Gotowa bibliografia na temat „Adenosine mediated cardioprotection”

All four adenosine receptor subtypes have been shown to play a role in cardioprotection, and there is evidence that all four subtypes may be expressed in cardiomyocytes. There is also increasing evidence that optimal adenosine cardioprotection requires the activation of more than one receptor subtype. The purpose of this study was to determine whether adenosine A2A and/or A2B receptors modulate adenosine A1 receptor-mediated cardioprotection. Isolated perfused hearts of wild-type (WT), A2A knockout (KO), and A2BKO mice, perfused at constant pressure and constant heart rate, underwent 30 min of global ischemia and 60 min of reperfusion. The adenosine A1 receptor agonist N6-cyclohexyladenosine (CHA; 200 nM) was administrated 10 min before ischemia and for the first 10 min of reperfusion. Treatment with CHA significantly improved postischemic left ventricular developed pressure (74 ± 4% vs. 44 ± 4% of preischemic left ventricular developed pressure at 60 min of reperfusion) and reduced infarct size (30 ± 2% with CHA vs. 52 ± 5% in control) in WT hearts, effects that were blocked by the A1 antagonist 8-cyclopentyl-1,3-dipropylxanthine (100 nM). Treatments with the A2A receptor agonist CGS-21680 (200 nM) and the A2B agonist BAY 60-6583 (200 nM) did not exert any beneficial effects. Deletion of adenosine A2A or A2B receptor subtypes did not alter ischemia-reperfusion injury, but CHA failed to exert a cardioprotective effect in hearts of mice from either KO group. These findings indicate that both adenosine A2A and A2B receptors are required for adenosine A1 receptor-mediated cardioprotection, implicating a role for interactions among receptor subtypes.

Prior activation of protein kinase C (PKC) can precondition the cardiac cell against injury during subsequent ischaemia. By using cultured chick ventricular cell model for simulated ischaemia and preconditioning, the present study investigated the biochemical mechanism underlying the PKC-mediated preconditioning. A 5 min exposure to PMA enhanced the ability of pinacidil to mediate cardioprotection during a subsequent 90 min period of ischaemia, which is consistent with a sustained activation of the KATP channel initiated by PKC. The brief prior exposure to PMA was also associated with an enhanced ability of the adenosine A1 or A3 receptor agonist 2-chloro-N6-cyclopentyladenosine or N6-(3-iodobenzyl)adenosine-5´-N-methyluronamide to elicit a cardioprotective response during the subsequent ischaemia. In myocytes pretreated with PMA, the cardioprotection mediated by receptor agonist was blocked by the concomitant presence of KATP-channel antagonists glibenclamide or 5-hydroxydecanoic acid during the ischaemia. Thus the KATP channel acts downstream of the adenosine A1 and A3 receptors in mediating the protective effect due to prior PMA exposure. KATP channel activation is responsible for the adenosine receptor-mediated effect. PMA treatment had no effect on other A1 or A3 receptor-mediated effects such as the inhibition of adenylate cyclase, ruling out a direct stimulation of the receptor or G-protein by PMA. The present results indicate that prior stimulation of PKC causes a sustained KATP channel activation, which in turn renders the myocyte more responsive to the protective action of adenosine A1 and A3 receptor agonists during the subsequent ischaemia.

The relative roles of mitochondrial (mito) ATP-sensitive K+ (mitoKATP) channels, protein kinase C (PKC), and adenosine kinase (AK) in adenosine-mediated protection were assessed in Langendorff-perfused mouse hearts subjected to 20-min ischemia and 45-min reperfusion. Control hearts recovered 72 ± 3 mmHg of ventricular pressure (50% preischemia) and released 23 ± 2 IU/g lactate dehydrogenase (LDH). Adenosine (50 μM) during ischemia-reperfusion improved recovery (149 ± 8 mmHg) and reduced LDH efflux (5 ± 1 IU/g). Treatment during ischemia alone was less effective. Treatment with 50 μM diazoxide (mitoKATP opener) during ischemia and reperfusion enhanced recovery and was equally effective during ischemia alone. A3 agonism [100 nM 2-chloro- N 6-(3-iodobenzyl)-adenosine-5′- N-methyluronamide], A1 agonism ( N 6-cyclohexyladenosine), and AK inhibition (10 μM iodotubercidin) all reduced necrosis to the same extent as adenosine, but less effectively reduced contractile dysfunction. These responses were abolished by 100 μM 5-hydroxydecanoate (5-HD, mitoKATP channel blocker) or 3 μM chelerythrine (PKC inhibitor). However, the protective effects of adenosine during ischemia-reperfusion were resistant to 5-HD and chelerythrine and only abolished when inhibitors were coinfused with iodotubercidin. Data indicate adenosine-mediated protection via A1/A3 adenosine receptors is mitoKATP channel and PKC dependent, with evidence for a downstream location of PKC. Adenosine provides additional and substantial protection via phosphorylation to 5′-AMP, primarily during reperfusion.

Ischemic heart diseases (IHD) are the leading cause of death worldwide. Although the principal form of treatment of IHD is myocardial reperfusion, the recovery of coronary blood flow after ischemia can cause severe and fatal cardiac dysfunctions, mainly due to the abrupt entry of oxygen and ionic deregulation in cardiac cells. The ability of these cells to protect themselves against injury including ischemia and reperfusion (I/R), has been termed “cardioprotection”. This protective response can be stimulated by pharmacological agents (adenosine, catecholamines and others) and non-pharmacological procedures (conditioning, hypoxia and others). Several intracellular signaling pathways mediated by chemical messengers (enzymes, protein kinases, transcription factors and others) and cytoplasmic organelles (mitochondria, sarcoplasmic reticulum, nucleus and sarcolemma) are involved in cardioprotective responses. Therefore, advancement in understanding the cellular and molecular mechanisms involved in the cardioprotective response can lead to the development of new pharmacological and non-pharmacological strategies for cardioprotection, thus contributing to increasing the efficacy of IHD treatment. In this work, we analyze the recent advances in pharmacological and non-pharmacological strategies of cardioprotection.

Background Preconditioning with isoflurane has been shown to confer cardioprotection via activation of mitochondrial adenosine triphosphate-sensitive K+ (mito K(ATP)) channels. However, the relative contribution of mito K(ATP) channel and non-mito K(ATP) channel mechanisms to isoflurane-mediated cardioprotection has not been investigated. Methods Isolated and buffer-perfused rat hearts were used. Flavoprotein fluorescence was monitored as an index for mito K(ATP) channel activity. Isovolumic left ventricular function and infarct size were measured as indices for cardioprotection. Results Flavoprotein fluorescence, which was monitored as an index for mito K(ATP) channel activity, was increased by isoflurane and a known mito K(ATP) channel opener, diazoxide, in a 5-hydroxydecanoate-sensitive manner. Although flavoprotein oxidation induced by diazoxide was dissipated soon after its removal from the buffer, flavoprotein oxidation induced by isoflurane was sustained after cessation of the treatment. The sustained increase in flavoprotein oxidation was associated with a significant reduction in infarct size after 30 min of ischemia followed by 120 min of reperfusion. Although adenosine and S-nitroso-N-acetyl-penicillamine each alone did not increase flavoprotein fluorescence, nor did they confer significant cardioprotection, coadministration of adenosine and S-nitroso-N-acetyl-penicillamine with isoflurane conferred a highly significant reduction of infarct size and improvement of left ventricular function without increasing flavoprotein oxidation over isoflurane alone. The early treatment with 5-hydroxydecanoate before and during preconditioning completely reversed flavoprotein oxidation and inhibited the infarct-sparing effect of isoflurane and combined preconditioning with isoflurane, adenosine, and S-nitroso-N-acetyl-penicillamine. The late treatment with 5-hydroxydecanoate after preconditioning abolished flavoprotein oxidation and the infarct-sparing effect of isoflurane but only partially inhibited cardioprotection conferred by the combined preconditioning, despite complete abrogation of flavoprotein oxidation. Conclusions Mito K(ATP) channel activation is the essential trigger of both preconditioning with isoflurane and combined preconditioning with isoflurane, adenosine, and S-nitroso-N-acetyl-penicillamine. Mito K(ATP) channel activation is also a crucial mediator of cardioprotection afforded by preconditioning with isoflurane. However, enhanced cardioprotection conferred by combined preconditioning is mediated through both mito K(ATP) channel-dependent and -independent mechanisms.

Ischaemic heart disease is a major contributor to premature death and heart failure in the Westernised world. Ischaemic injury within the heart may be beneficially modulated by the nucleoside adenosine. Derived from catabolism of ATP, adenosine was initially known as a potent bradycardic and hypotensive agent. However, more recently the protective function of adenosine has been investigated. The protective effects of adenosine are mediated via activation of adenosine receptors: A1, A2A, A2B, and A3 receptors. Activation of these potentially protective (or retaliatory) adenosine receptors hinges upon accumulation of adenosine during ischaemia-reperfusion. This Thesis examines the role and mechanisms of adenosine mediated cardioprotection in young and aged hearts, exploring endogenous and exogenous adenosine receptor activation, genetic manipulation of A1 receptors and adenosine deaminase and pharmacological manipulation of adenosine metabolism. The effects of age on ischaemic responses and adenosine handling and protection are also assessed. The core approach to assess each of the above issues involved the Langendorff isolated mouse heart preparation. Experiments within Chapter 3 focuses on the contractile effects of adenosine receptors in normoxic hearts. This study indicates A2A receptors have no direct effect on contractility, while adenosine exerts positive inotropy independently of coronary flow and perfusion pressure (i.e. Independent of the Gregg phenomenon). In addition, investigations in genetically modified hearts hint at positive inotropy in response to A1 receptors. Since the latter is only evidenced in modified lines, it is possible A1-mediated inotropy may be abnormal or supraphysiological. In Chapter 4 the impact of genetic 'deletion' of A1ARs and/or adenosine deaminase (ADA) on intrinsic tolerance to ischaemia were studied. Data demonstrate that genetic deletion of A1 receptors significantly limits the ability of the mouse myocardium to withstand injury during ischaemic insult. Thus, providing strong support for a role of A1ARs in determining intrinsic tolerance to ischaemia-reperfusion. ADA KO mice confirm protection afforded by endogenous adenosine and the notion of adenosine metabolism modification as a protective strategy. Interestingly, effects of A1AR KO differ from A1AR overexpression or A1AR agonism in that the latter decrease contractile diastolic dysfunction while A1AR KO selectively increase systolic dysfunction and increase oncosis without altering diastolic injury. This challenges current dogma regarding the action of A1 adenosine receptors on ischaemic injury. In Chapter 5 the effects of adenosine metabolism inhibition (via adenosine deaminase (ADA) and adenosine kinase (AK) inhibitors) were studied. Inhibition of adenosine deaminase with the drug EHNA, and adenosine phosphorylation with iodotubercidin significantly protected mouse hearts from ischaemia-reperfusion, reducing contractile dysfunction and cardiac enzyme efflux. However, inhibitors failed to improve the outcome of the aged myocardium. 8-SPT and 5-HD reduced the protective effects of EHNA and iodotubercidin demonstrating thus; cardioprotection via ADA and AK appears to rely on adenosine receptor activation and involves a mitoK ATP dependent mechanism. Since aging is of considerable importance with regard to outcomes of ischaemic heart disease, experiments in Chapter 6 focused on effects of aging (and gender) on cardiovascular function and injury during ischaemia-reperfusion. In assessing post ischaemic outcomes in hearts from young adult (2-4 months), mature adult (8 months), middle aged (12 months), aged (18 months) and senescent (24-28 months) C57/BL/6J mice, data reveal a substantial age-related decline in ischaemic tolerance (which appears selective for myocardial vs. vascular injury). The decline in ischaemic tolerance is expressed primarily within the initial 12 months in both males and females with relatively little further decline with continued aging. There is evidence of a modest improvement in tolerance in senescence vs. aged hearts possibly reflecting selection of a protected phenotype in senescent populations. In addition, mature and middle-aged female hearts showed improved tolerance to ischaemia-reperfusion compared to males, supporting a role for hormonal changes. Effects of aging and purine metabolism were studied in Chapter 7. Data suggest impaired tolerance to ischaemia-reperfusion in older hearts may stem in part from shifts in myocardial purine catabolism. Data reveal reduced accumulation of salvageable and cardioprotective adenosine and enhanced accumulation of poorly salvaged (and potentially injurious) hypoxanthine and xanthine. These changes may potentially predispose the aged myocardium to ischaemic injury and radical generation via the xanthine oxidase reaction. The final data Chapter of this Thesis describes preliminary data regarding aging, signalling and adenosine mediated protection. It was found that protein kinase C (PKC) and A1 receptors mediate protection in young hearts and also that A1 receptors appear to mediate protection via a PKC LindependentLi signalling cascade. In addition, experiments in aged hearts (attempting to elucidate mechanisms behind impaired adenosinergic protection with age) suggest a PKC-independent A1-mediated protection path may be preserved with aging, since A1 receptors continue to offer some protection while PKC activation does not. It is possible the failure of exogenous adenosine to offer protection in aged hearts may result from a lesion at or downstream of PKC.

Ischaemic heart disease is a major contributor to premature death and heart failure in the Westernised world. Ischaemic injury within the heart may be beneficially modulated by the nucleoside adenosine. Derived from catabolism of ATP, adenosine was initially known as a potent bradycardic and hypotensive agent. However, more recently the protective function of adenosine has been investigated. The protective effects of adenosine are mediated via activation of adenosine receptors: A1, A2A, A2B, and A3 receptors. Activation of these potentially protective (or retaliatory) adenosine receptors hinges upon accumulation of adenosine during ischaemia-reperfusion. This Thesis examines the role and mechanisms of adenosine mediated cardioprotection in young and aged hearts, exploring endogenous and exogenous adenosine receptor activation, genetic manipulation of A1 receptors and adenosine deaminase and pharmacological manipulation of adenosine metabolism. The effects of age on ischaemic responses and adenosine handling and protection are also assessed. The core approach to assess each of the above issues involved the Langendorff isolated mouse heart preparation. Experiments within Chapter 3 focuses on the contractile effects of adenosine receptors in normoxic hearts. This study indicates A2A receptors have no direct effect on contractility, while adenosine exerts positive inotropy independently of coronary flow and perfusion pressure (i.e. Independent of the Gregg phenomenon). In addition, investigations in genetically modified hearts hint at positive inotropy in response to A1 receptors. Since the latter is only evidenced in modified lines, it is possible A1-mediated inotropy may be abnormal or supraphysiological. In Chapter 4 the impact of genetic 'deletion' of A1ARs and/or adenosine deaminase (ADA) on intrinsic tolerance to ischaemia were studied. Data demonstrate that genetic deletion of A1 receptors significantly limits the ability of the mouse myocardium to withstand injury during ischaemic insult. Thus, providing strong support for a role of A1ARs in determining intrinsic tolerance to ischaemia-reperfusion. ADA KO mice confirm protection afforded by endogenous adenosine and the notion of adenosine metabolism modification as a protective strategy. Interestingly, effects of A1AR KO differ from A1AR overexpression or A1AR agonism in that the latter decrease contractile diastolic dysfunction while A1AR KO selectively increase systolic dysfunction and increase oncosis without altering diastolic injury. This challenges current dogma regarding the action of A1 adenosine receptors on ischaemic injury. In Chapter 5 the effects of adenosine metabolism inhibition (via adenosine deaminase (ADA) and adenosine kinase (AK) inhibitors) were studied. Inhibition of adenosine deaminase with the drug EHNA, and adenosine phosphorylation with iodotubercidin significantly protected mouse hearts from ischaemia-reperfusion, reducing contractile dysfunction and cardiac enzyme efflux. However, inhibitors failed to improve the outcome of the aged myocardium. 8-SPT and 5-HD reduced the protective effects of EHNA and iodotubercidin demonstrating thus; cardioprotection via ADA and AK appears to rely on adenosine receptor activation and involves a mitoK ATP dependent mechanism. Since aging is of considerable importance with regard to outcomes of ischaemic heart disease, experiments in Chapter 6 focused on effects of aging (and gender) on cardiovascular function and injury during ischaemia-reperfusion. In assessing post ischaemic outcomes in hearts from young adult (2-4 months), mature adult (8 months), middle aged (12 months), aged (18 months) and senescent (24-28 months) C57/BL/6J mice, data reveal a substantial age-related decline in ischaemic tolerance (which appears selective for myocardial vs. vascular injury). The decline in ischaemic tolerance is expressed primarily within the initial 12 months in both males and females with relatively little further decline with continued aging. There is evidence of a modest improvement in tolerance in senescence vs. aged hearts possibly reflecting selection of a protected phenotype in senescent populations. In addition, mature and middle-aged female hearts showed improved tolerance to ischaemia-reperfusion compared to males, supporting a role for hormonal changes. Effects of aging and purine metabolism were studied in Chapter 7. Data suggest impaired tolerance to ischaemia-reperfusion in older hearts may stem in part from shifts in myocardial purine catabolism. Data reveal reduced accumulation of salvageable and cardioprotective adenosine and enhanced accumulation of poorly salvaged (and potentially injurious) hypoxanthine and xanthine. These changes may potentially predispose the aged myocardium to ischaemic injury and radical generation via the xanthine oxidase reaction. The final data Chapter of this Thesis describes preliminary data regarding aging, signalling and adenosine mediated protection. It was found that protein kinase C (PKC) and A1 receptors mediate protection in young hearts and also that A1 receptors appear to mediate protection via a PKC LindependentLi signalling cascade. In addition, experiments in aged hearts (attempting to elucidate mechanisms behind impaired adenosinergic protection with age) suggest a PKC-independent A1-mediated protection path may be preserved with aging, since A1 receptors continue to offer some protection while PKC activation does not. It is possible the failure of exogenous adenosine to offer protection in aged hearts may result from a lesion at or downstream of PKC.
Thesis (PhD Doctorate)
Doctor of Philosophy (PhD)
School of Medical Science
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