Nordihydroguaiaretic acid activates hTRPA1 and modulates behavioral responses to noxious cold in mice.

Nordihydroguaiaretic acid (NDGA) is a major biologically active component of the creosote bush, Larrea tridentate, widely used in unregulated therapies. NDGA is a lipoxygenase inhibitor while a derivative, terameprocol, has been trialed as a chemotherapeutic agent. When investigating fatty acid activation of the human transient receptor potential cation channel subfamily A, member 1 (hTRPA1), we found that NDGA activated the channel. Here we investigate the actions of NDGA and terameprocol at hTRPA1 and the consequences of this for noxious cold sensitivity in mice. hTRPA1 was stably expressed in HEK 293 cells (HEK 293-TRPA1) and channel activity examined by measuring changes in intracellular calcium ([Ca]i) using a fluorescent dye and activation of membrane currents using patch clamp electrophysiology. The effects of local NDGA and terameprocol application on acetone-induced paw flinching were examined in mice. NDGA (pEC50 of 5.4 ± 0.1, maximum change in fluorescence of 385 ± 30%) and terameprocol (pEC50 4.5 ± 0.2, maximum 550 ± 75%) increased [Ca]i in HEK 293-hTRPA1 cells. NDGA also induced an increase in membrane conductance in HEK 293-hTRPA1 cells. These effects were prevented by the TRPA1 antagonist HC-030031, and were dependent on the presence of Cys621, Cys 641, and Cys 665 in hTRPA1. Neither NDGA nor terameprocol alone produced spontaneous pain behaviors in mice after hind paw injection, but both enhanced responses to acetone. NDGA and terameprocol are efficacious activators of TRPA1. NDGA should be used with care to probe lipoxygenase involvement in nociception while TRPA1 activity should be considered when considering use of these drugs in humans.

Ligand determinants of fatty acid activation of the pronociceptive ion channel TRPA1.

Background and purpose. Arachidonic acid (AA) and its derivatives are important modulators of cellular signalling. The transient receptor potential cation channel subfamily A, member 1 (TRPA1) is a cation channel with important functions in mediating cellular responses to noxious stimuli and inflammation. There is limited information about the interactions between AA itself and TRPA1, so we investigated the effects of AA and key ethanolamide and amino acid/neurotransmitter derivatives of AA on hTRPA1. Experimental approach. HEK 293 cells expressing hTRPA1 were studied by measuring changes in intracellular calcium ([Ca] i ) with a fluorescent dye and by standard whole cell patch clamp recordings. Key results. AA (30 µM) increased fluorescence in hTRPA1 expressing cells by 370% (notional EC 50 13 µM). The covalent TRPA1 agonist cinnamaldehyde (300 µM) increased fluorescence by 430% (EC 50, 11 µM). Anandamide (230%) and N-arachidonoyl tyrosine (170%) substantially activated hTRPA1 at 30 µM, however, N-arachidonoyl conjugates of glycine and taurine were less effective while N-acyl conjugates of 5-HT did not affect hTRPA1. Changing the acyl chain length or the number and position of double bonds reduced fatty acid efficacy at hTRPA1. Mutant hTRPA1 (Cys621, Cys641 and Cys665 changed to Ser) could be activated by AA (100 µM, 40% of wild type) but not by cinnamaldehyde (300 µM). Conclusions and implications. AA is a more potent activator of TRPA1 than its ethanolamide or amino acid/neurotransmitter derivatives and acts via a mechanism distinct from that of cinnamaldehyde, further underscoring the likelyhood of multiple pharmacologically exploitable sites on hTRPA1.