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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 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.

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.

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

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.

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.

Previously, we have shown that A2A adenosine receptor (A2AAR) knockout mice (KO) have increased contraction to adenosine. The signaling mechanism(s) for A2AAR is still not fully understood. In this study, we hypothesize that, in the absence of A2AAR, ω-hydroxylase (Cyp4a) induces vasoconstriction through mitogen-activated protein kinase (MAPK) via upregulation of adenosine A1 receptor (A1AR) and protein kinase C (PKC). Organ bath and Western blot experiments were done using isolated aorta from A2AKO and corresponding wild-type (WT) mice. Isolated aortic rings from WT and A2AKO mice were precontracted with submaximal dose of phenylephrine (10−6 M), and concentration responses for selective A1AR, A2AAR agonists, angiotensin II and cytochrome P-450-epoxygenase, 20-hydroxyeicosatrienoic acid (20-HETE) PKC, PKC-α, and ERK1/2 inhibitors were obtained. 2- p-(2-Carboxyethyl)-phenethylamino-5′- N-ethylcarboxamidoadenosine hydrochloride (CGS-21680, A2AAR agonist) induced concentration-dependent relaxation in WT, which was blocked by methylsulfonyl-propargyloxyphenylhexanamide (cytochrome P-450-epoxygenase inhibitor; 10−5 M) and also with removal of endothelium. A1 agonist, 2-chloro- N6-cyclopentyladenosine (CCPA) produced higher contraction in A2AKO aorta than WT (49.2 ± 8.5 vs. 27 ± 5.9% at 10−6 M, P < 0.05). 20-HETE produced higher contraction in A2AKO than WT (50.6 ± 8.8 vs. 21.1 ± 3.3% at 10−7 M, P < 0.05). Contraction to CCPA in WT and A2AKO aorta was inhibited by PD-98059 (p42/p44 MAPK inhibitor; 10−6 M), chelerythrine chloride (nonselective PKC blocker; 10−6 M), Gö-6976 (selective PKC-α inhibitor; 10−7 M), and HET0016 (20-HETE inhibitor; 10−5 M). Also, contraction to 20-HETE in WT and A2AKO aorta was inhibited by PD-98059 and Gö-6976. Western blot analysis indicated the upregulation of A1AR, Cyp4a, PKC-α, and phosphorylated-ERK1/2 in A2AKO compared with WT ( P < 0.05), while expression of Cyp2c29 was significantly higher in WT. CCPA (10−6 M) increased the protein expression of PKC-α and phosphorylated-ERK1/2, while HET0016 significantly reduced the CCPA-induced increase in expression of these proteins. These data suggest that, in the absence of A2AAR, Cyp4a induces vasoconstriction through MAPK via upregulation of A1AR and PKC-α.

High-salt intake can change the effect of adenosine on arterial tone in mice. The aim of this study was to clarify the mechanism by which this occurs. Using aortas from mice fed a 4% NaCl (HS) or 0.45% NaCl (NS) diet for 4–5 wks, concentration-response curves for ACh, 5′- N-ethylcarboxamidoadenosine (NECA; adenosine analog) and 2- p-(2-carboxyethyl)phenethylamino-5′- N-ethylcarboxamidoadenosine hydrochloride hydrate [CGS-21680; A2Aadenosine receptor (A2AAR) agonist] were obtained with Nω-nitro-l-arginine methyl ester (l-NAME; nitric oxide inhibitor, 10−4M), methylsulfonyl-propargyloxyphenylhexanamide [MS-PPOH; a CYP (cytochrome P-450) epoxygenase blocker, 10−5M including CYP2J2], 12-(3-adamantan-1-yl-ureido)dodecanoic acid [AUDA; soluble epoxide hydrolase (sEH) blocker, 10−5M], dibromo-dodecenyl-methylsulfimide [DDMS; CYP ω-hydroxylase (CYP4A blocker), 10−5M], glibenclamide (KATPchannel blocker; 10−5M) and 5-hydroxydecanoate (5-HD; mitochondrial-KATPchannel blocker, 10−4M). HS dose response to ACh (10−7− 10−5M) was not different from NS ( P > 0.05). Relaxation to 10−6M NECA was greater in the HS group (28.4 ± 3.9%) than in the NS group (4.1 ± 2.3%). Relaxation to 10−6M CGS-21680 was also greater in HS (27.9 ± 4.5%) than in NS (4.9 ± 2.2%). l-NAME was able to block the dose response of ACh (10−7− 10−5M) equally in both HS and NS ( P > 0.05), whereas l-NAME did not block CGS-21680-induced response in HS. In HS the CGS-21680 response was greatly reduced by MS-PPOH (to 4.7 ± 2.0%) and 5-HD (to 8.9 ± 2.2%), and also abolished by glibenclamide (−1.0 ± 5.9%). In NS, the CGS-21680 response was increased by AUDA (to 26.3 ± 3.4%) and DDMS (to 27.2 ± 3.0%). Compared with NS, HS vessels showed increased CYP2J2 and A2AAR expression (46 and 74% higher, respectively) but decreased sEH, CYP4A, and A1AR expression (75, 30, and 55% lower, respectively). These data suggest that in mice fed NS-containing diet, upregulation of arterial A1receptor causes vasoconstriction via increased sEH and CYP4A proteins. However, in mice fed HS-containing diet, upregulation of A2Areceptor protein triggers vascular relaxation through ATP-sensitive (K+) channels via upregulation of CYP2J2 enzyme.

We hypothesize that A2Aadenosine receptors (A2AAR) promote aortic relaxation in mice through cytochrome P450 (CYP)-epoxygenases and help to avoid salt sensitivity. Aortas from male mice maintained on a high-salt (HS; 7% NaCl) or normal-salt (NS; 0.45% NaCl) diet for 4–5 wks were used. Concentration-response curves (10−11–10−5M) for 5′- N-ethylcarboxamidoadenosine (NECA; a nonselective adenosine analog) and CGS 21680 (A2AAR agonist) were obtained with different antagonists including ZM 241385 (A2AAR antagonist; 10−6M), SCH 58261 (A2AAR antagonist; 10−6M), Nω-nitro-l-arginine methyl ester (l-NAME; endothelial nitric oxide synthase inhibitor; 10−4M) and inhibitors including methylsulfonyl-propargyloxyphenylhexanamide (MS-PPOH; CYP epoxygenases inhibitor; 10−5M), 14,15-epoxyeicosa-5(z)-enoic acid (14,15-EEZE; EET antagonist; 10−5M), dibromo-dodecenyl-methylsulfimide (DDMS; CYP4A inhibitor; 10−5M), and HET0016 (20-HETE inhibitor; 10−5M). At 10−7M of NECA, significant relaxation in HS (+22.58 ± 3.12%) was observed compared with contraction in NS (−10.62 ± 6.27%, P < 0.05). ZM 241385 changed the NECA response to contraction ( P < 0.05) in HS. At 10−7M of CGS 21680, significant relaxation in HS (+32.04 ± 3.08%) was observed compared with NS (+10.45 ± 1.34%, P < 0.05). SCH 58261, l-NAME, MS-PPOH, and 14,15-EEZE changed the CGS 21680-induced relaxation to contraction ( P < 0.05) in HS. Interestingly, DDMS and HET0016 changed CGS 21680 response to relaxation ( P < 0.05) in NS; however, there was no significant difference found between DDMS, HET0016-treated HS and NS vs. nontreated HS group ( P > 0.05). CYP2C29 protein was 55% and 74% upregulated in HS vs. NS ( P < 0.05) mice aorta and kidney, respectively. CYP4A protein was 30.30% and 35.70% upregulated in NS vs. HS ( P < 0.05) mice aorta and kidneys, respectively. A1AR was downregulated, whereas A2AAR was upregulated in HS compared with NS. These data suggest that HS may activate CYP2C29 via A2AAR, causing relaxation, whereas NS may contribute to the upregulation of CYP4A causing contraction.

We hypothesized that A2Aadenosine receptor (A2AAR) activation causes vasorelaxation through cytochrome P-450 (CYP) epoxygenases and endothelium-derived hyperpolarizing factors, whereas lack of A2AAR activation promotes vasoconstriction through Cyp4a in the mouse aorta. Adenosine 5′- N-ethylcarboxamide (NECA; 10−6M), an adenosine analog, caused relaxation in wild-type A2AAR (A2AAR+/+; +33.99 ± 4.70%, P < 0.05) versus contraction in A2AAR knockout (A2AAR−/−; −27.52 ± 4.11%) mouse aortae. An A2AAR-specific antagonist (SCH-58261; 1μM) changed the NECA (10−6M) relaxation response to contraction (−35.82 ± 4.69%, P < 0.05) in A2AAR+/+aortae, whereas no effect was noted in A2AAR−/−aortae. Significant contraction was seen in the absence of the endothelium in A2AAR+/+(−2.58 ± 2.25%) aortae compared with endothelium-intact aortae. An endothelial nitric oxide synthase inhibitor ( N-nitro-l-arginine methyl ester; 100 μM) and a cyclooxygenase inhibitor (indomethacin; 10 μM) failed to block NECA-induced relaxation in A2AAR+/+aortae. A selective inhibitor of CYP epoxygenases (methylsulfonyl-propargyloxyphenylhexanamide; 10 μM) changed NECA-mediated relaxation (−22.74 ± 5.11% at 10−6M) and CGS-21680-mediated relaxation (−18.54 ± 6.06% at 10−6M) to contraction in A2AAR+/+aortae, whereas no response was noted in A2AAR−/−aortae. Furthermore, an epoxyeicosatrienoic acid (EET) antagonist [14,15-epoxyeicosa-5( Z)-enoic acid; 10 μM] was able to block NECA-induced relaxation in A2AAR+/+aortae, whereas ω-hydroxylase inhibitors (10 μM dibromo-dodecenyl-methylsulfimide and 10 μM HET-0016) changed contraction into relaxation in A2AAR−/−aorta. Cyp2c29 protein was upregulated in A2AAR+/+aortae, whereas Cyp4a was upregulated in A2AAR−/−aortae. Higher levels of dihydroxyeicosatrienoic acids (DHETs; 14,15-DHET, 11,12-DHET, and 8,9-DHET, P < 0.05) were found in A2AAR+/+versus A2AAR−/−aortae. EET levels were not significantly different between A2AAR+/+and A2AAR−/−aortae. It is concluded that CYP epoxygenases play an important role in A2AAR-mediated relaxation, and the deletion of the A2AAR leads to contraction through Cyp4a.

Several anesthetics are known to cause respiratory and cardiovascular depression in humans and animals; but, these diverse effects have not been extensively investigated in laboratory rodents. The objective of this study is to choose a suitable anesthetic combination for use in surgical models eg. coronary artery ligation in rats. Male Wistar rats were anesthetized with three different drugs viz. diazepam-ketamine (DK) (2.5 mg/Kg, intraperitoneally (i.p); 50 mg/Kg, i.p), xylazine-ketamine (XK) (5 mg/Kg i.p; 50 mg/Kg i.p) and thiopentone (T) (40 mg/Kg i.p) and the respiratory and cardiovascular functions were assessed after coronary artery ligation. Heart rate (HR), mean arterial pressure (MAP), partial pressure of carbon dioxide (PaCO2), partial pressure of oxygen (PaO2), oxygen saturation percentage (O2 sat (%)), arterial blood pH and rectal body temperature were studied in detail. During the anesthetic regime, HR was lower till 60 min in XK and T ligated group (333 +/- 6; 304 +/- 8 beats/min) and it was near normalcy in the case of DK ligated group (394 +/- 6 beats/min). Significant respiratory depression was particularly reflected in the T ligated group with an increase in PaCO2 at 30 min (40.32 +/- 2.64 mmHg), which decreased to 38.2 +/- 2.23 mmHg at 60 min. Throughout the investigation, DK showed the least overall effects compared to XK and T on respiratory functions. Thus, DK could be considered to be a suitable anesthetic for use in a surgical model such as coronary artery ligation in albino rats.

Inducible nitric oxide synthase (iNOS) has been implicated as a mediator in myocardial protection, but this property of iNOS is still conflicting. Therefore, the present study was designed to assess whether iNOS really contributes to the ischemically and pharmacologically induced delayed cellular protection (DCP) in mice myocytes. The following groups of cultured iNOS gene-knockout (iNOS-/-), and its respective wild-type (wt) mice myocytes subjected to simulated ischemia (SI) at 20 h were studied: (a) wt + SI: with ischemia alone; (b) iNOS-/- + SI: with ischemia alone; (c) iNOS-/- + heat shock (HS): iNOS-/- and HS; (d) iNOS-/- + sub-lethal simulated ischemia (SSI): iNOS-/- and SSI; (e) iNOS-/- + A1AR agonist 2-chloro-N6-cyclopentyladenosine (CCPA): iNOS-/- and 1 microM CCPA; (f) iNOS-/- + A1AR agonist (2S)-N6-[2-endo-norbomyl]adenosine (S-ENBA): iNOS-/- and 1 nM S-ENBA; (g) iNOS-/- + K(ATP) channel opener pinacidil (Pin): iNOS-/- and 0.05 microM Pin, and (h) iNOS-/- + mitochondrial K(ATP) channel opener diazoxide (Diazo): iNOS-/- and 100 microM Diazo. The release of LDH into the medium as well as the amount of LDH remaining in the cells was used as a marker of cellular injury and cell viability. The cellular resistance was acquired by iNOS-/- mice myocytes due to HS, SSI, CCPA, S-ENBA, pinacidil and diazoxide treatment, which was evidenced by reduction of LDH (U/L) release from 51.14 +/- 1.35 (iNOS-/-) to 42.20 +/- 1.01 (iNOS-/- + HS); 45.57 +/- 0.75 (iNOS-/- + SSI); 42.87 +/- 0.87 (iNOS-/- + CCPA); 43.21 +/- 0.70 (iNOS-/- + S-ENBA); 37.81 +/- 0.99 (iNOS-/- + Pin) and 36.79 +/- 0.68 (iNOS-/- + Diazo), p0.01. Our data suggest that heat shock (HS), sub-lethal simulated ischemia (SSI), A1 adenosine agonists CCPA, S-ENBA and K(ATP) channel openers pinacidil (membrane K(ATP) channel), diazoxide (mitochondrial K(ATP) channel) induce delayed cellular protection in mice myocytes against subsequent sustained simulated ischemia without the involvement of iNOS. Further, our data also suggest that pinacidil and diazoxide are more potent inducers of delayed cellular protection among others in iNOS-/- mice myocytes against sustained simulated ischemia.

We have previously reported that prolonged exposure of porcine coronary arteries to adenosine agonists upregulates protein kinase C (PKC) through the activation of adenosine A1 receptor-coupled to pertussis toxin sensitive G-protein(s) [Am. J. Physiol. 264 (1993) H1465; Am. J. Physiol. 269 (1995) H1619]. The mechanism(s) by which A1 adenosine receptor upregulates PKC (isoforms) are not yet clearly understood. In the present study, we identified the alpha, beta 1, beta 2, gamma, epsilon, and zeta PKC isoforms that were upregulated by adenosine A1 receptor agonist as a possible mechanism(s) involved for this upregulation. Incubation of porcine coronary smooth muscle cells (PCSMC) with adenosine A1 receptor agonist (2s)-N6-[2-endo-norbornyl]adenosine (ENBA) caused an upregulation of PKC (isoforms), which were blocked by adenosine A1 receptor antagonist 1,3-dipropyl-8-cyclopentylxanthine (DPCPX). Western blot analysis using specific antibodies to PKC isoforms indicated that all the isoforms tested (alpha, beta I, beta II, mu, gamma, delta, epsilon, and zeta) were present in the primary cultured smooth muscle cells from porcine coronary artery. Western blot studies indicated that PKC alpha, beta I, beta II, gamma, epsilon, and zeta isoforms were upregulated in a dose dependent manner by adenosine agonist (ENBA) and PKC delta and mu were not altered.

This study examined the hypothesis that the activation of A1 adenosine receptor (A1AR) induces delayed cellular protection (DCP) in porcine coronary smooth muscle cells (PCSMC). The following groups of cultured PCSMC, subjected to simulated ischemia (SI) at 20 h were studied: (a) SI: with ischemia alone; (b) A1AR agonist chloro-N6-cyclopentyl adenosine (CCPA: CCPA (1 microM) alone; (c) CCPA + PKC inhibitor chelerythrine chloride (CCL): CCPA and 1 microM CCL; (d) CCPA + iNOS inhibitor S-methylthiourea (SMT): CCPA and 100 nM SMT; (e) CCPA + KATP channel blocker Glibenclamide (Glb): CCPA and 50 microM Glb; (f) CCPA + mitochondrial KATP channel blocker 5-hydroxydecanoate (5-HD): CCPA and 100 microM of 5-HD; (g) CCPA + A1AR antagonist 8-cyclopentyl-1,3-dipropylxanthine (DPCPX): CCPA and 1 microM DPCPX. The release of LDH into the medium as well as the amount of LDH remaining in the cells was used as a marker of cellular injury and cell viability. Up-regulation of A1AR, epsilon-PKC, iNOS and HSP 72i was detected through Westem blot analysis. The cellular resistance (%LDH remaining in the cells) acquired by PCSMC due to CCPA (59.42 +/- 1.57) was significantly blocked by CCL: 39.30 +/- 2.03; SMT: 41.37 +/- 1.98; Glb: 47.24 +/- 1.31; 5-HD: 47.69 +/- 1.40 and DPCPX: 42.92 +/- 0.79. CCPA increased the expression of A1AR (1.30 fold), epsilon-PKC (1.20 fold), iNOS (1.50 fold), and HSP 72i (1.70 fold) compared to the controls. CCPA-induced up-regulation of A1AR, epsilon-PKC, iNOS, HSP 72i, and the opening of both mitochondrial and sarcolemmal KATP channels may possibly participate in signaling cascade. Our study suggests that A1AR activation up-regulates iNOS, HSP 72i via epsilon-PKC signaling pathway to activate both mitochondrial and sarcolemmal KATP channels for cellular protection against SI in the cultured PCSMC.

This study has examined the hypotheses that, firstly, the ethanol induces delayed cellular protection in mouse cardiac myocytes against subsequent sustained simulated ischemia (SI). Secondly, the delayed cyto-protective effect induced by the ethanol depends more on time than the dose in mouse cardiac myocytes. Finally, ethanol-induced delayed cellular protection in mouse cardiac myocytes is mediated through inducible nitric oxide synthase (iNOS). Accordingly, we planned the following groups of BALB/c mouse cultured cardiac myocytes in our study: (a) SI, (b) 5 mM ethanol (ETOH)/15 min + SI, (c) 5 mM ETOH/30 min + SI, (d) 10 mM ETOH/15 min + SI, (e) 10 mM ETOH/30 min + SI, (f) 25 mM ETOH/15 min + SI, (g) 25 mM ETOH/30 min + SI, (h) 25 mM ETOH/60 min + SI, (i) 50 mM ETOH/15 min + SI, (j) 50 mM ETOH/30 min + SI, (k) 50 mM ETOH/60 min + SI, (l) 250 mM ETOH/15 min + SI and (m) 250 mM ETOH/30 min + SI. Another set of experiments we designed with iNOS-/- and its wild-type (iNOS+/+) mice cardiac myocytes as follows: SI, 5 mM ETOH/30 min + SI, 10 mM ETOH/30 min + SI, 25 mM ETOH/30 min + SI, 50 mM ETOH/30 min + SI and 250 mM ETOH/30 min + SI. Cellular injury was measured by the release of creatinine kinase (CK, U/l) into the medium. BALB/c mouse cardiac myocytes subjected to SI demonstrated significant increase in CK release as compared to the ethanol-treated cells. Ethanol-induced delayed cellular protection resulted in a significant (p0.001) attenuation in the cellular injury as indicated by reduction in the release of CK (U/l) from 9.25 +/- 0.52 to 5.16 +/- 0.44 (5 mM ETOH/30 min), from 7.50 +/- 0.43 (10 mM ETOH/15 min) to 4.16 +/- 0.64 (10 mM ETOH/30 min), from 5.91 +/- 0.41 (25 mM ETOH/15 min) to 2.50 +/- 0.58 (25 mM ETOH/30 min) and to 2.25 +/- 0.37 (25 mM ETOH/60 min), from 5.41 +/- 0.28 (50 mM ETOH/15 min) to 1.66 +/- 0.56(50 mM ETOH/30 min) and to 1.25 +/- 0.30 (50 mM ETOH/60 min), and from 5.25 +/- 0.21(250 mM ETOH/15 min) to 1.66 +/- 0.51(250 mM ETOH/30 min). Reduction in CK release from ethanol-treated iNOS-/-mouse cardiac myocytes was insignificant (p0.05) compared to non-treated wild-type (iNOS+/+) mouse cardiac myocytes subjected to SI alone. Our data suggest that ethanol induces delayed cellular protection in mouse cardiac myocytes against sustained simulated ischemia. Further, ethanol-induced delayed cellular protection depends more on time than the dose. Furthermore, ethanol-induced delayed cellular protection is dependent on iNOS.

In this study we examined the hypotheses that over-expression of the adenosine A1 receptor (A1AR) in transgenic mouse cardiac myocytes (A1AR-tgm) induces cellular protection against subsequent sustained simulated ischemia (SI); that the cellular protection induced by over-expression of A1AR in A1AR-tgm is mediated through inducible nitric oxide synthase (iNOS) and KATP channels. Sub-lethal simulated ischemia (SSI) and the A1AR agonists chloro-N 6-cyclopentyl adenosine (CCPA) or (2S)-N 6-[2-endo-norbornyl]adenosine (S-ENBA) induce further, delayed cytoprotection, additional to the existing protection in A1AR-tgm. Cellular injury and cell viability was measured by the release of lactate dehydrogenase (LDH) or creatine kinase (CK) into the medium and the amount remaining in the cells. The cellular resistance acquired by cardiac myocytes due to the over-expression of A1AR was reflected by the reduced release of LDH (in units/liter) from 44.94±1.46 (wild-type mouse cardiac myocytes, wt) to 29.59±2.83 (A1AR-tgm, P

The transgenic mice overexpressing heat shock protein 72 (HSP72) or antioxidants have been reported to be more resistant to myocardial ischemia/reperfusion injury. However, it remains unknown whether whole body heat stress (HS) which may induce HSP72 or endogenous antioxidants affords similar protection in the mouse heart. Adult male mice were treated with either HS (42 degrees C for 15 min) or anesthesia only (SC) against a group of non-stressed controls (NC). At 6 or 24 h later, the hearts were excised and perfused at a constant pressure of 55 mmHg in Langendorff mode. Following 30 min equilibration, hearts were subjected to 20 min of global ischemia and 30 min reperfusion (37 degrees C). Ventricular force was measured by a force-displacement transducer attached to the apex. Leakage of intracellular enzymes (CK, LDH) was measured in coronary efflux. Infarct size was determined by tetrazolium staining. The results showed that no significant differences between HS, SC, and NC groups in ventricular contractile function, CK and LDH release, or infarct size were observed at either time window. HS enhanced the expression of HSP72 in mouse hearts by two- to three-fold, whereas antioxidant enzyme activities (catalase and MnSOD) did not change significantly. We conclude that HS does not precondition the isolated perfused mice hearts against ischemia/reperfusion injury, despite induction of HSP72.

We investigated the protective effect of heat stress and metabolic preconditioning in cultured adult rat cardiac myocytes and correlated this effect with induction of heat shock proteins (HSP). Myocytes were preconditioned with sublethal heat shock or metabolic preconditioning for 30 min. Twenty hours later, preconditioned myocytes were subjected to lethal heat shock (46 degrees C for 2 h) or ischemia by incubation in ischemic buffer for 2 h. Cellular injury index was reduced from 69 +/- 4.0% in lethally heat-shocked cells to 27.0 +/- 1.6% with heat shock preconditioning (mean +/- SE; P < 0.01) and 19.0 +/- 3.0% with metabolic preconditioning (P < 0.01). Cellular injury index was 81.0 +/- 1.0% in ischemic myocytes and was reduced to 25.9 +/- 2.7 and 21.4 +/- 2.6% in heat shock- and metabolic-preconditioned myocytes, respectively (P < 0.01). A significant cross-tolerance of myocytes against lethal injury was observed with the two preconditioning methods. Western blot analysis revealed 3.3- and 2.5-fold increases in HSP 90 and 500- and 15-fold increases in HSP 70 with heat shock and metabolic preconditioning, respectively. HSP 27 expression remained unaltered relative to control cells. We conclude that heat shock and metabolic preconditioning induce delayed tolerance against lethal injuries in adult cardiac myocytes with elevated levels of HSP 70 and HSP 90.

Heat shock protects against myocardial ischemia-reperfusion injury possibly via increased expression of heat shock proteins. The direct evidence of heat shock protein protection in vivo remains circumstantial, and no other new mechanism of protection has been proposed. Recent studies suggest that opening of ATP-sensitive K+ channels (KATP channels) plays an important role in ischemic preconditioning; however, it is not known whether this channel is also important in delayed protection conferred by heat shock. Anesthetized rabbits underwent heat shock treatment by raising core temperature to 42°C for 15 min. Twenty-four hours later, the animals were reanesthetized and subjected to regional ischemia-reperfusion. The specific KATP channel blockers glibenclamide (0.3 mg/kg ip) and sodium 5-hydroxydecanoate (5HD; 5 mg/kg iv) were used to block the channel function. The drugs were administered at two different times, either pre-heat stress or preischemia. Infarct size was determined by triphenyltetrazolium chloride staining. The 72-kDa heat shock protein (HSP 72) was measured by Western blots. Our results show that heat shock produced a marked reduction in infarct size (39.4 ± 8.1 to 14.3 ± 2.5% of risk area, P < 0.05). Glibenclamide and 5HD completely abolished heat shock-induced reduction in infarct size (42.3 ± 0.32 and 33.7 ± 4.8%) when given before ischemia-reperfusion; however, these antagonists failed to block protection when administered before the onset of heat shock. Furthermore, the enhanced expression of HSP 72 in heat shock groups was not diminished by glibenclamide or 5HD, suggesting a lack of a direct role of this protein in conferring cardiac protection by heat shock. The complete blockade of cardiac protection by glibenclamide and 5HD strongly suggests that opening of this channel is a very important component of heat shock-induced ischemic protection in rabbit hearts.