Prior research by us has shown that the long-term exposure of hyperoxia impairs airway relaxation [4, 32] and enhances airway contraction in neonatal rat lung . In this study we have shown that hyperoxia decreases EFS-induced relaxant responses of ASM. Both of these processes complement each other in increasing airway hyperreactivity. Hyperoxia increases arginase activity, which limits the availability of L-arginine to NOS . Limitation of L-arginine, being a common substrate for arginase and NOS, disrupts the NO/cGMP signaling pathway, resulting in impaired relaxation [4, 32]. In our recent publication we have shown that production of NO in ASM cells is reduced in hyperoxia-exposed rat pups . In a lung cell, L-arginine is used as a substrate for NOS and is obtained from different sources for the production of NO, including the recycling of L-citrulline . Maarsingh et al. have shown the existence of an L-citrulline/L-arginine cycle in TSM. We have observed that the supplementation of L–citrulline reversed the hyperoxia-induced impaired relaxation in both in vivo (i.p. administration to animals) and in vitro (addition to organ bath) conditions. The in vitro study indicates that in rat TSM, there is an active L-citrulline/L-arginine cycle that pro-duces L-arginine. Since the systemic administration of L-citrulline via i.p. route is also effective, we assume that, unlike L-arginine, L-citrulline is not taken up by the liver from the portal circulation, but is metabolized in the kidney to L-arginine that increases the plasma concentration of L-arginine. For this reason, oral or systemic administration of L-citrulline could be a better substitute for L-arginine as it bypasses the liver .
The inhibition of the enzymes (ASL and ASS) involved in the recycling pathway of L-citrulline to L-arginine did not affect EFS-induced relaxation of TSM under basal conditions, indicating that L-arginine in basal conditions is not a limiting substrate. Furthermore, the supplementation of TSM with exogenous L-citrulline did not affect EFS-induced relaxation, indicating that the supply of L-arginine for NOS through L-citrulline cycling is not an important source for NOS substrate under these conditions. However, this pathway appears to play a key role once the NOS enzyme is blocked. Interestingly, L-citrulline supplementation fully restored the reduced relaxation caused by the blockage of NOS under basal conditions. The effect of L-citrulline was more prominent particularly at higher voltages when an extra supply of NO is required to compensate the changes. This observation suggests that L-citrulline competes with the NOS blocker (L-NAME) reversing its inhibitory effects. These findings are comparable with other studies performed in the tracheal rings of a guinea pig model of asthma and human ASM using single or multiple frequencies of EFS [3, 27], where the inhibitory effect of NOS inhibitor, Nω-nitro-L-arginine, on NO-mediated iNANC relaxation was restored by L-citrulline as well as L-arginine . In addition, the inhibition of ASL reversed this beneficial effect of L-citrulline supplementation, indicating the presence of an active L-citrulline/L-arginine cycle in the airways of rat pups under conditions of substrate limitation for NO production. Similar results were shown in the tracheal rings of guinea pigs .
In asthmatic patients, a decrease in plasma L-arginine levels and an increase in serum arginase activity was observed . Similarly, in the experimental BPD in neonatal rats, the plasma level of L-arginine was found to have decreased after hyperoxic exposure of animals , while arginase protein expression was upregulated in the airway epithelium, further depriving the adjacent ASM for L-arginine substrate. Furthermore, Malleske et al.  showed that hyperoxia diminishes NO production in mice because of the increase in hepatic arginase activity. L-citrulline also plays a key role in the reversal of impaired relaxation during hyperoxia that creates a L-arginine limiting condition. L-citrulline has been reported to be a noncompetitive inhibitor of arginase . Therefore, in hyperoxic conditions, L-citrulline appears to work through arginase inhibition rather than competition with NOS inhibitors as discussed above.
In a rat pup model of BPD, L-arginine becomes a limiting substrate and disrupts the NO-cGMP signaling pathway, due to the hyperoxia-induced upregulation of arginase activity. The disruption of NO-cGMP signaling reduces the relaxation of airways as a consequence [4, 32]. An increase of arginase activity has been linked to airway hyperreactivity under different conditions in different animal models, such as rat pups, mice, and guinea pigs, as well as in humans [34, 35, 37–39]. In the perfused trachea of guinea pigs, it was demonstrated that increased arginase activity contributes to the airway hyperresponsiveness after early asthmatic reaction and deficiency of cNOS-derived NO . Marsingh et al.  have shown that the increase of arginase activity after early asthmatic reaction in guinea pigs impairs the neuronal nitric oxide-mediated relaxation of ASM. Therefore, in this study the role of L-citrulline recycling in the airways under in vitro and in vivo conditions of TSM obtained from hyperoxia exposed animals was studied. Hyperoxia-induced impairment of the relaxant responses of TSM of rat pups was reversed by L-citrulline supplementation under both in vitro and in vivo conditions, indicating the role of L-citrulline/L-arginine recycling enzymes in hyperoxic conditions. In order to demonstrate that the recovery of the impaired relaxation of TSM was due to recycling of L-citrulline into L-arginine, the ASS and ASL were inhibited using specific inhibitors. Interestingly, the protective effect of L-citrulline in hyperoxic TSM was lost by the inhibition of ASS, as well as ASL activity, further confirming the role of the L-citrulline/L-arginine cycle and the substrate limitation to NOS. Maarsingh et al.  also have found that exogenous L-citrulline fully reversed the impaired iNANC relaxation in the guinea pig model of asthma. We have shown that after hyperoxic exposure, arginase activity increased in the experimental BPD model [4, 32]. This model demonstrates that the raised levels of arginase activity, which compete with NOS for common substrate, contribute to the impairment of relaxation in most distal airways because of the decrease of NO production [4, 32].
Since it has been shown that the limitation of L-arginine availability to NOS is involved in pathogenesis of several diseases involving NO deficiencies, L-arginine supplementation to the animals has been used to treat these disorders. We have observed that the supplementation of 300 mg/kg of L-arginine single dose given daily for seven days through the intraperitoneal route reversed the impaired relaxant responses in rat pups . Although L-arginine supplementation in rat pups exposed to hyperoxia seems to normalize impaired relaxation of lung parenchymal strips, it further increased arginase activity, thus reducing the effectiveness of L-arginine therapy. In addition, oral L-arginine administration failed to reduce airway hyperresponsiveness in a murine model of allergic asthma  or in asthmatic patients . This failure might be because of the presystemic elimination of L-arginine by increased intestinal or hepatic arginase activity .
In contrast, L-citrulline is not taken up by the liver, but is metabolized predominantly in the kidney to L-arginine and could be considered as a masked precursor of L-arginine bypassing the liver , thus providing plenty of substrate for NO-cGMP signaling pathway . The benefits of oral administration of L-citrulline have been shown in many diseases. Oral administration of L-citrulline in patients suffering from sickle cell disease raised the decreased plasma levels of L-arginine without any toxicity . Furthermore, L-citrulline decreased pulmonary hypertension after surgery in patients with congenital heart disease , and it also reduced blood pressure response to cold stress . Recently, Cormio et al.  have shown that oral L-citrulline supplementation improves erection hardness in men with mild erectile dysfunction. Another study revealed that supplementation of L-citrulline prevents hyperoxia-induced lung injury in newborn rats . L-citrulline also ameliorates development of pulmonary hypertension and increases NO production in piglets exposed to chronic hypoxia . These studies provide evidence that support the use of L-citrulline as a therapy in diseases characterized by NO deficiency.
The animal model used in this study has proven quite successful to characterize the role of the L-citrulline/L-arginine cycle under normal and limiting conditions of NOS substrate. We recognize that this model under in vitro conditions has limitations, as it is isolated from systemic circulation and lacks the connections from central nervous system, which also affect the physiology of airways.