TRPM8 Channel Activation Induced by Monoterpenoid Rotundifolone Underlies Mesenteric Artery Relaxation

<div><p>In this study, our aims were to investigate transient receptor potential melastatin-8 channels (TRPM8) involvement in rotundifolone induced relaxation in the mesenteric artery and to increase the understanding of the role of these thermosensitive TRP channels in vascular tissue. Thus, message and protein levels of TRPM8 were measured by semi-quantitative PCR and western blotting in superior mesenteric arteries from 12 week-old Spague-Dawley (SD) rats. Isometric tension recordings evaluated the relaxant response in mesenteric rings were also performed. Additionally, the intracellular Ca²⁺ changes in mesenteric artery myocytes were measured using confocal microscopy. Using PCR and western blotting, both TRPM8 channel mRNA and protein expression was measured in SD rat mesenteric artery. Rotundifolone and menthol induced relaxation in the isolated superior mesenteric artery from SD rats and improved the relaxant response induced by cool temperatures. Also, this monoterpene induced an increase in transient intracellular Ca²⁺. These responses were significantly attenuated by pretreatment with capsazepine or BCTC, both TRPM8 channels blockers. The response induced by rotundifolone was not significantly attenuated by ruthenium red, a non-selective TRP channels blocker, or following capsaicin-mediated desensitization of TRPV1. Our findings suggest that rotundifolone induces relaxation by activating TRPM8 channels in rat superior mesenteric artery, more selectively than menthol, the classic TRPM8 agonist, and TRPM8 channels participates in vasodilatory pathways in isolated rat mesenteric arteries.</p></div

TRPM8 channels are present in rat superior mesenteric artery.

<p>(A) TRPM8 mRNA expression by semi-quantitative PCR in superior mesenteric artery with endothelium removed (n = 3). C, control; P1, 1 μL prostate RT reaction; P2, 2 μL prostate RT reaction, Me, 2 μL mesenteric artery RT reaction. (B) Western blot analysis of TRPM8 protein expression in rat vascular tissue. Molecular weight of TRPM8 was 125 KDa. Bottom panel: β actin used to demonstrate protein loading (at 42kDa).</p

Rotundifolone and the cooling compound menthol enhanced the vasorelaxation effect induced by cold temperatures in mesenteric arteries.

<p>Bar graph summary showing that old temperature-induced vasodilatation is increased in the presence of rotundifolone (A) or menthol (3x10-4 M, 10–3 M and 3x10-3 M; n = 6) (B) in rat superior mesenteric arteries rings without vascular endothelium. Data are the mean ± SEM. The data were examined using one-way ANOVA followed by the Bonferroni post-test. *p < 0.05 and ***p < 0.001 rotundifolone or menthol vs control.</p

Vasorelaxation induced by rotundifolone is unaffected by capsaicin-evoked desensitization of TRPV1 channels.

<p>Relaxants responses induced by rotundifolone (A) and menthol (B) in denuded rat mesenteric arteries rings pre-contracted with phenylephrine in the presence of capsaicin (10⁻⁵ M). Data are the mean ± SEM (n = 6). The data were examined using unpaired Student's t tests and one-way ANOVA followed by the Bonferroni post-test.</p

Vasorelaxation induced by rotundifolone and menthol in rat mesenteric arteries submitted to cold temperatures involves TRPM8 activation.

<p>Bar graph summary shows vasodilatation induced by (A) rotundifolone (3x10^-3 M, n = 6) or (B) menthol (10⁻³ M, n = 6) in rat mesenteric arteries without vascular endothelium submitted to cold temperatures, was abolished in the presence of BCTC (2x10^-6 M). Neither ruthenium red (10⁻⁵ M) nor capsaicin-evoked desensitization (10⁻⁵ M) of TRPV1 channels modified the relaxation induced by menthol or rotundifolone. Data are presented as the mean ± SEM. The data were examined using one-way ANOVA followed by the Bonferroni post-test. *p<0.05, **p<0.01 and ***p<0.001 vs control; #p< 0.05 and ###p<0.001 vs rotundifolone or menthol.</p

Rotundifolone and the cooling compound menthol enhanced the vasorelaxation effect induced by cold temperatures in rat mesenteric arteries by increasing calcium influx.

<p>Bar graph summary showing that the response induced by 3x10-3 M rotundifolone (A) or 10–3 M menthol (B) (n = 6) in the vasodilatation induced by cold temperatures in rat superior mesenteric arteries rings without vascular endothelium was attenuated in the presence of EGTA. Data are the mean ± SEM. The data were examined using one-way ANOVA followed by the Bonferroni post-test. *p < 0.05; **p < 0.01 and ***p < 0.001 rotundifolone or menthol vs control; ###p < 0.001 vs rotundifolone or menthol.</p

Vasorelaxation induced by rotundifolone is affected by SOC-inhibition.

<p>Relaxations responses induced by rotundifolone (A) and menthol (B) in denuded rat mesenteric arteries rings, pre-contracted with phenylephrine in the presence of SKF 96365 (10⁻⁵ M). Data are the mean ± SEM (n = 6). The data were examined using one-way ANOVA followed by the Bonferroni post-test.</p

Influence of ruthenium red and BCTC in the menthol and Rotundifolone-induced dilation.

<p>Tension recording traces of relaxation responses induced by rotundifolone and menthol in denuded rat mesenteric arteries rings pre-contracted with phenylephrine in the absence (A, B) or in the presence of ruthenium red (10⁻⁵ M; C, D) or BCTC (2x10^-6 M; E, F). Collective data are represented as bar graphs (G, H). Data are the mean ± SEM (n = 6). Data were examined using unpaired Student's t tests and one-way ANOVA followed by Bonferroni post-test.</p