Salvinorin A regulates dopamine transporter function via a kappa opioid receptor and ERK1/2-dependent mechanism.

2.1.

Live Cell Imaging to Quantify DAT function

Experiments were conducted in HEK-293 (HEK) and EM4 cells, a HEK cell line expressing a macrophage scavenger receptor to increase adherence to tissue culture plastic. Cells were maintained in DMEM/Ham's F-12 medium (50:50; Mediatech Inc., Herndon, VA) supplemented with 10% FBS and grown in a humidified atmosphere (37°C and 5% CO 2 ). Cells were transfected with myc-rat KOR (KOR; 0.1 μg) and either 0.3 μg of YFP-human DAT (DAT), eGFP-rat DAT (rDAT), FLAG-human DAT (FLAG-DAT) or GFP-human norepinephrine transporter (NET), or human serotonin transporter (hSERT) 24 h after plating, using Lipofectamine™ LTX (Invitrogen, Carlsbad, CA). Experiments were performed 48 h later (cell confluency: 70-80%). Addition of these tags does not alter the trafficking, protein localization or function ( Jordan and Devi, 1999 ; Zapata et al., 2007 ). Time-resolved quantification of DAT function in single cells was achieved using the fluorescent, high affinity monoamine transporter substrate 4-(4-diethylaminostyryl)- N -methylpyridinium iodide (ASP + ) ( Schwartz et al., 2003 ). ASP + is a sensitive probe for monitoring monoamine transporter function ( Bolan et al., 2007 ; Schwartz et al., 2003 ; Zapata et al., 2007 ). ASP + accumulation is linear for 10 min after ASP + addition, inhibited by substrates and dependent on temperature and extracellular NaCl concentrations. Immediately before experiments, media was removed and cells washed in Krebs-Ringer/HEPES medium (KRH in mM: 130 NaCl, 1.3 KCl, 2.2 CaCl 2 , 1.2 MgS0 4 , 1.2 KH 2 PO 4 , 10 HEPES, and 1.8 g/liter glucose, pH 7.4). Fresh KRH was added, and the culture dish was mounted on an Ultra VIEW™ LCI spinning-disk (PerkinElmer Life Sciences (Grand Island, NY) or Olympus FV1000 (Olympus, Tokyo, Japan) confocal microscope (60x water objective lens). A within cell design was used to assess the effects of graded SalA concentrations (0.1-10 μM), and to determine ASP + uptake kinetics. The microscope was focused on a cell monolayer and background auto-fluorescence determined by collecting an image immediately prior to replacing buffer with that containing ASP + (10 μM). SalA, SalA, U69,593 or U50,488 or vehicle was added 5 min later. The slope of ASP + accumulation was determined before and after addition. Images were collected every 20 s for 10 min to capture YFP or GFP (excitation, 488 nm; emission, 525–575 nm) and ASP + fluorescence (excitation, 488 nm; emission, 607–652 nm). To determine ASP + uptake kinetics, a range of ASP + concentrations (0-16 μM) were used. The slope of uptake phase was determined following background subtraction (cells not expressing DAT) and normalization to cell surface expression of either DAT or SERT. For studies assessing the effects of the selective KOR antagonist, nor-binaltorphimine (BNI: 1 μM; 10 min), pertussis toxin (PTX: 100 ng/ml; 16-24 hr) or the selective extracellular signal regulated kinase 1/2 (ERK) inhibitor, PD98059 (10 μM; 15 min) or p38 MAP kinase inhibitor SB203580 (3 μM; 5 min) (Tocris; Minneapolis, MN) or dopamine D 2 receptor antagonist L-741,626 (10 μM; 5 min) (Tocris; Minneapolis, MN) on SalA-evoked alterations of ASP + uptake, cells were incubated with drug for the indicated times and the slope of ASP + accumulation quantified as above. Drug concentrations were chosen based on reported effective concentrations ( Alessi et al., 1995 ; Bolan et al., 2007 ; Dalman and O'Malley, 1999 ; Grilli et al., 2009 ; Zapata et al., 2007 ). Fluorescent images were processed using Velocity (PerkinElmer Life Sciences) and NIH ImageJ (version 1.32) software. Within cell fluorescent accumulation, defined by GFP or YFP plasma membrane fluorescence, was measured as average pixel intensity of time-resolved images. Data are expressed as arbitrary fluorescence units (AFU) or percent change in ASP + uptake rate after drug addition. Typically 30–100 cells from three separate transfections were used. 2.2. Quantification of Striatal DAT Function All Procedures with rodents were approved by the Institutional Animal Care and Use Committee in accordance with the National Institutes of Health Guide (NIH Publication No. 8023, revised 1978) for the Care and Use of Laboratory Animals and the Victoria University of Wellington Animal Ethics Committee. Rats were maintained in a temperature and humidity controlled room on a 12:12 h light/dark cycle. Food and water were supplied ad libitum. All efforts and care were taken to minimize animal suffering and to reduce the number of animals used. As alternatives to brain tissues, cell culture models were utilized. 2.2.1. Rotating Disk Electrode (RDE) Voltammetry RDE was used to determine the initial velocity of dopamine clearance in minces of the striatal tissue of rats as previously described using an electrode rotation rate of 4000 rpm and an applied potential of +450 mV versus Ag/AgCl reference electrode ( Thompson et al., 2000 ). Voltage output was monitored until stable baselines were obtained (≅10 min). SalA or U50,488 (3 μl; final cell concentration: 10 nM) or an equivalent volume of vehicle was added to the electrochemical cell 4-5 min prior to addition of dopamine (6 μl; final concentration: 2 μM). PD98050 (3 μl; final concentration: 10 μM) was added to the cell, followed 10 min later by SalA. The resultant signals were detected as changes in voltage output versus time using electrochemical detection. The initial rate of signal decay after dopamine addition was calculated for 10 s. Rates of nonspecific signal decay, defined as signal decay in the absence of tissue at the end of each experimental day, were subtracted from that in the presence of tissue to calculate initial velocity of DA clearance (pmoles/s/g wet weight tissue). DA-clearance in the presence of GBR12909 was subtracted from DA clearance in absence of GBR12909 to derive DAT-mediated DA clearance.. 2.2.2. [ 3 H]DA uptake assay Synaptosomes from striatum were prepared and [ 3 H]DA uptake was measured as described previously ( Tejeda et al., 2013 ). Briefly, rats were rapidly decapitated, and striatal regions were dissected and collected in 10 volumes (wt/vol) of cold 0.32 M sucrose. The tissue was immediately homogenized using a Teflon-glass homogenizer and centrifuged at 1000 × g for 15 min at 4°C. The resulting supernatant was centrifuged at 12,000 × g for 20 min and the pellet was washed by resuspending in 0.32 M sucrose. The synaptosomal preparation was used immediately for experiments. Protein concentration was determined by DC protein assay (BioRad) using bovine serum albumin as standard. Striatal synaptosomes (40 μg) were incubated in a total volume of 0.5 ml of Krebs-Ringer-HEPES (KRH) buffer consisting of 120 mM NaCl, 4.7 mM KCl, 2.2 mM CaCl 2 10 mM HEPES, 1.2 mM MgSO 4 , 1.2 mM KH 2 PO 4 , 5 mM Tris, 10 mM D- glucose, pH 7.4 containing 0.1 mM ascorbic acid, and 0.1 mM pargyline in the presence of SalA (10 μM) or appropriate vehicle at 37°C for 5 min. Uptake was initiated by the addition of 10 nM [ 3 H]DA (78 Ci/mmoldihydroxyphenylethylamine [2,5,6,7,8- 3 H], PerkinElmer, Santa Clara, CA). Unlabelled DA was used along with [ 3 H]DA from 0.01 nM to 2.0 μM for saturation analysis. Uptake was terminated with the addition of 3 ml ice-cold PBS followed by rapid filtration over 0.3% polyethylenimine coated GF-B filters on a Brandel Cell Harvester (Brandel Inc., Gaithersburg, MD). Filters were washed rapidly with 5 ml cold PBS and radioactivity bound to filter was counted by liquid scintillation counter. Nonspecific uptake, defined as the uptake in the presence of 100 μM cocaine, was subtracted from total accumulation of [ 3 H]DA to yield specific total DA uptake (representing DA uptake mediated by both DAT and NET). To isolate only DAT-mediated [ 3 H]DA uptake from total [ 3 H]DA uptake (DAT/NET-specific), NET-specific blocker nisoxetine (50 nM) was used to block NET-mediated DA transport as described earlier ( Tejeda et al., 2013 ). DAT specific DA uptake was further verified using specific DAT blocker GBR12909 (50 nM) or nomifensine (100 nM). DAT activity isolated in this manner was completely blocked by DAT blockers GB12909 or nomifensine. Thus, [ 3 H]DA uptake in the presence of nisoxetine corresponds to specific DA uptake through DAT. All uptake assays were performed in triplicates and expressed as mean values of specific uptake ± S.E.M. 2.3. Immunoblotting of ERK1/2 and p38 MAPK Immunoblotting of phosphorylated ERK (p-ERK) and p38 MAPK (phospho-p38 MAPK) were conducted in EM4 cells that are serum-starved for 2 h. Cells were incubated with SalA (10 μM) or vehicle for 5 min at 37 °C. PD98059 (10 μM) was preincubated for 15 min prior to SalA addition. Following the treatment, the media was aspirated and the cells were washed before solubilizing with RIPA buffer (10 mM Tris-HCl, pH 7.5, 150 mM NaCl, 1 mM EDTA, 1% Triton X-100, 0.1% SDS, and 1% sodium deoxycholate) supplemented with a cocktail of protease and phosphatase inhibitors. Equal amount of protein was incubated with Laemmli buffer (62.5 mM Tris, pH 6.8, 20% glycerol, 2% SDS, 5% β-mercaptoethanol, and 0.01% bromphenol blue) and proteins separated by SDS-PAGE and transferred onto polyvinylidene difluoride membranes (Millipore, Bellerica, MA). Membranes were blocked for 1 h at room temperature in TBS-T containing 5% nonfat milk. p-ERK and phospho-p38 MAPK were detected using rabbit polyclonal antibody specific for p44/42 MAPK phosphorylated at threonine 202 and tyrosine 204 and threonine 180 and tyrosine 182 respectively. To analyze total ERK and p38 MAPK levels, blots were stripped with 2% SDS and 100 mM β-mercaptoethanol in 62.5 mM Tris, pH 6.8, for 1 h at 50 °C and reprobed with polyclonal antibodies (Cell Signaling Technology, Inc. Beverly, MA) recognizing total ERK or total p38 MAPK. Blots were visualized using HRP-conjugated secondary antibody (Jackson ImmunoResearch Laboratories, West Grove, PA) with enhanced chemiluminescence reagents (Amersham Biosciences, GE healthcare, NJ). Multiple exposures were evaluated by digital quantitation using NIH ImageJ (version 1.32j) software to ensure that results were within the linear range of film exposure. Amounts of p-ERK and phospho-p38 MAPK were normalized to that of total ERK and total p38 MAPK respectively ( Bolan et al., 2007 ). 2.4.

Cell Surface Biotinylation and Immunoblotting

Cell surface biotinylation and immunoblotting was performed as described previously ( Zapata et al., 2007 ). Cells: Briefly, EM4 cells (100,000 cells/well) transfected with myc-KOR and FLAG-DAT were grown in 12 well plates were first washed PBS/Ca-Mg (138 mM NaCl, 2.7 mM KCl, 1.5 mM KH 2 PO 4 , 9.6 mM Na 2 HPO 4 , 1 mM MgCl 2 , 0.1 mM CaCl 2 , pH 7.3). Where indicated, cells were treated with different modulators at 37°C as described in figure legends. Then cells were incubated with EZ link NHS-Sulfo-SS-biotin (1 mg/ml) (Thermo Fisher Scientific, Rockford, IL) in cold PBS/Ca-Mg for 30 min on ice and then the excess biotinylating reagents were removed by two times wash with 100 mM glycine and further incubated with glycine for 20 min. The cells were solubilized using RIPA buffer supplemented with a cocktail of protease inhibitors. Synaptosomes: Briefly, Synaptosomes (300 to 500 μg) were incubated in Krebs-Ringer buffer with indicated modulators or vehicle at 37°C for the times indicated in figure legends ( Samuvel et al., 2008 ). The samples were washed quickly by centrifugation, and the pellets were treated with EZ link NHS-Sulfo-SS-biotin (1 mg/1 mg protein) for 30 min at 4°C in in cold Krebs-bicarbonate buffer. Subsequently, the samples were washed with the same buffer containing 100 mM glycine, and the pellet was resuspended in RIPA lysis buffer. The resuspended synaptosomes or solubilized cells were triturated 10 times through a 25 gauge needle and centrifuged at 25,000 × g for 30 min. Total cellular protein content was determined by the Bradford Protein Assay procedure. Using equal amounts of solubilized proteins, the biotinylated proteins were isolated using NeutrAvidin Agarose resins overnight at 4°C followed by washing. Bound proteins were eluted with 50 μl Laemmli sample buffer for 30 min at room temperature. Aliquots from total extracts, unbound fractions and all of the eluate were separated by SDS-PAGE, transferred to polyvinylidene difluoride membrane and probed with DAT specific (Santa Cruz Biotechnology, Inc. Dallas, TX) or other antibodies as indicated. The immunoreactive proteins were visualized using ECL or ECL plus reagent. Subsequently, the blots were stripped and reprobed with anti-calnexin antibody (AKELA Pharma Inc. Montreal, QC, Canada) to validate the surface biotinylation of plasma membrane proteins as well as protein loading levels. DAT densities from total and biotinylated (representing the surface pool) fractions were normalized using levels of calnexin in the total extract. Multiple exposures were evaluated by digital quantitation using NIH ImageJ (version 1.32j) software to ensure that results were within the linear range of the film exposure ( Samuvel et al., 2008 ; Zapata et al., 2007 ). 2.5. Identification of DAT-KOR Complexes 2.5.1.

Co-immunoprecipitation and Immunoblot Analysis

Co-immunoprecipitation experiments were conducted using EM4 cells or striatal synaptosomes. A. EM4 cells: Co-immunoprecipitation experiments were conducted as previously described ( Bolan et al., 2007 ) in EM4 cells expressing myc-KOR and FLAG-DAT alone or together. Protein samples were prepared by incubation of cells with RIPA buffer pH 7.4, for 30 min (4°C) followed by centrifugation (25,000 × g for 30 min). Complexes were immunoprecipitated with polyclonal anti-myc antibodies (rabbit, clone A-14, Santa Cruz Biotechnology, CA). After pull down with Protein A-linked agarose beads, they were washed three times with RIPA buffer and eluted at room temperature. Immunoprecipitates were separated by SDS-PAGE (4-20% Duramide gradient gel, Cambrex, Walkersville, MD) and blotted onto polyvinylidene difluoride membranes. DAT was detected using a monoclonal antibody raised against the N-terminal domain of human DAT (Chemicon, Temecula, CA) and secondary goat HRP-conjugated anti-rat antibody (Jackson ImmunoResearch, West Grove, PA). B. Striatal synaptosomes . Presence of KOR protein in immunoprecipitated DAT protein complex. Immunoprecipitation of DAT protein was carried out using two DAT antibodies from Chemicon (Millipore), Billerica, MA and Santa Cruz Biotechnology, Inc. Dellas, TX as described ( Chakrabarti et al., 2010 ). Synaptosomes (500-700 μg) were solubilized with 20 mM Hepes buffer, pH 7.4 containing 150 mM NaCl, 5 mM EDTA, 1% Triton X-100, 0.1% SDS supplemented with a cocktail of protease and phosphatase inhibitors. In parallel, irrelevant IgG was used as a control. Clear solubilized extract after centrifugation (25,000 g for 45 min) was incubated with DAT antibodies (4 μg) overnight at 4°C, and the immunocomplex was isolated using Protein A and G Sepharose. Immunoprecipitates eluted with Laemmli buffer with out β-mercaptoethanol. The eluted samples were then incubated with 5% β-mercaptoethanol, and 0.01% bromphenol blue for 30 min at 22°C, electrophoresed as given above. The presence of KOR protein from DAT-immunocomplex was detected by immunoblotting using rabbit polyclonal KOR-1 antibody (Santa Cruz Biotechnology, Inc. Dellas, TX). 2.5.2. Biolumiescence Resonance Energy Transfer (BRET) BRET studies were carried out with HEK cells transfected with luciferase tagged KOR (Luc-KOR) alone or with YFP-DAT as described previously using a FluoroMax-2 spectrometer and the Fusion Microplate Reader ( Bolan et al., 2007 ). BRET ratios were calculated as described by Angers et al., (2000) ( Angers et al., 2000 ). Cells transfected with Luc-KOR in combination with YFP-tagged delta opioid receptor (YFP-DOR) were used as a positive control since studies have shown that KOR and DOR functionally interact and are in close proximity ( Gomes et al., 2003 ). As a negative control, we used cells co-transfected with YFP-tagged chemokine receptors CCR5 (YFP-CCR5) and Luc-KOR. 2.5.3. Fluorescence Resonance Energy Transfer (FRET) FRET ( Schmid and Sitte, 2003 ) was measured with a Carl Zeiss Axiovert 200 epifluorescence microscope. The ‘three-filter method’ was performed as described in ( Bartholomaus et al., 2008 ). Images were taken using 63x oil immersion objectives and LUDL filter wheels allowing a rapid excitation and emission filter exchange. We used HEK293 cells which were maintained and transiently transfected with plasmid cDNA (1.7 μg) by means of the calcium phosphate co-precipitation method as described previously ( Sucic et al., 2010 ). The LUDL filter wheels were configured as follows: CFP ( I Donor ; excitation: 436nm, emission: 480nm, and dichroic mirror: 455nm), YFP ( I Acceptor ; excitation: 500nm, emission: 535nm, and dichroic mirror: 515nm) and FRET ( I FRET ; excitation: 436nm, emission: 535nm, and dichroic mirror: 455nm). Images were taken with a CCD camera (Coolsnap fx , Roper Scientific). Background fluorescence was subtracted from all images. We analyzed the images pixel by pixel using ImageJ (Wayne Rassband, National Institute of Health, version 1.43b) and the ImageJ plug-in PixFRET (Pixel by Pixel analysis of FRET with ImageJ, version 1.5.0; Feige et al., 2003) with which spectral bleed-through (SBT) parameters for the donor bleed through (BT) and the acceptor BT were determined and NFRET was calculated in the following way: nFRET = IFRET − BTDonor ∗ IDonor − BTAcceptor ∗ IAcceptor IDonor ∗ IAcceptor ∗ 100 The mean N FRET was measured at the plasma membrane (pre-defined as the region of interest ) using the computed N FRET -image. The regions of interest were selected in the CFP (Donor) or YFP (Acceptor) image (to avoid bleaching-associated bias) and transmitted to the N FRET -image by the ImageJ Multi Measure Tool. As negative control we employed the YFP-labeled form of KOR with a membrane-bound form of CFP (kindly provided by R.Y. Tsien). As positive controls for membrane proteins we used the SERT tagged with CFP and YFP on its cytoplasmic N- and C-termini, respectively (to yield C-SERT-Y; ( Just et al., 2004 )). We tagged DAT and GAT1 with CFP to reveal C-DAT and C-GAT1 ( Schmid et al., 2001 ) to compare the FRET values between Y-KOR and C-DAT or C-GAT1. To compare expression levels of CFP- or YFP-tagged proteins, the fluorescence intensity within a rim of a few pixels positioned over the membrane was measured and expressed in arbitrary fluorescence units (a.f.u.). 2.6.

Purification of SalA

SalA was isolated and purified from commercially available Salvia divinorum leaves as described previously ( Butelman et al., 2007 ; Tidgewell et al., 2004 ) and determined to be >98% pure by HPLC. 2.7.

Statistical Analysis

Values are expressed as mean ± S.E.M. As noted in the figure legends and in the result section, one-way analysis of variance was used followed by post hoc testing (Bonferroni, Dunnett and Tukey) for multiple comparisons. Two-tailed unpaired Student's t test analysis was performed for comparisons between two groups using Prism (GraphPad, San Diego, CA). A value of p ≤ 0.05 was considered statistically significant.