This study documents the discrete solute permeability mechanisms associated with physiologically high concentrations of human alpha-thrombin and bradykinin stimulation of bovine pulmonary artery endothelial cell (BPAEC) monolayers using fluorescein isothiocyanate-hydroxyethyl starch macromolecules. Agonist-induced alterations of intracellular free calcium ([Ca2+]i) using fura-2 acetoxymethyl ester were also measured. BPAEC monolayers showed restricted diffusion consistent with a small-pore (approximately 150 A) radius under baseline conditions. Thrombin produced a major increase in monolayer permeability that was greatest for solute molecular radii (ae) > 100 A. This effect was associated with the exposure of the large (approximately 2,000 A) pores of the filter support by 50- to 1,050-microns2 open areas between approximately 0.5% of the adjacent endothelial cells. This heterogeneous endothelial barrier of parallel large- and small-pore transport pathways permitted solute convection with free diffusion across a few large pores to dominate the restricted diffusion of most apparently unperturbed endothelial junctions. Bradykinin produced a small, transient elevation in monolayer permeability to ae < 35 A, consistent with an increase in the number of small pores or a decrease in path length of this transport pathway. The bradykinin- and thrombin-induced peak elevations in [Ca2+]i were inversely associated with the degree of increased monolayer solute permeability, and enzymatically inhibited thrombin produced none of these effects. These data show that bradykinin and human alpha-thrombin represent two distinct classes of endothelial cell agonists that initiate discrete solute permeability mechanisms.

We studied the size-selective permeability and restricted diffusion (Rd) properties of macromolecules across bovine pulmonary artery endothelial cell (BPAEC) or epithelial (LLC-PK1) monolayers grown on polycarbonate (PC) filter supports using fluorescein isothiocyanate-hydroxyethyl starch (FITC-HES, 16 A less than ae less than 180 A). Most BPAEC seeded at subconfluent density and grown for 3-6 days produced barriers with marked Rd. This characteristic was similar to that measured across PC filters with pore radii (rp) of 150 and 250 A without cells. Rd of LLC-PK1 monolayers was consistent with a transport pathway rp of much less than 75A. BPAEC monolayers prepared by supraconfluent seeding showed convective solute transport due to a significant number of incompletely formed intracellular junctions. Most monolayers grown to confluence, or a thin layer of collagen type I prevented this effect, Rd was enhanced when BPAEC monolalyers were grown on this collagen network. These data suggest that the subendothelial layers, which includes basal lamina, pericyte, and interstitial collagen, may act as series resistors with the endothelium to provide the Rd observed in the microvascular wall in vivo. This may explain why BPAEC monolayers grown to confluence without subendothelial layers in vitro showed Rd consistent with large (150-250 A) rp that was significantly greater than those modeled as the small (approximately 50 A) "pore" or 6-A fiber matrix of the in vivo capillary wall.

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Respiratory distress that leads to death is seen in patients with Lassa fever. The development of this respiratory problem was studied using a Pichinde virus model (10(4) plaque forming units, IP, survival time 20 +/- 1 days) in strain 13 guinea pigs (n = 35, 229-353 g) of this lethal human contagious infectious disease. Extravascular lung water to bloodless dry lung weight (EVLW/BDLW) ratio showed a modest yet significant increase in animals 13 and 18-21 days postinoculation (PI). In contrast, residual lung blood and lung radioactive 125I-labeled human serum albumin activity index were elevated only in the 18- to 21-day group. These data are consistent with the progressive severity of perivascular edema, lymphocytic pneumonitis, and some alveolar protein between days 13 and 18-21 PI. Lymphocytic pneumonitis appeared to be distributed near most airways and was proportional to the degree of Pichinde virus antigen staining of alveolar macrophages, large mononuclear cells within the pulmonary vascular and extravascular spaces, and alveolar-capillary membranes. These findings suggest that lymphocyte recruitment to the lung reflects the Pichinde virus-induced cell-mediated immune response. Obstructed small bronchi with some lumenal cell debris and hypertrophied epithelial cells were found associated with the areas of marked pneumonitis. The severe hypoxemia and modest anaerobic metabolism in association with marked tachypnea and normocapnia are consistent with small airway obstruction and wasted ventilation, since no change in arterial blood pressure, heart rate, hematocrit, hemoglobin, or blood volume was noted. These data suggest that Pichinde virus-induced respiratory failure was due to obstruction of the small airways with wasted ventilation in association with lymphocytic pneumonitis.