Keratinocyte growth factor protects against Pseudomonas aeruginosa-induced lung injury

We have previously reported that keratinocyte growth factor (KGF) attenuates alpha-naphthylthiourea-induced lung injury by upregulating alveolar fluid transport. The objective of this study was to determine the effect of KGF pretreatment in Pseudomonas aeruginosa pneumonia. A 5% bovine albumin solution with 1 microCi of (125)I-labeled human albumin was instilled into the air spaces 4 or 24 h after intratracheal instillation of P. aeruginosa, and the concentration of unlabeled and labeled proteins in the distal air spaces over 1 h was used as an index of net alveolar fluid clearance. Alveolocapillary barrier permeability was evaluated with an intravascular injection of 1 microCi of (131)I-albumin. In early pneumonia, KGF increased lung liquid clearance (LLC) compared with that in nonpretreated animals. In late pneumonia, LLC was significantly reduced in the absence of KGF but increased above the control value with KGF. KGF pretreatment increased the number of polymorphonuclear cells recovered in the bronchoalveolar lavage fluid and decreased bacterial pulmonary translocation. In conclusion, KGF restores normal alveolar epithelial fluid transport during the acute phase of P. aeruginosa pneumonia and LLC in early and late pneumonia. Host response is also improved as shown by the increase in the alveolar cellular response and the decrease in pulmonary translocation of bacteria.     

Clearance of different-sized proteins from the alveolar space in humans and rabbits.

Investigation of the clearance of proteins from the air spaces is important for an understanding of the resolution of pulmonary edema and also because of current interest in delivery of therapeutic peptides via the distal air spaces. Few experimental studies have examined the size dependence for alveolar clearance of large macromolecules; there have been no human studies. In anesthetized rabbits, we measured clearance of cyanocobalamin and different-sized human proteins instilled into the air spaces. After 8 h, the amounts of instilled tracer recovered in the lungs were [57Co]cyanocobalamin, 19.4 +/- 3.0% (Stokes radius 0.65 nm); 125I-labeled insulin, 64.6 +/- 3.9% (1.2 nm); 131I-labeled albumin, 87.0 +/- 4.0% (3.5 nm); and 125I-labeled immunoglobulin G, 91.8 +/- 3.3% (5.5 nm) (P < 0.05). Sieving of different-sized proteins occurred across the alveolar epithelial barrier because tracer concentrations in air space lavage fluid after 8 h were decreased more for the smaller tracers than the larger ones. Size selectivity for alveolar protein clearance in humans with resolving alveolar edema was investigated by measuring the changes in albumin and total protein concentration. The fraction of total protein concentration made up of albumin was greater in the edema fluid than in the plasma initially. The albumin fraction decreased with time in 9 of 10 patients with resolving edema, from 0.62 +/- 0.2 to 0.58 +/- 0.10 (P < 0.05) after 10 +/- 5 h. Thus both rabbit studies and human studies provide evidence for size-dependent clearance of protein from the air spaces of the lung.(ABSTRACT TRUNCATED AT 250 WORDS)     

Infarct-induced chronic heart failure increases bidirectional protein movement across the alveolocapillary barrier

Chronic heart failure (CHF) is associated with adaptive structural changes at the alveolocapillary barrier that may be associated with altered protein permeability. Bidirectional protein movement across the barrier was studied in anesthetized rats with infarct-induced CHF by following (125)I-labeled albumin ((125)I-albumin) flux into the alveoli and the leakage of surfactant protein (SP)-B from the alveoli into the circulation. Three groups were studied: controls [0% left ventricular (LV) infarction], moderate infarct (25-45% LV infarction), and large infarct (>46% LV infarction). Wet and dry lung weights increased in the large infarct group (both P < 0.001), consistent with increased lung water and solid lung tissue. (125)I-albumin flux increased across the endothelial (P < 0.001) and epithelial (P < 0.01) components of the alveolocapillary barrier in the large infarct group. Plasma SP-B increased 23% with moderate infarcts (P < 0.05) and 97% with large infarcts (P < 0.001), independent of alveolar levels. Lavage fluid immune cells (P < 0.01) and myeloperoxidase activity (P < 0.05) increased in the large infarct group, consistent with inflammation. Bidirectional protein movement across the alveolocapillary barrier is increased in CHF, and alveolar inflammation may contribute to this pathophysiological defect.     

Glucocorticoid receptor expression in human bronchial epithelial cells: effects of smoking and COPD.

Previously, we found that inflammatory mediators modulated the number and binding affinity of glucocorticoid receptors (GR) in human bronchial epithelial cell lines. In this study we investigated whether smoking and chronic obstructive pulmonary disease (COPD), both characterized by airway inflammation with increased levels of inflammatory mediators, affect GR characteristics in cultured human bronchial epithelial cells (HBEC). A statistically significant difference was found between the dissociation constant (Kd) values in HBEC from smoking (Kd = 0.98+/-0.08 nM; n = 6) and nonsmoking controls (Kd = 0.76+/-0.10 nM, P = 0.03; n = 5), but no significant difference was found between the mean number of binding sites. Our results are the first indication that cultured HBEC from smokers possess GR with a lower binding affinity. This may result from the inflammation found in the airways from smokers. Furthermore, these results provide further evidence that the bronchial epithelium may be an actual target for inhaled glucocorticoid therapy.     

Effects of protamine, heparinase, and hyaluronidase on endothelial permeability and surface charge

We undertook studies in the isolated perfused rat lung to determine 1) the effects of endothelial charge neutralization with the polycation protamine sulfate on microvascular permeability, lung water, and anionic ferritin binding to the endothelium and 2) the role of heparan sulfate and hyaluronate, negatively charged cell surface glycosaminoglycans, on permeability. Capillary permeability was determined by tissue 125I-albumin accumulation in isolated perfused rat lungs. In control lungs the 5-min albumin uptake was 0.50 +/- 0.05 cm3.s-1.g dry tissue-1 X 10(-3). It was increased by 132 +/- 7.8% (P less than 0.001) by protamine (0.08 mg/ml) and 65 +/- 12% (P less than 0.01) by heparinase (5 U/ml), whereas hyaluronidase (25 NFU/ml) was without effect. In control lungs total water was 4.83 +/- 0.15 ml g/dry tissue. Protamine increased lung water 12 +/- 2% (P less than 0.05). Heparinase caused a 9 +/- 3% increase (P less than 0.05), and hyaluronidase had no effect. Electron microscopy demonstrated that protamine increased anionic ferritin binding to the surface of endothelial cells. We conclude that protamine sulfate neutralization of negative charge in the pulmonary microcirculation leads to increased microvascular permeability. Heparin sulfate may be responsible for this charge effect.     

Thrombin-induced increase in albumin permeability across the endothelium

We studied the effect of thrombin on albumin permeability across the endothelial monolayer in vitro. Bovine pulmonary artery endothelial cells were grown on micropore membranes. Morphologic analysis confirmed the presence of a confluent monolayer with interendothelial junctions. Albumin permeability was measured by the clearance of 125I-albumin across the endothelial monolayer. The control 125I-albumin clearance was 0.273 +/- 0.02 microliter/min. The native enzyme, alpha-thrombin (10(-6) to 10(-10) M), added to the luminal side of the endothelium produced concentration-dependent increases in albumin clearance (maximum clearance of 0.586 +/- 0.08 microliter/min at 10(-6) M). Gamma (gamma) thrombin (10(-6) M and 10(-8) M), which lacks the fibrinogen recognition site, also produced a concentration-dependent increase in albumin clearance similar to that observed with alpha-thrombin. Moreover, the two proteolytically inactive forms of the native enzyme, i-Pr2 P-alpha-thrombin and D-Phe-Pro-Arg-CH2-alpha-thrombin, increased the 125I-albumin clearance (0.610 +/- 0.09 microliter/min and 0.609 +/- 0.02 microliter/min for i-Pr2 P-alpha-thrombin and D-Phe-Pro-Arg-CH2-alpha-thrombin at 10(-6) M, respectively). Since the modified forms of thrombin lack the fibrinogen recognition and active serine protease sites, the results indicate that neither site is required for increased albumin permeability. The increase in albumin clearance with alpha-thrombin was not secondary to endothelial cell lysis because lactate dehydrogenase concentration in the medium following thrombin was not significantly different from baseline values. There was also no morphological evidence of cell lysis. Moreover, the increase in 125I-albumin clearance induced by alpha-thrombin was reversible by washing thrombin from the endothelium. The basis for the increased albumin permeability following the addition of alpha-thrombin appears to be a reversible change in endothelial cell shape with formation of intercellular gaps.     

TNF-alpha induced bronchial vasoconstriction

The pro-inflammatory characteristics of tumor necrosis factor-alpha (TNF-alpha) have been extensively characterized in in vitro systems. Furthermore, this cytokine has been shown to play a pivotal role in airways inflammation in asthma. Since the airway vasculature also performs an essential function in inflammatory cell transit to the airways, experiments were performed to determine the effects of TNF-alpha on bronchial vascular resistance (BVR). In anesthetized, ventilated sheep, the bronchial artery (BA) was cannulated and perfused with autologous blood. BVR was defined as inflow pressure/flow and averaged 6.3 +/- 0.2 mmHg. ml(-1). min(-1) (+/-SE) for the 25 sheep studied. Recombinant human TNF-alpha (10 microg for 20 or 40 min) infused directly into the BA resulted in a significant decrease in BVR to 87% of baseline (P < 0.05). This vasodilation was followed by a reversal of tone by 120 min and a sustained increase in BVR to 126% of baseline (P < 0.05). Since others have shown TNF-alpha caused coronary vasoconstriction through endothelial release of endothelin-1 (ET-1), an ET-1 antagonist was used to block bronchial vasoconstriction. BQ-123, a selective ET(A) receptor antagonist, was delivered to the bronchial vasculature prior to TNF-alpha challenge. Attenuation of bronchial vasoconstriction was observed at 120 min (P < 0.03). Thus TNF-alpha causes bronchial vasoconstriction by the secondary release of ET-1. Although TNF-alpha exerts pro-inflammatory actions on most cells of the airways, vasoactive properties of this cytokine likely further contribute to the inflammatory status of the airways.     

Evaluation of basement membrane degradation during TNF-alpha-induced increase in epithelial permeability

We evaluated whether tumor necrosis factor (TNF)-alpha induces an increase in permeability of an alveolar epithelial monolayer via gelatinase secretion and basement membrane degradation. Gelatinase secretion and epithelial permeability to radiolabeled albumin under unstimulated and TNF-alpha-stimulated conditions of an A549 human epithelial cell line were evaluated in vitro. TNF-alpha induced both upregulation of a 92-kDa gelatinolytic activity (pro form in cell supernatant and activated form in extracellular matrix) and an increase in the epithelial permeability coefficient compared with the unstimulated condition (control: 1.34 +/- 0.04 x 10(-6) cm/s; 1 microg/ml TNF-alpha: 1.47 +/- 0.05 x 10(-6) cm/s, P < 0.05). The permeability increase in the TNF-alpha-stimulated condition involved both paracellular permeability, with gap formation visualized by actin cytoskeleton staining, and basement membrane permeability, with an increase in the basement membrane permeability coefficient (determined after cell removal; control: 2.58 +/- 0.07 x 10(-6) cm/s; 1 microg/ml TNF-alpha: 2.82 +/- 0.02.10(-6) x cm/s, P < 0.05). Because addition of gelatinase inhibitors [tissue inhibitor of metalloproteinase (TIMP)-1 or BB-3103] to cell supernatants failed to inhibit the permeability increase, the gelatinase-inhibitor balance in the cellular microenvironment was further evaluated by cell culture on a radiolabeled collagen matrix. In the unstimulated condition, spontaneous collagenolytic activity inhibited by addition to the matrix of 1 microg/ml TIMP-1 or 10(-6) M BB-3103 was found. TNF-alpha failed to increase this collagenolytic activity because it was associated with dose-dependent upregulation of TIMP-1 secretion by alveolar epithelial cells. In conclusion, induction by TNF-alpha of upregulation of both the 92-kDa gelatinase and its inhibitor TIMP-1 results in maintenance of the gelatinase-inhibitor balance, indicating that basement membrane degradation does not mediate the TNF-alpha-induced increase in alveolar epithelial monolayer permeability.     

Effect of short-duration constant exercise on permeability of cockerel aorta to 125I-albumin

To determine quantitatively the effect of short duration constant exercise on the rate of uptake (U) of intravenously injected 125I-labeled cockerel albumin (A) by the aorta of the adult cockerel, 24 birds divided into age-matched pairs, each pair consisting of an exercised and nonexercised control bird, were studied. The time period of heparinization, anesthesia, and time from injection of A (each member of each pair received about 50 microCi from the same batch) to the death of the animal (T) was identical for each member of each pair. The exercised animal was exercised at a constant speed of 3.2 kph at 0 degrees elevation for between 2 and 5 min on a treadmill. U was defined as accumulated wall radioactivity (dpm)/plasma radioactivity (dpm/ml) X endothelial surface area (cm2) X T (s). Free 125I in the injectate amounted to 1.29 +/- 0.31% (mean +/- SD). Free 125I in the plasma and the wall in the exercise and control animals was not significantly different: plasma 0.84 +/- 0.34% (mean +/- SD) and 0.55 +/- 0.18 (P less than 0.20); wall 3.38 +/- 5.64% and 6.42 +/- 4.72 (P less than 0.04). Injected A remaining in the blood at between 8 and 16 min after intravenous injection was 83 +/- 8.7% (n = 10) in the exercised and 82 +/- 10% (n = 7) in the control (P less than 0.2). U was greater in the exercise group in 9 out of 12 matched pairs (P less than 0.05). We conclude that U increases for short periods of constant exercise.     

Increased bronchovascular permeability after allergen exposure in sensitive asthmatics

To investigate the possibility that an increase in bronchovascular permeability is associated with allergen exposure in sensitive asthmatics we evaluated the amounts of serum proteins in bronchoalveolar lavage (BAL) effluents before and after local challenge with allergen. After exposure of sensitive asthmatic airways (n = 15) to allergen significant increases in total protein compared with controls were observed: 0.08 +/- 0.01 mg/ml in control airways and 0.13 +/- 0.02 mg/ml in challenged airways; P less than 0.05. The greatest changes induced by allergen exposure involved small-molecular-weight proteins (less than 345,000) and an inverse correlation was observed between log molecular weight and percent increase in the concentrations of the specific proteins; r = -0.61. BAL-serum distribution coefficients of serum proteins in airway fluids reflected a greater diffusability of low-molecular-weight proteins immediately after allergen exposure. We also evaluated the movement of serum proteins into lung after local allergen exposure using intravenously administered 99mTc-albumin (n = 10) and found an immediate 3.8-fold increase in amounts of radioactive albumin in allergen exposed airways compared with airways exposed to diluent. Most of the radioactivity was recovered in the first 5 ml of aliquot withdrawn, suggesting a marked increase in the permeability of the bronchial (large airway) vascular-epithelial membrane. An increase in serum proteins was also observed in BAL fluid of asthmatics 2-4 h after aerosol challenge (n = 4), including all proteins in the molecular weight range 45,000-900,000. These studies suggest that allergen exposure in sensitive asthmatics causes an acute increase in bronchovascular permeability to serum proteins.     

Effects of albumin supplementation on microvascular permeability in septic patients.

Albumin has a stabilizing effect on endothelium and helps maintain capillary permeability to macromolecules. Critically ill patients with sepsis may have profound hypoalbuminemia, but the effect of this hypoalbuminemia on microvascular permeability is unknown. To determine the degree and potential importance of this effect, we measured the transcapillary escape rate (TER) of (125)I-labeled albumin in 12 adult patients fulfilling American College of Chest Physicians/Society of Critical Care Medicine criteria for septic shock. We measured TER over a 90-min baseline period and then repeated these measurements immediately after the rapid infusion of 200 ml of 20% albumin. At baseline, patients had a mean serum albumin concentration of 10.3 +/- 3.8 g/l, which, at 30 min after the albumin infusion, was 18.5 +/- 3.7 g/l. The baseline TER was 6.7 +/- 1.5%/h, with a postinfusion TER of 6.4 +/- 2.1%/h (P = 0.550). Albumin supplementation sufficient to nearly double serum concentrations in profoundly hypoalbuminemic septic patients had no clinically significant effect in reducing microvascular permeability.     

Role of Rho-kinase in reexpansion pulmonary edema in rabbits

Reexpansion of a collapsed lung increases the microvascular permeability and causes reexpansion pulmonary edema. Neutrophils and their products have been implicated in the development of this phenomenon. The small GTP-binding proteins Rho and its target Rho-kinase (ROCK) regulate endothelial permeability, although their roles in reexpansion pulmonary edema remain unclear. We studied the contribution of ROCK to pulmonary endothelial and epithelial permeability in a rabbit model of this disorder. Endothelial and epithelial permeability was assessed by measuring the tissue-to-plasma (T/P) and bronchoalveolar lavage (BAL) fluid-to-plasma (B/P) ratios with (125)I-labeled albumin. After intratracheal instillation of (125)I-albumin, epithelial permeability was also assessed from the plasma leak (PL) index, the ratio of (125)I-albumin in plasma/total amount of instilled (125)I-albumin. T/P, B/P, and PL index were significantly increased in the reexpanded lung. These increases were attenuated by pretreatment with Y-27632, a specific ROCK inhibitor. However, neutrophil influx, neutrophil elastase activity, and malondialdehyde concentrations in BAL fluid collected from the reexpanded lung were not changed by Y-27632. In endothelial monolayers, Y-27632 significantly attenuated the H(2)O(2)-induced increase in permeability and mitigated the morphological changes in the actin microfilament cytoskeleton of endothelial cells. These in vivo and in vitro observations suggest that the Rho/ROCK pathway contributes to the increase in alveolar barrier permeability associated with reexpansion pulmonary edema.     

Structural mechanisms in the abolishment of VEGF-induced microvascular hyperpermeability by cAMP

To investigate the structural mechanisms by which elevation of the intraendothelial cAMP levels abolishes or attenuates the transient increase in microvascular permeability by vascular endothelial growth factor (VEGF), we examined cAMP effect on VEGF-induced hyperpermeability to small solute sodium fluorescein (Stokes radius = 0.45 nm) P(sodium fluorescein), intermediate-sized solute alpha-lactalbumin (Stokes radius = 2.01 nm) P(alpha-lactalbumin), and large solute albumin (BSA, Stokes radius = 3.5 nm) P(BSA) on individually perfused microvessels of frog mesenteries. After 20 min pretreatment of 2 mM cAMP analog, 8-bromo-cAMP, the initial increase by 1 nM VEGF was completely abolished in P(sodium fluorescein) (from a peak increase of 2.6+/-0.37 times control with VEGF alone to 0.96+/-0.07 times control with VEGF and cAMP), in P(alpha-lactalbumin) (from a peak increase of 2.7+/-0.33 times control with VEGF alone to 0.76+/-0.07 times control with VEGF and cAMP), and in P(BSA) (from a peak increase of 6.5+/-1.0 times control with VEGF alone to 0.97+/-0.08 times control with VEGF and cAMP). Based on these measured data, the prediction from our mathematical models suggested that the increase in the number of tight junction strands in the cleft between endothelial cells forming the microvessel wall is one of the mechanisms for the abolishment of VEGF-induced hyperpermeability by cAMP.     

Polymorphonuclear leukocyte cytoplasts mediate acute lung injury

Injection of phorbol 12-myristate 13-acetate (PMA) into polymorphonuclear leukocyte (PMN)-depleted, PMN cytoplast-repleted New Zealand White rabbits caused the development of acute lung injury in vivo. PMN cytoplasts are nucleus- and granule-free vesicles of cytoplasm capable of releasing toxic O2 radicals but incapable of releasing granule enzymes. PMN cytoplasts when activated by PMA reduced 66 +/- 12.7 nmol of cytochrome c compared with 2.6 +/- 0.7 nmol in their resting state and did not release a significant quantity of granule enzymes (P greater than 0.05). Injection of PMA into New Zealand White rabbits caused a significant decrease (P less than 0.05) in the number of circulating cytoplasts. Increases in lung weight-to-body weight ratios in PMA-treated rabbits (9.8 +/- 0.5 X 10(-3] compared with saline-treated rabbits (5.3 +/- 0.2 X 10(-3] were also noted. Levels of angiotensin-converting enzyme in lung lavage as well as the change in alveolar-arterial O2 ratio correlated with the numbers of cytoplasts in lung lavage (P = 0.001, r = 0.84 and P = 0.0166, r = 0.73, respectively). Albumin in lung lavage increased to 1,700 +/- 186 mg/ml in PMA-treated rabbits from 60 +/- 30 mg/ml in saline-treated rabbits. These changes were attenuated by pretreatment of rabbits with dimethylthiourea (DMTU). In vitro, cytoplasts were able to mediate increases in endothelial monolayer permeability. This was evidenced by increases in fractional transit of albumin across endothelial monolayers when treated with PMA-activated cytoplasts (0.08 +/- 0.01 to 0.28 +/- 0.02).(ABSTRACT TRUNCATED AT 250 WORDS)     

Sources of alveolar soluble TNF receptors during acute lung injury of different etiologies

Elevated soluble tumor necrosis factor-α receptor (sTNFR) levels in bronchoalveolar lavage fluid (BALF) are associated with poor patient outcome in acute lung injury (ALI). The mechanisms underlying these increases are unknown, but it is possible that pulmonary inflammation and increased alveolar epithelial permeability may individually contribute. We investigated mechanisms of elevated BALF sTNFRs in two in vivo mouse models of ALI. Anesthetized mice were challenged with intratracheal lipopolysaccharide or subjected to injurious mechanical ventilation. Lipopolysaccharide instillation produced acute intra-alveolar inflammation, but minimal alveolar epithelial permeability changes, with increased BALF sTNFR p75, but not p55. Increased p75 levels were markedly attenuated by alveolar macrophage depletion. In contrast, injurious ventilation induced substantial alveolar epithelial permeability, with increased BALF p75 and p55, which strongly correlated with total protein. BALF sTNFRs were not increased in isolated buffer-perfused lungs (devoid of circulating sTNFRs) subjected to injurious ventilation. These results suggest that lipopolysaccharide-induced intra-alveolar inflammation upregulates alveolar macrophage-mediated production of sTNFR p75, whereas enhanced alveolar epithelial permeability following mechanical ventilation leads to increased BALF p75 and p55 via plasma leakage. These data provide new insights into differential regulation of intra-alveolar sTNFR levels during ALI and may suggest sTNFRs as potential markers for evaluating the pathophysiology of ALI.     

Comparison of upper and lower airway responses of two sensitized rat strains to inhaled antigen.

The purpose of the study was to investigate the relationships between upper airways responses and pulmonary responses of two strains of highly inbred rats to inhaled antigen. To do this we measured the upper and lower airways resistance for 60 min after challenge of Brown-Norway rats (BN; n = 13) and an inbred rat strain (MF; n = 11), derived from Sprague-Dawley, with aerosolized ovalbumin (OA). Rats were actively sensitized with OA (1 mg sc) using Bordetella pertussis as an adjuvant. Two weeks later the animals were anesthetized and challenged. Tracheal pressure, esophageal pressure, and airflow were measured, from which total pulmonary resistance was partitioned into upper airway and lower pulmonary resistance (RL). The peak upper airway response to inhaled OA was similar in BN (1.89 +/- 0.66 cmH2O.ml-1.s; n = 7) and MF (2.85 +/- 0.68 cmH2O.ml-1.s; n = 6). The lower airway response to OA challenge was substantially greater in BN, and RL changed from 0.07 +/- 0.01 to 0.34 +/- 0.13 (n = 6; P < 0.05). The MF did not have any significant increase in RL after challenge; the baseline RL was 0.12 +/- 0.02 and only reached a peak value of 0.15 +/- 0.05 (n = 5; P = NS). Lower airway responsiveness of BN (n = 10) to serotonin, an important mediator early allergic airway responses, was similar to MF (n = 7).(ABSTRACT TRUNCATED AT 250 WORDS)     

Intrinsic and antigen-induced airway hyperresponsiveness are the result of diverse physiological mechanisms.

Airway hyperresponsiveness (AHR) is a defining feature of asthma. We have previously shown, in mice sensitized and challenged with antigen, that AHR is attributable to normal airway smooth muscle contraction with exaggerated airway closure. In the present study we sought to determine if the same was true for mice known to have intrinsic AHR, the genetic strain of mice, A/J. We found that A/J mice have AHR characterized by minimal increase in elastance following aerosolized methacholine challenge compared with mice (BALB/c) that have been antigen sensitized and challenged [concentration that evokes 50% change in elastance (PC(50)): 22.9 +/- 5.7 mg/ml for A/J vs. 3.3 +/- 0.4 mg/ml for antigen-challenged and -sensitized mice; P < 0.004]. Similar results were found when intravenous methacholine was used (PC(30) 0.22 +/- 0.08 mg/ml for A/J vs. 0.03 +/- 0.004 mg/ml for antigen-challenged and -sensitized mice). Computational model analysis revealed that the AHR in A/J mice is dominated by exaggerated airway smooth muscle contraction and that when the route of methacholine administration was changed to intravenous, central airway constriction dominates. Absorption atelectasis was used to provide evidence of the lack of airway closure in A/J mice. Bronchoconstriction during ventilation with 100% oxygen resulted in a mean 9.8% loss of visible lung area in A/J mice compared with 28% in antigen-sensitized and -challenged mice (P < 0.02). We conclude that the physiology of AHR depends on the mouse model used and the route of bronchial agonist administration.     

Urokinase binding and receptor identification in cultured endothelial cells.

Recombinant human single-chain urokinase (rscu-PA), two-chain urokinase (tcu-PA), and diisopropyl-fluorophosphate-treated tcu-PA (DFP-tcu-PA) bound to cultured human and porcine endothelial cells in a rapid, saturable, dose-dependent and reversible manner. Analysis of specific binding results in cultured human umbilical vein endothelial cells (HUVECs) gave the following estimated values for Kd and Bmax: 0.57 +/- 0.08 nM (mean +/- S.E.) and 188,000 +/- 18,000 sites/cell for 125I-labeled rscu-PA; 0.54 +/- 0.10 nM and 132,000 +/- 23,900 sites/cells for 125I-labeled tcu-PA; 0.89 +/- 0.14 nM and 143,000 +/- 30,300 sites/cell for 125I-labeled DFP-tcu-PA, respectively. Values for Kd were similar for primary and subcultured (six passages) HUVECs, but Bmax values were lower in subcultured HUVECs. Similar Kd values were found in cultured porcine endothelial cells; however, Bmax values varied depending on the endothelial cell type. All 125I-labeled urokinase forms yielded similar cross-linked approximately 110-kDa ligand-receptor complexes with cultured HUVECs, and 125I-labeled DFP-tcu-PA bound to a single major approximately 55-kDa protein in whole-cell lysates (ligand blotting/autoradiography), suggesting the presence of a single major approximately 55-kDa urokinase receptor in cultured HUVECs. The approximately 55-kDa urokinase receptor, isolated from several separate batches of cultured HUVECs (3-5 micrograms of protein, approximately 1 x 10(9) cells), by ligand affinity chromatography, exhibited the following properties: retained biologic activity as evidenced by its ability to bind 125I-labeled rscu-PA by ligand blotting/autoradiography and formation of a cross-linked 125I-labeled approximately 110-kDa rscu-PA-receptor complex; single-chain approximately 55-kDa protein, following reduction; complete conversion to and formation of a single major deglycosylated approximately 35-kDa protein, following treatment with N-glycanase.     

A transmural pressure gradient induces mechanical and biological adaptive responses in endothelial cells

A sudden increase in the transmural pressure gradient across endothelial monolayers reduces hydraulic conductivity (L(p)), a phenomenon known as the sealing effect. To further characterize this endothelial adaptive response, we measured bovine aortic endothelial cell (BAEC) permeability to albumin and 70-kDa dextran, L(p), and the solvent-drag reflection coefficients (sigma) during the sealing process. The diffusional permeability coefficients for albumin (1.33 +/- 0.18 x 10(-6) cm/s) and dextran (0.60 +/- 0.16 x 10(-6) cm/s) were measured before pressure application. The effective permeabilities (measured when solvent drag contributes to solute transport) of albumin and dextran (P(ealb) and P(edex)) were measured after the application of a 10 cmH(2)O pressure gradient; during the first 2 h of pressure application, P(ealb), P(edex), and L(p) were significantly reduced by 2.0 +/- 0.3-, 2.1 +/- 0.3-, and 3.7 +/- 0.3-fold, respectively. Immunostaining of the tight junction (TJ) protein zonula occludens-1 (ZO-1) was significantly increased at cell-cell contacts after the application of transmural pressure. Cytochalasin D treatment significantly elevated transport but did not inhibit the adaptive response, whereas colchicine treatment had no effect on diffusive permeability but inhibited the adaptive response. Neither cytoskeletal inhibitor altered sigma despite significantly elevating both L(p) and effective permeability. Our data suggest that BAECs actively adapt to elevated transmural pressure by mobilizing ZO-1 to intercellular junctions via microtubules. A mechanical (passive) component of the sealing effect appears to reduce the size of a small pore system that allows the transport of water but not dextran or albumin. Furthermore, the structures of the TJ determine transport rates but do not define the selectivity of the monolayer to solutes (sigma).     

Turnover of 125I-VLDL and 131I-LDL apolipoprotein B in rabbits fed diets containing casein or soy protein

Rabbits fed low-fat, cholesterol-free, semi-purified diets containing casein developed a marked hypercholesterolemia compared to rabbits fed a similar diet containing soy protein (plasma cholesterol 281 +/- 31 vs. 86 +/- 9 mg/dl; P less than 0.05). Turnover studies (three per dietary group) were carried out in which homologous 125I-labeled VLDL and 131I-labeled LDL were injected simultaneously into casein- (n = 8) or soy protein- (n = 9) fed rabbits. ApoB-specific activities were determined in VLDL, IDL and LDL isolated from the pooled plasma of two or three rabbits per dietary group. The production rate of VLDL apoB (1.20 +/- 0.3 vs. 1.09 +/- 0.1 mg/h per kg) was similar for the two dietary groups. The fractional catabolic rate of VLDL apoB was lower for the casein group (0.15 +/- 0.03 vs. 0.23 +/- 0.01.h-1; 0.05 less than P less than 0.10). Although the pool size of VLDL apoB was higher in the casein group (8 +/- 2 vs. 5 +/- 0.3 mg/kg), this value did not reach statistical significance. For LDL apoB, the increased pool size in casein-fed rabbits (30 +/- 5 vs. 5 +/- 1 mg/kg; P less than 0.01) was associated with a decreased fractional catabolic rate (0.03 +/- 0.005 vs. 0.08 +/- 0.008.h-1; P less than 0.01) and a 2-fold increase in the production rate of LDL apoB (1 +/- 0.3 vs. 0.4 +/- 0.06 mg/kg per h; 0.05 less than P less than 0.10) compared to rabbits fed soy protein. Analysis of precursor-product relationships between the various lipoprotein fractions showed that casein-fed rabbits synthesized a higher proportion of LDL apoB (95% +/- 2 vs. 67% +/- 2; P less than 0.001) independent of VLDL catabolism. These results support the concept that the hypercholesterolemia in casein-fed rabbits is associated with impaired LDL removal consistent with a down-regulation of LDL receptors. These changes do not occur when the casein is replaced by soy protein.