Cocaine hydrolase blocks cocaine‐induced dopamine transporter trafficking to the plasma membrane

Abstract

Cocaine blocks dopamine uptake via dopamine transporter (DAT) on plasma membrane of neuron cells and, as a result, produces the high and induces DAT trafficking to plasma membrane which contributes to the drug seeking or craving. In this study, we first examined the dose dependence of cocaine-induced DAT trafficking and hyperactivity in rats, demonstrating that cocaine at an intraperitoneal dose of 10 mg/kg or higher led to redistribution of most DAT to the plasma membrane while inducing significant hyperactivity in rats. However, administration of 5-mg/kg cocaine (ip) did not significantly induce DAT trafficking or hyperactivity in rats. So the threshold (intraperitoneal) dose of cocaine that can significantly induce DAT trafficking or hyperactivity should be between 5 and 10 mg/kg. These data suggest that when a cocaine dose is high enough to induce significant hyperactivity, it can also significantly induce DAT trafficking to the plasma membrane. Further, the threshold brain cocaine concentration required to induce significant hyperactivity and DAT trafficking was estimated to be $2.0 ± 0.8 μg/g. Particularly, for treatment of cocaine abuse, previous studies demonstrated that an exogenous cocainemetabolizing enzyme, for example, CocH3-Fc(M3), can effectively block cocaineinduced hyperactivity. However, it was unknown whether an enzyme could also effectively block cocaine-induced DAT trafficking to the plasma membrane. This study demonstrates, for the first time, that the enzyme is also capable of effectively blocking cocaine from reaching the brain even with a lethal dose of 60-mg/kg cocaine (ip) and, thus, powerfully preventing cocaine-induced physiological effects such as the hyperactivity and DAT trafficking.

Recovery of dopaminergic system after cocaine exposure and impact of a long‐acting cocaine hydrolase

“…Our previous study 11 reported that in normal rat brain, striatal DAT mainly existed in the intracellular fraction, with only a very small fraction (~5.7%) existed in the plasma membrane in rats. Notably, our finding of only ~5.7% of DAT in the plasma membrane surface in rat brain is far less than the previously reported surface DAT fraction in cultured PC12‐hDAT cells (~25–40%), 38–40 ) but is close to the synaptosomal surface faction (~7%) of DAT in rats reported by Johnson et al 41 Cocaine at an i.p.…”

“…We previously demonstrated 11 that cocaine at an intraperitoneal dose of 10 mg/kg or higher induced trafficking of most DAT to the plasma membrane (with only <5% remained in the intracellular) at 24 h after cocaine injection in rats. In current study, we aimed to reveal how long it could take for the dopaminergic system recovering to its normal status (in terms of intracellular and plasma membrane fractions of DAT) in rats.…”

“…Biotinylation and Western blotting assays were performed using a previously published method 11 . Briefly, rats ( n = 4 for each group) were given a single or multiple intraperitoneal dose of 20 mg/kg cocaine and then decapitated under deep anaesthesia with CO 2 (40% chamber replacement rate, 3 to 4 min) to collect the brain samples at different time‐points after cocaine exposure.…”

“…Biotinylation and Western blotting assays were performed using a previously published method. 11 Briefly, rats (n = 4 for each group) were given a single or multiple intraperitoneal dose of 20 mg/kg cocaine and then decapitated under deep anaesthesia with CO 2 (40% chamber replacement rate, 3 to 4 min) to collect the brain samples at different time-points after cocaine exposure. Striatum samples were quickly isolated on ice and immediately homogenized in 1-ml ice-cold sucrose solution (0.32 M sucrose, 5 mM sodium bicarbonate, 1-X phosphatase inhibitor cocktail and 1-X protease inhibitor cocktail, pH 7.4) of 1-min continuous homogenization with a cordless pestle motor in 1.5 ml microtubes on ice.…”

“…Long‐standing evidence supports that DAT presentation on neuron cell surface is highly plastic and is subject to regulated endocytic trafficking—it constitutively internalizes and recycles during the neurotransmission process 9,10 . Cocaine administration can significantly induce DAT trafficking to the plasma membrane, without significantly changing the total amount of DAT (which is a sum of the intracellular and plasma membrane DAT) 6,11 . One‐time use of cocaine increases the surface DAT expression (via DAT trafficking) for at least a month, 12 while abstinence from chronic use of cocaine would take a longer period of time for striatal DAT distribution to recover in monkey models, 13 as normalization of dopaminergic function is usually a slow process.…”