All the cell culture reagents were purchased from Invitrogen (Carlsbad, CA). and show that noxious cold activates both human and rat TRPA1. Further, we have used CHO cells expressing human TRPA1 to screen a small molecule compound library and discovered that ‘trichloro(sulfanyl)ethyl benzamides’ (AMG2504, AMG5445, AMG7160 and AMG9090) act as potent antagonists of Sigma-1 receptor antagonist 2 human TRPA1 activated by AITC and noxious cold. However, trichloro(sulfanyl)ethyl benzamides’ (TCEB compounds) displayed differential pharmacology at rat TRPA1. AMG2504 and AMG7160 Sigma-1 receptor antagonist 2 marginally inhibited rat TRPA1 activation by AITC, whereas AMG5445 and AMG9090 acted as partial agonists. In summary, we conclude that both human and rat TRPA1 channels show comparable AITC and noxious cold activation profiles, but TCEB compounds display species-specific differential pharmacology at TRPA1. Background The herb irritant materials such as mustard oil and wasabi are known to cause rapid intense burning sensation [1-3]. Mustard oil causes pain in humans and pain behavior in rodents by excitation of sensory nerve fibers in part due to neurogenic inflammation through release of neuropeptides such as material P and CGRP and other transmitters from activated nerve endings [3]. The active ingredient in mustard oil, allyl isothiocyanate (AITC) selectively activates a non-selective cation channel, transient receptor potential ankyrin 1 (TRPA1) expressed in the small neurons of the dorsal root and trigeminal ganglia [4,5]. Interestingly, other herb irritant compounds such as allicin from garlic and cinnamaldehyde from cinnamon also activate TRPA1 [5-7]. Since these compounds are capable of forming covalent adducts with thiols, other reactive compounds such as acrolein, iodo-acetamide, N-methylmaleimide, and several others were evaluated and shown to activate TRPA1 through reversible covalent modification of cystenies in the intracellular loops of TRPA1 [8-11]. These studies resulted in the proposal that TRPA1 acts as a sensor for reactive chemicals in the Rabbit Polyclonal to GPR113 body [12,13]. In agreement with this hypothesis, recently, it was reported that 4-hydroxynonenal, an endogenous aldehyde causes pain and neurogenic inflammation through activation of TRPA1 [14]. In addition to reactive Sigma-1 receptor antagonist 2 chemical activators, mechanical stimuli and noxious cold have been shown to activate TRPV1 in recombinant expression systems [15,16]. Reactive chemicals such as AITC did not cause pain behavior in TRPA1 knockout mice, unequivocally confirming that their actions are mediated exclusively by TRPA1 [9,17]. On the other hand, noxious cold effect in TRPA1 knockout mice from two different labs differed [9,17,18], questioning the validity of noxious cold activation of TRPA1. However, recent studies clearly showed that noxious cold indeed activates TRPA1 in calcium imaging experiments as well as in single channel recordings [19]. Formalin model is usually widely used to assess pain and to evaluate analgesic drugs in rodents. Recently, formalin was reported to directly activate TRPA1 and mediate the formalin-induced pain behaviors [20]. Both Phase I and Phase II pain behaviors were attenuated in TRPA1 knockout mice. In addition, TRPA1 expression induced in Sigma-1 receptor antagonist 2 sensory neurons was reported to contribute to cold hyperalgesia after inflammation and nerve injury [21], and antisense knock down of TRPA1 reported to alleviate cold hyperalgesia after spinal nerve ligation in rats [22]. In all, these studies suggest that TRPA1 is usually a target to identify potential novel analgesics. In our attempts to discover the TRPA1 antagonists, we have used CHO cells recombinantly expressing TRPA1 channels to screen a compound library and found that ‘trichloro(sulfanyl)ethyl benzamides’ (TCEB compounds; Fig. ?Fig.1)1) act as potent and selective antagonists of human TRPA1. Here, we report the pharmacological characterization of TCEB compounds effects on chemical ligand and noxious cold activation of human and rat TRPA1. Open in a separate window Physique 1 Chemical structures of compounds used in these studies. Results Characterization of CHO cells expressing human and rat TRPA1 To identify novel TRPA1 antagonists we have established high throughput luminescence readout based functional assays utilizing stable CHO cell lines expressing aequorin cDNA under control of constitutively active promoter and human or rat TRPA1 cDNAs under control of tetracycline inducible promoter. This enabled ad hoc expression of TRPA1 channels for cell based assays without the potential toxic effects of constitutive expression of TRPA1 during freezing and thawing of the cells. To characterize our cell lines we began by testing their functional activity in luminescence based Ca2+ influx assay. Addition of TRPA1 agonist AITC to the cells increased luminescence signal in a concentration-dependent manner (Fig. ?(Fig.2A).2A). EC50 values for AITC activation of human and rat TRPA1 channels were 20 5 and 14 3 M respectively. Based on these results we selected 80 M AITC to be used for activation of TRPA1 in all antagonist experiments. We then examined the ability of a pore blocker, ruthenium red, to inhibit AITC activation (Fig. ?(Fig.2B).2B). Ruthenium red inhibited AITC activation of both human and rat TRPA1 with IC50 values of 29 6 and.