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

Abstract

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 [subscript] BK Ca [/subscript] and [subscript] K V [/subscript] channels are not involved in that relaxant effect. However, in the presence of glibenclamide or BaCl2, [subscript] K ATP [/subscript] and [subscript] K ir [/subscript] 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 [subscript] K ATP [/subscript] and [subscript] K ir [/subscript] channels.

[ProQuest: [...] denotes non US-ASCII text; see PDF]

Martapolyana T. M. Silva 1 and Fernanda P. R. A. Ribeiro 1 and Maria Alice M. B. Medeiros 1 and Pedrita A. Sampaio 2 and Yonara M. S. Silva 2 and Morganna T. A. Silva 2 and Jullyana S. S. Quintans 3 and Lucindo J. Quintans-Júnior 3 and Luciano A. A. Ribeiro 4

Academic Editor:Xavier Norel

1, Programa de Pos-Graduação em Recursos Naturais do Semiarido (PGRNSA), Universidade Federal do Vale do São Francisco (UNIVASF), Campus Centro, 56304-205 Petrolina, PE, Brazil
2, Curso de Farmacia, Universidade Federal do Vale do São Francisco (UNIVASF), Campus Centro, 56304-205 Petrolina, PE, Brazil
3, Centro de Ciências Biologicas e da Saúde, Departamento de Fisiologia, Universidade Federal de Sergipe, Campus de São Cristovão, 49100-001 Aracaju, SE, Brazil
4, Colegiado de Ciências Farmacêuticas (CFARM), Universidade Federal do Vale do São Francisco (UNIVASF), Campus Centro, 56304-205 Petrolina, PE, Brazil

Received 25 July 2014; Revised 15 December 2014; Accepted 29 December 2014; 15 January 2015

This is an open access article distributed under the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.

1. 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 anti-inflammatory, analgesic, and antinociceptive activities [8-10]; immunomodulatory effect [11]; antibacterial activity 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.

2. Materials and Methods

2.1. 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 ( [figure omitted; refer to PDF] °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).

2.2. Chemicals and Krebs Solution

For chemicals, acetylcholine chloride (ACh), cesium chloride (CsCl), p -cymene (Figure 1), glibenclamide, dimethyl sulphoxide (DMSO), phenylephrine hydrochloride (PE), tetraethylammonium chloride (TEA), 4-aminopyridine (4-AP), and barium chloride (BaCl₂ ) were purchased from Sigma-Aldrich (St. Louis, MO, USA). All salts used in Krebs solution were purchased from Vetec QuimicaFina Ltda. (Duque de Caxias, RJ, Brazil). p -Cymene was dissolved in DMSO (100%) and diluted in distilled water according to the requirements of each experiment.

Figure 1: Chemical structure of p -cymene.

[figure omitted; refer to PDF]

For all experiments with isolated rat aorta, Krebs salt solution was used, with the composition and concentration (in mM) as follows: NaCl (118), KCl (4.6), MgSO₄ [figure omitted; refer to PDF] 7H₂ O (5.7), NaH₂ PO₄ [figure omitted; refer to PDF] H₂ O (1.1), CaCl₂ (2.5), NaHCO₃ (25), and glucose (11) with pH adjusted to 7.4 with 1 N HCl solution.

2.3. 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% O₂ plus 5% CO₂ ), 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 [figure omitted; refer to PDF] M) and the isometric tension was recorded. When a stable contraction was attained (plateau reached in 15-20 min), ACh (1 [figure omitted; refer to PDF] 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), BaCl₂ (0.1 mM), or glibenclamide (3 [figure omitted; refer to PDF] 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.

2.4. Statistics

All the data are expressed as the mean ± S.E.M. and [figure omitted; refer to PDF] refers to the number of animals used in each set of experiments. The EC₅₀ values (half-maximal effective concentration) were calculated through nonlinear regression of the concentration-response curves of p -cymene in each protocol. The [figure omitted; refer to PDF] 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 [figure omitted; refer to PDF] -test, where such differences were considered significant when [figure omitted; refer to PDF] values were less than 0.05. Statistical analyses were performed using the software Graph-Pad [figure omitted; refer to PDF] 5.1 (GraphPad Software Inc., San Diego, USA).

3. Results

3.1. 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 concentration-dependent manner, in the presence and absence of the endothelium (Figure 2), reaching an [figure omitted; refer to PDF] value of 100% of relaxation at the concentration of 10^-3 M (Figure 2). For rings with endothelium, the p -cymene presented an EC₅₀ value of [figure omitted; refer to PDF] M and for rings without endothelium, an EC₅₀ value of [figure omitted; refer to PDF] M (Figure 3). There was no statistical difference between values of [figure omitted; refer to PDF] and EC₅₀ presented by p -cymene in the presence and absence of the endothelium. The contractile effect of phenylephrine was completely reestablished after 30 min of washout of the organ with a fresh new Krebs solution, to remove the p -cymene of the organ chambers (data not shown). Concerning a possible direct effect of the solvent, the addition of DMSO (1% v/v), which was the maximum concentration of the solvent in the organ bath in our experiments, caused no significant effect on the tonus of the aortic rings constricted (data not shown).

Representative original record of the effect of p -cymene on the isolated rat aortic rings contracted by phenylephrine with (a) and without (b) endothelium. The arrows represent the time-course of the p -cymene administration (10^-6 to 10^-3 M).

(a) [figure omitted; refer to PDF]

(b) [figure omitted; refer to PDF]

Figure 3: Relaxant effect of p -cymene on the isolated rat aortic rings contracted by phenylephrine with ([...]) and without ([white square]) endothelium.

[figure omitted; refer to PDF]

3.2. 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 when compared to the control curve (Figure 4). Nevertheless, no change was observed in [figure omitted; refer to PDF] , which was reached at a concentration of 10^-3 M, similarly to the control. The EC₅₀ values were [figure omitted; refer to PDF] M and [figure omitted; refer to PDF] M for the control condition and in the presence of CsCl, respectively, showing statistical difference between them.

Figure 4: Relaxant effect of p -cymene on the isolated rat aortic rings without endothelium, contracted by phenylephrine, in the absence ([white square]) and presence of CsCl 5 mM ([...]). ^* [figure omitted; refer to PDF] (unpaired [figure omitted; refer to PDF] -test: absence [figure omitted; refer to PDF] presence of CsCl).

[figure omitted; refer to PDF]

3.3. Effect of p -Cymene in the Presence or Absence of TEA or 4-AP

The p -cymene relaxed the aortic rings without endothelium, in a concentration-dependent manner, in both the absence (control) and the presence of TEA (1 mM), TEA (10 mM), or 4-AP (3 mM), with EC₅₀ values of [figure omitted; refer to PDF] M, [figure omitted; refer to PDF] M, [figure omitted; refer to PDF] M, and [figure omitted; refer to PDF] M, respectively, showing no statistical differences between them ( [figure omitted; refer to PDF] ). In all experimental conditions, the [figure omitted; refer to PDF] value was 100% and reached 10^-3 M (Figure 5).

Figure 5: Relaxant effect of p -cymene on the isolated rat aortic rings without endothelium, contracted by phenylephrine, in the absence ([white square]) and presence of TEA 1 mM ([...]), TEA 10 mM ([white up triangle]), or 4-AP 3 mM ([...]).

[figure omitted; refer to PDF]

3.4. Effect of p -Cymene in the Presence or Absence of BaCl₂ 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 BaCl₂ . However, in the presence of BaCl₂ , the dose-response curve was significantly shifted to the right with a change in [figure omitted; refer to PDF] when compared to the control curve (Figure 6). The presence of BaCl₂ reduced the [figure omitted; refer to PDF] from [figure omitted; refer to PDF] % to [figure omitted; refer to PDF] % and increased the EC₅₀ value from [figure omitted; refer to PDF] M to [figure omitted; refer to PDF] M. Both values present a statistical difference between control values.

Figure 6: Relaxant effect of p -cymene on the isolated rat aortic rings without endothelium, contracted by phenylephrine, in the absence ([white square]) and presence of BaCl₂ 0.1 mM ([...]). [figure omitted; refer to PDF] (unpaired [figure omitted; refer to PDF] -test: absence [figure omitted; refer to PDF] presence of BaCl₂ ).

[figure omitted; refer to PDF]

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 potassium channel blocker, the dose-response curve was significantly shifted to the right with a decrease in [figure omitted; refer to PDF] value when compared to the control curve (Figure 7). The presence of glibenclamide reduced the [figure omitted; refer to PDF] from [figure omitted; refer to PDF] % to [figure omitted; refer to PDF] % and increased the EC₅₀ value from [figure omitted; refer to PDF] M to [figure omitted; refer to PDF] M. Both values present a statistical difference between control values.

Figure 7: Relaxant effect of p -cymene on the isolated rat aortic rings without endothelium, contracted by phenylephrine, in the absence ([white square]) and presence of glibenclamide 3 μ M ( [figure omitted; refer to PDF] ). [figure omitted; refer to PDF] (unpaired [figure omitted; refer to PDF] -test: absence [figure omitted; refer to PDF] presence of glibenclamide).

[figure omitted; refer to PDF]

4. Discussion

In this work, we demonstrated that p -cymene has a dose-dependent 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 (MLC₂₀ ), which can occur through the Ca²⁺ /calmodulin-dependent actions of myosin light chain kinase (MLCK) or through the Ca²⁺ -independent actions of many additional kinases. The main event that determines the activation of that Ca²⁺ /calmodulin pathway with a subsequent activation of MLCK, which leads to the contraction of the smooth muscle, is increased intracellular Ca²⁺ [16]. Two classical ways to increase the Ca²⁺ driving to muscle contraction are pharmacomechanical and electromechanical coupling mechanisms. The first mechanism involves Ca²⁺ mobilization of sarcoplasmic reticulum via G-protein-coupled receptors as well as Ca²⁺ entry by [figure omitted; refer to PDF] calcium channels; the second mechanism involves change in membrane potential and activation of [figure omitted; refer to PDF] channels, with a subsequent Ca²⁺ 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 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 (PGI₂ ), 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 [figure omitted; refer to PDF] and EC₅₀ 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 [figure omitted; refer to PDF] 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 [figure omitted; refer to PDF] 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 [figure omitted; refer to PDF] 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.

Several different types of potassium channels are present in the vascular smooth muscle, including the calcium-activated potassium ( [figure omitted; refer to PDF] or BK_Ca ), blocked by TEA 1-10 mM [22], voltage-gated potassium channels (K_V ), blocked by 4-AP [23], inward rectifying potassium channel (K_ir ), blocked by Ba²⁺ [24], and ATP-sensitive potassium channels (K_ATP ), blocked by glibenclamide [25].

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 [figure omitted; refer to PDF] and EC₅₀ 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 Ba²⁺ and glibenclamide, the vasorelaxant response to p -cymene was significantly reduced, with changes in the [figure omitted; refer to PDF] and EC₅₀ , 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.

Acknowledgments

The authors would like to thank UNIVASF and CNPq for the financial support to conduct this work.

Conflict of Interests

The authors declare that there is no conflict of interests regarding the publication of this paper.

References

[1] H. C. Joca, Y. Cruz-Mendes, K. Oliveira-Abreu, R. P. M. Maia-Joca, R. Barbosa, T. L. Lemos, P. S. Lacerda Beirão, J. H. Leal-Cardoso, "Carvacrol decreases neuronal excitability by inhibition of voltage-gated sodium channels," Journal of Natural Products , vol. 75, no. 9, pp. 1511-1517, 2012.

[2] G. Buchbauer, L. Jirovetz, W. Jager, C. Plank, H. Dietrich, "Fragrance compounds and essential oils with sedative effects upon inhalation," Journal of Pharmaceutical Sciences , vol. 82, no. 6, pp. 660-664, 1993.

[3] L. F. Leal Interaminense, J. H. Leal-Cardoso, P. J. Caldas Magalhães, G. P. Duarte, S. Lahlou, "Enhanced hypotensive effects of the essential oil of Ocimum gratissimum leaves and its main constituent, eugenol, in DOCA-salt hypertensive conscious rats," Planta Medica , vol. 71, no. 4, pp. 376-378, 2005.

[4] J. Grassmann, S. Hippeli, R. Spitzenberger, E. F. Elstner, "The monoterpene terpinolene from the oil of Pinus mugo L. in concert with α -tocopherol and β -carotene effectively prevents oxidation of LDL," Phytomedicine , vol. 12, no. 6-7, pp. 416-423, 2005.

[5] S. Lahlou, L. F. Interaminense, J. H. Leal-Cardoso, G. P. Duarte, "Antihypertensive effects of the essential oil of Alpinia zerumbet and its main constituent, terpinen-4-ol, in DOCA-salt hypertensive conscious rats," Fundamental & Clinical Pharmacology , vol. 17, no. 3, pp. 323-330, 2003.

[6] F. Bakkali, S. Averbeck, D. Averbeck, M. Idaomar, "Biological effects of essential oils-a review," Food and Chemical Toxicology , vol. 46, no. 2, pp. 446-475, 2008.

[7] M. Selvaraj, A. Pandurangan, K. S. Seshadri, P. K. Sinha, V. Krishnasamy, K. B. Lal, "Comparison of mesoporous Al-MCM-41 molecular sieves in the production of p-cymene for isopropylation of toluene," Journal of Molecular Catalysis A: Chemical , vol. 186, no. 1-2, pp. 173-186, 2002.

[8] P. Liapis, G. J. Pass, R. A. McKinnon, I. Stupans, "Characterisation of tolbutamide hydroxylase activity in the common brushtail possum, (Trichosurus vulpecula ) and koala (Phascolarctos cinereus ): inhibition by the Eucalyptus terpene 1,8-cineole," Comparative Biochemistry and Physiology C: Toxicology and Pharmacology , vol. 127, no. 3, pp. 351-357, 2000.

[9] L. R. Bonjardim, E. S. Cunha, A. G. Guimarães, M. F. Santana, M. G. B. Oliveira, M. R. Serafini, A. A. S. Araújo, Â. R. Antoniolli, S. C. H. Cavalcanti, M. R. V. Santos, L. J. Quintans-Júnior, "Evaluation of the anti-inflammatory and antinociceptive properties of p -Cymene in mice," Zeitschrift fur Naturforschung C , vol. 67, no. 1-2, pp. 15-21, 2012.

[10] L. Quintans-Júnior, J. C. F. Moreira, M. A. B. Pasquali, "Antinociceptive activity and redox profile of the monoterpenes (+)-camphene, p -cymene, and geranyl acetate in experimental models," ISRN Toxicology , vol. 2013, 2013.

[11] W. Zhong, G. Chi, L. Jiang, L. W. Soromou, N. Chen, M. Huo, W. Guo, X. Deng, H. Feng, " P -Cymene modulates in vitro and in vivo cytokine production by inhibiting MAPK and NF-κ B activation," Inflammation , vol. 36, no. 3, pp. 529-537, 2013.

[12] G. Kisko, S. Roller, "Carvacrol and p-cymene inactivate Escherichia coli O157:H7 in apple juice," BMC Microbiology , vol. 5, article 36, pp. 1-9, 2005.

[13] M. R. V. Santos, F. V. Moreira, B. P. Fraga, D. P. de Souza, L. R. Bonjardimand, L. J. Quintans-Junior, "Cardiovascular effects of monoterpenes: a review," Revista Brasileira de Farmacognosia , vol. 21, no. 4, pp. 764-771, 2011.

[14] Y. Yvon, E. Guy Raoelison, R. Razafindrazaka, A. Randriantsoa, M. Romdhane, N. Chabir, M. Guedri Mkaddem, J. Bouajila, "Relation between chemical composition or antioxidant activity and antihypertensive activity for six essential oils," Journal of Food Science , vol. 77, no. 8, pp. H184-H191, 2012.

[15] R. F. Furchgott, J. V. Zawadzki, "The obligatory role of endothelial cells in the relaxation of arterial smooth muscle by acetylcholine," Nature , vol. 288, no. 5789, pp. 373-376, 1980.

[16] K. M. Sanders, "Regulation of smooth muscle excitation and contraction,", supplement 1 Neurogastroenterology and Motility , vol. 20, pp. 39-53, 2008.

[17] A. P. Somlyo, A. V. Somlyo, "Signal transduction and regulation in smooth muscle," Nature , vol. 372, no. 6503, pp. 231-236, 1994.

[18] L. J. Ignarro, R. E. Byrns, G. M. Buga, K. S. Wood, "Mechanisms of endothelium-dependent vascular smooth muscle relaxation elicited by bradykinin and VIP," The American Journal of Physiology-Heart and Circulatory Physiology , vol. 253, no. 5, pp. H1074-H1082, 1987.

[19] S. Moncada, J. R. Vane, "The role of prostacyclin in vascular tissue," Federation Proceedings , vol. 38, no. 1, pp. 66-71, 1979.

[20] W. A. Catterall, E. Perez-Reyes, T. P. Snutch, J. Striessnig, "International Union of Pharmacology. XLVIII. Nomenclature and structure-function relationships of voltage-gated calcium channels," Pharmacological Reviews , vol. 57, no. 4, pp. 411-425, 2005.

[21] M. D. G. C. de Souza, E. Bouskela, "Arteriolar diameter and spontaneous vasomotion: importance of potassium channels and nitric oxide," Microvascular Research , vol. 90, pp. 121-127, 2013.

[22] R. Latorre, A. Oberhauser, P. Labarca, O. Alvarez, "Varieties of calcium-activated potassium channels," Annual Review of Physiology , vol. 51, pp. 385-399, 1989.

[23] H. El-Hajj, B. Chandrasekhar, E. A. Kadavil, M. A. Oriowo, "Interaction of BK _Ca channel modulators with adrenergic agonists in the rat aorta is influenced by receptor reserve," Vascular Pharmacology , vol. 41, no. 4-5, pp. 119-124, 2004.

[24] G. H. Liang, A. Adebiyi, M. Dennis Leo, E. M. McNally, C. W. Leffler, J. H. Jaggar, "Hydrogen sulfide dilates cerebral arterioles by activating smooth muscle cell plasma membrane KATP channels," American Journal of Physiology-Heart and Circulatory Physiology , vol. 300, no. 6, pp. H2088-H2095, 2011.

[25] P. D. Smith, S. E. Brett, K. D. Luykenaar, S. L. Sandow, S. P. Marrelli, E. J. Vigmond, D. G. Welsh, "KIR channels function as electrical amplifiers in rat vascular smooth muscle," Journal of Physiology , vol. 586, no. 4, pp. 1147-1160, 2008.

[26] P. Rattanachaikunsopony, P. Phumkhachorn, "Synergistic antimicrobial effect of nisin and p-cymene on salmonella enterica serovar typhi in vitro and on ready-to-eat food," Bioscience, Biotechnology and Biochemistry , vol. 74, no. 3, pp. 520-524, 2010.