Reverse‐mode NCX current in mouse airway smooth muscle: Na+ and voltage dependence, contributions to Ca2+ influx and contraction, and altered expression in a model of allergen‐induced hyperresponsiveness
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AbstractAim:We examined the electrophysiological properties of reverse‐mode Na+/Ca2+ exchange (NCX) in mouse airway smooth muscle (ASM), assessing its contributions to regulation of [Ca2+], and its expression in acute and chronic airway hyperresponsiveness (AHR).Methods:Membrane currents were studied in single murine ASM cells under voltage clamp at −60 mV using ramp depolarizing commands to +80 mV. Confocal fluorimetric and RT‐PCR techniques were used to monitor changes in cytosolic [Ca2+] and NCX expression, respectively.Results:With standard KCl‐containing electrode, 30 μm KB‐R7943 (an inhibitor of reverse‐mode NCX activity) exhibited variable effects on membrane current, indicating modulation of more than one conductance. KB‐R7943 activated outwardly rectifying current that was inhibited by 100 μm iberiotoxin (blocker of large‐conductance Ca2+‐dependent K+ channels), indicating a direct enhancing effect of KB‐R7943 on those K+ channels. After obviating K+ currents, we found that a current sensitive to 4‐4’‐diisothiocyanostilbene‐2,2’‐disulfonic acid (blocker of Ca2+‐dependent Cl‐ channels) was markedly increased by elevating [Na+] in the electrode solution to 13, 15.5 and 18 mm and suppressed by KB‐R7943, indicating Ca2+ influx via reverse‐mode NCX activity. With conditions preventing Ca2+ influx through voltage‐dependent Ca2+ channels but promoting that through NCX, we found that introduction of Ca2+ led to marked but transient KB‐R7943‐sensitive elevation of [Ca2+]. Additionally, KB‐R7943 suppressed cholinergically evoked Ca2+ waves. Finally, NCX1 expression was not significantly changed in allergen‐induced AHR acute model but increased approx. 2.5‐fold in a chronic model.Conclusion:Reverse‐mode NCX activity leads to a physiologically relevant increase in [Ca2+] even under control conditions, and this may be exaggerated in allergen‐induced AHR and asthma.
