5B, inset) Vehicle-treated CF HBECs had a significantly lower AS

5B, inset). Vehicle-treated CF HBECs had a significantly lower ASL height than NL controls. However, the ASL height in CF G22-A39 treated samples, 7.9 �� 0.6 ��m, was significantly higher than the CF controls, together 4.2 �� 0.6 ��m. This increase in ASL height in G22-A39 exposed CF HBECs was comparable to that observed in NL HBECs (~8 ��m). Next, a full dose response was completed in order to calculate the IC50 by measuring the ASL height of both NL and CF HBECs 6 h after addition of G22-A39 (Fig. 5C). The IC50 was not significantly different between NL and CF HBECs, 0.29 �� 0.19 and 0.52 �� 0.23 ��M, respectively. Figure 5. G22-A39 inhibits CF ASL hyperabsorption. A) Confocal micrographs of NL and CF ASL height 24 h after exposure to G22-A39 or vehicle (control). Scale bar = 7 ��m.

B) Mean ASL height over time in NL and CF HBECs with or without addition of G22-A39; … To test whether G22-A39 specifically affected ENaC in NL and CF HBECs, the 24 h transepithelial potential difference Vt was measured (Fig. 5D, E). In the NL HBECs, a basal Vt of ?6.6 �� 0.5 mV was observed, and this decreased to ?10.6 �� 0.7 mV following a brief exposure to trypsin, which is indicative of ENaC activation (15). G22-A39 had little additional effect on the 24 h Vt in NL HBECs. Consistent with our previous observation that CF ENaC remains fully active and nonresponsive to trypsin (15), the CF vehicle-exposed HBECs had an elevated Vt of ?15.2 �� 0.9 mV, and trypsin had no further effect. In contrast, after 24 h exposure to G22-A39, the CF HBEC Vt was significantly lowered to ?8.4 �� 0.

9 mV, and a 30-min exposure to trypsin changed the Vt to ?11 �� 0.7 mV, suggesting that G22-A39 works through ENaC in CF HBECs. In contrast, ADG had no effect on NL or CF HBEC Vt (both n=6). To further confirm that G22-A39 functions by inhibiting ENaC hyperabsorption, the ASL height of NL HBECs was measured over time in the presence of bumetanide with or without G22-A39 (Fig. 5F). Serosal bumetanide significantly decreased NL ASL height toward CF levels (i.e., <5 ��m). In contrast G22-A39 could maintain significantly greater ASL height in the presence of bumetanide, indicating that G22-A39 increases ASL height by inhibiting absorption not secretion. G22-A39 has no intrinsic structure To better understand how G22-A39 may interact with ENaC, we next looked for intrinsic structure in this peptide.

We first used the program PSIPRED to predict its structure (33, 34). Consistent with a GSK-3 lack of predicted structure with PSIPRED, we also failed to detect any secondary structure by circular dichroism (Fig. 6A). To functionally test this hypothesis, we heat denatured G22-A39 by incubating it at 67��C for 30 min and then added it to the mucosal surface of CF HBECs. The ASL height of the CF HBECs was measured 2 h later. The heat-denatured G22-A39 could prevent ASL hyperabsorption to the same extent as the non-heat-denatured peptide (Fig.

Leave a Reply

Your email address will not be published. Required fields are marked *

*

You may use these HTML tags and attributes: <a href="" title=""> <abbr title=""> <acronym title=""> <b> <blockquote cite=""> <cite> <code> <del datetime=""> <em> <i> <q cite=""> <strike> <strong>