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

Abstract

We examined the electrophysiological properties of reverse-mode Na(+) /Ca(2+) exchange (NCX) in mouse airway smooth muscle (ASM), assessing its contributions to regulation of [Ca(2+) ], and its expression in acute and chronic airway hyperresponsiveness (AHR). 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 [Ca(2+) ] and NCX expression, respectively. With standard KCl-containing electrode, 30μm 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 100μm iberiotoxin (blocker of large-conductance Ca(2+) -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 Ca(2+) -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 Ca(2+) influx via reverse-mode NCX activity. With conditions preventing Ca(2+) influx through voltage-dependent Ca(2+) channels but promoting that through NCX, we found that introduction of Ca(2+) led to marked but transient KB-R7943-sensitive elevation of [Ca(2+) ]. Additionally, KB-R7943 suppressed cholinergically evoked Ca(2+) waves. Finally, NCX1 expression was not significantly changed in allergen-induced AHR acute model but increased approx. 2.5-fold in a chronic model. Reverse-mode NCX activity leads to a physiologically relevant increase in [Ca(2+) ] even under control conditions, and this may be exaggerated in allergen-induced AHR and asthma.