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1.
J Mol Cell Cardiol ; 140: 1-9, 2020 Feb 11.
Artigo em Inglês | MEDLINE | ID: mdl-32057736

RESUMO

Diabetes is an important risk factor for the development of cardiovascular disease including atherosclerosis and ischemic heart disease. Vascular complications including macro- and micro-vascular dysfunction are the leading causes of morbidity and mortality in diabetes. Disease mechanisms at present are unclear and no ideal therapies are available, which urgently calls for the identification of novel therapeutic targets/agents. An altered nucleotide- and nucleoside-mediated purinergic signaling has been implicated to cause diabetes-associated vascular dysfunction in major organs. Alteration of both purinergic P1 and P2 receptor sensitivity rather than the changes in receptor expression accounts for vascular dysfunction in diabetes. Activation of P2X7 receptors plays a crucial role in diabetes-induced retinal microvascular dysfunction. Recent findings have revealed that both ecto-nucleotidase CD39, a key enzyme hydrolyzing ATP, and CD73, an enzyme regulating adenosine turnover, are involved in the renal vascular injury in diabetes. Interestingly, erythrocyte dysfunction in diabetes by decreasing ATP release in response to physiological stimuli may serve as an important trigger to induce vascular dysfunction. Nucleot(s)ide-mediated purinergic activation also exerts long-term actions including inflammatory and atherogenic effects in hyperglycemic and diabetic conditions. This review highlights the current knowledge regarding the altered nucleot(s)ide-mediated purinergic signaling as an important disease mechanism for the diabetes-associated vascular complications. Better understanding the role of key receptor-mediated signaling in diabetes will provide more insights into their potential as targets for the treatment.

2.
J Pharmacol Sci ; 141(1): 64-69, 2019 Sep.
Artigo em Inglês | MEDLINE | ID: mdl-31640919

RESUMO

Activation of both adenosine A2A and A2B receptors (A2BR) contributes to coronary vasodilation. We previously demonstrated that uridine adenosine tetraphosphate (Up4A) is a novel vasodilator in the porcine coronary microcirculation, acting mainly on A2AR in smooth muscle cells (SMC). We further investigated whether activation of A2BR is involved in Up4A-mediated coronary SMC relaxation. Both A2AR and A2BR may stimulate H2O2 production leading to activation of KATP channels in SMCs, we also studied the involvement of H2O2 and KATP channels in Up4A-mediated effect. Coronary small arteries dissected from the apex of porcine hearts were mounted on wire myograph for Up4A concentration responses. Up4A-induced coronary SMC relaxation was attenuated by A2AR but not A2BR antagonism or non-selective P2R antagonism, despite greater endogenous A2BR expression vs. A2AR in both coronary small arteries and primary cultured coronary SMCs. Moreover, Up4A-induced coronary SMC relaxation was blunted by H2O2 catabolism. This effect was not altered by KATP channel blockade. Combination of H2O2 catabolism and A2AR antagonism attenuated Up4A-induced coronary SMC relaxation to the similar extent as A2AR antagonism alone. Collectively, Up4A-induced porcine coronary SMC relaxation is mediated by activation of A2AR-H2O2 pathway. This process does not involve A2BR, P2R or KATP channels.

3.
Immunopharmacol Immunotoxicol ; 41(3): 428-437, 2019 Jun.
Artigo em Inglês | MEDLINE | ID: mdl-31062639

RESUMO

Objective: Angiotensin II (Ang II) exerts its effects through two G-protein coupled receptors: angiotensin II type 1 receptors (AT1) and type 2 receptors (AT2). Both these receptor subtypes are poorly understood in asthma. In this study, we investigated effects of AT1 receptor antagonist losartan, novel AT2 receptor agonist novokinin and AT2 receptor antagonist PD 123319 in a mouse model of asthma. Methods: Mice were divided into control (CON) and allergen sensitized (SEN) groups. SEN was sensitized with ovalbumin (OVA) on days 1 and 6 (30 µg; i.p.), followed by 5% OVA aerosol challenge (days 11-13). Treatments included (a) losartan (SEN + LOS; 20 mg/kg i.p., day 14), (b) novokinin (SEN + NOV; 0.3 mg/kg i.p., day 14), and (c) PD 123319 (SEN + PD; 5 mg/kg i.p., day 14). Experiments for airway responsiveness, bronchoalveolar lavage, and tracheal ring reactivity using isolated organ bath were performed. Results: Airway responsiveness to methacholine (MCh) (48 mg/mL) was significantly higher in SEN (563.71 ± 40% vs. 294.3 ± 123.84 in CON). This response was potentiated in SEN + PD group (757 ± 30%; p < .05 compared to SEN). SEN + LOS (247.61 ± 86.85%) and SEN + NOV (352 ± 11%) had significantly lower response compared to SEN. SEN + LOS (26.22 ± 0.29%) and SEN + NOV (46.20 ± 0.76%) treatment significantly (p < .001) attenuated total cell count and eosinophils compared to SEN group (69.38 ± 1.5%), while SEN + PD (73.04 ± 0.69%) had highest number of eosinophils. Tracheal response to MCh was significantly higher in SEN group compared to controls, and this response was significantly lowered with the losartan and novokinin treatments. Conclusions: These data suggest that AT1 and AT2 receptors have opposite effects in modulating airway hyperresponsiveness and inflammation in asthma.


Assuntos
Asma/imunologia , Receptor Tipo 1 de Angiotensina/imunologia , Receptor Tipo 2 de Angiotensina/imunologia , Bloqueadores do Receptor Tipo 1 de Angiotensina II/farmacologia , Bloqueadores do Receptor Tipo 2 de Angiotensina II/farmacologia , Animais , Asma/induzido quimicamente , Asma/tratamento farmacológico , Asma/patologia , Feminino , Imidazóis/farmacologia , Inflamação/induzido quimicamente , Inflamação/tratamento farmacológico , Inflamação/imunologia , Inflamação/patologia , Losartan/farmacologia , Masculino , Camundongos , Oligopeptídeos/farmacologia , Piridinas/farmacologia
4.
Pharmacol Res ; 141: 32-45, 2019 03.
Artigo em Inglês | MEDLINE | ID: mdl-30553823

RESUMO

Uridine adenosine tetraphosphate (Up4A), biosynthesized by activation of vascular endothelial growth factor receptor (VEGFR) 2, was initially identified as a potent endothelium-derived vasoconstrictor in perfused rat kidney. Subsequently, the effect of Up4A on vascular tone regulation was intensively investigated in arteries isolated from different vascular beds in rodents including rat pulmonary arteries, aortas, mesenteric and renal arteries as well as mouse aortas, in which Up4A produces vascular contraction. In contrast, Up4A produces vascular relaxation in porcine coronary small arteries and rat aortas. Intravenous infusion of Up4A into conscious rats or mice decreases blood pressure, and intravenous bolus injection of Up4A into anesthetized mice increases coronary blood flow, indicating an overall vasodilator influence in vivo. Although Up4A is the first dinucleotide described that contains both purine and pyrimidine moieties, its cardiovascular effects are exerted mainly through activation of purinergic receptors. These effects not only encompass regulation of vascular tone, but also endothelial angiogenesis, smooth muscle cell proliferation and migration, and vascular calcification. Furthermore, this review discusses a potential role for Up4A in cardiovascular pathophysiology, as plasma levels of Up4A are elevated in juvenile hypertensive patients and Up4A-mediated vascular purinergic signaling changes in cardiovascular disease such as hypertension, diabetes, atherosclerosis and myocardial infarction. Better understanding the vascular effect of the novel dinucleotide Up4A and the purinergic signaling mechanisms mediating its effects will enhance its potential as target for treatment of cardiovascular disease.


Assuntos
Fenômenos Fisiológicos Cardiovasculares , Fosfatos de Dinucleosídeos/fisiologia , Receptores Purinérgicos/fisiologia , Animais , Sistema Cardiovascular , Humanos , Transdução de Sinais
5.
Eur J Pharmacol ; 842: 111-117, 2019 Jan 05.
Artigo em Inglês | MEDLINE | ID: mdl-30347181

RESUMO

L-NAME-induced hypertension is commonly used to study endothelial dysfunction and related vascular effects. It has been reported that genetic deletion of A1 adenosine receptor (AR) reduces blood pressure (BP) increases in mice and thus, suggesting the involvement of A1AR. Thus, we sought to determine whether A1AR-induced vascular responses were altered in this mouse model of hypertension. L-NAME (1 mg/ml) was given in the drinking water for 28 days to mice. The BP was monitored using non-invasive tail-cuff system. Muscle tension studies were performed using DMT for mesenteric arteries (MAs) and organ bath for aorta. Protein expression was analyzed by western blot. Significantly, higher systolic and mean arterial blood pressure was noted in L-NAME mice. In MAs, higher 2-Chloro-N6-cyclopentyladenosine (CCPA, selective A1AR agonist) induced contractions in hypertensive mice were observed. This enhanced contraction was inhibited by HET0016 (Cytochrome 450 4A inhibitor, 10 µM, 15 min). Contrary, 5'-(N-Ethylcarboxamido) adenosine (NECA, non-selective AR agonist) induced vascular responses were comparable in both groups. Pinacidil (KATP channel opener) induced relaxation was significantly increased in hypertensive mice. In aorta, CCPA-induced contractions were enhanced and inhibited by HET0016 in hypertensive mice. Notably, NECA-induced contractions in aorta were enhanced in hypertensive mice. Higher expressions of A1AR and Cyp4A were noted in MAs of hypertensive mice. In addition, in aorta, higher A1AR and comparable Cyp4A levels were observed in hypertensive mice. A1AR-induced vascular contractions were enhanced in hypertensive mice aorta and MAs. Cyp4A plays a role in altered vascular responses in MAs.


Assuntos
Aorta/fisiopatologia , Hipertensão/fisiopatologia , Artérias Mesentéricas/fisiopatologia , NG-Nitroarginina Metil Éster/farmacologia , Receptor A1 de Adenosina/metabolismo , Vasoconstrição/efeitos dos fármacos , Vasodilatação/efeitos dos fármacos , Animais , Aorta/efeitos dos fármacos , Pressão Sanguínea/efeitos dos fármacos , Citocromo P-450 CYP4A/metabolismo , Modelos Animais de Doenças , Regulação Enzimológica da Expressão Gênica/efeitos dos fármacos , Masculino , Artérias Mesentéricas/efeitos dos fármacos , Camundongos , Camundongos Endogâmicos C57BL , Óxido Nítrico Sintase Tipo III/metabolismo
6.
Eur J Pharmacol ; 820: 191-197, 2018 Feb 05.
Artigo em Inglês | MEDLINE | ID: mdl-29269016

RESUMO

Activation of adenosine receptors has been implicated in several biological functions, including cardiovascular and renal function. Diabetes causes morphological and functional changes in the vasculature, resulting in abnormal responses to various stimuli. Recent studies have suggested that adenosine receptor expression and signaling are altered in disease states such as hypertension, diabetes. Using a streptozotocin (STZ) mouse model of type I diabetes (T1D), we investigated the functional changes in aorta and resistance mesenteric arteries to adenosine receptor agonist activation in T1D. Organ baths and DMT wire myographs were used for muscle tension measurements in isolated vascular rings, and western blotting was used for protein analysis. Concentration response curves to selective adenosine receptor agonists, including CCPA (A1 receptor agonist), Cl-IBMECA (A3 receptor agonist), CGS-21680 (A2A receptor agonist), and BAY 60-6583 (A2B receptor agonist), were performed. We found that diabetes did not affect adenosine receptor agonist-mediated relaxation or contraction in mesenteric arteries. However, aortas from diabetic mice exhibited a significant decrease (P < 0.05) in A1 receptor-mediated vasoconstriction. In addition, the aortas from STZ-treated mice exhibited an increase in phenylephrine-mediated contraction (EC50 7.40 ± 0.08 in STZ vs 6.89 ± 0.14 in vehicle; P < 0.05), while relaxation to A2A receptor agonists (CGS-21680) tended to decrease in aortas from the STZ-treated group (not statistically significant). Our data suggest that changes in adenosine receptor(s) vascular reactivity in T1D is tissue specific, and the decrease in A1 receptor-mediated aortic contraction could be a compensatory mechanism to counterbalance the increased adrenergic vascular contractility observed in aortas from diabetic mice.


Assuntos
Aorta/fisiopatologia , Diabetes Mellitus Tipo 1/metabolismo , Artérias Mesentéricas/fisiopatologia , Receptores Purinérgicos P1/metabolismo , Animais , Glicemia/metabolismo , Peso Corporal , Diabetes Mellitus Tipo 1/sangue , Diabetes Mellitus Tipo 1/fisiopatologia , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Vasoconstrição , Vasodilatação
7.
Purinergic Signal ; 13(4): 591-600, 2017 Dec.
Artigo em Inglês | MEDLINE | ID: mdl-28929376

RESUMO

Uridine adenosine tetraphosphate (Up4A) exerts potent relaxation in porcine coronary arteries that is reduced following myocardial infarction, suggesting a crucial role for Up4A in the regulation of coronary flow (CF) in cardiovascular disorders. We evaluated the vasoactive effects of Up4A on CF in atherosclerosis using ApoE knockout (KO) mice ex vivo and in vivo. Functional studies were conducted in isolated mouse hearts using the Langendorff technique. Immunofluorescence was performed to assess purinergic P2X1 receptor (P2X1R) expression in isolated mouse coronary arteries. In vivo effects of Up4A on coronary blood flow (CBF) were assessed using ultrasound. Infusion of Up4A (10-9-10-5 M) into isolated mouse hearts resulted in a concentration-dependent reduction in CF in WT and ApoE KO mice to a similar extent; this effect was exacerbated in ApoE KO mice fed a high-fat diet (HFD). The P2X1R antagonist MRS2159 restored Up4A-mediated decreases in CF more so in ApoE KO + HFD than ApoE KO mice. The smooth muscle to endothelial cell ratio of coronary P2X1R expression was greater in ApoE KO + HFD than ApoE KO or WT mice, suggesting a net vasoconstrictor potential of P2X1R in ApoE KO + HFD mice. In contrast, Up4A (1.6 mg/kg) increased CBF to a similar extent among the three groups. In conclusion, Up4A decreases CF more in ApoE KO + HFD mice, likely through a net upregulation of vasoconstrictor P2X1R. In contrast, Up4A increases CBF in vivo regardless of the atherosclerotic model.


Assuntos
Aterosclerose/metabolismo , Circulação Coronária/efeitos dos fármacos , Fosfatos de Dinucleosídeos/farmacologia , Receptores Purinérgicos P2X1/metabolismo , Animais , Preparação de Coração Isolado , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout para ApoE , Antagonistas do Receptor Purinérgico P2X/farmacologia
8.
Purinergic Signal ; 13(1): 27-49, 2017 03.
Artigo em Inglês | MEDLINE | ID: mdl-27696085

RESUMO

Influences of adenosine 2A receptor (A2AR) activity on the cardiac transcriptome and genesis of endotoxemic myocarditis are unclear. We applied transcriptomic profiling (39 K Affymetrix arrays) to identify A2AR-sensitive molecules, revealed by receptor knockout (KO), in healthy and endotoxemic hearts. Baseline cardiac function was unaltered and only 37 A2AR-sensitive genes modified by A2AR KO (≥1.2-fold change, <5 % FDR); the five most induced are Mtr, Ppbp, Chac1, Ctsk and Cnpy2 and the five most repressed are Hp, Yipf4, Acta1, Cidec and Map3k2. Few canonical paths were impacted, with altered Gnb1, Prkar2b, Pde3b and Map3k2 (among others) implicating modified G protein/cAMP/PKA and cGMP/NOS signalling. Lipopolysaccharide (LPS; 20 mg/kg) challenge for 24 h modified >4100 transcripts in wild-type (WT) myocardium (≥1.5-fold change, FDR < 1 %); the most induced are Lcn2 (+590); Saa3 (+516); Serpina3n (+122); Cxcl9 (+101) and Cxcl1 (+89) and the most repressed are Car3 (-38); Adipoq (-17); Atgrl1/Aplnr (-14); H19 (-11) and Itga8 (-8). Canonical responses centred on inflammation, immunity, cell death and remodelling, with pronounced amplification of toll-like receptor (TLR) and underlying JAK-STAT, NFκB and MAPK pathways, and a 'cardio-depressant' profile encompassing suppressed ß-adrenergic, PKA and Ca2+ signalling, electromechanical and mitochondrial function (and major shifts in transcripts impacting function/injury including Lcn2, S100a8/S100a9, Icam1/Vcam and Nox2 induction, and Adipoq, Igf1 and Aplnr repression). Endotoxemic responses were selectively modified by A2AR KO, supporting inflammatory suppression via A2AR sensitive shifts in regulators of NFκB and JAK-STAT signalling (IκBζ, IκBα, STAT1, CDKN1a and RRAS2) without impacting the cardio-depressant gene profile. Data indicate A2ARs exert minor effects in un-stressed myocardium and selectively suppress NFκB and JAK-STAT signalling and cardiac injury without influencing cardiac depression in endotoxemia.


Assuntos
Endotoxemia/metabolismo , Miocárdio/metabolismo , Receptor A2A de Adenosina/metabolismo , Animais , Endotoxemia/genética , Perfilação da Expressão Gênica , Inflamação/genética , Inflamação/metabolismo , Janus Quinase 1/metabolismo , Camundongos , Camundongos Knockout , NF-kappa B/metabolismo , Receptor A2A de Adenosina/genética , Fatores de Transcrição STAT/metabolismo , Transcriptoma
9.
Physiol Rep ; 4(11)2016 Jun.
Artigo em Inglês | MEDLINE | ID: mdl-27302991

RESUMO

Bolus injections of adenosine and the A2A adenosine receptor (AR) selective agonist (regadenoson) are used clinically as a substitute for a stress test in people who cannot exercise. Using isolated tissue preparations, our lab has shown that coronary flow and cardiac effects of adenosine are mostly regulated by the AR subtypes A1, A2A, and A2B In this study, we used ultrasound imaging to measure the in vivo effects of adenosine on coronary blood flow (left coronary artery) and cardiac function in anesthetized wild-type, A1 knockout (KO), A2AKO, A2BKO, A3KO, A1, and A3 double KO (A1/3 DKO) and A2A and A2B double KO (A2A/2B DKO) mice in real time. Echocardiographic and Doppler studies were performed using a Visualsonic Vevo 2100 ultrasound system. Coronary blood flow (CBF) baseline data were obtained when animals were anesthetized with 1% isoflourane. Diameter (D) and velocity time integral (VTI) were measured on the left coronary arteries (CBF = ((π/4) × D(2) × VTI × HR)/1000). CBF changes were the highest within 2 min of injection (about 10 mg/kg). Heart rate, cardiac output, and stroke volume were measured by tracing the left ventricle long axis. Our data support a role for the A2 AR in CBF and further support our conclusions of previous studies from isolated tissues. Adenosine-mediated decreases in cardiac output and stroke volume may be A2B and/or A3 AR-mediated; however, the A1 and A2 ARs also play roles in overall cardiac function. These data further provide a powerful translational tool in studying the cardiovascular effects of adenosine in disease states.


Assuntos
Agonistas do Receptor A2 de Adenosina/farmacologia , Adenosina/farmacologia , Velocidade do Fluxo Sanguíneo/efeitos dos fármacos , Vasos Coronários/efeitos dos fármacos , Receptores Purinérgicos P1/genética , Fluxo Sanguíneo Regional/efeitos dos fármacos , Animais , Débito Cardíaco/efeitos dos fármacos , Camundongos , Camundongos Knockout , Receptores Purinérgicos P1/metabolismo , Volume Sistólico/efeitos dos fármacos
10.
J Pharmacol Exp Ther ; 356(3): 673-80, 2016 Mar.
Artigo em Inglês | MEDLINE | ID: mdl-26718241

RESUMO

Activation of adenosine receptors (ARs) has been implicated in the modulation of renal and cardiovascular systems, as well as erectile functions. Recent studies suggest that adenosine-mediated regulation of erectile function is mainly mediated through A2BAR activation. However, no studies have been conducted to determine the contribution of AR subtype in the regulation of the vascular tone of the pudendal artery (PA), the major artery supplying and controlling blood flow to the penis. Our aim was to characterize the contribution of AR subtypes and identify signaling mechanisms involved in adenosine-mediated vascular tone regulation in the PA. We used a DMT wire myograph for muscle tension measurements in isolated PAs from wild-type, A2AAR knockout, A2BAR knockout, and A2A/A2BAR double-knockout mice. Real-time reverse transcription-polymerase chain reaction was used to determine the expression of the AR subtypes. Data from our pharmacologic and genetic approaches suggest that AR activation-mediated vasodilation in the PA is mediated by both the A2AAR and A2BAR, whereas neither the A1AR nor A3AR play a role in vascular tone regulation of the PA. In addition, we showed that A2AAR- and A2BAR-mediated vasorelaxation requires activation of nitric oxide and potassium channels; however, only the A2AAR-mediated response requires protein kinase A activation. Our data are complemented by mRNA expression showing the expression of all AR subtypes with the exception of the A3AR. AR signaling in the PA may play an important role in mediating erection and represent a promising therapeutic option for the treatment of erectile dysfunction.


Assuntos
Artérias/fisiologia , Receptor A2B de Adenosina/fisiologia , Vasodilatação/fisiologia , Agonistas do Receptor A2 de Adenosina/farmacologia , Animais , Artérias/efeitos dos fármacos , Relação Dose-Resposta a Droga , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Técnicas de Cultura de Órgãos , Períneo/irrigação sanguínea , Vasodilatação/efeitos dos fármacos
11.
J Mol Cell Cardiol ; 90: 30-7, 2016 Jan.
Artigo em Inglês | MEDLINE | ID: mdl-26654777

RESUMO

Adenosine A2A receptor (A2AAR) activation plays a major role in the regulation of coronary flow (CF). Recent studies from our laboratory and others have suggested that A2AAR expression and/or signaling is altered in disease conditions. However, the coronary response to AR activation, in particular A2AAR, in diabetes is not fully understood. In this study, we use an STZ mouse model of type 1 diabetes (T1D) to look at CF responses to the nonspecific AR agonist NECA and the A2AAR specific agonist CGS 21680 in-vivo and ex-vivo. Using immunofluorescence, we also explored the effect of diabetes on A2AAR expression in coronary arteries. NECA mediated increase in CF was significantly increased in hearts isolated from STZ-induced diabetic mice. In addition, both in in-vivo and ex-vivo responses to A2AAR activation using CGS 21680 were significantly higher in diabetic mice when compared to their controls. Immunohistochemistry showed an upregulation of A2AAR in both coronary smooth muscle and endothelial cells (~160% and ~140%, respectively). Our data suggest that diabetes resulted in an increased A2AAR expression in coronary arteries which resulted in enhanced A2AAR-mediated increase in CF observed in diabetic hearts. This is the first report implying that A2AAR has a role in the regulation of CF in diabetes, supporting recent studies suggesting that the use of adenosine and its A2A selective agonist (regadenoson, Lexiscan®) may not be appropriate for the detection of coronary artery diseases in T1D and the estimation of coronary reserve.


Assuntos
Circulação Coronária/efeitos dos fármacos , Diabetes Mellitus Experimental/metabolismo , Células Endoteliais/efeitos dos fármacos , Receptor A2A de Adenosina/genética , Receptor A2B de Adenosina/genética , Adenosina/análogos & derivados , Adenosina/farmacologia , Agonistas do Receptor A2 de Adenosina/farmacologia , Adenosina-5'-(N-etilcarboxamida)/farmacologia , Animais , Vasos Coronários/efeitos dos fármacos , Vasos Coronários/metabolismo , Vasos Coronários/patologia , Diabetes Mellitus Experimental/genética , Diabetes Mellitus Experimental/patologia , Diabetes Mellitus Tipo 1/genética , Diabetes Mellitus Tipo 1/metabolismo , Diabetes Mellitus Tipo 1/patologia , Células Endoteliais/metabolismo , Células Endoteliais/patologia , Regulação da Expressão Gênica , Coração/efeitos dos fármacos , Coração/fisiopatologia , Humanos , Masculino , Camundongos , Músculo Liso Vascular/efeitos dos fármacos , Músculo Liso Vascular/metabolismo , Músculo Liso Vascular/patologia , Técnicas de Cultura de Órgãos , Fenetilaminas/farmacologia , Receptor A2A de Adenosina/metabolismo , Receptor A2B de Adenosina/metabolismo , Transdução de Sinais , Vasodilatação/efeitos dos fármacos , Vasodilatadores/farmacologia
12.
Br J Pharmacol ; 172(20): 4959-69, 2015 Oct.
Artigo em Inglês | MEDLINE | ID: mdl-26227882

RESUMO

BACKGROUND AND PURPOSE: Stimulation of the A1 adenosine receptor and angiotensin II receptor type-1 (AT1 receptor) causes vasoconstriction through activation of cytochrome P450 4A (CYP4A) and ERK1/2. Thus, we hypothesized that acute angiotensin II activation alters the vasomotor response induced by the non-selective adenosine receptor agonist, NECA, in mouse mesenteric arteries (MAs). EXPERIMENTAL APPROACH: We used a Danish Myo Technology wire myograph to measure muscle tension in isolated MAs from wild type (WT), A1 receptor and A2B receptor knockout (KO) mice. Western blots were performed to determine the expression of AT1 receptors and CYP4A. KEY RESULTS: Acute exposure (15 min) to angiotensin II attenuated the NECA-dependent vasodilatation and enhanced vasoconstriction. This vasoconstrictor effect of angiotensin II in NECA-treated MAs was abolished in A1 receptor KO mice and in WT mice treated with the A1 receptor antagonist DPCPX, CYP4A inhibitor HET0016 and ERK1/2 inhibitor PD98059. In MAs from A2B receptor KO mice, the vasoconstrictor effect of angiotensin II on the NECA-induced response was shown to be dependent on A1 receptors. Furthermore, in A2B receptor KO mice, the expression of AT1 receptors and CYP4A was increased and the angiotensin II-induced vasoconstriction enhanced. In addition, inhibition of KATP channels with glibenclamide significantly reduced NECA-induced vasodilatation in WT mice. CONCLUSIONS AND IMPLICATIONS: Acute angiotensin II stimulation enhanced A1 receptor-dependent vasoconstriction and inhibited A2B receptor-dependent vasodilatation, leading to a net vasoconstriction and altered vasomotor response to NECA in MAs. This interaction may be important in the regulation of BP.


Assuntos
Adenosina/farmacologia , Angiotensina II/farmacologia , Artérias Mesentéricas/efeitos dos fármacos , Receptor A2B de Adenosina/fisiologia , Receptor Tipo 1 de Angiotensina/fisiologia , Adenosina-5'-(N-etilcarboxamida)/farmacologia , Animais , Citocromo P-450 CYP4A/fisiologia , Endotélio Vascular/fisiologia , Técnicas In Vitro , Masculino , Artérias Mesentéricas/fisiologia , Camundongos Endogâmicos C57BL , Camundongos Knockout , Receptor A2B de Adenosina/genética , Receptor Tipo 1 de Angiotensina/genética , Vasoconstrição/efeitos dos fármacos , Vasodilatação/efeitos dos fármacos , Vasodilatadores/farmacologia
13.
Vascul Pharmacol ; 73: 78-85, 2015 Oct.
Artigo em Inglês | MEDLINE | ID: mdl-25921923

RESUMO

Uridine adenosine tetraphosphate (Up4A), a novel endothelium-derived vasoactive agent, is proposed to play a role in cardiovascular disorders and induces aortic contraction through activation of cyclooxygenases (COXs). We and others demonstrated that activation of A1 or A3 adenosine receptors (ARs) results in vascular contraction via thromboxane (TX) A2 production. However, the mechanisms of Up4A-induced vascular contraction in mouse aorta are not understood. We hypothesize that Up4A-induced aortic contraction is through COX-derived TXA2 production, which requires activation of A1 and/or A3AR. Concentration responses to Up4A were conducted in isolated aorta. The TXB2 production, a metabolite of TXA2, was also measured. Up4A (10(-9)-10(-5)M) produced a concentration-dependent contraction >70%, which was markedly attenuated by COX and COX1 but not by COX2 inhibition. Notably, Up4A-induced aortic contraction was blunted by both TX synthase inhibitor ozagrel and TXA2 receptor (TP) antagonist SQ29548. Surprisingly, A3AR deletion had no effect on Up4A-induced contraction. Moreover, A1AR deletion or antagonism as well as A1/A3AR deletion potentiated Up4A-induced aortic contraction, suggesting a vasodilator influence of A1AR. In contrast, non-selective purinergic P2 receptor antagonist PPADS significantly blunted Up4A-induced aortic contraction to a similar extent as selective P2X1R antagonist MRS2159, the latter of which was further reduced by addition of ozagrel. Endothelial denudation almost fully attenuated Up4A-induced contraction. Furthermore, Up4A (3µM) increased TXB2 formation, which was inhibited by either MRS2159 or ozagrel. In conclusion, Up4A-induced aortic contraction depends on activation of TX synthase and TP, which partially requires the activation of P2X1R but not A1 or A3AR through an endothelium-dependent mechanism.


Assuntos
Aorta/efeitos dos fármacos , Fosfatos de Dinucleosídeos/farmacologia , Agonistas Purinérgicos/farmacologia , Receptores Purinérgicos P2X1/efeitos dos fármacos , Receptores de Tromboxano A2 e Prostaglandina H2/efeitos dos fármacos , Vasoconstrição/efeitos dos fármacos , Vasoconstritores/farmacologia , Animais , Aorta/metabolismo , Inibidores de Ciclo-Oxigenase/farmacologia , Relação Dose-Resposta a Droga , Endotélio Vascular/efeitos dos fármacos , Endotélio Vascular/metabolismo , Feminino , Masculino , Camundongos Endogâmicos C57BL , Camundongos Knockout , Receptor A1 de Adenosina/genética , Receptor A1 de Adenosina/metabolismo , Receptor A3 de Adenosina/genética , Receptor A3 de Adenosina/metabolismo , Receptores Purinérgicos P2X1/metabolismo , Receptores de Tromboxano A2 e Prostaglandina H2/metabolismo , Transdução de Sinais/efeitos dos fármacos , Tromboxano A2/metabolismo , Tromboxano B2/metabolismo , Tromboxano-A Sintase/antagonistas & inibidores , Tromboxano-A Sintase/metabolismo
14.
Purinergic Signal ; 11(2): 263-73, 2015 Jun.
Artigo em Inglês | MEDLINE | ID: mdl-25911169

RESUMO

Adenosine increases coronary flow mainly through the activation of A2A and A2B adenosine receptors (ARs). However, the mechanisms for the regulation of coronary flow are not fully understood. We previously demonstrated that adenosine-induced increase in coronary flow is in part through NADPH oxidase (Nox) activation, which is independent of activation of either A1 or A3ARs. In this study, we hypothesize that adenosine-mediated increase in coronary flow through Nox activation depends on A2A but not A2BARs. Functional studies were conducted using isolated Langendorff-perfused mouse hearts. Hydrogen peroxide (H2O2) production was measured in isolated coronary arteries from WT, A2AAR knockout (KO), and A2BAR KO mice using dichlorofluorescein immunofluorescence. Adenosine-induced concentration-dependent increase in coronary flow was attenuated by the specific Nox2 inhibitor gp91 ds-tat or reactive oxygen species (ROS) scavenger EUK134 in both WT and A2B but not A2AAR KO isolated hearts. Similarly, the A2AAR selective agonist CGS-21680-induced increase in coronary flow was significantly blunted by Nox2 inhibition in both WT and A2BAR KO, while the A2BAR selective agonist BAY 60-6583-induced increase in coronary flow was not affected by Nox2 inhibition in WT. In intact isolated coronary arteries, adenosine-induced (10 µM) increase in H2O2 formation in both WT and A2BAR KO mice was attenuated by Nox2 inhibition, whereas adenosine failed to increase H2O2 production in A2AAR KO mice. In conclusion, adenosine-induced increase in coronary flow is partially mediated by Nox2-derived H2O2, which critically depends upon the presence of A2AAR.


Assuntos
Vasos Coronários/efeitos dos fármacos , Miocárdio/metabolismo , NADPH Oxidases/metabolismo , Receptor A2A de Adenosina/metabolismo , Aminopiridinas/farmacologia , Animais , Coração/efeitos dos fármacos , Peróxido de Hidrogênio/farmacologia , Camundongos Endogâmicos C57BL , Camundongos Knockout , Receptor A2B de Adenosina/metabolismo , Vasodilatação/efeitos dos fármacos , Vasodilatação/fisiologia
15.
Mol Cell Biochem ; 404(1-2): 87-96, 2015 Jun.
Artigo em Inglês | MEDLINE | ID: mdl-25739357

RESUMO

This study aims to investigate the signaling mechanism involved in HS-induced modulation of adenosine-mediated vascular tone in the presence or absence of adenosine A2A receptor (A2AAR). We hypothesized that HS-induced enhanced vascular relaxation through A2AAR and epoxyeicosatrienoic acid (EETs) is dependent on peroxisome proliferator-activated receptor gamma (PPARγ) and ATP-sensitive potassium channels (KATP channels) in A2AAR(+/+) mice, while HS-induced vascular contraction to adenosine is dependent on soluble epoxide hydrolase (sEH) that degrades EETs in A2AAR(-/-) mice. Organ bath and Western blot techniques were conducted in HS (4 % NaCl) and normal salt (NS, 0.45 % NaCl)-fed A2AAR(+/+) and A2AAR(-/-) mouse aorta. We found that enhanced vasodilation to A2AAR agonist, CGS 21680, in HS-fed A2AAR(+/+) mice was blocked by PPARγ antagonist (T0070907) and KATP channel blocker (Glibenclamide). Also, sEH inhibitor (AUDA)-dependent vascular relaxation was mitigated by PPARγ antagonist. PPARγ agonist (Rosiglitazone)-induced relaxation in HS-A2AAR(+/+) mice was attenuated by KATP channel blocker. Conversely, HS-induced contraction in A2AAR(-/-) mice was attenuated by sEH inhibitor. Overall, findings from this study that implicates the contribution of EETs, PPARγ and KATP channels downstream of A2AAR to mediate enhanced vascular relaxation in response to HS diet while, role of sEH in mediating vascular contraction in HS-fed A2AAR(-/-) mice.


Assuntos
Aorta/fisiologia , Epóxido Hidrolases/metabolismo , Canais KATP/metabolismo , PPAR gama/metabolismo , Receptor A2A de Adenosina/genética , Animais , Aorta/efeitos dos fármacos , Ácidos Araquidônicos/metabolismo , Benzamidas/administração & dosagem , Inibidores Enzimáticos/administração & dosagem , Canais KATP/genética , Camundongos , PPAR gama/genética , Piridinas/administração & dosagem , Receptor A2A de Adenosina/metabolismo , Cloreto de Sódio/administração & dosagem , Vasodilatação/efeitos dos fármacos
16.
Am J Physiol Heart Circ Physiol ; 307(7): H1046-55, 2014 Oct 01.
Artigo em Inglês | MEDLINE | ID: mdl-25108010

RESUMO

We have previously demonstrated that adenosine-mediated H2O2 production and opening of ATP-sensitive K(+) (KATP) channels contributes to coronary reactive hyperemia. The present study aimed to investigate the roles of adenosine, H2O2, and KATP channels in coronary metabolic hyperemia (MH). Experiments were conducted on isolated Langendorff-perfused mouse hearts using combined pharmacological approaches with adenosine receptor (AR) knockout mice. MH was induced by electrical pacing at graded frequencies. Coronary flow increased linearly from 14.4 ± 1.2 to 20.6 ± 1.2 ml·min(-1)·g(-1) with an increase in heart rate from 400 to 650 beats/min in wild-type mice. Neither non-selective blockade of ARs by 8-(p-sulfophenyl)theophylline (8-SPT; 50 µM) nor selective A2AAR blockade by SCH-58261 (1 µM) or deletion affected MH, although resting flow and left ventricular developed pressure were reduced. Combined A2AAR and A2BAR blockade or deletion showed similar effects as 8-SPT. Inhibition of nitric oxide synthesis by N-nitro-l-arginine methyl ester (100 µM) or combined 8-SPT administration failed to reduce MH, although resting flows were reduced (by ∼20%). However, glibenclamide (KATP channel blocker, 5 µM) decreased not only resting flow (by ∼45%) and left ventricular developed pressure (by ∼36%) but also markedly reduced MH by ∼94%, resulting in cardiac contractile dysfunction. Scavenging of H2O2 by catalase (2,500 U/min) also decreased resting flow (by ∼16%) and MH (by ∼24%) but to a lesser extent than glibenclamide. Our results suggest that while adenosine modulates coronary flow under both resting and ischemic conditions, it is not required for MH. However, H2O2 and KATP channels are important local control mechanisms responsible for both coronary ischemic and metabolic vasodilation.


Assuntos
Circulação Coronária , Peróxido de Hidrogênio/metabolismo , Hiperemia/metabolismo , Canais KATP/metabolismo , Receptores Purinérgicos P1/metabolismo , Adenosina/metabolismo , Antagonistas do Receptor A2 de Adenosina/farmacologia , Animais , Depuradores de Radicais Livres/farmacologia , Glibureto/farmacologia , Coração/efeitos dos fármacos , Coração/fisiologia , Hiperemia/fisiopatologia , Técnicas In Vitro , Canais KATP/antagonistas & inibidores , Canais KATP/genética , Camundongos , Camundongos Endogâmicos C57BL , Contração Miocárdica , Miocárdio/metabolismo , NG-Nitroarginina Metil Éster/farmacologia , Bloqueadores dos Canais de Potássio/farmacologia , Pirimidinas/farmacologia , Receptores Purinérgicos P1/genética , Teofilina/análogos & derivados , Teofilina/farmacologia , Triazóis/farmacologia
17.
Cardiovasc Res ; 102(1): 157-65, 2014 Apr 01.
Artigo em Inglês | MEDLINE | ID: mdl-24525840

RESUMO

AIMS: The goal of this study was to determine whether the A1 adenosine receptor (AR) plays a role in atherosclerosis development and to explore its potential mechanisms. METHODS AND RESULTS: Double knockout (DKO) mice, deficient in the genes encoding A1 AR and apolipoprotein E (apoE), demonstrated reduced atherosclerotic lesions in aortic arch (en face), aortic root, and innominate arteries when compared with apoE-deficient mice (APOE-KO) of the same age. Treating APOE-KO with an A1 AR antagonist (DPCPX) also led to a concentration-dependent reduction in lesions. The total plasma cholesterol and triglyceride levels were not different between DKO and APOE-KO; however, higher triglyceride was observed in DKO fed a high-fat diet. DKO also had higher body weights than APOE-KO. Plasma cytokine concentrations (IL-5, IL-6, and IL-13) were significantly lower in DKO. Proliferating cell nuclear antigen expression was also significantly reduced in the aorta from DKO. Despite smaller lesions in DKO, the composition of the innominate artery lesion and cholesterol loading and efflux from bone marrow-derived macrophages of DKO were not different from APOE-KO. CONCLUSION: The A1 AR may play a role in the development of atherosclerosis, possibly due to its pro-inflammatory and mitogenic properties.


Assuntos
Apolipoproteínas E/deficiência , Aterosclerose/metabolismo , Colesterol/metabolismo , Receptor A1 de Adenosina/metabolismo , Animais , Aorta/metabolismo , Modelos Animais de Doenças , Inflamação/metabolismo , Macrófagos/metabolismo , Camundongos Endogâmicos C57BL , Camundongos Knockout , Receptor A1 de Adenosina/deficiência
18.
Physiol Rep ; 1(3): e00070, 2013 Aug 01.
Artigo em Inglês | MEDLINE | ID: mdl-24159377

RESUMO

The NADPH oxidase (Nox) subunits 1, 2 (gp91 phox) and 4 are the major sources for reactive oxygen species (ROS) in cardiovascular system. In conditions such as ischemia-reperfusion injury and hypoxia, both ROS and adenosine are released suggesting a possible interaction. We hypothesized that ROS generated through Nox is involved in adenosine-induced coronary flow (CF) responses. Adenosine (10-8-10-5.5 M) increased CF in isolated hearts from wild type (WT; C57/BL6), A1 adenosine receptor (AR) knockout (A1KO), A3AR KO (A3KO) and A1 and A3AR double KO (A1/A3DKO) mice. The Nox inhibitors apocynin (10-5 M) and gp91 ds-tat (10-6 M) or the SOD and catalase-mimicking agent EUK134 (50 µM) decreased the adenosine-enhanced CF in the WT and all the KOs. Additionally, adenosine increased phosphorylation of p47-phox subunit and ERK 1/2 without changing protein expression of Nox isoforms in WT. Moreover, intracellular superoxide production was increased by adenosine and CGS-21680 (a selective A2A agonist), but not BAY 60-6583 (a selective A2B agonist), in mouse coronary artery smooth muscle cells (CASMCs) and endothelial cells (CAECs). This superoxide increase was inhibited by the gp91 ds-tat and ERK 1/2 inhibitor (PD98059). In conclusion, adenosine-induced increase in CF in isolated heart involves Nox2-generated superoxide, possibly through ERK 1/2 phosphorylation with subsequent p47-phox subunit phosphorylation. This adenosine/Nox/ROS interaction occurs in both CASMCs and CAECs, and involves neither A1 nor A3 ARs, but possibly A2A ARs in mouse.

19.
Am J Physiol Heart Circ Physiol ; 305(11): H1668-79, 2013 Dec 01.
Artigo em Inglês | MEDLINE | ID: mdl-24043252

RESUMO

We previously demonstrated that A2A, but not A2B, adenosine receptors (ARs) mediate coronary reactive hyperemia (RH), possibly by producing H2O2 and, subsequently, opening ATP-dependent K(+) (KATP) channels in coronary smooth muscle cells. In this study, A1 AR knockout (KO), A3 AR KO, and A1 and A3 AR double-KO (A1/A3 DKO) mice were used to investigate the roles and mechanisms of A1 and A3 ARs in modulation of coronary RH. Coronary flow of isolated hearts was measured using the Langendorff system. A1 KO and A1/A3 DKO, but not A3 KO, mice showed a higher flow debt repayment [~30% more than wild-type (WT) mice, P < 0.05] following a 15-s occlusion. SCH-58261 (a selective A2A AR antagonist, 1 µM) eliminated the augmented RH, suggesting the involvement of enhanced A2A AR-mediated signaling in A1 KO mice. In isolated coronary arteries, immunohistochemistry showed an upregulation of A2A AR (1.6 ± 0.2 times that of WT mice, P < 0.05) and a higher magnitude of adenosine-induced H2O2 production in A1 KO mice (1.8 ± 0.3 times that of WT mice, P < 0.05), which was blocked by SCH-58261. Catalase (2,500 U/ml) and glibenclamide (a KATP channel blocker, 5 µM), but not N(G)-nitro-l-arginine methyl ester, also abolished the enhanced RH in A1 KO mice. Our data suggest that A1, but not A3, AR counteracts the A2A AR-mediated CF increase and that deletion of A1 AR results in upregulation of A2A AR and/or removal of the negative modulatory effect of A1 AR, thus leading to an enhanced A2A AR-mediated H2O2 production, KATP channel opening, and coronary vasodilation during RH. This is the first report implying that A1 AR has a role in coronary RH.


Assuntos
Circulação Coronária , Vasos Coronários/metabolismo , Peróxido de Hidrogênio/metabolismo , Hiperemia/metabolismo , Canais KATP/metabolismo , Receptor A1 de Adenosina/metabolismo , Receptor A2A de Adenosina/metabolismo , Vasodilatação , Antagonistas do Receptor A2 de Adenosina/farmacologia , Animais , Antioxidantes/farmacologia , Circulação Coronária/efeitos dos fármacos , Vasos Coronários/efeitos dos fármacos , Vasos Coronários/fisiopatologia , Feminino , Hiperemia/genética , Hiperemia/fisiopatologia , Canais KATP/antagonistas & inibidores , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Perfusão , Bloqueadores dos Canais de Potássio/farmacologia , Receptor A1 de Adenosina/deficiência , Receptor A1 de Adenosina/genética , Receptor A2A de Adenosina/efeitos dos fármacos , Receptor A3 de Adenosina/genética , Receptor A3 de Adenosina/metabolismo , Transdução de Sinais/efeitos dos fármacos , Fatores de Tempo , Vasodilatação/efeitos dos fármacos
20.
J Cardiovasc Pharmacol ; 62(1): 78-83, 2013 Jul.
Artigo em Inglês | MEDLINE | ID: mdl-23519140

RESUMO

Adenosine A1 receptor (A1AR) activation contracts smooth muscle, although signaling mechanisms are not thoroughly understood. Activation of A1AR leads to metabolism of arachidonic acid, including the production of 20-hydroxyeicosatetraenoic acid (20-HETE) by cytochrome P4504a (CYP4a). The 20-HETE can activate protein kinase C-α (PKC-α), which crosstalks with extracellular signal-regulated kinase (ERK1/2) pathway. Both these pathways can regulate smooth muscle contraction, we tested the hypothesis that A1AR contracts smooth muscle through a pathway involving CYP4a, PKC-α, and ERK1/2. Experiments included isometric tension recordings of aortic contraction and Western blots of signaling molecules in wild type (WT) and A1AR knockout (A1KO) mice. Contraction to the A1-selective agonist 2-chloro-N cyclopentyladenosine (CCPA) was absent in A1KO mice aortae, indicating the contractile role of A1AR. Inhibition of CYP4a (HET0016) abolished 2-chloro-N cyclopentyladenosine-induced contraction in WT aortae, indicating a critical role for 20-HETE. Both WT and A1KO mice aortae contracted in response to exogenous 20-HETE. Inhibition of PKC-α (Gö6976) or ERK1/2 (PD98059) attenuated 20-HETE-induced contraction equally, suggesting that ERK1/2 is downstream of PKC-α. Contractions to exogenous 20-HETE were significantly less in A1KO mice; reduced protein levels of PKC-α, p-ERK1/2, and total ERK1/2 supported this observation. Our data indicate that A1AR mediates smooth muscle contraction via CYP4a and a PKC-α-ERK1/2 pathway.


Assuntos
Citocromo P-450 CYP4A/fisiologia , Sistema de Sinalização das MAP Quinases/fisiologia , Contração Muscular/efeitos dos fármacos , Músculo Liso Vascular/efeitos dos fármacos , Proteína Quinase C-alfa/fisiologia , Receptor A1 de Adenosina/fisiologia , Adenosina/análogos & derivados , Adenosina/farmacologia , Agonistas do Receptor A1 de Adenosina/farmacologia , Animais , Western Blotting , Carbazóis/farmacologia , Citocromo P-450 CYP4A/antagonistas & inibidores , Inibidores Enzimáticos/farmacologia , Feminino , Flavonoides/farmacologia , Ácidos Hidroxieicosatetraenoicos/farmacologia , Contração Isométrica/efeitos dos fármacos , Sistema de Sinalização das MAP Quinases/efeitos dos fármacos , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Receptor A1 de Adenosina/efeitos dos fármacos
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