细胞内三磷酸腺苷和腺苷在细胞凋亡中的作用研究进展

众所周知,细胞内三磷酸腺苷(ATP)是维持细胞生命的重要能量物质,细胞内ATP缺失将导致细胞死亡[1]。近年的研究表明,ATP和腺苷还是细胞内外重要的信号分子,特别是在外周和神经系统中发挥着重要的作用[2]。此外,越来越多的研究表明,ATP和腺苷对神经系统、免疫系统以及内皮细胞和肿瘤细胞在内的多种细胞具有细胞毒作用[3],该作用主要是通过诱导细胞凋亡而实现。笔者近来的研究结果亦表明,ATP和腺苷对食管癌TE-13和胃癌HGC-27细胞具有抑制增殖、诱导凋亡的作用[4~6]。有关ATP和腺苷诱导细胞凋亡作用的研究目前多集中在嘌呤受体的介导…     

腺苷体外对人肝癌HepG2细胞凋亡的诱导作用及其作用机制

目的研究腺苷及其代谢途径对人肝癌HepG2细胞凋亡的诱导作用及其机制。方法将腺苷2.0mmol.L-1作用于HepG2细胞24和48h,采用流式细胞术(FCM)测定细胞周期及凋亡率;观察腺苷膜转运体抑制剂双嘧达莫、腺苷脱氨酶抑制剂红-9-(2-羟基-3-壬烷基)腺嘌呤(EHNA)和腺苷激酶抑制剂5′-氨基5′-脱氧腺苷(AMDA)对腺苷抑制细胞存活的影响,应用MTT法测定细胞存活率;应用Western蛋白印迹法检测P53和Bcl-2蛋白的表达。结果腺苷与HepG2细胞作用24和48h,HepG2细胞出现特征性的亚二倍体凋亡峰,细胞凋亡百分率分别由对照组的(1.2±0.4)%和(4.1±1.6)%增加到(24.3±4.8)%和(38.6±7.4)%,细胞周期阻滞于G0/G1期。腺苷与HepG2细胞作用24h明显抑制细胞存活,P53表达明显增强,Bcl-2表达降低。预先分别给予双嘧达莫,AMDA和AMDA+双嘧达莫处理后,各处理组HepG2细胞存活率较腺苷组升高,细胞凋亡百分率和P53表达降低,Bcl-2表达无明显变化;EHNA预处理组细胞存活率、细胞凋亡百分率、P53和Bcl-2表达与腺苷组比较均无明显变化。结论腺苷可诱导HepG2细胞凋亡,腺苷在胞内可能通过腺苷激酶而不是通过转氨酶的代谢途径参与诱导细胞凋亡的过程。腺苷对HepG2细胞凋亡的诱导作用还可能与其增加P53蛋白表达有关。     

外源性三磷酸腺苷和腺苷对肿瘤细胞增殖的影响

目的　研究三磷酸腺苷 (ATP)和腺苷 (ADO)对人红白血病细胞株K5 6 2、人胃中分化腺癌细胞株HGC 2 7、人食管低分化鳞癌细胞株TE 13细胞增殖的影响。方法　采用MTT法测定ATP和ADO抑制肿瘤细胞增殖的作用 ,Wright’s Giemsa染色观察细胞形态学的改变。结果　在 0 0 1～ 1 0mmol·L-1浓度范围内 ,ATP和ADO可不同程度地抑制TE 13、HGC 2 7和K5 6 2细胞的增殖 ,其中对TE 13的作用最强 ;ATP和ADO作用 72h后 ,对TE 13细胞的抑制率分别为80 5 2 %和 74 0 3%。细胞经高浓度 (1mmol·L-1)的ATP和ADO处理后 ,细胞形态出现了凋亡的特征。结论　ATP抗TE 13和HGC 2 7肿瘤细胞增殖的作用与其代谢产物ADO部分相关 ,ATP和ADO的作用机制可能涉及细胞凋亡     

外源性腺苷三磷酸和腺苷对食管癌TE-13细胞株凋亡的影响

目的　探讨腺苷三磷酸 (ATP)和腺苷 (ADO)是否具有诱导人食管低分化鳞癌细胞株TE 13凋亡的作用。方法　MTT法 ,AO/EB双荧光染色法 ,琼脂糖凝胶电泳 ,流式细胞仪方法。结果　ATP在0 .1～ 1.0mmol·L- 1或ADO在 0 .0 1～ 1mmol·L- 1浓度范围内 ,可不同程度地抑制TE 13细胞的增殖 ,ATP和ADO作用 72h后 ,对TE 13细胞的最大抑制率分别达 (80 .5± 6 .2 ) %和 (74 .0± 5 .3) %。细胞经0 .3mmol·L- 1的ATP或ADO处理 4 8h后 ,细胞形态出现了凋亡特征 ;电泳可见明显的“梯状”条带 ;ATP和ADO可浓度依赖性地诱导细胞的凋亡 ,1mmol·L- 1的ATP和ADO诱导细胞的凋亡率分别为 (16 .6± 1.1) %和 (16 .9± 1.2 ) %。结论　ATP和ADO均有诱导食管癌TE 13细胞凋亡的作用     

腺苷三磷酸通过P1受体影响兔窦房结起搏细胞电生理活动(英文)

目的:研究腺苷三磷酸(ATP)对兔离体窦房结起搏细胞动作电位的作用,分析相关受体。方法:利用细胞内微电极技术记录兔离体窦房结细胞跨膜电位。结果:ATP 0.1-3.0mmol/L浓度依赖性减慢窦房结自发搏动速率16％-43％,降低舒张期除极速率33％-67％,增大动作电位幅值6％9％,加快最大除极速率30％-76％,APD_(50)和APD_(90)分别缩短7％-12％和6.3％-9％,等浓度ATP、腺苷二磷酸(ADP)和腺苷(Ado)的效应相比时,各组间无显著性差异,尿苷三磷酸(UTP)和α,β-meATP对动作电位各参数均无影响,P1受体拮抗剂氨茶碱(0.1mmol/L)显著拮抗ATP和Ado的作用(P<0.05),且拮抗程度相同,P2受体拮抗剂反应兰2 (0.05mmol/L)不影响ATP的作用(P<0.05)。结论:兔窦房结不存在功能性P2X,和P2Y_2受体,ATP对兔窦房结的作用主要通过其降解产物Ado,由P1受体介导而发挥。     

P2X受体的结构解析进展

<正>三磷酸腺苷(adenosine triphosphate,ATP)配体门控P2X受体家族是由7个克隆亚型分别组装的同源或异源三聚体离子通道。研究显示,ATP不仅是细胞中主要的能量来源和核酸组分,在细胞间信息交流中也发挥重要作用。已证实[1-2],P2X受体为非选择性阳离子通道,存在于哺乳动物组织中并调节多种生理功能,可影响神经系统突触信号的快速传递,平滑肌细胞收缩、血小板聚集、巨噬细胞活化、细胞增殖和死亡等。P2X受体斑马鱼P2X4受体晶体结构的发现极大地推进了对其P2X受体分子和生理功能的理解,以及其药物靶点的预测和实验验证。本文探讨P2X受体结构和功能的关系,并简单介绍P2X受体选择性配体最新研究成果。     

内分泌胰腺嘌呤受体研究的新进展

嘌呤受体分为腺苷作用的P1受体和ATP作用的P2受体两大类;P2受体又分为P2X(离子通道受体)和P2Y(G蛋白耦联受体)。多种嘌呤受体亚型表达于内分泌胰腺,并参与调节胰岛素的分泌。嘌呤受体和配体与糖尿病及其并发症的发病机制密切相关,并可能成为糖尿病及其并发症治疗的新靶点。     

颈上交感神经节细胞P2X_3受体介导心肌痛伤害性信息作用的电生理研究

目的观察心肌缺血致心肌痛伤害性剌激后颈上神经节(superior cervical ganglion,SCG)细胞P2X3受体在心肌痛伤害性信息传递中的作用。方法用全细胞膜片钳技术记录心肌缺血组和对照组大鼠新鲜分离的SCG神经元P2X受体激动剂激活电流及P2X3受体特异性拮抗剂A-317491对P2X受体激动剂激活电流的作用。结果①大部分受检细胞对ATP(1～1000μmol.L-1)敏感,ATP-激活电流(IATP)显示快失敏和慢失敏两种形式的内向电流,心肌缺血组IATP明显高于同一浓度时对照组的IATP。②在心肌缺血组SCG细胞,P2X3受体选择性激动剂α、β亚甲三磷酸腺苷(α,β-meATP)产生激活电流,而在对照组SCG细胞只有极少数细胞有此反应,且电流幅度小。③P2X3受体特异性拮抗剂A-317491(100nmol.L-1)对心肌缺血组和对照组ATP(100μmol.L-1)-激活电流均出现抑制作用,且同一浓度的A-317491对心肌缺血组IATP的抑制作用较对照组IATP的作用更为明显。A-317491(100nmol.L-1)使心肌缺血组和对照组SCG细胞α,β-meATP(10μmol.L-1)-激活电流减小,对心肌缺血组的抑制作用更明显。④心肌缺血组和对照组SCG细胞ATP(100μmol.L-1)-激活电流的I-U曲线反转电位不变,均接近0mV。结论心肌缺血后P2X受体激动剂在SCG神经元激活电流增大,P2X3受体特异性拮抗剂A-317491可抑制P2X受体激动剂激活的电流。因此,颈上交感神经节细胞的P2X3受体可能参与心肌痛伤害性感受信息的传递。     

蜗牛多肽混合物抗过氧化氢诱导的SH-SY5Y细胞损伤的作用

目的观察蜗牛多肽混合物对过氧化氢(H2O2)诱导SH-SY5Y细胞氧化损伤的抑制作用及其可能机制。方法用H2O2诱导SH-SY5Y细胞损伤,MTT法测定细胞存活率;39～1250 mg·L-1蜗牛多肽混合物处理后,Hoechst染色检测细胞凋亡;罗丹明123染色检测线粒体通透性;细胞免疫化学技术和Western blot技术分别检测增殖细胞核抗原(PCNA)和脑源性神经营养因子(BDNF)的表达。结果 1.54 mmol·L-1H2O2可诱导SH-SY5Y细胞凋亡。用39～156 mg·L-1浓度的蜗牛多肽混合物处理后,H2O2诱导的SH-SY5Y细胞凋亡数量明显减少,同时可减少H2O2引起的线粒体通透性增加,升高PCNA与BDNF的表达(P﹤0.01)。结论蜗牛多肽混合物可抑制过氧化氢诱导的SH-SY5Y细胞凋亡,其作用机制可能与其增加细胞PCNA与BDNF的表达有关。     

腺苷诱导人食管癌EC109细胞凋亡及其内质网分子机制

目的探讨内质网应激途径在腺苷诱导食管癌EC109细胞凋亡中的作用。方法腺苷2 mmol.L-1作用于EC109细胞24～72 h或腺苷0.5～4 mmol.L-1作用于EC109细胞36 h,MTT法观察腺苷对EC109细胞存活率的时效和量效关系;免疫荧光法检测葡萄糖调节蛋白78(GRP78),胱天蛋白酶4,胱天蛋白酶3,转录因子CHOP和核因子κB(NF-κB)的表达及亚细胞定位;原位末端转移酶标记技术检测细胞凋亡;Western蛋白印迹法检测内质网应激相关蛋白表达。结果腺苷对EC109细胞生长具有明显的抑制作用。腺苷2 mmol.L-1与EC109细胞作用24,36,48和72 h,细胞存活率分别为(57.7±15.0)%,(56.5±11.1)%,(43.8±5.7)%和(28.8±4.1)%,呈时间依赖性下降(r=0.9192,P<0.01);腺苷0.5,1,2和4 mmol.L-1与EC109细胞作用36 h,随药物浓度增加,细胞存活率依次为(83.1±11.2)%,(67.9±6.7)%,(55.3±5.0)%和(45.4±5.4)%,呈浓度依赖性降低(r=0.8252,P<0.01)。腺苷0.5～4mmol.L-1与EC109细胞作用36 h,细胞凋亡率分别为(15.5±1.1)%,(28.2±0.8)%,(40.1±2.2)%和(50.6±1.3)%,与对照组(2.1±0.3)%相比均明显增加(P<0.05)。腺苷2 mmol.L-1与EC109细胞作用36 h,与正常对照组相比,GRP78、胱天蛋白酶4、胱天蛋白酶3和CHOP表达明显增强(P<0.05,P<0.01),胱天蛋白酶4,胱天蛋白酶3和CHOP发生核易位。GRP78、胱天蛋白酶4、胱天蛋白酶3、CHOP和NF-κB表达呈浓度依赖性增加(rGRP78=0.9471,r胱天蛋白酶4=0.8977,r胱天蛋白酶3=0.968,rCHOP=0.9762,rNF-κB=0.9471,P<0.05)。结论腺苷可诱导人食管癌EC109细胞凋亡,其分子机制可能与内质网应激凋亡途径的启动有关。     

Therapeutic potential of adenosine analogues and conjugates

This review summarizes current knowledge of adenosine analogues and conjugates with promising therapeutic properties. Adenosine is a signaling molecule that triggers numerous physiological responses. It acts through the adenosine receptors (ARs), belonging to the family of G-protein-coupled receptors and widely distributed throughout the body. Moreover, adenosine is involved in key biochemical processes as a part of ATP, the universal energy currency. Thus, compounds that are analogues of adenosine and its conjugates have been extensively studied as potential therapeutics. Many inhibitors of ARs are in clinical trials as promising agents in treatment of inflammation, type 2 diabetes, arrhythmia and as vasodilators used in the myocardial perfusion imaging (MPI) stress test. Furthermore, adenosine analogues revealed high efficacy as enzyme inhibitors, tested for antitrypanosomal action and as bivalent ligands and adenosine-oligoarginine conjugates as inhibitors of protein kinases.     

ATP- and adenosine-mediated signaling in the central nervous system: adenosine receptor activation by ATP through rapid and localized generation of adenosine by ecto-nucleotidases

Extracellular ATP is now recognized as a neurotransmitter or neuromodilator in the nervous system, producing diverse physiological effects by activating multiple P2 receptors. Although P2-receptor signaling is terminated by hydrolysis of ATP by the ecto-nucleotidase cascade, such a metabolic step leads to adenosine generation, thereby initiating adenosine (P1)-receptor activation. Because most cells and tissues co-express P1 and P2 receptors, ecto-nucleotidase on target tissues, especially enzymes catalyzing adenosine formation, are determinants of the cellular response to ATP. Ecto-5'-nucleotidase (E-5'-NT) has been considered to play a principal role in conversion of AMP to adenosine. In addition to E-5'-NT, we have recently demonstrated that ecto-alkaline phosphatase is also involved in ATP-induced P1-receptor activation through a rapid and localized adenosine production on the membrane surface. In this minireview, we describe the pharmacological profile of ecto-nucleotidase-dependent P1-receptor activation by ATP and molecular bases of preferential delivery of metabolically generated adenosine to P1 receptors. Several lines of evidence suggest that the close association between ecto-nucleotidases and P1 receptors may constitute a functional receptor for extracellular ATP, and some physiological responses to ATP would occur through this mechanism.     

ATP and adenosine inhibit transmitter release at the frog neuromuscular junction through distinct presynaptic receptors

1. The effects of exogenous ATP or adenosine on end-plate currents (e.p.cs; evoked by simultaneous action of a few hundred quanta of ACh) or on miniature e.p.cs (m.e.p.cs) were studied under voltage clamp conditions on frog sartorius muscle fibres.
2. ATP or adenosine (100 μM–1 mM) reduced the e.p.c. amplitude but did not affect m.e.p.c. amplitude, decay time constant and voltage-dependence of m.e.p.c., suggesting that e.p.c. depression induced by these purines had presynaptic origin only.
3. The action of ATP, unlike that of adenosine, was prevented by the P2-purinoceptor antagonist suramin (100 μM). The stable ATP analogue α,β-methylene ATP (100 μM), known to be desensitizing agent on P2X receptors, also abolished the depressant effect of ATP while sparing the action of adenosine. Concanavalin A, an inhibitor of ecto-5′-nucleotidase, did not affect the presynaptic action of exogenously applied ATP.
4. The presynaptic action of adenosine was prevented by theophylline (1 mM), a blocker of adenosine receptors, while the effect of ATP was not changed under these conditions. The selective blocker of A₁ adenosine receptors, 8-cyclopentyl-1,3,dipropylxanthine (DPCPX; 0.1 μM), abolished the presynaptic action of adenosine but did not prevent the depressant effect of ATP.
5. The effects of ATP and adenosine (at nearly saturating concentration) were additive suggesting that these purines activated not only distinct receptors but also different intracellular signalling mechanisms.
6. In contrast to the hypothesis that at the neuromuscular junction ATP reduces transmitter release via enzymatic degradation to presynaptically active adenosine, our data suggest that ATP (through its own presynaptic receptors) directly inhibits ACh release. Thus, ATP and adenosine might be almost equipotent as endogenous prejunctional neuromodulators at the neuromuscular junction.

     

Presynaptic adenosine and P2Y receptors

Adenine-based purines, such as adenosine and ATP, are ubiquitous molecules that, in addition to their roles in metabolism, act as modulators of neurotransmitter release through activation of presynaptic P1 purinoceptors or adenosine receptors (activated by adenosine) and P2 receptors (activated by nucleotides). Of the latter, the P2Y receptors are G protein-coupled, whereas the P2X receptors are ligand-gated ion channels and not covered in this review.     

Activation of adenosine and P2Y receptors by ATP in human peripheral nerve

Receptors for ATP in the peripheral nervous system may contribute to the transduction of sensory, including nociceptive, stimuli and are candidates in the pathogenesis of neuropathic pain. In a complex neural tissue, such as the human peripheral nerve trunk, ATP may activate P2X, P2Y, and adenosine receptors present on various cell types. Experiments were performed on segments of isolated human sural nerves. The experimental set-up enabled simultaneous recording of C fiber excitability, intracellular Ca(2+) ([Ca(2+)](i)) and extracellular K(+) activity (aK(e)). The increase in excitability of unmyelinated fibers seen during bath application of both ATP and adenosine was reversed to a reduction in axonal excitability in the presence of 4-(2-[7-amino-2-(2-furyl)[1,2,4]triazolol[2,3-a][1,3,5]triazin-5-ylamino]ethyl)phenol (ZM 241385), an antagonist of adenosine A2 receptors. The pharmacological profile of the axonal subexcitability indicates the presence and activation of adenosine A1 receptors. Intracellular Ca(2+) transients were observed during bath application of ATP but not of adenosine and were blocked by 2'-deoxy- N(6)-methyladenosine 3',5'-bisphosphate (MRS 2179), an antagonist at P2Y(1) receptors. K(+)-sensitive microelectrodes were used to search for a possible activation of P2X receptors by ATP. In isolated rat vagus nerve, activation of P2X receptors by alpha,beta-methylene-adenosine 5'-triphosphate (alpha,beta-meATP) and by diadenosine pentaphosphate (Ap5A) resulted in a rapid, transient rise in the extracellular K(+) activity. In contrast, in human nerve, application of P2X receptor agonists did not result in a detectable elevation of aK(e). The data suggest that ATP-induced changes in axonal excitability and of [Ca(2+)](i) result from activation of adenosine A2, A1 and P2Y nucleotide receptors in human nerve; a contribution of P2X receptors was not found with the methods used. It is suggested that antagonists of A2 receptors might suppress enhanced activity in human nociceptive afferent nerve fibers under conditions in which ATP and/or adenosine is released into the trunk of a human peripheral nerve.     

Evidence for separate receptors for ATP and adenosine in the guinea-pig taenia coli

Inhibitory actions of three pairs of congeneric ATP and adenosine analogues on the isolated guinea-pig taenia coli were compared with the actions of ATP and adenosine. 8-Bromoadenosine, and 9-beta-D-arabinofuranosyladenosine (ara-adenosine) were inactive; 2'-deoxyadenosine was a weak partial agonist. 8-Bromo-ATP, ara-ATP and 2'-deoxy-ATP behaved like ATP and elicited rapid relaxations of the taenia but were not potentiated by dipyridamole. The divergent effects of identical structural modifications to ATP and adenosine provide evidence for separate adenosine and ATP receptors in the taenia coli.     

New heterocyclic ligands for the adenosine receptors P1 and for the ATP receptors P2

Extracellular adenosine and adenine nucleotides induce various cellular responses through activation of P1 and P2 receptors. P1 receptors preferentially recognize adenosine and four different G protein-coupled receptors (A(1), A(2A), A(2B), and A(3) subtypes) have been identified. On the other hand, P2 receptors are activated by adenine and/or uridine nucleotides and classified into two families: ionotropic P2X and G protein-coupled P2Y receptors. In this article, we summarize our studies which led to development of new potent and selective heterocyclic ligands for the adenosine receptors P1 and for the ATP receptors P2X(7).     

ATP通过P2受体调节大鼠近端结肠纵行肌的舒张与收缩反应(英文)

目的腺苷三磷酸(ATP)对大鼠离体远端结肠纵行肌运动的影响已明确,对近端结肠纵行肌的影响可能不同,但未有报告,为此对此进行观察并探讨其受体机制。方法观察静息张力时或预收缩时0.1μmol·L-1～1mmol·L-1ATP和1～100μmol·L-1腺苷对大鼠近端结肠纵行肌的抑制和兴奋作用。结果在静息张力下,1μmol·L-1～1mmol·L-1ATP对大鼠近端结肠纵行肌产生3种效应,即抑制自发性收缩反应,一过性轻度降低基础张力(0.05～0.08g),随后产生浓度依赖性收缩反应(0.04～0.44g)。0.1μmol·L-1河豚毒素不影响ATP的上述作用。在静息张力下,1～100μmol·L-1腺苷对近端结肠纵行肌未产生明显的收缩反应。应用5羟色胺(5HT)或乙酰胆碱(ACh)预收缩标本时,1μmol·L-1～1mmol·L-1ATP产生明显的浓度依赖性舒张反应(23.2%～94.6%,5HT预收缩;24.8%～92.4%,ACh预收缩),而腺苷引起的舒张反应明显小于ATP。结论在大鼠离体近端结肠纵行肌,ATP主要通过嘌呤嘧啶(P)2受体介导收缩反应,部分通过P1受体介导舒张反应。     

Pharmacological properties of adenosine receptors and adenosine binding proteins

Two major subtypes of adenosine receptors occur in different tissues which have been distinguished by pharmacological and biochemical criteria. The A₁ adenosine receptor has a high-affinity for adenosine and mediates inhibition of adenylyl cyclase, whereas the A₂ adenosine receptor usually has a lower affinity and mediates stimulation of the enzyme. Furthermore, evidence has been obtained that A₁ receptors increase the conductance of receptor-regulated potassium channels, induce inactivation of calcium channels, and modulate the breakdown of phosphoinositides by phospholipase C. Selective agonists and antagonists have been developed for both receptor subtypes. In addition, both adenosine receptors have extensively been characterized by radioligand binding studies. Suitable radioligands for the A₁ receptor are the agonist [³H]2-chloro-N⁶-cyclopentyladenoisine (CCPA) and the antagonist [³H]8-cyclopentyl-1,3-dipropylxanthine (DPCPX)and for the A_2a receptor [³H]2-[p-(carboxyethyl)phenethylamino]- 5′-N-carboxamidoadenosine (CGS 21860). Furthermore, photoaffinity ligands were developed from adenosine derivatives, which can be covalently incorporated into the binding unit of both receptor subtypes. With this approach, it has been shown that the A₁ receptor has an apparent molecular weight of approximately 36 kDa and the A_2a receptor of 45 kDa. A second approach to elucidate the structure of adenosine receptors involves the purification of receptor protein by affinity chromatography. With this procedure, cerebral A₁ receptors have been purified to apparent homogeneity. More recently, the structure of receptor subtypes has been elucidated by cloning the receptors from a cDNA library. Furthermore, a novel adenosine binding with [³H] 5′ -N-ethylcarboxamidoadenosine ([³H]NECA). The pharmacological profile of this NECA-binding protein has been determined in competition experiments with adenosine receptor ligands. It can be distinguished from that of A_2a adenosine receptors and other adenosine binding proteins. We propose the name A_x for this unique adenosine binding protein. © 1993 Wiley-Liss, Inc.