Identifying TRPA1 Agonists by Monitoring Intracellular Calcium Levels in HEK Cells Paige Roe, Erik Johnson, and Wayne Silver Department of Biology, Wake Forest University Methods To maintain cell health, hTRPA1-HEK cells were cultured in a tetracycline-inducible system. In normal culture conditions, this system results in the repression of TRPA1 expression in the cell. Tetracycline binds a repressor allowing for TRPA1 expression to occur more abundantly. Both naive HEK and induced hTRPA1-HEK cells (tetracycline added) were allowed to grow in a black-walled, 96-well plate for a minimum of 24 hours. Intracellular calcium levels were monitored using the Ca +2 -sensitive fluorescent dye FLUO-3AM. Baseline fluorescence of each well was measured. A potential stimulus was then added to the well and fluorescence was measured again. The response elicited in hTRPA1-HEK cells by particular stimuli were normalized to the responses elicited in naïve HEK cells. The experiment was repeated using the TRPA1 inhibitor HC to confirm increases in intracellular calcium were due to TRPA1 activation. In the inhibitor assays, hTRPA1- HEK cell responses to stimulus addition were normalized to vehicle addition instead of naive HEK cells. Introduction Nasal irritation by ammonia, wasabi, onions and other irritants is due to stimulation of trigeminal nerves. These nerves appear to be stimulated by virtually all volatile compounds if presented in high concentrations (Bryant and Silver, 2000). Trigeminal nerve endings contain several types of receptors; however, the specific receptors stimulated by many trigeminal stimuli are unknown. Transient receptor potential (TRPs) proteins are non-specific cation channels with an affinity for calcium, and are associated with the trigeminal nerve. TRPA1, a TRP channel found in a subset of neurons in the trigeminal ganglion, is currently known to be activated by at least ninety different compounds. This study tested thirteen trigeminal stimuli to determine if they activate TRPA1.Three stimuli were previously shown to activate TRPA1 (AITC, CIN, EUG). Two do not activate TRPA1 (CAP, NIC). It was not known whether eight of the stimuli activated TRPA1 (ACA, AMA, ATER, BEN, LIM, DEN, CYC, TOL). Literature Cited Andre E., Campi B, Materazzi S, Trevisani M, Amadesi S, Massi D, Creminon C, Vaksman N, Nassini R, Civelli M, Baraldi PG, Poole DP, Bunnett NW, Geppetti P, and Patacchini R. (2008). Cigarette smoke-induced neurogenic inflammation is mediated by α, β- unsaturated aldehydes and the TRPA1 receptor in rodents. J Clin Invest 118: Bandell M, Story GM, Hwang SW, Wiswanath V, Eid SR, Petrus MJ, Early TJ, and Patapoutian A. (2004). Noxious cold ion channel TRPA1 is activated by pungent compounds and bradykinin." Neuron 41: Bautista DM, Movahed P, Hinman A, Axelsson HE, Sterner O, Hogestatt ED, Julius D, Jordt SE, and Zygmunt PM. (2005). Pungent products from garlic activate the sensory ion channel TRPA1. PNAS 102: Bryant, B. and Silver, W.L. (2000) Chemesthesis: The common chemical sense. In T.E. Finger, W.L. Silver, and D. Restrepo. (editors) Neurobiology of Taste and Smell 2nd Edition. Wiley- Liss, Inc. 479 pp Jordt, S.E., Bautista, D.M., Chuang, H.H., McKemy, D.D., Zygmunt, P.M., Hogestatt, E.D., Meng, I.D. and Julius, D. (2004) Mustard oils and cannabinoids excite sensory nerve fibres through the TRP channel ANKTM1. Nature. 427: Conclusions Based on both the dose response curves and inhibitor assays, the following conclusions were made about the tested stimuli: Stimulates TRPA1 (previously known agonists) ▫Allyl-Isothiocyanate (Jordt et al., 2004) ▫Cinnamaldehyde (Bautista et al., 2004) ▫Eugenol (Bandell et al., 2005) Stimulates TRPA1 (Newly identified agonists) ▫Benzaldehyde ▫Toluene Could stimulate TRPA1 (needs to be confirmed by additional assays) ▫alpha-Terpineol ▫Acetic Acid ▫Amyl Acetate ▫D-Limonene Does not stimulate TRPA1 ▫Capsaicin ▫Cyclohexanone ▫Denatonium benzoate ▫Nicotine (Andre et al., 2008) More experiments, both in this heterologous expression system and in whole animals, are currently underway to further characterize these stimuli. Hopefully, these additional assays will add insight and help classification of the four compounds where current results are not clear. * Acknowledgements Dr. David Julius (UCSF) for the hTRPA1-HEK cells. Dr. Gloria Muday for aid in data analysis Figure 1. Dose response curves for 13 tested stimuli. Relative fluorescence (RFU) for thirteen stimuli at various concentrations. The RFU calculation is shown in the bottom right corner of this section. n=9 for all concentrations. A two- tailed, independent T-test was conducted to determine statistical significance relative to the vehicle (p<0.05). Stars indicate statistical differences from vehicle addition. Error bars = standard error. Examples of where the stimulus occurs are denoted in parentheses. RFU = Final fluorescence value of well containing hTRPA1-HEK cells Final fluorescence value of well containing naive HEK cells Initial fluorescence value of well containing hTRPA1-HEK cells Initial fluorescence value of well containing naive HEK cells Figure 2. Responses to five stimuli are inhibited when using the TRPA1 inhibitor, HC Mean RFU for four stimuli when hTRPA1-HEK cells are either incubated with or without the inhibitor HC The RFU calculation is shown on the bottom side of this section. Note that the normalization calculations are different than those used for dose response curves above. Stimuli shown are 1 mM allyl isothiocyanate, 33 mM benzaldehyde, 33 mM cinnamaldehyde, 33 mM eugenol and 100 mM toluene. Error bars = standard error. A two- tailed, independent T-test was conducted to determine statistical significance between wells incubated with or without inhibitor (p<0.05). Other tested stimuli either showed no increase in fluorescence in hTRPA1-HEK cells without inhibitor or inconclusive results in this assay. RFU = Final fluorescence value of well receiving stimulus Final fluorescence value of well receiving vehicle Initial fluorescence value of well receiving stimulus Initial fluorescence value of well receiving vehicle