Transmembrane protein with unknown function 16/anoctamin-1 (ANO1) is a protein widely expressed in mammalian tissues, and it has the properties of the classic calcium-activated chloride channel (CaCC). This protein has been implicated in numerous major physiological functions. However, the lack of effective and selective blockers has hindered a detailed study of the physiological functions of this channel. In this study, we have developed a potent and selective blocker for endogenous ANO1 in Xenopus laevis oocytes (xANO1) using a drug screening method we previously established (Oh et al., 2008). We have synthesized a number of anthranilic acid derivatives and have determined the correlation between biological activity and the nature and position of substituents in these derived compounds. A structure-activity relationship revealed novel chemical classes of xANO1 blockers. The derivatives contain a 2NO 2 group on position 5 of a naphthyl group-substituted anthranilic acid, and they fully blocked xANO1 chloride currents with an IC 50 , 10 mM. The most potent blocker, N-((4-methoxy)-2-naphthyl)-5-nitroanthranilic acid (MONNA), had an IC 50 of 0.08 mM for xANO1. Selectivity tests revealed that other chloride channels such as bestrophin-1, chloride channel protein 2, and cystic fibrosis transmembrane conductance regulator were not appreciably blocked by 10∼30 mM MONNA. The potent and selective blockers for ANO1 identified here should permit pharmacological dissection of ANO1/CaCC function and serve as potential candidates for drug therapy of related diseases such as hypertension, cystic fibrosis, bronchitis, asthma, and hyperalgesia.

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Purpose:To evaluate the effects of trypan blue (TB) on the survival of cultured human trabecular meshwork cells (HTMCs). Methods: Primarily cultured HTMCs were exposed to 0.05, 0.10 or 0.50% TB for 1, 5 or 30 min. Cellular survival was assessed using the MTT assay and degree of apoptosis was analyzed with flow cytometry using annexin-V/propidium iodide double staining. Results: Long-term exposure or high concentration of TB decreased the survival of HTMCs (p < 0.05). In flow cytometric analysis, exposure to 0.50% TB for 30 min increased the degree of apoptosis (p < 0.05). Commercial TB decreased cell survival after exposure for 5 min and increased the degree of apoptosis after exposure for 30 min (p < 0.05). Conclusions: TB may cause cellular damage of cultured HTMCs and apoptosis could be the underlying mechanism. In TB-assisted cataract surgery, TB should be used for the shortest time possible and removed completely.

Purpose:To investigate the effects of amniotic membrane extract (AME) on the survival of cultured human nasal mucosa fibroblasts. Methods: Primary cultured human nasal mucosa fibroblasts were exposed to 0, 10, 20, or 30 μg/mL AME for 3 days. The survival of the human nasal mucosa fibroblasts was measured using the MTT assay and apoptosis was evaluated with flow cytometry using annexin-V/propidium iodide double staining. Results: AME decreased significantly in fibroblast proliferation after exposure to 10 μg/mL (p = 0.000), and caused significant apoptosis of the fibroblasts after exposure to 10 μg/mL (p = 0.024). Conclusions: AME decreased fibroblast proliferation in vitro at least through induction of apoptosis. Therefore, adjuvant use of AME during endonasal dacryocystorhinostomy may improve clinical outcomes.