L-citrulline supplementation reverses the impaired airway relaxation in neonatal rats exposed to hyperoxia

Abstract

Background: Hyperoxia is shown to impair airway relaxation via limiting L-arginine bioavailability to nitric oxide synthase (NOS) and reducing NO production as a consequence. L-arginine can also be synthesized by L-citrulline recycling. The role of L-citrulline supplementation was investigated in the reversing of hyperoxia-induced impaired relaxation of rat tracheal smooth muscle (TSM).

Methods: Electrical field stimulation (EFS, 2-20 V)-induced relaxation was measured under in vitro conditions in preconstricted tracheal preparations obtained from 12 day old rat pups exposed to room air or hyperoxia (>95% oxygen) for 7 days supplemented with L-citrulline or saline (in vitro or in vivo). The role of the L-citrulline/L-arginine cycle under basal conditions was studied by incubation of preparations in the presence of argininosuccinate synthase (ASS) inhibitor [α-methyl-D, L-aspartate, 1 mM] or argininosuccinate lyase inhibitor (ASL) succinate (1 mM) and/or NOS inhibitor [Nω-nitro-L-arginine methyl ester; 100 μM] with respect to the presence or absence of L-citrulline (2 mM).

Results: Hyperoxia impaired the EFS-induced relaxation of TSM as compared to room air control (p < 0.001; 0.5 ± 0.1% at 2 V to 50.6 ± 5.7% at 20 V in hyperoxic group: 0.7 ± 0.2 at 2 V to 80.0 ± 5.6% at 20 V in room air group). Inhibition of ASS or ASL, and L-citrulline supplementation did not affect relaxation responses under basal conditions. However, inhibition of NOS significantly reduced relaxation responses (p < 0.001), which were restored to control level by L-citrulline. L-citrulline supplementation in vivo and in vitro also reversed the hyperoxia-impaired relaxation. The differences were significant (p <0.001; 0.8 ± 0.3% at 2 V to 47.1 ± 4.1% at 20 V without L-citrulline; 0.9 ± 0.3% at 2 V to 68.2 ± 4.8% at 20 V with L-citrulline). Inhibition of ASS or ASL prevented this effect of L-citrulline.

Conclusion: The results indicate the presence of an L-citrulline/L-arginine cycle in the airways of rat pups. L-citrulline recycling does not play a major role under basal conditions in airways, but it has an important role under conditions of substrate limitations to NOS as a source of L-arginine, and L-citrulline supplementation reverses the impaired relaxation of airways under hyperoxic conditions.

Figure 1

Schematic presentation of the role of L-citrulline/L-arginine cycle in the regulation of airway function. The diagram integrates physiologic, biochemical and molecular mechanisms whereby neonatal hyperoxic exposure impairs NO-cGMP signaling and resultant airway smooth muscle relaxation. The L-arginine is a common substrate for NOS and arginase. Hyperoxia increases the lung arginase activity that reduces the bioavailability of L-arginine to NOS. L-citrulline is recycled to L-arginine through ASS and ASL enzymes. It is proposed that L-citrulline competes with NOS blockers (like L-NAME). However, it blocks arginase activity in hyperoxic conditions and thus increases the bioavailibility of L-arginine. The red lines with bars indicate inhibition. ASS, argininosuccinate synthase; ASL, argininosuccinate lyase; CATs, cationic amino acid transporters; cGMP, cyclic guanosine monophosphate; GDP, guanosine diphosphate; GTP, guanosine triphosphate; L-NAME, N^ω-nitro-L-arginine methyl ester; NO, nitric oxide; NOS, nitric oxide synthase; OAT, ornithine amino transferase; ODC, ornithine decarboxylase; PKG, protein kinase G; sGC, soluble guanylate cyclase.

Figure 2

Effect of hyperoxia on EFS-induced TSM relaxation: EFS-induced relaxation of TSM was impaired in tissues obtained from rat pups exposed to hyperoxia as compared to those from room air exposed rat pups. *Room air vs. hyperoxic rat pups (n = 10 for each group). *p < 0.05; ***p < 0.001.

Figure 3

Role of L-citrulline, α-MDLA, and succinate on EFS-induced relaxation of TSM of rat pups exposed to room air: No significant effect of L-citrulline, α-MDLA and L-succinate individually or in combination was observed in TSM of room air exposed rat pups (n = 7 for each protocol of the experiments).

Figure 4

Effect of L-citrulline and α-MDLA on TSM relaxation after NOS blockage: EFS-induced relaxation of TSM preparations obtained from room air-exposed rat pups was significanlty (p < 0.01) reduced by the addition of 100 μM L-NAME. The decrease in relaxation due to L-NAME was returned to normal levels by the addition of L-citrulline (2 mM). The argininosuccinate synthase (ASS) inhibitor – α-MDLA (1 mM) prevented the capability of L-citrulline to recover the diminished relaxation responses due to L-NAME. * Room air + L-NAME vs. Room air; § Room air + L-NAME + L-citrulline vs. Room air + L-NAME; † Room air + L-NAME + L-citrulline + α-MDLA vs. Room air + L-NAME + L-citrulline. **p < 0.01; §p < 0.05; §§p < 0.01; †p < 0.05; ††p < 0.01. (n = 8 for each protocol of the experiments).

Figure 5

Effect of L-citrulline and succinate on TSM relaxation after NOS blockage: EFS-induced relaxtion of TSM preparations obtained from room air-exposed rat pups was significanlty (p < 0.01) reduced by the addition of 100 μM L-NAME. The decrease in relaxation due to L-NAME was returned to normal levels by the addition of L-citrulline (2 mM). The argininosuccinate lyase (ASL) inhibitor – succinate (1 mM) prevented the capability of L-citulline to recover the diminished relaxation responces due to L-NAME. * Room air + L-NAME vs. Room air; § Room air + L-NAME + L-citrulline vs. Room air + L-NAME; † Room air + L-NAME + L-citrulline + succinate vs. Room air + L-NAME + L-citrulline. **p < 0.01; §p < 0.05; §§p < 0.01; †p < 0.05; ††p < 0.01. (n = 8 for each protocol of the experiments).

Figure 6

Effect of L-citrulline and inhibition of argininosuccinate synthase (ASS) on TSM relaxation in hyperoxia-exposed rat pups: L-citrulline supplementation (2 mM) reversed the hyperoxia-induced impairment of relaxation of TSM (p < 0.001). The effect of the L-citrulline was lost when TSM was incubated in the presence of argininosuccinate synthase (ASS) inhibitor, α-MDLA (1 mM) (p < 0.05). *Hyperoxia vs. Room air; §Hyperoxia + L-citrulline vs. Hyperoxia; †Hyperoxia + L-citrulline + α-MDLA vs. Hyperoxia + L-citrulline. *p < 0.05; ***p < 0.001; §§§p < 0.001; †p < 0.05 (n = 7 for each protocol of the experiments).

Figure 7

Effect of L-citrulline and inhibition of argininosuccinate lyase (ASL) on TSM relaxation in hyperoxia-exposed rat pups: The addition of 2 mM L-citrulline reversed the hyperoxia-induced impairment of relaxation of TSM (p < 0.001). The effect of the L-citrulline was lost when TSM was incubated in the presence of argininosuccinate lyase (ASL) inhibitor, L-succinate (1 mM) (p < 0.01). *Hyperoxia vs. Room air; §Hyperoxia + L-citrulline vs. Hyperoxia; †Hyperoxia + L-citrulline + succinate vs. Hyperoxia + L-citrulline. *p < 0.05; ***p < 0.001; §§p < 0.01; §§§p < 0.001; †p < 0.05; ††p < 0.01(n = 7 for each protocol of the experiments).

Figure 8

Effect of in vivo L-citrulline supplementation on TSM relaxation in hyperoxia-exposed rat pups: In vivo supplementation of rat pups with L-citrulline (200 mg/kg/day) reversed the hyperoxia-induced impairment of relaxant responses of TSM in the hyperoxic animals (p < 0.01). * Hyperoxia + L-citrulline vs. Hyperoxia; *p < 0.05; **p < 0.01 (n = 4 for each protocol of the experiments).