Carrier-mediated cocaine transport at the blood-brain barrier as a putative mechanism in addiction liability

Abstract

Background: The rate of entry of cocaine into the brain is a critical factor that influences neuronal plasticity and the development of cocaine addiction. Until now, passive diffusion has been considered the unique mechanism known by which cocaine crosses the blood-brain barrier.

Methods: We reassessed mechanisms of transport of cocaine at the blood-brain barrier using a human cerebral capillary endothelial cell line (hCMEC/D3) and in situ mouse carotid perfusion.

Results: Both in vivo and in vitro cocaine transport studies demonstrated the coexistence of a carrier-mediated process with passive diffusion. At pharmacological exposure level, passive diffusion of cocaine accounted for only 22.5% of the total cocaine influx in mice and 5.9% in hCMEC/D3 cells, whereas the carrier-mediated influx rate was 3.4 times greater than its passive diffusion rate in vivo. The functional identification of this carrier-mediated transport demonstrated the involvement of a proton antiporter that shared the properties of the previously characterized clonidine and nicotine transporter. The functionnal characterization suggests that the solute carrier (SLC) transporters Oct (Slc22a1-3), Mate (Slc47a1) and Octn (Slc22a4-5) are not involved in the cocaine transport in vivo and in vitro. Diphenhydramine, heroin, tramadol, cocaethylene, and norcocaine all strongly inhibited cocaine transport, unlike benzoylecgonine. Trans-stimulation studies indicated that diphenhydramine, nicotine, 3,4-methylenedioxyamphetamine (ecstasy) and the cathinone compound 3,4-methylenedioxypyrovalerone (MDPV) were also substrates of the cocaine transporter.

Conclusions: Cocaine transport at the BBB involves a proton-antiporter flux that is quantitatively much more important than its passive diffusion. The molecular identification and characterization of this transporter will provide new tools to understand its role in addictive mechanisms.

Keywords: biological transport; blood-brain barrier; cocaine; drug of abuse; pharmacokinetics..

Figure 1.

Passive and carrier-mediated flux of cocaine in hCMEC/D3 cells. A, Total uptake (nmol/min/mg; dashed line) was measured in hCMEC/D3 cells and plotted against total cocaine concentration in the KH incubation buffer at pH_e 7.40. The straight dotted line represents the passive transport of cocaine (K _passive of 1.09±0.09 µL/min/mg at pH 7.40). The solid line represents the curve obtained by subtracting the passive flux from the total flux and fitting to the carrier-mediated Michaelis-Menten term (see Equation 7) by nonlinear least-square regression. Estimated parameters for cocaine transport in hCMEC/D3 cells are: K _m, 0.123±0.023mM; V _max, 4.26±0.26 nmol/min/mg. Data represent means±SD of experiments performed in triplicate. B, Total (dashed line) and individual passive (dotted line) and carrier-mediated (solid line) cocaine transport into hCMEC/D3 cells fitted according to Equation 7 for concentrations <0.5mM. Data represent means±SD of experiments performed in triplicate.

Figure 2.

Passive and carrier-mediated flux of cocaine at the mouse luminal BBB. A, Total flux (J _in; nmol/sec/g; dashed line) measured in the right brain hemisphere of male Swiss mice and plotted against total cocaine concentration in the perfusion buffer (Krebs-carbonate) at pH_e 7.40 (Equation 6). The straight dotted line represents the passive diffusion flux of cocaine (K _passive of 4.5±1.0 µL/sec/g at pH 7.4). The solid line represents the curve obtained by subtracting the passive flux from the total flux and fitted to the carrier-mediated Michaelis-Menten term (see Equation 7) by nonlinear least-square regression. Estimated parameters for the brain transport of cocaine are: K _m, 4.5±1.8mM; V _max, 127.2±34.5 nmol/sec/g. Data represent means±SD of 4 to 5 mice. B, Total (dashed line), carrier-mediated (solid line), and passive (dotted line) cocaine fluxes at pharmacological concentrations at the mouse BBB, fitted according to Equation 7 for concentrations <0.1mM.

Figure 3.

Trans-stimulation studies of cocaine transport in hCMEC/D3 cells. hCMEC/D3 cells were loaded with [³H]-cocaine for 5 minutes and then incubated with KH buffer alone (control) or with 10 µM of unlabeled compound (TEA, benzoylecgonine, cocaine, MDPV, MDMA, diphenhydramine, or nicotine) in KH buffer. Data represent means±SD performed in quadruplicate. *** P<0.001 compared with controls.

Figure 4.

Effect of the modulation of extracellular (pH_e) and intracellular (pH_i) pH on cocaine transport in hCMEC/D3 cells. a, Effects of changes in the incubation buffer pH_e. [³H]-Cocaine uptake was measured for 5 minutes in KH buffer adjusted to pH 5.40, 6.40, or 7.40. *** P<0.001 compared with the pH 5.4 group, and ### P<0.001 for a comparison between pH 6.40 and 7.40 (n=3–4). b, Modulation of pH_i with NH₄Cl. In pulse condition (cellular alkalinization): after 30 minutes of the usual preincubation, a solution (pH_e 7.40) with NH₄Cl (30mM) and containing [³H]-cocaine was added for 5 minutes. The NH₄Cl prepulse condition (cellular acidification) was obtained by preincubating cells with the incubation buffer (pH_e 7.40) plus NH₄Cl (30mM) for 15 minutes. Then, the incubation medium was replaced by the usual KH buffer without NH₄Cl for 5 minutes. [³H]-Cocaine uptake was measured in the usual KH incubation buffer for 5 minutes (n=3–4).

Figure 5.

Effects of changes in the vascular perfusion medium pH_e, alteration of the BBB pH_i, and sodium dependency of [³H]-cocaine transport at the BBB in Swiss mice. a, Effects of the Krebs-carbonate perfusion buffer at a pH_e of 5.40, 6.40, or 7.40 on [³H]-cocaine brain transport (K _in; µL/sec/g), with (black column) or without (white column) co-perfusion with unlabeled cocaine (10mM), measured by in situ mouse brain perfusion for 60 seconds. Data represent means±SD (n=4 mice). * P<0.05, ** P<0.01, *** P<0.001 compared with the pH 5.4 group; + P<0.05, +++ P<0.001 for comparison (Student t test) between with and without unlabeled cocaine at the same pH; and ### P<0.001 for a comparison of pH 6.40 and 7.40 without unlabeled cocaine. b, Effects of altering pH_i (white columns) and removing sodium (grey column). The vascular perfusion media were Krebs-carbonate buffer plus NH₄Cl (30mM; “NH₄Cl pulse”), carbonate-free HEPES-buffered solution, and “mannitol” (sodium-free and chloride-free) Krebs-carbonate buffer to alter pH_i (white columns). The effect of Na⁺-free perfusion buffer (pH_e 7.40) was studied by replacing sodium with lithium (Li⁺; grey column). Data represent means±SD of 4 to 7 animals. * P<0.05, ** P<0.01, *** P<0.001 compared with the control group.