUSIONS: PTH-related protein gene expression in response to angiotensin II is impaired in SHR arteries. A deficiency in this potent local vasodilator may contribute to the development and/or maintenance of arterial hypertension in this model.
Asunto(s)
Angiotensina II/farmacología , Hipertensión/metabolismo , Músculo Liso Vascular/efectos de los fármacos , Proteínas/metabolismo , Vasoconstrictores/farmacología , Animales , Aorta Torácica/efectos de los fármacos , Aorta Torácica/metabolismo , Northern Blotting , Hemodinámica/efectos de los fármacos , Inyecciones Intravenosas , Masculino , Músculo Liso Vascular/metabolismo , Hormona Paratiroidea/metabolismo , Proteína Relacionada con la Hormona Paratiroidea , Proteínas/genética , Proteínas Proto-Oncogénicas c-myc/genética , Proteínas Proto-Oncogénicas c-myc/metabolismo , ARN Mensajero/metabolismo , Ratas , Ratas Endogámicas SHR , Ratas Endogámicas WKY , Ratas Sprague-Dawley , Receptor de Angiotensina Tipo 1 , Receptor de Angiotensina Tipo 2 , Receptores de Angiotensina/genética , Receptores de Angiotensina/metabolismoRESUMEN
[3H]Quinuclidinyl benzylate ([3H]QNB)-binding sites, showing similar properties to cholinergic muscarinic receptors in other tissues, were disclosed in the rat pineal gland. Functionality of these receptors was demonstrated, as in-vitro muscarinic activation by pilocarpine increased the pineal metabolic production of the hydroxyindole derivatives 5-hydroxytryptophan and serotonin, with a slight effect on melatonin biosynthesis. Electric-field stimulation of pineal slices caused similar metabolic effects. These effects were inhibited by muscarinic blockade with atropine and enhanced by neostigmine inactivation of acetylcholinesterase. These results suggest that acetylcholine is the neurotransmitter involved. Cholinergic activity may, therefore, regulate indole metabolism in the pineal gland.
Asunto(s)
Indoles/metabolismo , Glándula Pineal/metabolismo , Receptores Muscarínicos/fisiología , Animales , Membrana Celular/metabolismo , Colina O-Acetiltransferasa/metabolismo , Estimulación Eléctrica , Masculino , Glándula Pineal/efectos de los fármacos , Quinuclidinil Bencilato/metabolismo , Ratas , Ratas EndogámicasRESUMEN
Betamethasone was administered on alternate days to rats, and the role of the central cholinergic system in the development of hypertension assessed. After 15 days of treatment the systolic blood pressure of treated rats was significantly higher than that of control rats. Peripheral administration of atropine but not of methyl atropine reduced systolic pressure in glucocorticoid-treated rats and had no effect in controls. Therefore, [3H]quinuclidinyl benzylate binding, sodium-dependent high-affinity choline uptake and choline acetyltransferase studies were performed in the septal area, anteroventrolateral medulla (AVLM), anterior hypothalamic preoptic area (AH/PO) and hypothalamus. The density of muscarinic receptors was increased in the hypothalamus and AVLM of treated rats without significant changes in affinity. Choline acetyltransferase activity significantly decreased in the AVLM and increased in the AH/PO. In addition, a decrease in the hypothalamus and an increase in the AH/PO of sodium-dependent high-affinity choline uptake was observed in glucocorticoid-treated rats. These results suggest the presence of an enhanced muscarinic cholinergic activity in several brain nuclei in rats with glucocorticoid-induced hypertension. This activation could be due to pre- and post-synaptic hypersensitivity.