Figure 8. Chronic albuterol-induced CFTR impairment is rescued with application of exogenous cAMP.

wtCFTR⁺ and VX809-corrected F508del-CFTR⁺ CFBE41o- cells were exposed to albuterol and/or VX809 in media for 72 hours, then mounted in Ussing chambers and CFTR function was assessed under voltage clamp conditions. In contrast to previously demonstrated experiments, cell-permeant 8-bromoadenosine cAMP (8-Br cAMP) was used to stimulate CFTR function to bypass cellular mechanisms of cAMP generation. In wtCFTR⁺ cells (representative short-circuit current [I_sc] tracing in A, aggregate data in B; n = 4 inserts/condition. Circles represent total CFTR activity [cAMP + genistein], diamonds represent inhibited CFTR currents [Inh172]), maximal stimulation with 8-Br cAMP in place of forskolin/IBMX rescues the previously demonstrated albuterol-induced CFTR dysfunction. Stimulation with increasing doses of 8-Br cAMP (C, n = 3 inserts/data point) revealed a small increase in CFTR function in albuterol-pretreated cells at low stimulation doses. Squares, control conditions; triangles, albuterol pretreated. Similarly, in VX809-corrected F508del-CFTR⁺ cells (representative I_sc tracing in D, aggregate data in E; n = 4 inserts/condition), maximal stimulation with 8-Br cAMP rescues albuterol-induced CFTR dysfunction. Unlike wtCFTR⁺ cells, stimulation with increasing doses of 8-Br cAMP demonstrated no difference in VX809-corrected F508del-CFTR function following albuterol pretreatment at any stimulation dose (F, n = 3 inserts/data point. All indicators of statistical significance are comparisons with untreated cells; no difference was noted between VX809-corrected cells with or without albuterol). Circles, untreated; squares, VX809 alone; triangles, VX809 + albuterol. All studies are internally normalized as indicated to allow for comparisons. Stimulation protocol was as follows: amiloride (100 μM, not shown), cAMP (100 μM 8-Br cAMP; dark gray bars), CFTR potentiator (50 μM genistein for wtCFTR⁺; 1 μM VX770 for F508del-CFTR⁺ cells; light gray bars), and CFTR inhibition (10 μM Inh172; white bars). Both experiments are representative of studies repeated in duplicate with similar results. Data presented represent the mean ± SEM. *P < 0.05; ***P < 0.005; NS, non-significant by 2-way ANOVA with Tukey’s multiple comparisons test.