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Adenosine is a ubiquitous endogenous nucleoside or autacoid that affects the cardiovascular system through the activation of four G-protein coupled receptors: adenosine A

Previously, we have reported that the coronary reactive hyperemic response was reduced in adenosine A

Globally, cardiovascular diseases (CVDs) are the number one cause of mortality. Approximately 18 million people died from CVDs in 2015, representing more than 30% of all global deaths. New diagnostic tools and therapies are eagerly required to decrease the prevalence of CVDs related to mortality and/or risk factors leading to CVDs. Oxylipins are a group of metabolites, generated via oxygenation of polyunsaturated fatty acids that are involved in inflammation, immunity, and vascular functions, etc. Thus far, over 100 oxylipins have been identified, and have overlapping and interconnected roles. Important CVD pathologies such as hyperlipidemia, hypertension, thrombosis, hemostasis and diabetes have been linked to abnormal oxylipin signaling. Oxylipins represent a new era of risk markers and/or therapeutic targets in several diseases including CVDs. The role of many oxylipins in the progression or regression in CVD, however, is still not fully understood. An increased knowledge of the role of these oxygenated polyunsaturated fatty acids in cardiovascular dysfunctions or CVDs including hypertension could possibly lead to the development of biomarkers for the detection and their treatment in the future.

Previously, we showed that adenosine A(2A) receptor-induces relaxation independent of NO in soluble epoxide hydrolase-null mice (Am J Physiol Regul Integr Comp Physiol 304: R23–R32, 2013). Currently, we hypothesize that Ephx2-gene deletion affects acetylcholine (Ach)-induced relaxation which is independent of A(2A)AR but dependent on NO and CYP-epoxygenases. Ephx2(−/−) aortas showed a lack of sEH (97.1%, p0.05). Ach-induced response was tested with nitro-l-arginine methyl ester (l-NAME) NO-inhibitor; 10(−4) M, N-(methylsulfonyl)-2-(2-propynyloxy)-benzenehexanamide (MS-PPOH) (CYP-epoxygenase inhibitor; 10(−5) M), 14,15-epoxyeicosa-5(Z)-enoic acid (14,15-EEZE, an epoxyeicosatrienoic acid-antagonist; 10(−5) M), SCH-58261 (A(2A)AR-antagonist; 10(−6) M) and angiotensin-II (Ang-II, 10(−6) M). In Ephx2(−/−) mice, Ach-induced relaxation was not different from C57Bl/6 mice except at 10(−5) M (92.75 ± 2.41 vs. 76.12 ± 3.34, P0.05) vs. non-treated (P>0.05). Interestingly, Ang-II attenuates less relaxation in Ehx2(−/−) vs. C57Bl/6 mice (58.80 ± 7.81% vs. 45.92 ± 7.76, P

The signaling nucleoside adenosine is produced intra- and extracellularly under physiologic and, more importantly, under pathologic conditions. Adenosine modulates cellular functions involved in injury, metabolic derangement, energy perturbations, and inflammation. The biologic effects of adenosine are mediated by four adenosine receptor (AR) subtypes of the G-protein coupled receptors (GPCRs) family: A1AR, A2AAR, A2BAR and A3AR. In the cardiovascular (CV) system, adenosine and its receptors are intricately involved in the regulation of myocardial contraction, heart rate, sympathetic control, conductivity, vascular tone, cardiac and vascular growth, inflammation, injury and apoptosis. As such, the modulation of the adenosinergic system has therapeutic potential for cardiovascular diseases (CVDs) such as metabolic disorders, atherosclerosis, hypertrophy, ischemic heart diseases, and heart failure. Nevertheless, despite the many years of investigation and experimentation only a few drugs targeting the adenosinergic system were developed and actually have reached clinical application. This chapter outlines the unique role adenosine plays in the CV system in physiology, pathology, and potentially therapeutic pharmacology. It also presents an updated review of the different adenosine receptors ligands, and their clinical potential in different CVDs.

Adenosine is an endogenous mediator involved in a myriad of physiologic functions, including vascular tone regulation. It is also implicated in some pathologic conditions. Four distinct receptor subtypes mediate the effects of adenosine, such as its role in the regulation of the vascular tone. Vascular tone regulation is a complex and continuous process which involves many mechanisms and mediators that are not fully disclosed. The vascular endothelium plays a pivotal role in regulating blood flow to and from all body organs. Also, the vascular endothelium is not merely a physical barrier; it is a complex tissue with numerous functions. Among adenosine receptors,A2Areceptor subtype (A2AAR) stands out as the primary receptor responsible for the vasodilatory effects of adenosine. This review focuses on important effectors of the vascular endothelium, including adenosine, adenosine receptors, EETs (epoxyeicosatrienoic acids), HETEs (hydroxyeicosatetraenoic acids), PPARs (peroxisome proliferator-activated receptors), andKATPchannels. Given the impact of vascular tone regulation in cardiovascular physiology and pathophysiology, better understanding of the mechanisms affecting it could have a significant potential for developing therapeutic agents for cardiovascular diseases.

The relationship between soluble epoxide hydrolase (sEH) and coronary reactive hyperemia (CRH) response to a brief ischemic insult is not known. Epoxyeicosatrienoic acids (EETs) exert cardioprotective effects in ischemia/reperfusion injury. sEH converts EETs into dihydroxyeicosatrienoic-acids (DHETs). Therefore, we hypothesized that knocking out sEH enhances CRH through modulation of oxylipin profiles, including an increase in EET/DHET ratio. Compared with sEH+/+, sEH−/− mice showed enhanced CRH, including greater repayment volume (RV; 28% higher, P < 0.001) and repayment/debt ratio (32% higher, P < 0.001). Oxylipins from the heart perfusates were analyzed by LC-MS/MS. The 14,15-EET/14,15-DHET ratio was 3.7-fold higher at baseline ( P < 0.001) and 5.6-fold higher post-ischemia ( P < 0.001) in sEH−/− compared with sEH+/+ mice. Likewise, the baseline 9,10- and 12,13-EpOME/DiHOME ratios were 3.2-fold ( P < 0.01) and 3.7-fold ( P < 0.001) higher, respectively in sEH−/− compared with sEH+/+ mice. 13-HODE was also significantly increased at baseline by 71% ( P < 0.01) in sEH−/− vs. sEH+/+ mice. Levels of 5-, 11-, 12-, and 15-hydroxyeicosatetraenoic acids were not significantly different between the two strains ( P > 0.05), but were decreased postischemia in both groups ( P = 0.02, P = 0.04, P = 0.05, P = 0.03, respectively). Modulation of CRH by peroxisome proliferator-activated receptor gamma (PPARγ) was demonstrated using a PPARγ-antagonist (T0070907), which reduced repayment volume by 25% in sEH+/+ ( P < 0.001) and 33% in sEH−/− mice ( P < 0.01), and a PPARγ-agonist (rosiglitazone), which increased repayment volume by 37% in both sEH+/+ ( P = 0.04) and sEH−/− mice ( P = 0.04). l-NAME attenuated CRH in both sEH−/− and sEH+/+. These data demonstrate that genetic deletion of sEH resulted in an altered oxylipin profile, which may have led to an enhanced CRH response.

Soluble epoxide hydrolase (sEH) converts epoxyeicosatrienoic acids that are endothelium-derived hyperpolarizing factors into less active dihydroxyeicosatrienoic acids. Previously, we reported a decrease in adenosine A1 receptor (A1AR) protein levels in sEH knockout (sEH−/−) and an increase in sEH and A1AR protein levels in A2AAR−/− mice. Additionally, KATP channels are involved in adenosine receptor (AR)-dependent vascular relaxation. Thus, we hypothesize that a potential relationship may exist among sEH overexpression, A1AR up-regulation, inactivation of KATP channels and increased in vascular tone. We performed DMT myograph muscle tension measurements and western blot analysis in isolated mouse mesenteric arteries (MAs) from wild type (WT) and endothelial over-expression of sEH (Tie2-sEH Tr) mice. Our data revealed that NECA (a non-selective adenosine receptors agonist)-induced relaxation was significantly reduced in Tie2-sEH Tr mice, and CCPA (A1AR agonist)-induced contraction was increased in Tie2-sEH Tr mice. A1AR-dependent contraction in Tie2-sEH Tr mice was significantly attenuated by pharmacological inhibition of CYP4A (HET0016, 10 μM), PKCα (GO6976, 1 μM), and ERK1/2 (PD58059, 1 μM). Our western blot analysis revealed significantly higher basal protein expression of CYP4A, A1AR, and reduced p-ERK in MAs of Tie2-sEH Tr mice. Notably, pinacidil (KATP channel opener)-induced relaxation was also significantly reduced in MAs of Tie2-sEH Tr mice. Furthermore, KATP channel-dependent relaxation in MAs was enhanced by inhibition of PKCα and ERK1/2 in WT but not Tie2-sEH Tr mice. In conclusion, our data suggests that over-expression of sEH enhances A1AR-dependent contraction and reduces KATP channel-dependent relaxation in MAs. These results suggest a possible interaction between sEH, A1AR, and KATP channels in regulating vascular tone.

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.

Coronary reactive hyperemia (CRH) protects the heart against ischemia. Adenosine A2AAR-deficient (A2AAR-/-) mice have increased expression of soluble epoxide hydrolase (sEH); the enzyme responsible for breaking down the cardioprotective epoxyeicosatrienoic acids (EETs) to dihydroxyeicosatrienoic acids (DHETs). sEH-inhibition enhances CRH, increases EETs, and modulates oxylipin profiles. We investigated the changes of oxylipins and their impact on CRH in A2AAR-/- and wild type (WT) mice. We hypothesized that the attenuated CRH in A2AAR-/- mice is mediated by changes in oxylipin profiles, and that it can be reversed by either sEH- or ω-hydroxylases-inhibition. Compared to WT mice, A2AAR-/- mice had attenuated CRH and changed oxylipin profiles, which were consistent between plasma and heart perfusate samples, including decreased EET/DHET ratios, and increased hydroxyeicosatetraenoic acids (HETEs). Plasma oxylipns in A2AAR-/- mice indicated an increased proinflammatory state including increased ω-terminal HETEs, decreased epoxyoctadecaenoic/dihydroxyoctadecaenoic acids (EpOMEs/DiHOMEs) ratios, increased 9-hydroxyoctadecadienoic acid, and increased prostanoids. Inhibition of either sEH or ω-hydroxylases reversed the reduced CRH in A2AAR-/- mice. In WT and sEH-/- mice, blocking A2AAR decreased CRH. These data demonstrate that A2AAR-deletion was associated with changes in oxylipin profiles, which may contribute to the attenuated CRH. Also, inhibition of sEH and ω-hydroxylases reversed the reduction in CRH.

Cardiovascular diseases remain the number one diseases affecting patients' morbidity and mortality. The adenosine receptors are G-protein coupled receptors which have been of interest for drugs target for the treatment of multiple diseases ranging from cardiovascular to neurological. Adenosine receptors have been connected to several biological pathways affecting the physiology and pathology of the cardiovascular system. In this review, we will cover the different adenosine receptor ligands that have been identified to interact with adenosine receptors and affect the vascular system. These ligands will be evaluated from clinical as well as medicinal chemistry perspectives with more emphasis on how structural changes in structure translate into ligand potency and efficacy. Adenosine receptors represent a novel therapeutic target for development of treatment options treating a wide variety of diseases, including vascular disease and obesity.

High salt (4% NaCl, HS) diet modulates adenosine-induced vascular response through adenosine A(2A) receptor (A(2A)AR). Evidence suggests that A(2A)AR stimulates cyp450-epoxygenases, leading to epoxyeicosatrienoic acids (EETs) generation. The aim of this study was to understand the vascular reactivity to HS and underlying signaling mechanism in the presence or absence of A(2A)AR. Therefore, we hypothesized that HS enhances adenosine-induced relaxation through EETs in A(2A)AR⁺/⁺, but exaggerates contraction in A(2A)AR⁻/⁻. Organ bath and Western blot experiments were conducted in HS and normal salt (NS, 0.18% NaCl)-fed A(2A)AR⁺/⁺ and A(2A)AR⁻/⁻ mice aorta. HS produced concentration-dependent relaxation to non-selective adenosine analog, NECA in A(2A)AR⁺/⁺, whereas contraction was observed in A(2A)AR⁻/⁻ mice and this was attenuated by A₁AR antagonist (DPCPX). CGS 21680 (selective A(2A)AR agonist) enhanced relaxation in HS-A(2A)AR⁺/⁺ versus NS-A(2A)AR⁺/⁺, which was blocked by EETs antagonist (14,15-EEZE). Compared with NS, HS significantly upregulated the expression of vasodilators A(2A)AR and cyp2c29, whereas vasoconstrictors A₁AR and cyp4a in A(2A)AR⁺/⁺ were downregulated. In A(2A)AR⁻/⁻ mice, however, HS significantly downregulated the expression of cyp2c29, whereas A₁AR and cyp4a were upregulated compared with A(2A)AR⁺/⁺ mice. Hence, our data suggest that in A(2A)AR⁺/⁺, HS enhances A(2A)AR-induced relaxation through increased cyp-expoxygenases-derived EETs and decreased A₁AR levels, whereas in A(2A)AR⁻/⁻, HS exaggerates contraction through decreased cyp-epoxygenases and increased A₁AR levels.

Cytochromes P450 metabolize arachidonic acid (AA) into two vasoactive oxylipins with opposing biologic effects: epoxyeicosatrienoic acids (EETs) and omega-(ω)-terminal hydroxyeicosatetraenoic acids (HETEs). EETs have numerous beneficial physiological effects, including vasodilation and protection against ischemia/reperfusion injury, whereas ω-terminal HETEs induce vasoconstriction and vascular dysfunction. We evaluated the effect of these oxylipins on post-ischemic vasodilation known as coronary reactive hyperemia (CRH). CRH prevents the potential harm associated with transient ischemia. The beneficial effects of EETs are reduced after their hydrolysis to dihydroxyeicosatrienoic acids (DHETs) by soluble epoxide hydrolase (sEH). ω-terminal HETEs are formed by ω-hydroxylase family members. The relationship among endothelial over-expression of sEH (Tie2-sEH Tr), the changes in oxylipins it may produce, the pharmacologic inhibition of ω-hydroxylases, activation of PPARγ, and CRH response to a brief ischemia is not known. We hypothesized that CRH is attenuated in isolated mouse hearts with endothelial sEH over-expression through modulation of oxylipin profiles, whereas both inhibition of ω-hydroxylases and activation of PPARγ enhance CRH. Compared to WT mice, Tie2-sEH Tr mice had decreased CRH, including repayment volume, repayment duration, and repayment/debt ratio (P < 0.05), whereas inhibition of ω-hydroxylases increased these same CRH parameters in Tie2-sEH Tr mice. Inhibition of sEH with t-AUCB reversed the decreased CRH in Tie2-sEH Tr mice. Endothelial over-expression of sEH significantly changed oxylipin profiles, including decreases in DHETs, mid-chain HETEs, and prostaglandins (P < 0.05). Treatment with rosiglitazone, PPARγ-agonist, enhanced CRH (P < 0.05) in both Tie2-sEH Tr and wild type (WT) mice. These data demonstrate that endothelial over-expression of sEH (through changing the oxylipin profiles) attenuates CRH, whereas inhibition of ω-hydroxylases and activation of PPARγ enhance it.

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.

Arachidonic acid is metabolized to epoxyeicosatrienoic acids (EETs) by cytochrome (CYP) P450 epoxygenases, and to ω-terminal hydroxyeicosatetraenoic acids (HETEs) by ω-hydroxylases. EETs and HETEs often have opposite biologic effects; EETs are vasodilatory and protect against ischemia/reperfusion injury, while ω-terminal HETEs are vasoconstrictive and cause vascular dysfunction. Other oxylipins, such as epoxyoctadecaenoic acids (EpOMEs), hydroxyoctadecadienoic acids (HODEs), and prostanoids also have varied vascular effects. Post-ischemic vasodilation in the heart, known as coronary reactive hyperemia (CRH), protects against potential damage to the heart muscle caused by ischemia. The relationship among CRH response to ischemia, in mice with altered levels of CYP2J epoxygenases has not yet been investigated. Therefore, we evaluated the effect of endothelial overexpression of the human cytochrome P450 epoxygenase CYP2J2 in mice (Tie2-CYP2J2 Tr) on oxylipin profiles and CRH. Additionally, we evaluated the effect of pharmacologic inhibition of CYP-epoxygenases and inhibition of ω-hydroxylases on CRH. We hypothesized that CRH would be enhanced in isolated mouse hearts with vascular endothelial overexpression of human CYP2J2 through modulation of oxylipin profiles. Similarly, we expected that inhibition of CYP-epoxygenases would reduce CRH, whereas inhibition of ω-hydroxylases would enhance CRH. Compared to WT mice, Tie2-CYP2J2 Tr mice had enhanced CRH, including repayment volume, repayment duration, and repayment/debt ratio (P < 0.05). Similarly, inhibition of ω-hydroxylases increased repayment volume and repayment duration, in Tie2-CYP2J2 Tr compared to WT mice (P < 0.05). Endothelial overexpression of CYP2J2 significantly changed oxylipin profiles, including increased EETs (P < 0.05), increased EpOMEs (P < 0.05), and decreased 8-iso-PGF2α (P < 0.05). Inhibition of CYP epoxygenases with MS-PPOH attenuated CRH (P < 0.05). Ischemia caused a decrease in mid-chain HETEs (5-, 11-, 12-, 15-HETEs P < 0.05) and HODEs (P < 0.05). These data demonstrate that vascular endothelial overexpression of CYP2J2, through changing the oxylipin profiles, enhances CRH. Inhibition of CYP epoxygenases decreases CRH, whereas inhibition of ω-hydroxylases enhances CRH.

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.

High salt (HS) intake can change the arterial tone in mice, and the nitric oxide (NO) acts as a mediator to some of the receptors mediated vascular response. The main aim of this study was to explore the mechanism behind adenosine-induced vascular response in HS-fed eNOS(+/+) and eNOS(-/-) mice The modulation of vascular response by HS was examined using aortas from mice (eNOS(+/+) and eNOS(-/-)) fed 4% (HS) or 0.45% (NS) NaCl-diet through acetylcholine (ACh), NECA (adenosine-analog), CGS 21680 (A(2A) AR-agonist), MS-PPOH (CYP epoxygenase-blocker; 10(-5) M), AUDA (sEH-blocker; 10(-5) M), and DDMS (CYP4A-blocker; 10(-5) M). ACh-response was greater in HS-eNOS(+/+) (+59.3 ± 6.3%) versus NS-eNOS(+/+) (+33.3 ± 8.0%; P0.05). However, there was no response in both HS-eNOS(-/-) and NS-eNOS(-/-). NECA-response was greater in HS-eNOS(-/-) (+37.4 ± 3.2%) versus NS-eNOS(-/-) (+7.4.0 ± 3.8%; P0.05). CGS 21680-response was also greater in HS-eNOS(-/-) (+45.4 ± 5.2%) versus NS-eNOS(-/-)(+5.1 ± 5.0%; P0.05). In HS-eNOS(-/-), the CGS 21680-response was reduced by MS-PPOH (+7.3 ± 3.2%; P0.05). In NS-eNOS(-/-), the CGS 21680-response was increased by AUDA (+38.2 ± 3.3%; P0.05) and DDMS (+30.1 ± 4.1%; P0.05). Compared to NS, HS increased CYP2J2 in eNOS(+/+) (35%; P0.05) and eNOS(-/-) (61%; P0.05), but decreased sEH in eNOS(+/+) (74%; P0.05) and eNOS(-/-) (40%; P0.05). Similarly, CYP4A decreased in HS-eNOS(+/+) (35%; P0.05) and HS-eNOS(-/-) (34%; P0.05). These data suggest that NS causes reduced-vasodilation in both eNOS(+/+) and eNOS(-/-) via sEH and CYP4A. However, HS triggers possible A(2A)AR-induced relaxation through CYP epoxygenase in both eNOS(+/+) and eNOS(-/-).

Platelet-derived endothelial cell growth factor (PDECGF) is a potent angiogenic peptide with anti-apoptotic activity expressed widely in tumours. However, its expression in myocardial infarction (MI) is not yet established. This study aimed to assess the myocardial expression of PDECGF in rats after MI. Extracellular matrix (ECM) remodeling plays an important role in angiogenesis; hence, changes in the ECM components were investigated in the myocardium after MI, which was induced in rats by coronary artery ligation (CAL) and verified using biochemical markers and histopathology. Immunohistochemistry, RT-PCR, and activity assays identified the expression pattern of PDECGF on days 1, 2, 4, 8, 16, and 32 after CAL. The levels of TNF-α, MMP-2, collagen, and glycosaminoglycans in the ECM were assessed. Studies on immunohistochemistry, RT-PCR, and PDECGF activity demonstrated elevated levels of PDECGF expression from day 2 after CAL. Macrophages, endothelial cells, fibroblasts, and cardiomyocytes, especially at the border region of the lesion, showed an enhanced expression for PDECGF. Remodeling of the ECM was depicted by changes in the levels of TNF-α, MMP-2, collagen, and GAG. Hence, this study clearly indicated PDECGF as an important angiogenic molecule expressed during MI and the alterations in ECM components facilitated the process of angiogenesis.

The aim of this study was to evaluate the time course events of cellular damage during myocardial ischemia and reperfusion injury in rats and to find out a correlation between the structural alterations with respect to the biochemical changes. Cardiac biomarkers and lysosomal enzymes viz. cathepsin D, acid phosphatase and beta-glucuronidase and matrix metalloproteinases (MMPs) were evaluated at different time points, in response to ischemia-reperfusion induced oxidative stress in an isolated rat heart model perfused in Langendorff mode. Microscopically, changes in myocardial architecture, myofibrillar degradation, and collagen (COL) integrity were studied using hematoxylin-eosin, Masson's trichrome and toluidine blue staining techniques. A three-fold increase in the level of myoglobin was observed after 30 min of ischemia followed by 120 min of reperfusion as compared to 15 min ischemia, 120 min reperfusion. Similarly, a significant increase (P

Coronary reactive hyperemia (RH) is impaired in hypertrophic hearts. Epoxyeicosatrienoic acids (EETs) exert cardioprotective effects in ischemia / reperfusion injury. Soluble epoxide hydrolase (sEH...

Soluble epoxide hydrolase (sEH) degrades epoxyeicosatrienoic acids (EETs) to less active diols (dihydroxyeicosatrienoic acids; DHETs). EETs are endothelium-derived hyperpolarizing factors (EDHFs) w...

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 ...

This study attempted to probe the role of complement activation in promoting acute myocardial infarction (AMI) induced by coronary artery ligation (CAL) in rats. The surgical technique used in this study significantly reduced early mortality (95% survival rate) and also reduced the variation in infarct size (33+/-1.87%) at 32 h after surgery. Time course studies on the initiation of AMI at various time points were carried out using physiological, biochemical, histopathological and electron microscopical techniques. Serum markers and activities of lysosomal hydrolases were found to be significantly elevated at the 8th hour post ligation. Histological studies showed polymorphonuclear cells emigration and total coagulation necrosis. Transmission electron micrograph exhibited mild distortion of muscle fibres and mitochondrial rupture with disrupted cristae. Immunoblotting studies confirmed the presence of alpha2-macroglobulin which supported the inflammatory response at 8th h of post ligation. The initiation of the complement (C) activation was observed by the increase in the level of the soluble form of the membrane attack complex (sC5b-9) in serum and left ventricle. Immunoexpression studies confirmed the initiation of the terminal C activation as shown by the expression of C5, C6, C7, C8, C9 and sC5b-9 complex at the 8th h of AMI. This study conclusively demonstrated that initiation of the C activation was observed to be significant at the 8th h of AMI induced by CAL in rats.

In vivo models of myocardial infarction induced by coronary artery ligation (CAL) in rats usually suffer from high early mortality and a low rate of induction. This study investigated the time course initiation of chronic myocardial infarction (CMI) in albino rats and the possibility of reducing early mortality rate due to myocardial infarction by modification of the surgical technique. CAL was carried out by passing the suture through the epicardial layer around the midway of the left anterior descending coronary artery including a small area of the myocardium to avoid mechanical damage to the heart geometry. In addition, the role of endothelin-1 (ET-1) in rat heart with congestive heart failure was critically assessed. Time course initiation experiments were designed by sacrificing the animals at different time intervals and by carrying out physiological, biochemical, histopathological, electron microscopical and immunohistochemical studies. Specific markers of myocardial injury, viz. cardiac troponin-T (cTnT), high sensitivity C-reactive protein, lactate dehydrogenase and fibrinogen were measured at different time points. Serum marker enzymes and activities of lysosomal hydrolases were found to be elevated on the eighth day post-ligation. Histopathological studies demonstrated focal areas showing fibrovascular tissue containing fibroblasts, collagenous ground substance and numerous small capillaries replacing cardiac muscle fibers. Transmission electron micrographs exhibited mitochondrial changes of well-developed irreversible cardiac injury, viz. swelling, disorganization of cristae, appearance of mitochondrial amorphous matrix densities, significant distortion of muscle fibers and distinct disruption of the intercalated discs. Immunoblotting studies confirmed the presence of alpha 2-macroglobulin which supported the inflammatory response. The severity of the CMI was inferred by the measurement of the level of ET-1 in plasma and left ventricle which was significantly higher in the CMI rats than in the sham-operated rats. Immunohistochemical studies at different time intervals showed that there was a significant immunoexpression of ET-1 on the eighth day post-ligation. This study conclusively showed that ligation of left anterior descending artery minimized mortality and ET-1 was expressed during CMI.

In this study, we examined whether sublethal simulated ischemia (SSI) induces delayed cellular protection in mouse cardiac myocytes, and whether the delayed cellular protection depends on the activation of protein kinase C-epsilon (PKC-epsilon), inducible nitric oxide synthase (iNOS), and ATP-sensitive K(+) (K(ATP)) channels against subsequent sustained simulated ischemia (SI). The following groups of mouse cardiac myocytes were studied: (a) SI: incubation with SI buffer for 1 h; (b) SSI: incubation with SSI buffer for 30 min; (c) SSI + PKC inhibitor, chelerythrine chloride (CCl): SSI and 1 micro M CCl; (d) SSI + iNOS inhibitor, S-methylthiourea (SMT): SSI and 100 nM SMT; (e) SSI + K(ATP) channel blocker, glibenclamide (Glb): SSI and 50 micro M Glb; (f) SSI + mitochondrial K(ATP) channel blocker, 5-hydroxydecanoate (5-HD): SSI and 50 micro M 5-HD. The release of lactate dehydrogenase into the medium and the amount remaining in the cells was measured, and A(1) adenosine receptor, PKC-epsilon, and iNOS were detected through western blot analysis. The delayed cellular protection acquired due to SSI showed a decreased release of lactate dehydrogenase (%) from 46.51 +/- 1.60 to 37.00 +/- 1.34 (p0.001) and was blocked by CCl (47.08 +/- 0.95), SMT (48.08 +/- 1.18), Glb (45.88 +/- 1.31), and 5-HD (47.20 +/- 1.56). Simultaneously, SSI-induced up-regulation of A(1) adenosine receptor, PKC-epsilon, iNOS, and opening of both membrane and mitochondrial K(ATP) channels also was observed compared with controls.

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.

Coronary reactive hyperemia (RH) is impaired in hypertrophic hearts and in hearts with metabolic syndrome. Epoxyeicosatrienoic acids (EETs) exert cardioprotective effects against ischemia / reperfu...

Ischemic preconditioning (PC) induces delayed phase of protection, known as the second window of protection (SWOP). We investigated this phenomenon in rat and correlated it with the expression of 72-kDa heat shock protein (HSP 72). Rats were preconditioned with 1, 2, and 3 cycles of 5-min left anterior descending artery occlusions, each separated by a 10-min reperfusion (PC × 1, PC × 2 and PC × 3, respectively). Another group of rats was preconditioned with heat shock (HS) by raising temperature to 42°C for 15 min. Twenty-four hours later, rats were given sustained ischemia for 30 min and 90 min of reperfusion. Infarct sizes (%risk area) were 40.0 ± 7.5, 37.6 ± 5.6, and 47.6 ± 2.4 (mean ± SE) for PC × 1, PC × 2, and PC × 3 hearts, respectively, which were not different from the sham (49.9 ± 3.9, P > 0.05). In contrast, infarct size was reduced from 47.5 ± 3.8% in sham to 4.7 ± 2.3% ( P < 0.01) 24 h after HS. Additionally, early PC significantly reduced infarct size from 47.5 ± 3.8% in controls to 6.0 ± 1.2 and 5.0 ± 1.1% with PC × 1 and PC × 3. Repeated PC cycles induced over a threefold increase in HSP 70 mRNA after 2 h compared with sham ( P < 0.05). HSP 72, which increased 24 h after PC or HS, was not significantly different between the two PC stimuli. We conclude that PC does not induce SWOP in rat heart despite enhanced expression of HSP 72. In contrast, HS-induced delayed protection was associated with enhanced accumulation of HSP 72. It is possible that SWOP and HS have distinct mechanisms of protection that may not be exclusively related to HSP 72 expression.

High salt (4% NaCl, HS) diet modulates adenosine-induced vascular response through adenosine A(2A) receptor (A(2A)AR). Evidence suggests that A(2A)AR stimulates cyp450-epoxygenases, leading to epoxyeicosatrienoic acids (EETs) generation. The aim of this study was to understand the vascular reactivity to HS and underlying signaling mechanism in the presence or absence of A(2A)AR. Therefore, we hypothesized that HS enhances adenosine-induced relaxation through EETs in A(2A)AR⁺/⁺, but exaggerates contraction in A(2A)AR⁻/⁻. Organ bath and Western blot experiments were conducted in HS and normal salt (NS, 0.18% NaCl)-fed A(2A)AR⁺/⁺ and A(2A)AR⁻/⁻ mice aorta. HS produced concentration-dependent relaxation to non-selective adenosine analog, NECA in A(2A)AR⁺/⁺, whereas contraction was observed in A(2A)AR⁻/⁻ mice and this was attenuated by A₁AR antagonist (DPCPX). CGS 21680 (selective A(2A)AR agonist) enhanced relaxation in HS-A(2A)AR⁺/⁺ versus NS-A(2A)AR⁺/⁺, which was blocked by EETs antagonist (14,15-EEZE). Compared with NS, HS significantly upregulated the expression of vasodilators A(2A)AR and cyp2c29, whereas vasoconstrictors A₁AR and cyp4a in A(2A)AR⁺/⁺ were downregulated. In A(2A)AR⁻/⁻ mice, however, HS significantly downregulated the expression of cyp2c29, whereas A₁AR and cyp4a were upregulated compared with A(2A)AR⁺/⁺ mice. Hence, our data suggest that in A(2A)AR⁺/⁺, HS enhances A(2A)AR-induced relaxation through increased cyp-expoxygenases-derived EETs and decreased A₁AR levels, whereas in A(2A)AR⁻/⁻, HS exaggerates contraction through decreased cyp-epoxygenases and increased A₁AR levels.

We examined the in vitro preconditioning effect of non-toxic derivative of endotoxin, monophosphoryl lipid A (MLA) in adult rat cardiac myocytes. Cultured 5-7-day-old myocytes were preconditioned for 4 h by treatment with 200 ng/ml MLA. Twenty h later, cells were subjected to simulated ischemia by incubation in 0.75 mm sodium hydrosulfite, 12 mM KCl, 20 mM dl-lactic acid and 10 mM 2-deoxy-D-glucose (pH 6.5) for 2 h. MLA caused a significant reduction in the levels of LDH from 286+/-8 units/l in controls to 165+/-5 units/l (mean+/-s.e.m.; P

Adenosine A1 receptor (A1AR) activation contracts smooth muscle, although signaling mechanisms are not thoroughly understood. Activation of A1AR leads to metabolism of arachidonic acid, including the production of 20-hydroxyeicosatetraenoic acid (20-HETE) by cytochrome P4504a (CYP4a). The 20-HETE can activate protein kinase C-α (PKC-α), which crosstalks with extracellular signal-regulated kinase (ERK1/2) pathway. Both these pathways can regulate smooth muscle contraction, we tested the hypothesis that A1AR contracts smooth muscle through a pathway involving CYP4a, PKC-α, and ERK1/2. Experiments included isometric tension recordings of aortic contraction and Western blots of signaling molecules in wild type (WT) and A1AR knockout (A1KO) mice. Contraction to the A1-selective agonist 2-chloro-N cyclopentyladenosine (CCPA) was absent in A1KO mice aortae, indicating the contractile role of A1AR. Inhibition of CYP4a (HET0016) abolished 2-chloro-N cyclopentyladenosine-induced contraction in WT aortae, indicating a critical role for 20-HETE. Both WT and A1KO mice aortae contracted in response to exogenous 20-HETE. Inhibition of PKC-α (Go6976) or ERK1/2 (PD98059) attenuated 20-HETE-induced contraction equally, suggesting that ERK1/2 is downstream of PKC-α. Contractions to exogenous 20-HETE were significantly less in A1KO mice; reduced protein levels of PKC-α, p-ERK1/2, and total ERK1/2 supported this observation. Our data indicate that A1AR mediates smooth muscle contraction via CYP4a and a PKC-α-ERK1/2 pathway.