Vasorelaxation in rat pulmonary artery induced by the monoterpene thymol: evaluation of the endothelium derived relaxant factors dependence

Thymol and carvacrol are the main compounds found in Lippia mycrophylla essential oil (LM-OE) and have presented some spasmolytic effects. This work was designed to explore a possible vasorelaxant effect of LM-OE and its major monoterpenes constituents on rat pulmonary artery. For that, the organ was in vitro stimulated with phenylephrine (Phe) 3 M and over the tonic contraction the relaxant effect of LM-OE, carvacrol and thymol was observed in both intact and denuded-endothelium. Moreover, atropine, L-NAME, indomethacin, 2,3-O-isopropylidene adenosine, H-89 and Y-27632 were incubated before the relaxant curve of thymol over Phe-tonic contraction. Furthermore, the effects of thymol on KCl 30 or 80 mM and S-(−)-Bay K8644-induced tonic contractions were evaluated, as well as its inhibitory effect on CaCl2-induced cumulative contractions. LM-OE, carvacrol and thymol presented relaxant effect on pulmonary artery, thymol was the most potent and its relaxant potency in intactendothelium preparations was reduced by atropine, L-NAME, indomethacin, 2,3-O-isopropylidene adenosine and H89, despite there was not change on its maximum relaxat effect. Also, the monoterpene relaxed equipotently KCl 30 or 80 mM pre-contracted pulmonary artery, antagonized CaCl2-induced cumulative contractions and relaxed S-(−)-Bay K8644 pre-contracted organ. Ultimately, thymol relaxant potency was not modified by Y-27632. Therefore, thymol acts by endothelium-dependent and independent mechanisms, possibly positively modulating the endothelial cholinergic pathway, prostanoids release and further activation of AC/PKA and also inhibiting Ca influx through CaV. Research, Society and Development, v. 10, n. 4, e29010413971, 2021 (CC BY 4.0) | ISSN 2525-3409 | DOI: http://dx.doi.org/10.33448/rsd-v10i4.13971 2

The pulmonary artery is characterized as a thin, elastic vessel that branches to supply the various arteries of the pulmonary lobe, pulmonary arterioles and alveolar capillaries to promote pulmonary gas exchange (Singhal et al., 1973;Greyson, 2010;Zahid et al., 2020). Comparing with other vascular beds, the pulmonary circulation presents some particularities, being a system of low resistance and presenting different pattern of mediators and receptors, such as endothelin, endothelial nitric oxide synthase and prostanoids, acting itself as a system of own and differentiated functioning of the systemic circulation (Sommer et al., 2020;Wang et al., 2021).
Several pathological processes may result in acute or chronic increases in the afterload in the right ventricle, such as pulmonary arterial hypertension. As excess afterload increases, right heart failure may occur and sudden hemodynamic instability and death may occur (Greyson, 2010;Huang et al., 1996;Sommer et al., 2020;Zahid et al., 2020). Therefore, substances that relax smooth muscle of the pulmonary vascular bed can lead to a decrease in the afterload of the right ventricle and decreased symptoms of diseases, such as pulmonary arterial hypertension (PAH).
Accordingly, the pulmonary hypertension still has a lack of information about its pathophysiology and therapeutics, being responsible for high mortality worldwide (Lau et al., 2017). Therefore, this work was conceived in the hypothesis of a possible relaxant effect of LM-OE, thymol and carvacrol in healthy rat pulmonary artery and then further characterize the action mechanism as part of our research for new therapeutic candidates for the treatment of cardiopulmonary and circulatory disorders.

Animals
Male Wistar rats (Rattus norvegicus), weighing 270-310 g, were obtained from the animal Animal Production Unit of the Instituto de Pesquisa em Fármaco e Medicamentos (IPeFarM) of Federal University of Paraíba (UFPB). The animals had access to water and food (Labina ® ) ad libitum, were kept in rooms at 21 ± 1 °C and were subjected to a 12 h light-dark cycle (light from 6 to 18h00). All animal welfare and experimental procedures were undertaken following the Guidelines for the ethical use of animals in applied ethology studies (Sherwin et al., 2003) and were approved by the Ethics Committee on Animal Use (CEUA) of Federal University of Paraíba (UFPB) on July 16, 2013 by the certification number 0501/13.

Chemicals
The species Lippia microphylla Cham. was collected in June 2010 in the municipality of Serra Branca, state of Paraíba, Brazil, and the botanical material was identified by Maria de Fátima Agra, Ph.D., from Botany sector of UFPB. An exsiccate is deposited at the Herbarium Prof. Lauro Pires Xavier (JPB) of UFPB under identification code AGRA 6118.
The concentrations of thymol and carvacrol in the organ baths were expressed in µg/mL for better compare to the effect produced by the LM-OE.
Each preparation was equilibrated for 60 min, then a stimulus with Phe 3 μM was induced and over the tonic contraction (of amplitude equal to or greater than 0.5 g), acetylcholine (ACh) 1 M was added to verify the endothelium integrity (Furchgott;Zawadzki, 1980). Vascular endothelium was considered intact when the ACh-induced relaxation was > 60% of the Phe-induced contraction. When the ACh-induced relaxation was ≤ 10%, the preparations were considered without functional endothelium (Alencar et al., 2013).
The "n" value represents the number of rats from which the pulmonary artery was isolated for each experimental protocol.

Effect of LM-OE, carvacrol and thymol on isolated rat pulmonary artery
A stimulus was induced with Phe 3 μM and, over the tonic contraction, the test products or forskolin (positive control) were cumulatively added in different preparations. Relaxation was expressed as the percentage of the reverse contraction induced by Phe, and compared based on maximum effect (Emax), as efficacy parameter, and EC50 values (the concentration of a drug that provokes a response halfway between the baseline and maximum response) as potency parameter, obtained by non-linear regression from the concentration-response curves obtained for LM-OE, thymol and carvacrol in organ segments, in both intact and denuded endothelium (Alencar et al., 2013).

2.5
Characterization of the mechanism of action of thymol in rat pulmonary artery 2.5.1 Evaluation of the participation of endothelial muscarinic receptors (M3), nitric oxide synthase (eNOS) and

cyclooxygenase (COX) products
Intact endothelium pulmonary artery rings were pre-incubated with atropine1 nM, a non-specific muscarinic receptor antagonist (Choy;Wong;Kwan, 2002); L-NAME 0.1 mM, a competitive inhibitor of NOS (Rees et al., 1990); or indomethacin 10 μM, inhibitor of COX (Subramani;Leo;Kathirvel, 2010), for 30 min, in different preparations. Then, a stimulus was induced with Phe 3 μM and, over the tonic contraction, thymol was added in cumulative concentrations. The thymol relaxant efficacy and potency were compared in both the absence and presence of the pharmacological tools.

Effect of thymol on the adenylyl cyclase (AC) and protein kinase A (PKA) pathway
Intact endothelium pulmonary artery rings were pre-incubated with IPA10 μM, inhibitor of AC (Rodríguez-Ramos; González-Andrade; Navarrete, 2011); or H-89 0.5 μM, inhibitor of PKA (Zhang et al., 2010), for 30 min, in different preparations. Then, a stimulus was induced with Phe 3 μM and, over the tonic contraction, thymol was added in cumulative concentrations. Thymol relaxant efficacy and potency were compared in both the absence and presence of inhibitors.

Effect of thymol on pre-contracted pulmonary artery with 30 or 80 mM KCl
The pulmonary artery was stimulated with KCl 30 and 80 mM, in different preparations, and over the tonic contraction, thymol was added in cumulative concentrations. Relaxation was expressed as the reverse percentage of the initial contraction induced by KCl, and the efficacy and potency were compared between the two situations (Gurney, 1994).

Effect of thymol on Ca 2+ influx through CaV
After the stabilization period, the Krebs-Henseleit solution was replaced by the depolarizing solution (KCl 80 mM) nominally Ca 2+ -free for a period of 45 min. Following, two similar cumulative-concentrations curves to CaCl2 were obtained, referred as control. Then, thymol was incubated, in different concentrations and preparations, for 30 min, and a third contraction curve to CaCl2 was obtained. The contraction curves of CaCl2 in the presence of thymol was calculated based on the average amplitude of control contractions, and compared accordingly its Emax and EC50 values in the absence and presence of the monoterpene (Karasu-Minareci et al., 2011).

Effect of thymol on CaV1 channels
After the stabilization period, the pulmonary artery was partially depolarized by addition of KCl 10 mM for 10 min, and a stimulus was induced with S-(−)-Bay K8644 0.3 μM, a selective agonist of type 1 CaV. Over the tonic contraction, cumulative concentrations of thymol were added. EC50 values were obtained by non-linear regression from the concentrationresponse curve obtained for thymol and compared with its relaxant effect on Phe 3 μM or KCl 80 mM pre-contracted organ (Jeffery; Wanstall, 2011).

Thymol activity in RhoA/Rho kinase (ROCK) pathway
Intact endothelium pulmonary artery rings were pre-incubated with Y-27632 1 μM, an ROCK inhibitor, for 30 min.
Then, a stimulus with Phe 3 μM was induced and, over the tonic contraction, thymol was added in cumulative concentrations.
The thymol relaxant potency was compared in both the absence and presence of the inhibitor (Gao et al., 2007).

Statistical analysis
The values were expressed as mean and standard error of mean (S.E.M.). Differences between values were statistically compared using Student's t-test, for single comparison, or one-way ANOVA followed by Tukey's post-test, for multiple comparisons, and were considered to differ significantly when p < 0.05. All data were analyzed using GraphPad Prism software version 5.01 (GraphPad Software Inc., San Diego CA, USA).

Evaluation of the participation of endothelial muscarinic receptors (M3), nitric oxide synthase (eNOS) and cyclooxygenase (COX) products
Thymol (0.01-243 µg/mL, n = 5) relaxation curve was shifted to the right (p < 0.05) in the presence of atropine 1 nM, L-NAME 0.1 mM and indomethacin 10 μM, being 4-, 2-and 4-fold, respectively, less potent to relax the rat pulmonary artery pre-contracted by 3 μM Phe in the presence of these inhibitors, as indicated by the EC50 values ( Figure 3, Table 2).

Effect of thymol on the adenylyl cyclase (AC) and protein kinase A (PKA) pathway
Thymol (0.01-243 µg/mL, n = 5) relaxation curve was shifted to the right (p < 0.05) in the presence of IPA 10 μM or H-89 0.5 μM. The thymol relaxant potency was about 3-and 2-fold lower in the presence of these inhibitors, as can be seen by its EC50 values. In addition, thymol efficacy was also reduced by IPA (Fig. 4, Table 2). Data are expressed as the reverse percentage of maximal effect induced by phenylephrine. Symbols and vertical bars represent the mean and S.E.M, respectively (n = 5). One-way ANOVA followed by Tukey's post-test. * p < 0.05 (control vs. IPA + thymol). Source: Authors (2021).

Discussion
In this work, initially was performed a pharmacological analysis of the vasorelaxant effect of the essential oil from Lippia microphylla Cham., as well as its two major compounds, thymol and carvacrol, in rat pulmonary artery. The mechanism of action of thymol, the most potent of those, was investigated as well, showing a dependence of endothelial relaxing factors release, but also evidencing a relaxant effect independent of endothelial factors. The mechanisms explored showed a positive modulation of endothelial muscarinic receptors itself or downstream proteins, and consequent prostanoids and/or NO release, possibly mediated by activation of adenylyl cyclase and protein kinase A pathway in these cells, or directly in smooth muscle.
Moreover, it was showed an inhibitory effect on Ca 2+ influx through CaV1, that may be by direct or indirect mechanisms.
The chemical composition of LM-OE was previously determined (Xavier et al., 2011), presenting as the major constituents thymol (46.5%), carvacrol (31.7%), p-cymene (9%), and γ-terpinene (2.9%). Moreover, some pharmacological activities of LM-OE, thymol and carvacrol have been described, highlighting the vasorelaxant activity of carvacrol and thymol in rat thoracic aorta (Peixoto-Neves et al., 2010), and in guinea pig vena portae (Beer;Lukanov;Sagorchev, 2007). Despite this, the rationality of this study should be emphasized, which sought to investigate the relaxing effect of these compounds on the pulmonary artery, whose physiology differs in several aspects from that of the vessels in the systemic circulation, as discussed below.
Once thymol was more potent in relaxing the pulmonary artery when compared to LM-OE and carvacrol, and that the compound could be modulating endothelial factors, besides acting also in targets directly in smooth muscle, probably in higher concentrations, as it can be analyzed in the relaxant curves (Figure 1 and 2A-D, Table 1), we proceeded investigating which mechanisms would be involved in this effect.
It is well known that the ACh-induced relaxation response in blood vessels is mediated through the activation of endothelial muscarinic receptors and release of mediators, such as NO and PGI2 (Choy;Wong;Kwan, 2002;Norel et al., 1996). Thus, we evaluated the possible modulation of this pathway by thymol. Atropine, a non-selective muscarinic receptor antagonist (Choy;Wong;Kwan, 2002), attenuated the relaxant potency of this compound by about 4-fold, indicating that the monoterpene acts in this signaling pathway to promote the pulmonary artery relaxation (Figure 3, Table 2). However, more investigations are needed to deeply explore this hypothesis, e.g., by molecular docking, in the perspective to find some modulable binding sites by thymol.
The chemical identification of the endothelium-derived relaxant factor, nitric oxide (NO), allowed a better understanding of important physiological processes, especially the cardiovascular system, such as the regulation of vascular tone and platelet function (Dias-Junior;Cau;Tanus-Santos, 2008). In this sense, it was possible to observe that the inhibition of endothelial NO production induced by the false substrate L-NAME to eNOS reduced the relaxant potency of the compound about 2-fold, suggesting that the relaxant effect of this compound on the rat pulmonary artery may involve the release of NO from endothelium ( Figure 3, Table 2). Likewise, several terpenes have been described that have activities in different vascular beds by positively modulating the NO pathway, among them methyleugenol and α-terpineol, in rat aorta (Magalhães et al., 2008), and α-terpineol and citral, in rat superior mesenteric artery (Ribeiro et al., 2010;Moreira, 2013).
Prostanoids (e.g., PGI2 and TXA2) belong to the superfamily of eicosanoids, these being derivatives from arachidonic acid (AA) metabolism. Prostacyclin, or PGI2, is a member of the endogenous prostanoid family. In the pulmonary circulation, prostacyclin is released by the endothelial cells and is a potent endogenous vasodilator and inhibitor of platelet aggregation (Ruan et al., 2010).
According to this observation, we evaluated the possible action of thymol on the release of prostanoids, especially PGI2, to act in the pulmonary artery smooth muscle and induce relaxation. The inhibition of endothelial PGI2 precursor synthesis induced by the addition of indomethacin shifted the thymol concentration-response curve to the right about 4-fold, suggesting that the relaxant effect of this compound on rat pulmonary artery involves the release of this prostanoid ( Figure 3, Table 2). Furthermore, it is possible to observe that atropine and indomethacin were more able to reduce the thymol relaxant potency than L-NAME, indicating the major participation of the M3-PLA2-PGI2 than the NO pathway in the relaxant effect of the monoterpene. This may be explained by the fact that the NO signaling has not the great importance in regulating the pulmonary artery tonus as it has in the systemic circulation (Dias-Junior; Cau; Tanus-Santos, 2008).
The prostacyclin released by the endothelium activate its IP receptors in the smooth muscle cell, which couples to Gs protein and then activates adenylyl cyclase (AC), increasing cytosolic levels of cAMP that has been widely implicated in the control of pulmonary vascular tone (Hall, 2000;Sobolewski et al., 2004). After activation of AC, this enzyme will perform the conversion of ATP into cAMP, which, in turn, is a second messenger that produces the most diverse biological responses, where the activation of PKA is one of its most important targets (Billington;Hall, 2012). PKA is a key enzyme for the maintenance of tonus in the vascular smooth muscle, since it phosphorylates several cellular targets culminating in the decrease in the cytosolic Ca 2+ concentration with consequent relaxation of this muscle (Chen et al., 2011).
In this context the AC inhibitor, IPA, displaced the relaxation curve of thymol to the right, with the reduction of the relaxant potency by approximately 3-fold, in addition to a decrease in its efficacy. Moreover, in the presence of the PKA inhibitor, H-89, the relaxation curve of thymol was attenuated by approximately 2-fold, but with no change in efficacy ( Figure   4, Table 2). These results corroborate the previous results, thus confirming the positive modulation of the IP/AC/PKA pathway in the relaxant effect of thymol in rat pulmonary artery. Furthermore, in view the participation of this pathway in the release of endothelium-derived relaxant factors, such as prostanoids and NO, as previously discussed, we arise the possibility that thymol is activating AC/PKA in both endothelium and smooth muscle to lead pulmonary artery to relax.
Knowing that smooth muscle contractility depends on regulated changes in intracellular Ca 2+ concentration, and that changes in the conduction of potassium ions through the plasma membrane influence the Ca 2+ influx, we evaluated the possibility of thymol be modulating K + or Ca 2+ channels.
For this, the organ was contracted with 30 or 80 mM KCl, wherein in the first condition partial blockage of the K + ions efflux occurs (Clark;Fuchs, 1997), while in the second will occur a greater blockade of K + efflux. It is known that substances that promote relaxation for activation of K + channels are less effective in relaxant in the second condition, since the electrochemical gradient of K + difficulties the efflux of this ion and the smooth muscle relaxation (Gurney, 1994;Somlyo;Somlyo, 2003).
There was no difference in both the efficacy and potency of thymol when the pulmonary artery was contracted with 30 or 80 mM KCl, thus indicating that thymol would not be activating K + channels to exert its relaxant effect but inhibiting the Ca 2+ influx through CaV channels ( Figure 5). Confirming this hypothesis, we observed that the CaCl2 contraction curves were displaced to the right in a non-parallel manner, with a reduction in both the efficacy and potency of the contractile agent ( Figure 6, Table 3). Thus, these results confirm the hypothesis previously suggested that thymol is also exerting its vasodilatory effect in pulmonary artery by inhibiting the Ca 2+ influx through the CaV. Also, it is likely that the inhibition of Ca 2+ influx may be occurring at higher concentrations of the monoterpene, since there was overlap of thymol relaxation curves on both intact and denuded endothelium.
The CaV were initially classified as L, N, P/Q, R and T depending on some electrophysiological and pharmacological properties (Catterall, 2011). In the smooth muscle in general, the CaV1 (L type) are the best characterized and the main responsible for the Ca 2+ entry during contraction (Knot;Brayden;Nelson, 1996). We observed that thymol significantly and concentration-dependently relaxed the pulmonary artery contracted with the dihydropyridine S-(-)-Bay K8644. Additionally, the relaxant potency of the monoterpene was the same when the organ was contracted with Phe ( Figure 7), indicating that thymol acts in the common step of both contractile agents, in this case, the CaV1, to exert its vasorelaxant effect on pulmonary artery.
The main pathway of Ca 2+ sensitization is mediated by the small RhoA monomeric G protein and its Rho kinase target (ROCK). Recent studies have shown that inhibition of ROCK virtually normalizes high pressure and resistance of the pulmonary artery (AP) in rats with pulmonary arterial hypertension (Nagaoka et al., 2004). It is known that this pathway can be activated by GPCR signaling or by Ca 2+ influx through CaV, indicating a common signaling route for either GPCR agonists, such as Phe, or for CaV openers, as KCl and dihydropyridines. Moreover, thymol at 27 μg/mL reduced the Ca 2+ sensitivity in pulmonary artery, as shown by the reduced potency of CaCl2 in contract the organ.
Thus, we imply that thymol might be inhibiting this signaling pathway to exert its vasodilatory effect on pulmonary artery. In the presence of the ROCK blocker, Y-27632, there was no change in the thymol relaxant efficacy and potency, thus suggesting that this pathway is not involved in the relaxant effect of thymol in pulmonary artery ( Figure 8).

Conclusion
The essential oil from Lippia mycrophylla Cham., as well as its two major compounds, thymol and carvacrol, presents vasorelaxant effect in rat pulmonary artery, and the thymol mechanism of action involves the release of NO and relaxing prostanoids from endothelium and/or direct activation of AC/PKA pathway on smooth muscle, as well as the inhibition of Ca 2+ influx through CaV1, specially at higher concentrations. These data provide some new pharmacological evidence for the modulatory effect of thymol on the pulmonary vascular tone and its possible further application as an alternative for the treatment of diseases affecting the pulmonary vasculature, as pulmonary arterial hypertension.
Therefore, in the perspective as a new drug for pulmonary hypertension treatment, further studies in animal model of this disease are needed to explore this hypothesis.