Superoxide Anion Inhibits Intracellular Calcium Response in Porcine Airway Smooth Muscle Cells

BACKGROUND: Superoxide is implicated in lung disease, injury, and transplantation. In lung, many defense mechanisms, especially superoxide dismutase (SOD) and metallothionein, neutralize superoxide. Superoxide anions (O 2- ) have multiple effects on pulmonary parenchyma, altering cell proliferation, redox enzyme activation and smooth muscle contraction. Airway smooth muscle (ASM) contraction requires elevated intracellular Ca 2+ ([Ca 2+ ] i ). [Ca 2+ ] i release from intracellular stores also participates in contractile responses to multiple agonists. OBJECTIVE: We investigated the effects of O 2- on agonist-stimulated [Ca 2+ ] i responses in ASM cells. DESIGN/METHODS: Porcine ASM (PASM) cells were dissociated using collagenase and papain. Fura-2 AM-loaded PASM cells were used to examine [Ca 2+ ] i release in response to acetylcholine (ACh), histamine, endothelin combined with lanthanum, and no Ca 2+ . RESULTS: In PASM cells, agonist exposure generated a biphasic Ca 2+ response. Dihydrorhodamine-loaded cells exposed to xanthine and xanthine oxidase showed time-dependent generation of (O 2- ), which was inhibited by SOD. Pre-incubation with xanthine and xanthine oxidase for 15 or 45 min revealed signicant inhibition of net [Ca 2+ ] i responses to 100 nM and 1 M ACh and 50 M histamine. However, basal [Ca 2+ ] i was similar in cells exposed to O 2- and controls. Multiple agonists inhibited Ca 2+ release in the presence of O 2- . CONCLUSIONS: Superoxide impairs [Ca 2+ ] i release and may interfere with the contractile mechanism in ASM cells. Alteration of a common signaling pathway may be involved in [Ca 2+ ] i regulation. The effects of O 2- were not likely due to cell damage since basal [Ca 2+ ] i was unchanged. We need further experiments to identify the molecular targets of O 2- in Ca 2+ homeostasis. determine ROS alter airway epithelial Ca 2+ signaling properties and if so, to elucidate the mechanism, we studied freshly dissociated porcine ASM (PASM) cells in vitro using a hypoxanthine (HX)–xanthine oxidase (XO) system. We also examined the ability of superoxide dismutase (SOD) to reverse the effects of superoxide.


Introduction
Oxidative stress resulting from the toxic effects of reactive oxygen species (ROS) may contribute to the pathogenesis of various pulmonary disorders, such as acute respiratory distress syndrome, emphysema, and asthma [1], [2]. ROS, including superoxide anion (O 2 − ), hydrogen peroxide (H 2 O 2 ) and hydroxyl radical (OH − ), are in ammatory mediators of cell and tissue injury [3]. ROS exert their biological effects through lipid peroxidation of the cell membrane, alteration of enzyme activities, and breakage of DNA strands. Airway epithelial cells are vulnerable to ROS from endogenous and exogenous sources because in addition to their own oxygen metabolites, these cells are exposed to oxidant air pollutants, catalasenegative bacteria, and in ammatory cells [4]. Various oxidants induce epithelial damage and mucus hypersecretion, characteristics of asthma. In fact, ample evidence indicates ROS stimulate signal transduction related to epithelial cell dysfunction or injury, potentially resulting in the above pathophysiology [5], [6]. However, little is known about the intracellular regulatory mechanisms involved.  [7]. However, reports on the effects of oxidative stress, especially the mobilizing properties of Ca 2+ in airway smooth muscle (ASM) cells, are lacking.
[Ca 2+ ] i is essential in signal transduction and regulates numerous enzyme activities, such as proteases, phospholipases, and endonucleases [8]. Moreover, altered intracellular Ca 2+ homeostasis occurs early in the development of irreversible cell injury [9]. In airway epithelium, [Ca 2+ ] i mediates cell functions including ion transport [10] and mucus secretion [11]. Extracellularly applied ROS increase [Ca 2+ ] i in other cell types, such as vascular endothelial cells [12] and renal tubular epithelial cells [13]. Nevertheless, the effect of ROS on Ca 2+ dynamics in airway epithelium remains unexplored. The mechanisms underlying [Ca 2+ ] i oscillations are diverse and may be driven entirely by in ux of extracellular Ca 2+ across the plasma membrane, by the release of Ca 2+ from intracellular stores, or by an interaction between intra-and extracellular sources of Ca 2+ .Therefore, to determine whether ROS alter airway epithelial Ca 2+ signaling properties and if so, to elucidate the mechanism, we studied freshly dissociated porcine ASM (PASM) cells in vitro using a hypoxanthine (HX)-xanthine oxidase (XO) system. We also examined the ability of superoxide dismutase (SOD) to reverse the effects of superoxide.
Airway smooth muscle cell preparation PASM cells were isolated from the trachea as previously described ( [14]). Brie y, 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  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 lter changer (Sutter Instrument, Novato, CA), and emissions were collected using a 510 nm barrier lter. Fluorescence excitation, image acquisition, and real-time data analyses were controlled using a video uorescence imaging system (Meta uor; Universal Imaging, Bedford Hills, NY). Images were acquired using a Photometric Cool Snap 12-bit digital camera (Roper Scienti c, Teledyne Photometrics, Tucson, AZ) and transferred to a computer for subsequent analysis.
The ratio of uorescence intensities at 340 and 380 nm were calculated approximately every 0.75 s, and [Ca 2+ ] i was calculated from the ratio of intensities at 340 nm and 380 nm by extrapolation from a calibration curve as previously described [15].
Superoxide generation 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 uorometrically 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 uorescence 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 uorescence 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.

Experimental protocols
Agonist-induced intracellular calcium responses:

Statistical analysis
All experiments were repeated in at least 4-5 different cell preparations. Data were analyzed using oneway analysis of variance (ANOVA) using GraphPad Prism (GraphPad Software Inc., San Diego, CA) statistical software. Two means were considered signi cantly different when the p value was less than 0.05.

Results
Generation of superoxide: The X/X0 system generated superoxide in a time-dependent manner ( Figure 1).

Response to agonists:
[Ca 2+ ] i responses to agonists were attenuated with superoxide generated by the X/XO system (p<0.05), Effect of superoxide dismutase: SOD reversed the attenuation of [Ca 2+ ] i in a concentration-dependent manner (p<0.05) ( Figure 6).

Discussion
Fetal lung development involves multiple cell types and complex interaction of signaling mechanisms. Exposure to mechanical ventilation and high oxygen concentrations alter lung development. These complex processes are only partially understood [16]. ASM cells regulate airway muscle tone and contractility throughout life [17]. We are improving our understanding of [Ca 2+ ] i in ASM cells and the development of airway hyperreactivity and brosis after hyperoxia exposure in the developing lung [18,19].
Identifying the mechanisms underlying ASM structure and function early in development will help determine potential targets in disease. XO may be a source of biological free radical generation. This enzyme generates the superoxide radical and is widely used to obtain O 2 -. Superoxide and XO also contribute to myocardial reperfusion injury [20,21].
In our experiments, we generated superoxide with an in vitro system using X/XO. We previously showed  [25].
In airway epithelial cells, voltage-dependent Ca 2+ channels are absent, and mobilization of Ca 2+ is controlled mainly by Ca 2+ release from storage sites and CRAC [26]. The generation of ROS participates in normal cell signaling, but oxidative stress can damage cellular macromolecules such as lipids, protein, and DNA. These effects may contribute to the pathogenesis of severe lung disease in premature newborns and adults [27].
Superoxide is not freely diffusible but can cross membranes via ion channels. Extracellular superoxide enters the cell via anion blocker-sensitive chloride channel 3 [28]. Here, we showed superoxide could suppress Ca 2+ release from intracellular storage sites, while the addition of SOD reversed these effects. Major calcium release channels from the sarcoplasmic/endoplasmic reticulum (SR/ER) are ryanodine receptors (RyR) in excitable cells and inositol 1,4,5-trisphosphate receptors (IP 3 R) in non-excitable cells.
ROS can directly modulate RyR activity by oxidizing redox-sensing thiol groups [29]. In future experiments, we intend to study the effects of caffeine-induced IP 3