Dopamine release modifies intracellular calcium levels in tyrosine hydroxylase-transfected C6 cells

Glioma cell line C6, transfected with tyrosine hydroxylase (TH) cDNA under the control of the glial fibrillary acid protein promoter (C6-THA cells), elicited a reduction in the apomorphine-induced turning behavior when they are implanted in Parkinson's disease models. Nevertheless, dopamine (Da) release has not been explicitly demonstrated nor has a possible mechanism of release been implicated. In this study, the in vitro Da release by C6 and C6-THA cells after chemical stimulation with KCl or glutamate was quantified using HPLC. Modifications in intracellular calcium levels in response to KCl stimulation and participation of Da receptor-mediated feedback in calcium regulation were also studied using FLUO 3 as a calcium concentration indicator. C6-THA cells release dopamine in basal conditions, and increase its release after KCl or glutamic acid stimulation. In a fraction of C6 and C6-THA cells, a transient intracellular calcium increase was observed after KCl stimulation, but C6-THA cells demonstrated a faster rate of calcium removal. C6 cells express mRNA from all five subtypes of Da receptors as demonstrated by real time PCR. D1 receptors were most abundant in C6 cells and its expression was further increased in C6-THA cells. Blocking D1-like receptors in C6-THA cells with the specific antagonist drug SCH-23390 induced a decrease in intracellular calcium removal rate, resembling non-manipulated C6 cells’ calcium clearance. Da release by C6-THA cells could be related to calcium dependent mechanisms. Furthermore, production of Da by C6-THA cells seems to upregulate the expression of D1 receptors’ mRNA.