Rescue Treatment with L-Citrulline Inhibits Hypoxia-Induced Pulmonary Hypertension in Newborn Pigs

Abstract

Infants with cardiopulmonary disorders associated with hypoxia develop pulmonary hypertension. We previously showed that initiation of oral L-citrulline before and continued throughout hypoxic exposure improves nitric oxide (NO) production and ameliorates pulmonary hypertension in newborn piglets. Rescue treatments, initiated after the onset of pulmonary hypertension, better approximate clinical strategies. Mechanisms by which L-citrulline improves NO production merit elucidation. The objective of this study was to determine whether starting L-citrulline after the onset of pulmonary hypertension inhibits disease progression and improves NO production by recoupling endothelial NO synthase (eNOS). Hypoxic and normoxic (control) piglets were studied. Some hypoxic piglets received oral L-citrulline starting on Day 3 of hypoxia and continuing throughout the remaining 7 days of hypoxic exposure. Catheters were placed for hemodynamic measurements, and pulmonary arteries were dissected to assess NO production and eNOS dimer-to-monomer ratios (a measure of eNOS coupling). Pulmonary vascular resistance was lower in L-citrulline-treated hypoxic piglets than in untreated hypoxic piglets but was higher than in normoxic controls. NO production and eNOS dimer-to-monomer ratios were greater in pulmonary arteries from L-citrulline-treated than from untreated hypoxic animals but were lower than in normoxic controls. When started after disease onset, oral L-citrulline treatment improves NO production by recoupling eNOS and inhibits the further development of chronic hypoxia-induced pulmonary hypertension in newborn piglets. Oral L-citrulline may be a novel strategy to halt or reverse pulmonary hypertension in infants suffering from cardiopulmonary conditions associated with hypoxia.

Keywords: nitric oxide signaling; superoxide; uncoupled eNOS.

Figure 1.

Plasma L-citrulline levels after a single oral-gastric bolus of L-citrulline in treatment-naive normoxic control piglets. Eight hours after administration of an oral-gastric dose of L-citrulline (0.13 g/kg [circles; n = 6], 0.26 g/kg [squares; n = 4], or 0.5 g/kg [inverted triangles; n = 4]), plasma L-citrulline levels were 50 to 100% above baseline. All values are mean ± SEM. *Different from time 0 baseline level (P < 0.05; unpaired t test).

Figure 2.

(A) Pulmonary vascular resistance (PVR) in control (normoxic) and chronically hypoxic piglets. Low-dose (n = 6) and high-dose (n = 8) L-citrulline treatment reduced PVR in hypoxic piglets to values less than those in untreated (n = 8) hypoxic piglets but higher than values in normoxic controls (n = 7). PVR was less in hypoxic piglets treated with the high- versus low-dose L-citrulline. *Different from control (normoxia); ⁺different from untreated chronic hypoxia; ^!different from low-dose L-citrulline–treated chronic hypoxia (P < 0.05; ANOVA with post hoc comparison test). (B) Fulton index (i.e., right ventricular free wall divided by the weight of the left ventricular wall and septum [RV/LV+S]) measurements of right ventricular mass in control (normoxic) and chronically hypoxic piglets. All three groups of chronically hypoxic piglets developed increased right heart mass compared with normoxic controls (normoxic controls, n = 12; untreated chronic hypoxia, n = 12; low-dose L-citrulline–treated chronic hypoxia, n = 6; high-dose L-citrulline–treated chronic hypoxia, n = 10). High-dose, but not low-dose, L-citrulline treatment reduced right ventricular mass in chronically hypoxic piglets to values below those measured in untreated chronically hypoxic piglets. *Different from control (normoxia); ⁺different from untreated chronic hypoxia; ^!different from low-dose L-citrulline–treated chronic hypoxia (P < 0.05; ANOVA with post hoc comparison test).

Figure 3.

(A) Plasma L-citrulline levels in control (normoxic) and chronically hypoxic piglets. Plasma L-citrulline levels were greater in chronically hypoxic piglets receiving high-dose L-citrulline treatment (n = 10) than in normoxic control piglets (n = 12), in untreated chronically hypoxic piglets (n = 12), or in piglets receiving low-dose L-citrulline treatment (n = 6). *Different from control (normoxia); ⁺different from untreated chronic hypoxia; ^!different from low-dose L-citrulline–treated chronic hypoxia (P < 0.05; ANOVA with post hoc comparison test). (B) L-citrulline levels in small pulmonary arteries from control (normoxic) and chronically hypoxic piglets. L-citrulline levels were greater in small pulmonary arteries from chronically hypoxic piglets receiving high-dose L-citrulline treatment (n = 10) than in small pulmonary arteries from normoxic control piglets (n = 10), untreated chronically hypoxic piglets (n = 8), or piglets receiving low-dose L-citrulline (n = 6). *Different from control (normoxia); ⁺different from untreated chronic hypoxia; ^!different from low-dose L-citrulline–treated chronic hypoxia (P < 0.05; ANOVA with post hoc comparison test). (C) Plasma L-arginine levels in control (normoxic) and chronically hypoxic piglets. Plasma L-arginine levels were lower in untreated hypoxic animals (n = 12) than in normoxic control animals (n = 12). Plasma L-arginine levels were similar to control animals in piglets treated with high-dose (n = 10), but not low-dose (n = 6), L-citrulline. *Different from control (normoxia); ⁺different from untreated chronic hypoxia; ^!different from low-dose L-citrulline–treated chronic hypoxia (P < 0.05; ANOVA with post hoc comparison test). (D) L-arginine levels in small pulmonary arteries from control (normoxia) and chronically hypoxic piglets. L-arginine levels were similar in small pulmonary arteries from all groups of piglets (normoxic control piglets, n = 10; untreated chronically hypoxic piglets, n = 8; low-dose L-citrulline–treated piglets, n = 6; high-dose L-citrulline–treated piglets, n = 10).

Figure 4.

(A) Responses to the endothelium-dependent vasodilator acetylcholine (ACh) in small pulmonary arteries from control (normoxic) and chronically hypoxic piglets. Small pulmonary arteries from normoxic control piglets dilated to ACh (n = 10); ACh elicited a similar degree of constriction in small pulmonary arteries from all groups of hypoxic piglets, whether untreated (n = 6) or treated with low-dose (n = 3) or high-dose (n = 4) L-citrulline (Cit). *Different from control (normoxia) (P < 0.05; ANOVA with post hoc comparison test). (B) Responses to the nitric oxide (NO) synthase antagonist N^ω-nitro-L-arginine methylester (l-NAME) in small pulmonary arteries from control (normoxic) and chronically hypoxic piglets. Responses to l-NAME were less for small pulmonary arteries from the untreated group of hypoxic piglets (n = 8) than for any other group of piglets. Small pulmonary arteries from low-dose (n = 3) and high-dose (n = 10) L-citrulline–treated groups of chronically hypoxic piglets had responses to l-NAME that were similar to normoxic control piglets (n = 12). *Different from control (normoxia); ⁺different from untreated chronic hypoxia (P < 0.05; ANOVA with post hoc comparison test). (C) Responses to the NO donor S-nitroso-N-acetyl-penicillamine (SNAP) in small pulmonary arteries from control (normoxic) and chronically hypoxic piglets. Responses to SNAP were less for small pulmonary arteries from the untreated group of hypoxic piglets (n = 9) than for any other group of piglets. Small pulmonary arteries from low-dose (n = 5) and high-dose (n = 9) L-citrulline–treated groups of chronically hypoxic piglets had responses to SNAP that were greater than untreated hypoxic piglets. Responses to SNAP were similar for small pulmonary arteries from normoxic control piglets (n = 10) and high-dose L-citrulline–treated chronically hypoxic piglets. *Different from control (normoxia); ⁺different from untreated chronic hypoxia; ^!different from low-dose L-citrulline–treated chronic hypoxia (P < 0.05; ANOVA with post hoc comparison test).

Figure 5.

(A) NO production by small pulmonary arteries from control (normoxic) and chronically hypoxic piglets. NO production was greater for small pulmonary arteries from normoxic control piglets (n = 6) than from all groups of hypoxic piglets. Small pulmonary arteries from low-dose (n = 5) and high-dose (n = 10) L-citrulline–treated groups of piglets had greater NO production than untreated (n = 5) hypoxic animals. *Different from control (normoxia); ⁺different from untreated chronic hypoxia (ANOVA with post hoc comparison test). (B) Superoxide (O₂^∙−) production by small pulmonary arteries from control (normoxic) and chronically hypoxic piglets. O₂^∙− production was less for small pulmonary arteries from normoxic control piglets (n = 6) than from all groups of hypoxic piglets. Small pulmonary arteries from low-dose (n = 5) and high-dose (n = 10) L-citrulline–treated groups of piglets had less O₂^∙− production than untreated (n = 6) hypoxic animals. O₂^∙− production was less for pulmonary arteries from high-dose compared with low-dose–treated chronically hypoxic piglets. *Different from control (normoxia); ⁺different from untreated chronic hypoxia; ^!different from low-dose L-citrulline–treated chronic hypoxia (P < 0.05; ANOVA with post hoc comparison test). (C) Effect of l-NAME treatment on O₂^∙− production by small pulmonary arteries from control (normoxic) and chronically hypoxic piglets. O₂^∙− production was unchanged by ex vivo l-NAME (10⁻⁴ M) treatment in small pulmonary arteries from control (normoxic) piglets (n = 5). Ex vivo treatment with l-NAME reduced O₂^∙− generation in small pulmonary arteries from untreated (n = 5) and L-citrulline–treated (n = 5) groups of hypoxic piglets. *Different from control (normoxia); ⁺different from ex vivo l-NAME–treated control (normoxia); ^!different from chronic hypoxia; ^different from L-citrulline–treated chronic hypoxia (P < 0.05; ANOVA with post hoc comparison test).

Figure 6.

A representative Western blot for endothelial nitric oxide synthase (eNOS) dimer-to-monomer ratios with corresponding densitometry shows that small pulmonary arteries from low- and high-dose L-citrulline–treated groups of piglets had greater eNOS dimer-to-monomer ratios than untreated hypoxic animals. eNOS dimer-to-monomer ratios were greater for pulmonary arteries from high-dose compared with low-dose L-citrulline–treated chronically hypoxic piglets. *Different from control (normoxia); ⁺different from untreated chronic hypoxia; ^!different from low-dose L-citrulline–treated chronic hypoxia (P < 0.05; ANOVA with post hoc comparison test).

Figure 7.

Representative Western blot for phospho-eNOS (Ser¹¹⁷⁷) (p-eNOS), eNOS, and β-actin (A) and corresponding densitometry (B and C) show that small pulmonary arteries from low-dose and high-dose L-citrulline–treated groups of piglets had greater p-eNOS/eNOS ratios than untreated hypoxic animals. The p-eNOS/eNOS ratios were greater in pulmonary arteries from high-dose compared with low-dose L-citrulline–treated chronically hypoxic piglets. eNOS/β actin expression was similar in small pulmonary arteries from all groups of piglets. *Different from control (normoxia); ⁺different from untreated chronic hypoxia; ^!different from low-dose L-citrulline–treated chronic hypoxia (P < 0.05; ANOVA with post hoc comparison test).