The catecholaminergic RCSN-3 cell line: A model to study dopamine metabolism

Abstract

RCSN-3 cells are a cloned cell line derived from the substantia nigra of an adult rat. The cell line grows in monolayer and does not require differentiation to express catecholaminergic traits, such as (i) tyrosine hydroxylase; (ii) dopamine release; (iii) dopamine transport; (iv) norepinephrine transport; (v) monoamine oxidase (MAO)-A expression, but not MAO-B; (vi) formation of neuromelanin; (vii) VMAT-2 expression. In addition, this cell line expresses serotonin transporters, divalent metal transporter, DMT1, dopamine receptor 1 mRNA under proliferating conditions, and dopamine receptor 5 mRNA after incubation with dopamine or dicoumarol. Expression of dopamine receptors D(2), D(3) and D(4) mRNA were not detected in proliferating cells or when the cells were treated with dopamine, CuSO(4), dicoumarol or dopamine-copper complex. Angiotensin II receptor mRNA was also found to be expressed, but it underwent down regulation in the presence of aminochrome. Total quinone reductase activity corresponded 94% to DT-diaphorase. The cells also express antioxidant enzymes such as superoxide dismutase, catalase and glutathione peroxidase. This cell line is a suitable in vitro model for studies of dopamine metabolism, since under proliferating conditions the cells express all the pertinent markers.

Overexpression of VMAT-2 and DT-diaphorase protects substantia nigra-derived cells against aminochrome neurotoxicity

“…In addition, aminochrome inactivates parkin, that is a ubiquitin ligase 3 [35], tyrosine hydroxylase and human dopamine transporter [36,37]; and (ii) its one-electron reduction leukoaminochrome o -semiquinone radical with concomitant formation of superoxide, hydrogen peroxide and hydroxyl radical [16,23], inducing a redox cycling depleting NADH and oxygen required for ATP production. Aminochrome has been proposed to be the endogenous neurotoxin responsible of the slow and progressive neurodegeneration of dopaminergic neurons containing neuromelanin [23,38]. VMAT-2 mediated dopamine uptake into monoaminergic vesicles and therefore, will prevent dopamine oxidation and aminochrome neurotoxicity and DT-diaphorase prevents aminochrome one-electron reduction and the formation of adducts with proteins.…”

Copper·Dopamine Complex Induces Mitochondrial Autophagy Preceding Caspase-independent Apoptotic Cell Death

“…The cultures were kept in an incubator at 37°C with 100% humidity, and the cells grew well in atmospheres of both 5% or 10% CO 2 (6,19,21).…”

“…The purpose of this study was to examine the Cu⅐DA complex-induced cell death mechanism in RCSN-3 cells, a cell line that expresses dopamine, norepinephrine, and serotonin transporters (18,19).…”

Protective Effects of Nicotine Against Aminochrome-Induced Toxicity in Substantia Nigra Derived Cells: Implications for Parkinson’s Disease

“…For this purpose we utilized RCSN-3 cells, a cell line derived from rat SN with catecholaminergic features including synthesis and release of dopamine, expression of VMAT-2, dopamine, norepinephrine and serotonin transporter as well as production of neuromelanin (Paris et al2008). RCSN-3 cells grow in monolayer with a doubling time of 52 h at a plating efficiency of 21 % and a saturation density of 56,000 cells/cm 2 when kept in normal growth media composed of: DME/HAM-F12 (1:1), 10 % bovine serum, 2.5 % fetal bovine serum, 40 mg/l gentamicine sulfate (Arriagada et al 2004; Paris et al 2008, 2010; Muñoz et al 2012).…”

“…RCSN-3 cells grow in monolayer with a doubling time of 52 h at a plating efficiency of 21 % and a saturation density of 56,000 cells/cm 2 when kept in normal growth media composed of: DME/HAM-F12 (1:1), 10 % bovine serum, 2.5 % fetal bovine serum, 40 mg/l gentamicine sulfate (Arriagada et al 2004; Paris et al 2008, 2010; Muñoz et al 2012). Cultures were kept in an incubator at 37 °C with 100 % humidity and an atmosphere of 10 % CO 2 .…”