Open Access Research

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

Ramadan B Sopi12*, Syed IA Zaidi34*, Mitko Mladenov2, Hazbije Sahiti1, Zahide Istrefi1, Icko Gjorgoski2, Azem Lajçi1 and Muharrem Jakupaj1

Author Affiliations

1 Department of Pharmacy-Biology, Faculty of Medicine, University of Prishtina, St. Martyrs’ Boulevard n.n., Prishtina, 10000, Kosovo

2 Institute of Biology, Faculty of Natural Sciences and Mathematics, “Sts, Cyril and Methodius” University, Skopje, 1000, Macedonia

3 Department of Chemical Engineering, Case Western Reserve University, Cleveland, OH, 44106, USA

4 Department of Chemical Engineering, Case Western Reserve University, Cleveland, OH, 44106, USA

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Respiratory Research 2012, 13:68  doi:10.1186/1465-9921-13-68

Published: 7 August 2012



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).


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).


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.


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.

Airway relaxation; Airway smooth muscle; Argininosuccinate lyase; Argininosuccinate synthase; L-arginine; Nitric oxide; Nitric oxide synthase