Routinely used reagents, acetylcholine (ACh) and histamine were obtained from Sigma Chemical Company (St. Louis, MO). Fura-2 AM and dihydrorhodamine were purchased from Molecular Probes (Eugene, OR). Endothelin-1 (ET-1), SOD, xanthine (X) and XO were obtained from Calbiochem (La Jolla, CA).
Airway smooth muscle cell preparation
PASM cells were isolated from the trachea as previously described (). Briefly, 6- to 10-wk-old, outbred Yorkshire pigs (~10 -18 kg body weight) were anesthetized with an intramuscular injection of tiletamine hydrochloride-zolazepam (Telazol, 8 mg/kg; Fort Dodge Laboratories, Fort Dodge, IA) combined with xylazine (8 mg/kg). The animals were euthanized by barbiturate overdose in accordance with a protocol approved by the Institutional Animal Care and Use Committee (IACUC), University of Minnesota. Isolated tracheas were transferred to ice-cold HBSS containing 10 mM HEPES, 11 mM glucose, 2.5 mM CaCl2, and 1.2 mM MgCl2 (pH 7.4) and maintained in an oxygenated environment. Following removal of the epithelium from the trachea, the smooth muscle layer was dissected and used for cell dissociation. The tissue was initially minced in ice-cold HBSS and transferred to Earle’s balanced salt solution containing 20 U/ml papain and 0.005% DNase (Worthington Biochemical, Freehold, NJ) and incubated at 37°C for 2 hours. After the initial incubation, 0.4 mg/ml type IV collagenase and 0.3 U/ml elastase (Worthington Biochemical) were added and incubated at 37°C until the cells were completely dispersed (~15-30 min). Cell dispersion was aided by gentle trituration with a fire-polished glass pipette. The solution was centrifuged at 2,000 rpm for 5 min, and the pelleted cells were resuspended in HBSS. The cells were placed at 4°C overnight and subsequently prepared for plating. The cell suspension (200 µl) was beaded onto glass coverslips and allowed to attach at 37°C in 95% O2 and 5% CO2 for 30 min. Coverslips with attached cells were placed in HBSS containing 5 µM Fura-2 AM (Molecular Probes, Eugene, OR) and incubated at 37°C for 30 min. Coverslips were washed in HBSS, treated as described in the experimental protocols, and used to determine [Ca2+] i.
Digital video fluorescence imaging
Coverslips were mounted on a 150-µl open slide chamber (Warner Instruments, Hamden, CT) and placed on the stage of a Nikon Diaphot inverted microscope (Nikon, Tokyo, Japan). Cells were perfused with HBSS or agonists as described in the protocol. The cells were visualized using a Nikon Fluor ×40 oil immersion objective lens. Fura-2-loaded cells were excited at 340 and 380 nm using a Lambda DG-4 filter changer (Sutter Instrument, Novato, CA), and emissions were collected using a 510 nm barrier filter. Fluorescence excitation, image acquisition, and real-time data analyses were controlled using a video fluorescence imaging system (Metafluor; Universal Imaging, Bedford Hills, NY). Images were acquired using a Photometric Cool Snap 12-bit digital camera (Roper Scientific, Teledyne Photometrics, Tucson, AZ) and transferred to a computer for subsequent analysis.
The ratio of fluorescence intensities at 340 and 380 nm were calculated approximately every 0.75 s, and [Ca2+]i was calculated from the ratio of intensities at 340 nm and 380 nm by extrapolation from a calibration curve as previously described .
X/XO were used to generate superoxide in all the experiments. XO (10 mU/ml) was incubated with 100 mM X prepared in HBSS. Superoxide generation was determined fluorometrically using dihydrorhodamine. PASM cells were loaded with 5 mM dihydrorhodamine for 30 min and washed with HBSS to remove excess dye. The cells were resuspended in HBSS containing 100 mM X, and basal fluorescence was measured at 485 nm and 538 nm excitation and emission wavelengths, respectively. Then, XO was added to the cell suspension, and the change in the fluorescence was measured. In different experiments, cells were preincubated with HBSS containing SOD, followed by the addition of X/XO. The generation of superoxide with and without preincubation with SOD was determined.
Agonist-induced intracellular calcium responses:
PASM cells were perfused with HBSS containing no calcium and 1 mM lanthanum chloride (‘0’ Ca2+ HBSS). Basal [Ca2+]i was determined as described above. The cells were subsequently perfused with ‘0’ Ca2+ HBSS containing 100 nM ACh, 1 mM ACh, 50 mM histamine, or 200 nM ET-1 for at least 1 min. Changes in [Ca2+]i were monitored during stimulation of cells with agonists, and peak [Ca2+]i was determined. The net intracellular Ca2+ response to each agonist was calculated by subtracting basal from peak [Ca2+]i.
Effects of superoxide (O2-) on [Ca2+] i responses:
Unless otherwise noted, cells were perfused with ‘0’ Ca2+ HBSS. PASM cells were incubated with X/XO for 15, 30 or 45 min. The X/XO system generated superoxide anions over time as described above. The cells were washed with HBSS and used for [Ca2+]i measurements as described above. After determining basal [Ca2+]i, the cells were stimulated with 100 nM ACh, 1 mM ACh, 50 mM histamine, or 200 nM ET-1 for at least 1 min. The net [Ca2+]i responses to each of the agonists were calculated by subtracting basal from peak [Ca2+]i. The net intracellular Ca2+ responses in PASM cells exposed to X/XO were compared to those in control cells exposed to HBSS.
Effects of superoxide dismutase on superoxide (O2-)-mediated inhibition of [Ca2+]i responses:
Cells were preincubated with 250 or 500 U/ml SOD for 30 min before exposure to X/XO. These experiments were repeated to determine the net [Ca2+]i responses to agonists as described above. The net [Ca2+]i responses of PASM cells to multiple agonists upon exposure to X/XO with or without preincubation with SOD were compared.
All experiments were repeated in at least 4-5 different cell preparations. Data were analyzed using one-way analysis of variance (ANOVA) using GraphPad Prism (GraphPad Software Inc., San Diego, CA) statistical software. Two means were considered significantly different when the p value was less than 0.05.