We investigated effects of TMEM16A blockers benzbromarone, MONNA, CaCC_inhA01 and Ani9 on isometric contractions in mouse bronchial rings and on intracellular calcium in isolated bronchial myocytes. Separate concentrations of carbachol (0.1-10 μM) were applied for 10 min periods to bronchial rings, producing concentration-dependent contractions that were well maintained throughout each application period. Benzbromarone (1 μM) markedly reduced the contractions with a more pronounced effect on their sustained component (at 10 min) compared to their initial component (at 2 min). Iberiotoxin (0.3 μM) enhanced the contractions, but they were still blocked by benzbromarone. MONNA (3 μM) and CaCC_inhA01 (10 μM) had similar effects to benzbromarone, but were less potent. In contrast, Ani9 (10 μM) had no effect on carbachol-induced contractions. Confocal imaging revealed that benzbromarone (0.3 μM), MONNA (1 μM) and CaCC_inhA01 (10 μM) increased intracellular calcium in isolated myocytes loaded with Fluo-4AM. In contrast, Ani9 (10 μM) had no effect on intracellular calcium. Benzbromarone and MONNA also increased calcium in calcium-free extracellular solution, but failed to do so when intracellular stores were discharged with caffeine (10 mM). Caffeine was unable to cause further discharge of the store when applied in the presence of benzbromarone. Ryanodine (100 μM) blocked the ability of benzbromarone (0.3 μM) to increase calcium, while tetracaine (100 μM) reversibly reduced the rise in calcium induced by benzbromarone. We conclude that benzbromarone and MONNA caused intracellular calcium release, probably by opening ryanodine receptors. Their ability to block carbachol contractions was likely due to this off-target effect.

2023-03-01·British Journal of Pharmacology

Analysis of inhibitors of the anoctamin‐1 chloride channel (transmembrane member 16A, TMEM16A) reveals indirect mechanisms involving alterations in calcium signalling

Article

作者: De Cegli, Rossella ; di Bernardo, Diego ; Guidone, Daniela ; Buccirossi, Martina ; Genovese, Michele ; Galietta, Luis J. V. ; Sarnataro, Sergio

Background and PurposePharmacological inhibitors of TMEM16A (ANO1), a Ca2+‐activated Cl− channel, are important tools of research and possible therapeutic agents acting on smooth muscle, airway epithelia and cancer cells. We tested a panel of TMEM16A inhibitors, including CaCCinh‐A01, niclosamide, MONNA, Ani9 and niflumic acid, to evaluate their possible effect on intracellular Ca2+.Experimental ApproachWe recorded cytosolic Ca2+ increase elicited with UTP, ionomycin or IP3 uncaging.Key ResultsUnexpectedly, we found that all compounds, except for Ani9, markedly decreased intracellular Ca2+ elevation induced by stimuli acting on intracellular Ca2+ stores. These effects were similarly observed in cells with and without TMEM16A expression. We investigated in more detail the mechanism of action of niclosamide and CaCCinh‐A01. Acute addition of niclosamide directly increased intracellular Ca2+, an activity consistent with inhibition of the SERCA pump. In contrast to niclosamide, CaCCinh‐A01 did not elevate intracellular Ca2+, thus implying a different mechanism of action, possibly a block of inositol triphosphate receptors.Conclusions and ImplicationsMost TMEM16A inhibitors are endowed with indirect effects mediated by alteration of intracellular Ca2+ handling, which may in part preclude their use as TMEM16A research tools.

2022-06-01·Pharmacological Research2区 · 医学

A multi-parameter approach to measurement of spontaneous myogenic contractions in human stomach: Utilization to assess potential modulators of myogenic contractions

2区 · 医学

Article

作者: O'Grady, Greg ; Makwana, Raj ; Straface, Marilisa ; Andrews, Paul Lr ; Loy, John ; Cai, Weigang ; Palmer, Alexandra ; Adebibe, Miriam ; Gharibans, Armen ; Crawley, Ellie ; Kouassi, Marie-Ange ; Sanger, Gareth J

Electrical slow waves, generated by interstitial cells of Cajal (ICC), cause spontaneous contractions of human stomach. Software was developed to measure muscle tone and eleven different parameters defining these contractions in human stomach, displaying data as radar plots. A pilot study assessed the effects of potential modulators, selected from among compounds known to influence ICC activity; n = 4-7 each concentration tested/compound. Human distal stomach (corpus-antrum) muscle strips were suspended in tissue baths for measuring myogenic (non-neuronal) contractions in the presence of tetrodotoxin (10^-6 M). Initial characterization: Contractions (amplitude 4 ± 0.4mN, frequency 3 ± 0.1 min^-1, n = 49) were unchanged by ꭃ-conotoxin GVIA (10^-7 M) or indomethacin (10^-6 M) but abolished by nifedipine (10^-4 M). Carbachol (10^-7 M) increased contraction rate and amplitude; 10^-6-10^-5 M increased tone and caused large, irregular contractions. [Ca²⁺]_imodulators: Ryanodine (10^-5-10^-4 M) increased muscle tone accompanied by inhibition of myogenic contractions. Xestospongin-C (10^-6 M; IP₃ channel inhibitor) had no effects. SERCA pump inhibitors, 2-APB and cycloplazonic acid (10^-5-10^-4 M) increased tone and myogenic contraction amplitude before abolishing contractions; thapsigargin was weakly active. CaCC blockers: MONNA and CaCCinh-A01 had little-or-no effects on tone but reduced myogenic contractions; MONNA (10^-4 M) was more effective, reducing amplitude (77.8 ± 15.2%) and frequency. Ca_V3.1/3.2/3.3 channel block: Mibefradil reduced tone and myogenic contraction amplitude (pIC₅₀ 4.8 ± 0.9). Inward-rectifying K⁺-channel inhibitor: E-4031 (10^-4 M) increased contraction duration (17.4 ± 5.8%). Conclusions: (1) Measurement of multiple parameters of myogenic contractions identified subtle differences between compounds, (2) only E-4031 and CaCC blockers influenced myogenic contractions, not muscle tone, (3) studies are needed with compounds with known and/or improved selectivity/potency for human targets affecting ICC functions.

100 项与 MONNA 相关的药物交易

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