Darizy Flávia Silva

Calcium antagonism and the vasorelaxation of the rat aorta induced by rotundifolone

The vasorelaxing activity of rotundifolone (ROT), a major constituent (63.5%) of the essential oil of Mentha x villosa, was tested in male Wistar rats (300-350 g). In isolated rat aortic rings, increasing ROT concentrations (0.3, 1, 10, 100, 300, and 500 microg/ml) inhibited the contractile effects of 1 microM phenylephrine and of 80 or 30 mM KCl (IC50 values, reported as means +/- SEM = 184 +/- 6, 185 +/- 3 and 188 +/- 19 microg/ml, N = 6, respectively). In aortic rings pre-contracted with 1 microM phenylephrine, the smooth muscle-relaxant activity of ROT was inhibited by removal of the vascular endothelium (IC50 value = 235 +/- 7 microg/ml, N = 6). Furthermore, ROT inhibited (pD2 = 6.04, N = 6) the CaCl2-induced contraction in depolarizing medium in a concentration-dependent manner. In Ca2+-free solution, ROT inhibited 1 microM phenylephrine-induced contraction in a concentration-dependent manner and did not modify the phasic contractile response evoked by caffeine (20 mM). In conclusion, in the present study we have shown that ROT produces an endothelium-independent vasorelaxing effect in the rat aorta. The results further indicated that in the rat aorta ROT is able to induce vasorelaxation, at least in part, by inhibiting both: a) voltage-dependent Ca2 channels, and b) intracellular Ca2+ release selectively due to inositol 1,4,5-triphosphate activation. Additional studies are required to elucidate the mechanisms underlying ROT-induced relaxation.

Unravelling the cardiovascular effects induced by α-terpineol: a role for the nitric oxide-cGMP pathway.

1. α‐Terpineol is a monoterpene found in the essential oils of several aromatic plant species. In the present study, we investigated the mechanisms underlying the cardiovascular changes induced by α‐terpineol in rats.

Rotundifolone-Induced Relaxation is Mediated by BKCa Channel Activation and Cav Channel Inactivation

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 BK(Ca) channels. Furthermore, rotundifolone inhibited the vasoconstriction induced by CaCl(2) in depolarizing nominally Ca(2+) -free medium and antagonized the contractions elicited by an L-type Ca(2+) channel agonist, S(-)-Bay K 8644 (2 × 10(-7)  M), indicating that the vasodilatation involved inhibition of Ca(2+) influx through L-type voltage-dependent calcium channels (Ca(v) type-L). Additionally, rotundifolone inhibited L-type Ca(2+) currents (I(Ca) L), affecting the voltage-dependent activation of I(Ca) L 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 BK(Ca) channels and causes a shift in the steady-state inactivation relationship for Ca(v) type-L towards more hyperpolarized membrane potentials.

Resurgent Na+ current: a new avenue to neuronal excitability control.

Integrative and firing properties are important characteristics of neuronal circuits and these responses are determined in large part by the repertoire of ion channels they express, which can vary considerably between cell types. Recently, a new mode of operation of voltage dependent sodium channels has been described that generates a so-called resurgent Na+ current. Accumulating evidence suggests resurgent Na current participates in the generation of sub-threshold inward Na+ current causing membrane depolarization which provides the necessary drive to fire high-frequency action potentials. Recent studies indicate that resurgent Na+ current could be a more widespread feature than previously thought.

Cardiovascular Effects Elicited by Milonine, a New 8,14-Dihydromorphinandienone Alkaloid

The mechanisms underlying the cardiovascular responses evoked by milonine (i.v.), an alkaloid, were investigated in rats. In normotensive rats, milonine injections produced hypotension and tachycardia, which were attenuated after N(w) -nitro-L-arginine methyl esther (L-NAME; 20 mg/kg, i.v.). In phenylephrine (10 μM), pre-contracted mesenteric artery rings, milonine (10⁻¹⁰ M to 3 × 10⁻⁴ M) caused a concentration-dependent relaxation (EC₅₀ = 1.1 × 10⁻⁶ M, E(max) = 100 ± 0.0%) and this effect was rightward shifted after either removal of the vascular endothelium (EC₅₀ = 1.6 × 10⁻⁵, p < 0.001), or after L-NAME 100 μM (EC₅₀ = 6.2 × 10⁻⁵, p < 0.001), hydroxocobalamin 30 μM (EC₅₀ = 1.1 × 10⁻⁴, p < 0.001) or ODQ 10 μM (EC₅₀ = 1.9 × 10⁻⁴ p < 0.001). In addition, in rabbit aortic endothelial cells, milonine increased NO₃⁻ levels. The relaxant effect induced by milonine was attenuated in the presence of KCl (20 mM), a modulator efflux K(+) (EC₅₀ = 1.2 × 10⁻⁵, p < 0.001), or different potassium channel blockers such as glibenclamide (10 μM) (EC₅₀ = 6.3 × 10⁻⁵, p < 0.001), TEA (1 mM) (EC₅₀ = 2.3 × 10⁻⁵ M, n = 6) or Charybdotoxin (0.2 μM) plus apamin (0.2 μM) (EC₅₀ = 3.9 × 10⁻⁴ M, n = 7). In addition, pre-contraction with high extracellular potassium concentration prevented milonine-induced vasorelaxation (EC₅₀ = 1.0 × 10⁻⁴, p < 0.001). Milonine also reduced CaCl₂ -induced contraction in Ca²(+) -free solution containing KCl (60 mM). In conclusion, using combined functional and biochemical approaches, we demonstrated that the hypotensive and vasorelaxant effects produced by milonine are, at least in part, mediated by the endothelium, likely via nitric oxide release, activation of nitric oxide-cGMP pathway and opening of K(+) channels.

Participation of nitric oxide pathway in the relaxation response induced by E-cinnamaldehyde oxime in superior mesenteric artery isolated from rats.

Abstract: For many years, nitric oxide (NO) has been studied as an important mediator in the control of vascular tone. Endothelial deficiencies that diminish NO production can result in the development of several future cardiovascular diseases, such as hypertension and arteriosclerosis. In this context, new drugs with potential ability to donate NO have been studied. In this study, 3 aromatic oximes [benzophenone oxime, 4-Cl-benzophenone oxime, and E-cinnamaldehyde oxime (E-CAOx)] induced vasorelaxation in endothelium-denuded and intact superior mesenteric rings precontracted with phenylephrine. E-CAOx demonstrated the most potent effect, and its mechanism of action was evaluated. Vascular reactivity experiments demonstrated that the effect of E-CAOx was reduced by the presence of 2-phenyl-4,4,5,5-tetramethylimidazoline-1-oxyl-3-oxide, 1H[1,2,4,]oxadiazolo[4,3-a]quinoxalin-1-one, and (Rp)-8-(para-chlorophenylthio)guanosine-3′,5′-cyclic monophosphorothioate, suggesting the participation of NO/sGC/PKG pathway. NO donation seems to be mediated through nicatinamide adenine dinucleotide phosphate-dependent reductases because 7-ethoxyresorufin decreased the effect of E-CAOx on vascular reactivity and reduced NO formation as detected by flow cytometry using the NO indicator diaminofluorescein 4,5-diacetate. Further downstream of NO donation, K+ subtype channels were also shown to be involved in the E-CAOx vasorelaxant effect. The present study showed that E-CAOx acts like an NO donor, activating NO/sGC/PKG pathway and thus K+ channels.

Participation of the TRP channel in the cardiovascular effects induced by carvacrol in normotensive rat.

Carvacrol has been described as an agonist/antagonist of different transient receptor potential (TRP) channels and voltage-dependent calcium channels (Cavs). The aim of this study was to evaluate the role of Cav and TRP channels following carvacrol stimulation. Initially, in mesenteric artery rings carvacrol relaxed phenylephrine-induced contractions. Furthermore, carvacrol inhibited contraction elicited by CaCl2 in depolarizing nominally without Ca2+ medium and antagonized the contractions induced by S(-)-Bay K 8644 and inhibited Ca2+ currents indicating the inhibition of Ca2+ influx through L-type Cav. Additionally, carvacrol antagonized the contractions induced by CaCl2 in the presence of nifedipine/Cyclopiazonic acid/phenylephrine or nifedipine/Cyclopiazonic acid/KCl 60, suggesting a possible inhibition of calcium influx by store operated channels (SOCs), receptor operated channels (ROCs) and/or TRP channels. Interestingly, among the TRP channel blockers used, the effect induced by carvacrol was attenuated by Mg2+ and potentiated by La3+ and Gd3+, suggesting that TRP channels are involved in relaxation induced by carvacrol. Monoterpene also induced hypotension and bradycardia in non-anesthetized normotensive rats and negative inotropic and chronotropic effects. In conclusion, these results suggest that the hypotensive effect of carvacrol is probably due to bradycardia and a peripheral vasodilatation that involves, at least, the inhibition of the Ca2+ influx through Cav and TRP channels.

TRPM8 Channel Activation Induced by Monoterpenoid Rotundifolone Underlies Mesenteric Artery Relaxation.

In this study, our aims were to investigate transient receptor potential melastatin-8 channels (TRPM8) involvement in rotundifolone induced relaxation in the mesenteric artery and to increase the understanding of the role of these thermosensitive TRP channels in vascular tissue. Thus, message and protein levels of TRPM8 were measured by semi-quantitative PCR and western blotting in superior mesenteric arteries from 12 week-old Spague-Dawley (SD) rats. Isometric tension recordings evaluated the relaxant response in mesenteric rings were also performed. Additionally, the intracellular Ca2+ changes in mesenteric artery myocytes were measured using confocal microscopy. Using PCR and western blotting, both TRPM8 channel mRNA and protein expression was measured in SD rat mesenteric artery. Rotundifolone and menthol induced relaxation in the isolated superior mesenteric artery from SD rats and improved the relaxant response induced by cool temperatures. Also, this monoterpene induced an increase in transient intracellular Ca2+. These responses were significantly attenuated by pretreatment with capsazepine or BCTC, both TRPM8 channels blockers. The response induced by rotundifolone was not significantly attenuated by ruthenium red, a non-selective TRP channels blocker, or following capsaicin-mediated desensitization of TRPV1. Our findings suggest that rotundifolone induces relaxation by activating TRPM8 channels in rat superior mesenteric artery, more selectively than menthol, the classic TRPM8 agonist, and TRPM8 channels participates in vasodilatory pathways in isolated rat mesenteric arteries.

Warifteine, a bisbenzylisoquinoline alkaloid, induces relaxation by activating potassium channels in vascular myocytes

The present study used functional and electrophysiological approaches to investigate the mechanisms by which warifteine, a bisbenzylisoquinoline alkaloid isolated from Cissampelos sympodialis Eichl., causes vasorelaxation of the rat thoracic aorta. Warifteine (1 pmol/L–10 μmol/L) induced concentration‐dependent relaxation (pD2 = 9.40 ± 0.06; n = 5) of endothelium‐intact aortic rings precontracted with noradrenaline (10–100 μmol/L). The relaxation effects were not attenuated by removal of the endothelium. Warifteine also induced the relaxation of prostaglandin F2α (1–10 mmol/L)‐precontracted rings (pD2 = 9.2 ± 0.2; n = 8). In contrast, the relaxant activity of warifteine was nearly abolished in high K+ (80 mmol/L)‐precontracted aortic rings. In preparations incubated with 20 mmol/L KCl or with the K+ channel blockers tetraethylammonium (1, 3 and 5 mmol/L), iberiotoxin (20 nmol/L), 4‐aminopyridine (1 mmol/L) or glibenclamide (10 μmol/L), the vasorelaxant activity of warifteine was markedly reduced. However, BaCl2 (1 mmol/L) had no effect on the relaxant effects of warifteine. In vascular myocytes, warifteine (100 nmol/L) significantly increased whole‐cell K+ currents (at 70 mV). Under nominally Ca2+‐free conditions, warifteine did not reduce extracellular Ca2+‐induced contractions in rings precontracted with high K+ or noradrenaline (100 μmol/L). Together, the results of the present study indicate that warifteine induces potent concentration‐dependent relaxation in the rat aorta via an endothelium‐independent mechanism that involves the activation of K+ channels.

Involvement of Potassium Channels in Vasorelaxant Effect Induced by Valeriana prionophylla Standl. in Rat Mesenteric Artery

Assays in vitro and in vivo were performed on extract from roots and leaves from the Valeriana prionophylla Standl. (VPR and VPF, resp.). In phenylephrine (1 μM) precontracted rings, VPR (0.01–300 μg/mL) induced a concentration-dependent relaxation (maximum response (MR) = 75.4 ± 4.0%, EC50 = 5.97 (3.8–9.3) μg/mL, n = 6]); this effect was significantly modified after removal of the endothelium (EC50 = 39.6 (27.2–57.6) μg/mL, P < 0.05). However, VPF-induced vasorelaxation was less effective compared to VPR. When rings were preincubated with L-NAME (100 μM) or indomethacin (10 μM), the endothelium-dependent relaxation induced by VPR was significantly attenuated (MR = 20.9 ± 2.3%, 34.2 ± 2.9%, resp., P < 0.001). In rings denuded endothelium, precontracted with KCl (80 mM), or in preparations pretreated with KCl (20 mM) or tetraethylammonium (1 or 3 mM), the vasorelaxant activity of VPR was significantly attenuated (MR = 40.0 ± 8.2, n = 5; 50.5 ± 6.0%; 49.3 ± 6.4%; 46.8 ± 6.2%; resp., P < 0.01). In contrast, neither glibenclamide (10 μM), barium chloride (30 μM), nor 4-aminopyridine (1 mM) affected VPR-induced relaxation. Taken together, these results demonstrate that hypotension induced by VPR seems to involve, at least in part, a vascular component. Furthermore, endothelium-independent relaxation induced by VPR involves K+ channels activation, most likely due to BKCa channels, in the rat superior mesenteric artery.