Aristoteline, an Indole-Alkaloid, Induces Relaxation by Activating Potassium Channels and Blocking Calcium Channels in Isolated Rat Aorta

Abstract

Alkaloids derived from plants have shown great medicinal benefits, and are often reported for their use in cardiovascular disease management. Aristotelia chilensis (Molina) Stuntz (Maqui) has shown important medicinal properties in traditional useage. In this study, we evaluated the effect of the indole-alkaloid aristoteline (ARI), isolated from leaves of Maqui, on vascular reactivity of isolated aortic rings from normotensive rats. ARI induced relaxation (100%) in a concentration-dependent manner in intact or denuded-endothelium aortic rings pre-contracted with phenylephrine (PE; 1 μM). However, a specific soluble guanylyl cyclase inhibitor (ODQ; 1 μM) significantly reduced the relaxation to ARI in aortic rings pre-contracted with PE. In the presence of ARI, the contraction induced by KCl or PE was significantly (p < 0.05) decreased. Interestingly, the potassium channel blockade with 10 μM BaCl₂ (Kir), 10 μM glibenclamide (K_ATP), 1 mM tetraethylammonium (TEA; KCa1.1), or 1 mM 4-aminopyridine (4-AP; Kv) significantly (p < 0.05) reduced the ARI-induced relaxation. ARI significantly (p < 0.05) reduced the contractile response to agonist of Ca_V1.2 channels (Bay K8644; 10 nM), likely reducing the influx of extracellular calcium through plasma membrane. The mechanisms associated with this process suggest an activation of the potassium channels, a calcium-induced antagonism and endothelium independent vasodilation that possibly involves the nitric oxide-independent soluble guanylate cyclase pathway.

Keywords: Aristoteline; calcium channels; potassium channels; rat; vascular activity.

Figure 1

Chemical structure of aristoteline (ARI).

Figure 2

ARI directly causes relaxation on vascular smooth muscle of aorta. Vasodilatation response induced by ARI (1 nM to 100 μM) after the addition of phenylephrine (PE) (1 μM) is shown in intact aortic rings (endo or control) and denuded aorta (endo-denuded) (A), preincubate with L-NAME (100 μM) (B) or with 1H-(1,2,4)oxadiazolo[4,3-a]quinoxalin-1-one (ODQ) (1 μM) (C). Values are mean ± standard error of the mean of 6 experiments. The determination of IC₅₀ were performed using nonlinear regression and a repeated-measures two-way analysis of variance (ANOVA) to compare curves. Statistically significant difference *p < 0.05 vs. Control.

Figure 3

Original trace showing the time course of the concentration–response curves to KCl in intact aortic ring from rat in absence and presence of ARI (10⁻⁵ M). The aortic ring was pre-incubated with ARI for 20 min before KCl was added.

Figure 4

Effect of ARI on the contractile response to KCl (A) and PE (B) in rat aortic ring. The aorta was pre-incubated in absence (control) or presence of ARI (10 μM) or caffeine (10 mM) for 20 min. Values are the mean ± standard error of the means of 6 experiments. The asterisks in (A) refer to both, ARI and caffeine. The determination of EC₅₀ were performed using nonlinear regression and a repeated-measures two-way ANOVA to compare curves. Statistically significant difference *p < 0.05 vs. Control.

Figure 5

Effect of ARI on calcium release from the phenylephrine-sensitive intracellular calcium stores. The aortic rings were pre-incubated in a free calcium buffer for 10 min before PE was added (A), and then, the CaCl₂ (0.1, 0.3, 0.6, and 1.0 mM) was added to the bath (B). Vasoconstriction occurred just when the agonist of Ca_V1.2 channels (10 nM Bay K8644) was added with 15 mM KCl to the bath (C). The aorta was pre-incubated in absence (control) or presence of ARI (10 μM) for 20 min. Values are mean ± standard error of the mean of 6 experiments. Statistically significant difference *p < 0.05 vs. Control.

Figure 6

Original trace showing the time course of the concentration–response curves to ARI in intact aortic ring from rat in absence (A) and presence of BaCl₂ (10 μM; B). The aortic ring was pre-incubated with BaCl₂ for 20 min before PE (1 μM) was added.

Figure 7

Evaluation of the ARI vasodilatation mechanism associated with potassium channels. Effect of ARI after the addition of BaCl₂ (10 μM) as a blocker of inward rectifier potassium channels (Kir) is shown (A), glibenclamide (10 μM) as blocker of adenosine triphosphate (ATP)-sensitive K⁺ channels (B), 4-AP (1 mM) as a non-selective blocker of Kv channels (C) and tetraethylammonium (TEA) (1 mM) as a blocker of KCa channels (D). PE (1 μM) was used to induce the contractile responses to the aortic rings. The determination of IC₅₀ were performed using non-linear regression and a repeated-measures two-way ANOVA to compare curves; n = 6. Statistically significant difference *p < 0.05 vs. Control.

Figure 8

Original tracing showing the time course of the contractile response to KCl (60 mM) and TEA (1 mM) in intact aortic rings from rats. These are in the absence or presence of ARI (10^-5 M). The aortic rings were pre-incubated with ARI for 20 min before TEA was added.

Figure 9

Evaluation of the ARI contraction mechanism associated with potassium channels. The aortic rings were pre-contracted with KCl (15 mM) (A), BaCl₂ (1 mM) as a blocker of inward rectifier potassium channels (Kir) (B), TEA (1 mM) as a non-selective blocker of KCa1.1 channels (C). The vascular tissue was pre-incubated in absence (control) or presence of ARI (10 μM) for 20 min. Unpaired Student’s t-tests; n = 6. Statistically significant difference *p < 0.05 vs. Control.

Figure 10

Evaluation of the ARI vasodilatation mechanism associated with endothelial prostanoids. Data shows the effect of ARI after the addition of indomethacin (10 μM) as a non-selective cyclooxygenase inhibitor. PE (1 μM) was used to induce the contractile responses. The determination of IC₅₀ were performed using nonlinear regression and a repeated-measures two-way ANOVA to compare curves; n = 6. Statistically significant difference *p < 0.05 vs. Control.