Li Hong-zhu, Han Li-ping, Jiang Chun-ming, Li Hong, Zhao Ya-jun, Gao Jun, Lin Yan, Ma Shu-xia, Tian Ye, Yang Bao-feng, Xu Chang-qing
Department of Pathophysiology, Harbin Medical University, Harbin, China.
Basic Clin Pharmacol Toxicol. 2008 Mar;102(3):329-36. doi: 10.1111/j.1742-7843.2007.00177.x.
Dopamine receptors exist in many tissues, including rat cardiac tissue. However, the physiological importance of dopamine receptors in the homeostatic regulation of cardiac function is unclear. In this study, a model of ischaemia/reperfusion was established by culturing primary neonatal rat cardiomyocytes in ischaemia-mimetic solution for 2 hr, followed by incubation in normal culture medium for 24 hr. Lactate dehydrogenase activity, superoxide dismutase activity and malondialdehyde content were determined colorimetrically with a spectrophotometer. Apoptotic cell death was assayed by 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide assay, terminal deoxynucleotidyl transferase-mediated dUTP-biotin nick end labelling staining and flow cytometry, and morphological alterations were observed with transmission electron microscopy. The intracellular free calcium concentration ([Ca2+]i) was measured by confocal laser scanning microscopy. Finally, the expression of dopamine receptor 1 (DR1), caspase-3, -8 and -9, Fas, Fas ligand and Bcl-2 and the release of cytochrome c were analysed by Western blot. The results showed that DR1 expression was increased markedly during ischaemia/reperfusion. Treatment with 10 microM SKF-38393 (DR1 agonist) significantly increased lactate dehydrogenase activity, decreased superoxide dismutase activity and increased malondialdehyde content in the culture medium. The DR1 agonist promoted the release of cytochrome c, accumulation of [Ca2+]i, and apoptosis induced by ischaemia/reperfusion. Furthermore, SKF-38393 up-regulated the expression of caspase-3, -8 and -9, Fas and Fas ligand, and down-regulated Bcl-2 expression. In contrast, 10 microM SCH-23390 (DR1 antagonist) had no significant effects on the above indicators. In conclusion, DR1 activation is involved in the apoptosis of cultured neonatal rat cardiomyocytes in simulated ischaemia/reperfusion through the mitochondrial and death receptor pathways.
多巴胺受体存在于包括大鼠心脏组织在内的许多组织中。然而，多巴胺受体在心脏功能稳态调节中的生理重要性尚不清楚。在本研究中，通过将原代新生大鼠心肌细胞在模拟缺血溶液中培养2小时，然后在正常培养基中孵育24小时，建立了缺血/再灌注模型。用分光光度计通过比色法测定乳酸脱氢酶活性、超氧化物歧化酶活性和丙二醛含量。通过3-(4,5-二甲基噻唑-2-基)-2,5-二苯基四氮唑溴盐法、末端脱氧核苷酸转移酶介导的dUTP-生物素缺口末端标记染色和流式细胞术检测凋亡细胞死亡，并用透射电子显微镜观察形态学改变。通过共聚焦激光扫描显微镜测量细胞内游离钙浓度([Ca2+]i)。最后，通过蛋白质免疫印迹法分析多巴胺受体1(DR1)、半胱天冬酶-3、-8和-9、Fas、Fas配体和Bcl-2的表达以及细胞色素c的释放。结果表明，在缺血/再灌注期间DR1表达明显增加。用10微摩尔/升SKF-38393(DR1激动剂)处理显著增加了培养基中乳酸脱氢酶活性，降低了超氧化物歧化酶活性并增加了丙二醛含量。DR1激动剂促进了细胞色素c的释放、[Ca2+]i的积累以及缺血/再灌注诱导的细胞凋亡。此外，SKF-38393上调了半胱天冬酶-3、-8和-9、Fas和Fas配体的表达，并下调了Bcl-2表达。相比之下，10微摩尔/升SCH-23390(DR1拮抗剂)对上述指标没有显著影响。总之，DR1激活通过线粒体和死亡受体途径参与模拟缺血/再灌注时培养的新生大鼠心肌细胞的凋亡。
