Institute of Biophysics, Medical University of Graz, Graz, Austria.
Br J Pharmacol. 2012 Dec;167(8):1712-22. doi: 10.1111/j.1476-5381.2012.02126.x.
Pyrazole derivatives have recently been suggested as selective blockers of transient receptor potential cation (TRPC) channels but their ability to distinguish between the TRPC and Orai pore complexes is ill-defined. This study was designed to characterize a series of pyrazole derivatives in terms of TRPC/Orai selectivity and to delineate consequences of selective suppression of these pathways for mast cell activation.
Pyrazoles were generated by microwave-assisted synthesis and tested for effects on Ca(2+) entry by Fura-2 imaging and membrane currents by patch-clamp recording. Experiments were performed in HEK293 cells overexpressing TRPC3 and in RBL-2H3 mast cells, which express classical store-operated Ca(2+) entry mediated by Orai channels. The consequences of inhibitory effects on Ca(2+) signalling in RBL-2H3 cells were investigated at the level of both degranulation and nuclear factor of activated T-cells activation.
Pyr3, a previously suggested selective inhibitor of TRPC3, inhibited Orai1- and TRPC3-mediated Ca(2+) entry and currents as well as mast cell activation with similar potency. By contrast, Pyr6 exhibited a 37-fold higher potency to inhibit Orai1-mediated Ca(2+) entry as compared with TRPC3-mediated Ca(2+) entry and potently suppressed mast cell activation. The novel pyrazole Pyr10 displayed substantial selectivity for TRPC3-mediated responses (18-fold) and the selective block of TRPC3 channels by Pyr10 barely affected mast cell activation.
The pyrazole derivatives Pyr6 and Pyr10 are able to distinguish between TRPC and Orai-mediated Ca(2+) entry and may serve as useful tools for the analysis of cellular functions of the underlying Ca(2+) channels.
吡唑衍生物最近被认为是瞬时受体电位阳离子（TRPC）通道的选择性阻断剂，但它们区分 TRPC 和 Orai 孔复合物的能力尚未明确。本研究旨在描述一系列吡唑衍生物在 TRPC/Orai 选择性方面的特征，并阐明这些途径选择性抑制对肥大细胞激活的后果。
通过微波辅助合成生成吡唑，并通过 Fura-2 成像和膜电流记录检测 Ca(2+) 内流的作用。在过表达 TRPC3 的 HEK293 细胞和表达经典由 Orai 通道介导的储存操作 Ca(2+) 内流的 RBL-2H3 肥大细胞中进行实验。在 RBL-2H3 细胞中，研究了抑制作用对 Ca(2+) 信号的后果，既在脱颗粒水平，也在激活 T 细胞核因子水平。
Pyr3，先前被提议为 TRPC3 的选择性抑制剂，对 Orai1 和 TRPC3 介导的 Ca(2+) 内流和电流以及肥大细胞激活具有相似的抑制作用。相比之下，Pyr6 对 Orai1 介导的 Ca(2+) 内流的抑制作用比 TRPC3 介导的 Ca(2+) 内流的抑制作用高 37 倍，并且强烈抑制肥大细胞激活。新型吡唑 Pyr10 对 TRPC3 介导的反应具有显著的选择性（18 倍），并且 Pyr10 对 TRPC3 通道的选择性阻断几乎不影响肥大细胞激活。
吡唑衍生物 Pyr6 和 Pyr10 能够区分 TRPC 和 Orai 介导的 Ca(2+) 内流，可能成为分析潜在 Ca(2+) 通道细胞功能的有用工具。
