Blackwell Publishing LtdEffect of Dopamine Receptor 1 on Apoptosis of Cultured Neonatal RatCardiomyocytes in Simulated Ischaemia/Reperfusion
Dopamine receptors exist in many tissues, including rat cardiac tissue. However, the physiological importance ofdopamine 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, followedby incubation in normal culture medium for 24 hr. Lactate dehydrogenase activity, superoxide dismutase activity andmalondialdehyde content were determined colorimetrically with a spectrophotometer. Apoptotic cell death was assayed by3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide assay, terminal deoxynucleotidyl transferase-mediateddUTP-biotin nick end labelling staining and flow cytometry, and morphological alterations were observed with transmissionelectron microscopy. The intracellular free calcium concentration ([Ca2+]i) was measured by confocal laser scanningmicroscopy. Finally, the expression of dopamine receptor 1 (DR1), caspase-3, -8 and -9, Fas, Fas ligand and Bcl-2 and therelease of cytochromecwere analysed by Western blot. The results showed that DR1 expression was increased markedlyduring ischaemia/reperfusion. Treatment with 10M SKF-38393 (DR1 agonist) significantly increased lactate dehydrogenaseactivity, decreased superoxide dismutase activity and increased malondialdehyde content in the culture medium. The DR1agonist promoted the release of cytochromec, 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-regulatedBcl-2 expression. In contrast, 10M SCH-23390 (DR1 antagonist) had no significant effects on the above indicators. Inconclusion, DR1 activation is involved in the apoptosis of cultured neonatal rat cardiomyocytes in simulated ischaemia/reperfusion through the mitochondrial and death receptor pathways.