Isolation of darp (dopamine-releasing protein) from fetal rat brain and effects of darp immunoneutralization on fetal mesencephalic dopamine levels

Recent work from this laboratory suggests a role for a novel dopamine-releasing protein (DARP) during rat fetal development. We have previously shown that intrafetal administration of an anti-DARP monoclonal antibody (DARP mAb) at Embryonic Day 17 (E17) induces fetal resorption in a dose-dependent manner (Kuhananthan et al., Mol. Cell. Neurosci., 2, 410-417). In this study we present evidence that (1) DARP is present in the rat brain at E17 and (2) in vivo immunoneutralization of DARP at E17 alters dopamine (DA) levels selectively in the prenatal mesencephalon. Enzyme-linked immunosorbent assay of supernatants of crude fetal (E17) brain homogenates using DARP mAb as a probe detects the presence of a DARP-like immunoreactive protein in the E17 rat brain. Purification of these homogenates with concanavalin A and immunoaffinity chromatography yielded a single 60-kDa protein that displayed dopamine-releasing activity in an in vitro superfusion assay. In addition, intrafetal administration of DARP mAb at E17 significantly elevated DA levels in the mesencephalon 24 and 48 h postinjection, while decreasing these levels 72 h postinjection. No changes in DA levels were detected in the diencephalon or in the telencephalon. These findings indicate that DARP is present in the rat brain at E17 and may play a role in the development of dopaminergic neurons of the mesencephalon.