Basic & Clinical Pharmacology & Toxicology, 109, 465–475
Doi: 10.1111/j.1742-7843.2011.00749.x
Rotundifolone-Induced Relaxation is Mediated by BKCa Channel Activation and Cav Channel Inactivation Darzy F. Silva1, Islania G. A. Araffljo2, Jos G. F. Albuquerque1, Dayanne L. Porto1, Katy L. G. Dias1, Karla V. M. Cavalcante1, Robson C. Veras1, Xirley P. Nunes1, Jos M. Barbosa-Filho1, Demetrius A. M. Araffljo1, Jader S. Cruz2, Nadja A. Correia1 and Isac A. De Medeiros1 1
Laboratrio de Tecnologia FarmacÞutica (LTF), Universidade Federal da Paraba – UFPB, Jo¼o Pessoa, PB – Brazil, and 2Departamento de Bioqumica e Imunologia, Universidade Federal de Minas Gerais – UFMG, Belo Horizonte, MG – Brazil (Received 26 November 2010; Accepted 10 June 2011)
Abstract: Rotundifolone is the major constituent of the essential oil of Mentha x villosa Hudson. In preliminary studies, rotundifolone induced significant hypotensive, bradycardic and vasorelaxant effects in rats. Thus, to gain more insight into the pharmacology of rotundifolone, the aim of this study was to characterize the molecular mechanism of action involved in relaxation produced by rotundifolone. The relaxant effect was investigated in rat superior mesenteric arteries by using isometric tension measurements and whole-cell patch-clamp techniques. Rotundifolone relaxed phenylephrine-induced contractions in a concentration-dependent manner. Pre-treatment with KCl (20 mM), charybdotoxin (10)7 M) or tetraethylammonium (TEA 10)3 or 3 · 10)3 M) significantly attenuated the relaxation effect induced by rotundifolone. Additionally, whole-cell patch-clamp recordings were made in mesenteric smooth muscle cells and showed that rotundifolone significantly increased K+ currents, and this effect was abolished by TEA (10)3 M), suggesting the participation of BKCa channels. Furthermore, rotundifolone inhibited the vasoconstriction induced by CaCl2 in depolarizing nominally Ca2+-free medium and antagonized the contractions elicited by an L-type Ca2+ channel agonist, S(-)-Bay K 8644 (2 · 10)7 M), indicating that the vasodilatation involved inhibition of Ca2+ influx through L-type voltage-dependent calcium channels (Cav type-L). Additionally, rotundifolone inhibited L-type Ca2+ currents (ICaL), affecting the voltage-dependent activation of ICaL and steady-state inactivation. Our findings suggest that rotundifolone induces vasodilatation through two distinct but complementary mechanisms that clearly depend on the concentration range used. Rotundifolone elicits an increase in the current density of BKCa channels and causes a shift in the steady-state inactivation relationship for Cav type-L towards more hyperpolarized membrane potentials.
Terpenoids constitute the largest class of plant secondary metabolites and have been used in essential oils for centuries as therapeutically relevant compounds. However, little is known about their mechanism of action [1,2]. The monoterpenoids, such as camphor, borneol, citronellol, alpha-terpineol or menthol, compose a group of naturally occurring organic compounds derived from two isoprene units. They are the major components of some essential oils presenting anaesthetic and analgesic activities [3,4], anti-inflammatory effects [5], vasodilatation [6], hypotension [7], anti-pruritic applications [8,9], as well as agonist or antagonist actions on different members of the transient receptor potential (TRP) channel family, which are nonselective cationic channels [4,10–13]. Furthermore, it was reported that some monoterpenoids have specific action on biological systems: e.g., menthol modulates g-aminobutyric acid A channels [14]; camphor and borneol are non-competitive inhibitors of nicotinic acetylcholine receptors [15]; thymol blocks Ca2+ and K+ channels [16,17]; and carvacrol inhibits Drosophila TRPL (member of the Canonical or Classical TRPC subfamily, TRP-like) and mammalian TRPM7 (Melastatin related TRP) channels [18]. Author for correspondence: Darzy F. Silva, Laboratrio de Tecnologia FarmacÞutica (LTF), Universidade Federal da Paraba – UFPB, zip code 58051-970 – Jo¼o Pessoa, PB – Brazil (fax +55-83 32167366, e-mail
[email protected]).
Rotundifolone (C10H14O2; molecular weight 166) is a naturally occurring monoterpenic ketone of plant origin and an important chemical constituent of the essential oils of many Mentha species (Mentha rotundifolia, M. suaveolens, M. spicata L., M. longifolia and M. x villosa) [19,20]. A previous report from our group showed that rotundifolone induced marked hypotension and bradycardia in non-anaesthetized normotensive rats using an in vivo approach. Those effects were probably due to a decrease in peripheral vascular resistance [21], a hypothesis that was subsequently strengthened by studies from Guedes and collaborators [20], who demonstrated a vasorelaxant effect in rat aortas that was apparently mediated by an inhibitory effect on Ca2+ influx and inhibition of intracellular Ca2+ release from stores. However, the mechanism of molecular action remains elusive. New molecules have been harvested from natural sources to fulfil a need for new chemical designs with distinctive pharmacological activities. This study was designed to gain more insight into the pharmacology of rotundifolone and sought a more detailed mechanism of molecular action that leads to the vascular smooth muscle relaxation induced by monoterpenes. In this study, our group demonstrated through functional and electrophysiological studies that rotundifolone can cause vasorelaxation in the rat mesenteric artery by activation of BKCa channels and inhibition of the L-type Ca2+ channels in vascular smooth muscle.
2011 The Authors Basic & Clinical Pharmacology & Toxicology 2011 Nordic Pharmacological Society
DARZY F. SILVA ET AL.
466 Materials and Methods
Animals. Male Sprague Dawley (SD) or Lyon normotensive (LN) rats (10–12 weeks old, 250–300 g), were used for all experiments. The animals were housed under conditions of controlled temperature (21 € 1C) and lighting (lights on: 06:00–18:00 hr). In addition, they had access to animal chow (PURINA-Brazil) and tap water ad libitum. The study was carried out in accordance with the Guide for the Care and Use of Laboratory Animals as adopted by the U.S. National Institutes of Health, and it was approved by the Animal Care and Use Committees of the Federal University of Paraba and the Federal University of Minas Gerais. Isometric tension measurement. Lyon normotensive rats were euthanized by cervical dislocation, and the superior mesenteric arteries were cleaned of adherent tissue as described by Cavalcante and colleagues [22]. Briefly, the superior mesenteric artery was removed and cleaned from connective tissue and fat. Rings (1–2 mm) were obtained and placed in physiological Tyrode’s solution (composition in mM: NaCl 158.3; KCl 4.0; CaCl2Æ2H2O 2.0; MgCl2Æ6H2O 1.05; NaHCO3 10.0; NaH2PO4ÆH2O 0.42; glucose 5.6), maintained at 37C, gassed with a carbogenic mixture (95% O2 and 5% CO2) resulting pH 7.4. Rings were stabilized with an optimal resting tension of 0.75 g, which had been determined previously by length–tension relationship experiments and studies using the optimal contraction to 10)5 M phenylephrine against passive tension. The tissues were then allowed to equilibrate further for 60 min. The resting tension was readjusted to 0.75 g when necessary. The isometric contraction was recorded by a force transducer (FORT-10; WPI, Sarasota, FL, USA) coupled to an amplifier-recorder (Miobath-4; WPI) and to a personal computer equipped with an analogue-to-digital converter board. To determine the relaxant effect, each mesenteric ring was contracted with 10)5 M phenylephrine, 80 mM KCl or 2 · 10)7 M methyl-1,4-dihydro2,6-dimethyl-3-nitro-4-(2-trifluoromethylphenyl)-pyridine-5-carboxylate (Bay K 8644), and after a plateau was attained, rotundifolone (10)7 to 10)3 M) was added cumulatively. A Tyrode solution contained 20 mM of KCl (equimolar replacement of NaCl with KCl) was used to contract the vessels with 200 nM of the Ca2+ channel agonist Bay K 8644. This solution was used because it elicited a very stable contraction while keeping the K+ equilibrium potential (EK) sufficiently negative that K+ channel activation will cause hyperpolarization and relaxation. Furthermore, the pre-incubation of mesenteric arteries with 20-mM KCl solution led to a slight depolarization, favouring the action of dihydropyridine (Bay K 8644), an activator of Ca2+ channels that depends on the state [23,24]. For some experiments, the endothelium layer was removed by gently rubbing the intimal surface of the vessels with a cotton ball to determine whether the relaxant effect of rotundifolone was endothelium-dependent. Endothelial integrity was assessed qualitatively by the degree of relaxation caused by acetylcholine (10)5 M) in the presence of contractile tone induced by phenylephrine (10)5 M). Rings were considered to be endothelium-denuded when acetylcholineinduced relaxant effects were
