Mechanism of high-salt diet up-regulating TMEM16A-induced cerebral arterial remodeling in C57BL/6J mice / 中国药理学通报

Aim To investigate the mechanism of high salt-induced cerebral artery remodeling in mice by up-regulating TMEM16A. Methods Forty C57BL/6J mice were randomly divided into four groups (10 per group, 8 weeks of intervention), namely, blank control group (normal diet), low-salt group (2% high salt diet), medium-salt group (4% high salt diet) and high-salt group (8% high salt diet). HE staining was used to observe the morphological changes of cerebral arteries; blood vessel permeability test was used to compare the color and absorbance value of brain tissue. Immunofluorescence was employed to detect TMEM16A expression in cerebral arteries of mice in each group; PCR and Western blot were applied to detect the mRNA and protein expression of TMEM16A in cerebral arterial tissues; whole-cell patch clamp was use to record the calcium-activated chloride channel (CaCC) currents of mouse cerebral artery smooth muscle cells in each group. Results HE results showed that 2%, 4%, and 8% high salt diet could concentra-tion-dependently induce cerebral arterial wall thickening and lumen stenosis in C57BL/6J mice. The permeability test found that compared with the control group, the absorbance value of the brain tissue of the mice in the 2%, 4% and 8% high salt groups increased significantly. The results of isolated muscle tension showed that compared with the control group, the systolic response of isolated cerebral arteries to 60 mmol • L

Discovery of 4-arylthiophene-3-carboxylic acid as inhibitor of ANO1 and its effect as analgesic agent

Anoctamin 1 (ANO1) is a kind of calcium-activated chloride channel involved in nerve depolarization. ANO1 inhibitors display significant analgesic activity by the local peripheral and intrathecal administration. In this study, several thiophenecarboxylic acid and benzoic acid derivatives were identified as novel ANO1 inhibitors through the shape-based virtual screening, among which the 4-arylthiophene-3-carboxylic acid analogues with the best ANO1 inhibitory activity were designed, synthesized and compound

The expression of transmembrane protein 16A in guinea pig cochlea increases with years / 西安交通大学学报(医学版)

Objective To investigate the expression of transmembrane protein 16A (TMEM16A)in the cochlea of guinea pigs and its relationship with the age-related hearing loss.Methods We used auditory brainstem response (ABR)to explore the changes of hearing in guinea pigs of different age (groups of 2 w,3 m,1 y,and D-galactose).The distribution and expression of TMEM16A in the cochlea were detected by immunofluorescence and Western blot.Results ABR threshold was gradually increased,with significant difference between D-gal and the other three groups (P <0.01).TMEM16A was expressed in the cochlear striae vascularis at different ages,and the expression increased with age before 1 y (P <0.05). However, its level was increased in D-gal group and significantly differed from that in 3 m and 1 y groups (P < 0.05 ).Conclusion The change in TMEM16A expression in the cochlear striae vascularis of guinea pigs may be related to age-related hearing loss.

The expression of transmembrane protein 16A in guinea pig cochlea increases with years / 西安交通大学学报(医学版)

Objective To investigate the expression of transmembrane protein 16A (TMEM16A)in the cochlea of guinea pigs and its relationship with the age-related hearing loss.Methods We used auditory brainstem response (ABR)to explore the changes of hearing in guinea pigs of different age (groups of 2 w,3 m,1 y,and D-galactose).The distribution and expression of TMEM16A in the cochlea were detected by immunofluorescence and Western blot.Results ABR threshold was gradually increased,with significant difference between D-gal and the other three groups (P <0.01).TMEM16A was expressed in the cochlear striae vascularis at different ages,and the expression increased with age before 1 y (P <0.05). However, its level was increased in D-gal group and significantly differed from that in 3 m and 1 y groups (P < 0.05 ).Conclusion The change in TMEM16A expression in the cochlear striae vascularis of guinea pigs may be related to age-related hearing loss.

Expression and significance of PAR2 and TMEM16A on DRG rat modelin of neuropathic pain / 实用医学杂志

Objective To observe the expression of PAR2 and TMEM16A in the model of chronic constriction injury (CCI) in rat dorsal root ganglion (DRG) neurons,and to explore the role of it in the neuropathic pain.Methods Rats were divided into Sham operation group (Sham) and CCI group.Both groups were observed respectively to determine thermal withdrawal latency (TWL).The expression of PAR2 and TMEM16A in the dorsal root ganglion of the rat was analyzed using Western blot and immunofluorescence.Results The difference in preoperative TWL between CCI group and Sham group rats was not statistically significant (P ＜ 0.01).TWL was signifi cantly lower at all other time points after operation (P ＜ 0.01).Immunofluorescence results showed that PAR2 and TMEM16A coexisted in rat DRG neurons.Western blot results showed that,compared with Sham group,CCI group PAR2 and TMEM16A protein expression significantly increased after 7 d and 14 d (P ＜ 0.01),and the PAR2 and TMEM16A protein expression on 14 d is higher than that of 7 d (P ＜ 0.05).Conclusions Expression level of PAR2 and TMEM16A in CCI group was significantly higher than those in Sham group.The expression level of these proteins may be the cause of rat model of neuropathic pain.

TMEM16A-Mediated Mucin Secretion in IL-13-Induced Nasal Epithelial Cells From Chronic Rhinosinusitis Patients

PURPOSE: Chronic rhinosinusitis with nasal polyps (CRSwNP), a mainly Th2 cytokine-mediated disease, often involves mucus secretion. Recent evidence suggests that transmembrane protein 16A (TMEM16A), a calcium-activated Cl- channel (CaCC), can regulate mucus secretion from airway epithelium by transepithelial electrolyte transport and hydration. However, the role of TMEM16A in mucin production/secretion in the airway epithelium is not clear. This study was conducted to determine the role of TMEM16A in mediating mucin secretion in human nasal polyp epithelial cells (HNPECs) induced by IL-13. METHODS: Human sinonasal mucosa tissue and dissociated sinonasal epithelium from control subjects and patients with CRSwNP were assessed for the expression of TMEM16A and the secretion of human mucin 5AC (MUC5AC) by immunohistochemistry, Western blot analysis, and enzyme-linked immuno-sorbent assay (ELISA). A model of the Th2 inflammatory environment was created by exposure of primary air-liquid interface (ALI)-cultured HNPECs to interleukin-13 (IL-13) for 14 days, with subsequent assessment of TMEM16A expression in cell lysates by Western blotting and MUC5AC secretion in apical washings of cells by ELISA. RESULTS: The expressions of TMEM16A and MUC5AC were increased in human nasal polyp tissue and dissociated nasal polyp epithelium. TMEM16A was detected in IL-13-treated HNPECs, specifically in MUC5AC-positive cells but not in ciliated cells. IL-13 treatment increased percentages of TMEM16A-positive cells, MUC5AC-positive cells, and cells coexpressing TMEM16A/MUC5AC, the expression of TMEM16A protein, and the secretion of MUC5AC. T16Ainh-A01, a TMEM16A inhibitor, attenuated these IL-13-induced effects. CONCLUSIONS: The expression of TMEM16A and MUC5AC are increased in CRSwNP, which might be a direct effect of Th2 cytokines present in the sinonasal mucosa in CRSwNP. Down-regulation of TMEM16A expression and MUC5AC secretion in HNPECs by T16Ainh-A01 indicates that TMEM16A might play an important role in mucin secretion in upper airway inflammatory diseases.

The Significance of Interstitial Cells in Neurogastroenterology

Smooth muscle layers of the gastrointestinal tract consist of a heterogeneous population of cells that include enteric neurons, several classes of interstitial cells of mesenchymal origin, a variety of immune cells and smooth muscle cells (SMCs). Over the last number of years the complexity of the interactions between these cell types has begun to emerge. For example, interstitial cells, consisting of both interstitial cells of Cajal (ICC) and platelet-derived growth factor receptor alpha-positive (PDGFRalpha+) cells generate pacemaker activity throughout the gastrointestinal (GI) tract and also transduce enteric motor nerve signals and mechanosensitivity to adjacent SMCs. ICC and PDGFRalpha+ cells are electrically coupled to SMCs possibly via gap junctions forming a multicellular functional syncytium termed the SIP syncytium. Cells that make up the SIP syncytium are highly specialized containing unique receptors, ion channels and intracellular signaling pathways that regulate the excitability of GI muscles. The unique role of these cells in coordinating GI motility is evident by the altered motility patterns in animal models where interstitial cell networks are disrupted. Although considerable advances have been made in recent years on our understanding of the roles of these cells within the SIP syncytium, the full physiological functions of these cells and the consequences of their disruption in GI muscles have not been clearly defined. This review gives a synopsis of the history of interstitial cell discovery and highlights recent advances in structural, molecular expression and functional roles of these cells in the GI tract.

Developmental Role of Anoctamin-1/TMEM16A in Ca2+-Dependent Volume Change in Supporting Cells of the Mouse Cochlea

Mammalian cochlea undergoes morphological and functional changes during the postnatal period, around the hearing onset. Major changes during the initial 2 postnatal weeks of mouse include maturation of sensory hair cells and supporting cells, and acquisition of afferent and efferent innervations. During this period, supporting cells in the greater epithelial ridge (GER) of the cochlea exhibit spontaneous and periodic activities which involves ATP, increase in intracellular Ca2+, and cell volume change. This Ca2+-dependent volume change has been proposed to involve chloride channels or transporters. We found that the spontaneous volume changes were eliminated by anion channel blocker, 100 microM NPPB. Among candidates, expression of Anoctamin-1 (Ano1 or TMEM16A), bestriphin-1 and NKCC1 were investigated in whole-mount cochlea of P9-10 mice. Immunolabeling indicated high level of Ano1 expression in the GER, but not of betrophin-1 or NKCC1. Double-labeling with calretinin and confocal image analysis further elucidated the cellular localization of Ano1 immunoreactivity in supporting cells. It was tested if the Ano1 expression exhibits similar time course to the spontaneous activities in postnatal cochlear supporting cells. Cochlear preparations from P2-3, P5-6, P9-10, P15-16 mice were subjected to immunolabeling. High level of Ano1 immunoreactivity was observed in the GER of P2-3, P5-6, P9-10 cochleae, but not of P15-17 cochleae. Taken together, the localization and time course in Ano1 expression pattern correlates with the spontaneous, periodic volume changes recorded in postnatal cochlear supporting cells. From these results we propose that Ano1 is the pacemaker of spontaneous activities in postnatal cochlea.