The Vasorelaxant Effect of p-Cymene in Rat Aorta Involves Potassium Channels

The monoterpenes are the main constituents of most essential oils and p-cymene is a monoterpene commonly found in various species of aromatic herbs, which has been reported for anti-inflammatory, antinociceptive, and antimicrobial activities. However, there is no report concerning its pharmacological activity on the vascular smooth muscle. The aim of current work was to investigate the effects of p-cymene in isolated rat aorta and also study its mechanism of action. In this work, we show that p-cymene has a relaxant effect, in a dose-dependent way, on the vascular smooth muscle, regardless of the presence of the endothelium. Using a nonselective potassium channel blocker, the CsCl, the relaxant effect of p-cymene was attenuated. In the presence of more selective potassium channels blockers, such as TEA or 4-AP, no change in the relaxant effect of p-cymene was evidenced, indicating that BKCa and KV channels are not involved in that relaxant effect. However, in the presence of glibenclamide or BaCl2, KATP and Kir blockers, respectively, the relaxant effect of p-cymene was attenuated. The data presented indicate that p-cymene has a relaxing effect on rat aorta, regardless of the endothelium, but with the participation of the KATP and Kir channels.


Introduction
Essential oils are volatile, have strong smell, present a complex composition, and are formed by the secondary metabolism of aromatic plants [1]. Due to their characteristic odor, essential oils are widely used in fragrances, cosmetics, and sanitary products. Besides, they are used in holistic medicine therapies such as aromatherapy. Furthermore, due to the complexity of their constituents, these oils are used in medicines, in dentistry, in pest control, and in canned food preservatives [2].
Some studies indicate that many of these essential oils have biological activities, such as cardiovascular effects [3], inhibition of the oxidation of LDL cholesterol, and blood pressure lowering effect [4,5].
It is known that the major chemical components of the essential oils are monoterpenes and sesquiterpenes [6]. A monoterpene that is commonly found in various species of herbs is the p-cymene. Indeed, this monoterpene is present in the volatile oils of over 100 plant species and occurs naturally in more than 200 kinds of foods, such as orange juice, grapefruit, tangerine fruit, carrots, raspberries, butter, nutmeg, oregano, and most spices [7].
It has been demonstrated that the p-cymene has antiinflammatory, analgesic, and antinociceptive activities [8][9][10]; immunomodulatory effect [11]; antibacterial activity 2 The Scientific World Journal against Escherichia coli [12]; hypotension and bradycardia effects in urethane anaesthetized rats [13]; and antioxidant activity and relaxant effect in rat aorta [14]. Although there are reports on the relaxing activity in rat aorta, little is known about the mechanism of action of this monoterpene regarding its relaxing effect.
Concerning the aforementioned, the objective of this work was to access the role of endothelium and potassium channels in the relaxing effect of p-cymene in isolated rat aorta.

Animals and Ethics Considerations.
In all experiments, male Wistar rats were used, weighing 250-350 g, fed on standard rat chow with free access to food (PURINA, Brazil) and tap water ad libitum. The animals were maintained in a 12 h light-dark cycle (lights on: 06:00-18:00 h) under controlled temperature conditions (21 ± 1 ∘ C). All experimental procedures were performed in accordance with the guidelines proofed by the Ethics Committee on Animal Experimentation of UNIVASF (CEUA/UNIVASF number 0002/131211) and follow the recommendations of the National Council for Control of Animal Experimentation of Brazil (CONCEA).

Preparation and Protocols of Isolated Rat Aorta.
In order to record the isotonic contractions, isolated rat aorta rings (4-5 mm) were suspended in organ chambers (10 mL) of an isolated organ bath system, model EFF 321 from Insight Instruments (São Paulo, SP, Brazil), and attached to isometric transducers, model TRO015 from Panlab S.L.U. (Barcelona, Spain), coupled to a bridge system amplifier 321 from Insight Instruments (São Paulo, SP, Brazil) and connected to a computer.
The rats were euthanized following the principles of Laboratory Animal Care in accordance with the guidelines of bioethics committee. The thoracic aorta was removed from the animals, immediately immersed in Krebs solution bubbled with carbogen mixture (95% O 2 plus 5% CO 2 ), and cleaned up from fat and connective tissues. The aortas were cut into 5 mm long rings and transferred to an organ chamber with Krebs solution at 37 ∘ C. The aortic rings were allowed to stabilize for 60 min at preload tension of 1 g (baseline). During the resting time, the Krebs solution was changed every 15 min to avoid accumulation of metabolites. After that elapsed time, the aortic rings were contracted by the addition of PE (1 M) and the isometric tension was recorded. When a stable contraction was attained (plateau reached in 15-20 min), ACh (1 M) was added to the organ bath to confirm the presence or absence of functional endothelium, as described earlier by Furchgott and Zawadzki [15]. Aortic rings without endothelium were obtained by means of the mechanical removal of the endothelial layer. After 30 min, a second PE-induced stable contraction was induced and cumulative-concentration curves by p-cymene were performed in absence or in the presence of functional endothelium, CsCl (5 mM), TEA (1 mM or 10 mM), 4-AP (3 mM), BaCl 2 (0.1 mM), or glibenclamide (3 M) for the assessment of the participation of potassium channels in the relaxant effect of p-cymene. In experiments involving the use of potassium channel blockers, the absence of the endothelium was verified by the lack of relaxation induced by Ach. All potassium channels blockers were added 30 min before a second contraction induced by phenylephrine.

Statistics.
All the data are expressed as the mean ± S.E.M. and refers to the number of animals used in each set of experiments. The EC 50 values (half-maximal effective concentration) were calculated through nonlinear regression of the concentration-response curves of p-cymene in each protocol. The max value refers to a maximal effect induced by a substance in percentage, being equal to 0% in maximum contraction tension elicited by phenylephrine and 100% when initial preload tension level was reached (baseline). Differences between the means were statistically compared using nonpaired Student's -test, where such differences were considered significant when values were less than 0.05. Statistical analyses were performed using the software Graph-Pad Prism © 5.1 (GraphPad Software Inc., San Diego, USA).

Effect of p-Cymene in Aortic Rings with and without
Endothelium. The p-cymene relaxed the isolated rat aorta rings, precontracted by phenylephrine, in a concentrationdependent manner, in the presence and absence of the endothelium (Figure 2

Effect of p-Cymene in the Presence or Absence of CsCl.
In the presence of CsCl, p-cymene also relaxed the aortic rings without endothelium in a concentration-dependent manner. However, the dose-response curve was shifted to the right

Effect of p-Cymene in the Presence or Absence of BaCl 2
or Glibenclamide. The p-cymene relaxed the aortic rings without endothelium, in a concentration-dependent manner, in both the absence (control) and the presence of BaCl 2 . However, in the presence of BaCl 2 , the dose-response curve was significantly shifted to the right with a change in max when compared to the control curve ( Figure 6). In the same way, p-cymene relaxed the aortic rings without endothelium in both the absence (control) and the presence of glibenclamide, but in the presence of that

Discussion
In this work, we demonstrated that p-cymene has a dosedependent vasorelaxant effect, which was independent of the vascular endothelium with a marked involvement of potassium channels, especially the K IR and K ATP channels.
Smooth muscle contraction is ultimately determined by phosphorylation of the 20 kDa myosin light chain subunits (MLC 20 ), which can occur through the Ca 2+ /calmodulindependent actions of myosin light chain kinase (MLCK) or through the Ca 2+ -independent actions of many additional kinases. The main event that determines the activation of that Ca 2+ /calmodulin pathway with a subsequent activation of MLCK, which leads to the contraction of the smooth muscle, is increased intracellular Ca 2+ [16]. Two classical ways to increase the Ca 2+ driving to muscle contraction are pharmacomechanical and electromechanical coupling mechanisms. The first mechanism involves Ca 2+ mobilization of sarcoplasmic reticulum via G-protein-coupled receptors as well as Ca 2+ entry by Ca V1.2 calcium channels; the second mechanism involves change in membrane potential and activation of Ca V1.2 channels, with a subsequent Ca 2+ influx from extracellular medium and increase in its intracellular levels [17].
On the other hand, the mechanisms that lead to relaxation may involve multiple signaling pathways. In the vascular smooth muscle cells, both endothelium-derived factors and the membrane potential are important in the regulation of the vascular tone [18]. Thus, a possible role for the endothelium The Scientific World Journal 5 and K + channels in the relaxation induced by p-cymene was investigated in this work.
The endothelium produces and releases at least three important relaxant factors: nitric oxide (NO), prostacyclin (PGI 2 ), and the endothelium-derived hyperpolarizing factor (EDHF) [15,19,20]. Currently, it is generally accepted that endothelium-derived factors, such as NO, have an important role in modulating relaxation in the rat aorta. However, our results showed that p-cymene relaxes aortic rings in an endothelium-independent manner, with similar max and EC 50 values in the presence or the absence of endothelium. That suggests that the effect of p-cymene does not depend on the release of these endothelium-derived relaxant factors.
Since the Ca V1.2 is the main type of voltage-opened calcium channel present in smooth muscle cells [21], which is important for the development and maintenance of contraction in smooth muscle, it is known that the opening and closing of Ca V1.2 are closely related variations in membrane potential. Therefore, potassium channels play an important role in controlling this membrane potential and consequently control the function of Ca V1.2 channels [22]. Once determined that the relaxing effect of p-cymene is not dependent on the presence of the vascular endothelium, denoting a likely direct effect on the smooth muscle, investigation of the role of potassium channels in that effect was carried out in the absence of endothelium. In this work it was shown that, in the presence of CsCl, a well-known blocker of potassium channels [23], the vasorelaxant effect of p-cymene was attenuated, indicating that some types of potassium channels may be involved in its relaxant effect.
In this work, it was demonstrated that p-cymene relaxed aortic rings in the presence of TEA 1 or 10 mM, which in the lowest concentration is a selective BK Ca blocker and in higher concentration is a nonselective blocker for some types of potassium channels, as well as in the presence of K V blocker, the 4-AP, without significant differences in its max and EC 50 values. These data indicate that neither BK Ca nor K V would be involved in the relaxing effect of p-cymene. However, in the presence of both Ba 2+ and glibenclamide, the vasorelaxant response to p-cymene was significantly reduced, with changes in the max and EC 50 , indicating that K ir and K ATP could be involved in its relaxing effect on aortic rings. p-Cymene is a known monoterpene, which has some applications in the odorant industry and also many biological effects, such as the modulation of MAPK and NF-kappa B activation [11], anti-inflammatory and antinociceptive effects [9], and antimicrobial effects in food [26]. Here we demonstrate, for the first time in literature, the p-cymene effect on the vascular smooth muscle, trying to determine its relaxing mechanism of action. Despite our best efforts, further studies are necessary for a better comprehension of its effect on the smooth muscle.