Animal use/Ethics Statement
Male Sprague-Dawley rats (250–300 g; Charles River Laboratories, Inc., Portage, MI, USA) were used. Rats were anesthetized with pentobarbital (60 mg kg−1, i.p.) prior to removal of tissues. Procedures that involved animals were performed in accordance with the guidelines of Michigan State University, and approved by the Institutional Animal Use and Care Committee.
Paraffin-embedded tissue sections were dewaxed, unmasked and taken through a protocol as previously described 
. Primary antibodies used were: transglutaminase II (TGII; mouse monoclonal TG100, LabVision, Fremont CA, USA), N-epsilon gamma glutamyl lysine (mouse monoclonal, ab424, Abcam, Cambridge, MA, USA). In some experiments, primary antibodies were left out of the experiment, and tissues developed only in the presence of secondary antibody.
Protein isolation and western blotting procedures were performed as previously described 
using standard SDS-PAGE conditions and blotting proteins to nitrocellulose. Primary antibodies used were TGII (LabVision, Fremont CA, USA), smooth muscle cell α-actin (mouse monoclonal, Ab-2, EMD Biosciences, La Jolla, CA, USA) and 5-HT (AbD Serotec, Raleigh NC, USA). Films were scanned and placed within the figure without gamma modifications using Adobe Photoshop.
Protein homogenate (50 µg) was placed in transglutaminase reaction buffer (50 mM Tris-HCl, pH 7.5, 150 mM NaCl, 1 mM phenylmethylsulfonyl fluoride, 2 mg/ml aprotinin and leupeptin, 1 mM sodium orthovanadate, 5 mM calcium chloride) containing pentylamine biotin (BAP; 8 mM) or biotinylated amine (IBL, Hamburg, Germany). For biotinylation of 5-HT, 5-HT hydrochloride (Sigma Germany) and EZ-Link Sulfo-NHS-LC-LC-Biotin (Pierce) were used. Equimolar amounts of serotonin and NHS-LC-LC-Biotin were used with pyridine as solvent. After a period of several hours on a roller mixer, the turbid solution was stored over night. On next morning, solvent was evaporated in a vacuum centrifuge and the residue re-suspended in dimethylformamide (DMF). The coupling of the indole amine to biotin was checked through 5-HT enzyme immunoassay (ELISA) by running the ELISA according to manufacturer instructions (IBL International GmbH, Hamburg, Germany Serotonin ELISA cat. No. RE59121). Instead of Biotin required in the Instructions for Use included in the kit, freshly synthesized biotinylated serotonin in different dilutions was used. The purity was checked greater than 90%. Stock concentration was 1.59 mM.
Amines were incubated in the presence of vehicle or the TGII inhibitor cystamine (0.001–10 mM) at 37°C for one hour. An equal volume of 2× SDS sample buffer was added to stop the reaction and the samples were boiled for 10 minutes. Samples were separated on 10% polyacrylamide gels (Bio Rad CA, USA), and transferred to nitrocellulose. Samples were blocked overnight at 4°C in 4% chick egg ovalbulmin [TBS-0.1% Tween+0.025% NaN3,
], washed in TBS-Tween for 20 minutes, and incubated with streptavidin-linked, horseradish peroxidase-conjugated secondary antibody (1
2000, 1 hr, 4°C GE Healthcare, Piscataway NJ, USA). ECL® reagents (GE Healthcare, Piscataway NJ USA) were used to visualize bands. Films were scanned and placed within the figure without gamma modifications using Adobe Photoshop.
At room temperature, dissected and cleaned aorta were placed in 100 µL physiological salt solution [PSS: 103 mM NaCl; 4.7 mM KCl; 1.18 mM KH2PO4; 1.17 mM MgSO4-7H2O; 1.6 mM CaCl2-2H2O; 14.9 mM NaHCO3; 5.5 mM dextrose, and 0.03 mM CaNa2 EDTA]. Tissues were briefly dipped in fresh PSS and placed in tissue buffer (0.05 mM sodium phosphate and 0.03 mM citric acid buffer (pH 2.5) containing 15% methanol]. Tissue samples were frozen in −80°C until assay. Samples were thawed, sonicated for 3 seconds and centrifuged for 30 seconds (10,000 g). Supernatant was collected and transferred to new tubes. Tissue pellets were dissolved in 1.0 M NaOH and assayed for protein (Lowry assay). Concentrations of 5-HIAA and 5-HT in tissue supernatants were determined by isocratic high pressure liquid chromatography (HPLC/ESA; described below).
Cell culture and 5-HT uptake
Aortic cells were derived from explants of thoracic aorta. Cells were fed with DMEM supplemented with 10% fetal bovine serum and 1% penicillin/streptomycin. Cells were plated to P-60 dishes or coverslips for Western and immunocytochemical experiments, respectively. Cells used were between passages 2 and 9 and all explants stained positive for smooth muscle cell α-actin (EMD Biosciences, La Jolla, CA, USA). Cells were starved of serum 24 hours prior to experimentation because serum contains 5-HT (determined by HPLC). Cells were incubated for 1 hour in PSS and (+/−10 µM pargyline, a monoamine oxidase A inhibitor) with vehicle (0.01% DMSO) or fluoxetine (1 µM) prior to addition of 5-HT (10−8–10−5 M) or 5-HT-biotin (0–12.7 µM). Cells were placed on ice, washed with PSS, scraped in tissue (HPLC analyses) or lysis buffer (Western analyses), and centrifuged (14, 000 rpm, 10 minutes). The supernatant was removed for HPLC or Westerns (described above). Protein concentration of supernatant and pellet were measured using the Lowry assay.
Samples were thawed, sonicated for 3 seconds and centrifuged for 30 seconds (10,000 g). Supernatant was collected and transferred to new tubes. Tissue pellets were dissolved in 1.0 M NaOH and assayed for protein. Concentrations of 5-HIAA, 5-HTP and 5-HT in tissue supernatants were determined by isocratic high pressure liquid chromatography (HPLC/ESA Systems) using electrochemical detection. An ESA MD-150 C18 column was used at 0.4 V and 0.6–9 ml/min flow rate (mobile phase: 90 mM NaH2PO4, 50 mM citric acid, 1.7 mM 1-octanesulfonic acid, 50 µM EDTA, 10% acetonitrile) as compared to standards run daily. Data are reported as ng amine/ mg protein.
Cells adherent to cover slips were equilibrated in PSS 30 minutes (+10 µM pargyline) prior to a one hour incubation with 5-HT-biotin (12.7 µM) or 5-HT (10 µM) and vehicle or cystamine (10 mM). Cells were rinsed and fixed in 1 mL acetone (1 minute). Primary antibody used was anti-α-actin, smooth muscle specific (mouse monoclonal, Ab-2, EMD Biosciences, La Jolla CA, USA 1
100 in PBS) or anti-5-HT (rabbit polyclonal, 8250-0004, AbD Serotec, Raleigh NC, USA). Secondary antibodies used were: DyLight™ 488 streptavidin (1
2000, Rockland Inc, Gilbertsville, PA, USA) and Cy3-conjugated Affini Pure donkey anti-mouse (1
1000; IgG, Jackson, West Grove PA, USA) or Cy3-conjugated Affini Pure donkey anti-rabbit (1
1000; IgG, Jackson, West Grove PA, USA) in PBS. Following rinsing, cover slips were blotted dry and mounted on slides using Prolong Gold medium with DAPI (Invitrogen, Carlsbad, CA, USA). Slides were viewed and photographed on a Nikon TE2000 microscope using MetaMorph ® software (Molecular Devices, Sunnyvale CA, USA; all at 20°C). The light source was an X-Cite 120 fluorescence illumination system (EXFO, Mississauga Ontario, Canada), the camera a cool nap ES monochrome digital camera (Roper Scientific Photometrics, Pleasanton CA, USA). Slides were viewed under a 60× Nikon Plan Apo oil-immersion objective (Nikon Corporation, Toyko, Japan) using non-drying immersion oil, type a, formula code 1248 (Cargille, Cedar Grove, NJ, USA). At this magnification, 1 pixel is equivalent to 0.22 microns. Three Nikon filters were used: UV-2E/C96310M (lot number: C48793), Cy3 HYQ 96323 (lot number: C71280), and B-2E/C96311 (lot number: C57657). Excitation ranges were: UV-2E/C 340–380 nm, Cy5 HYQ 530–560 nm and B-2E/C465–495. Emission ranges were UV-2E/C435–C485, Cy3 HYA 573–648, and B-2E/C515–555 nM. Photograph bit depth was 12 nm. No neutral density filters or dichromic beamsplitters were utilized. The LUT was linear and covered the full range of data. Gamma values were equal to 1. Resolution of the photographs was 696×520 pixels (28.3 pixels/cm). There was no deconvolution, reconstruction, rendering or projection. Images were unaltered when combined into the overlay image.
Confluent petri dishes of rat aortic smooth muscle cells were rinsed of growth medium and equilibrated in physiological salt solution (PSS) for 30 minutes at 37°C and 5% CO2. Cells were incubated for 1 hour in 10 mM cystamine (Sigma-Aldrich, St. Louis, MO, USA) in PSS at 37°C and 5% CO2. The cystamine solution was gently removed but not rinsed from the dishes. Cells were incubated for 5 minutes with 0.05% trypsin-EDTA (Gibco, Invitrogen, Carlsbad, CA, USA). The cell/trypsin mixture was transferred to a conical tube and neutralized with 1.5 mL DMEM (Gibco, Invitrogen, Carlsbad, CA, USA) per 1 mL trypsin. This was centrifuged at 1400 RPM for 6 minutes. The supernatant was removed and the cell pellet was resuspended in 1 mL DMEM. Equal parts cell suspension and 0.4% trypan blue (Sigma-Aldrich, St. Louis, MO, USA) were mixed immediately before cell viability was ascertained using a Bright Line hemocytometer (Reichert, Buffalo, NY, USA) and Nikon TMS phase-contrast microscope (Nikon, Tokyo, Japan). Cells that took up the blue dye were deemed dead and percent viability was calculated by dividing the number of living cells (non-dye containing) by the total number of cells per square centimeter.
Tandem Mass Spectrometry
Tissue homogenates were taken through a transglutaminase activity assay with and without 5-HT-biotin (12.7 µM). Samples incubated with streptavidin-coated magnetic beads (25 µL, Invitrogen, Carlsbad CA, USA) for 1 hour at room temperature, with tumbling. Samples were magnetized to pull down biotin-labelled proteins and supernatant discarded. The magnetic beads were boiled in 2× SDS loading buffer, and separated on polyacrylamide gels (10%). Bands were excised and taken through tandem mass spectrometry by the proteomics core at Michigan State University. Those bands reported are those that only appeared in 5-HT-biotin-labelled samples.
Immunoprecipitation was carried out as previously described 
, using primary antibody against smooth muscle cell α-actin [(1 µg/200 µg protein) EMD Biosciences, La Jolla, CA, USA] in a phosphate-buffered saline based buffer. Samples were incubated overnight with protein A/G beads (25 µl/sample, Santa Cruz Biotechnologies, Santa Cruz, CA, USA), washed 3× with a protease-inhibitor rich phosphate buffered saline and then beads spun down. Captured beads were incubated in 2× SDS sample buffer, boiled for 10 minutes, centrifuged and the supernatant loaded onto standard SDS-PAGE gels (10%), at which point standard western protocol was used.
Helical strips of endothelial cell-intact strips were mounted in tissue baths for isometric tension recordings using Grass transducers and PowerLab data Acquisitions (Colorado Springs, CO, USA). Strips were placed under optimum resting tension (1500 milligrams) and equilibrated for one hour, with washing, before exposure to compounds. Tissue baths contained warmed (37°C), aerated (95% O2
) PSS. Administration of an initial concentration of 10 µM phenylephrine (PE) was used to test arterial strip viability. All tissues had an intact endothelial cell layer, evidenced by a robust (>50%) relaxation to acetylcholine (1 µM) in tissues contracted with a half-maximum concentration of PE. Tissues were incubated for one hour with vehicle (water) or cystamine (0.1–1 mM) for one hour prior to cumulative addition of 5-HT (10−9
M) or the non-receptor mediated agonist potassium chloride (KCl, 6–100 mM). Data are reported as the percentage of the initial contraction to PE 
All compounds were purchased from Sigma Chemical Company (St. Louis, MO, USA) unless otherwise noted.
All values are reported as means±standard error of the mean for the number of animals or explants (N) indicated. Data were analyzed by ANOVA with repeated test where more than two groups were compared (Graph Pad Prism), or two-tailed t test when two groups were compared. P values smaller than 0.05 were considered significant.