A dietary agonist of transient receptor potential cation channel V3 elicits endothelium-dependent vasodilation

Abstract

The Mediterranean diet may be responsible for lower cardiovascular disease rates in Southern versus Northern European countries. Oregano is used abundantly in Mediterranean cooking, but potential cardiovascular benefits have not been investigated. Carvacrol, present in oregano, activates the transient receptor potential (TRP) cation channels TRPA1 and TRPV3. We hypothesized that chemosensing of this dietary molecule by TRP channels in the endothelium promotes arterial relaxation. TRPA1 and TRPV3 were detected in the endothelium of intact arteries. Carvacrol causes concentration-dependent increases in the intracellular [Ca(2+)] of native cerebral artery endothelial cells and is more potent (EC(50) = 34 microM) than the TRPA1 agonist allyl isothiocyanate (EC(50) = 400 microM) or the TRPV3 agonist eugenol (EC(50) = 2.3 mM). Carvacrol also activates TRPV3-like cation currents in cerebral artery endothelial cells. Carvacrol elicits vasodilation of intact cerebral arteries (EC(50) = 4.1 microM) that is accompanied by smooth muscle hyperpolarization and a decrease in the intracellular [Ca(2+)] of arterial myocytes. Endothelium disruption inhibits carvacrol-induced vasodilation, but block of nitric-oxide synthase and cyclooxygenase activity does not alter the response. Vasodilation in response to carvacrol is inhibited when blockers of Ca(2+)-activated K(+) channels are present in the lumen or when the inwardly rectifying K(+) channel blocker BaCl(2) is present in the superfusion bath. Carvacrol-induced dilation is not diminished by a TRPA1 antagonist but is inhibited by the TRPV blocker ruthenium red. Our findings show that oregano can relax arteries by activating TRPV3 channels in the endothelium. This effect may account for some of the cardioprotective effects of the Mediterranean diet.

Fig. 1.

TRPV3 channels are present in the endothelium of cerebral and cerebellar arteries. A, RT-PCR demonstrating expression of TRPA1, TRPV3, eNOS, and smooth muscle α-actin in RNA isolated from cerebral and cerebellar arteries. The second lane for each PCR reaction is the no-template control. B, confocal images of superior cerebellar arteries that were cut longitudinally and pinned to Silgard blocks with the luminal surface facing up. Top, the autofluorescence of the IEL; middle, immunostaining for either TRPA1 or TRPV3; bottom, a merged image of the two. Images are representative of tissue from at least three animals. Scale bar, 50 μm.

Fig. 2.

Carvacrol elicits robust increases in intracellular [Ca²⁺] in native endothelial cells. A, representative recording of increases in endothelial cell [Ca²⁺] in response to carvacrol (100 μM). B, endothelial cell [Ca²⁺] before and after administration of carvacrol. *, P ≥ 0.05 versus baseline; n = 3 to 15 cell per group. C, fold increase in endothelial intracellular [Ca²⁺] in response to AITC (1000 μM; n = 24), eugenol (3000 μM; n = 15), or carvacrol (100 μM, n = 14). *, P ≤ 0.05 versus AITC and eugenol. D, change in endothelial cell [Ca²⁺] as a function of agonist concentration for AITC, eugenol, and carvacrol. Data are normalized to maximum response. EC₅₀ values: carvacrol, 34 μM; AITC, 400 μM; and eugenol, 2.3 mM.

Fig. 3.

Carvacrol stimulates TRPV3 cation currents in cerebral artery endothelial cells A, representative whole-cell currents recorded from human microvascular cerebral artery endothelial cells during voltage ramps from −100 to +100 mV. Current density is shown for cells under baseline conditions (B) and after first (1), second (2), and third (3) application of carvacrol (100 μM). B, summary data demonstrating sensitization of the carvacrol-induced current (n = 6). C, summary data showing the effects of the TRPA1 antagonist HC-030031 (HC, 3 μM) and the TRPV1–4 blocker RuR (10 μM) on carvacrol-induced currents recorded from cerebral artery endothelial cells; n = 5 for HC-030031, n = 4 for ruthenium red. Current magnitude was normalized to peak carvacrol-induced currents. *, P ≤ 0.05 versus control (C).

Fig. 4.

Carvacrol causes endothelium-dependent dilation of cerebral and cerebellar arteries. A, representative recording of simultaneous changes in luminal diameter and vessel wall [Ca²⁺] (expressed as the 340:380 ratio) in response to increasing concentrations of carvacrol. B, vasodilation normalized to the maximal response as a function of carvacrol concentration; EC₅₀ = 4.1 μM. C, representative recording of carvacrol-induced dilation before and after the endothelium was disrupted by passage of air and distilled water through the lumen. D, effects of endothelial disruption on carvacrol-induced dilation. *, P ≤ 0.05 versus endothelium-intact; n = 5.

Fig. 5.

Carvacrol-induced smooth muscle cell hyperpolarization and vasodilation requires IK_Ca, SK_Ca, and K_IR channel activity. A, representative recordings of smooth muscle membrane potential in isolated arteries pressured to 70 mm Hg under control conditions and in the presence of carvacrol (10 μM) before and after TRAM34 (1 μM) was introduced into the lumen. B, effects of luminal administration of TRAM34 (1 μM) on carvacrol-induced smooth muscle hyperpolarization. *, P ≤ 0.05 versus control; #, P ≤ 0.05 versus carvacrol-treated in the presence of TRAM34, n = 5. C, representative recordings of carvacrol (10 μM)-induced dilation under control conditions, when TRAM34 (1 μM) or apamin (1 μM) is present in the lumen, or when BaCl₂ (30 μM) is present in the superfusion bath. D, effects of luminal administration of TRAM34 (1 μM) on carvacrol-induced dilation. *, P ≤ 0.05 versus control, n = 6. E, effects of luminal administration of apamin (1 μM) on carvacrol-induced dilation. *, P ≤ 0.05 versus control, n = 7. F, effects of BaCl₂ (30 μM) in the superfusion bath on carvacrol-induced dilation. *, P ≤ 0.05 versus control, n = 5.

Fig. 6.

Carvacrol-induced dilation requires TRPV but not TRPA1 activity. A, representative recordings of carvacrol (10 μM)-induced dilation after the TRPA1 blocker HC-030031 (3 μM) or the TRPV blocker RuR (10 μM) was introduced to the lumen. B, effects of luminal administration of HC-030031 (3 μM) on carvacrol-induced dilation, n = 5. There were no significant differences. C, effects of luminal administration of RuR (10 μM) on carvacrol-induced dilation. *, P ≤ 0.05 versus control, n = 5.