Effect of L-citrulline on nitrate tolerance: role of arginase

Abstract

BACKGROUND: Chronic administration of nitroglycerin (GTN) leads to nitrate tolerance, which is associated with increased arginase II activity. Supplementation of L-arginine appears to reduce nitrate tolerance. Experiments were designed to examine whether or not L-citrulline, the precursor and the metabolite of L-arginine, prevents the development of nitrate tolerance, and whether or not arginase plays a role. METHODS: Human umbilical vein endothelial cells (HUVEC) were exposed to nitroglycerin (GTN; 10 lM), in the absence and presence of L-arginine (1 mM), L-citrulline (10 mM) and/or L-norvaline (10 mM), for one and 24 h. They were then stimulated with GTN (100 lM) and the amount of cyclic guanosine triphosphate (cyclic GMP) release was measured by enzyme immunoassay. The protein expressions and the activities of arginases were examined by Western blotting and colorimetric assay, respectively. RESULTS: Prolonged (24 h), but not short-term (1 h), incubation of HUVECs with exogenous GTN caused a significant reduction of the release of cyclic GMP caused by the acute administration of the nitrate. This reduction was not affected significantly by L-citrulline or L-norvaline, while L-arginine appeared to blunt the reduction. Prolonged incubation with exogenous GTN also caused a significant increase in arginase activity, which was prevented by L-norvaline, in control cell cultures or in those co-incubated with L-citrulline, but not with L-arginine. The different incubations did not significantly affect the protein presence of arginase II, in the absence of L-norvaline; the presence of arginase I was not detectable in HUVECs with Western blotting. CONCLUSIONS: These experiments show minimal involvement of arginase II, L-citrulline and L-arginine in tolerance that develops in HUVECs following 24 h of exposure to GTN.