Technical Reference #1649

1649.

Binding and Transport in Norepinephrine Transporters Joel Schwartz; Randy Blakely; Louis DeFelice, Vanderbilt univesity Medical Center, The Journal of Biological Chemistry, 278(1649), (2003)
Link To Paper

Abstract:
Monoamine transporters the molecular targets for drugs of abuse and antidepressants clear norepinephrinedopamine or serotonin from the synaptic cleft.Neurotransmitters amphetamines and neurotoxinsbind before being transported whereas cocaine

Materials & Methods:
Cell Culture—HEK-293 cells were maintained in Dulbecco’s modified Eagle’s medium with 10% fetal bovine serum (v/v) 2 mM glutamine 100 IU/ml penicillin and 100 g/ml streptomycin (Invitrogen). The stable cells lines that we used for the human norepinephrine transporter (HEK-hNET) and the human serotonin transporter (HEK-hSERT) were previously described (43 44). The stable cell lines that we used for the human dopamine transporter (HEK-hDAT) cells are a gift from Michelle Mazei. Radiolabeled Transport Assay—All experiments were performed at room temperature (22 °C) unless otherwise indicated. HEK-hNET cells were plated on poly-L-lysine-coated 24-well tissue culture plates at 105 cells per well 3 days prior to performing transport assays (90% confluence on the third day). The medium was removed by aspiration. Cells were then preincubated for 10 min in Krebs-Ringer-Hepes (KRH (in mM): 130 NaCl 1.3 KCl 2.2 CaCl2 1.2 MgSO4 1.2 KH2PO4 10 Hepes and 1.8 g/liter glucose pH 7.4) medium with or without 10 M desipramine. Desipramine a specific NET blocker was used to establish nonspecific activity in hNET cells. Pargyline (10 M) and ascorbic acid (10 M) were added to prevent metabolism and oxidation of NE respectively. The assay mixture was aspirated after 10 min and cells were washed three times with 4 °C KRH buffer. Accumulated [3H]NE was determined by liquid scintillation of 1% (w/v) SDS-solubilized cells. Primary Tissue Culture—SCG neurons were dissociated by trituration followed by digestion with 0.25% trypsin and 0.3% collagenase. Non-neuronal cells were removed by pre-plating on uncoated Falcon 60-mm plates. Neurons were cultured on poly-L-ornithine/laminin/poly- D-lysine-coated MatTek dishes at a density of 3000–4000 cells/well in F-14 media containing 5% fetal calf serum 2 mM L-glutamine 60 ng/ml progesterone 16 mg/ml putrescine 400 ng/ml L-thyroxine 38 ng/ml sodium selenite 340 ng/ml tri-iodothyroxine 5 mg/ml insulin/ penicillin/streptomycin 10 M fluorodeoxyuridine and 20 ng/ml nerve growth factor. The neurons were maintained for 3–5 days in the presence of nerve growth factor before use. Microscopy—HEK-hNET cells were plated on 35-mm glass bottom Petri dishes (MatTek Ashland MA) coated with poly-L-lysine 3 days prior to experimentation. The culture medium was aspirated cells were immediately mounted on a Zeiss 410 confocal microscope and the microscope was focused on the center of the monolayer of cells. During the confocal measurement cells remain without buffer for approximately 30 s. Background autofluorescence was established by collecting images for 10 s prior to the addition of KRH (see “Radiolabeled Transport”) 1.8 mg/liter glucose 10 M ascorbic acid 10 M pargyline 10 M tropolone (Sigma) and ASP . The argon laser was tuned to 488 nm; the emitted light was filtered with a 580- to 630-nm band pass filter (max 610 nm). The gain (contrast) and offset (brightness) for the photomultiplier tube was set to avoid detector saturation at the highest ASP concentration used in these experiments (10 M). The effects of photobleaching on ASP accumulation were determined by examining the rate of ASP accumulation and decay at various acquisition rates. Acquisition rates greater than 0.3 Hz degraded sequestered ASP (3.33 s/image). Image Analysis—The fluorescent images were processed using MetaMorph imaging software (Universal Imaging Corp. Downington PA). Fluorescent accumulation was established by measuring the average pixel intensity of time-resolved fluorescent images within a specified region identified by the differential interference contrast image. Average pixel intensity is used to normalize among cells. HEK-293 cells and SCG neurons possess endogenous mechanisms for ASP accumulation (35). NET-mediated ASP accumulation is defined as the fluorescence of HEK-hNET cells or neurons minus background fluorescence. Fluorescence Anisotropy—To evaluate ASP binding to the surface membranes HEK-hNET cells were exposed to 2 M ASP with horizontal polarizer (Fig. 5C) with the polarizer rapidly switching to the vertical position. Cells were imaged with alternating polarizations for 3 min to measure light intensity in the horizontal (Ih) and vertical (Iv) positions to calculate the anisotropy ratio r (Iv gIh)/(Iv 2gIh). The factor g was determined by using a half-wave plate as described by Blackman et al. (45). In this formulation r 0.4 implies an immobile light source (45). Surface anisotropy was measured at the cell circumference over 1 pixel width (0.625 m). Cytosolic anisotropy was measured near the center of the cell 5 pixel widths from the membrane.

Microscopic Technique
Fluorescence Microscopy

Cell Type(s)
HEK-293