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

Abstract

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.