Nitric oxide-dependent vasodilation and intracellular Ca 2+ concentration increase induced by 6,8-dihydroxy- 4'-methoxyflavone in rat aorta

In this study, we attempted to evaluate the role of endothelium-derived hyperpolarizing factor (EDHF) in the vasorelaxant response induced by 6,8-Dihydroxy-4’-methoxyflavone (DMF) in isolated rat aorta rings by using functional and biochemical approaches. Thoracic aortic rings were isolated and suspended in organ baths, and the effects of DMF were studied by means of isometric tension recording experiments. Nitric oxide (NO) wasdetected by ozone-induced chemiluminescence. The technique used to evaluate changes in intracellular ([Ca2+]i) in intact endothelium was determined by open 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 electrical conductivities (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. In aortic rings with intact endothelium pre-contracted with norepinephrine (NE) (10-4 M), the addition of DMF (10−8 to 10−4 M) induced vasorelaxation in a concentration-dependent manner; in endothelium-denuded rings, the relaxant response induced by DMF was almost completely abolished suggesting that vasorelaxation was endothelium-dependent. DMF (10−4 M) was able to significantly increase NOx levels. This effect was completely abolished after removal of the vascular endothelium. DMF (100 µM) caused a slow, long-lasting increase in [Ca2+]i. These results further support the hypothesis that DMF can induce activation of the NO/sGC/cGMP pathway, as suggested by functional studies. The results of the present study, using combined functional and biochemical in vitro approaches, indicated that DMF relaxes pre-contracted isolated rat aortic rings. Such a vasorelaxation was an endothelium-dependent effect, via the NO/sGC/cGMP pathway. This result also suggests that DMF causes a slow influx of extracellular Ca2+. Release from the intracellular Ca2+ stores and an inhibition of Ca2+extruding mechanisms.   Key words: 6,8-dihydroxy-4’-methoxyflavone, aorta, nitric oxide (NO), Ca2+ signalling, endothelium, NO–cGMP pathway, vasodilation.