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Animal models of asthma have shown that limonene, a naturally occurring terpene in citrus fruits, can reduce inflammation and airway reactivity. However, the mechanism of these effects is unknown. We first performed computational and molecular docking analyses that showed limonene could bind to both A2A and A2B receptors. The pharmacological studies were carried out with A2A adenosine receptor knock-out (A2AKO) and wild-type (WT) mice using ovalbumin (OVA) to generate the asthma phenotype. We investigated the effects of limonene on lung inflammation and airway responsiveness to methacholine (MCh) and NECA (nonselective adenosine analog) by administering limonene as an inhalation prior to OVA aerosol challenges in one group of allergic mice for both WT and KO. In whole-body plethysmography studies, we observed that airway responsiveness to MCh in WT SEN group was significantly lowered upon limonene treatment but no effect was observed in A2AKO. Limonene also attenuated NECA-induced airway responsiveness in WT allergic mice with no effect being observed in A2AKO groups. Differential BAL analysis showed that limonene reduced levels of eosinophils in allergic WT mice but not in A2AKO. However, limonene reduced neutrophils in sensitized A2AKO mice, suggesting that it may activate A2B receptors as well. These data indicate that limonene-induced reduction in airway inflammation and airway reactivity occurs mainly via activation of A2AAR but A2B receptors may also play a supporting role.

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 P2X(7) 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.

OBJECTIVE: Angiotensin II (Ang II) exerts its effect 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 PD123319 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 day 1 and 6 (30μg; 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) c) PD123319 (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 (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

Uridine adenosine tetraphosphate (Up(4)A), 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 Up(4)A 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 Up(4)A produces vascular contraction. In contrast, Up(4)A produces vascular relaxation in porcine coronary small arteries and rat aortas. Intravenous infusion of Up(4)A into conscious rats or mice decreases blood pressure, and intravenous bolus injection of Up(4)A into anesthetized mice increases coronary blood flow, indicating an overall vasodilator influence in vivo. Although Up(4)A 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 Up(4)A in cardiovascular pathophysiology, as plasma levels of Up(4)A are elevated in juvenile hypertensive patients and Up(4)A-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 Up(4)A and the purinergic signaling mechanisms mediating its effects will enhance its potential as target for treatment of cardiovascular disease.

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.

Adenosine is an ubiquitous extracellular signaling molecule and plays a fundamental role in the regulation of coronary microcirculation through activation of adenosine receptors (ARs). Adenosine is regulated by various enzymes and nucleoside transporters for its balance between intra- and extracellular compartments. Adenosine-mediated coronary microvascular tone and reactive hyperemia are through receptors mainly involving A2AR activation on both endothelial and smooth muscle cells, but also involving interaction among other ARs. Activation of ARs further stimulates downstream targets of H2O2, KATP, KV and KCa2+ channels leading to coronary vasodilation. An altered adenosine-ARs signaling in coronary microcirculation has been observed in several cardiovascular diseases including hypertension, diabetes, atherosclerosis and ischemic heart disease. Adenosine as a metabolite and its receptors have been studied for its both therapeutic and diagnostic abilities. The present review summarizes important aspects of adenosine metabolism and AR-mediated actions in the coronary microcirculation.

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

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.

Animal models of asthma have shown that limonene, a naturally occurring terpene in citrus fruits, can reduce inflammation and airway reactivity. However, the mechanism of these effects is unknown. We first performed computational and molecular docking analyses that showed limonene could bind to both A(2A) and A(2B) receptors. The pharmacological studies were carried out with A(2A) adenosine receptor knock-out (A(2A)KO) and wild-type (WT) mice using ovalbumin (OVA) to generate the asthma phenotype. We investigated the effects of limonene on lung inflammation and airway responsiveness to methacholine (MCh) and NECA (nonselective adenosine analog) by administering limonene as an inhalation prior to OVA aerosol challenges in one group of allergic mice for both WT and KO. In whole-body plethysmography studies, we observed that airway responsiveness to MCh in WT SEN group was significantly lowered upon limonene treatment but no effect was observed in A(2A)KO. Limonene also attenuated NECA-induced airway responsiveness in WT allergic mice with no effect being observed in A(2A)KO groups. Differential BAL analysis showed that limonene reduced levels of eosinophils in allergic WT mice but not in A(2A)KO. However, limonene reduced neutrophils in sensitized A(2A)KO mice, suggesting that it may activate A(2B) receptors as well. These data indicate that limonene-induced reduction in airway inflammation and airway reactivity occurs mainly via activation of A(2A)AR but A(2B) receptors may also play a supporting role. ELECTRONIC SUPPLEMENTARY MATERIAL: The online version of this article (10.1007/s11302-020-09697-z) contains supplementary material, which is available to authorized users.

Limonene a mono-terpene flavonoid that is anti-inflammatory in asthma. We have shown that limonene induces bronchodilation and lowers inflammation via A2A receptor activation (FASEB J April 2017 31...

L-NAME-induced hypertension is commonly used to study endothelial dysfunction and related vascular effects. It has been reported that genetic deletion of A(1) adenosine receptor (AR) reduces blood pressure (BP) increases in mice and thus, suggesting the involvement of A(1)AR. Thus, we sought to determine whether A(1)AR-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-N(6)-cyclopentyladenosine (CCPA, selective A(1)AR 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 (K(ATP) 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 A(1)AR and Cyp4A were noted in MAs of hypertensive mice. In addition, in aorta, higher A(1)AR and comparable Cyp4A levels were observed in hypertensive mice. A(1)AR-induced vascular contractions were enhanced in hypertensive mice aorta and MAs. Cyp4A plays a role in altered vascular responses in MAs.

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.

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.

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.

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.

Cardiac injury and dysfunction are contributors to disease progression and mortality in sepsis. This study evaluated the cardiovascular role of intrinsic A₂A adenosine receptor (A₂AAR) activity during lipopolysaccharide (LPS)-induced inflammation.We assessed the impact of 24 h of LPS challenge (20 mg/kg, IP) on cardiac injury, coronary function and inflammatory mediator levels in Wild-Type (WT) mice and mice lacking functional A₂AARs (A₂AAR KO).Cardiac injury was evident in LPS-treated WTs, with ~7-fold elevation in serum cardiac troponin I (cTnI), and significant ventricular and coronary dysfunction. Absence of A₂AARs increased LPS-provoked cTnI release at 24 h by 3-fold without additional demise of contraction function. Importantly, A₂AAR deletion per se emulated detrimental effects of LPS on coronary function, and LPS was without effect in coronary vessels lacking A₂AARs. Effects of A₂AAR KO were independent of major shifts in circulating C-reactive protein (CRP) and haptoglobin. Cytokine responses were largely insensitive to A₂AAR deletion; substantial LPS-induced elevations (up to 100-fold) in IFN-γ and IL-10 were unaltered in A₂AAR KO mice, as were levels of IL-4 and TNF-α. However, late elevations in IL-2 and IL-5 were differentially modulated by A₂AAR KO (IL-2 reduced, IL-5 increased). Data demonstrate that in the context of LPS-triggered cardiac and coronary injury, A₂AAR activity protects myocardial viability without modifying contractile dysfunction, and selectively modulates cytokine (IL-2, IL-5) release. A₂AARs also appear to be targeted by LPS in the coronary vasculature.These experimental data suggest that preservation of A₂AAR functionality might provide therapeutic benefit in human sepsis.

Ultrasound and Duplex ultrasonography in particular are routinely used to diagnose cardiovascular disease (CVD), which is the leading cause of morbidity and mortality worldwide. However, these techniques may not be able to characterize vascular tissue compositional changes due to CVD. This work describes an ultrasound-based hybrid imaging technique that can be used for vascular tissue characterization and the diagnosis of atherosclerosis. Ultrasound radiofrequency (RF) data were acquired and processed in time, frequency, and wavelet domains to extract six parameters including time integrated backscatter (TIB), time variance (Tvar), time entropy (TE), frequency integrated backscatter (FIB), wavelet root mean square value (Wrms), and wavelet integrated backscatter (WIB). Each parameter was used to reconstruct an image co-registered to morphological B-scan. The combined set of hybrid images were used to characterize vascular tissue in vitro and in vivo using three mouse models including control (C57BL/6), and atherosclerotic apolipoprotein E-knockout (APOE-KO) and APOE/A1 adenosine receptor double knockout (DKO) mice. The technique was tested using high-frequency ultrasound including single-element (center frequency = 55 MHz) and commercial array (center frequency = 40 MHz) systems providing superior spatial resolutions of 24 μm and 40 μm, respectively. Atherosclerotic vascular lesions in the APOE-KO mouse exhibited the highest values (contrast) of −10.11 ± 1.92 dB, −12.13 ± 2.13 dB, −7.54 ± 1.45 dB, −5.10 ± 1.06 dB, −5.25 ± 0.94 dB, and −10.23 ± 2.12 dB in TIB, Tvar, TE, FIB, Wrms, WIB hybrid images (n = 10, p < 0.05), respectively. Control segments of normal vascular tissue showed the lowest values of −20.20 ± 2.71 dB, −22.54 ± 4.54 dB, −14.94 ± 2.05 dB, −9.64 ± 1.34 dB, −10.20 ± 1.27 dB, and −19.36 ± 3.24 dB in same hybrid images (n = 6, p < 0.05). Results from both histology and optical images showed good agreement with ultrasound findings within a maximum error of 3.6% in lesion estimation. This study demonstrated the feasibility of a high-resolution hybrid imaging technique to diagnose atherosclerosis and characterize plaque components in mouse. In the future, it can be easily implemented on commercial ultrasound systems and eventually translated into clinics as a screening tool for atherosclerosis and the assessment of vulnerable plaques.

The interaction between adenosine and soluble epoxide hydrolase (sEH) in vascular response is not known. Therefore, we hypothesized that lack of sEH in mice enhances adenosine-induced relaxation through A2A adenosine receptors (AR) via CYP-epoxygenases and peroxisome proliferator-activated receptor γ (PPARγ). sEH−/− showed an increase in A2A AR, CYP2J, and PPARγ by 31%, 65%, and 36%, respectively, and a decrease in A1AR and PPARα (30% and 27%, respectively) vs. sEH+/+. 5′-N-ethylcarboxamidoadenosine (NECA, an adenosine receptor agonist), CGS 21680 (A2A AR-agonist), and GW 7647 (PPARα-agonist)-induced responses were tested with nitro-l-arginine methyl ester (l-NAME) (NO-inhibitor; 10−4 M), ZM-241385, SCH-58261 (A2A AR-antagonists; 10−6 M), 14,15-epoxyeicosa-5(Z)-enoic acid (14,15-EEZE, an epoxyeicosatrienoic acid-antagonist; 10−5 M), 12-(3-adamantan-1-yl-ureido) dodecanoic acid (AUDA; 10 μM) or trans-4-[4-(3-adamantan-1-yl-ureido)-cyclohexyloxy]-benzoic acid ( t-AUCB, sEH-inhibitors; 10−5 M), and T0070907 (PPARγ-antagonist; 10−7 M). In sEH−/− mice, ACh response was not different from sEH+/+ ( P > 0.05), and l-NAME blocked ACh-responses in both sEH−/− and sEH+/+ mice ( P < 0.05). NECA (10−6 M)-induced relaxation was higher in sEH−/− (+12.94 ± 3.2%) vs. sEH+/+ mice (−5.35 ± 5.2%); however, it was blocked by ZM-241385 (−22.42 ± 1.9%) and SCH-58261(−30.04 ± 4.2%). CGS-21680 (10−6 M)-induced relaxation was higher in sEH−/− (+37.4 ± 5.4%) vs. sEH+/+ (+2.14 ± 2.8%). l-NAME (sEH−/−, +30.28 ± 4.8%, P > 0.05) did not block CGS-21680-induced response, whereas 14,15-EEZE (−7.1 ± 3.7%, P < 0.05) did. Also, AUDA and t-AUCB did not change CGS-21680-induced response in sEH−/− ( P > 0.05), but reversed in sEH+/+ (from +2.14 ± 2.8% to +45.33 ± 4.1%, and +63.37 ± 7.2, respectively). PPARα-agonist did not relax as CGS 21680 (−2.48 ± 1.1 vs. +37.4 ± 5.4%) in sEH−/−, and PPARγ-antagonist blocked (from +37.4 ± 5.4% to +9.40 ± 3.1) CGS 21680-induced relaxation in sEH−/−. Our data suggest that adenosine-induced relaxation in sEH−/− may depend on the upregulation of A2A AR, CYP2J, and PPARγ, and the downregulation of A1 AR and PPARα.

Our recent finding showed that acute angiotensin (ANG) II stimulation enhanced A 1 adenosine receptor (AR) dependent cytochrome P450 (CYP) 4A mediated contraction in mouse mesenteric arteries (MA). In addition, A 2B AR dependent and partly K ATP channel mediated relaxation was also reduced with ANGII stimulation. These data suggest a possible interaction between ANG II and ARs at vascular level which may play a role in ANG II dependent hypertension. We developed ANG II (1000 ng/kg/min, 21 days) infused hypertension mouse model and tail cuff was used to measure blood pressure. Aorta as a conduit and MA (second order) as a resistance artery were chosen for muscle tension studies. Protein expressions in aortas and MAs were analyzed by western blot. Infusion of ANG II increased systolic arterial blood pressure (SAP) in mice. Particularly, in ANG II infused mice, SAP (in mmHg, mean ± SD) at day 0 was 101.6 ± 6.675 which increased to 156.7 ± 15.68 on day 21 (p Higher expression of A 1 AR (~155% over 100%) and CYP4A (~50%, ~0.6 over 0.3, ratio to actin) was present in MAs in hypertensive compared to control mice. Expression of A 1 AR and CYP4A was comparable in aortas of hypertensive and control mice. CCPA (A 1 AR agonist) -induced concentration dependent contraction (% normalize to phenylephrine contraction) was significantly reduced (~15% from 40% at 100 nM) in aorta of hypertensive mice compared to control. NECA (non-selective AR agonist) produced a significantly higher (~ -15% from 0% at 10 μM) relaxation in aorta of hypertensive mice. In MAs, CCPA-induced contraction was reduced in hypertensive mice. NECA induced relaxation was comparable in MAs. Pinacidil (K ATP channel opener) induced relaxation was significantly lower (~0% from -25% at 100 nM) in MAs of hypertensive mice. In conclusion, our data reveal important role for A 1 AR dependent signaling in ANG II induced hypertension. Higher A1AR and CYP4A may reduce K ATP channel dependent relaxation of MAs aiding vascular contraction and blood pressure. Further studies involving ARs pharmacological inhibitors and genetic models are required to fully understand the relationship between ANG II and ARs in the development of hypertension.

Previously, we have shown that A2A adenosine receptor (A2AAR) mediates aortic relaxation via cytochrome P-450 (CYP)-epoxygenases. However, the signaling mechanism is not understood properly. We hypothesized that ATP-sensitive K+ (KATP) channels play an important role in A2AAR-mediated relaxation. Organ bath and Western blot experiments were done using isolated aorta from A2AKO and corresponding wild-type (WT) mice. Aortic rings from WT and A2A knockout (KO) mice were precontracted with submaximal dose of phenylephrine (PE, 10−6 M), and concentration-response curves for pinacidil, cromakalim (nonselective KATP openers), and diazoxide (mitochondrial KATP opener) were obtained. Diazoxide did not have any relaxation effect on PE-precontracted tissues, whereas relaxation to pinacidil (48.09 ± 5.23% in WT vs. 25.41 ± 2.73% in A2AKO; P < 0.05) and cromakalim (51.19 ± 2.05% in WT vs. 38.50 ± 2.26% in A2AKO; P < 0.05) was higher in WT than A2AKO aorta. This suggested the involvement of sarcolemmal rather than mitochondrial KATP channels. Endothelium removal, treatment with SCH 58651 (A2AAR antagonist; 10−6 M), NG-nitro-l-arginine methyl ester (l-NAME, nitric oxide synthase inhibitor) and methylsulfonyl-propargyloxyphenylhexanamide (MS-PPOH, CYP-epoxygenases inhibitor; 10−5 M) significantly reduced pinacidil-induced relaxation in WT compared with controls, whereas these treatments did not have any effect in A2AKO aorta. Glibenclamide (KATP channel inhibitor, 10−5 M) blocked 2- p-(2-carboxyethyl)phenethylamino-5′ N-ethylcarboxamido adenosine hydrochloride (CGS 21680, A2AAR agonist)-induced relaxation in WT and changed 5′- N-ethylcarboxamide (NECA) (nonselective adenosine analog)-induced response to higher contraction in WT and A2AKO. 5-Hydroxydecanoate (5-HD, mitochondrial KATP channel inhibitor, 10−4 M) had no effect on CGS 21680-mediated response in WT aorta. Our data suggest that A2AAR-mediated vasorelaxation occurs through opening of sarcolemmal KATP channels via CYP-epoxygenases and possibly, nitric oxide, contributing to pinacidil-induced responses.

Endothelium-dependent, nitric oxide (NO)-mediated vasodilation can be impaired by reactive oxygen species (ROS), and this deleterious effect of ROS on NO availability may increase with aging. Endothelial function declines rapidly after menopause, possibly because of loss of circulating estrogen and its antioxidant effects. The purpose of the current study was to determine the role of O2−and H2O2in regulating flow-induced dilation in coronary arterioles of young (6-mo) and aged (24-mo) intact, ovariectomized (OVX), or OVX + estrogen-treated (OVE) female Fischer 344 rats. Both aging and OVX reduced flow-induced NO production, whereas flow-induced H2O2production was not altered by age or estrogen status. Flow-induced vasodilation was evaluated before and after treatment with the superoxide dismutase (SOD) mimetic Tempol (100 μM) or the H2O2scavenger catalase (100 U/ml). Removal of H2O2with catalase reduced flow-induced dilation in all groups, whereas Tempol diminished vasodilation in intact and OVE, but not OVX, rats. Immunoblot analysis revealed elevated nitrotyrosine with aging and OVX. In young rats, OVX reduced SOD protein while OVE increased SOD in aged rats; catalase protein did not differ in any group. Collectively, these studies suggest that O2−and H2O2are critical components of flow-induced vasodilation in coronary arterioles from female rats; however, a chronic deficiency of O2−buffering by SOD contributes to impaired flow-induced dilation with aging and loss of estrogen. Furthermore, these data indicate that estrogen replacement restores O2−homeostasis and flow-induced dilation of coronary arterioles, even at an advanced age.

The NADPH oxidase (Nox) subunits 1, 2 (gp91 phox), and 4 are the major sources for reactive oxygen species (ROS) in vascular tissues. In conditions such as ischemia-reperfusion and hypoxia, both ROS and adenosine are released, suggesting a possible interaction. Our aim in this study was to examine the A(3) adenosine receptor (A(3)AR)-induced vascular effects and its relation to ROS and Nox1, 2, and 4 using aortic tissues from wild-type (WT) and A(3)AR knockout (A(3)KO) mice. The selective A(3)AR agonist 2-chloro-N(6)-(3-iodobenzyl)-adenosine-5'-N-methyluronamide (Cl-IBMECA) (10(-10)-10(-5) M) induced contraction of the aorta from WT but not from A(3)KO mice, and this contraction was inhibited by the Nox inhibitor apocynin (10(-5) M) and the ROS scavengers superoxide dismutase-polyethylene glycol and catalase-polyethylene glycol (100 U/ml each). Cl-IBMECA-induced contraction was not affected by the mast cell degranulator compound 48/80 (100 μg/ml) or the stabilizer cromolyn sodium (10(-4) M). In addition, Cl-IBMECA (10(-7) M) increased intracellular ROS generation by 35 ± 14% in WT but not in A(3)KO aorta, and this increase was inhibited by apocynin (10(-5) M), diphenyleneiodonium chloride (10(-5) M), and the A(3)AR antagonist 3-propyl-6-ethyl-5-[(ethylthio)carbonyl]-2 phenyl-4-propyl-3-pyridine carboxylate (MRS1523) (10(-5) M). Furthermore, Cl-IBMECA selectively increased the protein expression of the Nox2 subunit by 150 ± 15% in WT but not in A(3)KO mice without affecting either Nox1 or 4, and this increase was inhibited by apocynin. The mRNA of Nox2 was unchanged by Cl-IBMECA in either WT or A(3)KO aortas. In conclusion, A(3)AR enhances ROS generation, possibly through activation of Nox2, with subsequent contraction of the mouse aorta.

The A1 adenosine receptor (A1AR) is coupled to Gi/Go proteins, but the downstream signaling pathways in smooth muscle cells are unclear. This study was performed in coronary artery smooth muscle cells (CASMCs) isolated from the mouse heart [A1AR wild type (A1WT) and A1AR knockout (A1KO)] to delineate A1AR signaling through the PKC pathway. In A1WT cells, treatment with (2 S)- N6-(2-endo-norbornyl)adenosine (ENBA; 10−5M) increased A1AR expression by 150%, which was inhibited significantly by the A1AR antagonist 1,3-dipropyl-8-cyclopentylxanthine (10−6M), but not in A1KO CASMCs. PKC isoforms were identified by Western blot analysis in the cytosolic and membrane fractions of cell homogenates of CASMCs. In A1WT and A1KO cells, significant levels of basal PKC-α were detected in the cytosolic fraction. Treatment with the A1AR agonist ENBA (10−5M) translocated PKC-α from the cytosolic to membrane fraction significantly in A1WT but not A1KO cells. Phospholipase C isoforms (βI, βIII, and γ1) were analyzed using specific antibodies where ENBA treatment led to the increased expression of PLC-βIII in A1WT CASMCs while having no effect in A1KO CASMCs. In A1WT cells, ENBA increased PKC-α expression and p42/p44 MAPK (ERK1/2) phospohorylation by 135% and 145%, respectively. These effects of ENBA were blocked by Gö-6976 (PKC-α inhibitor) and PD-98059 (p42/p44 MAPK inhibitor). We conclude that A1AR stimulation by ENBA activates the PKC-α signaling pathway, leading to p42/p44 MAPK phosphorylation in CASMCs.

We investigated whether A3 adenosine receptor (A3AR) is involved in endothelium-mediated contraction through cyclooxygenases (COXs) with the use of wild-type (WT) and A3 knockout (A3KO) mice aorta. A3AR-selective agonist, Cl-IBMECA, produced a concentration-dependent contraction (EC50: 2.9 ± 0.2 × 10−9 M) in WT mouse aorta with intact endothelium (+E) and negligible effects in A3KO +E aorta. At 10−7 M, contractions produced by Cl-IBMECA were 29% in WT +E, while being insignificant in A3KO +E aorta. Cl-IBMECA-induced responses were abolished in endothelium-denuded tissues (−E), in both WT and A3KO aorta. A3AR gene and protein expression were reduced by 74 and 72% ( P < 0.05), respectively, in WT −E compared with WT +E aorta, while being undetected in A3KO +E/−E aorta. Indomethacin (nonspecific COXs blocker, 10−5 M), SC-560 (specific COX-1 blocker, 10−8 M), SQ 29549 (thromboxane prostanoid receptor antagonist, 10−6 M), and furegrelate (thromboxane synthase inhibitor, 10−5 M) inhibited Cl-IBMECA-induced contraction significantly. Cl-IBMECA-induced thromboxane B2 production was also attenuated significantly by indomethacin, SC-560, and furegrelate in WT +E aorta, while having negligible effects in A3KO +E aorta. NS-398 (specific COX-2 blocker) produced negligible inhibition of Cl-IBMECA-induced contraction in both WT +E and A3KO +E aorta. Cl-IBMECA-induced increase in COX-1 and thromboxane prostanoid receptor expression were significantly inhibited by MRS1523, a specific A3AR antagonist in WT +E aorta. Expression of both A3AR and COX-1 was located mostly on endothelium of WT and A3KO +E aorta. These results demonstrate for the first time the involvement of COX-1 pathway in A3AR-mediated contraction via endothelium.

A2Aadenosine receptor (A2AAR) has potent anti-inflammatory properties, which may be important in the regulation of airway reactivity and inflammation. Inflammatory cells that possess A2AAR also produce nitrosative stress, which is associated with pathophysiology of asthma, so we hypothesized that A2AAR deficiency may lead to increased airway reactivity and inflammation through nitrosative stress. Thus the present study was carried out to investigate the role of A2AAR on airway reactivity, inflammation, NF-κB signaling, and nitrosative stress in A2AAR knockout (KO) and wild-type (WT) mice using our murine model of asthma. Animals were sensitized intraperitoneally on days 1 and 6 with 200 μg of ragweed, followed by aerosolized challenges with 0.5% ragweed on days 11, 12, and 13, twice a day. On day 14, airway reactivity to methacholine was assessed as enhanced pause (Penh) using whole body plethysmography followed by bronchoalveolar lavage (BAL) and lung collection for various analyses. Allergen challenge caused a significant decrease in expression of A2AAR in A2AWT sensitized mice, with A2AAR expression being undetected in A2AKO sensitized group leading to decreased lung cAMP levels in both groups. A2AAR deletion/downregulation led to an increase in Penh to methacholine and influx of total cells, eosinophils, lymphocytes, and neutrophils in BAL with highest values in A2AKO sensitized group. A2AKO sensitized group further had increased NF-κB expression and nitrosative stress compared with WT sensitized group. These data suggest that A2AAR deficiency leads to airway inflammation and airway hyperresponsiveness, possibly via involvement of nitrosative stress in this model of asthma.

Introduction: Up4A, a novel endothelium-derived vasoactive agent, is proposed to play a role in cardiovascular disorder via activation of P2 receptors (P2R). Here, we evaluated the vasoactive effect of Up4A on coronary flow (CF) in ApoE knockout mice (KO) with and without high fat diet (HFD). Methods: Functional studies were conducted in isolated mouse hearts using Langendorff and isolated coronary arteries (diameter≈100 μm) using wire Myograph. Immunofluorescence was performed for P2X1R expression in isolated coronary arteries. Results: There was no significant difference in baseline CF (ml/min/g) among wild type (WT, 18.4±0.5), ApoE KO (17.6±1.1) and ApoE KO+HFD (15.9±1.0). Infusion of Up4A (10-9-10-5 M) resulted in a dose-dependent reduction in CF in WT (by ~38%), which was similar to ApoE KO (by ~40%). In isolated coronary arteries of WT, Up4A produced a mild contraction up to ~20%, indicating a vasoconstrictor effect of Up4A. Notably, Up4A further decreased CF in ApoE+HFD as compared to ApoE. Moreover, selective P2X1R antagonist MRS2159 (10 μM) restored Up4A-mediated decrease in CF more in ApoE+HFD than ApoE (Figure). Finally, the percentage expression of vasoconstrictor P2X1R on smooth muscle cells (SMC) of coronary arteries was higher in ApoE+HFD than ApoE, which was due to a markedly reduced vasodilator P2X1R expression on EC in ApoE+HFD (8.7±0.6 fluorescence intensity in A.U.) than ApoE (22.1±0.5), while P2X1R expression on SMC was similar between ApoE+HFD (6.3±0.4) and ApoE (8.4±0.3). Conclusion: Up4A exerts a vasoconstrictor influence in mouse coronary microcirculation. Up4A decreases CF in WT to a similar extent in ApoE, whereas HFD exacerbates Up4A-decreased CF in ApoE possibly through downregulation of vasodilator P2X1R on EC. These findings indicate that Up4A alters CF in atherosclerosis and P2X1R may represent a potential therapeutic target.

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.

Previously, we have shown that adenosine-induced aortic relaxation occurs through activation of A2A adenosine receptor (AR) via opening of KATP channels (Ponnoth et al., 2009, 2012). In this study, we investigated whether H2S, an endogenous gaseous mediator of vasorelaxation, contributes to A2A-induced aortic relaxation. Organ bath and western blot experiments were done using isolated aortas from A2AKO and WT mice. H2S donor sodium hydrosulfide (NaHS; 10-3M) produced significantly higher relaxation in WT aorta compared to A2AKO (54.63±2.14% vs.17.16±5.84%). Removal of endothelium had no effect on NaHS responses in WT and A2AKO, neither were responses different in A1KO or A2BKO compared to WT. KATP blocker glibenclamide significantly inhibited NaHS responses in both WT (from relaxation of 54.63±2.14% to contraction of 17.76±6.46%) and A2AKO (from relaxation of 17.16±5.84% to contraction of 52.43±9.23%) and this effect was more pronounced in A2AKO. Non-selective adenosine analog NECA (10-5M) response in WT ...

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).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.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.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.

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 (COX). We and others demonstrated that activation of A1 or A3 adenosine receptors (AR) 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 (3 μM) 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 A3 AR through an endothelium-dependent mechanism.

Adenosine, a ubiquitous biological molecule elicits both pro- and anti-inflammatory effects in the lungs. Bronchoconstriction in subjects with asthma after adenosine inhalation involves a selective interaction with adenosine receptors, which is subsequently linked to the release of mast cell mediators. Animal studies also support a role for adenosine in inflammation and airway hyperresponsiveness. Therefore, selective blockade of adenosine receptors may be an attractive and valuable approach to the treatment of asthma.

To determine whether A1 adenosine receptors (AR) participate in adenosine-induced changes of coronary flow, isolated hearts from A1AR−/− and A1AR+/+ mice were perfused under constant pressure, and the effects of nonselective and selective agonists were examined. Adenosine, 5′- N-ethylcarboxamidoadenosine (NECA, nonselective), and the selective A2AAR agonist 2–2-carboxyethylphenethylamino-5′- N-ethylcarboxamidoadenosine (CGS-21680) augmented maximal coronary vasodilation in A1AR−/− hearts compared with A1AR+/+ hearts. Basal coronary flow was increased ( P < 0.05) in A1AR−/− hearts compared with A1AR+/+ hearts: 2.548 ± 0.1 vs. 2.059 ± 0.17 ml/min. In addition, selective activation of A1AR with 2-chloro- N6-cyclopentyladenosine (CCPA) at nanomolar concentrations (1–100 nM) did not significantly change coronary flow; at higher concentrations, CCPA increased coronary flow in A1AR−/− and A1AR+/+ hearts. Because deletion of A1AR increased basal coronary flow, it is speculated that this effect is due to removal of an inhibitory influence associated with A1AR. Adenosine and NECA at approximately EC50 (100 and 50 nM, respectively) increased coronary flow in A1AR+/+ hearts to 177.86 ± 8.75 and 172.72 ± 17% of baseline, respectively. In the presence of the selective A1AR antagonist 1,3-dipropyl-8-cyclopentylxanthine (DPCPX, 50 nM), the adenosine- and NECA-induced increase in coronary flow in A1AR+/+ hearts was significantly augmented to 216.106 ± 8.35 and 201.61 ± 21.89% of normalized baseline values, respectively. The adenosine- and NECA-induced increase in coronary flow in A1AR−/− hearts was not altered by DPCPX. These data indicate that A1AR may inhibit or negatively modulate coronary flow mediated by other AR subtypes (A2A and A2B).

In the present study, we examined dynamic changes in cellular profile of bronchoalveolar lavage (BAL) fluid after adenosine challenge in ragweed sensitized and challenged mice. Mice systemically sensitized and airway challenged with ragweed showed marked airway inflammation manifesting increased eosinophils, lymphocytes, neutrophils and activated macrophages in BAL. Adenosine challenge further enhanced influx of inflammatory cells into BAL, notably neutrophils from 1 to 72 h and eosinophils from 1 to 48 h time-points (p

The purpose of this study was to explore the involvement of adenosine receptor(s) in porcine coronary artery (PCA) relaxation and to define the role of MAPK signaling pathways. Isometric tensions were recorded in denuded PCA rings. 5′-( N-ethylcarboxamido)adenosine (NECA), a nonselective adenosine receptor agonist, induced a concentration-dependent relaxation (EC50 = 16.8 nM) of PGF2α (10 μM)-preconstricted arterial rings. NECA-induced relaxation was completely blocked by 0.1 μM SCH-58261 (A2A antagonist) at lower doses (1–40 nM) but not at higher doses (80–1,000 nM). MRS-1706 (1 μM, A2B antagonist) was able to shift the NECA concentration-response curve to the right. CGS-21680 (selective A2A agonist) induced responses similarly to NECA, whereas N6-cyclopentyladenosine (A1 agonist) and Cl-IB-MECA (A3 agonist) did not. Furthermore, the effect of NECA was attenuated by the addition of SB-203580 (10 μM, p38 MAPK inhibitor) but not by PD-98059 (10 μM, MEK inhibitor). Interestingly, SB-203580 had no effect on CGS-21680-induced relaxation. Western blot analysis demonstrated that PGF2α and adenosine agonists stimulated p38 MAPK at a concentration of 40 nM in PCA smooth muscle cells. MRS-1706 (1 μM) significantly reduced NECA-induced p38 MAPK phosphorylation. Addition of NECA and SB-203580 alone or in combination inhibited PGF2α-induced p38 MAPK. Western blot data were further confirmed by p38 MAPK activity measurement using activating transcription factor-2 assay. Our results suggest that the adenosine receptor subtype involved in causing relaxation of porcine coronary smooth muscle is mainly A2A subtype, although A2B also may play a role, possibly through p38 MAPK pathway.

Previous reports from our laboratory have demonstrated that ethanol- and cannabinoid-induced ataxia is modulated by cerebellar adenosine A(1) receptor because intracerebellar (i.c.b.) adenosine A(1) agonists potentiated and A(1) antagonist attenuated ataxia by these psychoactive drugs. In this study, the novel approach involving pretreatment with adenosine A(1) antisense oligodeoxynucleotide via multiple routes provided further direct evidence of mouse cerebellar A(1) modulation of ethanol- and cannabinoid-induced ataxia. Animal groups were pretreated with A(1) antisense and its mismatch by oral (p.o.) (3.12, 6.25, 12.5, 50 microg/12 h; total three treatments/each dose), intraperitoneal (i.p.) (3.12, 5, 10, 50 microg/12 h; total three treatments/each dose), and i.c.b. (2 microg/12 h; total three treatments) routes. Based on our standard rotorod test, marked antagonism to ethanol (2 g/kg; i.p.) and delta(9)-THC (15 microg; i.c.b)-induced ataxia was observed 12 h after the last antisense treatment. Pretreatment with A(1) receptor mismatch was without an effect. The antagonism following systemic (p.o.; i.p.) antisense pretreatment was dose-dependent. No change in the normal motor coordination was observed when the animals were pretreated with antisense or its mismatch followed by vehicle. Results of Western blotting using commercially available antibodies and cerebellar membranes from various animal groups which received antisense and its mismatch via three routes confirmed a significant decrease in the A(1) adenosine receptor protein. These results, for the first time, demonstrated an oral and systemic effectiveness of A(1) antisense towards adenosine receptors in the central nervous system.

Using various pharmacological methods, we previously demonstrated that the smooth muscle and endothelium of porcine coronary artery contain vasorelaxant adenosine A2 receptors, which are predominantly the A2A subtype. The present study was intended to investigate the effect of adenosine receptor stimulation on agonist-induced inositol 1,4,5-trisphosphate (IP3) generation in porcine coronary artery using the nonselective adenosine analogs, 2-chloroadenosine (CAD) and 5'-(N-ethylcarboxamido)adenosine (NECA), and the A2A selective analog 2-p-(2-carboxyethyl)-phenethylamino-5'-N-ethylcarboxamidoadenosine (CGS). In both endothelium-intact and denuded coronary artery rings, CAD, NECA and CGS elicited a dose-dependent inhibition of prostaglandin F2 alpha (PG)-induced IP3 production. However, the inhibitory effect of NECA was relatively less in endothelium-denuded preparations. The nonselective xanthine adenosine receptor antagonist, 8-sulfophenyltheophylline (8-SPT), significantly attenuated the IP3-inhibitory effect of CAD and, to a lesser extent, that of NECA. Further, the A2A selective nonxanthine antagonist, 5-amino-7-(2-phenylethyl)-2-(furyl)-pyrazolo[4,3]-1,2,4-triazolo[1,5-c] pyrimidine (SCH), markedly decreased the effects of all CAD, NECA and CGS on PG-induced IP3 generation. These results provide evidence that activation of adenosine A2 receptors by CAD, NECA and CGS in porcine coronary artery causes inhibition of agonist-induced IP3 production, and these receptors involve at least the A2A subtype.

To determine whether adenosine A3 receptors participate in adenosine-induced changes in coronary flow, isolated hearts from wild-type (WT) and A3 receptor knockout (A3KO) mice were perfused under constant pressure and effects of nonselective and selective agonists were examined. Adenosine and the selective A2A agonist 2-[ p-(2-carboxyethyl)]phenylethylamino-5′- N-ethylcarboxamidoadenosine (CGS-21680) produced augmented maximal coronary vasodilation in A3KO hearts compared with WT hearts. Selective activation of A3 receptors with 2-chloro- N 6-(3-iodobenzyl)-adenosine-5′- N-methyluronamide (Cl-IB-MECA) at nanomolar concentrations did not effect coronary flow, but at higher concentrations it produced coronary vasodilation both in WT and A3KO hearts. Cl-IB-MECA-induced increases in coronary flow were susceptible to both pharmacological blockade and genetic deletion of A2A receptors. Because deletion or blockade of adenosine A3 receptors augmented coronary flow induced by nonselective adenosine and the selective A2A receptor agonist CGS-21680, we speculate that this is due to removal of an inhibitory influence associated with the A3 receptor subtype. These data indicate that the presence of adenosine A3 receptors may either inhibit or negatively modulate coronary flow mediated by other adenosine receptor subtypes.

In this study, we studied the role of adenosine on airway responsiveness and airway inflammation using an allergic mouse model. Mice were sensitized by two i.p. injections of ragweed and three consecutive ragweed aerosol challenges. It was found that inhalation of adenosine causes a dose-related bronchoconstriction in this model. Ragweed sensitized and challenged mice showed increased sensitivity to airway challenge to adenosine compared to control animals. Theophylline, a non-selective adenosine receptor antagonist, blocked adenosine-induced bronchoconstriction, but was unable to inhibit bronchoconstrictor response to methacholine. Mice systemically sensitized and airway challenged with allergen showed a marked airway inflammation manifesting increases in eosinophils, lymphocytes and neutrophils, and decrease in macrophages. Twenty-four hours after airway challenge with allergen, aerosolization of adenosine further potentiated the allergen-induced airway inflammation. Cells in bronchoalveolar lavage fluid after adenosine aerosolization increased by 3.07-fold as compared to control mice, and by 1.8-fold compared to ragweed sensitized and challenged mice. The increases in eosinophils, lymphocytes, and neutrophils caused by allergen were potentiated after adenosine challenge. Unexpectedly, macrophages significantly decreased after adenosine challenge. Theophylline attenuated adenosine-enhanced airway inflammation, but could not reverse allergen-induced airway inflammation. These findings suggested that specific adenosine receptors contribute to airway responsiveness and airway inflammation associated with this model of allergic asthma.

The present study was designed to characterize and compare the vascular effects of adenosine and its analogs in the murine heart and aorta. Mouse hearts perfused under constant pressure in standard Langendorff fashion demonstrated concentration-dependent increases in coronary flow to adenosine, 2-chloradenosine (CAD), 5′-( N-ethyl-carboxamido)-adenosine (NECA), and 2- p-(2-carboxyethyl)phenethylamino-5′- N-ethylcarboxam-idoadenosine (CGS-21680). All agonists produced comparable increases in coronary flow with the following order of potency: CGS-21680 = NECA ≫ CAD ≥ adenosine. In l-phenylephrine hydrochloride (phenylephrine) precontracted aortic rings, all nonselective agonists (NECA, CAD, and adenosine) produced marked concentration-dependent relaxation, whereas the adenosine A2A selective agonist CGS-21680 did not. Adenosine receptor agonists were >100 times more potent for coronary vasodilation than aortic vasorelaxation. The selective A2A receptor antagonist 5-amino-7-(β-phenylethyl)-2-(8-furyl)pyrazolo-[4,3-e]-1,2,4-triazolo-[1,5-c]pyrimidine (SCH-58261) blocked both CGS-21680- and NECA-induced increases in coronary flow, whereas the A2B receptor antagonist benzo[g]pteridine-2,4(1H,3H)-dione (alloxazine) inhibited NECA-induced aortic relaxation. These data indicate a differential response to adenosine agonists in murine coronary vasculature and aorta where coronary vasodilation is mediated predominantly by activation of A2A adenosine receptors.