IJCS | Volume 32, Nº2, May/June 2019

214 Brozzo et al. Curcuma longa abolishes contractions in the aortic artery Int J Cardiovasc Sci. 2019;32(3)207-216 Original Article (L-NA) and the corresponding methyl ester, NG-nitro- L-arginine-methyl ester (L-NAME). 26 These analogues compete with L-arginine and act as stereospecific inhibitors of NOS. 21 After the blockade of NO production (Figure 2), it was verified that during the AECL effect there is an important involvement of NO in the vasorelaxant effect production. In addition to the involvement of NO in the AECL effect, it is scientifically obligatory to investigate the participation of other pathways in relation to the observed effects, especially since it is a crude extract and, thus, a compound consisting of many substances. Therefore, it is possible to suggest that other endothelium-derived vasorelaxant pathways and other independent ones may also be involved in the AECL vasorelaxant activity. The prostaglandins are synthesized from the arachidonic acid,which is released fromthephospholipids of the endothelial cell membrane by the activated phospholipases (A2 and C), and through the action of cyclooxygenase enzymes (COX-1 and COX-2). 27 Once released, primarily PGI2 and PGE2, they diffuse into smooth muscle cells, promoting the vasorelaxant effect through IP and EP4 receptors, increased cAMP and by reducing intracellular Ca 2+ . 28 The blockade of vasorelaxant prostaglandins synthesis occurs through the inactivation of arachidonic acid- degrading enzymes (COX-1 and COX2). 27 Indomethacin is a non-selective inhibitor of these prostaglandins. 29 In the presence of this blockade and the reduction of the vasorelaxant effect produced by the extract (shown in Figure 3), it is also possible to suggest the involvement of prostaglandins. The muscarinic receptors are also present in the vascular endothelium; they can be found in many cell types and participate in cell signaling as soon as they are activated by the ligand acetylcholine. 30 There are five subtypes of muscarinic receptors that are currently accepted, namely M1, M2, M3, M4 and M5, with the M3 receptors being the ones that contribute to smooth muscle contraction, glandular secretion and endothelial NO secretion. It is through the production of endothelial NO that ACh has vasodilatory effects in vivo . 31 Aiming to verify the involvement of the muscarinic pathway in the AECL relaxant effect, the antagonism of the muscarinic receptors was performed through incubation with atropine, a muscarinic receptor antagonist that acts through competitiveness in these receptors, preventing ACh from binding and exercising its activity. In this approach (Figure 4), where the vasorelaxant response of AECL was more attenuated, muscarinic receptors seem to be the most significantly implicated (p < 0.01) in the vasorelaxant effect induced by the AECL in the isolated thoracic aorta of rats. The relaxant response in annuli (E-) was also important in these approaches to evaluate the possible involvement of ion channels. The activity of K + channels is an essential mechanism for the regulation of the vascular muscle cell membrane potential, being an important determinant of vascular tone. 32 The opening of a potassium channel present in the membrane of vascular muscle cells causes an increase in ion output, from the intracellular medium into the extracellular medium, causing cell membrane hyperpolarization and then a blockade of voltage- dependent Ca 2+ channels and a consequent decrease in the input of Ca 2+ ions into the intracellular medium, causing vascular relaxation. 32,33 Conversely, the closing of potassium channels causes a state of depolarization, opening of voltage-dependent Ca 2+ channels, increase in intracellular Ca 2+ and vasoconstriction. 32 This mechanism of smooth-muscle relaxation and contraction differs regarding the PHE mechanism of action, which directly stimulates the α -adrenergic receptors of the G protein, acting via phospholipase C, increasing the levels of IP3, and resulting in the release of intracellular Ca 2+ , causing muscular contraction. 34 These results, shown in Figure 5, demonstrate that part of the AECL relaxant effect is related to the influence that the extract seems to have on the opening of ion channels, especially K + channels, since the hyperpolarization of the smooth muscle cell membrane can be produced by agents that open the K + channels, leading to the cell’s K + efflux. 35 K + -induced contraction in the aorta seems to be dependent on the influx of Ca 2+ through VOCCs, whereas the contraction is inhibited by the removal of Ca 2+ from the external environment and by the blockers of these channels. 36 At the same time that the opening of Ca 2+ channels may be required by a membrane depolarization process, the agents that produce membrane hyperpolarization can cause the Ca 2+ channels to close, reducing the Ca 2+ influx and promoting smooth muscle relaxation. 37 Ionic channels also seem to be involved in the AECL significant vasorelaxant activity, with a greater expression in the opening of K + channels than in the blockade to VOCCs.