Effects of 6-hydroxydopamine and inhibitors for vesicular monoamine transporter on monoamine neurotransmitter and rate-limiting enzyme gene of them in PC12 cells

Objective To study the effects of 6-hydroxydopamine (6-OHDA) , and inhibitors for vesicular monoamine transporter (VMAT) on 5-hydroxytryptamine (5-HT), norepinephrine (NE) and dopamine (DA) and the expressions of tryptophan hydroxylase（TpH) mRNA, dopamine-beta-hydroxylase (DβH) mRNA and tyrosine hydroxylase (TH) mRNA in PC12 cells. Methods The cell viability was determined using MTT assay, the density of 5-HT, NE and DA was detected using enzyme-linked immunosorbent assay, and the expressions of TpHmRNA, DβHmRNA and THmRNA were detected using RT-PCR in PC12 cells at different time points (0, 12, 24, 36, 48 h) after exposure to different concentrations of 6-OHDA(25, 50, 100, 200 μmol/L) , and VMAT inhibitors, reserpine (50, 100, 400, 1600 nmol/L), which combined with 6-OHDA(100 μmol/L). Results (1)The cell viability declined with the increasing concentration of 6-OHDA which showed time dependence. The cell viability in PC12 cell which treated with reserpine decreased significantly in the responding group. The density of 5-HT in PC12 cell did not decrease with the increasing concentration of 6-OHDA, but the change had the time dependence, and the density of 5-HT was lowest at 36 h. The density of NE decreased with the increasing concentration of 6-OHDA which showed time dependence. The density of DA in PC12 cell decreased with the increasing concentration of 6-OHDA, but the change did not have the time dependence. The density of 5-HT, NE and DA in PC12 cell which treated with reserpine decreased significantly in the responding group. (2) The expressions of TpHmRNA, DβHmRNA and THmRNA in PC12 cell decreased with the increasing concentration of 6-OHDA which showed time dependence. The expressions of TpHmRNA（0.006±0.001, 0.003±0.000, 0.003±0.000, 0.002±0.000）; DβHmRNA（0.005±0.002, 0.003±0.001, 0.002±0.001, 0.001±0.000）and THmRNA（0.005±0.002, 0.003±0.001, 0.002±0.001, 0.001±0.000）in PC12 cell which treated with reserpine decreased significantly in the responding group（F=13.336,9.000,9.393, all P=0.000）. Conclusions 6-OHDA can decrease the cell viability in PC12 cell, reduce the density of 5-HT, NE and DA and decrease the expressions of TpHmRNA, DβHmRNA and THmRNA, and the effects have dose and time dependence. Reserpine can aggravate this damage. Key words: Oxidopamine; PC12 cells; Reserpine; Vesicular monoamine transporter proteins; Dopamine beta-hydroxylase