Cellular localization of dopamine-releasing protein (DARP) in rat C6 glioma and primary mesencephalic cell cultures

Abstract

Dopamine-releasing protein (DARP) is a multisubunit protein shown to have dramatic effects on development, recovery, and function of the rat catecholaminergic (CA) system. This study details efforts to determine if glial cells are responsible for the production of DARP in the central nervous system (CNS). Enzyme-linked immunosorbent assays (ELISA), Western blotting, and immunocytochemical techniques were employed to measure DARP levels and identify DARP immunoreactive proteins in rat C6 glioma cells and medium, respectively. ELISA analysis of serum-free C6 culture media revealed a maximal concentration of DARP by culture day 1. However, ELISA analysis of C6 cultures grown in F-12K/serum medium revealed that maximal levels of DARP were detected on culture day 6 with a 108% increase in DARP immunoreactivity from culture day 1. These values were determined using a polyclonal antibody generated against DARP-36aa (anti-DARP-36aa), a synthetic peptide with dopamine (DA) releasing activity, and anti-DARP B9–B10, a monoclonal antibody generated against partially purified DARP. Western blot analysis revealed that anti-DARP B9–B10 recognized proteins of approximately 60, 50, and 45 kDa in C6 cell homogenates while anti-DARP-36aa had immunoreactivity with the 60-kDa protein alone. Immunocytochemical studies demonstrated that anti-DARP-36aa and anti-DARP B9–B10 had strong immunoreactivity with proteins throughout the cytosol and in several processes of C6 cells. These results reveal that DARP is detected in glioma cells and secreted in a time-dependent fashion during culture. Primary rat mesencephalic cultures were also examined using immunocytochemistry. Incubation with DARP antibodies and antisera against glial fibrillary acidic protein (GFAP) revealed that DARP and GFAP immunoreactivity co-localized in primary mesencephalic cultures. However, the majority of DARP immunoreactivity was localized to cells without GFAP staining. These findings reveal that DARP is detected in astrocytes although the majority of DARP immunoreactivity is found in non-astrocyte type cells.