Design of Novel TRPA1 Agonists Based on Structure of Natural Vasodilator Carvacrol-In Vitro and In Silico Studies.

Considering the escalating global prevalence and the huge therapeutic demand for the treatment of hypertension, there is a persistent need to identify novel target sites for vasodilator action. This study aimed to investigate the role of TRPA1 channels in carvacrol-induced vasodilation and to design novel compounds based on carvacrol structure with improved activities. In an isolated tissue bath experiment, it was shown that 1 µM of the selective TRPA1 antagonist A967079 significantly (p < 0.001) reduced vasodilation induced by 3 mM of carvacrol. A reliable 3D-QSAR model with good statistical parameters was created (R2 = 0.83; Q2 = 0.59 and Rpred2 = 0.84) using 29 TRPA1 agonists. Obtained results from this model were used for the design of novel TRPA1 activators, and to predict their activity against TRPA1. Predicted pEC50 activities of these molecules range between 4.996 to 5.235 compared to experimental pEC50 of 4.77 for carvacrol. Molecular docking studies showed that designed molecules interact with similar amino acid residues of the TRPA1 channel as carvacrol, with eight compounds showing lower binding energies. In conclusion, carvacrol-induced vasodilation is partly mediated by the activation of TRPA1 channels. Combining different in silico approaches pointed out that the molecule D27 (2-[2-(hydroxymethyl)-4-methylphenyl]acetamide) is the best candidate for further synthesis and experimental evaluation in in vitro conditions.

Effect of folic acid on isoprenaline-induced myocardial injury in rats.

Background: Isoprenaline (ISO), a synthetic catecholamine and a ß-adrenoceptor agonist, is widely used to develop an experimental model of myocardial injury (MI) in rats. The leading hypothesis for ISO-induced MI in rats is that it results from catecholamine overstimulation, oxidative stress, inflammatory responses, and development of cardiomyopathy during ISO administration. Folic acid (FA) reduces oxidative stress, improves endothelial function and prevents apoptosis, thereby contributing to cardiovascular protection. This study aimed to investigate the potentially protective effect of FA pretreatment on ISO-induced MI in rats. Methods: For 7 days, adult male Wistar albino rats were pretreated with 5 mg/kg/day of FA. On the sixth and seventh days, MI in rats was induced by administering 85 mg/kg/day of ISO. Prooxidant markers in plasma samples, antioxidant capacity in erythrocyte lysates, cardiac damage markers, lipid profile, electrocardiography (ECG) and histopathological analysis were evaluated. Results: FA pretreatment significantly alleviated changes induced by ISO; it decreased the homocysteine and high-sensitivity troponin I level. FA moderately decreased the reactive oxygen species (ROS) levels (superoxide anion radical, hydrogen peroxide and thiobarbituric acid reactive substances) and improved the antioxidant activities of catalase, superoxide dismutase and reduced glutathione. ISO reduced the nitrite level and FA significantly alleviated this change. Conclusion: It can be concluded that FA, as a mild antioxidant, could be an appropriate cardioprotective substance in the rat model of ISO-induced MI.

Vascular effects of midazolam, flumazenil, and a novel imidazobenzodiazepine MP-III-058 on isolated rat aorta.

Hypotensive influences of benzodiazepines and other GABAA receptor ligands, recognized in clinical practice, seem to stem from the existence of "vascular" GABAA receptors in peripheral blood vessels, besides any mechanisms in the central and peripheral nervous systems. We aimed to further elucidate the vasodilatatory effects of ligands acting through GABAA receptors. Using immunohistochemistry, the rat aortic smooth muscle layer was found to express GABAA γ2 and α1-5 subunit proteins. To confirm the role of "vascular" GABAA receptors, we investigated the vascular effects of standard benzodiazepines, midazolam, and flumazenil, as well as the novel compound MP-III-058. Using two-electrode voltage clamp electrophysiology and radioligand binding assays, MP-III-058 was found to have modest binding but substantial functional selectivity for α5ß3γ2 over other αxß3γ2 GABAA receptors. Tissue bath assays revealed comparable vasodilatory effects of MP-III-058 and midazolam, both of which at 100 µmol/L concentrations had efficacy similar to prazosin. Flumazenil exhibited weak vasoactivity per se, but significantly prevented the relaxant effects of midazolam and MP-III-058. These studies indicate the existence of functional GABAA receptors in the rat aorta, where ligands exert vasodilatory effects by positive modulation of the benzodiazepine binding site, suggesting the potential for further quest for leads with optimized pharmacokinetic properties as prospective adjuvant vasodilators.

Vasorelaxant effect of monoterpene carvacrol on isolated human umbilical artery.

Carvacrol (CRV) is the main compound of essential oils extracted primarily from Thymus and Origanum species. Its various biological activities were confirmed: antioxidant, anti-inflammatory, antibacterial, antifungal, anti-tumour, antinematodal, and vasorelaxant action. Although vasodilation mediated by CRV was previously described, the exact mechanism of its action has not yet been established. Hence, the aim of this study was to investigate CRV vasoactivity on human umbilical arteries (HUA) and the different pathways involved in its mechanism of action using the tissue bath methodology. CRV caused a significant decrease in vascular tension of 5-HT-pre-contracted umbilical arteries, with EC50 of 442.13 ± 33.8 µmol/L (mean ± standard error of the mean-SEM). At 300 µmol/L, CRV shifted downward the 5-HT concentration-response curve with a statistical significance of p < 0.001 obtained for the four highest concentrations. At a concentration of 1 mmol/L, CRV completely abolished BaCl2-induced contraction in Ca2+-free Krebs-Ringer bicarbonate solution and the BAY K 8644-induced contraction in Krebs-Ringer bicarbonate solution (p < 0.001). Isopentenyl pyrophosphate, the antagonist of TRPV3 channel, was able to decrease the efficacy of CRV (p < 0.001). The blocking of L-type Ca2+ channels on smooth muscle cells is the most probable mechanism of CRV-induced vasorelaxation. However, the role of TRPV3 channels in CRV-induced vasodilation of HUA cannot be excluded either.

Vasodilatory effects of a variety of positive allosteric modulators of GABAA receptors on rat thoracic aorta.

Different subtypes of GABAA (gamma-aminobutyric acid A) receptors, through their specific regional and cellular localization, are involved in the manifestation of various functions, both at the central and peripheral levels. We hypothesized that various non-neuronal GABAA receptors are expressed on blood vessels, through which positive allosteric modulators of GABAA receptors exhibit vasodilatory effects. This study involved two parts: one to determine the presence of α1-6 subunit GABAA receptor mRNAs in the rat thoracic aorta, and the other to determine the vasoactivity of the various selective and non-selective positive GABAA receptor modulators: zolpidem (α1-selective), XHe-III-074 (α4-selective), MP-III-022 (α5-selective), DK-I-56-1 (α6-selective), SH-I-048A and diazepam (non-selective). Reverse transcription-polymerase chain reaction (RT-PCR) analysis data demonstrated for the first time the expression of α1, α2, α3, α4 and α5 subunits in the rat thoracic aorta tissue. Tissue bath assays on isolated rat aortic rings revealed significant vasodilatory effects of diazepam, SH-I-048A, XHe-III-074, MP-III-022 and DK-I-56-1, all in terms of achieved relaxations (over 50% of relative tension decrease), as well as in terms of preventive effects on phenylephrine (PE) contraction. Diazepam was the most efficient ligand in the present study, while zolpidem showed the weakest vascular effects. In addition, diazepam-induced relaxations in the presence of antagonists PK11195 or bicuculline were significantly reduced (P < 0.001 and P < 0.05, respectively) at lower concentrations of diazepam (10-7 M and 3 × 10-7 M). The present work suggests that the observed vasoactivity is due to modulation of "vascular" GABAA receptors, which after further detailed research may provide a therapeutic target.