Nitric oxide-dependent vasodilation and Ca²⁺signalling induced by erythrodiol in rat aorta

Objective: To evaluate the pharmacological property of erythrodiol, a natural triterpenoid contained in propolis, as vasodilatory agent, and to determine its mechanism of action. Methods: Rats aortic rings were isolated and suspended in organ baths, and the effects of erythrodiol were studied by means of isometric tension recording experiments. Nitric oxide (NO) was detected by ozone-induced chemiluminescence. The technique used to evaluate changes in intracellular Ca2+ concentration in intact endothelium was opened aortic ring and loaded with 16 μmol Fura-2/AM for 60 min at room temperature, washed and fixed by small pins with the luminal face up. In situ, ECs were visualized by an upright epifluorescence Axiolab microscope (Carl Zeiss, Oberkochen, Germany) equipped with a Zeiss×63 Achroplan objective (water immersion, 2.0 mm working distance, 0.9 numerical apertures). ECs were excited alternately at 340 and 380 nm, and the emitted light was detected at 510 nm. Results: In aortic rings with intact endothelium pre-contracted with norepinephrine (10-4 mol/L), the addition of erythrodiol (10-8 -10 -4 mol/L) induced vasorelaxation in a concentration-dependent manner; in endothelium-denuded rings, the relaxant response induced by erythrodiol was almost completely abolished suggesting that vasorelaxation was endothelium-dependent. They had almost no relaxant effect on depolarised or endothelium-denuded aortic segments. The relaxation was significantly attenuated by pre-treatment with the NO synthase inhibitor Nv-nitro-L-arginine-methylester. Erythrodiol (10-4 mol/L) was able to significantly increase NOx levels. This effect was completely abolished after removal of the vascular endothelium. Erythrodiol (100 μmol/L) caused a slow, long-lasting increase in intracellular Ca2+ concentration. These results further supported the hypothesis that erythrodiol can induce activation of the NO/soluble guanylate cyclase/cyclic guanosine monophosphate pathway, as suggested by functional studies. Conclusions: The present results suggest that the mechanism of relaxation seems to be mainly mediated by the endothelial production of NO. Such a vasorelaxation was an endothelium-dependent effect, via the NO/soluble guanylate cyclase/cyclic guanosine monophosphate pathway. This result also suggests that erythrodiol causes a slow influx of extracellular Ca2+ release from the intracellular Ca2+ stores and an inhibition of Ca2+ extruding mechanism. It can be concluded that erythrodiol may have interesting therapeutic potential as a new vasodilator drug, for protecting the cardiovascular system.