RESUMO
Regulation of arterial tone by perivascular adipose tissue (PVAT) differs between sexes. In male SHRSP.Z-Leprfa/IzmDmcr rats (SHRSP.ZF), PVAT exerts a compensatory relaxation effect for the loss of endothelium-mediated vasorelaxation, which occurs during the early stages of metabolic syndrome. However, this effect deteriorates by 23 weeks of age. Here, therefore, we compared the effects of PVAT in female and male SHRSP.ZF. Acetylcholine-induced relaxation in superior mesenteric artery without PVAT did not differ between 23-week-old females and males. However, the presence of PVAT enhanced relaxation in 23-week-old females, but not in males. The mRNA levels of angiotensin II type 1 receptor (AT1R) in PVAT did not differ between sexes, but AT1R-associated protein (ATRAP) and apelin levels were higher in females than in males. We observed a positive relationship between differences in artery relaxation with and without PVAT and ATRAP or apelin mRNA levels. In 30-week-old females, PVAT-enhanced relaxation disappeared, and mRNA levels of AT1R increased, while apelin levels decreased compared to 23-week-old females. These results demonstrated that in SHRSP.ZF, PVAT compensation for endothelium dysfunction extended to older ages in females than in males. Apelin and AT1R/ATRAP expression in PVAT may be predictors of favorable effects.
Assuntos
Artéria Mesentérica Superior , Óxido Nítrico , Tecido Adiposo/metabolismo , Animais , Apelina/metabolismo , Apelina/farmacologia , Modelos Animais de Doenças , Feminino , Masculino , Artérias Mesentéricas , Artéria Mesentérica Superior/metabolismo , Óxido Nítrico/metabolismo , RNA Mensageiro/metabolismo , Ratos , Receptor Tipo 1 de Angiotensina/genética , Receptor Tipo 1 de Angiotensina/metabolismo , VasodilataçãoRESUMO
The selective ß 3-adrenoceptor agonist mirabegron, an established alternative to antimuscarinic therapy for patients with overactive bladder, induces additional effects against receptors, transporters, and hepatic enzymes. The present study aimed to elucidate the effects of mirabegron on muscarinic receptors in the rat bladder using radioligand binding and functional assays. Mirabegron (0.1-100 µM) inhibited specific [N-methyl-3H]scopolamine methyl chloride binding in the bladder and other tissues of rats in a concentration-dependent manner. Binding affinity in the bladder was similar to that in the heart and significantly higher than those in the submaxillary gland and brain. Mirabegron induced the concentration-dependent relaxation of carbachol-induced contractions in the rat isolated bladder. Further analyses using a two-site model revealed that the relative quantities of high- and low-affinity components for mirabegron were 44.5% and 55.5%, respectively. Respective pEC50 values were 7.06 and 4.97. Based on the receptor binding affinity and pharmacokinetics of mirabegron, muscarinic receptor occupancy in the human bladder for 24 hours after the administration of a single oral dose of 50 mg mirabegron was 37%-76%. The present results demonstrate for the first time that mirabegron may relax the detrusor smooth muscle not only by ß 3-adrenoceptor activation but also muscarinic receptor blockade. SIGNIFICANCE STATEMENT: Mirabegron, the first selective ß 3-adrenoceptor agonist, represents an alternative to antimuscarinic agents for management of overactive bladder (OAB). The present study aimed to clarify whether mirabegron directly binds to muscarinic receptors and affects cholinergic agonist-induced contractions in rat urinary bladder and to predict muscarinic receptor occupancy in human bladder after oral administration of mirabegron. The results demonstrated that mirabegron therapy for patients with OAB may be due not only to ß 3-adrenoceptor activation but also muscarinic receptor blockade.
Assuntos
Acetanilidas/farmacocinética , Agonistas de Receptores Adrenérgicos beta 3/farmacologia , Antagonistas Muscarínicos/farmacocinética , Tiazóis/farmacocinética , Bexiga Urinária Hiperativa/tratamento farmacológico , Agentes Urológicos/farmacocinética , Acetanilidas/administração & dosagem , Acetanilidas/uso terapêutico , Administração Oral , Agonistas de Receptores Adrenérgicos beta 3/administração & dosagem , Agonistas de Receptores Adrenérgicos beta 3/uso terapêutico , Animais , Encéfalo/metabolismo , Masculino , Antagonistas Muscarínicos/administração & dosagem , Antagonistas Muscarínicos/uso terapêutico , Contração Muscular , Ligação Proteica , Ratos , Ratos Sprague-Dawley , Receptores Muscarínicos/metabolismo , Glândula Submandibular/metabolismo , Tiazóis/administração & dosagem , Tiazóis/uso terapêutico , Bexiga Urinária/metabolismo , Agentes Urológicos/administração & dosagem , Agentes Urológicos/uso terapêuticoRESUMO
Metabolic syndrome (MetS) increases the risk of kidney disease. In SHRSP.Z-Leprfa /IzmDmcr (SHRSP.ZF) rats with MetS, protease-activated receptor 2 (PAR2)-mediated vasorelaxation is preserved in the aorta at 20 weeks of age (weeks) via enhancement of nitric oxide production but impaired at 30 weeks by oxidative stress. However, impairment of PAR2-mediated vasorelaxation of renal arteries and its possible implications for kidney disease are unclear. We used organ baths to assess PAR2-mediated vasorelaxation of isolated renal arteries, colorimetric methods to measure urinary protein levels as an index of renal function, and western blot to determine expression of PAR2 and nephrin proteins in the kidneys of SHRSP.ZF rats at 10, 20, and 30 weeks. We assessed renal arteries and kidney function for effects of orally administered GB88, a pathway-dependent PAR2 antagonist, from 10 to 18 weeks, and azilsartan, an angiotensin II type 1 receptor blocker, from 13 to 23 weeks. PAR2-mediated vasorelaxation was slightly lower at 20 weeks and attenuated significantly at 30 weeks compared with those at 10 weeks. Urinary protein levels were increased at 20 and 30 weeks. Decreased protein expression of PAR2 and nephrin in the kidney were observed at 30 weeks. Administration of GB88 increased blood pressure (BP) and proteinuria. Azilsartan reduced the high BP and the impaired PAR2-mediated vasorelaxation, but did not restore the increase in urinary protein levels and decreased PAR2 and nephrin protein expression in the kidney. PAR2 activation in the kidney may be associated with maintenance of BP and urinary protein excretion in MetS.
Assuntos
Síndrome Metabólica , Animais , Pressão Sanguínea , Ratos , Receptor PAR-2RESUMO
OBJECTIVES: To examine the effects of vibegron, a selective ß3 -adrenoceptor agonist, used to treat overactive bladder, on muscarinic receptors in the rat bladder, and to predict the occupancy levels of muscarinic receptors by vibegron in the bladders of humans orally administered a clinical dose. METHODS: Muscarinic receptors in the rat bladder and other tissues were examined by a radioligand binding assay using [N-methyl-3 H]scopolamine chloride. The occupancy levels of muscarinic receptors by vibegron in bladders of humans after its oral administration were predicted from the estimation of unbound concentrations in human plasma and urine in the literature. RESULTS: Vibegron (0.1-100 µmol/L) inhibited specific [N-methyl-3 H]scopolamine chloride binding in the bladder and other tissues of rats in a concentration-dependent manner. The 50% inhibitory concentration value of vibegron in the bladder was approximately twofold higher than that in the heart, and approximately 315- and 3.5-fold lower than those in the submaxillary gland and brain, respectively. Therefore, the binding affinity of vibegron for muscarinic receptors was higher in the heart and bladder than in the submaxillary gland and brain. By using the rat bladder receptor binding affinity, occupancy levels of muscarinic receptors in the human bladder were predicted to be 51-91% until 24 h after its oral administration at 50 mg of vibegron. CONCLUSIONS: This is the first study to suggest that vibegron binds to muscarinic receptors in the rat bladder and other tissues, with a potentially higher affinity for the M2 subtype than the M1 and M3 subtypes. These results might be clinically relevant for pharmacotherapy with vibegron for overactive bladder.
Assuntos
Bexiga Urinária Hiperativa , Bexiga Urinária , Animais , Humanos , Pirimidinonas , Pirrolidinas , Ratos , Receptores Muscarínicos , Bexiga Urinária Hiperativa/tratamento farmacológicoRESUMO
In vitro and in vivo binding sites of [3H]-labeled 5-hydroxymethyltolterodine (5-HMT), a new radioligand for labeling muscarinic receptors in rat tissues were characterized. Specific [3H]5-HMT binding in rat tissues was saturable and of high affinity in each tissue. The dissociation constant (Kd) was significantly lower in bladder and heart than in submaxillary gland. Significant levels of in vivo specific [3H]5-HMT binding by intravenous injection of the radioligand were detected in tissues, except for cerebral cortex. Thus, [3H]5-HMT was shown to specifically label muscarinic receptors in rat tissues, suggesting a useful radioligand for labeling muscarinic receptors with high affinity.
Assuntos
Compostos Radiofarmacêuticos/metabolismo , Receptores Muscarínicos/metabolismo , Bexiga Urinária/metabolismo , Animais , Glicina Hidroximetiltransferase , Técnicas In Vitro , Masculino , Ratos Sprague-DawleyRESUMO
Diarrhea is often caused by changes in lifestyle, stress, or side effects of drugs. Acanthopanax senticosus root extract (ASRE) has long been used as a functional food remedy with anti-fatigue, neuroprotective, and immunomodulatory activities. However, it is unclear whether ASRE has beneficial effects on gastrointestinal (GI) motility. Therefore, we first investigated whether ASRE directly affects contractile functions of the isolated mouse ileum, and then assessed its effects on GI transit of a charcoal meal in normal mice and a carbachol (CCh)-induced diarrhea mouse model. ASRE caused contraction of the isolated mouse ileum and the maximum contraction was approximately half of that induced by acetylcholine (ACh) administration. In the presence of atropine, this ASRE-induced contraction disappeared, while relaxation responses were observed. However, ASRE reduced potassium chloride- and ACh-induced contractions, and the inhibitory effect was not counteracted by a ß-blocker. Administration of a nitric oxide synthase inhibitor or potassium channel blockers did not affect the ASRE-induced relaxation. Oral administration of ASRE for 1 and 4 d reduced the increased GI transit in CCh-treated but did not affect the GI transit of normal mice. These results indicate that ASRE exhibited dual effects of contraction via muscarinic receptors and direct relaxation on mouse ileal function, and its relaxant effect could be useful in treating diarrhea symptoms, resulting in an increase in the parasympathetic nerve activities.
Assuntos
Eleutherococcus , Motilidade Gastrointestinal/efeitos dos fármacos , Íleo/efeitos dos fármacos , Músculo Liso/efeitos dos fármacos , Extratos Vegetais/farmacologia , Animais , Íleo/fisiologia , Técnicas In Vitro , Masculino , Camundongos Endogâmicos ICR , Contração Muscular/efeitos dos fármacos , Músculo Liso/fisiologia , Raízes de PlantasRESUMO
PURPOSE: Perivascular adipose tissues (PVAT) are involved in the regulation of vascular tone. In mesenteric arteries, the compensatory vasodilatory effects of PVAT appear when vascular relaxation is impaired and disappear at around 23 weeks of age in SHRSP.Z-Leprfa/IzmDmcr (SHRSP.ZF) rats with metabolic syndrome (MetS). The renin-angiotensin system is involved in the development of endothelium and vascular dysfunction. Therefore, we investigated whether azilsartan, a potent angiotensin II type 1 (AT1) receptor antagonist, can protect against the deterioration of the PVAT compensatory vasodilator function that occurs with aging in MetS. METHODS: Two age groups of SHRSP.ZF rats (13 and 20 weeks of age) were administered azilsartan or vehicle through oral gavage once daily for 10 weeks. The vasodilation response of the isolated superior-mesenteric arteries upon addition of endothelium-dependent and -independent agonists was determined in the presence or absence of PVAT using organ bath methods. RESULTS: In vivo treatment with azilsartan improved the acetylcholine-induced vasodilation in mesenteric arteries with and without PVAT at both time-points. The mRNA levels of AT1 receptor and AT1 receptor-associated protein were unchanged in PVAT upon azilsartan treatment. Furthermore, in vitro treatment with azilsartan (0.1 and 0.3 µM for 30 min) did not affect the compensatory effect of PVAT on vasodilation in response to acetylcholine in SHRSP.ZF rat mesenteric arteries. CONCLUSIONS: Our results provide evidence supporting the use of azilsartan for the long-term protection against vascular dysfunctions in MetS. Azilsartan did not improve the dysfunction of PVAT-mediated modulation of vascular tone during MetS. The protective effect of azilsartan is mediated by restoring the endothelium- and vascular smooth muscle-mediated mechanisms.
Assuntos
Tecido Adiposo/efeitos dos fármacos , Bloqueadores do Receptor Tipo 1 de Angiotensina II/farmacologia , Benzimidazóis/farmacologia , Artéria Mesentérica Superior/efeitos dos fármacos , Síndrome Metabólica/tratamento farmacológico , Oxidiazóis/farmacologia , Receptor Tipo 1 de Angiotensina/efeitos dos fármacos , Vasodilatação/efeitos dos fármacos , Vasodilatadores/farmacologia , Tecido Adiposo/metabolismo , Tecido Adiposo/fisiopatologia , Fatores Etários , Animais , Modelos Animais de Doenças , Progressão da Doença , Masculino , Artéria Mesentérica Superior/metabolismo , Artéria Mesentérica Superior/fisiopatologia , Síndrome Metabólica/metabolismo , Síndrome Metabólica/fisiopatologia , Ratos Endogâmicos SHR , Receptor Tipo 1 de Angiotensina/genética , Receptor Tipo 1 de Angiotensina/metabolismo , Transdução de Sinais , Fatores de TempoRESUMO
We evaluated the effects of anticholinergic drugs principally used for the therapy of overactive bladder (OAB) on the activity of P-glycoprotein, an efflux transport protein, in Caco-2 cells. The time-dependent changes in the fluorescence of residual rhodamine 123, a P-glycoprotein activity marker, in the apical region of Caco-2 cells were measured in the presence of anticholinergic drugs using time-lapse confocal laser scanning microscopy. The effect of anticholinergic drugs on human P-glycoprotein ATPase activity was also measured. The fluorescence of residual rhodamine 123 in untreated Caco-2 cells decreased over time. The gradual decrease in the fluorescence was significantly inhibited by treatment with cyclosporine A, darifenacin, and trospium. In contrast, oxybutynin, N-desethyl-oxybutynin (DEOB), propiverine, and its active metabolites (M-1, M-2), imidafenacin, solifenacin, or tolterodine had little effect on the efflux of rhodamine 123. P-Glycoprotein ATPase activity was increased by darifenacin. Darifenacin and trospium reduced the rhodamine 123 transfer across the apical cell membrane. These data suggest that darifenacin and trospium interact with P-glycoprotein. Additionally, darifenacin influenced P-glycoprotein ATPase activity. These results suggest that darifenacin may be a substrate of P-glycoprotein. This study is the first paper to test simultaneously the effects of 10 anticholinergic drugs used currently for the therapy of OAB, on the P-glycoprotein.
Assuntos
Subfamília B de Transportador de Cassetes de Ligação de ATP/metabolismo , Antagonistas Colinérgicos/farmacologia , Adenosina Trifosfatases/metabolismo , Células CACO-2 , Antagonistas Colinérgicos/uso terapêutico , Humanos , Bexiga Urinária Hiperativa/tratamento farmacológicoRESUMO
Although Acanthopanax senticosus root extract (ASRE), a functional food used in Japan, improves peripheral blood circulation and exerts vasorelaxant effects in rats under healthy conditions, the underlying mechanisms currently remain unclear. Therefore, we investigated the mechanisms responsible for ASRE-induced relaxation in isolated thoracic aortas using organ bath techniques and examined whether ASRE affects systemic and peripheral circulation using a photoplethysmographic tail-cuff system and noncontact laser tissue blood flow meter in Wistar rats. Similar to acetylcholine (ACh), ASRE induced dose-dependent relaxation in aortas pre-contracted with phenylephrine; however, in contrast to ACh, ASRE-induced relaxation was partially inhibited by treatments with antagonists of nitric oxide (NO) synthase and soluble guanylyl cyclase as well as by endothelium removal. Contractile responses to phenylephrine or potassium chloride were observed in the presence of ASRE. The oral administration of ASRE (900 mg/kg/d for 1 wk) decreased systolic blood pressure in rats 3 h after the treatment and did not affect heart rate, tail blood flow, mass, or velocity; this decreasing effect was not observed on day 2. A 1-wk treatment with ASRE did not affect vasorelaxation in response to ASRE. These results demonstrate that ASRE induces vasorelaxation via endothelial NO production and an NO-independent pathway in rats. Based on these findings, positive impacts of ASRE on blood pressure and peripheral blood circulation cannot be expected under healthy conditions as the systemic effects of ASRE are temporary. Instead, caution is needed to prevent the occurrence of side effects (i.e., orthostatic dizziness) at the beginning of ASRE dosing.
Assuntos
Eleutherococcus/química , Endotélio Vascular/efeitos dos fármacos , Músculo Liso Vascular/efeitos dos fármacos , Óxido Nítrico Sintase Tipo III/efeitos dos fármacos , Extratos Vegetais/farmacologia , Vasodilatação/efeitos dos fármacos , Animais , Aorta Torácica , Relação Dose-Resposta a Droga , Masculino , Óxido Nítrico Sintase Tipo III/fisiologia , Raízes de Plantas/química , Ratos , Ratos WistarRESUMO
α,ß-Unsaturated carbonyl compounds readily form adducts with SH or NH2 residues, which are nucleophilic agents, by Michael addition. Glutathione (GSH) is a tripeptide that contains an SH residue and functions as an antioxidant. We demonstrated previously that acrolein (ACR), crotonaldehyde (CA), and methyl vinyl ketone (MVK) are present in nicotine- and tar-removed cigarette smoke extract (CSE) and reacted with GSH in B16-BL6 mouse melanoma cells to form GSH-ACR, GSH-CA, and GSH-MVK adducts, suggesting a possible mechanism for CSE-induced cytotoxicity. In this study, we searched for novel α,ß-unsaturated carbonyl compounds other than ACR, CA, and MVK. We selected candidate compounds in CSE based on accurate mass values generated using LC/MS analysis of products formed between CSE and GSH, and identified these using GC/MS analysis and library screening. As a result, we isolated trans-2-methyl-2-butenal, 2-methyl-2-cyclopenten-1-one, 3-methyl-2-cyclopenten-1-one, and furfural, which were poorly reactive with GSH and only very weakly inhibited growth of Colon-26 mouse carcinoma cells and BALB/3T3 clone A31 mouse normal cells. We also isolated 2-cyclopenten-1-one, trans-2-pentenal, 3-methyl-2-butenal and ethyl vinyl ketone, which were highly reactive with GSH and significantly inhibited the growth of both cell lines. Our data suggest that the reactivity of compounds in CSE with GSH may be positively correlated with the effect on inhibiting cell growth. Notably, trans-2-pentenal showed marked inhibition of carcinoma cells growth, whereas this compound exhibited little inhibitory effect on normal cells. trans-2-Pentenal may be a potent candidate or seed for antitumor agents.
Assuntos
Aldeídos/química , Glutationa/química , Fumaça/análise , Aldeídos/toxicidade , Animais , Linhagem Celular Tumoral , Sobrevivência Celular/efeitos dos fármacos , Cromatografia Líquida de Alta Pressão , Cromatografia Gasosa-Espectrometria de Massas , Isomerismo , Espectrometria de Massas , CamundongosRESUMO
Perivascular adipose tissue (PVAT) can regulate vascular tone. In mesenteric arteries of SHRSP.Z-Leprfa/IzmDmcr rats (SHRSP.ZF) with metabolic syndrome, vascular dysfunction is compensated by PVAT-dependent mechanisms that disappear with increasing age. In this study, we investigated the mechanisms of the age-related changes and responsible factor(s) involved in the enhancing effects of mesenteric arterial PVAT in SHRSP.ZF. Acetylcholine- and sodium nitroprusside-induced relaxations of isolated arteries were greater with PVAT than without PVAT at 17 and 20 weeks of age (wks), and as expected, this enhancement by the presence of PVAT disappeared at 23 wks. PVAT mRNA levels of angiotensin II type 1 (AT1) receptor-associated protein was less and AT1 receptor was unchanged at 23 wks when compared to 20 wks. At 20 wks, the enhanced acetylcholine-induced relaxation by the presence of PVAT was inhibited by N-acetyl-l-cysteine (NAC). Acetylcholine-induced relaxation of arteries without PVAT was increased in the presence of exogenously added apelin. PVAT mRNA level of apelin was higher in SHRSP.ZF than in control Wistar-Kyoto rats, and the level was decreased with aging. These results suggest that AT1 receptor activation in PVAT, and changes in the regulation of apelin and a NAC-sensitive factor are related to the age-dependent deterioration of the vasodilation enhancing effects of mesenteric arterial PVAT in SHRSP.ZF.
Assuntos
Acetilcisteína/farmacologia , Tecido Adiposo/metabolismo , Apelina/metabolismo , Síndrome Metabólica/metabolismo , Vasodilatação/efeitos dos fármacos , Acetilcolina/farmacologia , Tecido Adiposo/efeitos dos fármacos , Animais , Apelina/farmacologia , Biomarcadores , Modelos Animais de Doenças , Relação Dose-Resposta a Droga , Artérias Mesentéricas/efeitos dos fármacos , Artérias Mesentéricas/metabolismo , Artérias Mesentéricas/fisiopatologia , Síndrome Metabólica/etiologia , Óxido Nítrico/metabolismo , RNA Mensageiro/genética , Ratos , Receptor Tipo 1 de Angiotensina/genética , Receptor Tipo 1 de Angiotensina/metabolismoRESUMO
Protease-activated receptor 2 (PAR2) is expressed in vascular endothelium. Nitric oxide (NO) - cyclic GMP-mediated vasodilation in response to 2-furoyl-LIGRLO-amide (2fLIGRLO), a PAR2-activating peptide, is impaired in aortas from aged SHRSP.Z-Leprfa/IzmDmcr (SHRSP.ZF) rats with metabolic syndrome. Here we investigated mechanisms linking PAR2's vascular effects to phenotypic characteristics of male SHRSP.ZF rats at 10, 20, and 30 weeks of age. We found vasodilation responses to either 2fLIGRLO or enzyme-mediated PAR2 activation by trypsin were sustained until 20 weeks and lessened at 30 weeks. PAR2 protein and mRNA levels were lower in aortas at 30 weeks than at 10 and 20 weeks. PAR2-mediated responses positively correlated with PAR2 protein and mRNA levels. Decreased cGMP accumulation in the presence of 2fLIGRLO paralleled the decreased relaxations elicited by nitroprusside and the cGMP analog 8-pCPT-cGMP, and the less soluble guanylyl cyclase protein at 30 weeks. 2fLIGRLO-induced relaxation was negatively correlated with serum thiobarbituric acid reactive substances, an index of oxidative stress, which increased with age. Forward stepwise data regression supported a model of age-related decreases in PAR2 function resulting from decreased PAR2 mRNA and increased oxidative stress. We conclude that decreased responsiveness of aortic smooth muscle to NO and downregulation of receptor expression impair PAR2 functions at later stages of metabolic syndrome in SHRSP.ZF rats.
Assuntos
Envelhecimento/metabolismo , Endotélio Vascular/metabolismo , Síndrome Metabólica/metabolismo , Estresse Oxidativo/fisiologia , Receptor PAR-2/metabolismo , Vasodilatação/fisiologia , Animais , Aorta/metabolismo , GMP Cíclico/análogos & derivados , GMP Cíclico/metabolismo , GMP Cíclico/farmacologia , Modelos Animais de Doenças , Regulação para Baixo , Expressão Gênica/fisiologia , Masculino , Óxido Nítrico/metabolismo , Nitroprussiato/farmacologia , Oligopeptídeos/farmacologia , RNA Mensageiro/metabolismo , Ratos , Ratos Endogâmicos , Receptor PAR-2/agonistas , Substâncias Reativas com Ácido Tiobarbitúrico/análise , Tionucleotídeos/farmacologia , Tripsina/farmacologia , Vasodilatadores/farmacologiaRESUMO
The present study aimed to directly characterize specific binding sites of tritium ([(3)H])-labeled imidafenacin, a new radioligand for labeling muscarinic receptors, in the bladder and other peripheral or central nervous tissues of rats. Muscarinic receptors in rat tissues were measured by radioligand binding assay using [(3)H]imidafenacin. Specific [(3)H]imidafenacin binding in rat tissues was saturable, reversible, and of high affinity. Estimated dissociation constants (Kd values) were significantly lower in submaxillary gland and prostate and higher in heart than in bladder, indicating lower Kd values in M1 and M3 subtype- than M2 subtype-dominating tissues. Unlabeled imidafenacin and clinically used antimuscarinic agents competed with [(3)H]imidafenacin for binding sites in bladder and other tissues in a concentration-dependent manner, which indicated pharmacological specificity of [(3)H]imidafenacin binding sites. Pretreatment with N-(2-chloroethyl)-4-piperidinyl diphenylacetate (4-DAMP mustard), an irreversible inactivating agent of M3 subtype, significantly decreased the number of [(3)H]imidafenacin binding sites in bladder, submaxillary gland, and colon, but not in heart. [(3)H]imidafenacin labeled muscarinic receptors in M1 and M3 subtype-dominating tissues with higher affinity than [N-methyl-(3)H]scopolamine methyl chloride (NMS). [(3)H]imidafenacin is a useful radioligand to label muscarinic receptors in M1- and M3-dominating tissues with high affinity.
Assuntos
Imidazóis/metabolismo , Receptores Muscarínicos/metabolismo , Bexiga Urinária/metabolismo , Animais , Imidazóis/farmacocinética , Masculino , Ensaio Radioligante , Ratos , Ratos Sprague-Dawley , Distribuição Tecidual , TrítioRESUMO
Panax notoginseng saponins (PNS) are major components of Panax notoginseng, a herb with established clinical efficacy against vascular diseases. SHRSP.Z-Lepr(fa) /IzmDmcr (SHRSP.ZF) rats, a new animal model for metabolic syndrome, display an impaired vasorelaxation response in aortas and mesenteric arteries that is mediated by nitric oxide (NO). This study investigated whether PNS and its components can ameliorate this vascular dysfunction in SHRSP.ZF rats. In an in vitro study, in the presence or absence of PNS and its components, vasodilation in response to nitroprusside was determined from myographs under isometric tension conditions in aortas and mesenteric arteries from male SHRSP.ZF rats at 18-20 weeks of age. In an in vivo study, PNS (30 mg/kg per day) was orally administered to SHRSP.ZF rats from 8 to 20 weeks of age. In vitro treatment with PNS and Ginsenoside Rb1 increased nitroprusside-induced relaxation of aortas and mesenteric arteries in SHRSP.ZF rats. The PNS-induced increase was not affected by a nitric oxide (NO) synthase inhibitor or endothelium denudation. Relaxation in response to a cell-permeable cGMP analogue was increased by PNS, but cGMP accumulation by nitroprusside was not altered. In vivo treatment with PNS in SHRSP.ZF rats lowered blood pressure and increased relaxation and the expression of soluble guanylyl cyclase protein in arteries, without affecting metabolic abnormalities. These results indicate that PNS causes an increase in vasodilation in response to NO and a decrease in blood pressure, resulting in protection against vascular dysfunction in SHRSP.ZF rats. PNS might be beneficial in alleviating impaired vasodilation in metabolic syndrome.
Assuntos
Síndrome Metabólica/tratamento farmacológico , Síndrome Metabólica/fisiopatologia , Panax notoginseng/química , Saponinas/farmacologia , Vasodilatação/efeitos dos fármacos , Animais , Aorta/efeitos dos fármacos , Aorta/fisiopatologia , Biomarcadores/sangue , Pressão Sanguínea/efeitos dos fármacos , GMP Cíclico/farmacologia , Modelos Animais de Doenças , Masculino , Artérias Mesentéricas/efeitos dos fármacos , Artérias Mesentéricas/fisiopatologia , Síndrome Metabólica/metabolismo , Síndrome Metabólica/patologia , Óxido Nítrico/metabolismo , Estresse Oxidativo/efeitos dos fármacos , Ratos , Saponinas/uso terapêutico , Transdução de Sinais/efeitos dos fármacosRESUMO
Endothelium-dependent vasodilation via protease-activated receptor 2 (PAR2) is preserved in mesenteric arteries from SHRSP.Z-Leprfa/IzmDmcr rats (SHRSP.ZF) with metabolic syndrome even though nitric oxide (NO)-mediated vasodilation is attenuated. Therefore, we examined the PAR2 mechanisms underlying metabolic syndrome-resistant vasodilation in SHRSP.ZF aortas with ageing. In isolated aortas, the PAR2 agonist 2-furoyl-LIGRLO-amide (2fly) caused vasodilation that was sustained in male SHRSP.ZF until 18 weeks of age, but was attenuated afterwards compared with age-matched Wistar-Kyoto rats (controls) at 23 weeks. In contrast, acetylcholine-induced vasodilation was impaired in SHRSP.ZF already at 18 weeks of age. Treatments of aortas with inhibitors of NO synthase and soluble guanylate cyclase abolished the sustained 2fly- and residual acetylcholine-induced vasodilation in SHRSP.ZF at 18 weeks of age. In the aortas of SHRSP.ZF, 8-bromo-cGMP-induced vasodilation, NO production and cGMP accumulation elicited by 2fly were not different from in the controls. PAR2 agonist increased phospho-Ser1177-eNOS protein content only in SHRSP.ZF aortas. These results indicate that vasodilation mediated by PAR2 is sustained even though NO-dependent relaxation is attenuated with ageing/exposure to metabolic disorders in large-caliber arteries from SHRSP.ZF. PAR2 stimulation of NO production via an additional pathway that targets phosphorylation of Ser1177-eNOS suggests a regulatory mechanism for sustaining agonist-mediated vasodilation in metabolic syndrome.
Assuntos
Aorta Torácica/enzimologia , Síndrome Metabólica/enzimologia , Óxido Nítrico Sintase Tipo III/metabolismo , Receptor PAR-2/metabolismo , Vasodilatação , Acetilcolina/farmacologia , Fatores Etários , Animais , Aorta Torácica/efeitos dos fármacos , Aorta Torácica/fisiopatologia , Modelos Animais de Doenças , Relação Dose-Resposta a Droga , Ativação Enzimática , Inibidores Enzimáticos/farmacologia , Masculino , Síndrome Metabólica/sangue , Síndrome Metabólica/fisiopatologia , Óxido Nítrico/metabolismo , Óxido Nítrico Sintase Tipo III/antagonistas & inibidores , Nitroprussiato/farmacologia , Oligopeptídeos/farmacologia , Fosforilação , Ratos Endogâmicos SHR , Ratos Endogâmicos WKY , Receptor PAR-2/agonistas , Transdução de Sinais , Vasodilatação/efeitos dos fármacos , Vasodilatadores/farmacologiaRESUMO
Cordyceps sinensis, a fungus that parasitizes on the larva of Lepidoptera, has been used as a valued traditional Chinese medicine. We investigated the effects of water extracts of Cordyceps sinensis (WECS), and particularly focused on its anticancer and antimetastatic actions. Based on in vitro studies, we report that WECS showed an anticancer action, and this action was antagonized by an adenosine A3 receptor antagonist. Moreover, this anticancer action of WECS was promoted by an adenosine deaminase inhibitor. These results suggest that one of the components of WECS with an anticancer action might be an adenosine or its derivatives. Therefore, we focused on cordycepin (3'-deoxyadenosine) as one of the active ingredients of WECS. According to our experiments, cordycepin showed an anticancer effect through the stimulation of adenosine A3 receptor, followed by glycogen synthase kinase (GSK)-3ß activation and cyclin D1 suppression. Cordycepin also showed an antimetastatic action through inhibiting platelet aggregation induced by cancer cells and suppressing the invasiveness of cancer cells via inhibiting the activity of matrix metalloproteinase (MMP)-2 and MMP-9, and accelerating the secretion of tissue inhibitor of metalloproteinase (TIMP)-1 and TIMP-2 from cancer cells. In conclusion, cordycepin, an active component of WECS, might be a candidate anticancer and antimetastatic agent.
Assuntos
Cordyceps/química , Desoxiadenosinas/uso terapêutico , Metástase Neoplásica/tratamento farmacológico , Pentostatina/farmacologia , Fitoterapia , Extratos Vegetais/farmacologia , Agonistas do Receptor A3 de Adenosina/farmacologia , Agonistas do Receptor A3 de Adenosina/uso terapêutico , Antagonistas do Receptor A3 de Adenosina/farmacologia , Animais , Antineoplásicos/administração & dosagem , Antineoplásicos/farmacologia , Antineoplásicos/uso terapêutico , Aterosclerose/tratamento farmacológico , Linhagem Celular Tumoral , Desoxiadenosinas/administração & dosagem , Desoxiadenosinas/antagonistas & inibidores , Desoxiadenosinas/farmacologia , Quimioterapia Combinada , Humanos , Células de Kupffer/efeitos dos fármacos , Medicina Tradicional Chinesa , Metotrexato/uso terapêutico , Modelos Biológicos , Pentostatina/administração & dosagem , Extratos Vegetais/química , Extratos Vegetais/uso terapêutico , Receptor A3 de Adenosina , Transdução de Sinais/efeitos dos fármacosRESUMO
Although hyperlipidemia, high blood pressure, and diabetes increase the risk of arteriosclerosis, it is not clear whether hyperuricemia increases the risk of arteriosclerosis or not. We examined the effects of uric acid and curative drugs for hyperuricemia on atherosclerosis-susceptible C57BL/6J apolipoprotein E-deficient (apoE(-/-)) mice. Male apoE(-/-) mice (age: 6 weeks) were fed a normal diet (normal diet group) or a uric acid-enriched diet. Mice fed the uric acid-enriched diet were divided into three groups and administered a drinking vehicle (high uric acid diet group), allopurinol (20 mg·kg(-1)·d(-1)), or benzbromarone (20 mg·kg(-1)·d(-1)) for 10 weeks. Serum uric acid concentrations were higher in the high uric acid diet group than in the normal diet group, and concentrations in the allopurinol and benzbromarone groups were lower than in the high uric acid diet group. Serum total cholesterol and triglyceride levels were lower in the allopurinol group than in the high uric acid diet group. Oxidative stress was lower in the benzbromarone group than in the high uric acid diet group. Atherosclerotic lesion areas were smaller in the allopurinol and benzbromarone groups than in the high uric acid diet group. Thus, hyperuricemia may not be an independent risk factor for arteriosclerosis; however, the administration of allopurinol and benzbromarone prevented the development of atherosclerosis in apoE(-/-) mice fed a uric acid-enriched diet. The anti-atherosclerotic effect was in part due to lower total cholesterol and oxidative stress in the serum. Other possible mechanisms underlying this effect should be investigated.
Assuntos
Apolipoproteínas E/metabolismo , Arteriosclerose/etiologia , Hiperuricemia/complicações , Hiperuricemia/tratamento farmacológico , Ácido Úrico/efeitos adversos , Envelhecimento , Alopurinol/uso terapêutico , Ração Animal/análise , Animais , Apolipoproteínas E/genética , Benzobromarona/uso terapêutico , Dieta , Regulação da Expressão Gênica , Supressores da Gota/uso terapêutico , Masculino , Camundongos , Camundongos Knockout , Fatores de Risco , Ácido Úrico/administração & dosagem , Uricosúricos/uso terapêuticoRESUMO
St. John's wort (SJW), or Hypericum perforatum, is a perennial herb that has been used in the treatment of depression in several countries. Though its therapeutic effect on depression has been extensively studied, its influence on metabolic syndrome is yet to be fully characterized. Therefore, we investigated the effect of SJW extract on adipocyte differentiation and its anti-inflammatory effects by using 3T3-L1 preadipocytes. Oil Red O staining indicated that SJW promotes adipocyte differentiation, while immunoblots indicated that SJW increases the expression of peroxisome proliferator activated receptor γ (PPARγ), a nuclear receptor regulating adipocyte differentiation, and adiponectin, an anti-inflammatory adipokine. Furthermore, the anti-inflammatory activity of SJW was demonstrated by its inhibition of the activation of nuclear factor-κB (NF-κB), an inflammatory transcription factor. Stimulation of mature 3T3-L1 adipocytes by tumor necrosis factor-α (TNF-α) decreased the expression of the NF-κB inhibitor IκBα, and increased its phosphorylation. Treatment with SJW further decreased the TNF-α-induced perturbation in IκBα expression and phosphorylation, which indicated that SJW mediated the inhibition of NF-κB activation. In addition, SJW decreased the TNF-α-induced increase in the mRNA levels of pro-inflammatory adipokines, interleukin-6 (IL-6), and monocyte chemoattractant protein-1 (MCP-1). Collectively, our results indicate that SJW treatment could promote adipocyte differentiation probably through its anti-inflammatory activity, which in turn suggests that SJW has the potential to minimize the risk factors of metabolic syndrome.
Assuntos
Adipócitos/efeitos dos fármacos , Anti-Inflamatórios/farmacologia , Diferenciação Celular/efeitos dos fármacos , Hypericum/química , NF-kappa B/antagonistas & inibidores , Extratos Vegetais/farmacologia , Células 3T3-L1 , Adipócitos/citologia , Adipócitos/imunologia , Adipócitos/metabolismo , Adiponectina/biossíntese , Animais , Anti-Inflamatórios/isolamento & purificação , Western Blotting , Técnicas de Cultura de Células , Sobrevivência Celular/efeitos dos fármacos , Flores/química , Síndrome Metabólica/prevenção & controle , Camundongos , PPAR gama/biossíntese , Extratos Vegetais/isolamento & purificação , Reação em Cadeia da Polimerase em Tempo RealRESUMO
Metabolic syndrome (MetS) is a combination of clinical disorders that together increase the risk for cardiovascular disease and diabetes. SHRSP.Z-Lepr(fa)/IzmDmcr (SHRSP.ZF) rats with MetS show impaired nitric oxide-mediated relaxation in coronary and mesenteric arteries, and angiotensin II receptor type 1 blockers protect against dysfunction and oxidative-nitrosative stress independently of metabolic effects. We hypothesize that superoxide contributes to functional deterioration in SHRSP.ZF rats. To test our hypothesis, we studied effects of treatment with tempol, a membrane-permeable radical scavenger, on impaired vasodilation in SHRSP.ZF rats. Tempol did not alter body weight, high blood pressure, or metabolic abnormalities, but prevented impairment of acetylcholine-induced and nitroprusside-induced vasodilation in the coronary and mesenteric arteries. Furthermore, tempol reduced the levels of serum thiobarbituric acid reactive substance (TBARS) and 3-nitrotyrosine content in mesenteric arteries. Systemic administration of tempol elevated the expression of soluble guanylate cyclase (sGC) above basal levels in mesenteric arteries of SHRSP.ZF rats. However, acute treatment with tempol or ebselen, a peroxynitrite scavenger, did not ameliorate impaired relaxation of isolated mesenteric arteries. No nitration of tyrosine residues in sGC was observed; however, sGC mRNA expression levels in the arteries of SHRSP.ZF rats were lower than those in the arteries of Wistar-Kyoto rats. Levels of Thr(496)- and Ser(1177)-phosphorylated endothelial nitric oxide synthase (eNOS) were lower in arteries of SHRSP.ZF rats, and acetylcholine decreased Thr(496)-phosphorylated eNOS levels. These results indicated that prolonged superoxide production, leading to oxidative-nitrosative stress, was associated with impaired vasodilation in SHRSP.ZF rats with MetS. Down-regulated sGC expression may be linked to dysfunction, while reduced NO bioavailability/eNOS activity and modified sGC activity due to superoxide production were excluded as pivotal mechanisms.
Assuntos
Vasos Coronários/patologia , Síndrome Metabólica/fisiopatologia , Nitrogênio/química , Estresse Oxidativo , Vasodilatação/efeitos dos fármacos , Animais , Azóis/farmacologia , Óxidos N-Cíclicos/farmacologia , Modelos Animais de Doenças , Regulação para Baixo , Sequestradores de Radicais Livres/farmacologia , Guanilato Ciclase/sangue , Coração/fisiopatologia , Isoindóis , Masculino , Óxido Nítrico/química , Óxido Nítrico Sintase Tipo III/metabolismo , Compostos Organosselênicos/farmacologia , Fosforilação , RNA Mensageiro/metabolismo , Ratos , Receptor Tipo 1 de Angiotensina/metabolismo , Marcadores de Spin , Superóxidos/química , Tirosina/análogos & derivados , Tirosina/químicaRESUMO
Metabolic syndrome is known to increase the risk of abnormal cardiac structure and function, which are considered to contribute to increased incidence of cardiovascular disease and mortality. We previously demonstrated that ventricular hypertrophy and diastolic dysfunction occur in SHRSP.Z-Lepr(fa)/IzmDmcr (SHRSP fatty) rats with metabolic syndrome. The aim of this study was to investigate the possible mechanisms underlying abnormal heart function in SHRSP fatty rats. The amount of sarcoplasmic reticulum Ca(2+)-ATPase (SERCA) 2a, phospholamban (PLB) protein, and Ser(16)-phosphorylated PLB was decreased in cardiomyocytes from SHRSP fatty rats compared with those from control Wistar-Kyoto rats at 18 weeks of age, and the PLB-to-SERCA2a ratio was increased. Left ventricular developed pressure was unchanged, and coronary flow rate and maximum rate of left ventricular pressure decline (-dP/dt) was decreased in SHRSP fatty rats. Treatment with telmisartan reversed the abnormalities of PLB amount, coronary flow rate, and -dP/dt in SHRSP fatty rats. These results indicate that abnormal amounts of intracellular Ca(2+) regulatory proteins in cardiomyocytes, leading to reduced intracellular Ca(2+) reuptake into the sarcoplasmic reticulum, may play a role in the diastolic dysfunction in SHRSP fatty rats and that these effects are partially related to decreased coronary circulation. Telmisartan may be beneficial in protecting against disturbances in cardiac function associated with metabolic syndrome.