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The present study was designed to investigate the contribution of endothelial cell caveolae to vascular relaxation in aortas from a normotensive (2K) and renal hypertensive (2K-1C) rat. For that purpose, concentration-effect curves to acetylcholine were constructed in 2K and 2K-1C intact endothelium aortic rings, in the absence or in the presence of the caveolae disassembler methyl-beta-ciclodextrin. The potency (pD(2)) and the maximum relaxant effect to acetylcholine were greater in 2K than in 2K-1C aortas. Methyl-beta-ciclodextrin reduced the pD(2) in 2K and the maximum relaxant effect in both 2K and 2K-1C. The quantification of the caveolae number by electronic microscopy has shown a larger number of caveolae in 2K than in 2K-1C endothelial cells, which was reduced by methyl-beta-ciclodextrin in both 2K and 2K-1C. The production of NO stimulated with acetylcholine was greater in 2K than in 2K-1C endothelial cells, and this effect was impaired by methyl-beta-ciclodextrin in both 2K and 2K-1C. The cytosolic Ca(2+) concentration ([Ca(2+)]c) was simultaneously measured in endothelial and smooth muscle cells stimulated with acetylcholine by confocal image of aortic slices. Acetylcholine produced a greater [Ca(2+)]c increase in 2K than in 2K-1C endothelial cells, which response was inhibited by methyl-beta-ciclodextrin only in 2K cells. In smooth muscle cells the reduction of [Ca(2+)]c was higher in 2K than in 2K-1C. This effect was inhibited by methyl-beta-ciclodextrin only in 2K cells. Taken together, our results suggest that the decreased number of caveolae in the endothelial cells from 2K-1C rat aortas is involved in the impaired effect of acetylcholine on [Ca(2+)]c and NO.

Abstract:The pharmacological effects on the cardiovascular system of yangambin, a lignan isolated from Ocotea duckei Vattimo (Lauraceae), were studied in rats using combined functional and biochemical approaches. In non-anaesthetized rats, yangambin (1, 5, 10, 20, 30 mg/kg, i.v.) induced hypotension (−3.5 ± 0.2; −7.1 ± 0.8; −8.9 ± 1.3; −14 ± 2.3, −25.5% ± 2.6%, respectively) accompanied by tachycardia (5.9 ± 0.5; 5.9 ± 1.6; 8.8 ± 1.4; 11.6, 18.8% ± 3.4%, respectively). In isolated rat atria, yangambin (0.1 µM-1 mM) had very slight negative inotropic (Emax = 35.6% ± 6.4%) and chronotropic effects (Emax = 10.2% ± 2.9%). In endothelium-intact rat mesenteric artery, yangambin (0.1 µM-1 mM) induced concentration-dependent relaxation (pD 2 = 4.5 ± 0.06) of contractions induced by phenylephrine and this effect was not affected by removal of the endothelium. Interestingly, like nifedipine, the relaxant effect induced by yangambin was OPEN ACCESSMolecules 2014, 19 6864 more potent on the contractile response induced by KCl 80 mM (pD 2 = 4.8 ± 0.05) when compared to that induced by phenylephrine. Furthermore, yangambin inhibited CaCl 2 -induced contractions in a concentration-dependent manner. This lignan also induced relaxation (pD 2 = 4.0 ± 0.04) of isolated arteries pre-contracted with S(−)-Bay K 8644. In fura-2/AMloaded myocytes of rat mesenteric arteries, yangambin inhibited the Ca 2+ signal evoked by KCl 60 mM. In conclusion, these results suggest that the hypotensive effect of yangambin is probably due to a peripheral vasodilatation that involves, at least, the inhibition the Ca 2+ influx through voltage-gated Ca 2+ channels.

Endothelium-derived relaxing factors contribute to smooth muscle relaxation. The aim of the present study was to investigate the contribution of nitric oxide (NO) produced in the endothelial cells to the vasodilatation stimulated with forskolin in rat aorta. Forskolin that directly activates adenylyl-cyclase, induced complete relaxation in phenylephrine-contracted aortas. Endothelium removal reduced the potency (pEC₅₀) of forskolin without changes in the maximum effect (Emax). However, the inhibitor of endothelial NO-synthase (10 μM L- NG-Nitroarginine, L-NNA) reduced both Emax and pEC₅₀ in intact endothelium aortic rings. L-NNA or L-NNA plus cyclooxygenase inhibitor indomethacin (10 μM) reduced both Emax and pEC₅₀ of forskolin. Forskolin increased both the cytosolic Ca⁺² concentration and the cytosolic NO concentration ([NO]c) in the endothelial cells. The PKA inhibitor KT5720 reduced the NO production activated by forskolin in the endothelial cells. The enhanced [NO]c in the endothelial cells increased cyclic guanosine-monophosphate (cGMP) in smooth muscle cells, which was abolished by L-NNA. Taken together, our results indicate that vasodilatation mediated by forskolin in rat aortic rings is potentiated by NO production in endothelial cells that increases the cGMP levels in the smooth muscle cells that along with cAMP contribute to the vasodilatation