Divergent coronary flow responses to uridine adenosine tetraphosphate in atherosclerotic ApoE knockout mice.

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.

Transcriptomic effects of adenosine 2A receptor deletion in healthy and endotoxemic murine myocardium.

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.

Enhanced A2A adenosine receptor-mediated increase in coronary flow in type I diabetic mice.

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.

Sex Difference in Coronary Endothelial Dysfunction in Apolipoprotein E Knockout Mouse: Role of NO and A2A Adenosine Receptor.

OBJECTIVE: Sex plays an important role in the pathophysiology of cardiovascular diseases. This study aims to investigate how sex impacts on the coronary flow regulation during atherosclerosis. METHODS: ApoE KO mouse fed with western diet were used for atherosclerosis model. Coronary RH and flow response were measured using Langendorff-perfused isolated hearts. RESULTS: Coronary RH and A23187-induced NO-dependent flow increases were significantly reduced in female (by ~28% and 48%, respectively), but not in male atherosclerotic mice. However, SNP-induced coronary vasodilation remains intact in both sexes of ApoE KO mice. L-NAME (NOS inhibitor) reduced baseline flow and RH to a lesser extent in ApoE KO (by ~19% and 31%) vs. WT (~30% and 59%, respectively), and abolished the sex difference in RH. In contrast, SCH58261 (a selective A2A AR antagonist) reduced the baseline flow and RH to a greater extent in atherosclerotic mice, but did not affect the sex difference. Immunofluorescent staining of coronary arteries showed a similar A2A AR upregulation in both sexes of ApoE KO mice. CONCLUSIONS: Our results suggest that during atherosclerosis, female mice are more susceptible to NO-dependent endothelial dysfunction and the upregulation of A2A AR may serve as a compensatory mechanism to counteract the compromised endothelial function.

Involvement of NADPH oxidase in A2A adenosine receptor-mediated increase in coronary flow in isolated mouse hearts.

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.

Metabolic hyperemia requires ATP-sensitive K+ channels and H2O2 but not adenosine in isolated mouse hearts.

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.

A1 adenosine receptor negatively modulates coronary reactive hyperemia via counteracting A2A-mediated H2O2 production and KATP opening in isolated mouse hearts.

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.

Interactions between A(2A) adenosine receptors, hydrogen peroxide, and KATP channels in coronary reactive hyperemia.

Myocardial metabolites such as adenosine mediate reactive hyperemia, in part, by activating ATP-dependent K(+) (K(ATP)) channels in coronary smooth muscle. In this study, we investigated the role of adenosine A(2A) and A(2B) receptors and their signaling mechanisms in reactive hyperemia. We hypothesized that coronary reactive hyperemia involves A(2A) receptors, hydrogen peroxide (H(2)O(2)), and KATP channels. We used A(2A) and A(2B) knockout (KO) and A(2A/2B) double KO (DKO) mouse hearts for Langendorff experiments. Flow debt for a 15-s occlusion was repaid 128 ± 8% in hearts from wild-type (WT) mice; this was reduced in hearts from A(2A) KO and A(2A)/(2B) DKO mice (98 ± 9 and 105 ± 6%; P < 0.05), but not A(2B) KO mice (123 ± 13%). Patch-clamp experiments demonstrated that adenosine activated glibenclamide-sensitive KATP current in smooth muscle cells from WT and A(2B) KO mice (90 ± 23% of WT) but not A(2A) KO or A(2A)/A(2B) DKO mice (30 ± 4 and 35 ± 8% of WT; P < 0.05). Additionally, H(2)O(2) activated KATP current in smooth muscle cells (358 ± 99%; P < 0.05). Catalase, an enzyme that breaks down H(2)O(2), attenuated adenosine-induced coronary vasodilation, reducing the percent increase in flow from 284 ± 53 to 89 ± 13% (P < 0.05). Catalase reduced the repayment of flow debt in hearts from WT mice (84 ± 9%; P < 0.05) but had no effect on the already diminished repayment in hearts from A(2A) KO mice (98 ± 7%). Our findings suggest that adenosine A(2A) receptors are coupled to smooth muscle KATP channels in reactive hyperemia via the production of H(2)O(2) as a signaling intermediate.

Functional and RNA expression profile of adenosine receptor subtypes in mouse mesenteric arteries.

Concentration-response curves (CRCs) of adenosine receptor (AR) agonists, NECA (nonspecific), CCPA (A1 specific), CGS-216870 (A2A specific), BAY 60-6583 (A2B specific), and Cl-IB-MECA (A3 specific) for mesenteric arteries (MAs) from 4 AR knockout (KO) mice (A1, A2A, A2B, and A3) and their wild type (WT) were constructed. The messenger RNA expression of MAs from KO mice and WT were also studied. Adenosine (10 to 10 M) and NECA (10 to 10 M) induced relaxation in all mice except A2B KO mice, which only showed constriction by adenosine at 10 to 10 and NECA at 10 to 10 M. The CCPA induced a significant constriction at 10 and 10 M in all mice, except A1KO. BAY 60-6583 induced relaxation (10 to 10 M) in WT and no response in A2BKO except at 10 M. The CRCs for BAY 60-6583 in A1, A2A, and A3 KO mice shifted to the left when compared with WT mice, suggesting an upregulation of A2B AR. No responses were noted to CGS-21680 in all mice. Cl-IB-MECA only induced relaxation at concentration greater than 10 M, and no differences were found between different KO mice. The CRC for Bay 60-6583 was not significantly changed in the presence of 10 M of L-NAME, 10 M of indomethacin, or both. Our data suggest that A2B AR is the predominant AR subtype and the effect may be endothelial independent, whereas A1 AR plays a significant modulatory role in mouse MAs.

Dynamic detection and reversal of myocardial ischemia using an artificially intelligent bioelectronic medicine.

Myocardial ischemia is spontaneous, frequently asymptomatic, and contributes to fatal cardiovascular consequences. Importantly, myocardial sensory networks cannot reliably detect and correct myocardial ischemia on their own. Here, we demonstrate an artificially intelligent and responsive bioelectronic medicine, where an artificial neural network (ANN) supplements myocardial sensory networks, enabling reliable detection and correction of myocardial ischemia. ANNs were first trained to decode spontaneous cardiovascular stress and myocardial ischemia with an overall accuracy of ~92%. ANN-controlled vagus nerve stimulation (VNS) significantly mitigated major physiological features of myocardial ischemia, including ST depression and arrhythmias. In contrast, open-loop VNS or ANN-controlled VNS following a caudal vagotomy essentially failed to reverse cardiovascular pathophysiology. Last, variants of ANNs were used to meet clinically relevant needs, including interpretable visualizations and unsupervised detection of emerging cardiovascular stress. Overall, these preclinical results suggest that ANNs can potentially supplement deficient myocardial sensory networks via an artificially intelligent bioelectronic medicine system.

Contributions of A2A and A2B adenosine receptors in coronary flow responses in relation to the KATP channel using A2B and A2A/2B double-knockout mice.

Adenosine plays a role in physiological and pathological conditions, and A(2) adenosine receptor (AR) expression is modified in many cardiovascular disorders. In this study, we elucidated the role of the A(2B)AR and its relationship to the A(2A)AR in coronary flow (CF) changes using A(2B) single-knockout (KO) and A(2A/2B) double-KO (DKO) mice in a Langendorff setup. We used two approaches: 1) selective and nonselective AR agonists and antagonists and 2) A(2A)KO and A(2B)KO and A(2A/2B)DKO mice. BAY 60-6583 (a selective A(2B) agonist) had no effect on CF in A(2B)KO mice, whereas it significantly increased CF in wild-type (WT) mice (maximum of 23.3 ± 9 ml·min(-1)·g(-1)). 5'-N-ethylcarboxamido adenosine (NECA; a nonselective AR agonist) increased CF in A(2B)KO mice (maximum of 34.6 ± 4.7 ml·min(-1)·g(-1)) to a significantly higher degree compared with WT mice (maximum of 23.1 ± 2.1 ml·min(-1)·g(-1)). Also, CGS-21680 (a selective A(2A) agonist) increased CF in A(2B)KO mice (maximum of 29 ± 1.9 ml·min(-1)·g(-1)) to a significantly higher degree compared with WT mice (maximum of 25.1 ± 2.3 ml·min(-1)·g(-1)). SCH-58261 (an A(2A)-selective antagonist) inhibited the NECA-induced increase in CF to a significantly higher degree in A(2B)KO mice (19.3 ± 1.6 vs. 0.5 ± 0.4 ml·min(-1)·g(-1)) compared with WT mice (19 ± 3.5 vs. 3.6 ± 0.5 ml·min(-1)·g(-1)). NECA did not induce any increase in CF in A(2A/2B)DKO mice, whereas a significant increase was observed in WT mice (maximum of 23.1 ± 2.1 ml·min(-1)·g(-1)). Furthermore, the mitochondrial ATP-sensitive K(+) (K(ATP)) channel blocker 5-hydroxydecanoate had no effect on the NECA-induced increase in CF in WT mice, whereas the NECA-induced increase in CF in WT (17.6 ± 2 ml·min(-1)·g(-1)), A(2A)KO (12.5 ± 2.3 ml·min(-1)·g(-1)), and A(2B)KO (16.2 ± 0.8 ml·min(-1)·g(-1)) mice was significantly blunted by the K(ATP) channel blocker glibenclamide (to 0.7 ± 0.7, 2.3 ± 1.1, and 0.9 ± 0.4 ml·min(-1)·g(-1), respectively). Also, the CGS-21680-induced (22 ± 2.3 ml·min(-1)·g(-1)) and BAY 60-6583-induced (16.4 ± 1.60 ml·min(-1)·g(-1)) increase in CF in WT mice was significantly blunted by glibenclamide (to 1.2 ± 0.4 and 1.8 ± 1.2 ml·min(-1)·g(-1), respectively). In conclusion, this is the first evidence supporting the compensatory upregulation of A(2A)ARs in A(2B)KO mice and demonstrates that both A(2A)ARs and A(2B)ARs induce CF changes through K(ATP) channels. These results identify AR-mediated CF responses that may lead to better therapeutic approaches for the treatment of cardiovascular disorders.

Aging and estrogen alter endothelial reactivity to reactive oxygen species in coronary arterioles.

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 O(2)(-) and H(2)O(2) in 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 H(2)O(2) production 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 H(2)O(2) scavenger catalase (100 U/ml). Removal of H(2)O(2) with 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 O(2)(-) and H(2)O(2) are critical components of flow-induced vasodilation in coronary arterioles from female rats; however, a chronic deficiency of O(2)(-) buffering by SOD contributes to impaired flow-induced dilation with aging and loss of estrogen. Furthermore, these data indicate that estrogen replacement restores O(2)(-) homeostasis and flow-induced dilation of coronary arterioles, even at an advanced age.

A(1) adenosine receptor-mediated PKC and p42/p44 MAPK signaling in mouse coronary artery smooth muscle cells.

The A(1) adenosine receptor (A(1)AR) is coupled to G(i)/G(o) 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 [A(1)AR wild type (A(1)WT) and A(1)AR knockout (A(1)KO)] to delineate A(1)AR signaling through the PKC pathway. In A(1)WT cells, treatment with (2S)-N(6)-(2-endo-norbornyl)adenosine (ENBA; 10(-5)M) increased A(1)AR expression by 150%, which was inhibited significantly by the A(1)AR antagonist 1,3-dipropyl-8-cyclopentylxanthine (10(-6)M), but not in A(1)KO CASMCs. PKC isoforms were identified by Western blot analysis in the cytosolic and membrane fractions of cell homogenates of CASMCs. In A(1)WT and A(1)KO cells, significant levels of basal PKC-alpha were detected in the cytosolic fraction. Treatment with the A(1)AR agonist ENBA (10(-5)M) translocated PKC-alpha from the cytosolic to membrane fraction significantly in A(1)WT but not A(1)KO cells. Phospholipase C isoforms (betaI, betaIII, and gamma(1)) were analyzed using specific antibodies where ENBA treatment led to the increased expression of PLC-betaIII in A(1)WT CASMCs while having no effect in A(1)KO CASMCs. In A(1)WT cells, ENBA increased PKC-alpha expression and p42/p44 MAPK (ERK1/2) phospohorylation by 135% and 145%, respectively. These effects of ENBA were blocked by Gö-6976 (PKC-alpha inhibitor) and PD-98059 (p42/p44 MAPK inhibitor). We conclude that A(1)AR stimulation by ENBA activates the PKC-alpha signaling pathway, leading to p42/p44 MAPK phosphorylation in CASMCs.

Adenosine receptors and the heart: role in regulation of coronary blood flow and cardiac electrophysiology.

Adenosine is an autacoid that plays a critical role in regulating cardiac function, including heart rate, contractility, and coronary flow. In this chapter, current knowledge of the functions and mechanisms of action of coronary flow regulation and electrophysiology will be discussed. Currently, there are four known adenosine receptor (AR) subtypes, namely A(1), A(2A), A(2B), and A(3). All four subtypes are known to regulate coronary flow. In general, A(2A)AR is the predominant receptor subtype responsible for coronary blood flow regulation, which dilates coronary arteries in both an endothelial-dependent and -independent manner. The roles of other ARs and their mechanisms of action will also be discussed. The increasing popularity of gene-modified models with targeted deletion or overexpression of a single AR subtype has helped to elucidate the roles of each receptor subtype. Combining pharmacologic tools with targeted gene deletion of individual AR subtypes has proven invaluable for discriminating the vascular effects unique to the activation of each AR subtype. Adenosine exerts its cardiac electrophysiologic effects mainly through the activation of A(1)AR. This receptor mediates direct as well as indirect effects of adenosine (i.e., anti-beta-adrenergic effects). In supraventricular tissues (atrial myocytes, sinuatrial node and atriovetricular node), adenosine exerts both direct and indirect effects, while it exerts only indirect effects in the ventricle. Adenosine exerts a negative chronotropic effect by suppressing the automaticity of cardiac pacemakers, and a negative dromotropic effect through inhibition of AV-nodal conduction. These effects of adenosine constitute the rationale for its use as a diagnostic and therapeutic agent. In recent years, efforts have been made to develop A(1)R-selective agonists as drug candidates that do not induce vasodilation, which is considered an undesirable effect in the clinical setting.

Enhanced A1 adenosine receptor-induced vascular contractions in mesenteric artery and aorta of in L-NAME mouse model of hypertension.

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.

Up-regulation of A 2B adenosine receptor in A 2A adenosine receptor knockout mouse coronary artery.

In this study, we looked into possible compensatory changes of other adenosine receptors (ARs) in A(2A) genetic knockout mice (A2AKO) as well as the functional role of nitric oxide (NO) in A(2A) AR-mediated vasodilation. Gene expression of ARs from coronary arteries of A(2A) AR wild type mice (A2AWT) and A2AKO was studied using real-time PCR. Functional studies were carried out in isolated heart and isolated coronary artery preparations. A(2B) AR was found to be 4.5 fold higher in A2AKO than in A2AWT, while A(2A) AR expression was absent in A2AKO. There was no difference in A(1) and A(3) ARs between WT and KO animals. The concentration-relaxation curve for adenosine-5'-N-ethylcarboxamide (NECA, non-selective AR agonist) in isolated coronary arterial rings in A2AKO was shifted to the left when compared to A2AWT. The concentration-response curve for A(2B) selective agonist (BAY 60-6583) was also shifted to the left in A2AKO hearts. L-NAME, a non-specific NO synthase inhibitor, did not affect baseline coronary flow (CF) until the concentration reached 10 microM in A2AWT (76.32+/-11.35% from baseline, n=5). In A2AKO, the CF decreased significantly by L-NAME only at a higher concentration (100 microM, 93.32+/-5.8% from baseline, n=5). L-NMA (1 microM, n=4), another non-specific NO synthase inhibitor, also demonstrated similar results in decreasing CF (59.66+/-3.23% from baseline in A2AWT, while 81.76+/-8.91% in A2AKO). It was further demonstrated that the increase in CF by 100 microM NECA was significantly blunted with 10 microM L-NAME (377.08+/-25.23% to 305.41+/-30.73%, n=9) in A2AWT but not in A2AKO (153.66+/-22.7% to 143.88+/-36.65%, n=5). Similar results were also found using 50 nM of CGS-21680 instead of NECA in A2AWT (346+/-22.85 to 277+/-31.39, n=6). No change in CF to CGS-21680 was noted in A(2A)AKO. Our data demonstrate, for the first time, that coronary A(2B) AR was up-regulated in mice deficient in A(2A) AR. We also provide direct evidence supporting a role for NO in A(2A) AR-mediated coronary vasodilation. The data further support the role for A(2A) AR in the regulation of basal coronary tone through the release of NO.

Functional changes in vascular reactivity to adenosine receptor activation in type I diabetic mice.

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.

Impaired Aortic Contractility to Uridine Adenosine Tetraphosphate in Angiotensin II-Induced Hypertensive Mice: Receptor Desensitization?

OBJECTIVE: We previously showed that uridine adenosine tetraphosphate (Up4A)-mediated aortic contraction is partly mediated through purinergic P2X1 receptors (P2X1R). It has been reported that the plasma level of Up4A is elevated in hypertensive patients, implying a potential role for Up4A-P2X1R signaling in hypertension. This study investigated the vasoactive effect of Up4A in aortas isolated from angiotensin (Ang) II-infused (21 days) hypertensive mice. METHODS: Blood pressure was measured by tail cuff plethysmography. Aortas were isolated for isometric tension measurements, and protein expression was analyzed by western blot. RESULTS: Mean and systolic arterial pressures were elevated by ~50% in Ang II-infused mice. Protein levels of both AT1R and P2X1R were upregulated in Ang II-infused aortas. Surprisingly, Up4A (10-9-10-5 M)-induced concentration-dependent contraction was significantly impaired in Ang II-infused mice. Studies in control mice revealed that both P2X1R (MRS2159) and AT1R (losartan) antagonists significantly attenuated Up4A-induced aortic contraction. In addition, desensitization of AT1R by prior Ang II (100 nM) exposure had no effect on Up4A-induced aortic contraction. However, subsequent serial exposure responses to Up4A-induced aortic contraction were markedly reduced, suggesting a desensitization of purinergic receptors. This desensitization was further confirmed in control mice by prior exposure of aortas to the P2X1R desensitizer α, ß-methylene ATP (10 µM). CONCLUSION: Despite upregulation of AT1R and P2X1R in hypertension, Up4A-mediated aortic contraction was impaired in Ang II-infused mice, likely through the desensitization of P2X1R but not AT1R. This implies that vascular P2X1R activity, rather than plasma Up4A level, may determine the role of Up4A in hypertension.

In vivo assessment of coronary flow and cardiac function after bolus adenosine injection in adenosine receptor knockout mice.

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.

Effects of xylazine on canine coronary artery vascular rings.

OBJECTIVE: To determine the effects of xylazine on canine coronary artery smooth muscle tone. SAMPLE POPULATION: Hearts of 26 healthy dogs. PROCEDURE: Dogs were anesthetized with pentobarbital, and vascular rings of various diameters were prepared from the epicardial coronary arteries. Vascular rings were placed in tissue baths to which xylazine was added (cumulative concentrations ranging from 10(-10) to 10(-4) M), and changes in vascular ring tension were continuously recorded. Effects of the nitric oxide inhibitor NG-nitro-L-arginine methyl ester (L NAME; 5 mM), the alpha1-adrenoceptor antagonist prazosin (10 mM), and the alpha2-adrenoceptor antagonist atipamezole (10 mM) on xylazine-induced changes in vascular ring tension were determined. Results were expressed as percentage of maximal contraction for each vascular ring preparation. RESULTS: Xylazine induced vasoconstriction of small (< 500-microm-diameter) and medium (500- to 1,000-microm-diameter) vascular rings but not of large (> 1,000-microm-diameter) rings. For large vascular rings, L-NAME, atipamezole, and prazosin did not significantly affect the contractile response to xylazine. For small vascular rings, the contractile response following addition of xylazine to rings treated with L-NAME was not significantly different from the contractile response following addition of xylazine to control rings, except at a xylazine concentration of 10(-6) M. Xylazine-induced vasoconstriction of small vascular rings was blocked by atipamezole, but the addition of prazosin had no effect on xylazine-induced vasoconstriction. CONCLUSIONS AND CLINICAL RELEVANCE: Results suggest that xylazine increases smooth muscle tone of small canine coronary arteriesand that this effect is predominantly mediated by stimulation of alpha2adrenoceptors.