Thus, Eact and Fact activate Pancreatic cancer TMEM16A by different mechanisms: Eact as a largely Ca2+-independent activator, and Fact as a potentiator of Ca2+ activation. T16Ainh-A01 completely blocked Cl? currents produced by Eact or Fact (Fig. 5D), as expected. Figure 5. Patch-clamp analysis of Ca2+ requirements for TMEM16A activation by Eact and Fact. A) Apical membrane current measured in TMEM16A-expressing FRT cells. ER calcium stores were depleted by CPA (50 ��M, 30 min) and 0 CaCl2 in bath. ATP (100 ��M), … Epithelial fluid secretion and intestinal smooth muscle contraction Prior studies using TMEM16A inhibitors and RNAi knockdown indicated that TMEM16A is a minor component of total CaCC conductance in human bronchial surface epithelial cell cultures under basal conditions, but that TMEM16A is strongly up-regulated after IL-4 treatment for 24 h (18).

Supporting this conclusion, we found here that Eact did not induce Cl? current in untreated human CF bronchial epithelial cells, whereas UTP, which elevates cytoplasmic Ca2+ and hence non-TMEM16A CaCC(s), produced a large Cl? current (Fig. 6A, left panel). Remarkably, Eact induced Cl? current in IL-4-treated CF bronchial cells, which was blocked by the TMEM16A-selective inhibitor T16Ainh-A01 (Fig. 6A, center and right panels). Figure 6. Airway epithelial chloride secretion. A) Short-circuit current in CF HBE cells. Left and center panels: Eact and UTP (100 ��M) were added in control (left) and IL-4 (10 ng/ml, 24 h, center) treated CF HBE cells. Right panels: summary of Eact-induced, …

Prior reports suggested the involvement of TMEM16A in CaCC activity in airway submucosal glands (30, 31). To verify TMEM16A function, short-circuit current measurements were done in primary cultures of human tracheal submucosal gland epithelial cells that were grown under conditions that preserve serous-type phenotype (26). Figure 6B (left panel) shows increased Cl? current in response to UTP and Eact, which was largely abolished by T16Ainh-A01 pretreatment. Figure 6B (middle panel) shows increased Cl? conductance with Eact in the absence of UTP pretreatment. Immunoblot (Fig. 6B, inset) confirmed TMEM16A protein in the glandular epithelial cell cultures. Averaged peak Cl? currents are summarized in Fig. 6B (right panel). Immunostaining of human non-CF and CF bronchi showed TMEM16A expression at the luminal membrane of submucosal gland serous cells, but not gland mucous cells or surface epithelial cells (Fig.

7A). Similar staining was found in non-CF and CF human bronchi. Fluid secretion was measured in individual submucosal glands from the increasing size of mucus (fluid) bubbles in which airway fragments were mounted in a 37��C perfusion chamber and Cilengitide the mucosal solution was covered with oil. Figure 7B (top panel) shows mucus bubbles following addition of carbachol and Eact in human bronchi.