Lipopolysaccharide (LPS), interferon-γ (IFN-γ) and forskolin were purchased from Sigma-Aldrich (St Louis, MO), and Angiotensin II (Ang II), CGP-42112A, Candesartan and PD123319 from Tocris Bioscience (Bristol, UK).
HEK-293T cells and primary cultures
Human embryonic kidney (HEK-293T) cells were grown in Dulbecco’s modified Eagle’s medium (DMEM) (Gibco) supplemented with 2 mM L-glutamine, 100 µg/ml sodium pyruvate, 100 U/ml penicillin/streptomycin, MEM Non-Essential Amino Acids Solution (1/100) and 5% (v/v) heat inactivated Fetal Bovine Serum (FBS) (all supplements were from Invitrogen, Paisley, Scotland, UK).
To prepare mice striatal primary microglial cultures, brain was removed from C57BL/6 mice of 2–4 days of age. Microglial cells were isolated following protocols described elsewhere [12–14] and grown in DMEM medium supplemented with 2 mM L-glutamine, 100 U/ml penicillin/streptomycin, MEM Non-Essential amino acids preparation (1/100) and 5% (v/v) heat inactivated Fetal Bovine Serum (FBS) (Invitrogen, Paisley, Scotland, UK). Briefly, striatum tissue was dissected, carefully stripped of its meninges and digested with 0.25% trypsin for 20 min at 37 ºC. Trypsinization was stopped by washing the tissue. Cells were brought to a cell suspension by repeated pipetting followed by passage through a 100 µm pore mesh. Glial cells were resuspended in medium and seeded at a density of 1 × 106 cells/ml in 6-well plates for cyclic adenylic acid (cAMP) assays, in 12-well plates with coverslips for in situ proximity ligation assays (PLA) and in 96-well plates for mitogen-activated protein kinase (MAPK) experiments. Cultures were maintained at 37ºC in humidified 5% CO2 atmosphere and, unless otherwise stated, medium was replaced once a week.
Parkinson’s disease (PD) model generation, levodopa treatment and dyskinesia assessment
All experiments were carried out in accordance with EU directives (2010/63/EU and 86/609/CEE) and were approved by the Ethical committee of the University of Santiago de Compostela. Animals, male Wistar rats, were divided into three groups as follows: non-lesioned rats, 6-hydroxydopamine (6-hydroxy-DA)-lesioned animals receiving vehicle, and 6-hydroxy-DA-lesioned animals receiving a chronic treatment with levodopa. A total of 15 animals were used. Details of model generation, protocol of drug administration and behavioral analysis, performed by a blinded investigator, are given elsewhere [1, 15]. In brief, surgery was performed on rats anesthetized with ketamine/xylazine (1% ketamine − 75 mg/kg-, and 2% xylazine − 10- mg/kg). Lesions were produced in the right medial forebrain bundle to achieve complete lesion of the nigrostriatal pathway. The rats were injected with 12 µg of 6-hydroxy-DA (to provide 8 µg of 6-hydroxy-DA free base; SigmaAldrich) in 4 µl of sterile saline containing 0.2% ascorbic acid.
Some animals were treated with levodopa by receiving a daily subcutaneous injection with levodopa methyl ester (6 mg/kg) plus benserazide (10 mg/kg) for 3 weeks (such treatment reliably induces dyskinetic movements). In order to discriminate dyskinetic from non-dyskinetic animals, the manifestation of levodopa-induced AIMS (abnormal involuntary movements) was evaluated according to the rat dyskinesia scale described in detail previously [15–17]. The severity of each AIM subtype (limb, orolingual, and axial) was assessed using scores from 0 to 4 (1, occasional, i.e., present < 50% of the time; 2, frequent, i.e., present > 50% of the time; 3, continuous, but interrupted by strong sensory stimuli; 4, continuous, not interrupted by strong sensory stimuli). Rats were classified as “dyskinetic” if they displayed a ≥ 2 score per monitoring period on at least two abnormal involuntary movement (AIM) subtypes. Animals classified as “non-dyskinetic” exhibited either no AIMs or very mild/occasional ones. Animals with low scores, i.e., either non-dyskinetic or dyskinetic, were discarded.
Human cDNAs for AT1, AT2 and σ1 receptors cloned into pcDNA3.1 were amplified without their stop codons using sense and antisense primers harboring either BamHI and HindIII restriction sites to amplify AT1R and AT2R or BamHI and EcoRI restriction sites to amplify σ1 receptor. Amplified fragments were then subcloned to be in frame with an enhanced yellow fluorescent protein (pEYFP-N1; Clontech, Heidelberg, Germany) or a Rluc (pRluc-N1; PerkinElmer, Wellesley, MA) on the C-terminal end of the receptor to produce AT1R-YFP, AT2R-RLuc, AT2R-YFP and σ1R-RLuc fusion proteins.
HEK-293T cells were transiently transfected with the corresponding cDNA by the PEI (PolyEthylenImine, Sigma-Aldrich, St. Louis, MO) method [18, 19]. Briefly, the corresponding cDNA diluted in 150 mM NaCl was mixed with PEI (5.5 mM in nitrogen residues) also prepared in 150 mM NaCl for 10 min. The cDNA-PEI complexes were transferred to HEK-293T cells and were incubated for 4 hours in a serum-starved medium. Then, the medium was replaced by fresh supplemented culture medium and cells were maintained at 37 ºC in a humid atmosphere of 5% CO2. 48 hours after transfection, cells were washed, detached, and resuspended in the assay buffer.
HEK-293T cells were seeded on glass coverslips in 12-well plates. On DIV 2, cells were transfected with AT1R-YFP cDNA (1 µg), AT2R-Rluc cDNA (1 µg) or both. On DIV 4, cells were fixed in 4% paraformaldehyde for 15 min and washed twice with PBS containing 20 mM glycine before permeabilization with PBS-glycine containing 0.2% Triton X-100 (5 min incubation). Cells were blocked during 1 hour with PBS containing 1% bovine serum albumin. HEK-293T cells were labeled with a mouse anti-Rluc antibody (1/100; Millipore, Darmstadt, Germany) and subsequently treated with Cy3 conjugated anti-mouse (1/200; Jackson ImmunoResearch (red)) IgG (1 hour each). The AT1R-YFP expression was detected by YFP´s own fluorescence. Nuclei were stained with Hoechst (1/100 from stock 1 mg/mL; SigmaAldrich). Samples were washed several times and mounted with 30% Mowiol (Calbiochem). Images were obtained in a Leica SP2 confocal microscope (Leica Microsystems) with the 63X oil objective.
Bioluminescence resonance energy transfer (BRET) assays
For BRET assays, HEK-293T cells were transiently cotransfected with a constant amount of cDNA encoding for AT2R-RLuc (0.9 µg) and with increasing amounts of cDNA corresponding to AT1R-YFP (0.5 to 4 µg). For negative control, HEK-293T cells were transiently cotransfected with a constant amount of cDNA encoding for σ1-RLuc (0.75 µg) and with increasing amounts of cDNA corresponding to AT2R-YFP (0.1 to 4 µg). To control the cell number, sample protein concentration was determined using a Bradford assay kit (Bio-Rad, Munich, Germany) using bovine serum albumin (BSA) dilutions to prepare the standard absorbance versus concentration relationship. To quantify fluorescent proteins, cells (20 µg protein) were distributed in 96-well microplates (black plates with a transparent bottom) and fluorescence was read in a Fluostar Optima Fluorimeter (BMG Labtech, Offenburg, Germany) equipped with a high-energy xenon flash lamp, using a 10 nm bandwidth excitation filter at 485 nm. For BRET measurements, the equivalent of 20 µg of cell suspension was distributed in 96-well white microplates with white bottom (Corning 3600, Corning, NY) and 5 µM coelenterazine H (Molecular Probes, Eugene, OR) was added. One minute after adding coelenterazine H, BRET was determined using a Mithras LB 940 reader (Berthold Technologies, DLReady, Germany), which allows the integration of the signals detected in the short-wavelength filter at 485 nm and the long-wavelength filter at 530 nm. To quantify AT2R-RLuc expression, luminescence readings were performed 10 minutes after the addition of 5 µM coelenterazine H. MilliBRET units (mBU) are defined as:
where Cf corresponds to [(long-wavelength emission)/(short-wavelength emission)] for the RLuc construct expressed alone in the same experiment.
Detection of cytoplasmic calcium ion
HEK-293T cells were cotransfected with the cDNA for the Ang receptors AT1 (1 µg) and/or AT2 (1 µg) and the GCaMP6 calcium sensor (1 µg)  by the use of PEI method (Section “Cell Transfection”). 48 hours after transfection, HEK-293T cells plated in 6-well black, clear bottom plates, were incubated with Mg2+-free Locke’s buffer (154 mM NaCl, 5.6 mM KCl, 3.6 mM NaHCO3, 2.3 mM CaCl2, 5.6 mM glucose, 5 mM HEPES, pH 7.4) supplemented with 10 µM glycine. Receptor antagonists were added 10 min before readings and receptor agonists just a few seconds before readings. Fluorescence emission intensity of GCaMP6 was recorded at 515 nm upon excitation at 488 nm on the EnSpire® Multimode Plate Reader for 150 s every 5 s at 100 flashes per well.
cAMP level determination
The analysis of cAMP levels was performed in HEK-293T cells transfected with cDNA for AT1 (1 µg) and/or AT2 (1 µg) receptors in primary cultures of striatal neurons or glia using the Lance Ultra cAMP kit (PerkinElmer). The optimal cell density to obtain an appropriate fluorescent signal was first established by measuring the TR-FRET signal as a function of forskolin concentration using different cell densities. Forskolin dose-response curves were related to the cAMP standard curve in order to establish which cell density provides a response that covers most of the dynamic range of cAMP standard curve. 2 hours before the experiment the medium was substituted by serum-starved DMEM medium. Cells (2,000 HEK-293T cells, 4,000 striatal neurons or glial cells by well in 384-well microplates) growing in medium containing 50 µM zardaverine were pre-treated with the AT1R or AT2R antagonists (Candesartan and PD123319 respectively) or the corresponding vehicle at 24 °C for 15 min, and stimulated with the AT1R and/or AT2R agonists (Ang II and CGP-42112A respectively) for 15 min before adding 0.5 µM forskolin or vehicle, and incubating for an additional 15 min period. After 1 hour, fluorescence at 665 nm was analyzed on a PHERAstar Flagship microplate reader equipped with an HTRF optical module (BMG Labtech). A standard curve for cAMP was obtained in each experiment.
Extracellular signal-regulated kinases 1/2 (ERK1/2) phosphorylation
To determine ERK1/2 phosphorylation, 40,000 HEK-293T cells transfected with cDNA for AT1R (1 µg) and/or AT2R (1 µg) or 50,000 striatal neurons or glial cells primary cultures were plated in each well of transparent Deltalab 96-well microplates. Two hours before the experiment, the medium was substituted by serum-starved DMEM medium. Then, cells were treated or not for 10 min with the selective antagonists Candesartan or PD123319 in serum starved DMEM medium followed by 7 min treatment with the selective agonists Ang II and/or CGP-42112A. Cells were then washed twice with cold PBS before the addition of lysis buffer (15 min treatment). 10 µL of each supernatant were placed in white ProxiPlate 384-well microplates and ERK1/2 phosphorylation was determined using AlphaScreen®SureFire® kit (Perkin Elmer) following the instructions of the supplier and using an EnSpire® Multimode Plate Reader (PerkinElmer, Waltham, MA, USA).
Dynamic Mass-Redistribution (DMR) label free assays
Cell signaling was explored using an EnSpire® Multimode Plate Reader (PerkinElmer, Waltham, MA, USA) by a label-free technology. Cellular cytoskeleton redistribution movement induced upon receptor activation were detected by illuminating the underside of the plate with polychromatic light and measured as changes in wavelength of the reflected monochromatic light that is a sensitive function of the index of refraction. The magnitude of this wavelength shift (in picometers) is directly proportional to the amount of DMR. To determine the label free-DMR signal, 10,000 HEK-293T cells transfected with cDNA for AT1R (1 µg) and/or AT2R (1 µg) receptors or 10,000 striatal neurons or glial cells primary cultures were plated on each well of transparent 384-well fibronectin coated microplates to obtain 70–80% confluent monolayers, and kept in the incubator for 24 h. Previous to the assay, cells were washed twice with assay buffer (HBSS with 20 mM HEPES, pH 7.15, 0.1% DMSO) and incubated in the reader for 2 hours in 30 µl/well of assay-buffer at 24 °C. Hereafter, the sensor plate was scanned and a baseline optical signature was recorded for 10 minutes before adding 10 µl of test antagonists (Candesartan or PD123319) dissolved in assay buffer, and 30 minutes later, followed by the addition of 10 µl of selective agonists (Ang II and/or CGP-42112A) also dissolved in assay buffer. DMR responses induced by the agonist were monitored for a minimum of 3,600 s.
Proximity Ligation Assay (PLA)
Detection in natural sources of clusters formed by AT1 and AT2 receptors was addressed in striatal neurons, primary cultures of glial cells and rat brain slices. Cells were grown on glass coverslips, fixed in 4% paraformaldehyde for 15 min, washed with PBS containing 20 mM glycine to quench the aldehyde groups, permeabilized with the same buffer containing 0.05% Triton X-100 for 5 to 15 min and washed with PBS. After 1 hour incubation at 37 °C with the blocking solution in a pre-heated humidity chamber, primary cultures were incubated overnight at 4ºC with a mixture of a mouse monoclonal anti-AT1R antibody (1/100, sc-515884, Santa Cruz Biotechnology, Texas, USA), a rabbit monoclonal anti-AT2R antibody (1/100, ab92445, Abcam, Cambridge, UK) and Hoechst (1/100 from stock 1 mg/mL; SigmaAldrich) to stain the nuclei. The antibodies were validated following the method in the technical brochure of the vendor with fairly similar results and also by immunofluorescence in HEK-293T cells (transfected versus untransfected). Samples from KO animals were not available for validation. Cells were further processed using the PLA probes detecting primary antibodies (Duolink In Situ PLA probe Anti-Mouse plus and Duolink In Situ PLA probe Anti-Rabbit minus) (1/5 v:v for 1-hour at 37ºC). Ligation and amplification were done as indicated by the supplier (SigmaAldrich) and cells were mounted using the mounting medium 30% Mowiol (Calbiochem). To detect red dots corresponding to AT1/2Hets, samples were observed in a Leica SP2 confocal microscope (Leica Microsystems, Mannheim, Germany) equipped with an apochromatic 63X oil-immersion objective (N.A. 1.4), and a 405 nm and 561 nm laser lines. For each field of view a stack of two channels (one per staining) and 3 Z-planes with a step size of 1 µm were acquired. The Andy’s Algorithm , a specific ImageJ macro for reproducible and high-throughput quantification of the total PLA foci dots and total nuclei, was used for data analysis.
The data in graphs are the mean ± SEM (n = 5, at least). GraphPad Prism software version 7 (San Diego, CA, USA) was used for data fitting and statistical analysis. One-way ANOVA followed by post-hoc Bonferroni’s test were used when comparing multiple values. When a pair of values were compared, the Student’s t test was used. Significant differences were considered when the p value was < 0.05.