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.

Activation of PI3K/Akt pathway mediated by estrogen receptors accounts for estrone-induced vascular activation of cGMP signaling.

Estrone (E1) produces remarkable vascular effects, including relaxation, modulation of proliferation, apoptosis and cell adhesion. This study investigated the role of estrogen receptors and endothelial signaling pathways in the vascular relaxation promoted by E1. Aortic rings from male Wistar rats (250-300 g) were contracted with phenylephrine and stimulated with graded concentrations of E1. The concentration-dependent relaxation induced by E1 was abolished after removal of the endothelium or incubation with the estrogen receptor antagonist ICI 182,780. G protein-coupled estrogen receptor antagonism did not alter the E1 effect. Pretreatment of endothelium-intact arteries with inhibitors of nitric oxide synthase, guanylyl cyclase, calmodulin (CaM) and PI3K reduced the E1-induced vasorelaxation. Incubation with inhibitors of the MEK/ERK1/2 or p38MAPK pathways did not alter the E1 vasorelaxation. Similarly, inhibition of cyclooxygenase or blockade of potassium channels did not change the E1 effect. Western blot analysis evidenced that E1 induces phosphorylation of eNOS, PI3K and Akt in rat aorta. Our data demonstrate that E1 induces aortic vascular relaxation through classic estrogen receptors activation on the endothelium. We also identify CaM and PI3K/Akt pathways as critical mediators of the NO-cGMP signaling activation by E1. These findings contribute to the notion that this estrogen regulates arterial function and represents another link, besides 17ß-estradiol (E2), between postmenopause and vascular dysfunction.

The vasorelaxant effect of gallic acid involves endothelium-dependent and -independent mechanisms.

The mechanisms of action involved in the vasorelaxant effect of gallic acid (GA) were examined in the isolated rat thoracic aorta. GA exerted a relaxant effect in the highest concentrations (0.4-10mM) in both endothelium-intact and endothelium-denuded aortic rings. Pre-incubation with L-NAME, ODQ, calmidazolium, TEA, 4-aminopyridine, and barium chloride significantly reduced the pEC50 values. Moreover, this effect was not modified by indomethacin, wortmannin, PP2, glibenclamide, or paxillin. Pre-incubation of GA (1, 3, and 10mM) in a Ca(2+)-free Krebs solution attenuated CaCl2-induced contractions and blocked BAY K8644-induced vascular contractions, but it did not inhibit a contraction induced by the release of Ca(2+) from the sarcoplasmatic reticulum stores. In addition, a Western blot analysis showed that GA induces phosphorylation of eNOS in rat thoracic aorta. These results suggest that GA induces relaxation in rat aortic rings through an endothelium-dependent pathway, resulting in eNOS phosphorylation and opening potassium channels. Additionally, the relaxant effect by an endothelium-independent pathway involves the blockade of the Ca(2+) influx via L-type Ca(2+) channels.

Endothelium-Dependent Vasorelaxant Effect of Butanolic Fraction from Caryocar brasiliense Camb. Leaves in Rat Thoracic Aorta.

Caryocar brasiliense Camb. "pequi" is a native plant from the Cerrado region of Brazil that contains bioactive components reported to be antioxidant agents. Previous work has demonstrated that dietary supplementation with pequi decreased the arterial pressure of volunteer athletes. We found that the crude hydroalcoholic extract (CHE) of C. brasiliense leaves relaxed, in a concentration-dependent manner, rat aortic rings precontracted with phenylephrine, and that the butanolic fraction (BF) produced an effect similar to that of the CHE. Aortic relaxation induced by BF was abolished by endothelium removal, by incubation of the nitric oxide synthase inhibitor L-NAME, or the soluble guanylatecyclase inhibitor ODQ. However, incubation with atropine and pyrilamine had no effect on the BF-induced vasorelaxation. Moreover, this effect was not inhibited by indomethacin and tetraethylammonium. The concentration-response curve to calcium in denuded-endothelium rings was not modified after incubation with BF, and the vasorelaxation by BF in endothelium-intact rings precontracted with KCl was abolished after incubation with L-NAME. In addition, administration of BF in anesthetized rats resulted in a reversible hypotension. The results reveal that C. brasiliense possesses both in vivo and in vitro activities and that the vascular effect of BF involves stimulation of the nitric oxide/cyclic GMP pathway.

Mesenteric microcirculatory dysfunctions and translocation of indigenous bacteria in a rat model of strangulated small bowel obstruction.

PURPOSE: Bacterial translocation has been shown to occur in critically ill patients after extensive trauma, shock, sepsis, or thermal injury. The present study investigates mesenteric microcirculatory dysfunctions, the bacterial translocation phenomenon, and hemodynamic/metabolic disturbances in a rat model of intestinal obstruction and ischemia. METHODS: Anesthetized (pentobarbital 50 mg/kg, i.p.) male Wistar rats (250-350 g) were submitted to intestinal obstruction or laparotomy without intestinal obstruction (Sham) and were evaluated 24 hours later. Bacterial translocation was assessed by bacterial culture of the mesenteric lymph nodes (MLN), liver, spleen, and blood. Leukocyte-endothelial interactions in the mesenteric microcirculation were assessed by intravital microscopy, and P-selectin and intercellular adhesion molecule (ICAM)-1 expressions were quantified by immunohistochemistry. Hematocrit, blood gases, lactate, glucose, white blood cells, serum urea, creatinine, bilirubin, and hepatic enzymes were measured. RESULTS: About 86% of intestinal obstruction rats presented positive cultures for E. coli in samples of the mesenteric lymph nodes, liver, and spleen, and 57% had positive hemocultures. In comparison to the Sham rats, intestinal obstruction induced neutrophilia and increased the number of rolling (approximately 2-fold), adherent (approximately 5-fold), and migrated leukocytes (approximately 11-fold); this increase was accompanied by an increased expression of P-selectin (approximately 2-fold) and intercellular adhesion molecule-1 (approximately 2-fold) in the mesenteric microcirculation. Intestinal obstruction rats exhibited decreased PaCO2, alkalosis, hyperlactatemia, and hyperglycemia, and increased blood potassium, hepatic enzyme activity, serum urea, creatinine, and bilirubin. A high mortality rate was observed after intestinal obstruction (83% at 72 h vs. 0% in Sham rats). CONCLUSION: Intestinal obstruction and ischemia in rats is a relevant model for the in vivo study of mesenteric microcirculatory dysfunction and the occurrence of bacterial translocation. This model parallels the events implicated in multiple organ dysfunction (MOD) and death.

Mesenteric microcirculatory dysfunctions and translocation of indigenous bacteria in a rat model of strangulated small bowel obstruction

PRUPOSE: Bacterial translocation has been shown to occur in critically ill patients after extensive trauma, shock, sepsis, or thermal injury. The present study investigates mesenteric microcirculatory dysfunctions, the bacterial translocation phenomenon, and hemodynamic/metabolic disturbances in a rat model of intestinal obstruction and ischemia. METHODS: Anesthetized (pentobarbital 50 mg/kg, i.p.) male Wistar rats (250-350 g) were submitted to intestinal obstruction or laparotomy without intestinal obstruction (Sham) and were evaluated 24 hours later. Bacterial translocation was assessed by bacterial culture of the mesenteric lymph nodes (MLN), liver, spleen, and blood. Leukocyte-endothelial interactions in the mesenteric microcirculation were assessed by intravital microscopy, and P-selectin and intercellular adhesion molecule (ICAM)-1 expressions were quantified by immunohistochemistry. Hematocrit, blood gases, lactate, glucose, white blood cells, serum urea, creatinine, bilirubin, and hepatic enzymes were measured. RESULTS: About 86 percent of intestinal obstruction rats presented positive cultures for E. coli in samples of the mesenteric lymph nodes, liver, and spleen, and 57 percent had positive hemocultures. In comparison to the Sham rats, intestinal obstruction induced neutrophilia and increased the number of rolling (~2-fold), adherent (~5-fold), and migrated leukocytes (~11-fold); this increase was accompanied by an increased expression of P-selectin (~2-fold) and intercellular adhesion molecule-1 (~2-fold) in the mesenteric microcirculation. Intestinal obstruction rats exhibited decreased PaCO2, alkalosis, hyperlactatemia, and hyperglycemia, and increased blood potassium, hepatic enzyme activity, serum urea, creatinine, and bilirubin. A high mortality rate was observed after intestinal obstruction (83 percent at 72 h vs. 0 percent in Sham rats). CONCLUSION: Intestinal obstruction and ischemia in rats is a relevant model for ...