Fig. 4
From: Soluble adenylyl cyclase mediates hydrogen peroxide-induced changes in epithelial barrier function
H2O2-induced changes in permeability and resistance are mediated by sAC. Panel a. Treatment with 400 μM H2O2 stimulated an increase in [Ca2+]i in NHBE cells loaded with fura-2 AM (traces from three regions of interest). Panel b. sAC, but not tmAC, is involved in the H2O2-induced change in resistance. Fully differentiated NHBE cells were mounted in Ussing chambers, treated with 10 μM amiloride and the sAC inhibitor KH7 (25 μM) or the tmAC inhibitor MDL-12,330A (25 μM) in the apical and basolateral compartments for 20 min. The cells were then stimulated with apical 1 mM H2O2 in the presence of current pulses to determine the membrane resistance before and after H2O2 exposure. Pre-treating NHBE cells with KH7 attenuated the H2O2-induced decrease in membrane resistance while the tmAC inhibitor did not have an effect (n = 6 cultures from 6 donors, * = p < 0.05). Panel c. Inhibition of sAC attenuated the increase in permeability associated with prolonged H2O2 exposure (n = 4 lungs). Panel d. Lower concentrations of KH7 (10 μM) also had a significant effect on the H2O2-induced change in resistance (n = 6 cultures from 6 donors, * = p < 0.05). Panel e. NHBE cells were mounted in Ussing chambers and pretreated with 15 μM H89 for 20 min. The cells were then exposed to apical 1 mM H2O2 for 15 min. Inhibition of PKA attenuates the H2O2 induced decrease in resistance (n = 3 cultures from 3 donors, * = p < 0.05)