The following drugs were purchased or obtained from the indicated sources: (-) epinephrine (EPI), (±)norepinephrine (NE), sodium ascorbate, UK14,304 (Sigma-Aldrich, St. Louis, MO.); cell culture media (Gibco, Grand Island, NY); fetal bovine serum (Atlanta Biologicals, Norcross, GA); and antibiotics (Mediatech, Inc., Herndon, VA). GRK2 (C-15) and GRK3 (C-14) primary antibodies and horseradish peroxidase-conjugated secondary antibody (Santa Cruz Biotechnology, Inc., Santa Cruz, CA); anti-glyceraldehyde-3-phosphate dehydrogenase (GADPH, Research Diagnostics, Inc., Flanders, NJ).
SH-SY5Y (passages 37–55) human neuroblastoma cells (Dr. Robert A. Ross, Fordham University, Bronx, NY) were maintained in a humidified atmosphere (6% CO2:94% air) in a 1:1 mixture of Eagle's minimum essential medium with non-essential amino acids and Ham's F-12 that contains 10% fetal bovine serum, 100 U/ml penicillin G and 0.1 mg/ml streptomycin sulfate. Plates of cells greater than 60% confluence were used throughout the study.
Plasmid cDNA with the human β2-AR gene (provided by Dr. Brian Knoll; University of Houston, Houston, TX) or vector alone was stably transfected into SH-SY5Y cells with the fuGENE 6 Transfecting Reagent (Roche). Ten positive clones were isolated by their resistance to 800 μg/mL of G418 and maintained in media containing 600 μg/mL of G418. SHβ2AR4 was selected for use in all experiments because it expressed similar levels of β2-ARs as that expressed natively in BE(2)-C cells; SHβ2AR4 expressed 14.78 ± 4.19 fmol/mg protein while BE(2)-C express 18.5 ± 6.2 fmol/mg protein . This β2-AR level remained consistent to passage 12 in SHβ2AR4 cells. After passage 12, SHβ2ARs neither expressed β2-ARs nor maintained resistance to G418, suggesting that the cells no longer expressed the transfected plasmid.
RNA isolation and RT-PCR
Total RNA was isolated from several different passages of freshly harvested SH-SY5Y cells by the guanidinium isothiocyanate/phenol-chloroform extraction method . Total RNA concentrations were determined by UV spectroscopy; integrity of each isolate was determined by electrophoresis through a 1% agarose gel in the presence of 0.01 M sodium phosphate buffer. Poly(A) mRNA was isolated using a Dynabead oligo(dT)25 Kit (Dynal, Oslo, Norway) and was used for RT-PCR reactions. Each RT reaction (20 μL) contained 5–10 μg total or poly(A) RNA preincubated with 5 ng/μL oligo(dT)12–18, for 10 min at 70°C. The reaction mixture contained 80 μM each of deoxynucleotides (dATP, dCTP, dGTP and dTTP), RT buffer (50 mM Tris-HCl, pH 8.3, 75 mM KCl, 3 mM MgCl2), and 5 mM dithiothreitol, and was preincubated for 2 min at 42°C before the addition of Moloney Murine Leukemia Virus reverse transcriptase (200 U/μl) for 60 min at 42°C; a 5 min incubation at 95°C terminated the reactions.
ODNs [29, 30] corresponded to sequences for the various human α2-AR (α2A antisense: 5'-AGA CGA GCT CTC CTC CAG GT-3'; sense: 5'-AAA CCT CTT CCT GGT GTC TC-3'), α2A/2C-(antisense: 5'-GTG CGC TTC AGG TTG TAC TC-3'; sense: 5'-AAA CCT CTT CCT GGT GTC TC-3'), or α2C-AR (antisense: 5'-CGT TTT CGG TAG TCG GGG AC-3'; sense: 5'-GTG GTG ATC GCC GTG CTG AC-3'). The contents of each RT reaction tube were diluted to a final volume of 50 μL with 10% DMSO, 80 μM each of dATP, dCTP, dGTP and dTTP, 8 μM each of the appropriate sense/antisense primer pair, 1.5 mM MgCl2, and magnesium free buffer [containing 10 mM Tris-HCl (pH 9.0), 0.1% Triton X-100, 50 mM KCl] in sterile distilled water. Reaction mixtures were overlayed with mineral oil and subjected to a hot start for 5 min at 95°C. DNA polymerase (2.5 U taq, 5 U/μl, Promega, Madison, WI) was added to each reaction tube after the hot start, and the tubes were subjected to a PCR reaction of 30 cycles in a thermal cycler (MJ Research Inc., Watertown, MA) for 1 min at 94°C, 1.5 min at 55°C, and 2 min at 72°C with a final elongation step at 72°C for 7 min. Reaction products were separated by electrophoresis through 2% agarose gels and visualized by ethidium bromide staining. PCR products were isolated from the gel using a DNA extraction kit (Amicon Inc., Bedford, MA). Identity of the purified PCR products was confirmed by their susceptibility to digestion with restriction enzymes specific for each reaction product (see Table 1; ).
To determine the effects of α2-AR agonists on forskolin-induced cAMP accumulation, intact cells were incubated for 5 minutes at 37°C in HBSS buffer (in mM): NaCl (137), KCl (5), Na2HPO4 (0.6), KH2PO4 (0.4), NaHCO3 (4), D-glucose (6), MgCl2 (0.5), MgSO4 (0.4) and CaCl2 (1), containing the phosphodiesterase inhibitor IBMX (0.5 mM). In some experiments, antagonists also were included in this step. To prohibit oxidation, sodium ascorbate (0.11 mM) was included when assaying catecholamines. Upon addition of forskolin (10 μM) and agonist, assay tubes were incubated for an additional 10 min at 37°C. Removing the tubes to a boiling water bath for 5 min terminated the assay. All assays were performed in duplicate in a total volume of 0.5 ml. After boiling, samples were centrifuged for 5 min at 14000 × g, and cAMP levels from the supernatant fractions were determined in a [3H]cAMP (0.8 pmol) binding assay as previously described . β-AR-mediated stimulation of cAMP accumulation was performed in the same manner except that forskolin was not included in the assay mixture. Forskolin (10 μM) stimulated cAMP accumulation to 587 ± 88 pmol/mg protein (n = 46), 15-fold over basal levels (40.5 ± 2 pmol/mg protein).
Preparation of cell membranes
Cells were homogenized in 20 volumes of Tris-HCl buffer (50 mM, pH 7.4) containing NaCl (100 mM), Na2 EDTA (1 mM) and PMSF (0.1 mM), and the membranes sedimented by centrifugation for 30 minutes at 34000 × g at 4°C. Pellets were resuspended in 0.32 M sucrose, and aliquots of the membrane fractions were stored frozen (-80°C) until use.
The level of α2-ARs in SH-SY5Y cell membranes (0.5 mg/ml) was determined with various concentrations of [3H]rauwolscine (60–80 Ci/mmol, 0.3 – 12 nM) in a total volume of 1–2 ml in potassium phosphate buffer (50 mM, pH 7.4) containing MgSO4 (5 mM) at 37°C for 45 min. Thereafter, 2 ml Tris-HCl (5 mM, pH 7.4, 4°C) was added to the homogenate to terminate the binding reaction and the contents of the tubes was filtered over #32 glass fiber filter strips (Schleicher & Schuell, Keene, NH) using a PHD cell harvester (Cambridge Technology, Cambridge, MA). The reaction tubes and the filter strips were rinsed twice with a further 2–3 ml of buffer. Levels of radioactivity were determined by scintillation spectroscopy in a Beckman LS6000 liquid scintillation counter. All assays were performed in triplicate, and specific binding was determined by subtracting the binding in the presence of yohimbine or phentolamine (10 μM; nonspecific) from the binding in its absence.
Previously we have shown that agonist treatments do not alter the Kd of the ligand for the α2-AR . Therefore, levels of α2-ARs in SHβ2AR4 cell membranes (0.1 – 0.2 mg/mL) were determined using a single concentration (2 nM) of either [3H]rauwolscine or [3H]RX821002 following catecholamine treatment.
β2-AR binding was performed with [3H]CGP-12177. For saturation studies, cell membranes (0.5 mg/mL) were incubated with [3H]CGP-12177 (0.2 to 40 nM) in Tris-HCl buffer (50 mM, pH 7.5) containing MgCl2 (0.5 mM) at 37°C for 30 min. Specific binding was determined by subtracting the binding in the presence and absence of propranolol (1 μM).
Cell membrane fractions were incubated as described above except that the concentration of [3H]rauwolscine was fixed (1 nM), and various (4–9) concentrations of unlabeled drugs were included.
Membrane proteins were separated from cytosolic proteins by centrifugation, were resolved by SDS-PAGE through 10% gels and relative levels of GRK2 and GRK3 determined by immunoblotting as described previously . Briefly, proteins were transferred to PVDF membrane, blocked with 5% nonfat dried milk in PBS containing 0.1% Tween (PBS/T) and incubated overnight at 4°C with dilutions of a rabbit polyclonal antibody directed against GRK2 (1:1000), GRK3 (1:1000), or both GRK2 and GRK3 (GRK2/3; 1:1000; wt SH-SY5Y). Blots were subjected to 4 washes before incubating for 60 min at room temperature with a goat anti-rabbit horseradish peroxidase-conjugated secondary antibody (1:2000) in PBS/T. Immunoreactive bands were visualized by enhanced chemiluminescence (Amersham Corp., Arlington Heights, IL or Santa Cruz Biotechnology, Inc., Santa Cruz, CA). The intensity of each immunoreactive band was determined using a Nucleovision Imaging Workstation (Nucleotech Corp., San Carlos, CA), and normalized to the GAPDH loading control (1:5000).
Bovine serum albumin was used as a standard in the determination of protein levels in intact cells and cell membranes as described .
Kd, Bmax, IC50 and LogEC50 values were determined by nonlinear regression analysis using GraphPad Prism (GraphPad Software http://www.graphpad.com). The Ki values were calculated according to the Cheng-Prusoff equation  in which Ki = (IC50)/(1+S), where S = [concentration of radioligand]/[KD of radioligand]. Comparisons between groups were made by two-way Student's t-tests or ANOVA and Tukey's or Dunnett's post hoc test (where appropriate; GraphPad Software, San Diego, CA), and groups were considered significantly different if p ≤ 0.05.