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

Aim: 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 -60mV using ramp depolarizing commands to +80mV. 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 mu m 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 mu m 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 18mm 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.