Caryocar brasiliense induces vasorelaxation through endothelial Ca2+/calmodulin and PI3K/Akt/eNOS-dependent signaling pathways in rats

ABSTRACT Caryocar brasiliense Cambess., Caryocaraceae (pequi) is a typical Brazilian Cerrado tree. A previous study showed that the butanolic fraction of pequi leaves promotes endothelium-dependent relaxation mediated by nitric oxide and that it causes reversible hypotension in rats. In the present study, we investigated the cell signaling pathways associated with the butanolic fraction-induced nitric oxide release, and we characterized the chemical composition of its fraction. Vascular reactivity tests, a western blotting analysis, and a chemiluminescence assay were used to investigate the signaling pathways involved in the vasorelaxant effect of the butanolic fraction. Electrospray Ionization Fourier Transform Ion Cyclotron Resonance Mass Spectrometry was used to characterize the butanolic fraction chemical composition. Vasorelaxation was mediated through the activation of the calmodulin and phosphatidylinositol 3-kinase (PI3K)/Akt signaling pathways, leading to subsequent endothelial nitric oxide synthase phosphorylation and nitric oxide production, as evidenced by western blotting and chemiluminescence assays, respectively. The chemical characterization of the butanolic fraction revealed the presence of 72 oxygenated compounds, whose molecular formulae are compatible with phenolic compounds, suggesting a potential contribution of these compounds for the butanolic fraction vasorelaxant effect. These findings show that the calmodulin and phosphatidylinositol 3-kinase (PI3K)/Akt signaling pathways are involved in the butanolic fraction-induced endothelial nitric oxide synthase activation and are promoted by polyphenol compounds present in the C. brasiliense leaves.

Vasorelaxant activity of Euphorbia furcillata Kunth mainly by activation of NO/cGMP pathway and calcium channel blockade / Actividad vasorrelajante de Euphorbia furcillata Kunth principalmente por activación de la vía NO/GMPc y bloqueo de canales de calcio]

The aim of current study was to determinate ex vivo and chromatographic fingerprint by HPLC of four extracts of Euphorbia furcillata K. Ethyl acetate extract of Euphorbia furcillata (EaEEf) was the most effective and potent extract (Emax=98.69±1.24%) and its effect was partially endothelium-dependent. Functional vasorelaxant mechanism of action of EaEEf was determinate, EaEEf showed efficient relaxation of KCl [80 mM]-induced contraction and norepinephrine and CaCl2 contraction curves showed diminution of maximal contraction in the presence of EAEEf and EaEEf-relaxation curve was shifted to the right in the presence of L-NAME (nitric oxide synthase inhibitor) and ODQ (guanylate cyclase inhibitor). Chromatographic fingerprints analysis suggests presence of diterpenoid such as abietane, tigliane, and ingenane skeletons. Our experiments suggest the EaEEf vasorelaxant activity could be attributed to diterpenoid molecules whose mechanism involves nitric oxide production and calcium channel blockade.

Se determinó el efecto vasorrelajante ex vivo y los perfiles cromatográficos mediante HPLC de cuatro extractos de Euphorbia furcillata K.. El extracto de acetato de etilo de E. furcillata (EaEEf) fue el más eficaz y potente en la contracción inducida por norepinefrina (Emax=98.69±1.24%) y el efecto fue parcialmente dependiente del endotelio vascular. Se determinó el mecanismo de acción vasorrelajante para EaEEf, este mostró ser eficaz sobre la contracción inducida por KCl [80 mM] y la curva de contracción en respuesta a norepinefrina y CaCl2 en presencia de EaEEf mostró disminución en la contracción máxima, mientras que la curva de relajación de EaEEf en presencia de L-NAME (inhibidor de óxido nítrico sintasa) y ODQ (inhibidor de guanilato ciclasa) se desplazó hacia la derecha. El análisis cromatográfico de EaEEf sugiere la presencia de moléculas diterpenoides como abietano, tigliano y esqueletos de ingenano. Nuestros resultados sugieren que el efecto vasorrelajante de EaEEf podría atribuirse a moléculas diterpenoides, cuyo mecanismo de acción involucra la producción de óxido nítrico y bloqueo de canales de calcio.

The lectin isolated from Lonchocarpus araripensis seed elicits endothelium-dependent vasorelaxation / A lectina isolada de sementes de Lonchocarpus araripensis promove efeito vasorelaxante dependente de endotélio

Background: The vasorelaxant effect of lectins from leguminous plants (Diocleinae subtribe) is well described. However, this effect has been little explored for lectins isolated from Dalbergieae tribe, except for that of Vatairea guianensis, that induces vasorelaxation involving nitric oxide and the lectin domain. Objective: To evaluate the vasorelaxant effect of a lectin isolated from Lonchocarpus araripensis (LAL), Dalbergieae tribe, and the involvement of the lectin domain and endothelium derived relaxing factors. Methods: Aortic rings of Wistar rats (250 - 300 g) were mounted in organ bath and mantained in physiological conditions (CEUA No. 10130208-8/40). LAL (0.1­100 µg/ml) was added to phenylephrine (0.1 µM)-contracted tissues with either endothelium intact or denuded. In order to investigate the mechanisms of LAL relaxation, inhibitors of NOS (L-NAME: 100 µM), cyclooxygenase (indomethacin: 10 µM), or potassium channels (TEA: 5 mM) were added to endothelized tissues 30 min before contraction. The involvement of lectin domain was assessed by previous incubation of LAL (30 µg/ml) with GlcNAc (0.1 M). Results: LAL (0.1-100 µg/ml) induced relaxation only in endothelized aorta, being maximal at 100 µg/ml (62.57 ± 7.8%). The relaxant effect induced by LAL at 30 µg/ml (52.49 ± 10.32%) was abolished by previous incubation with GlcNAc. LAL relaxant effect (IC50 9.75 ± 7.1) was partially reversed by indomethacin (IC50 LAL + indomethacin: 30.47 ± 10.93) and was abolished by L-NAME or TEA. Conclusion: LAL exhibits vasorelaxant activity in contracted endothelized aorta of rats, involving the lectin domain, muscarinic receptor of acetylcholine and endothelial derived relaxing factors. (AU)

Introdução: O efeito vasorrelaxante de lectinas de plantas leguminosas (Subtribo Diocleinae) já é bem descrito, embora pouco explorado para lectinas isoladas da tribo Dalbergieae, com exceção da lectina de Vatairea guianensis, que induz relaxamento com envolvimento de óxido nítrico e do domínio lectínico. Objetivo: Avaliar o efeito vasorrelaxante da lectina isolada de Lonchocarpus araripensis (LAL), tribo Dalbergieae, e o envolvimento do domínio lectínico e de fatores relaxantes derivados do endotélio (EDRF). Métodos: Anéis de aorta de ratos Wistar (250-300 g) foram montados em banho de órgãos em condições fisiológicas (Tyrode, 37 ° C, 95% de O2 e 5% de CO2, pH = 7,4) (CEUA No. 10130208-8/40). LAL (0,1-100 µg/ml) foi adicionada a tecidos pré-contraídos com fenilefrina (0,1 µM) com ou sem endotélio. Para investigar os mecanismos de relaxamento, foram adicionados inibidores de NOS (L-NAME: 100 µM), guanilato ciclase (ODQ: 10 µM), receptor muscarínico (atropina: 1 µM), ciclooxigenase (indometacina: 10 µM) ou canais de potássio (TEA: 5 mM) aos tecidos endotelizados 30 minutos antes da contração. O envolvimento do domínio lectínico foi avaliado por incubação prévia da LAL (30 µg/ml) com GlcNAc (0,1 M). Resultados: LAL (0,1-100 µg/ml) relaxou apenas anéis de aorta endotelizadas, com efeito máximo na dose de 100 µg/ml (62,57 ± 7,8%). O efeito relaxante da LAL a 30 µg/ml (52,49 ± 10,32%) foi abolido por incubação prévia com GlcNAc, atropina ou ODQ. O relaxamento da LAL (IC50 9,75 ± 7,1) a 10, 30 e 100 µg/ml foi parcialmente revertido por indometacina (IC50 LAL + indometacina: 30,47 ± 10,93) e abolido por L-NAME e TEA. Conclusão: A LAL exibe atividade vasorrelaxante em aorta endotelizada de ratos, no estado contraído, envolvendo o domínio lectínico, receptor muscarínico e fatores relaxantes derivados do endotélio. (AU)

Erythroxylum pungens elicits vasorelaxation by reducing intracellular calcium concentration in vascular smooth muscle cells of rats

The cardiovascular effects elicited by the ethanolic extract obtained from the roots of Erythroxylum pungens O.E. Schulz, Erythroxylaceae (EEEP) and the vasorelaxant effect induced by its main tropane alkaloid (pungencine) were investigated. In normotensive rats, administration of EEEP (1, 10, 30 and 60 mg/kg i.v., randomly) produced dose-dependent hypotension (-2±1, -7±0.5 -17.6±1, -24±1 Δ mmHg, n=5) followed by tachycardia (3±0.5, 7±2, 7.1±1, 10±5 Δ bpm, n=5). In intact phenylephrine (Phe, 10 µM)-pre-contracted rings, EEEP (0.01-500 µg/mL) induced concentration-dependent vasorelaxation (EC50 13.7±5.5 µg/mL, Maximal Response= 92±2.6%), and this effect was unchanged after the removal of the vascular endothelium (EC50 27.2±4.7 µg/ml, Maximal Response= 88.3±3.3 %). In KCl (80 mM)-pre-contracted-endothelium-denuded rings, EEEP elicited concentration-dependent relaxation (EC50= 128.2±11.2 µg/mL, Maximal Response 76.8±3.4%). Vasorelaxation has also been achieved with tonic contractions evoked by the L-type Ca2+ channel agonist Bay K 8644 (EC50 80.2±9.1 µg/mL, Maximal Response 86.3±8.3%). In addition, in a depolarizing medium, EEEP inhibited CaCl2 (30-500 µg/mL) induced contractions and caused a concentration-dependent rightward shift of the relaxation curves. Lastly, the tropane alkaloid pungencine caused vasorelaxation in mesenteric arteries resembling to the EEEP responses. These results suggests that EEEP induces hypotension and vasorelaxation, at least in part, due to the reduction in [Ca2+]i in vascular smooth muscle cells.

Vasorelaxant effect of Hyptis fruticosa Salzm. ex Benth., Lamiaceae, dichloromethane extract on rat mesenteric artery / Efeito vasorelaxante do extrato diclorometano de Hyptis fruticosa Salzm. ex Benth., Lamiaceae, em artéria mesentérica de ratos

Vasorelaxant effect of Hyptis fruticosa dichloromethane extract (HFDE) on isolated rings of rat mesenteric artery was evaluated in this study. In intact rings, HFDE (0.1-3000 µg/mL) induced concentration-dependent vasorelaxations (Emax = 119±14 percent; n = 6) of phenylephrine tonus that were not modified after endothelium removal (Emax = 116±6 percent; n = 6), after KCl 20 mM (Emax = 135±9 percent; n = 6) or in rings pre-contracted with KCl 80 mM (Emax = 125±4 percent; n = 6). In endothelium denuded rings, HFDE (300 or 1000 µg/mL) inhibited contractions induced by CaCl2 (maximal inhibition = 25±7 percent and 95±1 percent; respectively). Furthermore, HFDE promoted an additional vasorelaxation (15±3 percent; n = 7) after maximal response of 10 µM nifedipine (78±3 percent; n = 7). In conclusion, HFDE induces vasorelaxant effect through an endothelium-independent pathway, which appears to be due in major part to inhibition of the Ca2+ influx through voltage-operated Ca2+ channels.

O efeito vasorelaxante do extrato diclorometano de Hyptis fruticosa Salzm. ex Benth., Lamiaceae (HFDE), em anéis isolados de artéria mesentérica de ratos foi avaliado nesse estudo. Em anéis intactos, pré-contraídos com fenilefrina (10 µM), HFDE (0,1-3000 µg/mL) induziu vasorelaxamento de maneira dependente de concentração (Emax = 119±14 por cento; n = 6), o qual não foi afetado após remoção do endotélio (Emax = 116±6 por cento; n = 6), após KCl 20 mM (Emax = 135±9 por cento; n = 6) ou em anéis pré-contraídos com KCl 80 mM (Emax = 125±4 por cento; n = 6). Em anéis sem endotélio, HFDE (300 ou 1000 µg/mL) inibiu as contrações induzidas por CaCl2 (inibição máxima = 25±7 por cento e 95±1 por cento, respectivamente). Além disso, HFDE promoveu um vasorelaxamento adicional (15±3 por cento; n = 7) sobre o relaxamento máximo de 10 µM de nifedipina (78±3 por cento, n = 7). Em conclusão, HFDE induz efeito vasorelaxante através de uma via independente de endotélio, possivelmente devido à inibição do influxo de Ca2+ através de canais de Ca2+ operados por voltagem.