FIGURE 9.

TMEM16A antagonists fully bronchodilate airways and resist use- and inflammatory-desensitization pathways that limit β-agonist efficacy. Treatment of mouse tracheal rings overnight with a cytokine cocktail of IL-β, IL-13, and TNFα strongly reduced isoproterenol bronchodilation of carbachol pre-contracted airways (A), while the TMEM16A antagonists benzbromarone (B) and niclosamide (C) remained effective in fully relaxing the cytokine treated airways (n = 4; 2 rings/group, 2 experiments). As a model of β-adrenergic receptor use-dependent desensitization, carbachol pre-contracted (EC₅₀ or EC₇₅ level of CCh) mouse tracheal rings were treated with the β-agonist isoproterenol (round 1), washed, re-contracted with carbachol and tested again (round 2) for bronchodilation. Isoproterenol showed a rightward shift in potency after the second treatment with the β-agonist and a reduction in maximal efficacy in bronchodilation (D). Airway rings showing desensitization and only partial bronchodilation after this second treatment and highest isoproterenol dose, were fully relaxed by add-on of the TMEM16A antagonist Compound 4 (% relaxation shifts from 78.6% → 98.9% at EC₅₀ and from 42.7% → 95.4% at EC₇₅); see arrows to open cross-hatched circle and square (D). ^∗P < 0.05, ^∗∗P < 0.01, #P < 0.001, $P < 0.0001 from two-way ANOVA with Sidak’ s multiple comparison test for differences between untreated and cytokine treated airway rings for each compound treatment group; all other data points showed no significant (ns) difference (p > 0.05). Mean ± SEM.