Effect of L-citrulline on nitrate tolerance in human umbilical vein endothelial cells

Abstract

The primary functions of the cardiovascular system are to transport the required nutrients to sustain the metabolic activity of tissues and to remove the metabolic waste products from tissue. Therefore, a steady supply of oxygen for the cells is crucial. Vascular endothelium synthesizes both relaxing and contracting factors to regulate the local tissue blood flow. Nitric oxide (NO) is one of the major endothelium-derived relaxing factor. It is synthesized by endothelial nitric oxide synthase (eNOS), which interacts with soluble guanylyl cyclase in smooth muscle to produce cyclic guanosine monophosphate (cGMP) resulting in vascular relaxation.

Nitroglycerin (GTN) is an organic nitrate used for the management of angina pectoris, hypertension and congestive heart failure, it is converted to NO or closely related moleculesin the vasculature resulting in vasodilatation. It has been demonstrated that chronic administration of GTN leads to nitrate tolerance, which is associated with increased arginase activity and reactive oxidative species (ROS)production. Supplement of L-arginine appears to reduce nitrate tolerance, however, the bioavailability of L-arginine is limited. It has been reported that L-citrulline supplement in human results in elevated plasma level of L-arginine.

This study was designed to investigate the effect of long-term GTN treatment on the expressions and activities of arginase I, arginase II and eNOS, and on the production of cGMP inhuman umbilical vein endothelial cells (HUVECs).Moreover, the effect of L-citrulline on nitrate tolerance due to GTN treatment was examined. HUVECs were treated with GTN (10 µM), thrombin, L-arginine, L-citrulline and L-norvaline, alone or in combinations, for 1 hour (short-term treatment) or 24 hours(long-term treatment) followed by 30 minutes stimulation by GTN (100 µM).Western immunoblotting was used to measure the protein expression levelsof eNOS, arginase I and arginase II. Enzyme immunoassay and colorimetric assay were performed to determine the cGMP level and the arginase activities, respectively.

Our results suggested that the amount of cGMP release in response to acute (30 minutes) GTN stimulation (100 µM)was reduced in HUVECs that were pre-treated with24 hour GTN(10 µM), and this is associated with an increased arginase activity. L-arginine,L-citrullineor L-norvaline alone was not able to prevent this reduction in cGMP release (nitrate tolerance), although L-arginine and L-norvaline, but not L-citrulline, prevented the increase in arginase activity. The combination of L-arginine or L-citrulline with L-norvaline is effective to protect HUVECs against nitrate tolerance. On the other hand, thrombin (a stimulator of eNOS, 1 U/ml) pre-treatment for 24 hours also reduced cGMP production toacute GTN stimulation. Similar to long-term GTN-induced nitrate tolerance, long-term thrombin-induced nitrate tolerance was not prevented by concomitant presence of L-arginine, L-citrulline or L-norvaline. Long-term thrombinpre-treatment also increased arginase activity and this effect was inhibited by L-arginine, L-citrulline and L-norvaline. The increased arginase activity by long-term GTN or thrombin pre-treatment was not associated with increased arginase protein expression.

In conclusion, the present data suggested that prolonged pre-exposure to exogenous GTN or endogenous increase NO by thrombin induced nitrate tolerance in HUVECs, and this is unlikely the consequence of the increased arginase activity. In addition, neither L-arginine nor L-citrulline were effective in protecting HUVECs against nitrate tolerance, and only the combination of L-citrulline and L-norvaline or L-arginine and L-norvaline were effective.