adenosine-kinase and Inflammation

Gestational diabetes mellitus (GDM) is a detrimental condition for human pregnancy associated with endothelial dysfunction and endothelial inflammation in the fetoplacental vasculature and leads to increased cardio-metabolic risk in the offspring. In the fetoplacental vasculature, GDM is associated with altered adenosine metabolism. Adenosine is an important vasoactive molecule and is an intermediary and final product of transmethylation reactions in the cell. Adenosine kinase is the major regulator of adenosine levels. Disruption of this enzyme is associated with alterations in methylation-dependent gene expression regulation mechanisms, which are associated with the fetal programming phenomenon. Here we propose that cellular and molecular alterations associated with GDM can dysregulate adenosine kinase leading to fetal programming in the fetoplacental vasculature. This can contribute to the cardio-metabolic long-term consequences observed in offspring after exposure to GDM.

Topics: Adenosine; Adenosine Kinase; Animals; Cardiovascular System; Diabetes Mellitus; Diabetes, Gestational; DNA Methylation; Endothelium; Epigenomics; Female; Fetal Development; Gene Expression Regulation; Humans; Inflammation; Mice; Placenta; Pregnancy

Adenosine (ADO) is an endogenous inhibitory neuromodulator that increases nociceptive thresholds in response to tissue trauma and inflammation. Adenosine kinase (AK) is a key intracellular enzyme regulating intra- and extracellular concentrations of ADO. AK inhibition selectively amplifies extracellular ADO levels at cell and tissue sites where accelerated release of ADO occurs. AK inhibitors have been shown to provide effective antinociceptive, antiinflammatory and anticonvulsant activity in animal models, thus suggesting their potential therapeutic utility for pain, inflammation, epilepsy and possibly other central and peripheral nervous system diseases associated with cellular trauma and inflammation. This beneficial outcome may potentially lack nonspecific effects associated with the systemic administration of ADO receptor agonists. Until recently all of the reported AK inhibitors contained adenosine-like structural motif. The present review will discuss design, synthesis and analgesic and antiinflammatory properties of the novel nonnucleoside AK inhibitors that do not have close structural resemblance with the natural substrate ADO. Two classes of the nonnucleoside AK inhibitors are built on pyridopyrimidine and alkynylpyrimidine cores.

Topics: Adenosine Kinase; Animals; Drug Design; Enzyme Inhibitors; Epilepsy; Humans; Inflammation; Molecular Structure; Pain; Pyrimidines; Structure-Activity Relationship

Adenosine kinase (AK; EC 2.7.1.20) is a key intracellular enzyme regulating intra and extracellular concentrations of adenosine (ADO), an endogenous modulator of intercellular signalling that reduces cell excitability during tissue stress and trauma. The inhibitory effects of ADO are mediated by interactions with specific cell-surface G-protein coupled receptors (GPCR), which regulate membrane cation flux, membrane polarisation and the release of excitatory neurotransmitters. Inhibition of AK potentiates local extracellular ADO levels at cell and tissue sites which are undergoing accelerated ADO release. Thus, AK inhibition represents a mechanism to selectively enhance the endogenous protective actions of ADO during cellular stress while potentially minimising the non-specific effects associated with the systemic administration of ADO receptor agonists. Novel, potent AK inhibitors have recently been synthesised that demonstrate high specificity for this particular enzyme as compared to other ADO metabolic enzymes, transporters and receptors. AK inhibitors have been shown to increase ADO concentrations in various systems in vitro, as well as in an in vivo model of neurotoxicity. In addition, AK inhibitors have demonstrated efficacy in animal models of epilepsy, cerebral ischaemia as well as pain and inflammation, thus suggesting their potential therapeutic utility for these conditions.

Topics: Adenosine Kinase; Animals; Brain Ischemia; Disease Models, Animal; Enzyme Inhibitors; Epilepsy; Humans; Inflammation; Pain; Purinergic P1 Receptor Antagonists; Receptors, Cell Surface

Adenosine (ADO) is an endogenous modulator of intercellular signaling that provides homeostatic reductions in cell excitability during tissue stress and trauma. The inhibitory actions of ADO are mediated by interactions with specific cell-surface G-protein coupled receptors regulating membrane cation flux, polarization, and the release of excitatory neurotransmitters. ADO kinase (AK; EC 2.7.1.20) is the key intracellular enzyme regulating intra- and extracellular ADO concentrations. Inhibition of AK produces marked increases in extracellular ADO levels that are localized to cells and tissues undergoing accelerated ADO release. Thus AK inhibition represents a mechanism to selectively enhance the protective actions of ADO during tissue trauma without producing the nonspecific effects associated with the systemic administration of ADO receptor agonists. During the last 10 years, specific inhibitors of AK based on the endogenous purine nucleoside substrate, ADO, have been developed. Potent AK inhibitors have recently been synthesized that demonstrate high specificity for this enzyme as compared to other ADO metabolic enzymes, transporters, and receptors. In both in vitro and in vivo models, AK inhibitors have been shown to potently increase ADO concentrations in a tissue and event specific fashion and to demonstrate potential clinical utility in animal models of epilepsy, ischemia, pain, and inflammation. AK inhibitors have demonstrated superior efficacy in these models as compared to other mechanisms of modulating ADO availability, and these agents exhibit reduced side-effect liabilities compared to direct acting ADO receptor agonists. The preclinical profile of AK inhibitors indicate that these agents may have therapeutic utility in a variety of central and peripheral diseases associated with cellular trauma and inflammation. Clinical trials are currently underway to evaluate the efficacy of AK inhibitors in seizure disorders and pain.

Topics: Adenosine; Adenosine Kinase; Animals; Chemistry, Pharmaceutical; Enzyme Inhibitors; Humans; In Vitro Techniques; Inflammation; Pain; Wounds and Injuries

Nonalcoholic fatty liver disease is highly associated with obesity and progresses to nonalcoholic steatohepatitis when the liver develops overt inflammatory damage. While removing adenosine in the purine salvage pathway, adenosine kinase (ADK) regulates methylation reactions. We aimed to study whether hepatocyte ADK functions as an obesogenic gene/enzyme to promote excessive fat deposition and liver inflammation.. Liver sections of human subjects were examined for ADK expression using immunohistochemistry. Mice with hepatocyte-specific ADK disruption or overexpression were examined for hepatic fat deposition and inflammation. Liver lipidomics, hepatocyte RNA sequencing (RNA-seq), and single-cell RNA-seq for liver nonparenchymal cells were performed to analyze ADK regulation of hepatocyte metabolic responses and hepatocyte-nonparenchymal cells crosstalk.. Whereas patients with nonalcoholic fatty liver disease had increased hepatic ADK levels, mice with hepatocyte-specific ADK disruption displayed decreased hepatic fat deposition on a chow diet and were protected from diet-induced excessive hepatic fat deposition and inflammation. In contrast, mice with hepatocyte-specific ADK overexpression displayed increased body weight and adiposity and elevated degrees of hepatic steatosis and inflammation compared with control mice. RNA-seq and epigenetic analyses indicated that ADK increased hepatic DNA methylation and decreased hepatic Ppara expression and fatty acid oxidation. Lipidomic and single-cell RNA-seq analyses indicated that ADK-driven hepatocyte factors, due to mitochondrial dysfunction, enhanced macrophage proinflammatory activation in manners involving increased expression of stimulator of interferon genes.. Hepatocyte ADK functions to promote excessive fat deposition and liver inflammation through suppressing hepatocyte fatty acid oxidation and producing hepatocyte-derived proinflammatory mediators. Therefore, hepatocyte ADK is a therapeutic target for managing obesity and nonalcoholic fatty liver disease.

Topics: Adenosine Kinase; Animals; Diet, High-Fat; Fatty Acids; Hepatitis; Hepatocytes; Humans; Inflammation; Liver; Mice; Mice, Inbred C57BL; Non-alcoholic Fatty Liver Disease; Obesity

Acute kidney injury (AKI) has a high morbidity and mortality, and there is no targeted treatment yet. One of the main causes of AKI is ischemia-reperfusion (IR). Increased release of adenosine under stress and hypoxia exerts anti-inflammatory and antioxidant effects. Adenosine kinase (ADK) is an important enzyme that eliminates adenosine in cells, and can maintain low adenosine concentration in cells. Our previous studies have shown that pretreatment of adenosine kinase inhibitor ABT-702 could markedly attenuate cisplatin-induced nephrotoxicity both in vivo and in vitro. This study is designed to investigate the effect of ADK inhibition on IR-induced AKI. The results showed that ADK expression was positively correlated with the degree of renal tubular injury, which suggested that the degree of ADK inhibition reflected the severity of acute tubular necrosis. In vivo, ADK inhibitor could reduce IR-induced renal injury, which might play a protective role by increasing tissue adenosine level, inhibiting oxidative stress, and reducing cell apoptosis. In HK2 cells, cobaltous dichloride (CoCl

Topics: Acute Kidney Injury; Adenosine Kinase; Adult; Animals; Apoptosis; Cell Line; Cobalt; Enzyme Inhibitors; Female; Humans; Inflammation; Inosine; Kidney Tubules; Male; Mice, Inbred C57BL; Morpholines; Necrosis; Oxidative Stress; Pyrimidines; Reperfusion Injury

Insulin resistance-related disorders are associated with endothelial dysfunction. Accumulating evidence has suggested a role for adenosine signaling in the regulation of endothelial function. Here, we identified a crucial role of endothelial adenosine kinase (ADK) in the regulation of insulin resistance. Feeding mice with a high-fat diet (HFD) markedly enhanced the expression of endothelial Adk. Ablation of endothelial Adk in HFD-fed mice improved glucose tolerance and insulin sensitivity and decreased hepatic steatosis, adipose inflammation and adiposity, which were associated with improved arteriole vasodilation, decreased inflammation and increased adipose angiogenesis. Mechanistically, ADK inhibition or knockdown in human umbilical vein endothelial cells (HUVECs) elevated intracellular adenosine level and increased endothelial nitric oxide synthase (NOS3) activity, resulting in an increase in nitric oxide (NO) production. Antagonism of adenosine receptor A2b abolished ADK-knockdown-enhanced NOS3 expression in HUVECs. Additionally, increased phosphorylation of NOS3 in ADK-knockdown HUVECs was regulated by an adenosine receptor-independent mechanism. These data suggest that Adk-deficiency-elevated intracellular adenosine in endothelial cells ameliorates diet-induced insulin resistance and metabolic disorders, and this is associated with an enhancement of NO production caused by increased NOS3 expression and activation. Therefore, ADK is a potential target for the prevention and treatment of metabolic disorders associated with insulin resistance.

Topics: Adenosine Kinase; Adipose Tissue; Animals; Cells, Cultured; Diet, High-Fat; Endothelium, Vascular; Fatty Liver; Human Umbilical Vein Endothelial Cells; Humans; Inflammation; Insulin Resistance; Male; Mice, Knockout; Mice, Transgenic; Nitric Oxide Synthase Type III; Obesity; Phosphorylation

The molecular mechanisms underlying vascular inflammation and associated inflammatory vascular diseases are not well defined. Here we show that endothelial intracellular adenosine and its key regulator adenosine kinase (ADK) play important roles in vascular inflammation. Pro-inflammatory stimuli lead to endothelial inflammation by increasing endothelial ADK expression, reducing the level of intracellular adenosine in endothelial cells, and activating the transmethylation pathway through increasing the association of ADK with S-adenosylhomocysteine (SAH) hydrolase (SAHH). Increasing intracellular adenosine by genetic ADK knockdown or exogenous adenosine reduces activation of the transmethylation pathway and attenuates the endothelial inflammatory response. In addition, loss of endothelial ADK in mice leads to reduced atherosclerosis and affords protection against ischemia/reperfusion injury of the cerebral cortex. Taken together, these results demonstrate that intracellular adenosine, which is controlled by the key molecular regulator ADK, influences endothelial inflammation and vascular inflammatory diseases.The molecular mechanisms underlying vascular inflammation are unclear. Here the authors show that pro-inflammatory stimuli lead to endothelial inflammation by increasing adenosine kinase expression, and that its knockdown in endothelial cells inhibits atherosclerosis and cerebral ischemic injury in mice.

Topics: Adenosine; Adenosine Kinase; Adenosylhomocysteinase; Animals; Atherosclerosis; Blood Vessels; Cerebral Cortex; Endothelial Cells; Epigenesis, Genetic; Gene Expression Regulation; Gene Knockdown Techniques; Inflammation; Mice; Mice, Knockout, ApoE; Reperfusion Injury

Hypoxic-ischemic (HI) injury perinatal brain is a major contributor to morbidity and mortality to infants and children. Adenosine may play a role in the pathophysiology of HI, since it modulates the inflammatory process and the release of several neurotransmitters. Thus, the aim of this study was to identify the isoforms of adenosine deaminase (ADA) responsible for the enzymatic activity as well as the adenosine kinase (ADK) and A1 adenosine receptor (A1R) expression in the cerebral cortex eight days after HI. Myeloperoxidase (MPO) and N-acetyl-glucosaminidase (NAG) were assessed as inflammation markers. ADA activity was analyzed, in the presence or absence of a specific ADA1 inhibitor, erythro-9-(2-hydroxy-3-nonyl) adenine. The ADA1 activity (92.6%) was significantly higher than ADA2 (7.4%) activity in the cerebral cortex eight days after HI. A1Rs and ADK protein expression showed decreased 8 days after insult. Interestingly, the ADA1, MPO, and NAG activities were correlated positively. In view of this, we conclude that the inhibitor of ADA1, in in vitro conditions, was effective in decreasing the ADA activity, and that mainly ADA1 isoform is responsible for the increase in the ADA activity 8 days after HI insult. Therefore, HI neonatal was able to alter the ADK and A1R expression. Thus, due to the importance of adenosine signaling in the regulation of inflammatory and immune process and the crucial role of ADA in the postischemic homeostase of adenosine as well as during inflammatory process, we suggest that ADA1 inhibitors may play an important role in the regulation of events that follow the HI insult, favoring the increase in the adenosine in the sites of tissue injury. Together, these results highlight a role of the purinergic signaling cascade in the pathophysiology of HI neonatal.

Topics: Acetylglucosaminidase; Adenine; Adenosine Deaminase; Adenosine Kinase; Animals; Animals, Newborn; Blotting, Western; Brain; Cerebral Cortex; Hypoxia-Ischemia, Brain; Inflammation; Isoenzymes; Male; Peroxidase; Purines; Rats, Wistar; Receptor, Adenosine A1

Traumatic optic neuropathy (TON) is associated with apoptosis of retinal ganglion cells. Local productions of reactive oxygen species and inflammatory mediators from activated microglial cells have been hypothesized to underlie apoptotic processes. We previously demonstrated that the anti-inflammatory effect of adenosine, through A2A receptor activation had profound protective influence against retinal injury in traumatic optic neuropathy. This protective effect is limited due to rapid cellular re-uptake of adenosine by equilibrative nucleotside transporter-1 (ENT1) or break down by adenosine kinase (AK), the key enzyme in adenosine clearance pathway. Further, the use of adenosine receptors agonists are limited by systemic side effects. Therefore, we seek to investigate the potential role of amplifying the endogenous ambient level of adenosine by pharmacological inhibition of AK. We tested our hypothesis by comparing TON-induced retinal injury in mice with and without ABT-702 treatment, a selective AK inhibitor (AKI). The retinal-protective effect of ABT-702 was demonstrated by significant reduction of Iba-1, ENT1, TNF-α, IL-6, and iNOS/nNOS protein or mRNA expression in TON as revealed by western blot and real time PCR. TON-induced superoxide anion generation and nitrotyrosine expression were reduced in ABT-702 treated mice retinal sections as determined by immunoflourescence. In addition, ABT-702 attenuated p-ERK1/2 and p-P38 activation in LPS induced activated mouse microglia cells. The results of the present investigation suggested that ABT-702 had a protective role against marked TON-induced retinal inflammation and damage by augmenting the endogenous therapeutic effects of site- and event-specific accumulation of extracellular adenosine.

Topics: Adenosine; Adenosine Kinase; Animals; Anti-Inflammatory Agents; Calcium-Binding Proteins; Cells, Cultured; Disease Models, Animal; Enzyme Inhibitors; Equilibrative Nucleoside Transporter 1; Imidazoles; Inflammation; Lipopolysaccharides; Male; Mice; Mice, Inbred C57BL; Microfilament Proteins; Microglia; Morpholines; Neurotoxicity Syndromes; Nitric Oxide Synthase Type I; Optic Nerve Injuries; Oxidative Stress; Pyridines; Pyrimidines; Retina

Recent studies suggest AMP-activated protein kinase (AMPK), an enzyme involved in energy homeostasis, might be a novel signaling pathway in regulating inflammatory response, but the precise intracellular mechanisms are not fully understood. In this study, we have demonstrated that 5-aminoimidazole-4-carboxamide riboside (AICAR), an activator of AMPK, inhibited lipopolysaccharide (LPS)-induced protein expression of inducible nitric oxide synthase (iNOS) and cyclooxygenase-2 (COX-2) in macrophages and microglial cells at the gene transcription level. Data obtained from electrophoretic mobility shift assay (EMSA) and promoter activity assay have further confirmed the ability of AICAR to block LPS-mediated NF-kappaB, AP-1, CREB, and C/EBPbeta activation. However, AICAR did not affect LPS-mediated IKK, ERK, and p38 activation. Regardless of the ability of AICAR to activate AMPK, the inhibitory effects of AICAR on iNOS and COX-2 expression were not associated with AMPK. An adenosine kinase inhibitor 5'-iodotubercidin, which effectively abolished AMPK activation caused by AICAR, did not reverse the anti-inflammatory effect of AICAR. Moreover, another AMPK activator metformin was not able to mimic the effects of AICAR. Direct addition of AICAR in EMSA assay interrupted binding of NF-kappaB, CREB, and C/EBPbeta to specific DNA elements. Taken together, this study demonstrates that the anti-inflammatory effects of AICAR against LPS-induced iNOS and COX-2 gene transcription are not associated with AMPK activation, but might be resulting from the direct interference with DNA binding to transcription factors.

Topics: Adenosine Kinase; Aminoimidazole Carboxamide; AMP-Activated Protein Kinases; Animals; Anti-Inflammatory Agents; Cyclooxygenase 2; Electrophoretic Mobility Shift Assay; Enzyme Activation; Gene Expression; Genes, Reporter; Inflammation; Lipopolysaccharides; Macrophages; Mice; Microglia; Multienzyme Complexes; Nitric Oxide Synthase Type II; Protein Binding; Protein Serine-Threonine Kinases; Ribonucleosides; RNA, Messenger; Signal Transduction; Transcription Factors; Transcription, Genetic

The present study investigated 1) antihyperalgesic actions of a novel and selective adenosine kinase (AK) inhibitor, A-134974 (IC(50) = 60 pM), in the carrageenan model of thermal hyperalgesia; 2) effects of A-134974 on locomotor activity; and 3) relative contributions of supraspinal, spinal, and peripheral sites to the actions of A-134974. Systemic A-134974 (i.p.) dose dependently reduced hyperalgesia (ED(50) = 1 micromol/kg) and at higher doses, reduced locomotor activity (ED(50) = 16 micromol/kg). Administration of A-134974 intrathecally (i.t.) was more potent (ED(50) = 6 nmol) at producing antihyperalgesia than delivering the compound by intracerebralventricular (ED(50) = 100 nmol, i.c.v.) or intraplantar (ED(50) >300 nmol) routes. In contrast, i.c.v. administration of A-134974 was more effective in reducing locomotor activity than i.t. administration (ED(50) values were 1 and >100 nmol, respectively). Increasing the pretreatment time for i.t.-delivered A-134974 caused a greater reduction in locomotor activity (ED(50) = 10 nmol). This was due to diffusion of A-134974 (i.t.) to supraspinal sites. The antihyperalgesic effects of systemic A-134974 were antagonized by the adenosine receptor antagonist theophylline (THEO, 30-500 nmol) administered i.t., but not i.c.v. In the locomotor assay, i.t.-injected THEO did not antagonize hypomobility caused by systemic or i.t. administration of A-134974. However, i.c.v. infusion of THEO did block the hypomotive actions of i.c.v.-, i.t.-, and i.p.-administered A-134974. These data demonstrate that the novel AK inhibitor A-134974 potently reduces thermal hyperalgesia primarily through interactions with spinal sites, whereas its ability to depress locomotor activity is predominantly mediated by supraspinal sites.

Topics: Adenosine Kinase; Animals; Anti-Inflammatory Agents, Non-Steroidal; Carrageenan; Central Nervous System; Enzyme Inhibitors; Hyperalgesia; Inflammation; Injections, Intraventricular; Injections, Spinal; Male; Motor Activity; Nucleosides; Peripheral Nervous System; Rats; Rats, Sprague-Dawley; Spinal Cord

1. Adenosine (ADO) receptor activation modulates sensory transmission in the dorsal horn. Little is known about the circumstances underlying release of the purine. The present study was conducted to investigate the effect of a novel and potent non-nucleoside adenosine kinase (AK) inhibitor, ABT-702, on the responses of dorsal horn neurones to selected peripheral stimuli. ABT-702 is orally effective to reduce behavioural signs of nociception in models of acute, inflammatory, and neuropathic pain. 2. Electrophysiological recordings were made from wide dynamic range (WDR) neurones in halothane-anaesthetized rats. ABT-702 was given subcutaneously following either carrageenan inflammation or peripheral nerve injury (L5/L6 spinal nerve ligation). Comparisons were made between carrageenan and uninjected control animals, and similarly between spinal nerve ligated (SNL) and sham operated animals. 3. ABT-702 produced inhibition of the postdischarge, wind-up and C-fibre evoked responses in both carrageenan and nerve-injured animals. Furthermore, the mechanical and thermal evoked responses were similarly reduced in SNL rats. Overall, ABT-702 produced a significantly greater inhibition of these responses in SNL rats as compared to sham controls. Similarly ABT-702 tended to produce greater effects after carrageenan inflammation, however this did not reach significance. 4. Protection of endogenous adenosine by ABT-702 therefore produces a marked inhibition of the noxious evoked neuronal activity in inflamed and neuropathic rats. Our results demonstrate a plasticity in the endogenous adenosine-mediated inhibitory system following SNL and provide a possible basis for the use of this compound for the treatment of neuropathic and other persistent pain states.

Topics: Adenosine Kinase; Animals; Behavior, Animal; Carrageenan; Dose-Response Relationship, Drug; Electrophysiology; Enzyme Inhibitors; Hot Temperature; Inflammation; Ligation; Male; Morpholines; Neurons; Pain; Peripheral Nerve Injuries; Peripheral Nerves; Posterior Horn Cells; Pyrimidines; Rats; Rats, Sprague-Dawley; Spinal Cord; Spinal Nerves

Adenosine (Ado) triggers numerous protective mechanisms in the heart that may attenuate ischemia-reperfusion injury in cardiac grafts. We aimed to establish whether sustained increase in endogenous Ado production by the combined application of Ado metabolism inhibitors and nucleotide precursors attenuates reperfusion injury in transplanted hearts.. Rat hearts were collected after the infusion of St Thomas' Hospital cardioplegic solution, stored at 4 degrees C for 4 hours, and heterotopically transplanted into the abdomen of recipient rats. A solution containing Ado deaminase inhibitor erythro-9(2-hydroxy-3-nonyl)adenine, Ado kinase inhibitor 5'-aminoadenosine, and nucleotide precursors adenine and ribose was administered at the time of reperfusion in the treated group, whereas saline was administered to control animals. After 1 or 24 hours, mechanical function of the transplanted hearts was evaluated in an ex vivo perfusion system followed by the determination of myocardial ATP with related metabolites and measurement of the activity of neutrophil-specific enzyme myeloperoxidase in cardiac homogenates. After 24 hours of reperfusion, maximum left ventricular developed pressure increased from 87.0+/-6.8 mm Hg (mean+/-SEM) in controls to 118.1+/-8.2 mm Hg in the treated group (P<0.05), ATP increased from 11.0+/-0.8 micromol/g dry wt in controls to 15.1+/-1.2 micromol/g dry wt in the treated group (P<0.01), and myeloperoxidase activity decreased from 2.23+/-0.60 U/g wet wt in controls to 0.58+/-0.12 U/g wet wt in the treated group (P<0.001). No differences in cardiac function, ATP, or myeloperoxidase activity were observed between the treated group and controls after 1 hour of reperfusion.. The administration of Ado metabolism inhibitors with nucleotide precursors causes a sustained increase in endogenous Ado production and exerts a potent protective effect against reperfusion injury in transplanted hearts. Improved cardiac function and elevated ATP concentration were accompanied by complete amelioration of neutrophil infiltration in treated hearts, suggesting that reduction in postischemic inflammation could be an important mechanism of this protective effect.

Topics: Adenine; Adenosine; Adenosine Deaminase; Adenosine Deaminase Inhibitors; Adenosine Kinase; Animals; Cardioplegic Solutions; Creatinine; Deoxyadenosines; Enzyme Inhibitors; Heart Transplantation; In Vitro Techniques; Inflammation; Male; Myocardial Reperfusion Injury; Myocardium; Neutrophil Infiltration; Perfusion; Rats; Rats, Sprague-Dawley; Ribose; Treatment Outcome