The NADPH oxidase inhibitor diphenyleneiodonium activates the human TRPA1 nociceptor

Abstract

Transient receptor potential ankyrin 1 (TRPA1) is a Ca 2ϩ -permeable nonselective cation channel expressed in neuronal and nonneuronal cells and plays an important role in acute and inflammatory pain. Here, we show that an NADPH oxidase (NOX) inhibitor, diphenyleneiodonium (DPI), functions as a TRPA1 activator in human embryonic kidney cells expressing human TRPA1 (HEK-TRPA1) and in human fibroblast-like synoviocytes. Application of DPI at 0.03-10 M induced a Ca 2ϩ response in HEK-TRPA1 cells in a concentration-dependent manner. The Ca 2ϩ response was effectively blocked by a selective TRPA1 antagonist, HC-030031 (HC). In contrast, DPI had no effect on HEK cells expressing TRPV1-V4 or TRPM8. Four other NOX inhibitors, apocynin (APO), VAS2870 (VAS), plumbagin, and 2-acetylphenothiazine, also induced a Ca 2ϩ response in HEK-TRPA1 cells, which was inhibited by pretreatment with HC. In the presence of 5 mM glutathione, the Ca 2ϩ response to DPI was effectively reduced. Moreover, mutation of cysteine 621 in TRPA1 substantially inhibited the DPIinduced Ca 2ϩ response, while it did not inhibit the APO-and VASinduced responses. The channel activity was induced by DPI in excised membrane patches with both outside-out and inside-out configurations. Internal application of neomycin significantly inhibited the DPI-induced inward currents. In inflammatory synoviocytes with TRPA1, DPI evoked a Ca 2ϩ response that was sensitive to HC. In mice, intraplantar injection of DPI caused a pain-related response which was inhibited by preadministration with HC. Taken together, our findings demonstrate that DPI and other NOX inhibitors activate human TRPA1 without mediating NOX. calcium channel; transient receptor potential; NADPH oxidase inhibitors TRANSIENT RECEPTOR POTENTIAL ANKYRIN 1 (TRPA1), a Ca 2ϩ -permeable nonselective cation channel widely expressed in neuronal and nonneuronal cells, is activated by noxious cold, mechanical stimulation, irritant chemicals, and some clinical drugs (1,7,12,17,26,28,30,36). Because transgenic mice lacking TRPA1 have suppressed sensitivity to mechanical stimulation, cold stimuli, and TNF␣-induced mechanical hyperalgesia (5,8,20,23,33), the channel has been proposed to be a nociceptor mediating acute and inflammatory pain (3,23,33). Mutagenesis studies have revealed that the cysteine residues 414, 421, and 621 of human TRPA1 are targeted by physiological and nonphysiological electrophilic compounds that activate the channel (14, 26). In contrast, nonelectrophilic compounds, such as ⌬9-tetrahydrocannabinol, nicotine, and menthol, activate TRPA1 via unknown mechanisms (17,18,38). Because numerous structurally unrelated compounds stimulate TRPA1, greater attention should be given to whether common clinical drugs and experimental pharmacological agents activate the channel.The NADPH oxidases (NOXs), a family of transmembrane proteins comprising seven members (NOX1-NOX5, DUOX1, and DUOX2), function as transmembrane electron transporters that use cytosolic NADPH as electron donor and oxygen as electron a...

An environmental pollutant, 9,10‐phenanthrenequinone, activates human TRPA1 via critical cysteines 621 and 665

“…B) was similar to that evoked by 30 μ mol/L AITC (−2.64 ± 0.28 pA, n = 5) under experimental conditions that were similar to our previous study (Suzuki et al. ). Moreover, the channel activity was reversibly inhibited by the addition of HC (Fig.…”

“…2B), and the unit current amplitude at À50 mV (À2.64 AE 0.19 pA and À2.47 AE 0.15 pA by 1 and 3 lmol/L 9,10-PQ, see also Fig. 2B) was similar to that evoked by 30 lmol/L AITC (À2.64 AE 0.28 pA, n = 5) under experimental conditions that were similar to our previous study (Suzuki et al 2014). Moreover, the channel activity was reversibly inhibited by the addition of HC ( Fig.…”

“…Expression of wild‐type and mutant TRPA1 in HEK cells was confirmed using the following three assays as described previously (Suzuki et al. ): TRPA1 mRNA transcript expression (not shown), TRPA1 protein expression (Fig. A), and TRPA1 channel function (Figs.…”

“…dose-independent variable size and were transient even in the presence of 9,10-PQ. When 2.89 mmol/L TPP, a stabilizer of TRPA1 (Kim and Cavanaugh 2007;Suzuki et al 2014), was included in the pipette solution, application of 9,10-PQ significantly induced inward currents at À50 mV, which were still variable and sometimes transient (lower panel of Fig. 1D and F).…”

Advanced Oxidative Protein Products Cause Pain Hypersensitivity in Rats by Inducing Dorsal Root Ganglion Neurons Apoptosis via NADPH Oxidase 4/c-Jun N-terminal Kinase Pathways

“…Notably, Nox enzymes are distinct from most other ROS sources in that ROS generation is their major function and not a byproduct of other biological reactions. In general, 4 rodent genes of the catalytic subunit Nox (Nox1–Nox4) have been identified, which are expressed in a tissue-specific manner (Suzuki et al, 2014). For example, Nox1 is a source of ROS implicated in the development of thermal hypersensitivity (Ibi et al, 2008).…”