小鼠脑组织腺苷A2A受体缺失模型的建立与评价

制备了脑组织腺苷A2A受体基因缺失的小鼠模型并对该模型进行评价。在采用2次6.2 Gy X线间隔照射对敲除A2A受体基因的雌性C57BL/6小鼠进行清髓处理后, 将野生型雄性C57BL/6小鼠骨髓细胞移植到其体内, 使其脑组织的A2A受体仍保持缺失型。然后对移植效果进行鉴定, 并对模型的生理指标进行观察和评价。结果发现: 骨髓移植6周后, 受体小鼠的白细胞性染色体基因由雌性变为雄性; 骨髓中A2A受体阳性细胞率为94.85%, 而脑内A2A受体mRNA与A2A受体基因敲除小鼠比较, 无显著差异。模型小鼠除心率略低于野生型小鼠外, 在呼吸频率、脑含水量以及脑内谷氨酸含量等生理指标上均与野生型小鼠和A2A基因敲除小鼠无显著差异。     

小鼠脑组织腺苷A2A受体缺失模型的建立与评价

制备了脑组织腺苷A2A受体基因缺失的小鼠模型并对该模型进行评价。在采用2次6.2 Gy X线间隔照射对敲除A2A受体基因的雌性C57BL/6小鼠进行清髓处理后, 将野生型雄性C57BL/6小鼠骨髓细胞移植到其体内, 使其脑组织的A2A受体仍保持缺失型。然后对移植效果进行鉴定, 并对模型的生理指标进行观察和评价。结果发现: 骨髓移植6周后, 受体小鼠的白细胞性染色体基因由雌性变为雄性; 骨髓中A2A受体阳性细胞率为94.85%, 而脑内A2A受体mRNA与A2A受体基因敲除小鼠比较, 无显著差异。模型小鼠除心率略低于野生型小鼠外, 在呼吸频率、脑含水量以及脑内谷氨酸含量等生理指标上均与野生型小鼠和A2A基因敲除小鼠无显著差异。     

腺苷A2a受体的矛盾作用:保护还是加重损伤?

腺苷广泛存在于细胞外液中,通过A1、A2a、A2b和A34种受体发挥多种不同效应。其中A2a受体由于其作用的复杂性受到了广泛的关注。该受体的活化在神经系统中具有保护和加重损伤两种效应。在神经系统以外的组织器官,激活A2a受体可有效减轻组织损伤。目前认为,A2a受体激动剂和拈抗剂对于治疗心血管疾病及帕金森病具有良好的应用前景,因此对该受体的矛盾效应及产生的机制进行深入研究,将有助于促进A2a受体调节药物在临床治疗中的应用。     

腺苷A2a受体在博莱霉素诱导的小鼠肺纤维化中的作用

目的：观察腺苷A_2a受体（A_2aR）在小鼠肺纤维化形成中的调控作用。方法：30只雄性SPF级野生型BALB/C小鼠和20只A_2aR基因敲除BALB/C小鼠,随机分为以下5组：野生型小鼠对照组（A组）、野生型小鼠纤维化组（B组）、A_2aR基因敲除小鼠对照组（C组）、A_2aR基因敲除小鼠纤维化组（D组）、野生型小鼠纤维化+A_2aR激动剂（CGS21680）组（E组）,每组各10只。纤维化组小鼠气管内注入博莱霉素（bleomycin,BLM）溶液50μl （5 mg/kg体重）,对照组在气管内注入等体积生理盐水,注射后立即将小鼠直立旋转3~5 min,使其均匀分布于两肺。A、B、C、D各组每天腹腔注射生理盐水0.5 ml,E组每天腹腔注射A_2aR激动剂（CGS21680）0.5 ml （0.25 mg/kg体重）,连续28 d。第29天取血检测血清转化生长因子β1（TGF-β1）含量;检测肺组织中羟脯氨酸（hydroxyproline,Hyp）、TGF-β1和A_2aR蛋白质含量,TGF-β1 mRNA、A_2aR mRNA的表达,并观察肺组织光镜和超微结构。结果：①光镜和超微结构提示,B组肺泡壁增厚破坏,肺泡腔狭窄或部分陷闭,纤维增生,炎细胞浸润,I型、Ⅱ型肺泡上皮细胞明显空泡化,D组较B组明显,E组表现较B组减轻。Masson染色提示B组、D组小鼠肺组织纤维增生明显,E组明显减少,提示基因缺失加重了小鼠肺纤维化的形成。②与A组比较,B组血清TGF-β1含量,肺组织Hyp含量,TGF-β1、A_2aR蛋白质含量和TGF-β1 mRNA、A_2aR mRNA的表达均明显增高（P<0.01,P<0.05）。与C组比较,D组血清TGF-β1含量、肺组织Hyp、TGF-β1含量和TGF-β1 mRNA表达明显增高（P<0.01,P<0.05）,提示肺纤维化小鼠肺组织Hyp含量增高,TGF-β1表达上调。③与B组相比,D组血清TGF-β1、肺组织Hyp、TGF-β1含量和TGF-β1 mRNA表达明显增高（P<0.01）。与B组比较,E组A_2aR蛋白质含量和A_2aR mRNA的表达均增高（P<0.01）,而血清TGF-β1、肺组织Hyp和TGF-β1蛋白含量及TGF-β1 mRNA表达较B组明显下降（P<0.01,P<0.05）,提示A_2aR基因敲除小鼠肺组织Hyp含量增高、TGF-β1表达上调;A_2aR激动剂可使A_2aR表达上调、TGF-β1表达下降。结论：A_2aR基因敲除小鼠的肺纤维化明显增加。A_2aR在肺纤维化形成中反应性增高,可通过降低TGF-β1的表达而抑制肺纤维化形成。     

重组人源胶原蛋白对激光损伤小鼠皮肤修复作用及机制研究

本研究旨在观察重组人源胶原蛋白(recombinant human collagen,r-hc)对小鼠皮肤激光损伤的修复作用,初步探索其作用机制。应用458~514 nm激光照射小鼠背部皮肤制作皮肤损伤模型,将r-hc外涂于创伤皮肤,剂量为8 mg/mL(生理盐水配制),每天涂抹1次,连续给药14 d。分别于给药后1、4、7、14 d用双光子显微镜收集二次谐波(second harmonic generation,SHG)信号检测伤口真皮中的胶原纤维,并进行常规HE染色,观察伤口局部的病理学改变。体外试验检测r-hc对人皮肤角质形成细胞和成纤维细胞增殖活力的影响,计算细胞存活率。在小鼠模型上显示,与对照组相比,r-hc可明显加速伤口的愈合,缩短伤口愈合时间; SHG显示r-hc能够促进创伤局部胶原的产生;体外试验也显示它有促进人皮肤成纤维细胞和角质形成细胞增殖的能力。由此可见,r-hc(8 mg/mL)对小鼠激光损伤皮肤有修复作用,可能是通过促进表皮角质细胞和真皮成纤维细胞的增殖,促进胶原的沉积而发挥修复作用的。     

两种不同mGluR5抑制剂对创伤后Homerla蛋白表达水平影响的研究

目的:研究代谢型谷氨酸受体5(mGluR5)的激动剂依赖性活性和激动剂非依赖性对创伤性脑损伤后Homerla蛋白表达水平的影响.方法:离体培养神经元2w,采用mGluR5抑制剂2-甲基-6-苯基乙炔基嘧啶(MPEP)和α-甲基-4-羧苯基甘氨酸(MCPG)两种不同剂量预处理神经元细胞1h,神经元机械性损伤模型划伤细胞;利用立体定向仪固定大鼠,在大鼠脑皮层注射两种不同剂量的抑制剂,1h后采用自由落体损伤模型造成大鼠颅脑损伤.通过Westem blot分别检测离体和在体Homerla蛋白表达水平的变化.结果:两种不同剂量MPEP预处理后,大鼠脑皮层和离体培养的神经元细胞中Homerla蛋白表达水平表达明显减少,具有统计学差异(P＜0.05);而两种不同剂量的MCPG预处理后,Homerla蛋白表达水平的改变不明显.结论:颅脑损伤后内源性Homerla的产生是与代谢型谷氨酸受体激动剂非依赖性活性密切相关.     

Toll样受体在创伤后炎症反应中的作用

创伤后发生炎症反应以促进损伤恢复,反映了天然免疫的活化。Toll样受体家族(TLRs)不仅能识别病原体相关分子模式(PAMPs),近来发现其某些成员,尤其是TLR4对于无菌性损伤后释放的众多内源性分子即损伤相关分子模式(damage-associated molecular patterns,DAMPs)也有反应。通过基因突变或基因敲除动物证实,在局部损伤、失血性休克、股骨骨折等模型中,TLRs在局部炎症反应、远隔器官损害、全身炎症反应中也有重要作用。通过研究TLRs,对于深入理解创伤后局部及全身炎症反应发生、发展动态过程具有重要意义,有助于阐释创伤后并发症发生的机制,甚至可能寻找到调节创伤早期炎症反应的治疗靶点。     

基于转录组测序分析美洲大蠊提取物促进小鼠创面愈合的分子机制

美洲大蠊Periplaneta americana是传统的中药材,其提取物已在临床上广泛用于创面治疗,但对其中的分子机制知之甚少。本文通过构建小鼠皮肤创伤模型,并基于转录组测序(RNA-seq)深入分析美洲大蠊提取物促进小鼠创面愈合的分子机制。首先构建C57小鼠皮肤全层切除模型,并将其分为3组,其中实验组分别以2种美洲大蠊提取物(康复新液、精粉)为敷料,而对照组以75%乙醇为敷料。用药处理3 d后取伤口皮肤组织送样进行RNAseq,然后分析转录组数据找出与伤口愈合相关的差异性表达基因,并利用荧光定量PCR(Q-PCR)对伤口组织中相关基因的表达量进一步验证。实验结果显示,实验组小鼠伤口的结痂速度比对照组快,这表明了小鼠创面模型中,美洲大蠊提取物能促进伤口愈合。经转录组分析,得到显著性差异表达基因数:对照组vs.精粉组为545,对照组vs.康复新液组为938。结合生物信息分析结果和已发表的文献,发现3个可能参与调节伤口愈合的关键基因:表皮调节素(Ereg)、Gli-kruppel家族成员(Gli2)和表皮型转谷氨酰胺酶3(Tgm3)。Q-PCR实验表明,这3个基因在美洲大蠊提取物加药组中均高表达。本文研究结果表明,2种美洲大蠊提取物:康复新液、精粉可能通过诱导Ereg、Tgm3和Gli2的高表达来促进皮肤创面的愈合。     

α2A肾上腺素受体激动剂guanfacine增强大鼠空间学习能力而不影响条件性

α2A肾上腺素受体选择性激动剂guanfacine对空间工作记忆和选择性注意等前额叶皮层认知功能有重要的、有益的影响.然而,激活α2A受体对于依赖杏仁体和海马回路的恐惧记忆条件反射是否有影响,目前尚不清楚.本研究结果显示,全身给予guanfacine显著提高大鼠在Lashley迷宫中的空间学习能力:guanfacine组大鼠达到学会标准所需要的训练次数和所犯错误的次数显著少于生理盐水对照组大鼠.然而,guanfacine组大鼠场景和声音恐惧记忆的获得/巩固与对照组大鼠相比没有显著差异.结果提示,刺激α2A受体产生的有益效应是任务依赖的:guanfacine改善空间学习能力,但不影响恐惧记忆的获得/巩固.     

跑台运动对攻击行为大鼠内侧下丘脑和中脑导水管周围灰质5-HT1A受体、5-HT2A受体蛋白表达的影响

目的:探讨跑台运动对攻击行为大鼠内侧下丘脑(MH)和中脑导水管周围灰质(PAG)5-HT1A受体、5-HT2A受体蛋白表达的影响,为研究运动对攻击行为改善的神经生物学机制提供实验基础.方法:3月龄雄性SD大鼠40只,体重160～180 g,随机分为4组:安静组(A)、攻击模型组(G)、攻击跑台组(GP)、入侵组(R)....     

Adenosine A2A receptors control the extracellular levels of adenosine through modulation of nucleoside transporters activity in the rat hippocampus

Adenosine, a neuromodulator of the CNS, activates inhibitory-A1 receptors and facilitatory-A2A receptors; its synaptic levels are controlled by the activity of bi-directional equilibrative nucleoside transporters. To study the relationship between the extracellular formation/inactivation of adenosine and the activation of adenosine receptors, we investigated how A1 and A2A receptor activation modifies adenosine transport in hippocampal synaptosomes. The A2A receptor agonist, CGS 21680 (30 nm), facilitated adenosine uptake through a PKC-dependent mechanism, but A1 receptor activation had no effect. CGS 21680 (30 nm) also increased depolarization-induced release of adenosine. Both effects were prevented by A2A receptor blockade. A2A receptor-mediated enhancement of adenosine transport system is important for formatting adenosine neuromodulation according to the stimulation frequency, as: (1) A1 receptor antagonist, DPCPX (250 nm), facilitated the evoked release of [(3)H]acetylcholine under low-frequency stimulation (2 Hz) from CA3 hippocampal slices, but had no effect under high-frequency stimulation (50 Hz); (2) either nucleoside transporter or A2A receptor blockade revealed the facilitatory effect of DPCPX (250 nm) on [3H]acetylcholine evoked-release triggered by high-frequency stimulation. These results indicate that A2A receptor activation facilitates the activity of nucleoside transporters, which have a preponderant role in modulating the extracellular adenosine levels available to activate A1 receptors.     

Co-localization and functional interaction between adenosine A(2A) and metabotropic group 5 receptors in glutamatergic nerve terminals of the rat striatum

The anti-Parkinsonian effect of glutamate metabotropic group 5 (mGluR5) and adenosine A(2A) receptor antagonists is believed to result from their ability to postsynaptically control the responsiveness of the indirect pathway that is hyperfunctioning in Parkinson's disease. mGluR5 and A(2A) antagonists are also neuroprotective in brain injury models involving glutamate excitotoxicity. Thus, we hypothesized that the anti-Parkinsonian and neuroprotective effects of A(2A) and mGluR5 receptors might be related to their control of striatal glutamate release that actually triggers the indirect pathway. The A(2A) agonist, CGS21680 (1-30 nM) facilitated glutamate release from striatal nerve terminals up to 57%, an effect prevented by the A(2A) antagonist, SCH58261 (50 nM). The mGluR5 agonist, CHPG (300-600 mum) also facilitated glutamate release up to 29%, an effect prevented by the mGluR5 antagonist, MPEP (10 microm). Both mGluR5 and A(2A) receptors were located in the active zone and 57 +/- 6% of striatal glutamatergic nerve terminals possessed both A(2A) and mGluR5 receptors, suggesting a presynaptic functional interaction. Indeed, submaximal concentrations of CGS21680 (1 nM) and CHPG (100 microm) synergistically facilitated glutamate release and the facilitation of glutamate release by 10 nM CGS21680 was prevented by 10 microm MPEP, whereas facilitation by 300 microm CHPG was prevented by 10 nM SCH58261. These results provide the first direct evidence that A(2A) and mGluR5 receptors are co-located in more than half of the striatal glutamatergic terminals where they facilitate glutamate release in a synergistic manner. This emphasizes the role of the modulation of glutamate release as a likely mechanism of action of these receptors both in striatal neuroprotection and in Parkinson's disease.     

Study of Adenosine A2 Receptors in Membrane Preparations from Optic Tectum of Chicks

Binding properties of the subtypes of adenosine A2 receptors in membrane preparations and the effects of adenosine receptor ligands on cAMP accumulation in slices from the optic tectum of neonatal chicks have been investigated. [³H]2-[4-(2-p-carboxyethyl)phenylamino]-5'-N-ethylcarboxaminoadenosine (CGS 21680), a selective ligand for adenosine A2a receptors, did not bind to optic tectal membranes, as observed with rat striatal membranes. CGS 21680 also did not induce cyclic AMP accumulation in optic tectum slices. However, 5'-N-ethylcarboxamidoadenosine (NECA), 2-chloro-adenosine or adenosine induced a 2.5- to 3-fold increase on cyclic AMP accumulation in this preparation. [³H]NECA binds to fresh non-washed-membranes obtained from optic tectum of chicks, displaying one population of binding sites, which can be displaced by NECA, 8-phenyltheophylline, 2-chloro-adenosine, but is not affected by CGS 21680. The estimated K_D value was 400.90 ± 80.50 nM and the B_max was estimated to be 2.51 ± 0.54 pmol/mg protein. Guanine nucleotides, which modulate G-proteins activity intracellularly, are also involved in the inhibition of glutamate responses by acting extracellularly. Moreover, we have previously reported that guanine nucleotides potentiate, while glutamate inhibits, adenosine-induced cyclic AMP accumulation in slices from optic tectum of chicks. However, the guanine nucleotides, GMP or GppNHp and the metabotropic glutamate receptors agonist, 1S,3R-ACPD did not alter the [³H]NECA binding observed in fresh non-washed-membranes. Therefore, the adenosine A2 receptor found in the optic tectum must be the adenosine A2b receptor which is available only in fresh membrane preparations, and its not modulated by guanine nucleotides or glutamate analogs.     

Adenosine enhances glial glutamate efflux via A2a adenosine receptors

The present study investigated the effect of adenosine on glial glutamate efflux. Adenosine (from 1 nM to 100 microM) enhanced the release from cultured rat glial cells in a bell-shaped dose-responsive manner for the hippocampus and in a dose-dependent manner for the superior colliculus, and a similar increase was obtained with the A2a adenosine receptor agonist, 2-p-(2-carboxyethyl) phenethylamino-5'-N-ethylcarboxamidoadenosine hydrochloride (CGS21680), but not with the A1 adenosine receptor agonist, N6-cyclohexyladenosine (CHA). Adenosine and CGS21680 also enhanced glutamate efflux from Xenopus oocytes injected with the poly (A)+ mRNAs derived from cultured glial cells for the hippocampus and the superior colliculus together with and without the A2a adenosine receptor mRNA, but instead such increase was not found in oocytes expressing A2a adenosine receptors alone. The results of the present study thus suggest that adenosine enhances glutamate efflux from glial cells via A2a adenosine receptors, and this may represent a mechanism underlying the facilitatory action of adenosine on hippocampal and superior colliculus neurotransmissions.     

Modulation of A2A adenosine receptor(s) by K+(ATP) channels in bovine brain striatal membranes

The modulation of adenosine receptor with K+(ATP) channel blocker, glibenclamide, was investigated using the radiolabeled A2A-receptor selective agonist [3H]CGS 21680. Radioligand binding studies in bovine brain striatal membranes (BBM) indicated that unlabeled CGS 21680 displaced the bound [3H]CGS 21680 in a concentration-dependent manner with a maximum displacement being approximately 65% at 10(-4) M. In the presence of 10(-5) M glibenclamide, unlabeled CGS 21680 increased the displacement of bound [3H]CGS 21860 by approximately 28% at 10(-4) M. [3H]CGS 21680 bound to BBM in a saturable manner to a single binding site (Kd = 10.6+/-1.71 nM; Bmax = 221.4+/-6.43 fmol/mg of protein). In contrast, [3H]CGS 21680 showed saturable binding to two sites in the presence of 10(-5) M glibenclamide; (Kd = 1.3+/-0.22 nM; Bmax = 74.3+/-2.14 fmol/mg protein; and Kd = 8.9+/-0.64 nM; Bmax = 243.2+/-5.71 fmol/mg protein), indicating modulation of adenosine A2A receptors by glibenclamide. These studies suggest that the K+(ATP) channel blocker, glibenclamide, modulated the adenosine A2A receptor in such a manner that [3H]CGS 21680 alone recognizes a single affinity adenosine receptor, but that the interactions between K+(ATP) channels and adenosine receptors.     

Adenosine inhibits the release of interleukin-1beta in activated human peripheral mononuclear cells

The effects of adenosine and subtype-specific activators of adenosine receptors (A1, A2A, A2B and A3) were studied on the release of interleukin-1beta (IL-1beta) from peripheral mononuclear cells, monocytes and lymphocytes. In the cells activated by the protein kinase C specific phorbol ester (phorbol 12-myristate 13-acetate) and Ca(2+) ionophore (A23187) both adenosine and the subtype-specific receptor agonists, CPA (A1), CGS 21680 (A2A) and IB-MECA (A3) induced a concentration-dependent inhibition of IL-1beta release. The rank order of potency in the inhibition of IL-1beta release was CPA=CGS 21680>IB-MECA>adenosine>NECA (in the presence of A1, A2A and A3 receptor inhibitors). The inhibitory actions of CPA, CGS 21680 or IB-MECA were significantly reduced in the presence of DPCPX, ZM 243185 or MRS 1191 as subtype-specific antagonists on A1, A2A and A3 adenosine receptors, respectively. It can be concluded that adenosine inhibits the release of IL-1beta from the activated human peripheral mononuclear cells. In this process A1, A2A and A3 receptors are involved.     

Adenosine receptor-mediated modulation of dopamine release in the nucleus accumbens depends on glutamate neurotransmission and N-methyl-D-aspartate receptor stimulation

Adenosine, by acting on adenosine A(1) and A(2A) receptors, exerts opposite modulatory roles on striatal extracellular levels of glutamate and dopamine, with activation of A(1) inhibiting and activation of A(2A) receptors stimulating glutamate and dopamine release. Adenosine-mediated modulation of striatal dopaminergic neurotransmission could be secondary to changes in glutamate neurotransmission, in view of evidence for a preferential colocalization of A(1) and A(2A) receptors in glutamatergic nerve terminals. By using in vivo microdialysis techniques, local perfusion of NMDA (3, 10 microm), the selective A(2A) receptor agonist 2-p-(2-carboxyethyl)phenethylamino-5'-N-ethylcarboxamidoadenosine (CGS 21680; 3, 10 microm), the selective A(1) receptor antagonist 8-cyclopentyl-1,3-dimethylxanthine (CPT; 300, 1000 microm), or the non-selective A(1)-A(2A) receptor antagonist in vitro caffeine (300, 1000 microm) elicited significant increases in extracellular levels of dopamine in the shell of the nucleus accumbens (NAc). Significant glutamate release was also observed with local perfusion of CGS 21680, CPT and caffeine, but not NMDA. Co-perfusion with the competitive NMDA receptor antagonist dl-2-amino-5-phosphonovaleric acid (APV; 100 microm) counteracted dopamine release induced by NMDA, CGS 21680, CPT and caffeine. Co-perfusion with the selective A(2A) receptor antagonist MSX-3 (1 microm) counteracted dopamine and glutamate release induced by CGS 21680, CPT and caffeine and did not modify dopamine release induced by NMDA. These results indicate that modulation of dopamine release in the shell of the NAc by A(1) and A(2A) receptors is mostly secondary to their opposite modulatory role on glutamatergic neurotransmission and depends on stimulation of NMDA receptors. Furthermore, these results underscore the role of A(1) vs. A(2A) receptor antagonism in the central effects of caffeine.     

Sensitization by chronic diazepam treatment of A2A adenosine receptor-mediated relaxation in rat pulmonary artery

The effects of a 10-day i.p. treatment of rats with diazepam on responses to subtype selective adenosine receptor agonists were studied 3 h, 2 and 8 days after termination of diazepam treatment in isolated cardiovascular tissues possessing distinct adenosine receptors. After long-lasting diazepam exposure, the relaxation elicited by the specific A2A receptor agonist CGS 21680 was enhanced in rat main pulmonary arteries (a tissue containing A2A adenosine receptors). The increased sensitivity of A2A receptors observed 3 h and 2 days after withdrawal of diazepam was completely restored by the 8th day of the wash-out period. N6-cyclopentyladenosine (CPA)-induced suppression in mechanical activity of electrically stimulated rat atrial myocardium (a tissue containing A1 adenosine receptors) was not altered following diazepam treatment. In order to reveal the possible role of inhibition of membrane adenosine transport in the effects of diazepam (a moderate inhibitor of membrane adenosine transport), the action of a 10-day treatment with dipyridamole or S-(p-nitrobenzyl)-6-thioinosine (NBTI; prototypic adenosine uptake inhibitors) was also studied. Dipyridamole or NBTI treatment, like diazepam, increased the responsiveness of rat pulmonary artery to CGS 21680, but did not influence the cardiodepressive effect of CPA in electrically driven left atrial myocardium. The CGS 21680-induced relaxations were significantly antagonized by 10 nM ZM 241385 (a selective A2A adenosine receptor antagonist) in vessels of diazepam-treated rats. The relaxation responses to verapamil were unaltered in pulmonary arteries obtained from animals chronically treated with diazepam, dipyridamole or NBTI. These results suggest that chronic diazepam treatment is able to enhance the A2A adenosine receptor-mediated vascular functions, but does not modify the responses mediated via A1 receptors of rat myocardium, where nucleoside transport inhibitory sites of membrane are of a very low density. It is possible that sensitization of A2A adenosine receptor-mediated vasorelaxation is due to a long-lasting inhibition of membrane adenosine transporter during diazepam treatment.     

Interaction of Adenosine and Guanine Derivatives in the Rat Hippocampus: Effects on Cyclic AMP Levels and on the Binding of Adenosine Analogues and GMP

Guanine nucleotides (GN) have been implicated in many intracellular mechanisms. Extracellular actions, probably as glutamate receptor antagonists, have also been recently attributed to these compounds. GN may have a neuroprotective role by inhibiting excitotoxic events evoked by glutamate. Effects of extracellular GN on adenosine-evoked cellular responses have also been reported. However, the exact mechanism of such interaction is not known. In the present study, we showed that GN potentiated adenosine-induced cAMP accumulation in slices of hippocampus from young rats. However, neither GMP nor the metabotropic glutamate receptor agonist, 1S,3R-ACPD, inhibited the binding of the adenosine receptor agonist [³H]NECA (when binding to adenosine A2 receptors), or the binding of the adenosine A2a receptor agonist [³H]CGS 21680 in hippocampal membrane preparations. GppNHp, probably by interacting with G-proteins, decreased [³H]CGS 21680 binding. [³H]GMP binding was assayed in order to evaluate the GN sites which are not G-proteins. [³H]GMP binding was inhibited by GMP and GppNHp, but not by 1S,3R-ACPD. The interaction of endogenous adenosine with the GMP-binding sites was determined by incubating membranes in the presence or absence of adenosine deaminase (ADA). NECA, CADO, CGS 21680 and CPA (only at the highest concentration used) increased GMP binding in the presence of ADA. However, in the absence of ADA, the control levels of GMP binding were as high as in the presence of added ADA plus adenosine agonists, indicating that endogenous adenosine modulates the binding of GMP. If this site has a neuroprotective role, adenosine may be increasing its neuromodulator and proposed protective action.     

Adenosine A2A receptor activation is determinant for BDNF actions upon GABA and glutamate release from rat hippocampal synaptosomes

Adenosine, through A_2A receptor (A_2AR) activation, can act as a metamodulator, controlling the actions of other modulators, as brain-derived neurotrophic factor (BDNF). Most of the metamodulatory actions of adenosine in the hippocampus have been evaluated in excitatory synapses. However, adenosine and BDNF can also influence GABAergic transmission. We thus evaluated the role of A_2AR on the modulatory effect of BDNF upon glutamate and GABA release from isolated hippocampal nerve terminals (synaptosomes). BDNF (30 ng/ml) enhanced K⁺-evoked [³H]glutamate release and inhibited the K⁺-evoked [³H]GABA release from synaptosomes. The effect of BDNF on both glutamate and GABA release requires tonic activation of adenosine A_2AR since for both neurotransmitters, the BDNF action was blocked by the A_2AR antagonist SCH 58261 (50 nM). In the presence of the A_2AR agonist, {"type":"entrez-protein","attrs":{"text":"CGS21680","term_id":"878113053","term_text":"CGS21680"}}CGS21680 (30 nM), the effect of BDNF on either glutamate or GABA release was, however, not potentiated. It is concluded that both the inhibitory actions of BDNF on GABA release as well as the facilitatory action of the neurotrophin on glutamate release are dependent on the activation of adenosine A_2AR by endogenous adenosine. However, these actions could not be further enhanced by exogenous activation of A_2AR.