Vasoconstrictor and hemodynamic effects of a methanolic extract from Rhinella marina toad poison

Abstract

Rhinella marina toad is abundant in Brazil. Its poison contains cardiac glycosides called bufadienolides, which are extensively investigated for their bioactivity. Our aim was to characterize the vasoactivity of Rhinella marina poison (RmP) on the aorta of male Wistar rats. For this, the RmP was first collected and processed to obtain an alcoholic extract. To determine cardiovascular effects of RmP, we performed in vivo tests by administering RmP intravenously in doses of 0.1-0.8 mg/kg. Vascular reactivity was also performed through concentration-response curves to RmP (10 ng/mL to 200 μg/mL) in aortic segments with and without endothelium. RmP induced a concentration-dependent contraction in rat aorta which was partly endothelium-mediated. Nitric oxide contributes with this response in view that incubation with L-NAME increased the contractile response. Additionally, treatment with indomethacin [cyclooxygenase, (COX) inhibitor], nifedipine (L-type voltage-gated calcium channels blocker), and BQ-123 (ET_A receptors antagonist) decreased maximum response, and ketanserin (5-HT₂ receptors antagonist) decreased pEC₅₀, suggesting active participation of these pathways in the contractile response. On the other hand, apocynin (NADPH oxidase inhibitor) did not alter contractility. Incubation with prazosin (α₁-adrenergic receptor antagonist) abolished the contractile response, suggesting that the RmP-induced contraction is dependent on the adrenergic pathway. In the Na⁺/K⁺ ATPase protocol, a higher Emax was observed in the RmP experimental group, suggesting that RmP potentiated Na⁺/K⁺ATPase hyperpolarizing response. When this extract was injected (i.v.) in vivo, increase in blood pressure and decrease in heart rate were observed. The results were immediate and transitory, and occurred in a dose-dependent manner. Overall, these data suggest that the poison extract of R. marina toad has an important vasoconstrictor action and subsequent vasopressor effects, and its use can be investigated to some cardiovascular disorders.

Keywords: Amphibian poisons; Aorta; Rhinella marina; Vascular reactivity; blood pressure.

Fig. 1.

HPLC-UV chromatogram of the RmP methanolic extract Analysis method: Separation was performed on Phenomenex Luna C18 Column (250.0 × 4.6 mm, 5 μm), samples were eluted using water (solution A) and acetonitrile (solution B) as mobile phase. Elution varied according to gradient and solution flow as follows: 0–45 min with 0–70% solvent B, flow 0.80 mL/min, injection volume was 20 μL and detection at 296 nm.

Fig. 2.

Effect of RmP on aorta segments with and without endothelium. A: Concentration-response curves to RmP in segments of aorta in the presence and absence of endothelium. Some rings were incubated with L-NAME (100 μM) prior to concentration-response curves. B: Emax and C: pEC₅₀ from concentration-response curves. Each point represents mean ± SEM. *p < 0.05 versus E+.

Fig. 3.

Contribution of COX, L-type calcium channel and ROS in the vasoconstriction effect of RmP on aorta segments. A: Concentration-response curves to RmP in segments of aorta in the presence and absence of indomethacin (100 μM), nifedipine (100 μM) or apocynin (30 μM). B: Emax and C: pEC₅₀ from concentration-response curves. Each point represents mean ± SEM. *p < 0.05 versus E+.

Fig. 4.

Contribution of endothelin, serotonin and α-adrenergic pathway in the effect of RmP on aorta segments. A: Concentration-response curves to RmP in segments of aorta in the presence and absence of BQ-123 (1 μM), ketanserin (30 nM) or prazosin (1 μM). B: Emax and C: pEC₅₀ from concentration-response curves. Each point represents mean ± SEM. *p < 0.05 versus E+.

Fig. 5.

Indirect effect of RmP on Na+/K + -ATPase pump in aorta segments. A: Concentration-response curves to KCl (1–10 mM) in segments of aorta (n = 8) after pre-contraction with phenylephrine 1 μM (control) or RmP 2 μg/mL. B: Emax and C: pEC₅₀ from concentration-response curves (A). Each point represents mean ± SEM. *p < 0.05 versus control.

Fig. 6.

In vivo effect of RmP injection in blood pressure. (A) Mean arterial pressure (MAP - mmHg) over time after intravenous injection of RmP (0.1 mg/kg – 0.8 mg/kg). (B) Area under the curve (AUC) of mean arterial pressure (MAP) curves after intravenous injection of RmP (0.1 mg/kg – 0.8 mg/kg). Each point represents mean ± SEM. *p < 0.05 versus control.

Fig. 7.

In vivo effect of RmP injection in heart rate. Heart rate (beats per minute – BPM) over time (s) after intravenous injection of RmP (0.1 mg/kg – 0.8 mg/kg). Each point represents mean ± SEM. *p < 0.05 versus control.