The L-arginine precursor, L-citrulline, re-couples endothelial nitric oxide synthase (eNOS), increases nitric oxide (NO) production, and ameliorates chronic hypoxia-induced pulmonary hypertension (PH) in newborn pigs. L-arginine can induce arginase, which, in turn, may diminish NO production. Our major purpose was to determine if L-citrulline increases arginase activity in hypoxic piglet pulmonary arterial endothelial cells (PAECs), and if so, concomitantly impacts the ability to increase eNOS re-coupling and NO production. Piglet PAECs were cultured in hypoxic conditions with L-citrulline (0-3 mM) and/or the arginase inhibitor S-(2-boronoethyl)-L-cysteine, BEC. We measured arginase activity and NO production. We assessed eNOS coupling by measuring eNOS dimers and monomers. L-citrulline concentrations > 0.5 mM increased arginase activity in hypoxic PAECs. L-citrulline concentrations > 0.1 mM increased NO production and concentrations > 0.5 mM elevated eNOS dimer-to-monomer ratios. Co-treatment with L-citrulline and BEC elevated eNOS dimer-to-monomer ratios more than sole treatment. Despite inducing arginase, L-citrulline increased NO production and eNOS coupling in hypoxic piglet PAECs. However, these dose-dependent findings raise the possibility that there could be L-citrulline concentrations that elevate arginase to levels that negate improvements in eNOS dysfunction. Moreover, our findings suggest that combining an arginase inhibitor with L-citrulline merits evaluation as a treatment for chronic hypoxia-induced PH.

No abstract

Concomitant with developing pulmonary hypertension (PH), newborn piglets exposed to chronic hypoxia develop pulmonary vascular NO signaling impairments. PH is reduced and NO signaling is improved in chronically hypoxic piglets treated with the NO‐arginine precursor, L‐citrulline. Folic acid positively impacts NO signaling. We evaluated whether the effect on NO signaling and PH is greater using co‐treatment with folic acid and L‐citrulline than either alone. From day 3 to day 10 of hypoxia, piglets were treated solely with folic acid, solely with L‐citrulline, or co‐treated with both. Catheters were placed to measure in vivo hemodynamics. NO production was measured in vitro in dissected pulmonary arteries. Compared to normoxic piglets, pulmonary vascular resistance (PVR) was elevated and NO production was reduced in untreated hypoxic piglets. Regardless of treatment strategy, PVR was less in all three treated groups of hypoxic piglets when compared to the untreated hypoxic group. In addition, for all three groups of treated hypoxic piglets, NO production was higher than the untreated group. Improvements in PVR and NO production did not differ between piglets co‐treated with folic acid and L‐citrulline and those treated solely with either. Thus, the impact on NO production and PVR was not augmented by combining folic acid and L‐citrulline treatments. Nonetheless, treatment with folic acid, either singly or when combined with L‐citrulline, increases NO production and inhibits PH in chronically hypoxic newborn piglets. Folic acid merits consideration as a therapy for PH in human infants with chronic heart and lung conditions that are associated with chronic hypoxia.

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.