RESUMO
Rheumatoid arthritis (RA) is one of over 100 different types of autoimmune conditions. In RA, the cells of the immune system attack the tissue lining the joints, triggering inflammation. A large body of research suggests that the underlying trigger(s) of RA are unique to an individual. For example, increased risk of RA can be driven by smoking tobacco in one individual and mercury exposure in another. Due to the development of next-generation sequencing technology, the critical role of the microbiota in shaping RA risk has been elucidated. Therefore, it is surprising that diet, arguably the most important lever in shaping the gut microbiota, is ineffective in the treatment of RA, even in a sub-set of patients. To attempt to rationalise this apparent paradox, we conducted an umbrella review to address the question as to whether diet can affect outcomes in RA.
Assuntos
Artrite Reumatoide , Microbioma Gastrointestinal , Artrite Reumatoide/dietoterapia , Artrite Reumatoide/imunologia , Humanos , Preferências Alimentares , DietaRESUMO
Cerebrovascular disease is a group of conditions characterized by disorders of the cerebral vessels. Endothelial dysfunction renders the vasculature at risk of impaired blood flow and increases the potential of developing cerebrovascular disease. The gut microbiota has been recently identified as a possible risk factor of cerebrovascular disease. However, a direct link between gut microbiota and cerebral vascular function has not been established. Therefore, the aim of this study was to determine the effect of gut bacterial disruption on cerebral endothelial function. Male inbred Sprague-Dawley rats were randomly assigned to receive either drinking water with (n = 4) or without (n = 4) a cocktail of nonabsorbable broad-spectrum antibiotics (streptomycin, neomycin, bacitracin, and polymyxin B). Three weeks of antibiotic treatment resulted in a significant reduction in bacterial load and shifts within the bacterial sub-populations as assessed using flow cytometry. Using pressure myography, we found that spontaneous tone significantly increased and L-NAME-induced vasoconstriction was significantly blunted in middle cerebral arteries (MCAs) harvested from antibiotic-treated rats. ATP-mediated dilations were significantly blunted in MCAs from antibiotic-treated rats compared to their control counterparts. Immunoblotting revealed that the eNOS-P/total eNOS ratio was significantly reduced in cerebral artery lysates from antibiotic-treated rats compared to controls. Our findings suggest that disruption of the gut microbiota leads to cerebral endothelial dysfunction through reduction of eNOS activity. This study highlights the potential of the microbiota as a target to reverse endothelial dysfunction and a preventative approach to reducing risk of stroke and aneurysms.
Assuntos
Eixo Encéfalo-Intestino , Encéfalo/irrigação sanguínea , Endotélio Vascular/metabolismo , Microbioma Gastrointestinal , Animais , Endotélio Vascular/fisiologia , Masculino , Óxido Nítrico/metabolismo , Óxido Nítrico Sintase Tipo III/metabolismo , Ratos , Ratos Sprague-Dawley , Transdução de Sinais , VasoconstriçãoRESUMO
Rapamycin (sirolimus) is employed as an immunosuppressant following liver transplant, to inhibit the re-growth of cancer cells following liver resection for hepatocellular carcinoma (HCC), and for the treatment of advanced HCC. Rapamycin also induces the expression of antioxidant enzymes in the liver, suggesting that pretreatment with the drug could provide a potential strategy to reduce ischemia reperfusion injury following liver surgery. The aim of this study was to further investigate the actions of rapamycin in inducing expression of the antioxidant enzymes heme oxygenase-1 (HO-1) and peroxiredoxin-1 (Prx-1) in normal liver and in tumorigenic liver cells. A rat model of segmental hepatic ischemia and reperfusion, cultured freshly-isolated rat hepatocytes, and tumorigenic H4IIE rat liver cells in culture were employed. Expression of HO-1 and Prx-1 was measured using quantitative PCR and western blot. Rapamycin pre-treatment of normal liver in vivo or normal hepatocytes in vitro led to a substantial induction of mRNA encoding HO-1 and Prx-1. The dose-response curve for the action of rapamycin on mRNA expression was biphasic, showing an increase in expression at 0 - 0.1 µM rapamycin but a decrease from maximum at concentrations greater than 0.1 µM. By contrast, in H4IIE cells, rapamycin inhibited the expression of HO-1 and Prx-1 mRNA. Oltipraz, an established activator of transcription factor Nrf2, caused a large induction of HO-1 and Prx-1 mRNA. The dose response curve for the inhibition by rapamycin of HO-1 and Prx-4 mRNA expression, determined in the presence of oltipraz, was monophasic with half maximal inhibition at about 0.01 µM. It is concluded that, at concentrations comparable to those used clinically, pre-treatment of the liver with rapamycin induces the expression of HO-1 and Prx-1. However, the actions of rapamycin on the expression of these two antioxidant enzymes in normal hepatocytes are complex and, in tumorigenic liver cells, differ from those in normal hepatocytes. Further studies are warranted to evaluate preconditioning the livers of patients subject to liver resection or liver transplant with rapamycin as a viable strategy to reduce IR injury following liver surgery.
Assuntos
Heme Oxigenase-1/biossíntese , Hepatócitos/efeitos dos fármacos , Imunossupressores/farmacologia , Neoplasias Hepáticas/metabolismo , Peroxirredoxinas/biossíntese , Sirolimo/farmacologia , Animais , Linhagem Celular Tumoral , Hepatócitos/metabolismo , Masculino , Ratos , Ratos ZuckerRESUMO
Rapamycin is employed as an immunosuppressant following organ transplant and, in patients with hepatocellular carcinoma, to inhibit cancer cell regrowth following liver surgery. Preconditioning the liver with rapamycin to induce the expression of antioxidant enzymes is a potential strategy to reduce ischemia reperfusion (IR) injury. However, pre-treatment with rapamycin inhibits bile flow, especially following ischemia. The aim was to investigate the mechanisms involved in this inhibition. In a rat model of segmental hepatic ischemia and reperfusion, acute administration of rapamycin by intravenous injection did not inhibit the basal rate of bile flow. Pre-treatment of rats with rapamycin for 24â¯h by intraperitoneal injection inhibited the expression of mRNA encoding the sinusoidal influx transporters Ntcp, Oatp1 and 2 and the canalicular efflux transporter Bsep, and increased expression of canalicular Mrp2. Dose-response curves for the actions of rapamycin on the expression of Bsep and Ntcp in cultured rat hepatocytes were biphasic, and monophasic for effects on Oatp1. In cultured tumorigenic H4IIE liver cells, several bile acid transporters were not expressed, or were expressed at very low levels compared to hepatocytes. In H4IIE cells, rapamycin increased expression of Ntcp, Oatp1 and Mrp2, but decreased expression of Oatp2. It is concluded that the inhibition of bile flow recovery following ischemia observed in rapamycin-treated livers is principally due to inhibition of the expression of sinusoidal bile acid transporters. Moreover, in tumorigenic liver tissue the contribution of tumorigenic hepatocytes to total liver bile flow is likely to be small and is unlikely to be greatly affected by rapamycin.
Assuntos
Ductos Biliares/metabolismo , Proteínas de Transporte/metabolismo , Colestase/induzido quimicamente , Imunossupressores/efeitos adversos , Glicoproteínas de Membrana/metabolismo , Sirolimo/efeitos adversos , Animais , Bile/metabolismo , Ductos Biliares/efeitos dos fármacos , Carcinoma Hepatocelular/cirurgia , Linhagem Celular Tumoral , Colestase/patologia , Modelos Animais de Doenças , Relação Dose-Resposta a Droga , Hepatócitos/metabolismo , Humanos , Isquemia/complicações , Fígado/citologia , Fígado/efeitos dos fármacos , Fígado/metabolismo , Neoplasias Hepáticas/cirurgia , Transplante de Fígado/efeitos adversos , Masculino , Ratos , Ratos Sprague-Dawley , Ratos Zucker , Traumatismo por Reperfusão/complicações , Traumatismo por Reperfusão/prevenção & controle , Condicionamento Pré-Transplante/efeitos adversos , Condicionamento Pré-Transplante/métodosRESUMO
AIMS: Type 2 diabetes (T2D) increases the risk of death associated with cardiovascular complications. However, a complete understanding of protein changes within the diabetic vasculature is still lacking. METHODS: Herein, we utilized mass spectrometry to perform vascular and urinary proteome analysis using a rat model of high-fat feeding and low-dose streptozotocin to simulate late-stage T2D. The purpose of this study was to identify aortic and urine proteins that are differentially expressed in normal and T2D rats. RESULTS: High-fat feeding and low-dose streptozotocin resulted in hyperglycemia, hypoinsulinemia and high levels of circulating free fatty acids. Using a shotgun proteomic approach, high-mobility-group protein B1 and spondin-1 were significantly increased in T2D aorta compared to control aorta, suggesting vascular inflammation and smooth muscle proliferation, respectively. However, the majority of differentially expressed aortic proteins were downregulated in T2D, including proteins associated with coagulation, cell differentiation and redox homeostasis. Strikingly, we report a significant downregulation of commonly used cytoskeletal housekeeping proteins in T2D aorta. Urine from T2D rats displayed increased expression of proteins involved in inflammation and oxidative stress and decreased expression of proteins associated with lipid metabolism and cell adhesion. A number of differentially expressed proteins in urine of T2D rats have previously been reported in human T2D, thereby supporting this animal model as a good representation of human T2D. CONCLUSIONS: Our data offer new information regarding key pathways that could be therapeutically targeted to combat the cardiovascular complications of T2D.
Assuntos
Aorta/metabolismo , Diabetes Mellitus Experimental/metabolismo , Genes Essenciais , Proteoma/genética , Animais , Biomarcadores/sangue , Biomarcadores/metabolismo , Biomarcadores/urina , Diabetes Mellitus Experimental/sangue , Diabetes Mellitus Experimental/urina , Regulação para Baixo , Metabolismo dos Lipídeos , Masculino , Estresse Oxidativo , Proteoma/química , Proteoma/metabolismo , Ratos , Ratos WistarRESUMO
Vascular dysfunction is an early feature of diabetic vascular disease, due to increased oxidative stress and reduced nitric oxide (NO) bioavailability. This can lead to endothelial cell senescence and clinical complications such as stroke. Cells can become senescent by shortened telomeres and oxidative stress is known to accelerate telomere attrition. Sirtuin 1 (SIRT1) has been linked to vascular health by upregulating endothelial nitric oxide synthase (eNOS), suppressing oxidative stress, and attenuating telomere shortening. Accelerated leukocyte telomere attrition appears to be a feature of clinical type 2 diabetes (T2D) and therefore the telomere system may be a potential therapeutic target in preventing vascular complications of T2D. However the effect of T2D on vascular telomere length is currently unknown. We hypothesized that T2D gives rise to shortened leukocyte and vascular telomeres alongside reduced vascular SIRT1 expression and increased oxidative stress. Accelerated telomere attrition was observed in circulating leukocytes, but not arteries, in T2D compared to control rats. T2D rats had blunted arterial SIRT1 and eNOS protein expression levels which were associated with reduced antioxidant defense capacity. Our findings suggest that hyperglycemia and a deficit in vascular SIRT1 per se are not sufficient to prematurely shorten vascular telomeres.
Assuntos
Artérias/patologia , Diabetes Mellitus Tipo 2/sangue , Encurtamento do Telômero , Telômero/ultraestrutura , Animais , Antioxidantes/metabolismo , Pressão Sanguínea , Modelos Animais de Doenças , Endotélio Vascular/metabolismo , Hiperglicemia/patologia , Leucócitos/citologia , Leucócitos/metabolismo , Masculino , Óxido Nítrico/metabolismo , Óxido Nítrico Sintase Tipo III/metabolismo , Estresse Oxidativo , Fenótipo , Ratos , Ratos Wistar , Sirtuína 1/metabolismo , Superóxidos/metabolismoRESUMO
OBJECTIVE: NO and a non-NO/prostacyclin EDH mechanism are major contributors of vascular tone and cerebral blood flow. However, the effect of metabolic syndrome on EDH-mediated responses in cerebral vessels remains unknown and may offer another avenue for therapeutic targeting. The purpose of this study was to investigate EDH-dependent responses in cerebral arteries during metabolic syndrome. METHODS: EDH-dependent dilations were assessed in MCAs isolated from nondiabetic obese and lean Zucker rats in the presence and absence of NS309, an activator of SKCa and IKCa channels. IKCa channel expression and activity were assessed by western blotting and pressure myography, respectively. RESULTS: EDH-mediated dilations were significantly attenuated in the obese compared to the lean Zucker rat MCA. Luminal delivery of 1 µM NS309 enhanced EDH-mediated responses in lean and obese Zucker cerebral vessels. Both dose-dependent dilations to luminal NS309 and IKCa protein expression in pooled cerebral arteries were comparable between the two groups. CONCLUSIONS: Our results suggest that pharmacological targeting of IKCa channels can rescue EDH-mediated dilations in obese Zucker rat MCAs. Compromised EDH-mediated dilations in obesity are not due to impaired IKCa channel expression or activity.
Assuntos
Endotélio Vascular , Indóis/farmacologia , Canais de Potássio Ativados por Cálcio de Condutância Intermediária/biossíntese , Obesidade , Oximas/farmacologia , Canais de Potássio Ativados por Cálcio de Condutância Baixa/biossíntese , Vasodilatação/efeitos dos fármacos , Animais , Endotélio Vascular/metabolismo , Endotélio Vascular/patologia , Endotélio Vascular/fisiopatologia , Regulação da Expressão Gênica/efeitos dos fármacos , Masculino , Obesidade/metabolismo , Obesidade/patologia , Obesidade/fisiopatologia , Ratos , Ratos ZuckerRESUMO
Many diseases, including metabolic syndrome, are characterised by endothelial dysfunction mediated by reduced nitric oxide bioavailability and oxidative stress. Sirtuin 1 is a protein deacetylase that targets endothelial nitric oxide synthase resulting in enhanced nitric oxide bioavailability. Although it has been highlighted as a potential therapeutic target, we still have no understanding of vascular SIRT1 changes during obesity. Therefore, the aim of the present study was to measure vascular function, SIRT1 protein levels of expression and markers of oxidative stress in obese Zucker rats. Middle cerebral arteries from nondiabetic obese and lean Zucker rats were mounted in a pressure myograph to assess nitric oxide-dependent dilations. Western blotting was used to measure protein levels of SIRT1, p53, acetylated p53, eNOS, phosphorylated eNOS and markers of oxidative stress (nitrotyrosine, Nox4 and SOD2) in cerebral vascular tissue. SIRT1 expression was two-fold greater in both cerebral arteries and aorta from obese compared to lean Zucker rats. Acetylation of p53 at the SIRT1-specific lysine 379 site was markedly decreased. At the same time, there was noted cerebral vascular impairment however markers of oxidative stress were not increased. In fact, Nox4 appeared to be downregulated in obesity. Thus, SIRT1 protein levels within the vasculature are greater in obese compared to lean Zucker rats and are associated with higher SIRT1 activity and lower Nox4 expression. We propose that the increased expression and activity of SIRT1 may be a vascular adaptive mechanism in obesity, aiming to prevent oxidative stress.
Assuntos
Artérias Cerebrais/metabolismo , Obesidade/enzimologia , Sirtuína 1/metabolismo , Regulação para Cima , Animais , Biomarcadores/metabolismo , Análise Química do Sangue , Peso Corporal , Artérias Cerebrais/fisiopatologia , Endotélio Vascular/fisiopatologia , Regulação Enzimológica da Expressão Gênica , Masculino , Óxido Nítrico Sintase Tipo III/metabolismo , Obesidade/sangue , Obesidade/metabolismo , Obesidade/fisiopatologia , Estresse Oxidativo , Ratos , Ratos Zucker , VasodilataçãoRESUMO
OBJECTIVE: Endothelial dysfunction, associated with reduced nitric oxide bioavailability and oxidative stress, is a common feature of vascular-related diseases. Sirtuin 1 (SIRT1) is a protein deacetylase that has been shown to target endothelial nitric oxide synthase in large arteries and is protective during oxidative stress. However, within resistance-sized vessels, the expression and functional effects of SIRT1 remain unknown. METHODS: Immunoblotting and immunohistochemistry were used to determine SIRT1 expression and localization in cultured brain endothelial cells and intact rat middle cerebral artery. The influence of SIRT1 on vascular function was then studied in intact middle cerebral arteries using pressure myography. RESULTS: We report for the first time that SIRT1 is expressed in the resistance-sized vessels in the brain and is present in both the endothelium and smooth muscle. Pharmacological inhibition of SIRT1 demonstrated reduced endothelium-dependent dilation mediated by nitric oxide. However, endothelium-independent dilations were comparable in the presence and absence of SIRT1 block. CONCLUSIONS: Our results support a role for SIRT1 in endothelium-dependent relaxation in the cerebral vasculature and reveal a potential for SIRT1 as a therapeutic target in vascular-related diseases by restoring endothelial function.
Assuntos
Circulação Cerebrovascular/fisiologia , Endotélio Vascular/enzimologia , Regulação Enzimológica da Expressão Gênica/fisiologia , Músculo Liso Vascular/enzimologia , Sirtuína 1/biossíntese , Vasodilatação/fisiologia , Animais , Células Cultivadas , Endotélio Vascular/citologia , Masculino , Camundongos , Músculo Liso Vascular/citologia , Óxido Nítrico Sintase Tipo III/biossíntese , Ratos , Ratos Wistar , Sirtuína 1/antagonistas & inibidoresRESUMO
BACKGROUND: Connexin proteins are well known to participate in cell-to-cell communication within the cerebral vasculature. Pannexins are a recently discovered family of proteins that could potentially be involved in cell-to-cell communication. Herein, we sought to determine whether pannexins are expressed in rat middle cerebral artery (MCA). METHODS: A combination of RT-PCR, immunoblotting and immunohistochemistry techniques was used to characterize the expression pattern of pannexins in rat MCA. A fluorescent dye uptake approach in cultured smooth muscle cells was used to determine whether these cells have functional hemichannels. RESULTS: We report for the first time that pannexins are expressed in the cerebral vasculature. We reveal that pannexin 1 is expressed in smooth muscle but not in endothelium and pannexin 2 is expressed in both endothelium and smooth muscle. Fluorescent dye entered cultured smooth muscle cells in the absence of extracellular calcium or when the cells were depolarized, which was prevented by the putative hemichannel blocker carbenoxolone. CONCLUSIONS: The identification of pannexins in rat MCA indicates that pannexin expression is not restricted to neuronal cells. Dye uptake in cultured smooth muscle cells exhibited properties similar to those of connexin and pannexin hemichannels, which may represent another form of cell-to-cell communication within the vasculature.
Assuntos
Conexinas/biossíntese , Artéria Cerebral Média/metabolismo , Proteínas do Tecido Nervoso/biossíntese , Animais , Células Cultivadas , Corantes Fluorescentes/metabolismo , Imuno-Histoquímica , Isoquinolinas , Masculino , Músculo Liso Vascular/citologia , Ratos , Ratos Long-Evans , Reação em Cadeia da Polimerase Via Transcriptase ReversaRESUMO
Previous findings from our laboratory have shown that dilations to endothelium-derived hyperpolarizing factor (EDHF) in rat middle cerebral artery (MCA) are less in females compared to males. Myoendothelial gap junctions (MEGJs) appear to mediate the transfer of hyperpolarization between endothelium and smooth muscle in males. In the present study, we hypothesized that MEGJs are the site along the EDHF pathway which is compromised in female rat MCA. Membrane potential in endothelium was measured using the voltage-sensitive dye di-8-ANEPPS and in smooth muscle using intracellular glass microelectrodes in the presence of l-NAME (3x10(-5 )M) and indomethacin (10(-5 )M). Electron microscopy was used to assess MEGJ characteristics. In endothelial cells, the di-8-ANEPPS fluorescence ratio change to 10(-5 )M UTP was similar in males (-2.9+/-0.5%) and females (-3.2+/-0.2%), indicating comparable degrees of endothelial cell hyperpolarization. However, smooth muscle cell hyperpolarization to 10(-5 )M UTP was significantly attenuated in females (0 mV hyperpolarization; -31+/-1.5 mV resting) compared to males (8 mV hyperpolarization; -28+/-1.7 mV resting). Ultrastructural evidence suggested that MEGJ frequency and area of contact were comparable between males and females. Taken together, our data suggest that in rat MCA, MEGJ frequency does not account for the reduced EDHF responses observed in females compared to males. We conclude that reduced myoendothelial coupling and/or homocellular coupling within the media may account for these differences.
Assuntos
Fatores Biológicos/metabolismo , Células Endoteliais/metabolismo , Junções Comunicantes/fisiologia , Artéria Cerebral Média/fisiologia , Músculo Liso Vascular/metabolismo , Animais , Combinação de Medicamentos , Células Endoteliais/efeitos dos fármacos , Células Endoteliais/ultraestrutura , Feminino , Junções Comunicantes/efeitos dos fármacos , Indometacina/farmacologia , Masculino , Potenciais da Membrana/efeitos dos fármacos , Microscopia Eletrônica de Transmissão , Artéria Cerebral Média/efeitos dos fármacos , Músculo Liso Vascular/efeitos dos fármacos , Músculo Liso Vascular/ultraestrutura , NG-Nitroarginina Metil Éster/farmacologia , Compostos de Piridínio/metabolismo , Ratos , Ratos Long-Evans , Fatores SexuaisRESUMO
Dilations to endothelium-derived hyperpolarizing factor (EDHF) are significantly attenuated in the middle cerebral artery (MCA) isolated from female compared to male rats. Since gap junctions appear to be involved in the EDHF pathway and cAMP has been shown to enhance gap junction permeability, we tested the hypothesis that elevation of cAMP would enhance EDHF-mediated dilations in female rat MCA. Vascular diameter was measured in perfused MCA segments using videomicroscopy in the presence and absence of IBMX, an inhibitor of cAMP phosphodiesterase. In the presence of L-NAME and indomethacin, dilation to 10(-4) M ATP was significantly reduced in females (48+/-12%) compared to males (92+/-2%). IBMX, an inhibitor of cAMP phosphodiesterase, had no significant effect on ATP-mediated dilations in both males and females. Basal cAMP levels were comparable in male and female MCAs (1.7 pmol/mg protein). Incubation with IBMX (2 x 10(-4) M) significantly elevated cAMP in both male (12.8 pmol/mg protein) and female (11.2 pmol/mg protein) MCAs. Our results demonstrate that reduced EDHF dilations in female rat MCA cannot be solely attributed to impaired cAMP signaling. Future studies will target other potential sites along the EDHF pathway in order to identify why EDHF dilations are reduced in the female compared to the male rat MCA.
Assuntos
Fatores Biológicos/fisiologia , AMP Cíclico/metabolismo , Artéria Cerebral Média/fisiologia , Vasodilatação/fisiologia , Animais , Feminino , Junções Comunicantes/fisiologia , Masculino , Análise por Pareamento , Ratos , Ratos Long-Evans , Sistemas do Segundo Mensageiro/fisiologia , Fatores Sexuais , Transdução de Sinais/fisiologiaRESUMO
BACKGROUND: Unlike in peripheral vessels, the endothelium-derived hyperpolarizing factor (EDHF)-mediated component to P2Y(2) receptor-mediated dilations is significantly attenuated in the middle cerebral artery (MCA) of female rats compared to male rats. One aspect to the EDHF phenomenon is activation of the intermediate calcium-sensitive potassium (IK(Ca)) channels located on the endothelium. In an attempt to pinpoint the site along the EDHF pathway that is compromised in females, we tested the hypothesis that direct activation of IK(Ca) channels with DCEBIO would elicit attenuated hyperpolarization in the endothelium and smooth muscle of females compared to males. METHODS: Inhibitors of nitric oxide synthase and cyclooxygenase were present throughout all experiments. Vessel diameter changes were assessed in pressurized and luminally perfused MCAs. Membrane potential changes in the endothelium and smooth muscle were measured using the perforated patch clamp method and sharp electrodes, respectively. RESULTS AND CONCLUSIONS: The maximum vasodilation to 3 x 10(-4)M DCEBIO was significantly reduced in females (37 +/- 9%) compared to intact males (70 +/- 4%). Endothelial cell hyperpolarization to DCEBIO was similar in both males and females. Smooth muscle cell hyperpolarization was attenuated in females (2 +/- 1 mV) compared to males (15 +/- 3 mV). Taken together, our data suggest that the transfer of hyperpolarization from the endothelium to the smooth muscle is impeded in the female rat MCA.
Assuntos
Benzimidazóis/farmacologia , Ativação do Canal Iônico/efeitos dos fármacos , Artéria Cerebral Média/efeitos dos fármacos , Canais de Potássio Cálcio-Ativados/agonistas , Vasodilatação/efeitos dos fármacos , Vasodilatadores/farmacologia , Animais , Fatores Biológicos/metabolismo , Relação Dose-Resposta a Droga , Endotélio Vascular/efeitos dos fármacos , Feminino , Junções Comunicantes/metabolismo , Técnicas In Vitro , Masculino , Potenciais da Membrana/efeitos dos fármacos , Artéria Cerebral Média/metabolismo , Músculo Liso Vascular/efeitos dos fármacos , Técnicas de Patch-Clamp , Canais de Potássio Cálcio-Ativados/metabolismo , Ratos , Ratos Long-Evans , Fatores Sexuais , Fatores de TempoRESUMO
Cephalic elevations in arterial pressure associated with microgravity and prolonged bed rest alter cerebrovascular autoregulation in humans. Using the head-down tail-suspended (HDT) rat to chronically induce headward fluid shifts and elevate cerebral artery pressure, previous work has likewise shown cerebral perfusion to be diminished. The purpose of this study was to test the hypothesis that 2 wk of HDT reduces cerebral artery vasodilation. To test this hypothesis, dose-response relations for endothelium-dependent (2-methylthioadenosine triphosphate and bradykinin) and endothelium-independent (nitroprusside) vasodilation were determined in vitro in middle cerebral arteries (MCAs) from HDT and control rats. All in vitro measurements were done in the presence and absence of the nitric oxide synthase inhibitor N(G)-nitro-L-arginine methyl ester (10(-5) M) and cyclooxygenase inhibitor indomethacin (10(-5) M). MCA caveolin-1 protein content was measured by immunoblot analysis. Endothelium-dependent vasodilation to 2-methylthioadenosine triphosphate and bradykinin were both lower in MCAs from HDT rats. These lower vasodilator responses were abolished with N(G)-nitro-L-arginine methyl ester but were unaffected by indomethacin. In addition, HDT was associated with lower levels of MCA caveolin-1 protein. Endothelium-independent vasodilation was not altered by HDT. These results indicate that chronic cephalic fluid shifts diminish endothelium-dependent vasodilation through alterations in the endothelial nitric oxide synthase signaling mechanism. Such decrements in endothelium-dependent vasodilation of cerebral arteries could contribute to the elevations in cerebral vascular resistance and reductions in cerebral perfusion that occur after conditions of simulated microgravity in HDT rats.
Assuntos
Fatores Biológicos/fisiologia , Artérias Cerebrais/fisiologia , Endotélio Vascular/fisiologia , Óxido Nítrico Sintase Tipo III/metabolismo , Vasodilatação/fisiologia , Ausência de Peso , Trifosfato de Adenosina/análogos & derivados , Trifosfato de Adenosina/fisiologia , Animais , Fatores Biológicos/análise , Bradicinina/fisiologia , Caveolina 1/análise , Caveolina 1/fisiologia , Artérias Cerebrais/química , Inibidores de Ciclo-Oxigenase/farmacologia , Endotélio Vascular/química , Inibidores Enzimáticos/farmacologia , Regulação da Expressão Gênica/fisiologia , Elevação dos Membros Posteriores/fisiologia , Indometacina/farmacologia , Masculino , NG-Nitroarginina Metil Éster/farmacologia , Óxido Nítrico Sintase Tipo III/antagonistas & inibidores , Nitroprussiato/farmacologia , Prostaglandina-Endoperóxido Sintases/fisiologia , Ratos , Ratos Sprague-Dawley , Transdução de Sinais/fisiologia , Tionucleotídeos/fisiologia , Vasodilatação/efeitos dos fármacos , Vasodilatadores/farmacologiaRESUMO
The mechanisms underlying endothelium-dependent hyperpolarizing factor (EDHF) in the middle cerebral artery (MCA) remain largely unresolved. In particular, very little is known regarding the way in which the signal is transmitted from endothelium to smooth muscle. The present study tested the hypothesis that direct communication via myoendothelial gap junctions contributes to the EDHF response in the male rat MCA. EDHF-mediated dilations were elicited in rat MCAs by luminal application of ATP or UTP in the presence of Nomega-nitro-L-arginine methyl ester and indomethacin. Maximum dilation to luminal ATP (10(-4) M) was reduced significantly after incubation with a gap peptide cocktail (9 +/- 4%, n = 6) compared with a scrambled gap peptide cocktail (99 +/- 1%, n = 6, P < 0.05). A gap peptide cocktail had no effect on amplitude of endothelial cell hyperpolarization in response to 3 x 10(-5) M UTP (22 +/- 3 vs. 22 +/- 1 mV, n = 4), whereas smooth muscle cell hyperpolarization was significantly attenuated (17 +/- 1 vs. 6 +/- 1 mV, n = 4, P = 0.004). Connexin (Cx) 37 was localized to smooth muscle and Cx43 to endothelium, whereas Cx40 was found in endothelium and smooth muscle. Electron microscopy revealed the existence of frequent myoendothelial junctions. The total number of myoendothelial junctions per 5 microm of MCA sectioned was 2.5 +/- 0.5. Our results suggest that myoendothelial communication contributes to smooth muscle cell hyperpolarization and EDHF dilation in male rat MCA.