Background Superoxide anions (O2−) have multiple effects on pulmonary parenchyma altering cell proliferation, cellular metabolism, and airway smooth muscle (ASM) contraction. Intracellular calcium ([Ca2+]i) concentration plays a significant role in the regulation of ASM contraction, relaxation, proliferation, and gene expression.

Objective We investigated the effects of O2− on agonist-stimulated changes in [Ca2+]i in ASM cells.

Design/Methods Fura-2 AM-loaded, freshly isolated porcine ASM (PASM) cells were used to examine [Ca2+]i release in response to acetylcholine (ACh), histamine, endothelin, caffeine, and thapsigargin (TPG) in the presence or absence of extracellular Ca2+.

Results Exposure of PASM cells to xanthine and xanthine oxidase (X + XO) resulted in a time-dependent generation of O2−, inhibited by superoxide dismutase (SOD). Preincubating PASM cells with X + XO for 15- or 45-minute inhibited net [Ca2+]i responses to ACh, histamine, caffeine, and TPG compared with control cells. Pretreating PASM cells with SOD for 30 minutes mitigated the inhibitory effect of X + XO treatment on ACh-induced Ca2+ elevation suggesting role of O2−. X + XO treatment also inhibited caffeine- and TPG-induced Ca2+ elevation suggesting effect of O2− on [Ca2+]i release and reuptake mechanisms.

Conclusion Superoxide attenuates [Ca2+]i release, reuptake, and may interfere with physiological functions of ASM cells.

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

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

R.K. performed experiments, analyzed data, and prepared figures and manuscript. M.K. contributed to study design, data statistical analysis, interpretation, and manuscript writing and editing. D.D. contributed to study methods, data analysis, and manuscript writing and editing.

Received: 18 December 2023

Accepted: 25 April 2024

Accepted Manuscript online:
02 May 2024

Article published online:
23 May 2024

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