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Studies of the biogenic amine transporters. 1. Dopamine reuptake blockers inhibit [3H]mazindol binding to the dopamine transporter by a competitive mechanism: Preliminary evidence for different binding domains

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Abstract

The present study addressed the hypothesis that the DA transporter ligand, [3H]mazindol, labels multiple sites/states associated with the dopamine (DA) transporter in striatal membranes. Incubations with [3H]mazindol proceeded for 18–24 hr at 4δC in 55.2 mM sodium phosphate buffer, pH 7.4, with a protease inhibitor cocktail. In order to obtain data suitable for quantitative curve fitting, it was necessary to repurify the [3H]mazindol by HPLC before a series of experiments. Under these conditions, we observed greater than 80% specific binding. The method of binding surface analysis was used to characterize the interaction of GBR12935, BTCP, mazindol, and CFT with binding site/sites labeled by [3H]mazindol. A one site model fit the data as well as the two site model: Bmax=16911 fmol/mg protein, Kd of [3H]mazindol=75 nM, Ki of GBR12935 =8.1 nM, Ki of CFT=50 nM and Ki of BTCP=44 nM. The inhibitory mechanism (competitive or noncompetitive) of several drugs (GBR12935, CFT, BTCP, cocaine, cis-flupentixol, nomifensine, WIN35,065-2, bupropion, PCP, and benztropine) was determined. All drugs inhibited [3H]mazindol binding by a competitive mechanism. Although the ligand-selectivity of the [3H]mazindol binding site indicates that it is the uptake inhibitor recognition site of the classic DA transporter, the quantitative differences among the ligand-selectivities of different radioligands for the same site suggest that each radioligand labels different overlapping domains of the DA uptake inhibitor recognition site. It is likely that development of domain-selective drugs may further our under-standing of the DA transporter.

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References

  1. Wise, R. A. 1981. Brain dopamine and reward. Pages 103–122,in Cooper, S. J., (ed.), Theory in psychopharmacology, Academic Press, London, NY.

    Google Scholar 

  2. Ritz, M. C., Lamb, R. J., Goldberg, S. R., and Kuhar, M. J. 1987. Cocaine receptors on dopamine transporters are related to self-administration of cocaine. Science 237:1219–1223.

    Google Scholar 

  3. Kuhar, M. J., Ritz, M. C., and Boja, J. W. 1991. The dopamine hypothesis of the reinforcing properties of cocaine. Trends. Neurosci. 14:299–302.

    Google Scholar 

  4. Spealman, R. D., Madras, B. K., and Bergman, J. 1989. Effects of cocaine and related drugs in nonhuman primates. II. Stimulant effects on schedule-controlled behavior. J. Pharmacol. Exp. Ther. 251:142–149.

    Google Scholar 

  5. Bergman, J., Madras, B. K., Johnson, S. E., and Spealman, R. D. 1989. Effects of cocaine and related drugs in nonhuman primates. III. Self-administration by squirrel monkeys. J. Pharmacol. Exp. Ther. 251:150–155.

    Google Scholar 

  6. Carroll, F. I., Lewin, A. H., Boja, J. W., and Kuhar, M. J. 1992. Cocaine receptor: biochemical charaterization and structure-activity relationships of cocaine analogues at the dopamine transporter. J. Med. Chem. 35:969–981.

    Google Scholar 

  7. Calligaro, D. O., and Eldefrawi, M. E. 1987. Central and peripheral cocaine receptors. J. Pharmacol. Exp. Ther. 243:61–68.

    Google Scholar 

  8. Schoemaker, H., Pimoule, C., Arbilla, S., Scatton, B., Javoy-Agid, F., and Langer, S. Z. 1985. Sodium dependent [3H]cocaine binding associated with dopamine uptake sites in the rat striatum and human putamen decrease after dopaminergic denervation and in Parkinsons disease. Naunyn Schmiedeberg' Arch. Pharmacol. 329:227–235.

    Google Scholar 

  9. Madras, B. K., Fahey, M. A., Bergman, J., Canfield, D. R., and Spealman, R. D. 1989. Effects of cocaine and related drugs in nonhuman primates. I. [3H]cocaine binding sites in caudate-putamen. J. Pharmacol. Exp. Ther. 251:131–141.

    Google Scholar 

  10. Bonnet, J. J., Protais, P., Chagraoui, A., and Costentin, J. 1986. High-affinity [3H]GBR 12783 binding to a specific site associated with the neuronal dopamine uptake complex in the central nervous system Eur. J. Pharmacol. 126:211–222.

    Google Scholar 

  11. Berger, P., Janowsky, A. Vocci, F., Skolnik, P., Schweri, M. M., and Paul, S. M. 1985. [3H]GBR 12935: a specific high affinity ligand for labeling the dopamine transport complex. Eur. J. Pharmacol. 107:289–290.

    Google Scholar 

  12. Andersen, P. H. 1987. Biochemical and pharmacological characterization of [3H]GBR 12935 binding in vitro to rat striatal membranes: labeling of the dopamine uptake complex. J. Neurochem. 48:1887–1896.

    Google Scholar 

  13. Izenwasser, S., Werling, L. L., Rosenberger, J. G., and Cox, B. M. 1990. Characterization of binding of GBR 12935 (1-[2-(diphenylmethoxy)ethyl]-4-(3-phenylpropylpiperazine) to membranes and to solubilized membrane extracts from terminal field regions of mesolimbic, mesocortical and nigrostriatal dopamine pathways. Neuropharmacology. 29:1017–1024.

    Google Scholar 

  14. Javitch, J. A., Blaustein, R. O., and Snyder, S. H. 1984. [3H]mazindol binding associated with neuron dopamine and norepinephrine uptake sites. Mol. Pharmacol. 26:35–44.

    Google Scholar 

  15. Reith, M. E. A., Meisler, B. H., Sershen, H., and Lajtha, A. 1986. Structural requirements for cocaine congeners to interact with dopamine and serotonin uptake sites in mouse brain and to induce stereotyped behavior. Biochem. Pharmacol. 35:1123–1129.

    Google Scholar 

  16. Dubocovich, M. L., and Zahniser, N. R. 1985. Binding characteristics of the dopamine uptake inhibitor [3H]nomifensine to striatal membranes. Biochem. Pharmacol. 34:1137–1144.

    Google Scholar 

  17. Vignon, J., Pinet, V., Cerruti, C., Kamenka, J-M., and Chicheportiche, R. 1988. [3H]-[1-(2-benzo[b]thiophenyl)cyclohexyl]piperidine [3H]BTCP): a new phencyclidine analog selective for the dopamine uptake complex. Eur. J. Pharmacol. 148:427–436.

    Google Scholar 

  18. Madras, B. K., Spealman, R. D., Fahey, M. A., Neumeyer, J. L., Saha, J. K., and Milius, R. A. 1989. Cocaine receptors labeled by [3H]2β-carbomethoxy-3-β-(4-fluorophenyl)tropane. Mol. Pharmacol. 36:518–524.

    Google Scholar 

  19. Schweri, M. M., Skolnick, P., Rafferty, M. F., Rice, K. C., Janowsky, A. J., and Paul, S. M. 1985. [3H]Threo-(+/−)-methylphenidate binding to 3,4-dihydroxyphenylethylamine uptake sites in corpus striatum: correlation with the stimulant properties of ritalinic acid esters. J. Neurochem. 45:1062–1070.

    Google Scholar 

  20. Boja, J. W., Patel, A., Carroll, F. I., Rahman, M. A., Philip, A., Lewin, A. H., Kopajtic, T. A., and Kuhar, M. J. 1991. [125I]RTI-55: a potent ligand for dopamine transporters. Eur. J. Pharmacol. 194:133–134.

    Google Scholar 

  21. Kennedy, L. T., and Hanbauer, I. 1983. Sodium-sensitive cocaine binding to rat striatal membrane: possible relationship to dopamine uptake sites. J. Neurochem. 41:172–178.

    Google Scholar 

  22. Calligaro, D. O., and Eldefrawi, M. E. 1988. High affinity stereospecific binding of [3H]cocaine in striatum and its relationship to the dopamine transporter. Memb. Biochem. 7:87–106.

    Google Scholar 

  23. Reith, M. E. A., and Selmeci, G. 1992. Radiolabeling of dopamine uptake sites in mouse striatum: comparison of binding sites for cocaine, mazindol, and GBR 12935. Naunyn. Schmiedebergs. Arch. Pharmacol. 345:309–318.

    Google Scholar 

  24. Berger, P., Elsworth, J. D., Arroyo, J., and Roth, R. H. 1990. Interaction of [3H]GBR 12935 and GBR 12909 with the dopamine uptake complex in nucleus accumbens. Eur. J. Pharmacol. 177:91–94.

    Google Scholar 

  25. Janowsky, A., Berger, P., Vocci, F., Labarca, R., Skolnick, P., and Paul, S. M. 1986. Characterization of sodium-dependent [3H]GBR-12935 binding in brain: a radioligand for selective labeling of the dopamine transport complex. J. Neurochem. 46:1272–1276.

    Google Scholar 

  26. Sharif, N. A., Nunes, J. L., Michel, A. D., and Whiting, R. L. 1989. Comparative properties of the dopamine transport complex in dog and rodent brain: striatal [3H]GBR12935 binding and [3H]dopamine uptake. Neurochem. Int. 15:325–332.

    Google Scholar 

  27. Hitri, A, Venable, D., Nguyen, H. Q., Casanova, M. F., Kleinman, J. E., and Wyatt, R. J. 1991. Characteristics of [3H]GBR12935 binding in the human and rat frontal cortex. J. Neurochem. 56:1663–1672.

    Google Scholar 

  28. Richfield, E. K. 1991. Quantitative autoradiography of the dopamine uptake complex in rat brain using GBR 12935: binding characteristics. Brain Res. 540:1–13.

    Google Scholar 

  29. Niznik, H. B., Tyndale, R. F., Sallee, F. R., Gonzalez, F. J., Hardwick, J. P., Inaba, T., and Kalow, W. 1990. The dopamine transporter and cytochrome P450IID1 (debrisoquine 4-hydroxylese) in brain: resolution and identification of two distinct [3H]GBR-12935 binding proteins. Arch. Biochem. Biophys. 276:424–432.

    Google Scholar 

  30. Boja, J. W., Mitchell, W. M., Patel, A., Kopajtic, T. A., Carroll, F. I., Lewin, A. H., Abraham, P., and Kuhar, M. J. 1992. High affinity binding of [125I]RTI-55 to dopamine and serotonin transporters in rat brain. Synapse 12:27–36.

    Google Scholar 

  31. Ritz, M. C., Boja, J. W., Grigoriadis, D., Zaczek, R., Carroll, F. I., Lewis, A. H., and Kuhar, M. J. 1990. [3H]WIN 35,065-2:a ligand for cocaine receptors in striatum. J. Neurochem. 55:1556–1562.

    Google Scholar 

  32. Boja, J. W., Rahman, M. A., Philip, A., Lewin, A. H., Carroll, F. I., and Kuhar, M. J. 1991. Isothiocyanate derivatives of cocaine: irreversible inhibition of ligand binding at the dopamine transporter. Mol. Pharmacol. 39:339–345.

    Google Scholar 

  33. Boja, J. W., Markham, L., Patel, A., Uhl, G., and Kuhar, M. J. 1992. Expression of a single dopamine transporter cDNA can confer two cocaine binding sites. Neuroscience/Neuro Report 3:247–248.

    Google Scholar 

  34. Lowry, O. H., Rosenbrough, N. H., Farr, A. L., and Randall, P. J. 1951. Protein measurement with the Folin phenol reagent. J. Biol. Chem. 193:265–275.

    Google Scholar 

  35. Akunne, H. C., de Costa, B. R., Jacobson, A. E., Rice, K. C., and Rothman, R. B. 1992. [3H]Cocaine labels a binding site associated with the serotonin transporter in guinea pig brain: allosteric modulation by paroxetine. Neurochem. Res. 17:1275–1283.

    Google Scholar 

  36. Rothman, R. B. 1983. Analysis of binding surfaces: a methodology appropriate for the investigation of complex receptor mechanisms and multiple neurotransmitter receptors. Neuropeptides. 4:41–44.

    Google Scholar 

  37. Rothman, R. B. 1986. Binding surface analysis: an intuitive yet quantitative method for the design and analysis of ligand binding studies. Alcohol and Drug Res. 6:309–325.

    Google Scholar 

  38. Rothman, R. B., Reid, A. A., Mahboubi, A., Kim, C.-H. de Costa, B. R., Jacobson, A. E., and Rice, K. C. 1991. Labeling by [3H]1,3-Di(2-tolyl)guanidine of two high affinity binding sites in guinea pig brain: evidence for allosteric regulation by calcium channel antagonists and pseudoallosteric modulation by ligands. Mol. Pharmacol. 39:222–232.

    Google Scholar 

  39. McGonigle, P., Neve, K. A., and Molinoff, P. B. 1986. A quantitative method of analyzing the interaction of slightly selective radioligands with multiple receptor subtypes. Mol. Pharmacol. 30:329–333.

    Google Scholar 

  40. Rovati, G. E., Rodbard, D., and Munson, P. J. 1990. DESIGN: computerized optimization of experimental design for estimating Kd and Bmax in ligand binding experiments. II Simultaneous analysis of homologous and heterologous competition curves and analysis blocking and of “multiligand” dose-response surfaces. Anal. Biochem. 184:172–183.

    Google Scholar 

  41. Munson, P. J., and Rodbard, D. 1980. LIGAND: a versatile computerized approach for characterization of ligand-binding systems. Anal. Biochem. 107:220–239.

    Google Scholar 

  42. Rothman, R. B., Bykov, V., de Costa, B. R., Jacobson, A. E., Rice, K. C., and Brady, L. S. 1990. Interaction of endogenous opioid peptides and other drugs with four kappa opioid binding sites in guinea pig brain. Peptides 11:311–331.

    Google Scholar 

  43. Reid, A. A., Mattson, M. V., de Costa, B. R., Thurkauf, A., Jacobson, A. E., Monn, J. A., Rice, K. C., and Rothman, R. B. 1990. Specificity of phencyclidine-like drugs and benzomorphan opiates for NMDA-coupled and dopamine uptake carrier associated phencyclidine binding sites in guinea pig brain. Neuropharmacology. 29:811–817.

    Google Scholar 

  44. Rothman, R. B., Reid, A. A., Silverthorn, M., de Costa, B. R., Monn, J. A., Thurkauf, A., Jacobson, A. E., Rice, K. C., and Rogawski, M. A. 1992. Structure-activity studies on the interaction of biogenic amine reuptake inhibitors and potassium channel blockers with MK-801-sensitive (PCP site 1) and-insensitive (PCP site 2) [3H]TCP binding sites in guinea pig brain. Pp. 137–146, in Domino, E. F. and Kamenka, J. M., (eds.), Multiple Sigma and PCP Receptor Ligands: Mechanisms for Neuromodulation and Protection, NPP Books, Ann Arbor, MI.

    Google Scholar 

  45. Neve, K. A., McGonigle, P., and Molinoff, P. B. 1986. Quantitativa analysis of the selectivity of radioligands for subtypes of beta adrenergic receptors. J. Pharmacol. Exp. Ther. 238:46–53.

    Google Scholar 

  46. Rothman, R. B., Bykov, V., Mahboubi, A., Long, J. B., Jiang, Q., Porreca, F., de Costa, B. R., Jacobson, A. E., Rice, K. C., and Holaday, J. W. 1991. Interaction of beta-funaltrexamine with [3H]cycloFOXY binding in rat brain: further evidence that beta-FNA alkylates the opioid receptor complex. Synapse 8:86–99.

    Google Scholar 

  47. de Costa, B. R. 1990. Synthesis of high specific activity (3,4-[3H]Cyclohexyl)-N-[1-(2-benzo[b]cyclohexyl]piperid ine ([3H]BTCP): a selective probe for the dopamine reuptake complex. J. Labeled. Comp. Radiopharm. 29:165–173.

    Google Scholar 

  48. Van der Zee, P., Koger, H. S., Gootjes, J., and Hespe, W. 1980. Aryl 1,4-dialk(en)ylpiperazines as selective and very potent inhibitors of dopamine uptake. Eur. J. Med. Chem. 15:363–370.

    Google Scholar 

  49. Akunne, H. C., Dersch, H. S., Cadet, J. L., Char, G. U., Partilla, J. S., de Costa, B. R., Rice, K. C., Carroll, F. I., and Rothman, R. B. 1993. Studies of the biogenic amine transporters. III. Demonstration of two binding sites for [3H]GBR12935 and [3H]BTCP in rat caudate membranes. J. Pharmacol. Exp. Ther. (submitted)

  50. Kitayama, S. Shimada, S., Xu, H., Markham, L., Donovan, D. M., and Uhl, G. R. 1992. Dopamine transporter site-directed mutations differentially alter substrate transport and cocaine binding. Proc. Natl. Acad. Sci. U.S.A. 89:7782–7785.

    Google Scholar 

  51. O'Dowd, B. F., Hantowich, M., Regan, J. W., Leader, W. M., and Caron, M. G. 1988. Site-directed mutagenesis of the cytoplasmic domains of the human beta-2-adrenergic receptor localization of region involved in G protein-receptor coupling. J. Biol. Chem. 263:15985–15992.

    Google Scholar 

  52. Guan, X.-M., Peroutka, S. J., and Kobilka, B. K. 1992. Identification of a single amino acid residue responsible for the binding of a class of β-adrenergic receptor antagonists to 5-hydroxytryptamine1a receptors. Mol. Pharmacol. 41:695–698.

    Google Scholar 

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Dersch, C.M., Akunne, H.C., Partilla, J.S. et al. Studies of the biogenic amine transporters. 1. Dopamine reuptake blockers inhibit [3H]mazindol binding to the dopamine transporter by a competitive mechanism: Preliminary evidence for different binding domains. Neurochem Res 19, 201–208 (1994). https://doi.org/10.1007/BF00966817

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