Uptake and Release of Neurotransmitters

Aaron Janowsky1, Kim Neve1, Amy J. Eshleman1

1 Oregon Health Sciences University and Veterans Affairs Medical Center, Portland, Oregon
Publication Name:  Current Protocols in Neuroscience
Unit Number:  Unit 7.9
DOI:  10.1002/0471142301.ns0709s02
Online Posting Date:  May, 2001
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Abstract

The availability of clonal cell lines for norepinephrine, dopamine, and serotonin transporters allows the characterization of drug interactions with transporter recognition sites using radioligands, as well as the characterization of drug effects on selective transporter‐mediated uptake and release of substrate. In addition to clonal cell lines, synaptosomes prepared from specific brain regions can be used to conduct these studies without interference by endogenous transporters or binding proteins that are present in other tissues. This unit presents protocols for uptake and release of tritiated substrates using intact cells (either detached or in suspension) or synaptosomes. An HPLC procedure for electrochemical detection of nonradiolabeled substrates is also provided. Time‐dependent release can also be measured in assays involving real‐time sampling.

     
 
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Table of Contents

  • Basic Protocol 1: Study of Uptake and Release of Dopamine in Intact Attached Cells Expressing the Recombinant Dopamine Receptor
  • Alternate Protocol 1: Study of Dopamine Uptake in Detached Cells
  • Basic Protocol 2: Study of Dopamine Uptake in Synaptosomes
  • Basic Protocol 3: Study of Dopamine Release from Synaptosomes
  • Alternate Protocol 2: Detection of Uptake or Release of Dopamine by HPLC with Electrochemical Detection (HPLC‐EC)
  • Alternate Protocol 3: Using a Superfusion Apparatus for Time Sampling
  • Basic Protocol 4: Examination of the Dopamine Transporter with Radioligands
  • Support Protocol 1: Establishing Initial Binding‐Assay Parameters
  • Reagents and Solutions
  • Commentary
  • Literature Cited
  • Figures
  • Tables
     
 
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Materials

Basic Protocol 1: Study of Uptake and Release of Dopamine in Intact Attached Cells Expressing the Recombinant Dopamine Receptor

  Materials
  • Cells stably expressing the recombinant transporter (see Eshleman et al., ; contact authors at )
  • Uptake buffer (see recipe)
  • Drug stock solutions to be tested (at concentrations 10× desired final levels)
  • 50 µM mazindol (Research Biochemicals) in uptake buffer recipe (prepare from 10 mM stock in 0.1 N HCl)
  • 200 nM [3H]dopamine (40 to 60 Ci/mmol; NEN Life Sciences)
  • Phosphate‐buffered saline (PBS; see recipe), ice‐cold
  • 3% (w/v) trichloroacetic acid (TCA)
  • Release buffer: uptake buffer (see recipe) without calcium (ice‐cold)
  • 24‐well tissue culture plates
  • 25°C water bath
  • Scintillation vials and cocktail
  • Software for analyzing radioligand binding data (unit 7.5)
CAUTION: Radioactive materials require special handling; all supernatants must be considered radioactive waste and disposed of accordingly.NOTE: All culture incubations are performed in a humidified 37°C, 5% CO 2 incubator unless otherwise specified.

Alternate Protocol 1: Study of Dopamine Uptake in Detached Cells

  • HEK‐hDAT cells or other poorly adherent cells expressing the dopamine transporter
  • 0.05% (w/v) polyethylenimine
  • 0.9% NaCl
  • 150‐mm tissue culture plates
  • Cell scrapers
  • 96‐well arrays of assay tubes or microvials appropriate for use with cell harvester
  • Whatman GF/C glass fiber filters (or equivalent) appropriate for use with cell harvester
CAUTION: Radioactive materials require special handling; all supernatants must be considered radioactive waste and disposed of accordingly.NOTE: All culture incubations are performed in a humidified 37°C, 5% CO 2 incubator unless otherwise specified.

Basic Protocol 2: Study of Dopamine Uptake in Synaptosomes

  Materials
  • Rats or other suitable animals
  • 0.32 M sucrose, ice‐cold
  • Uptake buffer (see recipe)
  • Drug stock solutions to be tested (at concentrations such that desired final levels can be achieved by adding 50 µl per well)
  • 50 µM mazindol (Research Biochemicals) in uptake buffer recipe (prepare from 10 mM stock in 0.1 N HCl)
  • 200 nM [3H]dopamine (40 to 60 Ci/mmol; NEN Life Sciences)
  • 0.9% NaCl
  • 0.05% (w/v) polyethylenimine
  • Glass Potter‐Elvehjem tissue homogenizer with Teflon pestle
  • 96‐well arrays of assay tubes or microvials appropriate for use with cell harvester
  • Whatman GF/C glass fiber filters (or equivalent) suitable for use with cell harvester
  • Scintillation vials and cocktail
  • Software for analyzing radioligand binding data (unit 7.5)
CAUTION: Radioactive materials require special handling; all supernatants must be considered radioactive waste and disposed of accordingly.NOTE: All protocols using live animals must first be reviewed and approved by an Institutional Animal Care and Use Committee (IACUC) and must follow officially approved procedures for the care and use of laboratory animals.NOTE: Conduct experiments with fresh tissue and perform all steps for tissue preparation at 0° to 4°C.

Basic Protocol 3: Study of Dopamine Release from Synaptosomes

  Materials
  • Rats or other suitable animals
  • Uptake buffer (see recipe)
  • 200 nM [3H]dopamine (40 to 60 Ci/mmol; NEN Life Sciences)
  • 0.32 M sucrose
  • Release buffer: uptake buffer (see recipe) without calcium (CaCl 2)
  • Test drugs in release buffer
  • 0.05% (w/v) polyethylenimine
  • 0.9% NaCl, 4°C
  • Glass Potter‐Elvehjem tissue homogenizer with Teflon pestle
  • Refrigerated centrifuge
  • Whatman GF/C glass fiber filters (or equivalent) appropriate for use with cell harvester
  • Scintillation vials and cocktail
  • Software for analyzing radioligand binding data (unit 7.5)
CAUTION: Radioactive materials require special handling; all supernatants must be considered radioactive waste and disposed of accordingly.NOTE: All protocols using live animals must first be reviewed and approved by an Institutional Animal Care and Use Committee (IACUC) and must follow officially approved procedures for the care and use of laboratory animals.

Alternate Protocol 2: Detection of Uptake or Release of Dopamine by HPLC with Electrochemical Detection (HPLC‐EC)

  • Dopamine⋅HCl (unlabeled)
  • Solvent for dopamine standards (see recipe)
  • 3% (w/v) trichloroacetic acid (TCA)
  • HPLC mobile phase (see recipe)
  • HPLC microvials (Hewlett‐Packard)
  • HPLC/EC system (see unit 7.4 for more detail) including:
  •  Reversed‐phase C18 column (3‐µm particle size; ESA HR‐80)
  •  ESA Coulochem electrochemical detector
  • Additional reagents and equipment for HPLC‐EC (unit 7.4)

Alternate Protocol 3: Using a Superfusion Apparatus for Time Sampling

  Materials
  • Rats
  • Modified Krebs‐HEPES buffer (see recipe)
  • 200 nM [3H]dopamine (40 to 60 Ci/mmol; NEN Life Sciences)
  • Test drugs: transporter inhibitors
  • 20 mM K+/low Na+ buffer: modified Krebs‐HEPES buffer (see recipe) with KCl increased to 20 mM and NaCl reduced to 112 mM
  • Transporter substrates, e.g.:
  • S‐(+)‐Amphetamine (Research Biochemicals)
  •  (+)‐Methamphetamine (Research Biochemicals)
  •  Tyramine (Sigma)
  • 0.2 N HCl
  • Krebs‐bicarbonate buffer (see recipe)
  • Pargyline (Research Biochemicals)
  • Despiramine (Research Biochemicals)
  • Fluoxetine (Research Biochemicals)
  • 1 N NaOH
  • McIlwain tissue chopper (Brinkmann)
  • Superfusion apparatus including 12 superfusion chambers, tubing, and pump (Brandel SF12)
  • Glass fiber filter discs
  • Scintillation vials and cocktail
  • 95% O 2/5% CO 2 gas mixture in cylinder, with regulator and tubes
  • Electrical pulse generator (Brandel ES12955)
CAUTION: Radioactive materials require special handling; all supernatants must be considered radioactive waste and disposed of accordingly.NOTE: All protocols using live animals must first be reviewed and approved by an Institutional Animal Care and Use Committee (IACUC) and must follow officially approved procedures for the care and use of laboratory animals.

Basic Protocol 4: Examination of the Dopamine Transporter with Radioligands

  Materials
  • Cells expressing the dopamine transporter (see Eshleman et al., ; contact authors at )
  • recipePhosphate‐buffered saline (PBS; see recipe)
  • Lysis buffer: 2 mM HEPES/1 mM EDTA (ice‐cold)
  • 0.32 M sucrose
  • Test drugs
  • 400 to 600 pM [125I]3β‐(4‐iodophenyl)tropane‐2β‐carboxylic acid methyl ester ([125I]RTI‐55; 2200 Ci/mmol; NEN Life Sciences)
  • 50 µM mazindol
  • 0.9% NaCl, ice‐cold
  • Uptake buffer (see recipe)
  • Unlabeled 3β‐(4‐iodophenyl)tropane‐2β‐carboxylic acid methyl ester (RTI‐55; Research Triangle Institute)
  • 150‐mm tissue culture plates
  • Cell scrapers
  • Refrigerated centrifuge
  • Polytron tissue homogenizer (Brinkmann)
  • Whatman GF/C glass‐fiber filters (or equivalent)
  • Scintillation vials and cocktail
  • Software for analyzing radioligand binding data (unit 7.5)
CAUTION: Radioactive materials require special handling; all supernatants must be considered radioactive waste and disposed of accordingly.NOTE: All culture incubations are performed in a humidified 37°C, 5% CO 2 incubator unless otherwise specified.
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Figures

  •   FigureFigure 7.9.1 Uptake of [3H]dopamine by C6‐hDAT cells.
  •   FigureFigure 7.9.2 Substrate‐induced release of [3H]dopamine from COS‐7 cells transfected with cDNA for dopamine transporter.
  •   FigureFigure 7.9.3 Association (A) and dissociation (B) of [125I]RTI‐55 binding to membranes from COS‐7 cells transiently expressing the dopamine transporter. Be is the amount bound at equilibrium; Bt is the amount bound at time t.
  •   FigureFigure 7.9.4 Binding data analyzed using GraphPad Prism (also see UNIT).

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Literature Cited

Literature Cited
   Axelrod, J., Weil‐Malherbe, H., and Tomchick, R. 1959. The physiological disposition of [3H]epinephrine and its metabolite metanephrine. J. Pharmacol. Exp. Ther. 127:251‐256.
   Berger, P., Elsworth, J., Reith, M., Tanen, D., and Roth, R. 1990. Complex interaction of cocaine with the dopamine uptake carrier. Eur. J. Pharmacol. 176:251‐252.
   Blakely, R.D., Berson, H., Fremeau, R.T., Caron, M., Peek, M.M., Prince, H.K., and Bradley, C.C. 1991. Cloning and expression of a functional serotonin transporter from rat brain. Nature 354:66‐70.
   Buck, K.J. and Amara, S.G. 1995. Structural domains of catecholamine transporter chimeras involved in selective inhibition by antidepressants and psychomotor stimulants. Mol. Pharmacol. 48:1030‐1037.
   Chen, N.‐H. and Reith, M.E.A. 1993. [3H]Dopamine and [3H]serotonin release in vitro induced by electrical stimulation in A9 and A10 dopamine regions of rat brain: Characterization and responsiveness to cocaine. J. Pharmacol. Exp. Ther. 267:379‐389.
   Cheng, Y. and Prusoff, W.H. 1973. Relationship between the inhibition constant (Ki) and the concentration of inhibitor which causes 50 percent inhibition (I50) of an enzymatic reaction. Biochem. Pharmacol. 22:3099‐3108.
   Eshleman, A.J., Calligaro, D.O., and Eldefrawi, M.E. 1993. Allosteric regulation by sodium of the binding of [3H]cocaine and [3H]GBR 12935 to rat and bovine striata. Membr. Biochem. 10:129‐144.
   Eshleman, A.J., Henningsen, R.A., Neve, K.A., and Janowsky, A. 1994. Release of dopamine via the human transporter. Mol. Pharmacol 45:312‐316.
   Eshleman, A.J., Neve, R.L., Janowsky, A., and Neve, K.A. 1995. Characterization of a recombinant human dopamine transporter in multiple cell lines. J. Pharmacol. Exp. Ther. 274:276‐283.
   Eshleman, A.J., Stewart, E., Evanson, A., Janowsky, A., and Neve, K. 1997. Metabolism of dopamine by catechol‐O‐methyltransferase in multiple cell lines expressing a recombinant transporter. J. Neurochem. 69:1459‐1466.
   Fischer, J.F. and Cho, A.K. 1979. Chemical release of dopamine from striatal homogenates: Evidence for an exchange diffusion model. J. Pharmacol. Exp. Ther. 208:203‐209.
   Giros, B., Wang, Y.‐M., Suter, S., Mclesdy, S.B., Pifl, C., and Caron, M.G. 1994. Delineation of discrete domains for substrate, cocaine and tricyclic antidepressant interactions using chimeric dopamine‐norepinephrine transporters. J. Biol. Chem. 269:7124‐7130.
   Gray, E.C. and Whittaker, V.J. 1962. The isolation of nerve endings from brain: An electron‐microscope study of cell fragments derived by homogenization and centrifugation. J. Anat. 96:79‐87.
   Jacocks, H.M. and Cox, B.M. 1992. Serotonin‐stimulated release of [3H]dopamine via reversal of the dopamine transporter in rat striatum and nucleus accumbens: A comparison with release elicited by potassium, N‐methyl‐D‐aspartic acid, glutamic acid and D‐amphetamine. J. Pharmacol. Exp. Ther. 262:356‐364.
   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 labelling of the dopamine transport complex. J. Neurochem. 46:1272‐1276.
   Javitch, J.A., Blaustein, R.O., and Snyder, S.H. 1984. [3H]Mazindol binding associated with neuronal dopamine and norepinephrine uptake sites. Mol. Pharmacol. 26:35‐44.
   Johnson, R.A., Eshleman, A.J., Meyers, T., Neve, K.A., and Janowsky, A. 1998. [3H]substrate‐ and cell‐specific effects of uptake inhibitors on human dopamine and serotonin transporter‐mediated efflux. Synapse. In press.
   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.
   Kilty, J., Lorang, D., and Amara, S. 1991. Cloning and expression of a cocaine‐sensitive rat dopamine transporter. Science 254:578‐579.
   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.
   Little, K.Y., Kirkman, J.A., Carroll, F.I., Breese, G.R., and Duncan, G.E. 1993. [125I]RTI‐55 binding to cocaine‐sensitive dopaminergic and serotonergic uptake sites in the human brain. J. Neurochem. 61:1996‐2006.
   Madras, B.K., Fahey, M.A., Bergman, J., Canfield, D.R., and Spealman, R.D. 1989a. Effects of cocaine and related drugs in nonhuman primates. 1. [3H]cocaine binding sites in caudate‐putamen. J. Pharmacol. Exp. Ther. 251:131‐141.
   Madras, B.K., Spealman, R.D., Fahey, M.A., Neumeyer, J.L., Saha, J.K., and Milius, R.A. 1989b. Cocaine receptors labeled by [3H]2β‐carbomethoxy‐3β‐(4‐fluorophenyl)tropane. Mol. Pharmacol. 36:518‐524.
   Pacholczyk, T., Blakely, R., and Amara, S. 1991. Expression cloning of a cocaine‐ and antidepressant‐sensitive human noradrenaline transporter. Nature 350:350‐354.
   Pristupa, Z.B., Wilson, J.M., Hoffman, B.J., Kish, S.J., and Niznik, H.B. 1994. Pharmacological heterogeneity of the cloned and native human dopamine transporter: Dissociation of [3H]WIN 35,428 and [3H]GBR 12,935 binding. Mol. Pharmacol. 45:125‐135.
   Reith, M., de Costa, B., Rice, K., and Jacobson, A.E. 1992. Evidence for mutually exclusive binding of cocaine, BTCP, GBR 12935, and dopamine to the dopamine transporter . Eur J. Pharmacol. Mol. Pharmacol. Sect. 227:417‐425.
   Rudnick, G. 1997. Mechanisms of biogenic amine neurotransmitter transporters. In Neurotransmitter Transporters: Structure, Function and Regulation. (M.E.A. Reith, ed.) pp. 73‐100. Humana Press, Totowa, N.J.
   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's Arch Pharmakol. 329:227‐235.
   Schweri, M.M., Skolnick, P., Rafferty, M.F., Rice, K.C., Janowski, A.J., and Paul, S.M. 1985. Threo‐(±)‐methylphenidate binding to 3,4‐dihydroxyphenylethylamine uptake sites in corpus striatum: Correlation with the stimulant properties acid esters. J. Neurochem. 45:1062‐1070.
   Shimada, S., Kitayama, S., Lin, C.‐L., Patel, A., Nanthakumar, E., Gregor, P., Kuhar, M., and Uhl, G. 1991. Cloning and expression of a cocaine‐sensitive dopamine transporter complementary DNA. Science 254:576‐578.
   Shimizu, I. and Prasad, C. 1991. Relationship between [3H]mazindol binding to dopamine uptake sites and [3H]dopamine uptake in rat striatum during aging. J. Neurochem. 56:575‐579.
   Vaughan, R.A. 1995. Photoaffinity‐labeled ligand binding domains on dopamine transporters identified by peptide mapping. Mol. Pharmacol. 47:956‐964.
   Vaughan, R.A. and Kuhar, M.J. 1996. Dopamine transporter ligand binding domains. J. Biol. Chem. 35:21672‐21680.
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