Impact of l‐citrulline on nitric oxide signaling and arginase activity in hypoxic human pulmonary artery endothelial cells

Impaired nitric oxide (NO) signaling contributes to the development of pulmonary hypertension (PH). The l‐arginine precursor, l‐citrulline, improves NO signaling and has therapeutic potential in PH. However, there is evidence that l‐citrulline might increase arginase activity, which in turn, has been shown to contribute to PH. Our major purpose was to determine if l‐citrulline increases arginase activity in hypoxic human pulmonary artery endothelial cells (PAECs). In addition, to avoid potential adverse effects from high dose l‐citrulline monotherapy, we evaluated whether the effect on NO signaling is greater using co‐treatment with l‐citrulline and another agent that improves NO signaling, folic acid, than either alone. Arginase activity was measured in human PAECs cultured under hypoxic conditions in the presence of l‐citrulline (0–1 mM). NO production and endothelial nitric oxide synthase (eNOS) coupling, as assessed by eNOS dimer‐to‐monomer ratios, were measured in PAECs treated with l‐citrulline and/or folic acid (0.2 μM). Arginase activity increased in hypoxic PAECs treated with 1 mM but not with either 0.05 or 0.1 mM l‐citrulline. Co‐treatment with folic acid and 0.1 mM l‐citrulline increased NO production and eNOS dimer‐to‐monomer ratios more than treatment with either alone. The potential to increase arginase activity suggests that there might be plasma l‐citrulline concentrations that should not be exceeded when using l‐citrulline to treat PH. Rather than progressively increasing the dose of l‐citrulline as a monotherapy, co‐therapy with l‐citrulline and folic acid merits consideration, due to the possibility of achieving efficacy at lower doses and minimizing side effects.