Endothelial-dependent relaxation of α-pinene and two metabolites, myrtenol and verbenol, in isolated murine blood vessels.

Epidemiological evidence shows that residential proximity to greenspaces is associated with lower risk of all-cause and cardiovascular mortality; however, the mechanism(s) underlying this link remains unclear. Plants emit biogenic volatile organic compounds such as α-pinene that could elicit beneficial cardiovascular effects. To explore the role of α-pinene more directly, we studied the metabolism and the vascular effects of α-pinene. We found that exposure of mice to α-pinene (1 ppm, 6 h) generated two phase I oxidation metabolites, cis- and trans-verbenol [(1R,2R,5R)-verbenol and (1 R,2S,5R)-verbenol)] and myrtenol [(1S,5R)-(+)-myrtenol] that were identified in urine by GC-MS. Precontracted naïve murine male and female aorta and superior mesenteric artery (SMA) were relaxed robustly (60% tension reduction) by increasing concentrations of α-pinene, myrtenol, and verbenol to 0.3 mM, whereas 1 mM α-pinene was vasotoxic. The SMA was six times more sensitive than the aorta to α-pinene. Both myrtenol and verbenol were equally potent and efficacious as parent α-pinene in male and female SMA. The sensitive portion of the α-pinene-, myrtenol-, and verbenol-induced relaxations in male SMA was mediated by 1) endothelium, 2) eNOS-derived NO, and 3) guanylyl cyclase (GC) activity. Moreover, α-pinene activated the transient receptor potential ankyrin-1 (TRPA1) channel whereas the metabolites did not. Endothelial-derived NO regulates blood flow, blood pressure, and thrombosis, and it is plausible that inhaled (and ingested) α-pinene (or its metabolites) augments NO release to mediate the cardiovascular benefits of exposure to greenness.NEW & NOTEWORTHY A common plant-derived biogenic volatile organic compound, α-pinene, and two of its metabolites, myrtenol and verbenol, stimulate vasorelaxation in murine superior mesenteric artery. Both α-pinene- and its metabolites induce vasorelaxation by activation of the endothelium, nitric oxide, and guanylyl cyclase. α-Pinene also activates the transient receptor potential ankyrin-1. Positive associations between greenness exposure and human cardiovascular health may be a result of the vascular action of α-pinene and its metabolites, a novel consideration.

Acrolein but not its metabolite, 3-Hydroxypropylmercapturic acid (3HPMA), activates vascular transient receptor potential Ankyrin-1 (TRPA1): Physiological to toxicological implications.

Acrolein, an electrophilic α,ß-unsaturated aldehyde, is present in foods and beverages, and is a product of incomplete combustion, and thus, reaches high ppm levels in tobacco smoke and structural fires. Exposure to acrolein is linked with cardiopulmonary toxicity and cardiovascular disease risk. The hypothesis of this study is the direct effects of acrolein in isolated murine blood vessels (aorta and superior mesenteric artery, SMA) are transient receptor potential ankyrin-1 (TRPA1) dependent. Using isometric myography, isolated aorta and SMA were exposed to increasing levels of acrolein. Acrolein inhibited phenylephrine (PE)-induced contractions (approximately 90%) in aorta and SMA of male and female mice in a concentration-dependent (0.01-100 µM) manner. The major metabolite of acrolein, 3-hydroxypropylmercapturic acid (3HPMA), also relaxed PE-precontracted SMA. As the SMA was 20× more sensitive to acrolein than aorta (SMA EC50 0.8 ± 0.2 µM; aorta EC50 > 29.4 ± 4.4 µM), the mechanisms of acrolein-induced relaxation were studied in SMA. The potency of acrolein-induced relaxation was inhibited significantly by: 1) mechanically-impaired endothelium; 2) Nω-Nitro-L-arginine methyl ester hydrochloride (L-NAME); 3) guanylyl cyclase (GC) inhibitor (ODQ); and, 4) a TRPA1 antagonist (A967079). TRPA1 positive immunofluorescence was present in the endothelium. Compared with other known TRPA1 agonists, including allyl isothiocyanate (AITC), cinnamaldehyde, crotonaldehyde, and formaldehyde, acrolein stimulated a more potent TRPA1-dependent relaxation. Acrolein, at high concentration [100 µM], induced tension oscillations (spasms) independent of TRPA1 in precontracted SMA but not in aorta. In conclusion, acrolein is vasorelaxant at low levels (physiological) yet vasotoxic at high levels (toxicological).

Formaldehyde Induces Mesenteric Artery Relaxation via a Sensitive Transient Receptor Potential Ankyrin-1 (TRPA1) and Endothelium-Dependent Mechanism: Potential Role in Postprandial Hyperemia.

Formaldehyde (FA), the smallest aldehyde, is generated endogenously, and is widespread in the environment in foods, beverages and as a gas phase product of incomplete combustion. The main metabolite of FA, formate, was increased significantly in murine urine (∼3×) after overnight feeding. Because feeding increases mesenteric blood flow, we explored the direct effects of FA in isolated murine superior mesenteric artery (SMA). Over the concentration range of 30-1,200 µM, FA strongly and reversibly relaxed contractions of SMA induced by three different agonists: phenylephrine (PE), thromboxane A2 analog (U46,619) and high potassium (60K, 60 mM K+). Formate (to 1.5 mM) induced a modest relaxation. FA (>1,500 µM) irreversibly depressed vascular function in SMA indicating vasotoxicity. The sensitivity (EC50) but not the efficacy (% relaxation) of FA-induced relaxations was dependent on blood vessel type (SMA << aorta) and contractile agonist (PE, EC50= 52 ± 14 µM; U46,619, EC50= 514 ± 129 µM; 60K, EC50= 1,093 ± 87 µM). The most sensitive component of FA vasorelaxation was within physiological levels (30-150 µM) and was inhibited significantly by: (1) mechanically impaired endothelium; (2) Nω-Nitro-L-arginine methyl ester hydrochloride (L-NAME); (3) transient receptor potential ankyrin-1 (TRPA1) antagonist (A967079); (4) guanylyl cyclase (GC) inhibitor (ODQ); and, (5) K+ channel inhibitor (BaCl2). A similar mechanism of SMA vasorelaxation was stimulated by the TRPA1 agonist cinnamaldehyde. Positive TRPA1 immunofluorescent staining and gene-specific sequence were present in SMA but not in aorta. These data indicate FA, but not formate, robustly relaxes SMA via a sensitive TRPA1- and endothelium-dependent mechanism that is absent in aorta. Thus, as FA levels increase with feeding, FA likely contributes to the physiological reflex of post-prandial hyperemia via SMA vasodilatation.

Analysis of Stable Isotope Assisted Metabolomics Data Acquired by High Resolution Mass Spectrometry.

Stable isotope assisted metabolomics (SIAM) uses stable isotope tracers to support studies of biochemical mechanisms. We report a suite of data analysis algorithms for automatic analysis of SIAM data acquired on a high resolution mass spectrometer. To increase the accuracy of isotopologue assignment, metabolites detected in the unlabeled samples were used as reference metabolites to generate possible isotopologue candidates for analysis of peaks detected in the labeled samples. An iterative linear regression model was developed to deconvolute the overlapping isotopic peaks of isotopologues present in a full MS spectrum, where the threshold for the weight factor was determined by a simulation study assuming different levels of Gaussian white noise contamination. A normalization method enabling isotope ratio-based normalization was implemented to study the difference of isotopologue abundance distribution between sample groups. The developed method can analyze SIAM data acquired by direct infusion MS and LC-MS, and can handle metabolite tracers containing different tracer elements. Analysis of SIAM data acquired from mixtures of known compounds showed that the developed algorithms accurately identify metabolites and quantify stable isotope enrichment. Application of SIAM data acquired from a biological study further demonstrated the effectiveness and accuracy of the developed method for analysis of complex samples.