Combined l-citrulline and tetrahydrobiopterin therapy improves NO signaling and ameliorates chronic hypoxia-induced pulmonary hypertension in newborn pigs.

Newborn pigs with chronic hypoxia-induced pulmonary hypertension (PH) have evidence of endothelial nitric oxide synthase (eNOS) uncoupling. In this model, we showed that therapies that promote eNOS coupling, either tetrahydrobiopterin (BH(4)), a NOS cofactor, or l-citrulline, a NO-l-arginine precursor, inhibit PH. We wanted to determine whether cotreatment with l-citrulline and a BH(4) compound, sapropterin dihydrochloride, improves NO signaling and chronic hypoxia-induced PH more markedly than either alone. Normoxic (control) and hypoxic piglets were studied. Some hypoxic piglets received sole treatment with l-citrulline or BH(4), or were cotreated with l-citrulline and BH(4), from day 3 through day 10 of hypoxia. Catheters were placed for hemodynamic measurements, and pulmonary arteries were dissected to assess eNOS dimer-to-monomer ratios and NO production. In untreated hypoxic piglets, pulmonary vascular resistance (PVR) was higher and NO production and eNOS dimer-to-monomer ratios were lower than in normoxic piglets. Compared with the untreated hypoxic group, PVR was lower in hypoxic piglets cotreated with l-citrulline and BH(4) and in those treated with l-citrulline alone but not for those treated solely with BH(4). NO production and eNOS dimer-to-monomer ratios were greater for all three treated hypoxic groups compared with the untreated group. Notably, greater improvements in PVR, eNOS dimer-to-monomer ratios, and NO production were found in hypoxic piglets cotreated with l-citrulline and BH(4) than in piglets treated with either alone. Cotreatment with l-citrulline and BH(4) more effectively improves NO signaling and inhibits chronic hypoxia-induced PH than either treatment alone. Combination therapies may offer enhanced therapeutic capacity for challenging clinical conditions, such as chronic neonatal PH.