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Biomedical subjects

M A Matthay

Publications and source records attributed to M A Matthay.

At least 19 recordsLinked to original sources
PubMed2006

Activation of the stress protein response prevents the development of pulmonary edema by inhibiting VEGF cell signaling in a model of lung ischemia-reperfusion injury in rats.

Lung endothelial damage is a characteristic morphological feature of ischemia-reperfusion (I/R) injury, although the molecular steps involved in the loss of endothelial integrity are still poorly understood. We tested the hypothesis that the activation of vascular endothelial growth factor (VEGF) cell signaling would be responsible for the increase in lung vascular permeability seen early after the onset of I/R in rats. Furthermore, we hypothesized that the I/R-induced pulmonary edema would be significantly attenuated in rats by the activation of the stress protein response. Pretreatment with Ad Flk-1, an adenovirus encoding for the soluble VEGF receptor type II, prevented I/R-mediated increase in lung vascular permeability in rats. Furthermore, the I/R-induced lung injury was significantly decreased by prior activation of the stress protein response with geldanamycin or pyrrolidine dithiocarbamate. In vitro studies demonstrated that VEGF caused an increase in protein permeability across primary cultures of bovine macro- and microvascular lung endothelial cell monolayers that were associated with a phosphorylation of VE- and E-cadherin and the formation of actin stress fibers. Activation of the stress protein response prevented the VEGF-mediated changes in protein permeability across these cell monolayers and reduced the phosphorylation of VE-and E-cadherins, as well as the formation of actin stress fibers in these cells.

Animals↗
PubMed2005

VEGF levels in the alveolar compartment do not distinguish between ARDS and hydrostatic pulmonary oedema.

Although overexpression of vascular endothelial growth factor (VEGF) 165 in the lung causes pulmonary oedema, its role in human acute lung injury (ALI) is unclear. VEGF levels are reported to be lower in bronchoalveolar lavage from ALI patients compared with normals, but these studies did not include a comparably ill control group with noninflammatory pulmonary oedema. The current authors hypothesised that VEGF levels in pulmonary oedema fluid would be lower in ALI patients compared with control patients with severe hydrostatic pulmonary oedema. VEGF was measured in pulmonary oedema fluid and plasma from 56 patients with ALI and 46 controls with severe hydrostatic pulmonary oedema. Pulmonary oedema fluid levels of VEGF did not differ between patients with hydrostatic oedema (median 799 pg x mL(-1), interquartile range (IQR) 226-2,281) and ALI (median 507, IQR 0.8-1,031). Plasma levels were also the same (median 20.5 pg x mL(-1), IQR 0-152 versus 4.8, IQR 0-99.8). There was no association between plasma or oedema fluid VEGF levels and outcomes including mortality. Vascular endothelial growth factor levels in pulmonary oedema fluid were depressed both in acute lung injury and hydrostatic pulmonary oedema. The decrease in air space concentrations of vascular endothelial growth factor in acute lung injury may not be a function of the degree of lung injury, but rather may result from alveolar flooding.

Adult↗
PubMed2004

HO-1 induction restores c-AMP-dependent lung epithelial fluid transport following severe hemorrhage in rats.

Inhibition of cAMP-dependent stimulation of the vectorial fluid transport across the lung epithelium following hemorrhagic shock is mediated by NO released within the airspaces of the lung. We tested here the hypothesis that prior induction of HO-1 would attenuate the release of NO in the airspaces, thus preventing the inhibition of the c-AMP stimulation of alveolar fluid clearance (ALC) in rats. Indeed, HO-1 induction restored the cAMP-mediated up-regulation of ALC after hemorrhage by decreasing NO released within the airspaces of the lung. In vitro studies demonstrated that HO-1 induction significantly reduced the iNOS-mediated release of NO by alveolar macrophages stimulated with endotoxin for 24 h. This effect is explained in part by a HO-1-dependent attenuation of the LPS-mediated nuclear translocation of NF-kappaB. In addition, HO-1 induction also significantly reduced the iNOS-mediated release of NO by MH-S cells that were stimulated with interferon-gamma by decreasing the phosphorylation of STAT 1, another transcription factor important for the activation of the iNOS promoter. In contrast, HO-1 induction did not affect the production of NO by rat alveolar epithelial type II cells that were stimulated with cytomix (a mixture of TNF-alpha, IL-1beta, and IFN-gamma) for 24 h. In summary, these results provide the first in vivo evidence that the induction of HO-1 in the lung restores a normal fluid transport capacity of the alveolar epithelium following hemorrhagic shock by inhibiting the iNOS-mediated release of NO by alveolar macrophages.

Animals↗
PubMed2004

The ADP ribosyltransferase domain of Pseudomonas aeruginosa ExoT contributes to its biological activities.

ExoT is a type III secreted effector protein found in almost all strains of Pseudomonas aeruginosa and is required for full virulence in an animal model of acute pneumonia. It is comprised of an N-terminal domain with GTPase activating protein (GAP) activity towards Rho family GTPases and a C-terminal ADP ribosyltransferase (ADPRT) domain with minimal activity towards a synthetic substrate in vitro. Consistent with its activity as a Rho family GTPase, ExoT has been shown to inhibit P. aeruginosa internalization into epithelial cells and macrophages, disrupt the actin cytoskeleton through a Rho-dependent pathway, and inhibit wound repair in a scrape model of injured epithelium. We have previously shown that mutation of the invariant arginine of the GAP domain to lysine (R149K) results in complete loss of GAP activity in vitro but only partially inhibits ExoT anti-internalization and cell rounding activity. We have constructed in-frame deletions and point mutations within the ADPRT domain in order to test whether this domain might account for the residual activity observed in ExoT GAP mutants. Deletion of a majority of the ADPRT domain (residues 234 to 438) or point mutations of the ADPRT catalytic site (residues 383 to 385) led to distinct changes in host cell morphology and substantially reduced the ability of ExoT to inhibit in vitro epithelial wound healing over a 24-h period. In contrast, only subtle effects on the efficiency of ExoT-induced bacterial internalization were observed in the ADPRT mutant forms. Expression of each domain individually in Saccharomyces cerevisiae was toxic, whereas expression of each of the catalytically inactive mutant domains was not. Collectively, these data demonstrate that the ADPRT domain of ExoT is active in vivo and contributes to the pathogenesis of P. aeruginosa infections.

ADP Ribose Transferases↗
PubMed2003

Plasma surfactant protein levels and clinical outcomes in patients with acute lung injury.

BACKGROUND: Because injury to the alveolar epithelial barrier is a characteristic feature of acute lung injury (ALI) and the acute respiratory distress syndrome (ARDS), plasma surfactant protein levels may have prognostic value. To test this hypothesis plasma surfactant proteins A and D (SP-A and SP-D) levels were measured in patients with ALI or ARDS enrolled in the NHLBI sponsored multicentre ARDS Network randomised controlled trial of a 6 ml/kg v 12 ml/kg tidal volume strategy. METHODS: Data from 565 participants in the clinical trial were used. Plasma levels of SP-A and SP-D were measured at baseline and on day 3 after the start of the mechanical ventilation protocol. The longitudinal impact of baseline plasma surfactant protein levels on clinical outcomes was examined by multivariate analysis, controlling for mechanical ventilation group, APACHE III score, and other clinical covariates. The effect of 6 ml/kg tidal volume ventilation on plasma SP-A and SP-D levels was evaluated using analysis of covariance. RESULTS: Baseline plasma SP-A levels were not related to any clinical outcome. In contrast, higher baseline plasma SP-D levels were associated with a greater risk of death (OR 1.21 per 100 ng/ml increment; 95% CI 1.08 to 1.35), fewer ventilator-free days (mean decrease -0.88 days; p=0.001), and fewer organ failure-free days (mean decrease -1.06 days; p<0.0001). The 6 ml/kg tidal volume strategy had no effect on the rise in plasma SP-A levels (p=0.91) but attenuated the rise in plasma SP-D levels (p=0.0006). CONCLUSIONS: Early in the course of ALI/ARDS an increased level of plasma SP-D is associated with a worse clinical outcome. The 6 ml/kg tidal volume strategy attenuated the rise of SP-D early in the clinical course. Taken together, these observations indicate that plasma SP-D, a product of alveolar type II cells, is a valuable biomarker in ALI/ARDS.

Enzyme-Linked Immunosorbent Assay↗
PubMed2002

Stress preconditioning attenuates oxidative injury to the alveolar epithelium of the lung following haemorrhage in rats.

Inhibition of cAMP-dependent stimulation of vectorial fluid transport across the alveolar epithelium following haemorrhagic shock is mediated by reactive nitrogen species released within the airspaces of the lung. We tested here the hypothesis that the prior activation of the cellular heat shock or stress response, via exposure to either heat or geldanamycin, would attenuate the release of airspace nitric oxide (NO) responsible for the shock-mediated failure of the alveolar epithelium to respond to catecholamines in rats. Rats were haemorrhaged to a mean arterial pressure of 30-35 mmHg for 60 min, and then resuscitated with a 4 % albumin solution. Alveolar fluid clearance was measured by change in concentration of a protein solution instilled into the airspaces 5 h after the onset of haemorrhage. Stress preconditioning restored the cAMP-mediated upregulation of alveolar liquid clearance after haemorrhage. The protective effect of stress preconditioning was mediated in part by a decrease in the expression of iNOS in the lung. Specifically, stress preconditioning decreased the production of nitrite by endotoxin-stimulated alveolar macrophages removed from haemorrhaged rats or by A549 and rat alveolar epithelial type II cell monolayers stimulated with cytomix (a mixture of TNF-alpha, IL-1beta and IFN-gamma) for 24 h. In summary, these results provide the first in vivo evidence that stress preconditioning restores a normal fluid transport capacity of the alveolar epithelium in the early phase following haemorrhagic shock by attenuating NO-mediated oxidative stress to the lung epithelium.

Animals↗
PubMed2002

Novel role for CFTR in fluid absorption from the distal airspaces of the lung.

The active absorption of fluid from the airspaces of the lung is important for the resolution of clinical pulmonary edema. Although ENaC channels provide a major route for Na(+) absorption, the route of Cl(-) transport has been unclear. We applied a series of complementary approaches to define the role of Cl(-) transport in fluid clearance in the distal airspaces of the intact mouse lung, using wild-type and cystic fibrosis Delta F508 mice. Initial studies in wild-type mice showed marked inhibition of fluid clearance by Cl(-) channel inhibitors and Cl(-) ion substitution, providing evidence for a transcellular route for Cl(-) transport. In response to cAMP stimulation by isoproterenol, clearance was inhibited by the CFTR inhibitor glibenclamide in both wild-type mice and the normal human lung. Although isoproterenol markedly increased fluid absorption in wild-type mice, there was no effect in Delta F508 mice. Radioisotopic clearance studies done at 23 degrees C (to block active fluid absorption) showed approximately 20% clearance of (22)Na in 30 min both without and with isoproterenol. However, the clearance of (36)Cl was increased by 47% by isoproterenol in wild-type mice but was not changed in Delta F508 mice, providing independent evidence for involvement of CFTR in cAMP-stimulated Cl(-) transport. Further, CFTR played a major role in fluid clearance in a mouse model of acute volume-overload pulmonary edema. After infusion of saline (40% body weight), the lung wet-to-dry weight ratio increased by 28% in wild-type versus 64% in Delta F508 mice. These results provide direct evidence for a functionally important role for CFTR in the distal airspaces of the lung.

Absorption↗
PubMed2002

The pulmonary physician in critical care. 5: Acute lung injury and the acute respiratory distress syndrome: definitions and epidemiology.

An understanding of the epidemiology of ALI/ARDS and the effects of treatment have been hampered by the lack of a uniform definition of the syndrome. Various definitions have been proposed, and these are reviewed with particular attention to how changes in definition have affected our understanding of the natural history and treatment options for the condition.

Adult↗
PubMed2002

Selected contribution: long-term effects of beta(2)-adrenergic receptor stimulation on alveolar fluid clearance in mice.

Stimulation of active fluid transport with beta-adrenergic receptor (betaAR) agonists can accelerate the resolution of alveolar edema. However, chronic betaAR-agonist administration may cause betaAR desensitization and downregulation that may impair physiological responsiveness to betaAR-agonist stimulation. Therefore, we measured baseline and terbutaline- (10(-3) M) stimulated alveolar fluid clearance in mice that received subcutaneously (miniosmotic pumps) either saline or albuterol (2 mg. kg(-1). day(-1)) for 1, 3, or 6 days. Continuous albuterol administration increased plasma albuterol levels (10(-5) M), an effect that was associated with 1) a significant decrease in betaAR density and 2) attenuation, but not ablation, of maximal terbutaline-induced cAMP production. Forskolin-mediated cAMP-release was unaffected. Continuous albuterol infusion stimulated alveolar fluid clearance on day 1 but did not increase alveolar fluid clearance on days 3 and 6. However, terbutaline-stimulated alveolar fluid clearance in albuterol-treated mice was not reduced compared with saline-treated mice. Despite significant reductions in betaAR density and agonist-mediated cAMP production by long-term betaAR-agonist exposure, maximal betaAR-agonist-mediated increase in alveolar fluid clearance is not diminished in mice.

Adrenergic beta-Agonists↗
PubMed2002

Alveolar epithelial fluid transport in acute lung injury: new insights.

Pulmonary oedema is a life-threatening condition that frequently leads to acute respiratory failure. From a physiological perspective, pulmonary oedema develops either because of an increase in lung vascular hydrostatic pressure or an increase in lung vascular permeability. Resolution of alveolar oedema depends on the active removal of salt and water from the distal air spaces of the lung across the distal lung epithelial barrier. Much has been learned about the molecular and cellular basis for oedema fluid reabsorption, including the role of apical ion transporters for sodium (epithelial sodium channel) and chloride (cystic fibrosis transmembrane conductance regulator), as well as the central importance of the sodium pump. The rate of fluid clearance can be upregulated by both catecholamine-dependent and -independent mechanisms. Injury to the alveolar epithelium can disrupt the integrity of the alveolar barrier or downregulate ion transport pathways, thus, reducing net alveolar fluid reabsorption and enhancing the extent of alveolar oedema. Endogenous catecholamines upregulate alveolar fluid clearance in several experimental models of acute lung injury, but this upregulation may be short term and insufficient to counterbalance alveolar flooding. There is new evidence, however, that pharmacological treatment with beta2-adrenergic agonists and/or epithelial growth factors may influence a more sustained stimulation of alveolar fluid reabsorption and in turn facilitate recovery from experimental pulmonary oedema. Similar results have been achieved experimentally by gene transfer to enhance the abundance of sodium transporters in the alveolar epithelium. Clinical studies show that impaired alveolar fluid transport mechanisms contribute to the development, severity and outcome of pulmonary oedema in humans. Very recent data suggest that mechanisms that augment transepithelial sodium transport and enhance the clearance of alveolar oedema may lead to more effective prevention or treatment for some types of pulmonary oedema.

Adrenergic beta-Agonists↗
PubMed2001

Efficacy of low tidal volume ventilation in patients with different clinical risk factors for acute lung injury and the acute respiratory distress syndrome.

In patients with acute lung injury (ALI) and acute respiratory distress syndrome (ARDS), a recent ARDS Network randomized controlled trial demonstrated that a low tidal volume (VT) mechanical ventilation strategy (6 ml/kg) reduced mortality by 22% compared with traditional mechanical ventilation (12 ml/kg). In this study, we examined the relative efficacy of low VT mechanical ventilation among 902 patients with different clinical risk factors for ALI/ARDS who participated in ARDS Network randomized controlled trials. The clinical risk factor for ALI/ARDS was associated with substantial variation in mortality. The risk of death (before discharge home with unassisted breathing) was highest in patients with sepsis (43%); intermediate in subjects with pneumonia (36%), aspiration (37%), and other risk factors (35%); and lowest in those with trauma (11%) (p < 0.0001). Despite these differences in mortality, there was no evidence that the efficacy of the low VT strategy varied by clinical risk factor (p = 0.76, for interaction between ventilator group and risk factor). There was also no evidence of differential efficacy of low VT ventilation in the other study outcomes: proportion of patients achieving unassisted breathing (p = 0.59), ventilator-free days (p = 0.58), or development of nonpulmonary organ failure (p = 0.44). Controlling for demographic and clinical covariates did not appreciably affect these results. After reclassifying the clinical risk factors as pulmonary versus nonpulmonary predisposing conditions and infection-related versus non-infection-related conditions, there was still no evidence that the efficacy of low VT ventilation differed among clinical risk factor subgroups. In conclusion, we found no evidence that the efficacy of the low VT ventilation strategy differed among clinical risk factor subgroups for ALI/ARDS.

Adult↗
PubMed2001

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.

Adenylyl Cyclases↗
PubMed2001

Transepithelial sodium and water transport in the lung. Major player and novel therapeutic target in pulmonary edema.

Active transepithelial transport of sodium from the airspaces to the lung interstitium is a primary mechanism driving alveolar fluid clearance. This mechanism depends on sodium uptake by amiloride-sensitive sodium channels on the apical membrane of alveolar type II cells followed by extrusion of sodium on the basolateral surface by the Na-K-ATPase. Injury to the alveolar epithelium can disrupt the integrity of the alveolar barrier or downregulate ion transport pathways thus reducing net alveolar fluid reabsorption, and enhancing the extent of alveolar edema. Endogenous catecholamines upregulate alveolar fluid clearance in several experimental models of acute lung injury, but this upregulation is short-term and often not sufficient to counterbalance alveolar flooding. There is new evidence, however, that pharmacological treatment with beta-adrenergic agonists and/or epithelial growth factors may induce a more sustained stimulation of alveolar fluid reabsorption and in turn facilitate recovery from experimental pulmonary edema. Similar results have been achieved experimentally by gene transfer enhancing the abundance of sodium transporters in the alveolar epithelium. Clinical studies show that impaired alveolar fluid transport mechanisms contribute to the development, severity and outcome of pulmonary edema in humans. Very recent data suggest that mechanisms that augment transepithelial sodium transport and enhance the clearance of alveolar edema may lead to more effective prevention or treatment for pulmonary edema and acute lung injury.

Animals↗
PubMed2001

Pseudomonas aeruginosa ExoT inhibits in vitro lung epithelial wound repair.

The nosocomial pathogen Pseudomonas aeruginosa causes clinical infection in the setting of pre-existing epithelial tissue damage, an association that is mirrored by the increased ability of P. aeruginosa to bind, invade and damage injured epithelial cells in vitro. In this study, we report that P. aeruginosa inhibits the process of epithelial wound repair in vitro through the type III-secreted bacterial protein ExoT, a GTPase-activating protein (GAP) for Rho family GTPases. This inhibition primarily targets cells at the edge of the wound, and causes actin cytoskeleton collapse, cell rounding and cell detachment. ExoT-dependent inhibition of wound repair is mediated through the GAP activity of this bacterial protein, as mutations in ExoT that alter the conserved arginine (R149) within the GAP domain abolish the ability of P. aeruginosa to inhibit wound closure. Because ExoT can also inhibit P. aeruginosa internalization by phagocytes and epithelial cells, this protein may contribute to the in vivo virulence of P. aeruginosa by allowing organisms both to overcome local host defences, such as an intact epithelial barrier, and to evade phagocytosis by immune effector cells.

Actin Cytoskeleton↗
PubMed2001

Role of aquaporin water channels in airway fluid transport, humidification, and surface liquid hydration.

Several aquaporin-type water channels are expressed in mammalian airways and lung: AQP1 in microvascular endothelia, AQP3 in upper airway epithelia, AQP4 in upper and lower airway epithelia, and AQP5 in alveolar epithelia. Novel quantitative methods were developed to compare airway fluid transport-related functions in wild-type mice and knockout mice deficient in these aquaporins. Lower airway humidification, measured from the moisture content of expired air during mechanical ventilation with dry air through a tracheotomy, was 54-56% efficient in wild-type mice, and reduced by only 3-4% in AQP1/AQP5 or AQP3/AQP4 double knockout mice. Upper airway humidification, measured from the moisture gained by dry air passed through the upper airways in mice breathing through a tracheotomy, decreased from 91 to 50% with increasing ventilation from 20 to 220 ml/min, and reduced by 3-5% in AQP3/AQP4 knockout mice. The depth and salt concentration of the airway surface liquid in trachea was measured in vivo using fluorescent probes and confocal and ratio imaging microscopy. Airway surface liquid depth was 45 +/- 5 microm and [Na(+)] was 115 +/- 4 mM in wild-type mice, and not significantly different in AQP3/AQP4 knockout mice. Osmotic water permeability in upper airways, measured by an in vivo instillation/sample method, was reduced by approximately 40% by AQP3/AQP4 deletion. In doing these measurements, we discovered a novel amiloride-sensitive isosmolar fluid absorption process in upper airways (13% in 5 min) that was not affected by aquaporin deletion. These results establish the fluid transporting properties of mouse airways, and indicate that aquaporins play at most a minor role in airway humidification, ASL hydration, and isosmolar fluid absorption.

Adsorption↗
PubMed2001

von Willebrand factor antigen is an independent marker of poor outcome in patients with early acute lung injury.

OBJECTIVE: The primary objective of this study was to test the hypothesis that the degree of systemic endothelial activation, as measured by the release of von Willebrand factor antigen into the circulation and pulmonary edema fluid, is an important determinant of outcome from acute lung injury and acute respiratory distress syndrome. DESIGN: Observational study. SETTING: Intensive care unit patients in a tertiary university hospital and a university-affiliated city hospital. PATIENTS: Fifty-one intubated, mechanically ventilated intensive care unit patients with acute lung injury or acute respiratory distress syndrome as defined by the North American European Consensus Conference definitions. INTERVENTIONS: Undiluted pulmonary edema fluid and plasma were collected within 1 hr of endotracheal intubation in all patients. MEASUREMENTS: von Willebrand factor antigen concentrations and protein concentration were measured in pulmonary edema fluid and in plasma. MAIN RESULTS: At the time of intubation, median plasma von Willebrand factor antigen was 251%, two-fold higher than the median pulmonary edema fluid von Willebrand factor antigen of 130%. Median edema fluid and plasma von Willebrand factor antigen concentrations were significantly higher in patients who did not survive hospitalization. Plasma von Willebrand factor antigen was also higher in those patients who had a longer duration of mechanical ventilation (as measured by ventilator-free days). Plasma von Willebrand factor antigen was also significantly higher in patients with sepsis and two or more organ system failures. According to stepwise logistic regression analysis, plasma von Willebrand factor antigen was independently associated with in hospital death. The positive predictive value for death if the plasma von Willebrand factor antigen concentration was >450% was 83%. A plasma von Willebrand factor antigen concentration of >450% previously has been shown to predict the development of acute respiratory distress syndrome. CONCLUSIONS: These findings suggest that the degree of systemic endothelial activation and injury at the onset of acute lung injury is an important determinant of the outcome from acute lung injury.

Antigens↗
PubMed2001

Mechanisms of TNF-alpha stimulation of amiloride-sensitive sodium transport across alveolar epithelium.

Because tumor necrosis factor (TNF)-alpha can upregulate alveolar fluid clearance (AFC) in pneumonia or septic peritonitis, the mechanisms responsible for the TNF-alpha-mediated increase in epithelial fluid transport were studied. In rats, 5 microg of TNF-alpha in the alveolar instillate increased AFC by 67%. This increase was inhibited by amiloride but not by propranolol. We also tested a triple-mutant TNF-alpha that is deficient in the lectinlike tip portion of the molecule responsible for its membrane conductance effect; the mutant also has decreased binding affinity to both TNF-alpha receptors. The triple-mutant TNF-alpha did not increase AFC. Perfusion of human A549 cells, patched in the whole cell mode, with TNF-alpha (120 ng/ml) resulted in a sustained increase in Na(+) currents from 82 +/- 9 to 549 +/- 146 pA (P < 0.005; n = 6). The TNF-alpha-elicited Na(+) current was inhibited by amiloride, and there was no change when A549 cells were perfused with the triple-mutant TNF-alpha or after preincubation with blocking antibodies to the two TNF-alpha receptors before perfusion with TNF-alpha. In conclusion, although TNF- alpha can initiate acute inflammation and edema formation in the lung, TNF-alpha can also increase AFC by an amiloride-sensitive, cAMP-independent mechanism that enhances the resolution of alveolar edema in pathological conditions by either binding to its receptors or activating Na(+) channels by means of its lectinlike domain.

Adrenergic beta-Agonists↗