L-Citrulline ameliorates chronic hypoxia-induced pulmonary hypertension in newborn piglets

Abstract

Newborn piglets develop pulmonary hypertension and have diminished pulmonary vascular nitric oxide (NO) production when exposed to chronic hypoxia. NO is produced by endothelial NO synthase (eNOS) in the pulmonary vascular endothelium using l-arginine as a substrate and producing l-citrulline as a byproduct. l-Citrulline is metabolized to l-arginine by two enzymes that are colocated with eNOS in pulmonary vascular endothelial cells. The purpose of this study was to determine whether oral supplementation with l-citrulline during exposure of newborn piglets to 10 days of chronic hypoxia would prevent the development of pulmonary hypertension and increase pulmonary NO production. A total of 17 hypoxic and 17 normoxic control piglets were studied. Six of the 17 hypoxic piglets were supplemented with oral l-citrulline starting on the first day of hypoxia. l-Citrulline supplementation was provided orally twice a day. After 10 days of hypoxia or normoxia, the animals were anesthetized, hemodynamic measurements were performed, and the lungs were perfused in situ. Pulmonary arterial pressure and pulmonary vascular resistance were significantly lower in hypoxic animals treated with l-citrulline compared with untreated hypoxic animals (P < 0.001). In vivo exhaled NO production (P = 0.03) and nitrite/nitrate accumulation in the perfusate of isolated lungs (P = 0.04) were significantly higher in l-citrulline-treated hypoxic animals compared with untreated hypoxic animals. l-Citrulline supplementation ameliorated the development of pulmonary hypertension and increased NO production in piglets exposed to chronic hypoxia. We speculate that l-citrulline may benefit neonates exposed to prolonged periods of hypoxia from cardiac or pulmonary causes.

Fig. 1.

A: mean pulmonary arterial pressure measurements in normoxic control (n = 6), chronically hypoxic (n = 11), and l-citrulline-treated chronically hypoxic (n = 6) piglets. B: calculated pulmonary vascular resistance in normoxic control (n = 6), chronically hypoxic (n = 11), and l-citrulline-treated chronically hypoxic (n = 6) piglets. Values are means ± SD. Significantly different from normoxic control (*) and chronically hypoxic (⁺) (P < 0.05; ANOVA with post hoc comparison test).

Fig. 2.

A: exhaled nitric oxide in normoxic control (n = 6), chronically hypoxic (n = 11), and l-citrulline-treated chronically hypoxic (n = 5) piglets. B: nitrite/nitrate accumulation in lung perfusate in normoxic control (n = 17), chronically hypoxic (n = 9), and l-citrulline-treated chronically hypoxic (n = 5) piglets. Values are means ± SD. Significantly different from *normoxic control (*) and chronically hypoxic (⁺) (P < 0.05; ANOVA with post hoc comparison test).

Fig. 3.

A: immunoblot for eNOS protein reprobed for beta actin for lung tissue from normoxic controls (n = 3), chronic hypoxic (n = 3), and l-citrulline-treated chronic hypoxic (n = 3) piglets. B: densitometry of eNOS normalized to beta actin for lung tissue from normoxic controls (n = 3), chronic hypoxic (n = 3), and l-citrulline-treated chronic hypoxic (n = 3) piglets Values are means ± SD. *Significantly different from normoxic control (P < 0.05; ANOVA with post hoc comparison test).

Fig. 4.

A: immunoblot for nNOS protein reprobed for beta actin for lung tissue from normoxic controls (n = 3), chronic hypoxic (n = 3), and l-citrulline-treated chronic hypoxic (n = 3) piglets. B: densitometry of nNOS normalized to beta actin for lung tissue from normoxic controls (n = 3), chronic hypoxic (n = 3), and l-citrulline-treated chronic hypoxic (n = 3) piglets. Values are means ± SD.