Rapid substrate‐induced down‐regulation in function and surface localization of dopamine transporters: rat dorsal striatum versus nucleus accumbens

Abstract

Dopamine (DA), d‐amphetamine (AMPH) and methamphetamine (METH) are DA transporter (DAT) substrates that also rapidly and transiently reduce cell surface expression of DATs in dorsal striatum (dSTR) and cells expressing cloned DATs. However, it is not clear if DATs are regulated similarly by substrates in nucleus accumbens (NAc). Here, we investigated whether brief in vitro exposure to DAT substrates differentially regulates DATs in synaptosomes prepared from dSTR or NAc of male Sprague Dawley rats. Synaptosomes were pre‐incubated with DA, AMPH, METH (3–30 μM) or buffer for 5–30 min at 37oC and washed twice at 4oC; specific uptake of 0.5 nM [3H]DA, defined with 100 μM cocaine, was measured for 3 min at 37oC. As expected, [3H]DA uptake was 54% lower in NAc than in dSTR, and substrate pre‐incubation significantly reduced [3H]DA uptake in dSTR in a time‐ and concentration‐dependent manner. Although in NAc [3H]DA uptake was also reduced by pre‐incubation with DA, AMPH or METH, statistical significance was not reached with any pre‐incubation time or concentration. Kinetic analyses after a 15 min pre‐incubation with 10 μM AMPH confirmed a lower maximal DAT velocity in dSTR, but not in NAc. Our results suggest that brief substrate exposure more efficaciously down‐regulates DATs in dSTR; however, surface biotinylation experiments are needed to confirm these observations. Supported by DA004216, DA014204, AA007464, DA015050.

Acute Administration of Haloperidol Does Not Influence 123I-FP-CIT Binding to the Dopamine Transporter

“…If one assumes that there is a linear relationship between DAT downregulation and dopamine concentration, it is not likely that such a small increase in dopamine concentrations may lead to large changes in DAT levels. In line with this possibility, we did not observe even a trend for a change in DAT binding in the dorsal striatum, an area that may be more prone to downregulation of DAT than the ventral striatum (12). In addition, Hadlock et al showed that sole administration of the dopamine D 2 antagonist eticlopride (0.5 mg/kg intraperitoneally) also did not influence DAT expression in rats (15).…”

“…Similarly, effects of dopamine on the DAT expression are reported only after administration of high doses of dopamine. Richards and Zahniser showed significant decreases in DAT cell surface expression of 49% in the dorsal striatum and 22% in the nucleus accumbens after a 1-h preincubation period with 100 mM dopamine (12). However, in monkeys, the baseline striatal extracellular dopamine concentration is around 5-6 nM (14).…”

“…We looked into the dorsal and ventral parts (12). As the outcome measure, ratios of total to nonspecific binding were calculated for the striatum and nucleus accumbens.…”

Palmitoylation Controls Dopamine Transporter Kinetics, Degradation, and Protein Kinase C-dependent Regulation

“…As high sucrose blocks endocytosis, our findings demonstrate that downregulation of DAT by PKC in synaptosomes is achieved independently of internalization. We previously identified a nonendocytotic component to PKC-induced DA transport down-regulation in LLCPK 1 cells associated with cholesterolrich microdomains (21), and trafficking independent regulation of DAT by amphetamine has also been reported (44), further supporting kinetic mechanisms in regulation of transport.…”

Regulation of the Dopamine and Vesicular Monoamine Transporters: Pharmacological Targets and Implications for Disease

“…By contrast, releasers, such as AMPH, biphasically alter DAT surface expression. AMPH increases DAT cell surface expression within the first minutes of exposure (1-3 minutes), followed by DAT internalization and accumulation within endocytic structures (15-60 minutes; Saunders et al, 2000;Johnson et al, 2005;Furman et al, 2009;Richards and Zahniser, 2009) (Fig. 3B).…”