Urmaliya Vijay B, Church Jarrod E, Coupar Ian M, Rose'Meyer Roselyn B, Pouton Colin W, White Paul J
Medicinal Chemistry and Drug Action, Monash Institute of Pharmaceutical Sciences, Monash University, Victoria, Australia.
J Cardiovasc Pharmacol. 2009 May;53(5):424-33. doi: 10.1097/FJC.0b013e3181a443e2.
Extracellular adenosine concentrations increase within the heart during ischemia, and any exogenous adenosine receptor agonists therefore work in the context of significant local agonist concentrations. We evaluated the interactions between A1, A2A, A2B, and A3 receptors in the presence and absence of adenosine deaminase (ADA, which is used to remove endogenous adenosine) in a cardiac cell ischemia model. Simulated ischemia (SI) was induced by incubating H9c2(2-1) cells in SI medium for 12 hours in 100% N2 gas before assessment of necrosis using propidium iodide (5 microM) or apoptosis using AnnexinV-PE flow cytometry. N6-Cyclopentyladenosine (CPA; 10(-7)M) and N6-(3-iodobenzyl) adenosine-5'-N-methyluronamide (IB-MECA; 10(-7)M) reduced the proportion of nonviable cells to 30.87 +/- 2.49% and 35.18 +/- 10.30%, respectively (% of SI group). In the presence of ADA, the protective effect of CPA was reduced (62.82 +/- 3.52% nonviable), whereas the efficacy of IB-MECA was unchanged (35.81 +/- 3.84% nonviable; P < 0.05, n = 3-5, SI vs. SI + ADA). The protective effects of CPA and IB-MECA were abrogated in the presence of their respective antagonists DPCPX (8-cyclopentyl-1,3-dipropylxanthine) and MRS1191 [3-ethyl-5-benzyl-2-methyl-4-phenylethynyl-6-phenyl-1,4-(+/-)-dihydropyridine-3,5-dicarboxylate], whereas A2A and A2B agonists had no significant effect. CPA-mediated protection was abrogated in the presence of both A2A (ZM241385, 4-(2-[7-amino-2-(2-furyl)[1,2,4]triazolo[2,3-a][1,3,5]triazin-5-lamino]ethyl)phenol; 50 nM) and A2B (MRS1754, 8-[4-[((4-cyanophenyl)carbamoylmethyl)oxy]phenyl]-1,3-di(n-propyl)xanthine; 200 nM) antagonists (n = 3-5, P < 0.05). In the absence of endogenous adenosine, significant protection was observed with CPA in presence of CGS21680 (4-[2-[[6-amino-9-(N-ethyl-b-D-ribofuranuronamidosyl)-9H-purin-2-yl]amino]ethyl]benzenepropanoic acid) or LUF5834 [2-amino-4-(4-hydroxyphenyl)-6-(1H-imidazol-2-ylmethylsulfanyl)pyridine-3,5-dicarbonitrile] (P < 0.05 vs. SI + ADA + CPA). Apoptosis (14.35 +/- 0.15% of cells in SI + ADA group; P < 0.05 vs. control) was not significantly reduced by CPA or IB-MECA. In conclusion, endogenous adenosine makes a significant contribution to A1 agonist-mediated prevention of necrosis in this SI model by cooperative interactions with both A2A and A2B receptors but does not play a role in A3 agonist-mediated protection.
在缺血期间，心脏内细胞外腺苷浓度会升高，因此任何外源性腺苷受体激动剂都是在局部激动剂浓度较高的情况下发挥作用。我们在心肌细胞缺血模型中，评估了腺苷脱氨酶（ADA，用于去除内源性腺苷）存在和不存在时，A1、A2A、A2B和A3受体之间的相互作用。在使用碘化丙啶（5 microM）评估坏死或使用膜联蛋白V-PE流式细胞术评估凋亡之前，将H9c2(2-1)细胞在100%氮气中的缺血模拟（SI）培养基中孵育12小时，以诱导模拟缺血（SI）。N6-环戊基腺苷（CPA；10(-7)M）和N6-(3-碘苄基)腺苷-5'-N-甲基脲苷（IB-MECA；10(-7)M）分别将非存活细胞比例降低至30.87±2.49%和35.18±10.30%（相对于SI组的百分比）。在ADA存在的情况下，CPA的保护作用降低（非存活细胞为62.82±3.52%），而IB-MECA的疗效未改变（非存活细胞为35.81±3.84%；P<0.05，n = 3-5，SI组与SI + ADA组）。CPA和IB-MECA的保护作用在其各自的拮抗剂DPCPX（8-环戊基-1,3-二丙基黄嘌呤）和MRS1191 [3-乙基-5-苄基-2-甲基-4-苯基乙炔基-6-苯基-1,4-(±)-二氢吡啶-3,5-二羧酸]存在时被消除，而A2A和A2B激动剂没有显著影响。在同时存在A2A（ZM241385，4-(2-[7-氨基-2-(2-呋喃基)[1,2,4]三唑并[2,3-a][1,3,5]三嗪-5-氨基]乙基)苯酚；50 nM）和A2B（MRS1754，8-[4-[((4-氰基苯基)氨基甲酰基甲基)氧基]苯基]-1,3-二（正丙基）黄嘌呤；200 nM）拮抗剂时，CPA介导的保护作用被消除（n = 3-5，P<0.05）。在没有内源性腺苷的情况下，在CGS21680（4-[2-[[6-氨基-9-(N-乙基-b-D-核糖呋喃糖酰胺基)-9H-嘌呤-2-基]氨基]乙基]苯丙酸）或LUF5834 [2-氨基-4-(4-羟基苯基)-6-(1H-咪唑-2-基甲基硫烷基)吡啶-3,5-二腈]存在时，CPA观察到显著的保护作用（相对于SI + ADA + CPA组，P<0.05）。CPA或IB-MECA并未显著降低凋亡（SI + ADA组中细胞的14.35±0.15%；相对于对照组，P<0.05）。总之，在该SI模型中，内源性腺苷通过与A2A和A2B受体的协同相互作用，对A1激动剂介导的坏死预防做出了重大贡献，但在A3激动剂介导的保护中不起作用。
