Reactive nitrogen species inhibit alveolar epithelial fluid transport after hemorrhagic shock in rats.

Our recent experimental work demonstrated that a neutrophil-dependent inflammatory response in the lung prevented the normal up-regulation of alveolar fluid clearance by catecholamines following hemorrhagic shock. In this study, we tested the hypothesis that the release of NO within the airspaces of the lung was responsible for the shock-mediated failure of the alveolar epithelium to respond to catecholamines in rats. Hemorrhagic shock was associated with an inducible NO synthase (iNOS)-dependent increase in the lung production of NO and a failure of the alveolar epithelium to up-regulate vectorial fluid transport in response to beta-adrenergic agonists. Inhibition of iNOS restored the normal catecholamine-mediated up-regulation of alveolar liquid clearance. Airspace instillation of dibutyryl cAMP, a stable analog of cAMP, restored the normal fluid transport capacity of the alveolar epithelium after prolonged hemorrhagic shock, whereas direct stimulation of adenyl cyclase by forskolin had no effect. Pretreatment with pyrrolidine dithiocarbamate or sulfasalazine attenuated the iNOS-dependent production of NO in the lung and restored the normal up-regulation of alveolar fluid clearance by catecholamines after prolonged hemorrhagic shock. Based on in vitro studies with an alveolar epithelial cell line, A549 cells, the effect of sulfasalazine appeared to be mediated in part by inhibition of NF-kappaB activation, and the protective effect was mediated by the inhibition of IkappaBalpha protein degradation. In summary, these results provide the first in vivo evidence that NO, released within the airspaces of the lung probably secondary to the NF-kappaB-dependent activation of iNOS, is a major proximal inflammatory mediator that limits the rate of alveolar epithelial transport after prolonged hemorrhagic shock by directly impairing the function of membrane proteins involved in the beta-adrenergic receptor-cAMP signaling pathway in alveolar epithelium.

Ethanol ingestion via glutathione depletion impairs alveolar epithelial barrier function in rats.

We determined that rats fed a liquid diet containing ethanol (36% of calories) for 6 wk had decreased (P < 0.05) net vectorial fluid transport and increased (P < 0.05) bidirectional protein permeability across the alveolar epithelium in vivo compared with rats fed a control diet. However, both groups increased (P < 0.05) fluid transport in response to epinephrine (10(-5) M) stimulation, indicating that transcellular sodium transport was intact. In parallel, type II cells isolated from ethanol-fed rats and cultured for 8 days formed a more permeable monolayer as reflected by increased (P < 0.05) leak of [(14)C]inulin. However, type II cells from ethanol-fed rats had more sodium-permeant channels in their apical membranes than type II cells isolated from control-fed rats, consistent with the preserved response to epinephrine in vivo. Finally, the alveolar epithelium of ethanol-fed rats supplemented with L-2-oxothiaxolidine-4-carboxylate (Procysteine), a glutathione precursor, had the same (P < 0.05) net vectorial fluid transport and bidirectional protein permeability in vivo and permeability to [(14)C]inulin in vitro as control-fed rats. We conclude that chronic ethanol ingestion via glutathione deficiency increases alveolar epithelial intercellular permeability and, despite preserved or even enhanced transcellular sodium transport, renders the alveolar epithelium susceptible to acute edematous injury.

Interleukin-8 mediates injury from smoke inhalation to both the lung endothelial and the alveolar epithelial barriers in rabbits.

Although prior studies have shown that smoke inhalation causes lung endothelial injury and formation of pulmonary edema, there is no information about the effect of smoke inhalation on the function of the alveolar epithelial barrier. Therefore, the primary objective of this study was to determine the effect of smoke-induced lung injury on the alveolar epithelial barrier in a rabbit experimental model. The second objective was to investigate whether pretreatment with a monoclonal anti-interleukin (IL)-8 antibody prevented alveolar epithelial barrier injury after smoke inhalation. Anesthetized rabbits were tracheotomized and were insufflated with cooled smoke generated from burning cotton cloth (75 breaths). In some experiments, anti-IL-8 antibody or an irrelevant antibody (2 mg/¿g) was given intravenously 5 min before insufflation of cotton smoke. Smoke inhalation caused a significant increase in the alveolar epithelial permeability to protein and a 40% reduction in the fluid transport capacity of the alveolar epithelium. Pretreatment with anti-IL-8 antibody, but not with an irrelevant-isotype antibody, significantly reduced the smoke-mediated increase in bidirectional transport of protein across the alveolar epithelium, and restored alveolar liquid clearance to a normal level. The results of the study show that smoke inhalation causes injury to both the alveolar epithelial barrier and the lung endothelium, and that IL-8 is an important mediator of this injury.

Acid-induced lung injury. Protective effect of anti-interleukin-8 pretreatment on alveolar epithelial barrier function in rabbits.

Although prior experimental work has demonstrated that anti-interleukin-8 (anti-IL-8) therapy reduces lung endothelial injury after acid instillation, there is no information regarding the effect of anti-IL-8 on the function of the alveolar epithelial barrier after acid-induced lung injury. Therefore, the primary objective of this study was to determine the effect of acid-induced lung injury on the function of the alveolar epithelium, and secondly to determine whether pretreatment with anti-IL-8 attenuates acid-induced injury to the lung epithelial barrier. Hydrochloric acid (pH = 1.5 in 1/3 normal saline) was instilled into the lungs of anesthetized, ventilated rabbits. Anti-IL-8 monoclonal antibody (2 mg/kg) or saline was given intravenously 5 min before acid instillation. Acid instillation into the distal airspaces caused an increase in the alveolar epithelial permeability to protein and an approximately 50% reduction in net alveolar fluid clearance. Because a decrease in net alveolar fluid clearance could be due to lung endothelial injury and increased fluid flux from the blood into the airspaces, additional experiments were carried out in which pulmonary blood flow was eliminated. In the absence of pulmonary blood flow, acid instillation led to a 50% decrease in net alveolar fluid clearance. Pretreatment with anti-IL-8 antibody significantly reduced the acid-mediated increase in bi-directional transport of protein across the alveolar epithelium and restored alveolar fluid clearance to normal. The results indicate that acid instillation causes injury to the alveolar epithelial barrier that can be distinguished from the injury to the lung endothelium. Furthermore, pretreatment with anti-IL-8 therapy prevents acid-induced alveolar epithelial injury, a finding of potential clinical importance.

alpha-adrenergic blockade restores normal fluid transport capacity of alveolar epithelium after hemorrhagic shock.

Activation of beta-adrenergic receptors in the lung is an important mechanism that can prevent alveolar flooding after brief but severe hemorrhagic shock. However, a neutrophil-dependent oxidant injury to the alveolar epithelium prevents the normal upregulation of alveolar fluid clearance by catecholamines after prolonged hemorrhagic shock. Because hemorrhage increases proinflammatory cytokine expression in the lung partly through the activation of alpha-adrenergic receptors, the objective of this study was to determine whether alpha-adrenergic blockade would restore the normal fluid transport capacity of the alveolar epithelium after hemorrhagic shock. Hemorrhagic shock was associated with a significant increase of interleukin-1beta (IL-1beta) concentration in the lung and a failure of the alveolar epithelium to respond to beta-adrenergic agonists, with the upregulation of vectorial fluid transport despite intra-alveolar administration of exogenous catecholamines. In contrast, catecholamine-mediated upregulation of alveolar liquid clearance was restored by pretreatment with phentolamine, an alpha-adrenergic-receptor antagonist. Phentolamine pretreatment also significantly attenuated the shock-mediated increase of IL-1beta concentration in the lung. Additional experiments demonstrated that the inhibition of IL-1beta binding to its receptor by the administration of IL-1-receptor antagonist restored the normal fluid transport capacity of the alveolar epithelium after hemorrhagic shock. In summary, the results of these studies indicate that the activation of alpha-adrenergic receptors after hemorrhagic shock prevents the beta-adrenergic-dependent upregulation of alveolar fluid clearance by modulating the severity of the pulmonary inflammatory response.

Inhibition of beta-adrenergic-dependent alveolar epithelial clearance by oxidant mechanisms after hemorrhagic shock.

Endogenous release of catecholamines is an important mechanism that can prevent alveolar flooding after brief but severe hemorrhagic shock. The objective of this study was to determine whether this catecholamine-dependent mechanism upregulates alveolar liquid clearance after prolonged hemorrhagic shock. Rats were hemorrhaged to a mean arterial pressure of 30-35 mmHg for 60 min and then resuscitated with a 4% albumin solution. Alveolar liquid clearance was measured 5 h later as the concentration of protein in the distal air spaces over 1 h after instillation of a 5% albumin solution into one lung. There was no upregulation of alveolar liquid clearance after prolonged hemorrhagic shock and fluid resuscitation despite a significant increase in plasma epinephrine levels. The intravenous or intra-alveolar administration of exogenous catecholamines did not upregulate alveolar liquid clearance. In contrast, catecholamine-mediated upregulation of alveolar liquid clearance was restored either by depletion of neutrophils with vinblastine, by the normalization of the concentration of reduced glutathione in the alveolar epithelial lining fluid by N-acetylcysteine, or by the inhibition of the conversion from xanthine dehydrogenase to xanthine oxidase. These experiments provide the first in vivo evidence that a neutrophil-dependent oxidant injury to the alveolar epithelium prevents the upregulation of alveolar fluid clearance by catecholamines in the absence of a major alteration in paracellular permeability to protein after prolonged hemorrhagic shock.

Upregulation of alveolar liquid clearance after fluid resuscitation for hemorrhagic shock in rats.

The primary objective of this study was to test the hypothesis that a catecholamine-dependent mechanism would upregulate alveolar liquid clearance after fluid resuscitation from 15 min of hemorrhagic shock. Anesthetized rats were hemorrhaged to a mean arterial pressure of 35 mmHg for 15 min and were resuscitated with a 4% albumin solution. Alveolar liquid clearance was measured 5 h later by the concentration of protein in the distal airspaces over 1 h after instillation of a 5% albumin solution into one lung. Hemorrhaged rats developed a severe metabolic acidosis that was associated with a significant rise-in plasma epinephrine levels throughout the study. There was a 60% increase in alveolar liquid clearance in hemorrhaged and resuscitated rats compared with control rats. Amiloride (10(-4) or 10(-6) M), propranolol (10(-4) M), or bilateral adrenalectomy inhibited the increase in alveolar liquid clearance. This effect was reproduced by the intravenous administration of epinephrine in adrenalectomized and hemorrhaged rats. Thus these data provide evidence for a catecholamine-dependent regulation of sodium transport that protects the airspaces against flooding several hours after fluid resuscitation from hemorrhagic shock.

Nitric oxide attenuates lung endothelial injury caused by sublethal hyperoxia in rats.

Inhaled nitric oxide (NO) has been shown to prevent oxidant-induced lung injury in isolated-perfused lung models, whereas NO-derived oxidants may contribute to acute lung injury secondary to hyperoxia. Whether inhaled NO improves or contributes to oxidant-mediated lung injury may depend on the timing of NO administration or how lung injury is assessed. The objective of these studies was to determine whether inhaled NO (20 ppm) was protective or harmful to the different lung barriers when it was administered with 95% O2 for 60 h in Sprague-Dawley rats by measuring fluid transport and permeability to protein across the lung endothelium and the alveolar epithelium. Inhaled NO significantly attenuated the O2-mediated lung endothelial injury and abolished the increase in the bronchoalveolar lavage fluid content of rTI40, a specific and sensitive marker of alveolar epithelial type I cell injury, that occurs secondary to hyperoxia. In conclusion, inhaled NO administered with high concentrations of O2 may protect the lung endothelium and the alveolar epithelium against O2-mediated injury.

Alveolar liquid clearance is increased by endogenous catecholamines in hemorrhagic shock in rats.

The primary objective of this study was to test the hypothesis that hemorrhagic shock would stimulate alveolar liquid clearance by a catecholamine-dependent mechanism. Anesthetized rats were hemorrhaged to a mean arterial pressure of 30 mmHg for 90 min, but they were not resuscitated. Alveolar liquid clearance was measured by the concentration of labeled and unlabeled protein over 2 h in an isosmolar physiological solution of 5% albumin that had been instilled into one lung. Hemorrhaged rats developed a severe metabolic acidosis that was associated with a 5- to 10-fold rise in plasma epinephrine levels. There was a 60% increase in alveolar liquid clearance in the hemorrhaged rats compared with control rats (55 +/- 6 vs. 34 +/- 7%; P < 0.05). Amiloride (10(-4) M) or propranolol (10(-4)M) inhibited the increase in alveolar liquid clearance. Thus the endogenous release of catecholamines associated with hemorrhagic shock markedly stimulates alveolar fluid clearance by a beta-adrenergic-mediated stimulation of active sodium transport. These data suggest a new, previously unrecognized mechanism that may protect against alveolar flooding in the acute phase of hemorrhagic shock.