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. Author manuscript; available in PMC: 2011 Aug 1.

Published in final edited form as: Pulm Pharmacol Ther. 2010 Mar 10;23(4):268–278. doi: 10.1016/j.pupt.2010.02.001

Panel A. Comparison of cAMP levels in Calu-3, CFBE41o⁻, and FRT cells. Total cAMP levels (normalized for total protein) were measured as described in the Methods. Forskolin raised cAMP in all cell types in a dose dependent fashion‥ CFBE41o⁻ cells demonstrated the strongest response compared with FRT and Calu-3 cells (*p<0.01 versus control-treated cells of the same cell type; † p<0.001 versus FRT cells at the same concentration of forskolin; n = 4 per condition, ± SEM). Panel B. Phosphodiesterase inhibition does not rescue forskolin-stimulated I_sc in temperature corrected ΔF508 CFBE41o⁻ monolayers. ΔF508 CFBE41o⁻ cells were grown in polarizing conditions and studied in modified Ussing chambers following temperature correction (27°C x 48 hours). After placing cells in chambers with a basolateral to apical low Cl⁻ gradient and blocking apical Na⁺ transport with amiloride (100 µM), monolayers were stimulated with papaverine 100µM (or vehicle) followed by forskolin (20 µM) and then genistein (50 µM – all agonists added to the apical and basolateral compartments). Papaverine alone (100 µM) activated I_sc [(6.8 ± 0.4 µA/cm² versus 0.1 ± 0.1 µA/cm² for vehicle treated cells], **p<0.001), but had no effect on subsequent forskolin-mediated I_sc (p=NS). Genistein (50 µM) further stimulated I_sc, although this was blunted in cells pretreated with papaverine (*p<0.05; n=5 ± SEM). CFBE41o⁻ monolayers expressing wtCFTR had a robust Cl⁻ secretory response to 100 µM papaverine that was sensitive to glybenclamide blockade (59.8 ± 14.3 vs. 1.3 ± 0.7 µA/cm² with vehicle, p<0.005, n = 8, data not shown). Panel C. Phosphatase inhibition does not rescue forskolin-stimulated I_sc in temperature corrected ΔF508 CFBE41o monolayers. ΔF508 CFTR-transduced CFBE41o⁻ cells were treated as described above in Panel 6B, except that the phosphatase inhibitor endothall (400 µM, apical and basolateral addition) was used in place of papaverine. Endothall alone did not activate Cl⁻ conductance (1.6 ± 0.4 vs. 1.1 ± 1.3 µA/cm² in vehicle treated cells, p=NS). Endothall pretreatment had no effect on I_sc following addition of forskolin (20 µM) or genistein (50 µM), n=5, ±SEM. CFBE41o⁻ monolayers expressing wtCFTR had a robust Cl⁻ secretory response to 400 µM endothall that was sensitive to glybenclamide blockade (40.1 ± 14.4 vs. 8.8 ± 4.8 µA/cm² with vehicle, p=0.05, n=8, data not shown).

Panel A. Comparison of cAMP levels in Calu-3, CFBE41o⁻, and FRT cells. Total cAMP levels (normalized for total protein) were measured as described in the Methods. Forskolin raised cAMP in all cell types in a dose dependent fashion‥ CFBE41o⁻ cells demonstrated the strongest response compared with FRT and Calu-3 cells (*p<0.01 versus control-treated cells of the same cell type; † p<0.001 versus FRT cells at the same concentration of forskolin; n = 4 per condition, ± SEM). Panel B. Phosphodiesterase inhibition does not rescue forskolin-stimulated I_sc in temperature corrected ΔF508 CFBE41o⁻ monolayers. ΔF508 CFBE41o⁻ cells were grown in polarizing conditions and studied in modified Ussing chambers following temperature correction (27°C x 48 hours). After placing cells in chambers with a basolateral to apical low Cl⁻ gradient and blocking apical Na⁺ transport with amiloride (100 µM), monolayers were stimulated with papaverine 100µM (or vehicle) followed by forskolin (20 µM) and then genistein (50 µM – all agonists added to the apical and basolateral compartments). Papaverine alone (100 µM) activated I_sc [(6.8 ± 0.4 µA/cm² versus 0.1 ± 0.1 µA/cm² for vehicle treated cells], **p<0.001), but had no effect on subsequent forskolin-mediated I_sc (p=NS). Genistein (50 µM) further stimulated I_sc, although this was blunted in cells pretreated with papaverine (*p<0.05; n=5 ± SEM). CFBE41o⁻ monolayers expressing wtCFTR had a robust Cl⁻ secretory response to 100 µM papaverine that was sensitive to glybenclamide blockade (59.8 ± 14.3 vs. 1.3 ± 0.7 µA/cm² with vehicle, p<0.005, n = 8, data not shown). Panel C. Phosphatase inhibition does not rescue forskolin-stimulated I_sc in temperature corrected ΔF508 CFBE41o monolayers. ΔF508 CFTR-transduced CFBE41o⁻ cells were treated as described above in Panel 6B, except that the phosphatase inhibitor endothall (400 µM, apical and basolateral addition) was used in place of papaverine. Endothall alone did not activate Cl⁻ conductance (1.6 ± 0.4 vs. 1.1 ± 1.3 µA/cm² in vehicle treated cells, p=NS). Endothall pretreatment had no effect on I_sc following addition of forskolin (20 µM) or genistein (50 µM), n=5, ±SEM. CFBE41o⁻ monolayers expressing wtCFTR had a robust Cl⁻ secretory response to 400 µM endothall that was sensitive to glybenclamide blockade (40.1 ± 14.4 vs. 8.8 ± 4.8 µA/cm² with vehicle, p=0.05, n=8, data not shown).