Effect of flavonols on the activity of arginase and the action of nitroglycerin in endothelial cells with and without previous exposure to nitroglycerin

Abstract

Organic nitrates have been effective treatment for ischemic heart disease for over 100 years. However, there is limitation in their clinical utility since prolonged use of these drugs results in rapid development of nitrate tolerance, which is associated with increased arginase activity and production of reactive oxygen species. Quercetin, a flavonol abundantly found in fruits and vegetables, has been shown to reduce nitrate tolerance in vitro. The objective of this study is to investigate the potential effect of quercetin and three other flavonols (namely, kaempferol, myricetin and rutin) on the development of nitrate tolerance and the activity of arginase in endothelial cells.

Human umbilical vein endothelial cells (HUVECs) were incubated with or without nitroglycerin (GTN) and/or flavonols followed by an acute stimulation with GTN. Level of cyclic guanosine monophosphate (cGMP) released into the cell medium was measured by an enzyme immunoassay. Activity of arginase in cell lysate was measured by a quantitative colorimetric arginase determination assay.

Prior treatment with GTN at 〖10〗^(-5)M for either one or 24 hours did not affect the level of cGMP released from HUVECs induced by subsequent stimulation with GTN. On the other hand, arginase activity was significantly decreased in HUVECs pre-treated with GTN at 〖10〗^(-5)M alone for 24 hours and the decrease was not affected by the concomitant presence of the flavonols during the incubation period. However, the data obtained in HUVECs pre-treated with GTN for 24 hours is questionable due to the lack of a corresponding control (i.e. cells incubating with medium for 24 hours) for proper comparison.

Pre-treatment for one hour with myricetin (〖10〗^(-5) M) and rutin (〖10〗^(-5) M), alone but not in combination with GTN (〖10〗^(-5) M), appears to increase the release of cGMP to subsequent stimulation by GTN (〖10〗^(-5) M). Rutin pre-treatment for one hour also seems to decrease the activity of arginase in HUVECs. However, these effects of myricetin and rutin were significant only when compared to the control group (without pre-treatment with GTN) but not when comparison was made to the vehicle-treated group, while there is no significant difference between the control and the vehicle group in both the cGMP release and arginase activity. As such, these potential effects of myricetin and rutin are inconclusive.

The inability to induce nitrate tolerance in the present experimental condition does not allow further investigation on the potential effect of flavonols on nitrate tolerance. In addition, there are limitations in the present study [namely, lack of corresponding control for the 24 hour incubation groups and small sample size (n = 2-3)]. Therefore, the findings need to be interpreted with cautions; improvements of the present experimental design and increasing the number of experiments are needed in order to obtain more conclusive findings. Future experiments should also be performed in other vascular cells in addition to endothelial cells, as flavonols may exert their beneficial effect in an endothelium-independent manner.