RESUMEN
BACKGROUND: Prostaglandin I2 synthesized by endothelial COX (cyclooxygenase) evokes potent vasodilation in some blood vessels but is paradoxically responsible for endothelium-dependent constriction (EDC) in others. Prostaglandin I2 production and EDC may be enhanced in diseases such as hypertension. However, how PGIS (prostaglandin I2 synthase) deficiency affects EDC and how this is implicated in the consequent cardiovascular pathologies remain largely unknown. METHODS: Experiments were performed with wild-type, Pgis knockout (Pgis-/-) and Pgis/thromboxane-prostanoid receptor gene (Tp) double knockout (Pgis-/-Tp-/-) mice and Pgis-/- mice transplanted with unfractionated wild-type or Cox-1-/- bone marrow cells, as well as human umbilical arteries. COX-derived prostanoids were measured by high-performance liquid chromatography-mass spectrometry. Vasomotor responses of distinct types of arteries were assessed by isometric force measurement. Parameters of hypertension, vascular remodeling, and cardiac hypertrophy in mice at different ages were monitored. RESULTS: PGF2α, PGE2, and a trace amount of PGD2, but not thromboxane A2 (TxA2), were produced in response to acetylcholine in Pgis-/- or PGIS-inhibited arteries. PGIS deficiency resulted in exacerbation or occurrence of EDC ex vivo and in vivo. Endothelium-dependent hyperpolarization was unchanged, but phosphorylation levels of eNOS (endothelial nitric oxide synthase) at Ser1177 and Thr495 were altered and NO production and the NO-dependent relaxation evoked by acetylcholine were remarkably reduced in Pgis-/- aortas. Pgis-/- mice developed high blood pressure and vascular remodeling at 16 to 17 weeks and subsequently cardiac hypertrophy at 24 to 26 weeks. Meanwhile, blood pressure and cardiac parameters remained normal at 8 to 10 weeks. Additional ablation of TP (TxA2 receptor) not only restrained EDC and the downregulation of NO signaling in Pgis-/- mice but also ameliorated the cardiovascular abnormalities. Stimulation of Pgis-/- vessels with acetylcholine in the presence of platelets led to increased TxA2 generation. COX-1 disruption in bone marrow-derived cells failed to affect the development of high blood pressure and vascular remodeling in Pgis-/- mice though it largely suppressed the increase of plasma TxB2 (TxA2 metabolite) level. CONCLUSIONS: Our study demonstrates that the non-TxA2 prostanoids/TP axis plays an essential role in mediating the augmentation of EDC and cardiovascular disorders when PGIS is deficient, suggesting TP as a promising therapeutic target in diseases associated with PGIS insufficiency.
Asunto(s)
Endotelio Vascular , Oxidorreductasas Intramoleculares , Ratones Endogámicos C57BL , Ratones Noqueados , Prostaglandinas , Vasoconstricción , Animales , Oxidorreductasas Intramoleculares/genética , Oxidorreductasas Intramoleculares/deficiencia , Oxidorreductasas Intramoleculares/metabolismo , Ratones , Endotelio Vascular/metabolismo , Endotelio Vascular/fisiopatología , Prostaglandinas/metabolismo , Humanos , Sistema Enzimático del Citocromo P-450/metabolismo , Sistema Enzimático del Citocromo P-450/genética , Sistema Enzimático del Citocromo P-450/deficiencia , Tromboxano A2/metabolismo , Enfermedades Cardiovasculares/genética , Enfermedades Cardiovasculares/metabolismo , Enfermedades Cardiovasculares/fisiopatología , Enfermedades Cardiovasculares/etiología , Masculino , Receptores de Tromboxanos/metabolismo , Receptores de Tromboxanos/genética , Vasodilatación , Cardiomegalia/genética , Cardiomegalia/metabolismo , Cardiomegalia/fisiopatología , Remodelación Vascular , Transducción de Señal , Ciclooxigenasa 1/deficiencia , Ciclooxigenasa 1/genética , Ciclooxigenasa 1/metabolismoRESUMEN
Dysregulation of formaldehyde (FA) has been implicated in the development of Alzheimer's Disease (AD). Elevated FA levels in Alzheimer's patients and animal models are associated with impaired cognitive functions. However, the exact role of FA in AD remains unknown. We now identified that oxidative demethylation at serine8/26 of amyloid-beta protein (Aß) induced FA generation and FA cross-linked with the lysine28 residue in the ß-turn of Aß monomer to form Aß dimers, and then accelerated Aß oligomerization and fibrillogenesis in vitro. However, Aß42 mutation in serine8/26, lysine28 abolished Aß self-aggregation. Furthermore, Aß inhibited the activity of formaldehyde dehydrogenase (FDH), the enzyme for FA degradation, resulting in FA accumulation. In turn, excess of FA stimulated Aß aggregation both in vitro and in vivo by increasing the formation of Aß oligomers and fibrils. We found that degradation of FA by formaldehyde scavenger-NaHSO3 or coenzyme Q10 reduced Aß aggregation and ameliorated the neurotoxicity, and improved the cognitive performance in APP/PS1 mice. Our study provides evidence that endogenous FA is essential for Aß self-aggregation and scavenging FA could be an effective strategy for treating AD.
Asunto(s)
Enfermedad de Alzheimer , Enfermedad de Alzheimer/genética , Péptidos beta-Amiloides , Precursor de Proteína beta-Amiloide/genética , Animales , Modelos Animales de Enfermedad , Formaldehído/toxicidad , Humanos , Ratones , Ratones Transgénicos , FenotipoRESUMEN
The vasoconstrictor and/or pressor effects of prostaglandin (PG)F2α participate in the development of vascular pathologies and limit the clinical use of the agent. This study aimed to determine the receptor types responsible for the vasoconstrictor activity of PGF2α and whether they mediate the pressor response evoked by the prostanoid under in vivo conditions. Experiments were performed on genetically altered mice and/or on vessels from these mice or humans. Here we show that deletion of the thromboxane-prostanoid receptor (TP-/-) abolished or drastically diminished the contraction to PGF2α in isolated mouse vessels (some of which were resistance arteries) and reduced the elevation in blood pressure evoked by the prostanoid under in vivo conditions. In accordance, TP antagonism abolished the contraction in small arteries of human omentum. Further deletion of E prostanoid receptor type 3 (EP3-/-) removed the PGF2α-evoked contraction that remained in some TP-/- arteries and added to the effect of TP-/- on the elevation in blood pressure evoked by the prostanoid under in vivo conditions. In contrast, the uterine contraction to PGF2α mediated via the F prostanoid receptor (FP) was unaltered in TP-/-/EP3-/- mice. These results demonstrate that the non-FP receptors TP and/or EP3 mediate the vasoconstrictor and pressor effects of PGF2α, which are still of concern under clinical conditions.-Liu, B., Li, J., Yan, H., Tian, D., Li, H., Zhang, Y., Guo, T., Wu, X., Luo, W., Zhou, Y. TP and/or EP3 receptors mediate the vasoconstrictor and pressor responses of prostaglandin F2α in mice and/or humans.
Asunto(s)
Dinoprost/farmacología , Arterias Mesentéricas/efectos de los fármacos , Subtipo EP3 de Receptores de Prostaglandina E/fisiología , Receptores de Tromboxanos/fisiología , Vasoconstricción/fisiología , Vasoconstrictores/farmacología , Animales , Presión Sanguínea , Células Cultivadas , Femenino , Humanos , Masculino , Arterias Mesentéricas/metabolismo , Arterias Mesentéricas/patología , Ratones , Ratones Endogámicos C57BL , Ratones Noqueados , Músculo Esquelético/efectos de los fármacos , Músculo Esquelético/metabolismo , Músculo Esquelético/patología , Útero/efectos de los fármacos , Útero/metabolismo , Útero/patología , Vasoconstricción/efectos de los fármacosRESUMEN
An ultra-high performance liquid chromatography tandem mass spectrometry method was developed for determination of homocysteine (HCY) in human plasma. The HCY was derivatized with 2-chloro-1-methylquinolinium tetrafluoroborate and isolated using solid-phase extraction. Derivatization, isolation and detection procedures were optimized. Satisfactory linearity was obtained with determination coefficients (r2 ) >0.999. The intra- and inter-day precisions were in the interval of 1.2-5.1% and accuracy was within ±7%. Mean recoveries were close to 100%. The limit of detection and the limit of quantification were 0.46 and 1.38 µmol/L, respectively. The method was then applied to investigate the relationship between plasma HCY and whole blood 5-methyltetrahydrofolate levels in healthy volunteers. The results revealed that the plasma level of HCY was significantly negatively correlated to whole blood 5-methyltetrahydrofolate in volunteers.
Asunto(s)
Cromatografía Líquida de Alta Presión/métodos , Homocisteína/sangre , Espectrometría de Masas en Tándem/métodos , Tetrahidrofolatos/sangre , Adulto , Estabilidad de Medicamentos , Femenino , Humanos , Límite de Detección , Modelos Lineales , Masculino , Reproducibilidad de los Resultados , Adulto JovenRESUMEN
Patients with type 2 diabetes mellitus (T2DM) often develop cognitive impairments and have an increased risk of developing Alzheimer's disease. Hyperglycemia is a major characteristic of T2DM, but how elevated glucose levels lead to cognitive decline remains elusive. Here, we report that patients with T2DM and mutations in the formaldehyde (FA)-degrading enzyme aldehyde dehydrogenase 2 ( ALDH2) gene had higher levels of FA and more severe dementia. Injection of FA induced hyperglycemia and cognitive deficits in rats. Ablation of gene expression of ALDH2, the main enzyme to oxidize FA, resulted in abnormally high levels of hippocampal FA, leading to hyperglycemia and cognitive impairments as well as potentiating streptozotocin-induced diabetes development in ALDH2 knockout mice. We found that FA interacts with insulin to form FA-insulin adducts, and these FA-insulin adducts caused insulin deficiency, contributing to memory decline in diabetic rodent models. Reduction of FA by transgenic overexpression of human ALDH2 attenuates hyperglycemia and alleviates cognitive deficits in diabetic mouse models. These findings suggest that excess FA plays a critical role in mediating diabetes-related dementia. Targeting FA and its metabolizing enzyme ALDH2 may be a valid approach for preventing and treating dementia in diabetes mellitus.-Tan, T., Zhang, Y., Luo, W., Lv, J., Han, C., Hamlin, J. N. R., Luo, H., Li, H., Wan, Y., Yang, X., Song, W., Tong, Z. Formaldehyde induces diabetes-associated cognitive impairments.
Asunto(s)
Aldehído Deshidrogenasa Mitocondrial/genética , Disfunción Cognitiva/etiología , Diabetes Mellitus Tipo 2/complicaciones , Formaldehído/toxicidad , Anciano , Aldehído Deshidrogenasa Mitocondrial/metabolismo , Animales , Femenino , Formaldehído/sangre , Formaldehído/orina , Humanos , Insulina/metabolismo , Masculino , Ratones , Ratones Endogámicos C57BL , Mutación , Ratas , Ratas Sprague-DawleyRESUMEN
BACKGROUND/AIMS: Hydrazone and acylhydrazone derivatives, which are produced from aldehyde reacting with hydrazine or acylhydrazine, have been reported to exhibit antitumor activities. However, the angionenic effects of this kind of derivatives haven't been elucidated. Here, we synthesized 12 pyridoxal hydrazone and acylhydrazone compounds and investigated their antiangiogenic effects and the underlying mechanisms. METHOD: 3-(4,5-Dimethylthiazol-2-yl)-2, 5-dipheyltetrazolium bromide assay was used to screen the inhibitory effects of the synthesized compounds on endothelial cells (ECs) proliferation. The compound with best inhibitory effect was further evaluated with wound-healing assay and tube formation assay. Calcein-Am assay was carried out to determine the content of intracellular labile iron pool (LIP). Intracellular reduced glutathione (GSH) was determined by spectrophotometry. Flow cytometry was used to determine cell cycle and apoptosis. RESULTS: Compound 10 (3-hydroxy-5-[hydroxymethyl]-2-methyl-pyridine-4-carbaldehyde-2-naphthalen-1-acetyl hydrazone) showed the best inhibitory effect on human umbilical vascular ECs proliferation, with IC50 value of 25.4 µmol/L. It not only inhibited wound-healing and tube formation of ECs, but also decreased the content of intracellular LIP and GSH. Furthermore, it arrested ECs cycle at S phase and induced cell apoptosis. CONCLUSIONS: Compound 10 exhibits antiangiogenic effects by reducing the content of intracellular LIP and GSH, and subsequently arresting cell cycle and inducing cell apoptosis.
Asunto(s)
Inhibidores de la Angiogénesis/síntesis química , Inhibidores de la Angiogénesis/farmacología , Células Endoteliales de la Vena Umbilical Humana/efectos de los fármacos , Hidrazonas/síntesis química , Hidrazonas/farmacología , Apoptosis/efectos de los fármacos , Ciclo Celular/efectos de los fármacos , Movimiento Celular/efectos de los fármacos , Proliferación Celular/efectos de los fármacos , Células Cultivadas , Glutatión/metabolismo , Células Endoteliales de la Vena Umbilical Humana/metabolismo , Células Endoteliales de la Vena Umbilical Humana/fisiología , Humanos , Cicatrización de Heridas/efectos de los fármacosRESUMEN
Administration of mannitol with high dose could induce extensive isometric renal proximal tubular vacuolization and acute renal failure in clinic. We previously demonstrated that mannitol-induced human kidney tubular epithelial cell (HK-2) injury. The objective of our present work was to further study the cytotoxicity of mannitol in HK-2 cells and its potential mechanism. Cell viability was assessed by an MTT method. Cell morphological changes were observed. Furthermore, levels of malondialdehyde (MDA) and glutathione (GSH) were measured. Flow cytometry was performed to determine cell apoptosis by using Annexin V-FITC and PI. In addition, the F-actin of cells was labeled by FITC-Phalloidin for observation of cytoskeleton. The MTT assay displayed that the cell viability decreased significantly in a dose- and time-dependent manner. The morphological changes were observed, including cell membrane rapture and cell detachment. The GSH concentration in HK-2 cells decreased dramatically in mannitol treatment group, while MDA content increased significantly. The results of flow cytometry indicated that apoptotic percentages of HK-2 cells increased in 250 mmol/L mannitol treatment group. After treatment with 250 mmol/L mannitol for 48 h, HK-2 cells showed disorganization of cytoskeleton and even exhibited a totally destroyed cytoskeleton. Therefore, high dose of mannitol has a toxic effect on renal tubular epithelial cells, which might be attributed to oxidative stress, destroyed cellular cytoskeleton and subsequent cell apoptosis.
Asunto(s)
Supervivencia Celular/efectos de los fármacos , Diuréticos Osmóticos/farmacología , Células Epiteliales/efectos de los fármacos , Túbulos Renales Proximales/efectos de los fármacos , Manitol/farmacología , Lesión Renal Aguda/inducido químicamente , Apoptosis/efectos de los fármacos , Línea Celular , Membrana Celular/efectos de los fármacos , Membrana Celular/patología , Citoesqueleto/efectos de los fármacos , Citoesqueleto/patología , Células Epiteliales/metabolismo , Células Epiteliales/patología , Citometría de Flujo , Glutatión/metabolismo , Humanos , Túbulos Renales Proximales/citología , Malondialdehído/metabolismo , Estrés Oxidativo/efectos de los fármacosRESUMEN
The objective of this study was to determine the role of cyclooxygenase (COX)-1 or -2 in endothelium-dependent contraction under atherosclerotic conditions. Atherosclerosis was induced in apoE knockout (apoE(-/-)) mice and those with COX-1(-/-) (apoE(-/-)-COX-1(-/-)) by feeding with high fat and cholesterol food. Aortas (abdominal or the whole section) were isolated for functional and/or biochemical analyses. As in non-atherosclerotic conditions, the muscarinic receptor agonist acetylcholine (ACh) evoked an endothelium-dependent, COX-mediated contraction following NO synthase (NOS) inhibition in abdominal aortic rings from atherosclerotic apoE(-/-) mice. Interestingly, COX-1 inhibition not only abolished such a contraction in rings showing normal appearance, but also diminished that in rings with plaques. Accordingly, only a minor contraction (<30% that of apoE(-/-) counterparts) was evoked by ACh (following NOS inhibition) in abdominal aortic rings of atherosclerotic apoE(-/-)-COX-1(-/-) mice with plaques, and none was evoked in those showing normal appearance. Also, the contraction evoked by ACh in apoE(-/-)-COX-1(-/-) abdominal aortic rings with plaques was abolished by non-selective COX inhibition, thromboxane-prostanoid (TP) receptor antagonism, or endothelial denudation. Moreover, it was noted that ACh evoked a predominant production of the prostacyclin (PGI2, which mediates abdominal aortic contraction via TP receptors in mice) metabolite 6-keto-PGF1α, which was again sensitive to COX-1 inhibition or COX-1(-/-). Therefore, in atherosclerotic mouse abdominal aortas, COX-1 can still be the major isoform mediating endothelium-dependent contraction, which probably results largely from PGI2 synthesis as in non-atherosclerotic conditions. In contrast, COX-2 may have only a minor role in such response limited to areas of plaques under the same pathological condition.
Asunto(s)
Aorta Abdominal/fisiopatología , Aterosclerosis/fisiopatología , Ciclooxigenasa 1/metabolismo , Ciclooxigenasa 2/metabolismo , Endotelio Vascular/metabolismo , Vasoconstricción , Acetilcolina/farmacología , Animales , Aorta Abdominal/efectos de los fármacos , Aorta Abdominal/metabolismo , Apolipoproteínas E/deficiencia , Apolipoproteínas E/genética , Aterosclerosis/genética , Aterosclerosis/metabolismo , Ciclooxigenasa 1/deficiencia , Ciclooxigenasa 1/genética , Ciclooxigenasa 2/genética , Inhibidores de la Ciclooxigenasa/farmacología , Dinoprostona/biosíntesis , Endotelio Vascular/efectos de los fármacos , Epoprostenol/biosíntesis , Epoprostenol/metabolismo , Regulación Enzimológica de la Expresión Génica/efectos de los fármacos , Técnicas de Inactivación de Genes , Masculino , Ratones , Ratones Endogámicos C57BL , Agonistas Muscarínicos/farmacología , Óxido Nítrico/metabolismo , Receptores Muscarínicos/metabolismo , Vasoconstricción/efectos de los fármacosRESUMEN
This study aimed to determine whether prostacyclin (PGI2) functions as an endothelium-derived contracting factor (EDCF) in young rat renal arteries, and, if so, we wanted to examine the underlying mechanism(s) and how it changes in prehypertensive conditions. Vessels from Wistar-Kyoto (WKY) and prehypertensive spontaneously hypertensive rats (SHRs) of 25-28 days of age were isolated for functional and biochemical analyses. Result showed that following NO synthase (NOS) inhibition PGI2 and the thromboxane-prostanoid (TP) receptor agonist U-46619 evoked contractions in young WKY renal arteries that were similar to those in prehypertensive SHRs. Meanwhile, the endothelial muscarinic receptor agonist ACh evoked an endothelium-dependent contraction under NOS-inhibited conditions and a production of the PGI2 metabolite 6-keto-PGF1α; both were sensitive to cyclooxygenase (COX) and/or COX-1 inhibition but higher in prehypertensive SHRs than in young WKYs. Interestingly, in WKY renal arteries PGI2 did not evoke relaxation even after TP receptor antagonism that diminished the contraction evoked by the agonist. Indeed, PGI2 (IP) receptors were not detected in the vessel with Western blot. Moreover, we noted that treatment with the nonselective COX inhibitor indomethacin, which was started at the prehypertensive stage, blunted the elevation of systolic blood pressure and reduced the heart-to-body ratio in SHR within 2 mo of treatment. These results demonstrate that due to scarcity of IP receptors, PGI2, which is derived mainly from COX-1-mediated metabolism, acts as an EDCF in young WKY renal arteries, and it increases in prehypertensive conditions. Also, our data revealed that COX inhibition starting from the prehypertensive stage has an antihypertensive effect in young SHRs.
Asunto(s)
Epoprostenol/metabolismo , Hipertensión/metabolismo , Arteria Renal/metabolismo , Vasoconstricción , Ácido 15-Hidroxi-11 alfa,9 alfa-(epoximetano)prosta-5,13-dienoico/farmacología , Animales , Ciclooxigenasa 1/metabolismo , Inhibidores de la Ciclooxigenasa/farmacología , Hipertensión/fisiopatología , Proteínas de la Membrana/metabolismo , Agonistas Muscarínicos/farmacología , Ratas , Ratas Endogámicas SHR , Ratas Endogámicas WKY , Receptores de Tromboxano A2 y Prostaglandina H2/agonistas , Arteria Renal/efectos de los fármacos , Arteria Renal/crecimiento & desarrollo , Arteria Renal/fisiopatologíaRESUMEN
This study tested the hypothesis that in diabetic arteries, cyclooxygenase (COX)-1 mediates endothelial prostacyclin (PGI2) synthesis, which evokes vasoconstrictor activity under the pathological condition. Non-insulin-dependent diabetes was induced to C57BL/6 mice and those with COX-1 deficiency (COX-1(-/-) mice) using a high-fat diet in combination with streptozotocin injection. In vitro analyses were performed 3 mo after. Results showed that in diabetic aortas, the endothelial muscarinic receptor agonist ACh evoked an endothelium-dependent production of the PGI2 metabolite 6-keto-PGF1α, which was abolished in COX-1(-/-) mice. Meanwhile, COX-1 deficiency or COX-1 inhibition prevented vasoconstrictor activity in diabetic abdominal aortas, resulting in enhanced relaxation evoked by ACh. In a similar manner, COX-1 deficiency increased the relaxation evoked by ACh in nitric oxide synthase-inhibited diabetic renal arteries. Also, in diabetic abdominal aortas and/or renal arteries, both PGI2 and the COX substrate arachidonic acid evoked contractions similar to those of nondiabetic mice. However, the contraction to arachidonic acid, but not that to PGI2, was abolished in vessels from COX-1(-/-) mice. Moreover, we found that 3 mo after streptozotocin injection, systemic blood pressure increased in diabetic C57BL/6 mice but not in diabetic COX-1(-/-) mice. These results explicitly demonstrate that in the given arteries from non-insulin-dependent diabetic mice, COX-1 remains a major contributor to the endothelial PGI2 synthesis that evokes vasoconstrictor activity under the pathological condition. Also, our data suggest that COX-1 deficiency prevents or attenuates diabetic hypertension in mice, although this could be related to the loss of COX-1-mediated activities derived from both vascular and nonvascular tissues.
Asunto(s)
Aorta Abdominal/enzimología , Ciclooxigenasa 1/metabolismo , Diabetes Mellitus Experimental/enzimología , Diabetes Mellitus Tipo 2/enzimología , Dieta Alta en Grasa , Epoprostenol/metabolismo , Proteínas de la Membrana/metabolismo , Arteria Renal/enzimología , Transducción de Señal , Vasoconstricción , 6-Cetoprostaglandina F1 alfa/metabolismo , Acetilcolina/metabolismo , Animales , Aorta Abdominal/efectos de los fármacos , Aorta Abdominal/fisiopatología , Presión Sanguínea , Ciclooxigenasa 1/deficiencia , Ciclooxigenasa 1/genética , Complicaciones de la Diabetes/enzimología , Complicaciones de la Diabetes/fisiopatología , Complicaciones de la Diabetes/prevención & control , Diabetes Mellitus Experimental/genética , Diabetes Mellitus Experimental/fisiopatología , Diabetes Mellitus Tipo 2/genética , Diabetes Mellitus Tipo 2/fisiopatología , Relación Dosis-Respuesta a Droga , Inhibidores Enzimáticos/farmacología , Hipertensión/enzimología , Hipertensión/fisiopatología , Hipertensión/prevención & control , Proteínas de la Membrana/deficiencia , Proteínas de la Membrana/genética , Ratones Endogámicos C57BL , Ratones Noqueados , Agonistas Muscarínicos/farmacología , Óxido Nítrico/metabolismo , Óxido Nítrico Sintasa/antagonistas & inhibidores , Óxido Nítrico Sintasa/metabolismo , Arteria Renal/efectos de los fármacos , Arteria Renal/fisiopatología , Transducción de Señal/efectos de los fármacos , Factores de Tiempo , Vasoconstricción/efectos de los fármacos , Vasoconstrictores/farmacologíaRESUMEN
This study was to determine whether prostacyclin [prostaglandin I2 (PGI2)] evokes mouse renal vasoconstriction and, if so, the underlying mechanism(s) and how its synthesis via cyclooxygenase-1 (COX-1) influences local vasomotor reaction. Experiments were performed on vessels from C57BL/6 mice and/or those with COX-1 deficiency (COX-1(-/-)). Results showed that in renal arteries PGI2 evoked contraction more potently than in carotid arteries, where COX-1 is suggested to mediate prominent endothelium-dependent contraction. A similar result was observed with the thromboxane-prostanoid (TP) receptor agonist U46619. However, in renal arteries TP receptor antagonism, which inhibited the contraction, did not result in any relaxation in response to PGI2. Moreover, we noted that the endothelial muscarinic receptor agonist ACh evoked an increase in the production of the PGI2 metabolite 6-keto-PGF1α, which was prevented by endothelial denudation or COX-1(-/-). Interestingly, COX-1(-/-) was further found to abolish a force development that was sensitive to TP receptor antagonism and result in enhanced relaxation evoked by ACh following NO synthase inhibition. Also, in renal arteries the COX substrate arachidonic acid evoked a vasoconstrictor response, which was again abolished by COX-1(-/-). Meanwhile, nonselective COX inhibition did not show any effect in vessels from COX-1(-/-) mice. Thus, in mouse renal arteries, high expression of TP receptors together with little functional involvement from the vasodilator PGI2 receptors results in a potent vasoconstrictor effect evoked by PGI2. Also, our data imply that endogenous COX-1-mediated PGI2 synthesis leads to vasoconstrictor activity and this could be an integral part of endothelium-derived mechanisms in regulating local renal vascular function.
Asunto(s)
Ciclooxigenasa 1/fisiología , Epoprostenol/biosíntesis , Receptores de Epoprostenol/metabolismo , Arteria Renal/enzimología , Vasoconstricción , Ácido 15-Hidroxi-11 alfa,9 alfa-(epoximetano)prosta-5,13-dienoico , Animales , Ciclooxigenasa 1/genética , Sistema Enzimático del Citocromo P-450/metabolismo , Femenino , Oxidorreductasas Intramoleculares/metabolismo , Masculino , Ratones , Ratones Endogámicos C57BL , Sistema Vasomotor/fisiologíaRESUMEN
Vascular adhesion protein-1 (VAP-1) is both an endothelial adhesion molecule involved in leukocytes emigration, and an oxidase belonging to the family of semicarbazide-sensitive amine oxidases (SSAOs). The enzyme activity of VAP-1 plays an important role in the migration of myeloid-derived suppressor cells (MDSCs) into tumor site, and SSAO inhibitors can block the function of VAP-1. The effects of SSAO inhibitors on leukocyte infiltration and tumor progression were evaluated in H22 hepatocellular carcinoma-bearing C57BL/6 mice. Tumor weight and volume were measured after SSAO inhibitor treatment. Then, MDSCs recruitment and neo-angiogenesis were determined using immunostaining. SSAO inhibitors significantly blocked the catalytic activity of VAP-1 in tumor, attenuated tumor progression, and reduced neo-angiogenesis. CD11b(+) and Gr-1(+) MDSCs, which normally infiltrate into tumors, were significantly diminished in tumor-bearing mice treated with SSAO inhibitors. The present study demonstrated that SSAO inhibitors might have an anti-tumor effect on hepatocellular carcinoma by inhibiting recruitment of CD11b(+) and Gr-1(+) cells and hindering angiogenesis, which could be attributed to impairing the catalytic activity of VAP-1.
Asunto(s)
Amina Oxidasa (conteniendo Cobre)/antagonistas & inhibidores , Antineoplásicos/farmacología , Carcinoma Hepatocelular/patología , Neoplasias Hepáticas/patología , Animales , Western Blotting , Modelos Animales de Enfermedad , Inhibidores Enzimáticos/farmacología , Inmunohistoquímica , Ratones , Ratones Endogámicos C57BLRESUMEN
This study aimed to determine whether the cyclo-oxygenase (COX) substrate arachidonic acid (AA) evokes endothelium-dependent contraction and, if so, the specific COX isoform(s) involved and whether prostacyclin (prostaglandin I2; PGI2), a mediator of endothelium-derived vasoconstrictor activity, can be generated in medial smooth muscle from the intermediate COX product prostaglandin H2 (PGH2) that might diffuse from the endothelium. Aortae and/or carotid arteries were isolated from C57BL/6 mice or those lacking one of the two COX isoforms (COX-1(-/-) or COX-2(-/-)) for functional and/or biochemical analyses. Results showed that in vessels from C57BL/6 mice, exogenous AA evoked not only endothelium-dependent production of the PGI2 metabolite 6-keto-PGF1α, but also contractions reduced by thromboxane-prostanoid receptor antagonism or endothelial denudation. The minimal concentration for AA to evoke contraction was 0.3 µm, a level thought to activate only COX-2. However, neither the contraction nor 6-keto-PGF1α production was altered in vessels from COX-2(-/-) mice, while both were reduced in COX-1(-/-) counterparts. In vessels from COX-1(-/-) mice, AA also caused minor contractions that were sensitive to non-selective COX inhibition. Real-time PCR showed that like COX-1, COX-2 mainly existed in the endothelium, but it was unaltered in COX-1(-/-) mice. Also, we noted that in endothelium-denuded aortae, PGH2 generated PGI2 as in intact vessels. These results demonstrate a predominant role for COX-1 and suggest that in the given mouse arteries, metabolites from either COX isoform cause contraction. Moreover, our results imply that some of the PGI2 involved in vasoconstrictor activity of endothelial COX-mediated metabolism could possibly be generated from PGH2 in medial smooth muscle.
Asunto(s)
Aorta Abdominal/metabolismo , Aorta Abdominal/fisiología , Ácido Araquidónico/metabolismo , Ciclooxigenasa 1/metabolismo , Ciclooxigenasa 2/metabolismo , Endotelio Vascular/metabolismo , Endotelio Vascular/fisiología , Proteínas de la Membrana/metabolismo , 6-Cetoprostaglandina F1 alfa/metabolismo , Animales , Epoprostenol/metabolismo , Masculino , Ratones , Ratones Endogámicos C57BL , Músculo Liso/metabolismo , Músculo Liso/fisiología , Isoformas de Proteínas/genética , Vasoconstricción/fisiología , Sistema Vasomotor/metabolismo , Sistema Vasomotor/fisiologíaRESUMEN
The heme-based oxygen-sensor phosphodiesterase from Escherichia coli (Ec DOS), is composed of an N-terminal heme-bound oxygen sensing domain and a C-terminal catalytic domain. Oxygen (O2) binding to the heme Fe(II) complex in Ec DOS substantially enhances catalysis. Addition of hydrogen sulfide (H2S) to the heme Fe(III) complex in Ec DOS also remarkably stimulates catalysis in part due to the heme Fe(III)-SH and heme Fe(II)-O2 complexes formed by H2S. In this study, we examined the roles of the heme distal amino acids, M95 (the axial ligand of the heme Fe(II) complex) and R97 (the O2 binding site in the heme Fe(II)-O2 complex) of the isolated heme-binding domain of Ec DOS (Ec DOS-PAS) in the binding of H2S under aerobic conditions. Interestingly, R97A and R97I mutant proteins formed an oxygen-incorporated modified heme, verdoheme, following addition of H2S combined with H2O2 generated by the reactions. Time-dependent mass spectroscopic data corroborated the findings. In contrast, H2S did not interact with the heme Fe(III) complex of M95H and R97E mutants. Thus, M95 and/or R97 on the heme distal side in Ec DOS-PAS significantly contribute to the interaction of H2S with the Fe(III) heme complex and also to the modification of the heme Fe(III) complex with reactive oxygen species. Importantly, mutations of the O2 binding site of the heme protein converted its function from oxygen sensor to that of a heme oxygenase. This study establishes the novel role of H2S in modifying the heme iron complex to form verdoheme with the aid of reactive oxygen species.
Asunto(s)
Hemo Oxigenasa (Desciclizante)/metabolismo , Hemo/metabolismo , Sulfuro de Hidrógeno/química , Oxígeno/análisis , Hidrolasas Diéster Fosfóricas/genética , Hidrolasas Diéster Fosfóricas/metabolismo , Biocatálisis , Escherichia coli/enzimología , Escherichia coli/metabolismo , Compuestos Férricos/química , Compuestos Férricos/metabolismo , Hemo/química , Hemo Oxigenasa (Desciclizante)/química , Peróxido de Hidrógeno/química , Peróxido de Hidrógeno/metabolismo , Sulfuro de Hidrógeno/metabolismo , Modelos Moleculares , Estructura Molecular , Mutación , Oxígeno/metabolismo , Hidrolasas Diéster Fosfóricas/química , Especies Reactivas de Oxígeno/metabolismoRESUMEN
Homocysteine is an amino acid containing a free sulfhydryl group, making it probably contribute to the antioxidative capacity in the body. We recently found that plasma total homocysteine (total-Hcy) concentration increased with time when whole blood samples were kept at room temperature. The present study was to elucidate how increased plasma total-Hcy is produced and explore the potential physiological role of homocysteine. Erythrocytes and leukocytes were separated and incubated in vitro; the amount of total-Hcy released by these two kinds of cells was then determined by HPLC-MS. The effects of homocysteine and methionine on reactive oxygen species (ROS) production, osmotic fragility, and methemoglobin formation in erythrocytes under oxidative stress were studied. The reducing activities of homocysteine and methionine were tested by ferryl hemoglobin (Hb) decay assay. As a result, it was discovered that erythrocytes metabolized methionine to homocysteine, which was then oxidized within the cells and released to the plasma. Homocysteine and its precursor methionine could significantly decrease Rosup-induced ROS production in erythrocytes and inhibit Rosup-induced erythrocyte's osmotic fragility increase and methemoglobin formation. Homocysteine (but not methionine) was demonstrated to enhance ferryl Hb reduction. In conclusion, erythrocytes metabolize methionine to homocysteine, which contributes to the antioxidative capability under oxidative stress and might be a supplementary protective factor for erythrocytes against ROS damage.
RESUMEN
This study aimed to determine whether PGI(2) would be evoked by the endogenous endothelial B(2) receptor agonist bradykinin (BK) in the porcine interlobular renal artery and, if so, to determine how it would influence the vasomotor reaction, and the specific cyclooxygenase (COX) isoform(s) involved in its synthesis. The production of the PGI(2) metabolite 6-keto-PGF(1α) was analyzed with HPLC-mass spectroscopy, while vasomotor reaction to PGI(2) or BK was determined with isometric force measurement. Results showed that BK evoked an increase in the production of 6-keto-PGF(1α), which was abolished by endothelial denudation that removed COX-1 expression, or was reduced by COX-1 inhibition. Interestingly, PGI(2) evoked a potent contraction, which was prevented by antagonizing thromboxane-prostanoid (TP) receptors and was not enhanced by antagonizing the vasodilator PGI(2) (IP) receptors. The IP receptor agonists MRE-269 and iloprost did not induce any relaxation. Moreover, iloprost, which is also a PGI(2) analog, caused a contraction, which was sensitive to TP receptor antagonism, but was to a significantly lesser extent than that of PGI(2). Indeed, IP receptors were not detected by RT-PCR or Western blotting in the vessel. Following nitric oxide synthase (NOS) inhibition, BK also evoked an endothelium-dependent contraction, which was blocked by TP receptor antagonism. In addition, inhibition of COX-1 (but not COX-2) impeded the vasoconstrictor activity of BK and expedited the relaxation induced by the agonist in NOS-intact vessels. These results demonstrate that in the porcine interlobular renal artery BK evokes endothelial COX-1-mediated PGI(2) synthesis, which mainly leads to the activation of TP receptors and a vasoconstrictor response, possibly due to a scarcity of vasodilator activity mediated by IP receptors. Also, our data suggested that the effect of a PGI(2) analog on TP receptors could be reduced compared with that of PGI(2) due to modified structure as with iloprost.
Asunto(s)
Ciclooxigenasa 1/metabolismo , Endotelio Vascular/metabolismo , Epoprostenol/metabolismo , Arteria Renal/metabolismo , Vasoconstricción/fisiología , Animales , Bradiquinina/farmacología , Ciclooxigenasa 1/efectos de los fármacos , Endotelio Vascular/efectos de los fármacos , Inhibidores Enzimáticos/farmacología , Iloprost/farmacología , Modelos Animales , NG-Nitroarginina Metil Éster/farmacología , Arteria Renal/efectos de los fármacos , Porcinos , Vasoconstricción/efectos de los fármacosRESUMEN
This study was to determine whether the endothelium of mouse major arteries produces prostacyclin (PGI(2)) and, if so, to determine how PGI(2) affects vasomotor reactivity and whether cyclo-oxygenase-1 (COX-1) contributes to PGI(2) synthesis. Abdominal aortas, carotid and femoral arteries were isolated from wild-type mice and/or those with COX-1 or -2 deficiency (COX-1(-/-); COX-2(-/-)) for biochemical and/or functional analyses. The PGI(2) metabolite 6-keto-PGF(1α) was analysed with high-performance liquid chromatography-mass spectroscopy, while vasoreactivity was determined with isometric force measurement. Results showed that in the abdominal aorta, ACh evoked endothelium-dependent production of 6-keto-PGF(1α), which was abolished by COX-1(-/-), but not by COX-2(-/-). Interestingly, COX-1(-/-) enhanced the dilatation in response to ACh, while PGI(2), which evoked relaxation of the mesenteric artery, caused contraction that was abolished by antagonizing thromboxane prostanoid (TP) receptors in the abdominal aorta. However, the TP receptor agonist U46619 evoked similar contractions in the abdominal aorta and mesenteric artery. Also, antagonizing TP receptors enhanced the relaxation in response to PGI(2) in mesenteric arteries. Real-time PCR showed that the PGI(2) (IP) receptor mRNA level was lower in the abdominal aorta than in mesenteric arteries. In addition, COX-1(-/-) not only abolished the contraction in response to ACh following NO inhibition in abdominal aorta, but also those in the carotid and femoral arteries. These results demonstrate an explicit role for endothelial COX-1 in PGI(2) synthesis and suggest that in given mouse arteries, PGI(2) mediates not dilatation but rather vasoconstrictor activity, possibly due to a low expression or functional presence of IP receptors, which enables PGI(2) to act mainly on TP receptors.
Asunto(s)
Acetilcolina/farmacología , Ciclooxigenasa 1/metabolismo , Epoprostenol/biosíntesis , Proteínas de la Membrana/metabolismo , Arterias Mesentéricas/efectos de los fármacos , Arterias Mesentéricas/metabolismo , Vasodilatación/efectos de los fármacos , Ácido 15-Hidroxi-11 alfa,9 alfa-(epoximetano)prosta-5,13-dienoico/farmacología , 6-Cetoprostaglandina F1 alfa/metabolismo , Animales , Ciclooxigenasa 1/genética , Ciclooxigenasa 2/genética , Ciclooxigenasa 2/metabolismo , Sistema Enzimático del Citocromo P-450/genética , Sistema Enzimático del Citocromo P-450/metabolismo , Endotelio Vascular/efectos de los fármacos , Endotelio Vascular/metabolismo , Epoprostenol/metabolismo , Oxidorreductasas Intramoleculares/genética , Oxidorreductasas Intramoleculares/metabolismo , Contracción Isométrica/efectos de los fármacos , Contracción Isométrica/fisiología , Masculino , Proteínas de la Membrana/genética , Ratones , Ratones Endogámicos C57BL , Ratones Noqueados , Óxido Nítrico/metabolismo , Óxido Nítrico Sintasa/antagonistas & inhibidores , Óxido Nítrico Sintasa/metabolismo , ARN Mensajero/efectos de los fármacos , ARN Mensajero/genética , Receptores de Tromboxanos/metabolismo , Vasoconstrictores/farmacología , Vasodilatación/genética , Vasodilatación/fisiologíaRESUMEN
This study aimed to determine whether cyclo-oxygenase-1 (COX-1) mediates dilatation of mouse arteries via synthesis of prostacyclin (PGI(2)) and, if so, how PGI(2) (IP) receptors contribute and whether thromboxane prostanoid (TP) receptors are implicated in the process. Mesenteric arteries were isolated from wild-type mice or mice with COX-1 deficiency (COX-1(-/-)). The vasomotor reaction to the COX substrate arachidonic acid (AA) was determined with isometric force measurement, while the in vitro production or the plasma level of the PGI(2) metabolite 6-keto-PGF(1α) was analysed with high-performance liquid chromatography-mass spectroscopy or enzyme immunoassay, respectively. Results showed that AA, which evoked endothelium-dependent 6-keto-PGF(1α) production, elicited relaxation that was inhibited or enhanced by antagonizing IP or TP receptors, respectively. Also, IP receptor blockade resulted in contraction in response to AA (following NO synthase inhibition), which was prevented by a concomitant TP receptor antagonism. Meanwhile, COX-1(-/-) or COX-1 inhibition abolished the in vitro 6-keto-PGF(1α) production and reduced the relaxation or contraction observed with AA. Real-time PCR showed that whereas TP receptor mRNAs were detected at similar levels, IP receptor mRNAs were present at higher levels in the branches than in the main stem of the mesenteric artery. In addition, antagonizing the IP receptors enhanced the contraction evoked by PGI(2) in the carotid artery. Also, we noted that COX-1(-/-) mice had a reduced basal plasma 6-keto-PGF(1α) level. These results demonstrate an explicit vasodilator role for COX-1-mediated endothelial PGI(2) synthesis and suggest that the functionally opposing IP and TP receptors concomitantly mediate the vasomotor reaction to PGI(2), with the dilator activity of IP receptors being compromised by the vasoconstrictor effect of TP receptors and vice versa.
Asunto(s)
Ácido Araquidónico/farmacología , Receptores de Prostaglandina/metabolismo , Receptores de Tromboxanos/metabolismo , 6-Cetoprostaglandina F1 alfa/metabolismo , Animales , Ácido Araquidónico/metabolismo , Ciclooxigenasa 1/deficiencia , Ciclooxigenasa 1/metabolismo , Epoprostenol/biosíntesis , Epoprostenol/metabolismo , Masculino , Arterias Mesentéricas/metabolismo , Ratones , Ratones Endogámicos C57BL , Receptores de Epoprostenol/efectos de los fármacos , Receptores de Prostaglandina/efectos de los fármacos , Receptores de Tromboxanos/efectos de los fármacos , Vasoconstricción/efectos de los fármacos , Vasodilatación/efectos de los fármacosRESUMEN
The poor bioavailability and rapid metabolism of curcumin (CUR) restrict its clinical application. Piperine (PIP), which was extracted from natural compounds, can increase the plasma concentration of curcumin in humanidad. As an artificial synthetic piperine analog, silepcimide (ILE) has significant advantages because of the low price and simple synthesis process. In this study, a simple and rapid HPLC-UV method was developed for determination of the plasma concentration of CUR, PIP,ILE and dihydrocurcumin (DHC, a metabolite of CUR) simultaneously. Meanwhile, the effects of PIP and ILE on the plasma concentration and pharmacokinetics of DHC in SD rats was studied to explore whether ILE could serve as a CUR bioavailability enhancer. The metabolic pathway of CUR was studied by comparing the differences of CUR plasma concentration between intravenous injection and oral administration over the same time period, and reacting with small intestine homogenate without microbes of SD rats. The results of drug-time curve showed that combined administration of ILE and CUR had significant effect on plasma concentrations of DHC. Repeated administration of PIP or ILE could significantly increase the plasma concentration of DHC. Plasma CUR could be detected in the samples of from intravenous injection of CUR rats, whereas, it couldn't be detected in the plasma sample form oral administration rats. CUR incubated with intestinal homogenate without intestinal bacteria could not be transformed into DHC. In conclusion, our results show that ILE can improve the bioavailability of CUR. Additionally, it was inferred that most of the CUR was reduced to DHC by NADPH when it was absorbed from gastrointestinal tract, and our results demonstrated that this pathway might be mediated by gastrointestinal microorganisms.
RESUMEN
This study was to determine how endothelium-dependent contractions (EDCs) change in iliac arteries of Wistar-Kyoto (WKYs) and spontaneously hypertensive rats (SHRs) during the transition from adolescence to adulthood and the underlying mechanism(s). We also aimed to elucidate effects of L-798106, an EP3 receptor antagonist, on EDCs and the blood pressure increase in adolescent SHRs. Blood vessels were isolated for functional and biochemical analyses. EDCs were comparable in adolescent iliac arteries of both strains, and contractions to ACh, prostacyclin (PGI2), the EP3 receptor agonist sulprostone and the TP receptor agonist U46619 in adult vessels were less prominent compared with those in the adolescents, while the attenuation of vasoconstrictions to ACh, PGI2 or U46619 with age was to a lesser extent in SHRs. PGI2 production was decreased to a similar level in adult arteries. TP and EP3 expressions were downregulated in adult vessels, whereas the extent of TP downregulation was less in SHRs. L-798106 partially suppressed the vasoconstrictions to U46619 and attenuated EDCs to a greater extent than SQ29548, and administration of L-798106 blunted the blood pressure increase with age in prehypertensive SHRs. These results demonstrate the comparable EDCs in iliac arteries of the adolescents are decreased in the adults, but relatively larger EDCs in adult SHRs can be a reflection of differential downregulation of TP and EP3 receptors during the transition from adolescence to adulthood. Also, our data suggest that blockade of both TP and EP3 receptors starting from the prehypertensive stage suppresses EDCs and the development of hypertension in SHRs.