Carrier-mediated release, transport rates, and charge transfer induced by amphetamine, tyramine, and dopamine in mammalian cells transfected with the human dopamine transporter.
Amphetamine and related substances induce dopamine release. According to a traditional explanation, this dopamine release occurs in exchange for amphetamine by means of the dopamine transporter (DAT). We tested this hypothesis in human embryonic kidney 293 cells stably transfected with the human DAT by measuring the uptake of dopamine, tyramine, and D- and L-amphetamine as well as substrate-induced release of preloaded N-methyl-4-[3H]phenylpyridinium ([3H]MPP+). The uptake of substrates was sodium-dependent and was inhibited by ouabain and cocaine, which also prevented substrate-induced release of MPP+. Patch-clamp recordings revealed that all four substrates elicited voltage-dependent inward currents (on top of constitutive leak currents) that were prevented by cocaine. Whereas individual substrates had similar affinities in release, uptake, and patch-clamp experiments, maximal effects displayed remarkable differences. Hence, maximal effects in release and current induction were approximately 25% higher for D-amphetamine as compared with the other substrates. By contrast, dopamine was the most efficacious substrate in uptake experiments, with its maximal initial uptake rate exceeding those of amphetamine and tyramine by factors of 20 and 4, respectively. Our experiments indicate a poor correlation between substrate-induced release and the transport of substrates, whereas the ability of substrates to induce currents correlates well with their releasing action.
Institute of Biochemical Pharmacology, University of Vienna, Austria.
H H
HH
Sitte
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S
Huck
H
H
Reither
S
S
Boehm
E A
EA
Singer
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C
Pifl
D000661:Amphetamine / Q000493:pharmacokinetics / Q000494:pharmacology*; D001692:Biological Transport / Q000502:physiology; D002352:Carrier Proteins / Q000235:genetics / Q000502:physiology*; D002461:Cell Line, Transformed; D004298:Dopamine / Q000493:pharmacokinetics / Q000494:pharmacology*; D050483:Dopamine Plasma Membrane Transport Proteins; D004594:Electrophysiology; D006801:Humans; D008562:Membrane Glycoproteins; D026901:Membrane Transport Proteins; D009419:Nerve Tissue Proteins; D018408:Patch-Clamp Techniques; D014162:Transfection; D014439:Tyramine / Q000493:pharmacokinetics / Q000494:pharmacology*
D002352:Carrier Proteins; D050483:Dopamine Plasma Membrane Transport Proteins; D008562:Membrane Glycoproteins; D026901:Membrane Transport Proteins; D009419:Nerve Tissue Proteins; C493214:SLC6A3 protein, human; D000661:Amphetamine; D004298:Dopamine; D014439:Tyramine