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

doi:10.21203/rs.3.rs-223666/v1

Download PDF

Research

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

https://doi.org/10.21203/rs.3.rs-223666/v1

This work is licensed under a CC BY 4.0 License

Version 1

posted

You are reading this latest preprint version

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 (O₂^-) have multiple effects on pulmonary parenchyma, altering cell proliferation, redox enzyme activation and smooth muscle contraction. Airway smooth muscle (ASM) contraction requires elevated intracellular Ca²⁺ ([Ca²⁺]_i). [Ca²⁺]_i release from intracellular stores also participates in contractile responses to multiple agonists.

OBJECTIVE: We investigated the effects of O₂^- on agonist-stimulated [Ca²⁺]_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 [Ca²⁺]_i release in response to acetylcholine (ACh), histamine, endothelin combined with lanthanum, and no Ca²⁺.

RESULTS: In PASM cells, agonist exposure generated a biphasic Ca²⁺ response. Dihydrorhodamine-loaded cells exposed to xanthine and xanthine oxidase showed time-dependent generation of (O₂^-), which was inhibited by SOD. Pre-incubation with xanthine and xanthine oxidase for 15 or 45 min revealed significant inhibition of net [Ca²⁺]_i responses to 100 nM and 1 M ACh and 50 M histamine. However, basal [Ca²⁺]_i was similar in cells exposed to O₂^- and controls. Multiple agonists inhibited Ca²⁺ release in the presence of O₂^-.

CONCLUSIONS: Superoxide impairs [Ca²⁺]_i release and may interfere with the contractile mechanism in ASM cells. Alteration of a common signaling pathway may be involved in [Ca²⁺]_i regulation. The effects of O₂^- were not likely due to cell damage since basal [Ca²⁺]_i was unchanged. We need further experiments to identify the molecular targets of O₂^- in Ca²⁺homeostasis.

Pulmonology

Health Economics & Outcomes Research

Superoxide

airway smooth muscle cell

intracellular calcium response

superoxide dismutase

oxidative lung injury

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 (O₂⁻), hydrogen peroxide (H₂O₂) and hydroxyl radical (OH⁻), are inflammatory 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, catalase-negative bacteria, and inflammatory 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.

Elevated intracellular Ca²⁺ ([Ca²⁺] _i) is an initial step in smooth muscle contraction. The main Ca²⁺ mobilizing pathways of smooth muscle cells are Ca²⁺ entry through voltage-dependent Ca²⁺ channels (VDCCs), agonist-induced Ca²⁺ entry, and Ca²⁺ release from intracellular Ca²⁺ stores evoked by D-myo-inositol 1,4,5- trisphosphate [Ins(1,4,5)P₃] (10). Ca²⁺ release also activates Ca²⁺ release activated Ca²⁺ entry (CRAC) in vascular smooth muscle cells. Impairments in any of these Ca²⁺-[1] mobilizing pathways in smooth muscle cells will affect vascular contraction, so oxidative stress-induced inhibition of Ca²⁺ pathways should be considered a potential contributor to vascular diseases. In bovine aortic endothelial cells (BAEC), superoxide inhibits CRAC and Ca²⁺ extrusion and accelerates Ca²⁺ leakage from intracellular Ca²⁺ stores [7]. However, reports on the effects of oxidative stress, especially the mobilizing properties of Ca²⁺ in airway smooth muscle (ASM) cells, are lacking.

[Ca²⁺]_i is essential in signal transduction and regulates numerous enzyme activities, such as proteases, phospholipases, and endonucleases [8]. Moreover, altered intracellular Ca²⁺ homeostasis occurs early in the development of irreversible cell injury [9]. In airway epithelium, [Ca²⁺]_i mediates cell functions including ion transport [10] and mucus secretion [11]. Extracellularly applied ROS increase [Ca²⁺ ]_i in other cell types, such as vascular endothelial cells [12] and renal tubular epithelial cells [13]. Nevertheless, the effect of ROS on Ca²⁺ dynamics in airway epithelium remains unexplored. The mechanisms underlying [Ca²⁺]_i oscillations are diverse and may be driven entirely by influx of extracellular Ca²⁺ across the plasma membrane, by the release of Ca²⁺ from intracellular stores, or by an interaction between intra- and extracellular sources of Ca²⁺.Therefore, to determine whether ROS alter airway epithelial Ca²⁺ 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.

Materials

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 ([14]). 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 [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 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.

Experimental protocols

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.

Statistical analysis

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.

Generation of superoxide:

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

Response to agonists:

[Ca²⁺]_i responses to agonists were attenuated with superoxide generated by the X/XO system (p<0.05), (Figure 2-5). This attenuation of [Ca²⁺]_i responses to agonists was also time dependent and varied with agonist concentrations (Figure 2-3).

Effect of superoxide dismutase:

SOD reversed the attenuation of [Ca²⁺]_i in a concentration-dependent manner (p<0.05) (Figure 6).

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 [Ca²⁺] _i in ASM cells and the development of airway hyperreactivity and fibrosis 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 O₂-. 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 O₂- attenuates agonist-induced [Ca²⁺]_i mobilizing pathways. SOD reversed these effects in a time-dependent manner. Cholinergic receptors activated by the endogenous agonist ACh elevate [Ca²⁺]_i in many cell types. Muscarinic ACh receptors, found on glands, smooth muscle, cardiac muscle, and neurons, elevate [Ca²⁺]_i by stimulating release from intracellular stores. Our PASM cells were perfused with HBSS containing no calcium and 1 mM lanthanum chloride (‘0’ Ca²⁺ HBSS) to prevent entry of extracellular calcium. Thus, O₂- influences molecular mechanisms potentially involved in calcium release from intracellular stores. O₂- exerts similar effects in vascular smooth muscle cells [22]. In this study, ACh induced [Ca²⁺]_i release in a concentration-dependent manner, which was quenched in a time-dependent manner by incubating ASM cells in O₂- generated by the X/XO system.

Exposure of ASM cells to contractile agonists results in biphasic elevation of [Ca²⁺]_i characterized by a rapid, transient rise in Ca²⁺, followed by a decline to a lower steady-state concentration sustained above the basal level [23, 24]. This biphasic [Ca²⁺]_i response results from calcium influx from the extracellular space and release of calcium from intracellular stores (i.e., the sarcoplasmic reticulum [SR]). Real-time confocal imaging also revealed calcium oscillations originate from one end of the cell and propagate toward the other end [25].

In airway epithelial cells, voltage-dependent Ca²⁺channels are absent, and mobilization of Ca²⁺ is controlled mainly by Ca²⁺ 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 Ca²⁺ 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 (IP₃R) 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 IP₃R-mediated release and plan further experiments to identify the molecular targets of O₂⁻ in Ca²⁺ homeostasis.

PASM: Porcine Airway Smooth Muscle

ACh: Acetylcholine

ET-1: Endothelin 1

[Ca²⁺]_i : intracellular calcium

ROS: Reactive oxygen species

SOD: Superoxide dismutase

X/XO: Xanthine / Xanthine oxidase

ETHICS AND ANIMAL USE APPROVAL

The Institutional Animal Care and Use Committees of the University of Minnesota approved the study protocols for the animal care, harvest, and sampling of pigs (Sus scrofa) tissue.

AVAILABILITY OF DATA AND MATERIALS

The authors are willing to share the raw data and details of experimental materials used as per appropriate request.

FUNDING

This study was supported in part by a grant from Academic Health Center, University of

Minnesota, and a grant from the National Institutes of Health to Mathur Kannan, PhD.

None of the other authors have financial relationships to disclose.

COMPETING INTERESTS

The authors declare that they have no competing interests.

AUTHOR CONTRIBUTIONS

RK: contributed to study design, methods, data and statistical analysis, interpretation, and manuscript writing.

DD: contributed to study methods, data analysis, manuscript review and editing.

MK: contributed study design, methods, data and statistical analysis, interpretation, manuscript review and editing.

ACKNOWLEDGMENTS

The authors would like to acknowledge Dr. Courtney Bricker-Anthony, Scientific Editor at Le Bonheur Children’s Foundation Research Institute, for her help with scientific editing of this manuscript.

Liochev, S.I., Reactive oxygen species and the free radical theory of aging. Free Radic Biol Med, 2013. 60: p. 1-4.
Bast, A., G.R. Haenen, and C.J. Doelman, Oxidants and antioxidants: state of the art. Am J Med, 1991. 91(3c): p. 2s-13s.
Cochrane, C.G., Cellular injury by oxidants. Am J Med, 1991. 91(3c): p. 23s-30s.
Wright, D.T., et al., Interactions of oxygen radicals with airway epithelium. Environmental health perspectives, 1994. 102 Suppl 10(Suppl 10): p. 85-90.
Yamaya, M., et al., Differentiated structure and function of cultures from human tracheal epithelium. Am J Physiol, 1992. 262(6 Pt 1): p. L713-24.
Wright, D.T., et al., Oxidant stress stimulates mucin secretion and PLC in airway epithelium via a nitric oxide-dependent mechanism. Am J Physiol, 1996. 271(5 Pt 1): p. L854-61.
Kimura, C., M. Oike, and Y. Ito, Acute Glucose Overload Abolishes Ca<sup>2+</sup> Oscillation in Cultured Endothelial Cells From Bovine Aorta. Circulation Research, 1998. 82(6): p. 677-685.
Kuriyama, H., et al., Physiological features of visceral smooth muscle cells, with special reference to receptors and ion channels. Physiol Rev, 1998. 78(3): p. 811-920.
Trump, B.F. and I.K. Berezesky, Calcium-mediated cell injury and cell death. The FASEB Journal, 1995. 9(2): p. 219-228.
Bagur, R. and G. Hajnóczky, Intracellular Ca(2+) Sensing: Its Role in Calcium Homeostasis and Signaling. Molecular cell, 2017. 66(6): p. 780-788.
Leybaert, L. and M.J. Sanderson, Intercellular Ca(2+) waves: mechanisms and function. Physiological reviews, 2012. 92(3): p. 1359-1392.
Moccia, F., R. Berra-Romani, and F. Tanzi, Update on vascular endothelial Ca(2+) signalling: A tale of ion channels, pumps and transporters. World journal of biological chemistry, 2012. 3(7): p. 127-158.
Udagawa, T., et al., Characteristics of spontaneous calcium oscillations in renal tubular epithelial cells. Clin Exp Nephrol, 2012. 16(3): p. 389-98.
White, T.A., M.S. Kannan, and T.F. Walseth, Intracellular calcium signaling through the cADPR pathway is agonist specific in porcine airway smooth muscle. Faseb j, 2003. 17(3): p. 482-4.
Deshpande, D.A., et al., CD38/cyclic ADP-ribose-mediated Ca2+ signaling contributes to airway smooth muscle hyper-responsiveness. Faseb j, 2003. 17(3): p. 452-4.
Warburton, D., Overview of Lung Development in the Newborn Human. Neonatology, 2017. 111(4): p. 398-401.
Prakash, Y.S., Airway smooth muscle in airway reactivity and remodeling: what have we learned? Am J Physiol Lung Cell Mol Physiol, 2013. 305(12): p. L912-33.
Prakash, Y.S., Emerging concepts in smooth muscle contributions to airway structure and function: implications for health and disease. Am J Physiol Lung Cell Mol Physiol, 2016. 311(6): p. L1113-l1140.
Vogel, E.R., et al., Moderate hyperoxia induces extracellular matrix remodeling by human fetal airway smooth muscle cells. Pediatr Res, 2017. 81(2): p. 376-383.
McCord, J.M., Oxygen-derived free radicals in postischemic tissue injury. N Engl J Med, 1985. 312(3): p. 159-63.
Thompson-Gorman, S.L. and J.L. Zweier, Evaluation of the role of xanthine oxidase in myocardial reperfusion injury. J Biol Chem, 1990. 265(12): p. 6656-63.
Touyz, R.M., et al., Vascular smooth muscle contraction in hypertension. Cardiovasc Res, 2018. 114(4): p. 529-539.
Sims, S.M., Y. Jiao, and Z.G. Zheng, Intracellular calcium stores in isolated tracheal smooth muscle cells. Am J Physiol, 1996. 271(2 Pt 1): p. L300-9.
Murray, R.K. and M.I. Kotlikoff, Receptor-activated calcium influx in human airway smooth muscle cells. J Physiol, 1991. 435: p. 123-44.
Jude, J.A., et al., Calcium Signaling in Airway Smooth Muscle. Proceedings of the American Thoracic Society, 2008. 5(1): p. 15-22.
Muallem, S., et al., Regulation of intracellular calcium in epithelial cells. Semin Cell Biol, 1990. 1(4): p. 305-10.
Thannickal, V.J. and B.L. Fanburg, Reactive oxygen species in cell signaling. American Journal of Physiology-Lung Cellular and Molecular Physiology, 2000. 279(6): p. L1005-L1028.
Hawkins, B.J., et al., Superoxide flux in endothelial cells via the chloride channel-3 mediates intracellular signaling. Mol Biol Cell, 2007. 18(6): p. 2002-12.
Kourie, J.I., Interaction of reactive oxygen species with ion transport mechanisms. Am J Physiol, 1998. 275(1): p. C1-24.

Download PDF

Version 1

posted

You are reading this latest preprint version

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

Status:

Version 1

Abstract

Figures

Introduction

Materials And Methods

Materials

Airway smooth muscle cell preparation

Digital video fluorescence imaging

Superoxide generation

Experimental protocols

Agonist-induced intracellular calcium responses:

Effects of superoxide (O₂^-) on [Ca²⁺]_i responses:

Effects of superoxide dismutase on superoxide (O₂^-)-mediated inhibition of [Ca²⁺]_i responses:

Statistical analysis

Results

Generation of superoxide:

Response to agonists:

Effect of superoxide dismutase:

Discussion

Abbreviations

Declarations

ETHICS AND ANIMAL USE APPROVAL

AVAILABILITY OF DATA AND MATERIALS

FUNDING

COMPETING INTERESTS

AUTHOR CONTRIBUTIONS

ACKNOWLEDGMENTS

References

Status:

Version 1

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

Status:

Version 1

Abstract

Figures

Introduction

Materials And Methods

Materials

Airway smooth muscle cell preparation

Digital video fluorescence imaging

Superoxide generation

Experimental protocols

Agonist-induced intracellular calcium responses:

Effects of superoxide (O2-) on [Ca2+] i responses:

Effects of superoxide dismutase on superoxide (O2-)-mediated inhibition of [Ca2+]i responses:

Statistical analysis

Results

Generation of superoxide:

Response to agonists:

Effect of superoxide dismutase:

Discussion

Abbreviations

Declarations

ETHICS AND ANIMAL USE APPROVAL

AVAILABILITY OF DATA AND MATERIALS

FUNDING

COMPETING INTERESTS

AUTHOR CONTRIBUTIONS

ACKNOWLEDGMENTS

References

Status:

Version 1

Effects of superoxide (O₂^-) on [Ca²⁺]_i responses:

Effects of superoxide dismutase on superoxide (O₂^-)-mediated inhibition of [Ca²⁺]_i responses: