It is well established that NO, through the activity of the NOS enzymes, contributes to the development of ALI in sheep after burn and smoke inhalation injury (27
). In the first 48 hours following burn and inhalation injury, there are significant increases in pulmonary fluid flux and edema that are associated with increases in oxidative stress (28
). Although this inflammatory picture exits for the early changes observed in ALI, burn survivors are reported to have restrictive lung diseases in an 8-year follow-up study (2
). Consequently, we decided to determine the etiology of excess collagen deposition in a long-term, 2 and 3 week ovine model of burn and smoke inhalation injury. NO is involved in various pulmonary processes, including vascular tone and anti-inflammatory properties (29
). The recent report that the lung is a major source of the NOS inhibitor, ADMA, and that elevated circulating levels of ADMA can contribute to abnormal airway physiology suggests that ADMA may play a role in arginine metabolism in the pathophysiology of ALI (18
). In the present study, we explored the effects of burn and smoke inhalation injury on ADMA levels, DDAH-2 expression, and NOS activity. Results from this study demonstrate that increased collagen deposition is associated with increased oxidant stress, increased ADMA, and increased arginase activity.
The iNOS isoform produces large quantities of NO in the presence of pro-inflammatory cytokines, which contribute to ARDS. Our results show a significant increase and peak of nitric oxide products at 6 hrs, followed by their steady decline (). Our previous studies have shown that the use of the potent and highly selective iNOS inhibitor, BBS-2, after 48 hrs significantly attenuated the signs of ARDS after burn and inhalation injury including increased pulmonary edema, abnormal lung compliance, and extensive airway obstruction (30
). The present findings are consistent with our previous studies because a NOS inhibitor initially attenuated the injury in the short term when NO products are high. However, after NO products decrease, collagen deposition becomes problematic because of decreased NO and increased arginase. We found that as the NOS inhibitor ADMA increases, lung NO products decrease and arginase activity and collagen deposition are significantly increased (, , ). Another possible explanation of the indirect relationship between NOS and arginase is that ADMA blocks the formation of N (omega)-hydroxy-nor-L-arginine (NOHA), which is a potent inhibitor of arginase and an intermediate in the formation of NO. If NO is not formed, it is probable that NOHA will not form as well.
Acute respiratory distress syndrome (ARDS) is characterized by a PaO2
ratio below 200. The PaO2
ratio in our study significantly decreases from 6 hours up to 1 week after the burn and smoke inhalation injury compared to baseline values. However, the ratio begins to increase 2 weeks post-injury. A possible explanation is that the significant increase in NO causes a loss in hypoxic pulmonary vasoconstriction (HPV) in the ovine model (31
). From 6 hours to 1 week, NO remains increased, which leads to the loss of HPV. After NO decreases to baseline values after 2 and 3 weeks, HPV is restored. In addition, Westphal, et.al
. administered the neuronal NOS inhibitor, 7-nitroindazole, and significantly attenuated the loss of HPV. Because NO is heavily involved in the pathogenesis of burn and smoke inhalation injury, the decrease in NO and subsequent restoration of HPV allows the PaO2
ratio to increase. Also, the sheep are currently limited on their movement. Future studies will more accurately assess pulmonary function while the animals are engaged in physical activity.
Based on our previous studies and our current findings, we propose a mechanism () whereby DDAH-2 is inactivated by oxidation, resulting in increased ADMA accumulation and a decrease in NOS activity. As a consequence, L-arginine has an increased availability for metabolism by arginase and arginase activity is increased, resulting in elevated ODC and OAT expression, hydroxyproline generation, and collagen deposition. This mechanism stresses that the combined effects of increased arginase activity and increased oxidative stress result in altered pulmonary structure.
Figure 5 Model for the Role of Arginase Activity in Burn and Inhalation Injury. In this model, the burn and smoke inhalation injury and elevated oxidative stress decreases lung DDAH-2 expression, which (1) increases lung ADMA concentration, and (2) decreases the (more ...)
Inhalation injuries are associated with increased levels of ROS and RNS (32
). ROS and RNS modify cysteine and tyrosine residues and contribute to cell death, tissue injury, and organ dysfunction (33
). Their detrimental sequelae can be reversed by gamma-tocopherol nebulization in the lung because it is a scavenger of ROS and RNS (17
). Future studies will utilize gamma-tocopherol nebulization after burn and smoke inhalation injury as a tool to manage pulmonary changes and excess collagen deposition in the lung because of its actions as an ROS and RNS scavenger (35
In summary, we present evidence that arginase activity is significantly increased in an ovine model of burn and inhalation injury, which is associated with increased collagen deposition in the lung. Although it is well known that the arginase and NOS enzymes compete for arginine, this is the first report of the arginase pathway being involved in burn and inhalation injury in the lung. These findings also provide the first evidence that elevated oxidative stress may contribute to lung dysfunction and structural changes and suggest that they may play a role in the excess deposition of collagen.