Figure 5.

Human airway bronchodilation in precision-cut human lung slices (PCLS) by TAS2R agonist is ablated by knock-down of cofilin or Par3. (A) Human PCLS are studied by obtaining a core from a healthy donor lung that encompasses bronchi in parallel. Microtome cutting of the cores results in thin slices, which are placed in culture, with selected intact airways oriented parallel to the plating surface together with intact epithelium and interstitial tissue. Control (scrambled) or gene-specific siRNAs were transfected as described in Methods. Repeated images are acquired in response to potential bronchoactive agents and luminal area calculated using ImageJ software. (B) Gene-specific siRNA transfections of slices decreases cofilin or Par3 in protein derived from whole slices (representative western blots and results from multiple experiments are shown). *P < 0.01 versus control siRNA. (C) Representative images showing the same airway at baseline (vehicle) and in response to histamine or histamine with the TAS2R14 agonist AA. In the control siRNA condition (column 1), histamine constricts, while AA bronchodilates, as indicated by the increased airway area compared with the constriction evoked by histamine. In contrast, the cofilin (column 2) or Par3 (column 3) knock-downs show no effects of AA. (D) Results from measurements from four separate donor lungs, with a total of 12–15 slices per condition. *P < 0.05 versus a “0” baseline and **P < 0.01 versus responses from the respective doses under conditions of cofilin or Par3 knock-down. Scale bars, 300 μm; magnification, 40× .