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Interrogating the Activity of Ligands at Monoamine Transporters in Rat Brain Synaptosomes

  • John S. Partilla
  • Michael H. BaumannEmail author
  • Ann M. Decker
  • Bruce E. Blough
  • Richard B. Rothman
Protocol
Part of the Neuromethods book series (NM, volume 118)

Abstract

The plasma membrane transporters for dopamine (DAT), norepinephrine (NET), and serotonin (SERT) are the main sites of action for therapeutic and abused stimulant drugs. As a means to identify novel medications for stimulant addiction and other psychiatric disorders, we developed in vitro assays in rat brain tissue that can be used to determine structure–activity relationships for test compounds at these monoamine transporters. Uptake inhibition assays measure the ability of drugs to block the transporter-mediated uptake of [3H]neurotransmitters into synaptosomes, whereas release assays measure the ability of drugs to serve as transporter substrates that evoke efflux (i.e., release) of [3H]neurotransmitters from synaptosomes by reverse transport. These assays can be used to rapidly determine the potency of test compounds at DAT, NET, and SERT under similar conditions, establishing the selectivity of drugs across all three transporters. The combined results from uptake and release assays can discriminate whether a compound is a transporter inhibitor or substrate (i.e., releaser). Our assay procedures have been used to characterize the molecular mechanism of action for older amphetamine-type medications and newer transporter ligands with therapeutic potential. The data from these assays can also predict the addictive and neurotoxic properties of abused stimulants. Information provided by these assays continues to provide insight into monoamine transporter structure and function.

Key words

Transporter Synaptosomes Amphetamine Stimulants Neurotransmitter Uptake Release 

References

  1. 1.
    Gorman JM, Kent JM (1999) SSRIs and SMRIs: broad spectrum of efficacy beyond major depression. J Clin Psychiatry 60(Suppl 4):33–38PubMedGoogle Scholar
  2. 2.
    Iversen L (2006) Neurotransmitter transporters and their impact on the development of psychopharmacology. Br J Pharmacol 147(Suppl 1):S82–S88PubMedPubMedCentralGoogle Scholar
  3. 3.
    Rothman RB et al (2001) Amphetamine-type central nervous system stimulants release norepinephrine more potently than they release dopamine and serotonin. Synapse 39:32–41CrossRefPubMedGoogle Scholar
  4. 4.
    Howell LL, Kimmel HL (2008) Monoamine transporters and psychostimulant addiction. Biochem Pharmacol 75:196–217CrossRefPubMedGoogle Scholar
  5. 5.
    Rothman RB, Baumann MH (2003) Monoamine transporters and psychostimulant drugs. Eur J Pharmacol 479:23–40CrossRefPubMedGoogle Scholar
  6. 6.
    Sitte HH, Freissmuth M (2015) Amphetamines, new psychoactive drugs and the monoamine transporter cycle. Trends Pharmacol Sci 36:41–50CrossRefPubMedGoogle Scholar
  7. 7.
    Baumann MH, Wang X, Rothman RB (2007) 3,4-Methylenedioxymethamphetamine (MDMA) neurotoxicity in rats: a reappraisal of past and present findings. Psychopharmacology (Berl) 189:407–424CrossRefGoogle Scholar
  8. 8.
    Fleckenstein AE et al (2007) New insights into the mechanism of action of amphetamines. Annu Rev Pharmacol Toxicol 47:681–698CrossRefPubMedGoogle Scholar
  9. 9.
    Gray EG, Whittaker VP (1962) The isolation of nerve endings from brain: an electron-microscopic study of cell fragments derived by homogenization and centrifugation. J Anat 96:79–88PubMedPubMedCentralGoogle Scholar
  10. 10.
    Wilhelm BG et al (2014) Composition of isolated synaptic boutons reveals the amounts of vesicle trafficking proteins. Science 344:1023–1027CrossRefPubMedGoogle Scholar
  11. 11.
    Rothman RB et al (1993) Identification of a GBR12935 homolog, LR1111, which is over 4,000-fold selective for the dopamine transporter, relative to serotonin and norepinephrine transporters. Synapse 14:34–39CrossRefPubMedGoogle Scholar
  12. 12.
    Rothman RB et al (2015) Studies of the biogenic amine transporters 15. Identification of novel allosteric dopamine transporter ligands with nanomolar potency. J Pharmacol Exp Ther 353(3):529–538CrossRefPubMedPubMedCentralGoogle Scholar
  13. 13.
    Rothman RB et al (2000) Methamphetamine dependence: medication development efforts based on the dual deficit model of stimulant addiction. Ann N Y Acad Sci 914:71–81CrossRefPubMedGoogle Scholar
  14. 14.
    Baumann MH et al (2013) Powerful cocaine-like actions of 3,4-methylenedioxypyrovalerone (MDPV), a principal constituent of psychoactive ‘bath salts’ products. Neuropsychopharmacology 38(4):552–562CrossRefPubMedGoogle Scholar
  15. 15.
    Rudnick G, Clark J (1993) From synapse to vesicle: the reuptake and storage of biogenic amine neurotransmitters. Biochim Biophys Acta 1144:249–263CrossRefPubMedGoogle Scholar
  16. 16.
    Rothman RB, Baumann MH (2006) Therapeutic potential of monoamine transporter substrates. Curr Top Med Chem 6:1845–1859CrossRefPubMedGoogle Scholar
  17. 17.
    Rothman RB et al (2003) In vitro characterization of ephedrine-related stereoisomers at biogenic amine transporters and the receptorome reveals selective actions as norepinephrine transporter substrates. J Pharmacol Exp Ther 307:138–145CrossRefPubMedGoogle Scholar
  18. 18.
    Scholze P et al (2000) Transporter-mediated release: a superfusion study on human embryonic kidney cells stably expressing the human serotonin transporter. J Pharmacol Exp Ther 293:870–878PubMedGoogle Scholar
  19. 19.
    Rothman RB et al (2003) (+)-Fenfluramine and its major metabolite, (+)-norfenfluramine, are potent substrates for norepinephrine transporters. J Pharmacol Exp Ther 305:1191–1199CrossRefPubMedGoogle Scholar
  20. 20.
    Yu H et al (2000) Uptake and release effects of diethylpropion and its metabolites with biogenic amine transporters. Bioorg Med Chem 8:2689–2692CrossRefPubMedGoogle Scholar
  21. 21.
    Rothman RB et al (2012) Studies of the biogenic amine transporters. 14. Identification of low-efficacy “partial” substrates for the biogenic amine transporters. J Pharmacol Exp Ther 341(1):251–262CrossRefPubMedPubMedCentralGoogle Scholar
  22. 22.
    Baumann MH et al (2012) The designer methcathinone analogs, mephedrone and methylone, are substrates for monoamine transporters in the brain. Neuropsychopharmacology 37(5):1192–1203CrossRefPubMedGoogle Scholar
  23. 23.
    Saha K et al (2015) ‘Second-generation’ mephedrone analogs, 4-MEC and 4-MePPP, differentially affect monoamine transporter function. Neuropsychopharmacology 40(6):1321–1331CrossRefPubMedPubMedCentralGoogle Scholar
  24. 24.
    Marusich JA et al (2014) Pharmacology of novel synthetic stimulants structurally related to the “bath salts” constituent 3,4-methylenedioxypyrovalerone (MDPV). Neuropharmacology 87:206–213CrossRefPubMedPubMedCentralGoogle Scholar
  25. 25.
    Bonano JS et al (2015) Quantitative structure-activity relationship analysis of the pharmacology of para-substituted methcathinone analogues. Br J Pharmacol 172(10):2433–2444CrossRefPubMedPubMedCentralGoogle Scholar
  26. 26.
    Sakloth F et al (2015) Steric parameters, molecular modeling and hydropathic interaction analysis of the pharmacology of para-substituted methcathinone analogues. Br J Pharmacol 172(9):2210–2218CrossRefPubMedPubMedCentralGoogle Scholar

Copyright information

© Springer Science+Business Media New York 2016

Authors and Affiliations

  • John S. Partilla
    • 1
  • Michael H. Baumann
    • 2
    Email author
  • Ann M. Decker
    • 3
  • Bruce E. Blough
    • 3
  • Richard B. Rothman
    • 1
  1. 1.Medicinal Chemistry Section, Molecular Targets and Medications Discovery Branch, Intramural Research ProgramNational Institute on Drug Abuse, National Institutes of HealthBaltimoreUSA
  2. 2.Medicinal Chemistry Section, IRPNIDA, NIHBaltimoreUSA
  3. 3.Center for Drug DiscoveryResearch Triangle InstituteResearch Triangle ParkUSA

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