Pungent products from garlic activate the sensory ion channel TRPA1

Abstract

Garlic belongs to the Allium family of plants that produce organosulfur compounds, such as allicin and diallyl disulfide (DADS), which account for their pungency and spicy aroma. Many health benefits have been ascribed to Allium extracts, including hypotensive and vasorelaxant activities. However, the molecular mechanisms underlying these effects remain unknown. Intriguingly, allicin and DADS share structural similarities with allyl isothiocyanate, the pungent ingredient in wasabi and other mustard plants that induces pain and inflammation by activating TRPA1, an excitatory ion channel on primary sensory neurons of the pain pathway. Here we show that allicin and DADS excite an allyl isothiocyanate-sensitive subpopulation of sensory neurons and induce vasodilation by activating capsaicin-sensitive perivascular sensory nerve endings. Moreover, allicin and DADS activate the cloned TRPA1 channel when expressed in heterologous systems. These and other results suggest that garlic excites sensory neurons primarily through activation of TRPA1. Thus different plant genera, including Allium and Brassica, have developed evolutionary convergent strategies that target TRPA1 channels on sensory nerve endings to achieve chemical deterrence.

Fig. 1.

Chemical structures of allyl isothiocyanate (mustard oil), and the garlic derivatives, allicin, DADS, DAS, and dipropyl disulfide.

Fig. 2.

Garlic extracts and derivatives excite a subset of primary sensory neurons responses of dissociated rat trigeminal neurons to garlic extracts and derivatives as measured by fura-2 ratiometric imaging. (A) Graph displays intracellular calcium responses to garlic extracts (1:10,000 dilution) as a function of time (Left). Pseudocolor images of fura-2-loaded sensory neurons before and after application of garlic extract [scale bar indicates the intracellular calcium concentration in μM (Right)]. (B) Calcium responses of sensory neurons to 40 μM allicin, followed by subsequent application of 100 μM AITC. (C) Calcium responses of sensory neurons to 200 μM DADS, followed by subsequent application of 100 μM AITC. All graphs represent an average of 100 responsive cells. (D) Allicin/AITC activate a subset of capsaicin-responsive cells. Percentage of rat neurons exhibiting an agonist-evoked rise in intracellular calcium to 100 μM AITC, 100 μM allicin, or 1 μM capsaicin. All AITC-responsive cells were also responsive to both allicin and capsaicin. (E) Allicin evokes the similar calcium responses in neurons isolated from TRPV1^+/+ and TRPV^-/- mice. Percentage of TRPV1^+/+ and TRPV^-/- neurons exhibiting an agonist-evoked rise in intracellular calcium to 100 μM AITC and 100 μM allicin. Rat and mouse sensory neurons were insensitive to DAS (data not shown).

Fig. 3.

Garlic extracts and pungent garlic derivatives activate TRPA1 in transfected HEK293t cells and Xenopus oocytes. Garlic extract (1:10,000 dilution) (A), 40 μM allicin (B), and 200 μM DADS (C) activate calcium influx into human TRPA1-expressing HEK293t cells, as measured by fura-2/AM imaging. Subsequent exposure to 100 μM AITC elicited a slight increase in intracellular calcium in all allicin/DADS-responsive cells. Cells transfected with rat TRPA1, but not pcDNA3 alone, were also responsive to garlic extract, allicin, and DADS (data not shown). Traces represent an average of 100 responsive cells. (D) Representative trace from an oocyte expressing human TRPA1 recorded in ND-96. Allicin (3 μM, 40 s) and AITC (200 μM, 40 s) evoke large, reversible inward currents at -80 mV. No responses were observed in control injected oocytes (data not shown). (E) Dose-response curves for activation of human TRPA1 in oocytes by 20-sec application of allicin (•) or DADS (○). Currents were normalized to maximal response evoked by 100 μM allicin or 1 mM DADS. Half-maximal activates (EC₅₀) occurred at 7.5±0.34 μM and 192 ± 2.6 μM, for allicin and DADS, respectively.

Fig. 4.

Vascular relaxation induced by AITC, garlic extract, allicin, and DADS. (A) Schematic diagram of vascular relaxation mediated by activation of sensory neurons. Inflammatory mediators, such as capsaicin and allicin, activate TRP channels (TRPV1 and TRPA1, respectively), on sensory neurons in blood vessels. TRP channel activation triggers local release of neuropeptides, such as the potent vasodilator CGRP. Concentration-response curves for AITC (B), garlic extracts (C), allicin (D), and DADS (E). Vasorelaxation was recorded in phenylephrine (PhE)-contracted mesenteric arterial segments in the presence of ruthenium red (3 μM, ▵), capsazepine (3 μM, ▾), 8-37 CGRP (3 μM, •), or vehicle (○), or after pretreatment with capsaicin (10 μM for 30 min; ⋄). Dipropyl disulfide (DPDS) (F) and DAS (G) failed to induce relaxation. DADS was applied as positive control. Force of vessel contraction is plotted as a function of time. Dashed line indicates basal tension before addition of drugs. Data are expressed as mean ± SEM (n = 6-8 in B and D, 5-6 in C, 5-8 in E, and 4-5 in F and G).

Fig. 5.

TRPA1 immunoreactivity in sensory neurons and vascular nerve fibers coincides with the presence of CGRP and TRPV1. (A) Immunohistochemical staining of a rat DRG section with anti-TRPA1 Ab. The Ab stains cells of smaller diameter. (B) Immunohistochemical staining of a rat DRG section with anti-TRPV1 Ab. (C) Percentage of TRPA1- and TRPV1-positive cells in rat DRG sections. (D) Localization of TRPA1 in TRPV1-expressing cells. Staining of TRPA1 (Left, red) and TRPV1 (Center, green) in the same rat DRG section. The overlay (Right) indicates localization of TRPA1 in a subset of TRPV1-positive cells. (E) Partial overlap of TRPA1 and CGRP expression in DRG neurons. The same rat DRG section has been stained with TRPA1 (Left, red) and CGRP (Center, green). The overlay (Right) shows partial coexpression of TRPA1 and CGRP in the same neurons (yellow). (F) Expression of TRPA1 in adventitial nerve fibers in rat mesenteric artery. Confocal images of whole mount preparations are shown. TRPA1 immunoreactivity is indicated in green (Left). The same preparation was stained with anti-CGRP Ab (Center, red). The overlay (Right) indicates partial overlap between TRPA1 and CGRP on the same fibers (yellow). [Scale bars: 250 μm (A and B), 10μm (D and E), and 2.5 μm (F).]