Uptake and Release of Neurotransmitters

互联网2013-12-31

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Abstract
Table of Contents
Materials
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Literature Cited

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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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 [³ H]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% CO₂ 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

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 [³ H]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 [³ H]dopamine (40 to 60 Ci/mmol; NEN Life Sciences)
0.32 M sucrose
Release buffer: uptake buffer (see recipe ) without calcium (CaCl₂ )
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 )

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 [³ H]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% O₂ /5% CO₂ gas mixture in cylinder, with regulator and tubes
Electrical pulse generator (Brandel ES12955)

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 [¹²⁵ I]3β‐(4‐iodophenyl)tropane‐2β‐carboxylic acid methyl ester ([¹²⁵ I]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 )

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Figures

Figure 7.9.1 Uptake of [³ H]dopamine by C6‐hDAT cells.

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Figure 7.9.2 Substrate‐induced release of [³ H]dopamine from COS‐7 cells transfected with cDNA for dopamine transporter.

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Figure 7.9.3 Association (A ) and dissociation (B ) of [¹²⁵ I]RTI‐55 binding to membranes from COS‐7 cells transiently expressing the dopamine transporter. B _e is the amount bound at equilibrium; B _t is the amount bound at time t.

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Figure 7.9.4 Binding data analyzed using GraphPad Prism (also see UNIT ).

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Videos

Literature Cited

Literature Cited
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Uptake and Release of Neurotransmitters

Abstract

Table of Contents

Materials

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

Figures

Videos

Literature Cited