A1 adenosine receptors and muscarinic cholinoceptors in myocardial ischemia

The regulation of cardiac A₁ adenosine receptors and M₂ muscarinic cholinoceptors was investigated in ischemic rat hearts. Ischemia was induced in isolated, perfused hearts either by stop (stop-flow) or by reduction (low-flow) of perfusion flow. Receptor densities and affinities were determined by radioligand binding. The mRNA concentrations of the receptors and of control messages were measured by quantitative polymerase chain reactions (PCR). Second messenger coupling of the receptors was evaluated by measuring their inhibition of adenylate cyclase activity.Up to 60 min of stop-flow ischemia and 6 h of lowflow ischemia, cardiac A₁ adenosine receptor density and affinity, and adenosine receptor-mediated inhibition of adenylate cyclase, did not change significantly, compared to non-ischemic hearts. Receptor down-regulation, however, could be induced by perfusion with the A₁ receptor agonist R-phenyl-isopropyl-adenosine (R-PIA) during normal flow. After 6 h of perfusion with R-PIA (0.1 mol/l), A₁ adenosine receptor density was reduced. Agonist-induced receptor down-regulation was not found after perfusion with R-PIA in low-flow ischemia. The density and the affinity of muscarinic cholinoceptors were not affected during stop-flow ischemia up to 1 h either, whereas the density was down-regulated to 75% of controls (P<0.05) after 6 h of low-flow ischemia. This intervention also reduced inhibition of adenylate cyclase via muscarinic cholinoceptors. In non-ischemic hearts, perfusion with carbachol (10 µmol/l) suppressed receptor densities to 72% of control values.No significant changes in the concentration of A₁ adenosine receptor or M₂ cholinoceptor mRNAs occurred during normal flow, stop-flow and low-flow ischemia. Likewise, agonist stimulation with R-PIA or carbachol during normal flow did not change the respective receptor mRNA concentrations significantly. Conclusion: Although a down-regulation of A₁ adenosine receptor density was demonstrated after receptor agonist perfusion with normal flow, adenosine did not affect the density or functional activity of cardiac A₁ adenosine receptors in the ischemic myocardium. In contrast, muscarinic cholinoceptor density and function was down-regulated after prolonged ischemia. The lack of an agonist-induced down-regulation of A₁ adenosine receptors in the presence of decreasing activity of m-cholinoceptors suggests a growing importance of the adenosine system in myocardial ischemia.     

Continuous adenosine A2A receptor antagonism after focal cerebral ischemia in spontaneously hypertensive rats

Antagonism of the adenosine A_2A receptor (A_2AR) has been shown to elicit substantial neuroprotective properties when given immediately after cerebral ischemia. We asked whether the continuous application of a selective A_2AR antagonist within a clinically relevant time window will be a feasible and effective approach to treat focal cerebral ischemia. To answer this question, we subjected 20 male spontaneously hypertensive rats to permanent middle cerebral artery occlusion and randomized them equally to a verum and a control group. Two hours after stroke onset, the animals received a subcutaneous implantation of an osmotic minipump filled with 5 mg kg^?1 day^?1 8-(3-chlorostyryl) caffeine (CSC) or vehicle solution. The serum level of CSC was measured twice a day for three consecutive days. The infarct volume was determined at days 1 and 3 using magnetic resonance imaging. We found the serum level of CSC showing a bell-shaped curve with its maximum at 36 h. The infarct volume was not affected by continuous CSC treatment. These results suggest that delayed and continuous CSC application was not sufficient to treat acute ischemic stroke, potentially due to unfavorable hepatic elimination and metabolization of the pharmaceutical.     

Regulation of adenosine levels during cerebral ischemia

Adenosine is a neuromodulator with its level increasing up to 100-fold during ischemic events, and attenuates the excitotoxic neuronal injury. Adenosine is produced both intracellularly and extracellularly, and nucleoside transport proteins transfer adenosine across plasma membranes. Adenosine levels and receptor-mediated effects of adenosine are regulated by intracellular ATP consumption, cellular release of ATP, metabolism of extracellular ATP (and other adenine nucleotides), adenosine influx, adenosine efflux and adenosine metabolism. Recent studies have used genetically modified mice to investigate the relative contributions of intra- and extracellular pathways for adenosine formation. The importance of cortical or hippocampal neurons as a source or a sink of adenosine under basal and hypoxic/ischemic conditions was addressed through the use of transgenic mice expressing human equilibrative nucleoside transporter 1 (hENT1) under the control of a promoter for neuron-specific enolase. From these studies, we conclude that ATP consumption within neurons is the primary source of adenosine in neuronal cultures, but not in hippocampal slices or in vivo mice exposed to ischemic conditions.     

Blockade of A(2A) adenosine receptors leads to c-fos inhibition in a rat model of brain ischemia.

Adenosine plays an important role in cerebral ischemia by acting on its own receptors, in particular the A(2A)receptor. Its activation leads to excitatory amino acid release thus contributing to the ischemic damage. Blockade by specific antagonists may protect against cytotoxic injury. Our study was aimed to investigate the effect of the blockade of A(2A)receptors, by Sch 58261, on the expression of the early gene c-fos, in a model of permanent middle cerebral artery occlusion (pMCAo), in rats. In the pMCAo model, ischemia was induced in the right hemisphere whereas the contralateral one was considered the control. In our study, we have compared pMCAo rats, pMCAo rats treated with Sch 58261 and sham operated ones.C-fos was markedly expressed in the ischemic hemispheres, whereas lower levels were detected in the contralateral ones of the ischemic animals. The lowest bands were observed in sham operated rats. After treatment with Sch 58261, a considerable reduction in c-fos expression was observed in the ischemic hemispheres, whereas a limited effect was detected in the others.Our results suggest that inhibition of immediate-early gene expression by the A(2A)receptors antagonist Sch 58261 may contribute to its neuroprotective activity.     

Behavioral effects of adenosine analogs in squirrel monkeys: relation to adenosine A2 receptors

The behavioral effects of seven metabolically stable analogs of adenosine were studied in squirrel monkeys responding under a fixed-interval (FI) schedule of stimulus-shock termination. All drugs produced dose-related decreases in response rate, but differed in potency by up to three orders of magnitude. The 5-carboxamine and 2-chlorine substituted analogs were more potent than the N⁶-substituted analogs. The potencies of the adenosine analogs in decreasing schedule-controlled behavior correlate well with their reported affinities for adenosine A₂, but not A₁, recognition sites as determined by displacement of bound ligands or modulation of adenylate cyclase activity. The results suggest that the behavioral effects of adenosine analogs in squirrel monkeys are linked to their actions at adenosine A₂ receptors.Animals used in this study were maintained in accordance with the guidelines of the Committee on Animals of the Harvard Medical School and of the Guide for Care and Use of Laboratory Animals of the Institute of Laboratory Animal Resources, National Research Council, Department of Health, Education and Welfare, Publication No. (NIH)85-23, revised 1985     

Influence of the selective ligands of dopamine and 5-hydroxytryptamine receptors on the tolerance to global cerebral ischemia

Selective dopamine (D) receptor agonists either slightly improve (D2 and D3) or do not affect (D1 and D4) the tolerance of the brain to global ischemia. As for D and 5-HT (hydroxytryptamine) antagonists, only D1 antagonist SCH 23390 and 5-HT2 antagonist ketanserin produce a small neuroprotective effect, while D2, D4, 5-HT(2B) and 5-HT(2C) antagonists are not active. Simultaneous injection of D2 (raclopride), D3 (GR 103691), and D4 (L 745870) receptor blockers also does not protect the brain. These results are not at variance with a widespread hypothesis that the accumulation of extracellular 5-HT and especially D in the brain causes the neuron damage. The effect of ketanserine is not increased by D2 or D4 blockers, but the introduction of D3 blocker GR 103691 (+88%) and especially the simultaneous injection of D2,3,4 antagonists improve the effect of ketanserine (+134%). The neuroprotective effect of the last combination is not lower but even exceeds that of some neuroleptics. This fact shows the possibility to increase the tolerance to cerebral ischemia by simultaneously blocking D and 5-HT-receptors.     

5-hydroxytryptamine and 5-methoxytryptamine increase tolerance to global cerebral ischemia

In the global cerebral ischemia, 5-hydroxytryptamine (serotonin, 5-HT) and 5-methoxytryptamine (5-MT) produce a significant neuroprotector effect closely correlated with their hypothermic action. This expands the spectrum of cerebral functions controlled by 5-HT. At the same time, melatonin and 2-iodmelatonin, which are chemically (but not pharmacologically) close to 5-HT and 5-MT, do not exhibit such protective effects. Probably, an important role in the interaction with 5-HT receptors belongs to the NH2 group of the indole ring.     

Neuroprotective effects of trolox in global cerebral ischemia in gerbils

Stroke is a third leading cause of death and oxygen free radicals have been shown to be involved in its pathophysiology. In the present study, we have investigated neuroprotective potential of trolox, a free radical scavenger in bilateral carotid arteries occlusion (5 min) model of global cerebral ischemia in Mongolian gerbils. Gerbils were treated with trolox (3, 10 or 30 mg/kg, i.p.) 30 min prior to occlusion. There was a significant increase in neurological symptoms and locomotor activity in ischemic animals as compared with the sham-operated animals. These effects were attenuated by trolox 30 mg/kg, i.p. Significant increase in the number of the surviving neurons in the hippocampal CA1 pyramidal region was observed in ischemic animals treated with trolox 30 mg/kg, i.p. There was significant increase in the level of malondialdehyde (MDA) in ischemic animals indicating oxidative stress. Elevated levels of MDA in ischemic animals (25.79+/-3.34 microM/mg of protein) were reduced (16.43+/-3.32 microM/mg of protein) and (8.98+/-0.89 microM/mg of protein) by trolox 10 and 30 mg/kg, i.p., respectively. This study demonstrates the neuroprotective potential of trolox in global cerebral ischemia in gerbils.     

Neuroprotective effect of L-kynurenine sulfate administered before focal cerebral ischemia in mice and global cerebral ischemia in gerbils

Excessive stimulation of N-methyl-D-aspartate (NMDA) receptors during ischemia contributes to apoptotic and excitotoxic nerve cell death. Kynurenic acid is a selective antagonist at the glycine co-agonist site of the NMDA receptor complex at low concentration, and it is a broad-spectrum excitatory amino acid receptor blocker at high concentration. Kynurenic acid provides neuroprotection in animal models of cerebral ischemia only at very high doses as it hardly crosses the blood-brain barrier. The neuroprotective effect of L-kynurenine sulfate, a precursor of kynurenic acid, was therefore studied because L-kynurenine readily crosses the blood-brain barrier. L-kynurenine sulfate was administered 15 min before permanent focal cerebral ischemia produced by electrocoagulation of the distal middle cerebral artery in mice. L-kynurenine sulfate induced a small decrease in the surface area of the brain infarction (10%, P<0.05) at 30 mg/kg i.p., and it caused strong reductions in infarct size (24-25%, P<0.01) at 100 and 300 mg/kg i.p. Treatment of gerbils with L-kynurenine sulfate at 300 mg/kg i.p. 2 h before a 3-min bilateral carotid occlusion decreased (40%, P<0.01) the pyramidal cell loss in the CA1 area of the hippocampus. Furthermore, L-kynurenine sulfate inhibited the ischemia-induced hypermotility (77%, P<0.001), and decreased (50%, P<0.01) the ischemia-induced deterioration of spontaneous alternation, a measure of spatial memory, without altering the rectal temperature. In conclusion, the administration of L-kynurenine can elevate the brain concentration of kynurenic acid to neuroprotective levels, suggesting the potential clinical usefulness of L-kynurenine for the prevention of neuronal loss.     

Prevention by macrocortin of global cerebral ischemia in Sprague-Dawley rats

We have previously shown that dexamethasone protects female Sprague-Dawley CFY rats against global cerebral ischemia induced by bilateral carotid artery ligation. In the present study, macrocortin derived from rat peritoneal cells exposed to dexamethasone was found to exhibit antiphospholipase A2 activity and to provide significant protection against the fatal consequences of carotid artery ligation. These results suggest that the cerebroprotective effect of glucocorticoids may be related to macrocortin production.     

Pharmacology of adenosine A2A receptors and therapeutic applications

Adenosine A(2A) receptors were cloned about ten years ago and are known to be well conserved among mammals. Rather selective agonists and antagonists are available. In addition, two different knock-out mice have been prepared and extensively characterized. A(2A) receptors are highly enriched in the basal ganglia and on cells involved in inflammatory reactions. At these sites they are likely to play physiologically important roles. Efforts to develop new therapies based on A(2A) receptors have focused on these topics. However, A(2A) receptors are found on many other cell types and on them as well agonists can exert effect.     

脑缺血再灌注损伤大鼠大脑皮质NR2A及NR2B受体表达变化

目的探讨N-甲基-D-天冬氨酸受体亚单位NR2A/2B表达与缺血再灌注损伤的关系。方法建立局灶性大脑中动脉阻塞大鼠模型观察缺血2 h再灌6～96 h的组织病理学改变,实时荧光定量PCR及Western印迹法测定大脑皮质NR2A/2B mRNA及蛋白表达。结果大鼠缺血再灌注后6 h,皮质开始出现明显病理学改变,12 h可见血管内有淤血,24 h梗死区锥体细胞出现严重的核固缩、核溶解,几乎看不到正常神经元,48 h出现大面积角质化,96 h可见炎症细胞浸润。与假手术组相比,再灌组NR2A/2B mRNA于再灌注6 h即开始一直持续明显下降(P<0.01),再灌12和24 hNR2A/2B mRNA比值均为1∶2,偏离正常的1∶1,48 h两者的表达开始上调,至96 h NR2A/2B mRNA比值达到1∶1;再灌24 h后NR2A蛋白表达显著降低(P<0.05);NR2B蛋白于再灌6 h开始明显降低,一直持续到24 h(P<0.01),48 h开始上调,96 h后蛋白表达接近假手术组水平。结论缺血2 h再灌注24 h后神经元损伤最严重,并与NR2A/2B表达改变存在时间一致性和受体亚型选择性。     

Potential therapeutic interest of adenosine A2A receptors in psychiatric disorders

The interest on targeting adenosine A(2A) receptors in the realm of psychiatric diseases first arose based on their tight physical and functional interaction with dopamine D(2) receptors. However, the role of central A(2A) receptors is now viewed as much broader than just controlling D(2) receptor function. Thus, there is currently a major interest in the ability of A(2A) receptors to control synaptic plasticity at glutamatergic synapses. This is due to a combined ability of A(2A) receptors to facilitate the release of glutamate and the activation of NMDA receptors. Therefore, A(2A) receptors are now conceived as a normalizing device promoting adequate adaptive responses in neuronal circuits, a role similar to that fulfilled, in essence, by dopamine. This makes A(2A) receptors particularly attractive targets to manage psychiatric disorders since adenosine may act as go-between glutamate and dopamine, two of the key players in mood processing. Furthermore, A(2A) receptors also control glia function and brain metabolic adaptation, two other emerging mechanisms to understand abnormal processing of mood, and A(2A) receptors are important players in controlling the demise of neurodegeneration, considered an amplificatory loop in psychiatric disorders. Current data only provide an indirect confirmation of this putative role of A(2A) receptors, based on the effects of caffeine (an antagonist of both A(1) and A(2A) receptors) in psychiatric disorders. However, the introduction of A(2A) receptors antagonists in clinics as anti-parkinsonian agents is hoped to bolster our knowledge on the role of A(2A) receptors in mood disorders in the near future.     

Importance of ERK1/2 in upregulation of endothelin type B receptors in cerebral arteries

This study examines the importance of mitogen-activated protein kinases (MAPKs) in upregulation of endothelin type B (ETB) receptors. Rat middle cerebral arteries (MCAs) were incubated for 24 h with or without kinase inhibitors. Vessel segments were mounted in myographs and the contractile responses to endothelin-1 (ET-1; ETA and ETB receptor agonist) and sarafotoxin 6c (S6c; ETB receptor agonist) were studied. We used real-time PCR to measure the receptor mRNA levels. An ELISA assay showed the activation of ERK1/2 kinases after 3 h. Immunohistochemistry revealed the presence of ETB receptors on the vessels. After organ culture, S6c induced vasoconstriction. Incubation with the MEK/ERK inhibitors U0126 and SB386023 diminished the contractile response to S6c. The p38 MAPK inhibitor SB239063 did not affect the S6c-induced contraction. The ET-1-induced vasoconstriction was increased after incubation with SB386023 or SB239063, while unaffected by U0126. The ETB receptor mRNA levels were diminished by SB386023 and U0126. The ETA receptor mRNA levels were unaffected. The levels of activated ERK1/2 kinases were significantly higher after 3 h of organ culture as compared to fresh vessels. The level of ETB receptor protein on the smooth muscle cells of the MCA, visualised by immunohistochemistry, was somewhat diminished by SB386023. Our results show that the ERK1/2 MAPK is important in the upregulation of contractile ETB receptors in MCA after organ culture. Since there is a similar upregulation in models of focal ischaemia and subarachnoid haemorrhage, this may be an important pathophysiological event.     

A2b adenosine receptors can change their spots

Recently, a central role for the A_2b adenosine receptor in a variety of cardiovascular functions including inflammation, erectile function, coronary artery dilation, asthma and cardioprotection has been demonstrated. Despite this evidence, the low-affinity A_2b adenosine receptor is still poorly understood. This receptor appears to be very promiscuous in its coupling. In most tissues, it couples to G_s much like its cousin, the A_2a adenosine receptor, but in mast cells and now, most recently, in cardiac fibroblasts, the A_2b receptor also couples to G_q. Because of its low affinity, this receptor was originally thought unlikely to play any important physiological role. But the sensitivity of A_2b adenosine receptors can be greatly increased by interaction with protein kinase C (PKC) making this receptor, under various conditions, both an activator and a target of PKC. We have recently documented a third coupling involving G_i. This plasticity and versatility of A_2b adenosine receptors position them as potential triggers of signalling in multiple signalling cascades in many physiological responses, making this a most interesting receptor indeed.This article is a commentary on Feng et al., pp. 1598–1607 of this issue. To view this paper visit http://dx.doi.org/10.1111/j.1476-5381.2009.00558.x     

Molecular characterization of A1 and A2a adenosine receptors

Detailed amino acid sequence analyses of A₁ and A_2a adenosine receptors were assembled by analogy to other G-protein-coupled receptors and correlated with pharmacological observations. Sites for phosphorylation, palmitoylation, and sodium binding have been proposed. Striatal A_2a receptors from human and other species were photoaffinity-labeled using the selective, radioiodinated agonist PAPA-APEC. Selective chemical affinity labels for A₁ and A_2a receptors have been introduced. For example, an isothiocyanate, ρ-DITC-APEC (100 nM), irreversibly diminished the B_max for [³H]CGS 21680 (2-[4-[(2-carboxyethyl) phenyl] ethylamino]-5′-N-ethylcarboxamido-adenosine) binding in rabbit striatal membranes by 71% (K_d unaffected), suggesting a direct modification of the ligand binding site. Novel trifunctional affinity labels have been designed. Rabbit and human A_2a receptors were characterized using [³H]XAC binding in the presence of 50 or 25 nM CPX (8-cyclopentyl-1,3-dipropylxanthine), respectively. The inhibition of A₂ radioligand binding by the histidyl-modifying reagent diethylpyrocarbonate suggested the involvement of His residues in interactions with adenosine agonists and antagonists. Properties of transiently expressed mutants of bovine A₁ receptors in which either His²⁵¹ or His²⁷⁸ residues have been substituted with Leu suggest that both histidines are important in binding. © 1993 Wiley-Liss, Inc.     

Characterization of adenosine receptors in isolated cerebral arteries of cat.

The effect of some adenosine analogues and xanthine derivatives were studied on isolated cerebral arteries from cats. The adenosine analogues caused an almost complete relaxation of cerebral arteries contracted by prostaglandin F2 alpha (PGF2 alpha, 30 microM). The order of potency was: 5-N-ethylcarboxamide adenosine (NECA) greater than 2-chloroadenosine greater than adenosine greater than L-N6-phenylisopropyl adenosine (L-PIA). The analogue D-PIA was very weak and its maximum effect was small. NECA and L-PIA enhanced [3H]-cyclic AMP accumulation in [3H]-adenine labelled feline pial vessels with similar absolute and relative potency to their relaxant effects. The relaxant effects of adenosine and of NECA were competitively antagonized by 8-phenyl-theophylline (pA2 = 6.5). The effect of theophylline and enprofylline could not be tested in higher concentrations than 30 or 10 microM because they affected the vessels directly. At these concentrations they were essentially inactive as adenosine antagonists. The non-xanthine phosphodiesterase inhibitor rolipram (0.1 and 100 microM) caused a slight but non-significant potentiation of the relaxant effect of adenosine. The results are compatible with the opinion that adenosine relaxes cerebral vessels by an action on adenosine A2-receptors. The effect may be linked to adenylate cyclase and can be antagonized by 8-phenyl-theophylline.     

美洛昔康对全脑缺血/再灌注大鼠脑损伤的作用观察

目的探讨美洛昔康对大鼠全脑缺血/再灌注损伤的保护作用。方法采用双侧颈总动脉夹闭合并低血压方法建立全脑缺血/再灌注大鼠模型。美洛昔康(1、3和5mg·kg-1)在缺血前30min腹腔注射给药。Morris水迷宫测定大鼠空间学习能力,病理切片HE染色观察海马神经元形态结构,免疫组化检测海马组织核转录因子NF-κB p65蛋白表达,生物酶学方法观察超氧歧化酶活性和丙二醛含量。结果美洛昔康能明显缩短全脑缺血/再灌注大鼠的寻台潜伏期,减轻全脑缺血/再灌注大鼠海马神经元损伤,降低海马神经元NF-κB p65蛋白表达,明显阻遏全脑缺血/再灌注大鼠海马MDA含量的升高和SOD活性的降低。结论美洛昔康对全脑缺血/再灌注致大鼠脑损伤有明显的保护作用,其机制可能与其抑制COX-2活性,减少PGs等代谢产物的产生,抑制NF-κB活性,从而抑制炎症反应和氧化应激功能有关。