Induction of dopamine biosynthesis by l-DOPA in PC12 cells: Implications of l-DOPA influx and cyclic AMP

The effects of 3,4-dihydroxyphenylalanine (l-DOPA) on dopamine biosynthesis and cytotoxicity were investigated in PC12 cells. l-DOPA treatment (20–200 μM) increased the levels of dopamine by 226%–504% after 3–6 h of treatment and enhanced the activities of tyrosine hydroxylase (TH) and aromatic l-amino acid decarboxylase (AADC). l-DOPA (20–200 μM) treatment led to a 562%–937% increase in l-DOPA influx at 1 h, which inhibited the activity of TH, but not AADC, during the same period. The extracellular releases of dopamine were also increased by 231%–570% after treatment with 20 and 200 μM l-DOPA for 0.5–3 h. l-DOPA at a concentration of 100–200 μM, but not 20 μM, exerted apoptotic cytotoxicity towards PC12 cells for 24–48 h. l-DOPA (20–200 μM) increased the intracellular cyclic AMP levels by 318%–557% after 0.5–1 h in a concentration-dependent manner. However, the elevated cyclic AMP levels by l-DOPA could not protect against l-DOPA (100–200 μM)-induced cytotoxicity after 24–48 h. In addition, l-DOPA (20–200 μM)-induced increases in cyclic AMP and dopamine were significantly reduced by treatment with SCH23390 (dopamine D1 receptor antagonist). The increased levels of dopamine by l-DOPA were also reduced by H89 (protein kinase A, PKA, inhibitor) and GF109203X (protein kinase C inhibitor); however, the reduction by GF109203X was not significant. l-DOPA at 20–200 μM stimulated the phosphorylation of PKA and cyclic AMP-response element binding protein and induced the biosynthesis of the TH protein. These results indicate that 20–200 μM l-DOPA induces dopamine biosynthesis by two pathways. One pathway involves l-DOPA directly entering the cells to convert dopamine through AADC activity (l-DOPA decarboxylation). The other pathway involves l-DOPA and/or released dopamine activating TH to enhance dopamine biosynthesis by the dopamine D1 receptor-cyclic AMP–PKA signaling system (dopamine biosynthesis by TH).