Endothelial nitric oxide production is tightly coupled to the citrulline–NO cycle

Nitric oxide (NO) is an important vasorelaxant produced along with l-citrulline from l-arginine in a reaction catalyzed by endothelial nitric oxide synthase (eNOS). Previous studies suggested that the recycling of l-citrulline to l-arginine is essential for NO production in endothelial cells. However, there is no direct evidence demonstrating the degree to which the recycling of l-citrulline to l-arginine is coupled to NO production. We hypothesized that the amount of NO formed would be significantly higher than the amount of l-citrulline formed due to the efficiency of l-citrulline recycling via the citrulline–NO cycle. To test this hypothesis, endothelial cells were incubated with [14C]-l-arginine and stimulated by various agents to produce NO. The extent of NO and [14C]-l-citrulline formation were simultaneously determined. NO production exceeded apparent l-citrulline formation of the order of 8 to 1, under both basal and stimulated conditions. As further support, α-methyl-dl-aspartate, an inhibitor of argininosuccinate synthase (AS), a component of the citrulline–NO cycle, inhibited NO production in a dose-dependent manner. The results of this study provide evidence for the essential and efficient coupling of l-citrulline recycling, via the citrulline–NO cycle, to endothelial NO production.