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In isolated perfused rat lungs we have examined the effect of inhibition of receptor-mediated endocytosis and of intracellular acidic compartments activity on adsorptive and fluid-phase uptake and transport of macromolecules by capillary endothelium. Cationized and native ferritin were used as electron-dense tracers of adsorptive and fluid-phase transport, respectively. Luminal endothelial vesicles were labeled with tracers at 4 degrees C for 2 minutes and the density of ferritin particles in endothelial multivesicular bodies and in the capillary basement membrane, after a 60-minute chase with tracer-free perfusate at 37 degrees C, was determined by electron microscopic morphometry. Fluid-phase transport of native ferritin in 60-minute perfusions at 37 degrees C was also investigated. Adsorptive endocytosis was inhibited in chase experiments with hypertonic (800 mOsm) perfusate. Endothelial acidic compartments activity was decreased by adding either dextran sulfate or chloroquine to the chase media. We found that hypertonic solutions, dextran sulfate, and chloroquine inhibit cationized ferritin accumulation in multivesicular bodies and in the extravascular space. Hypertonic buffer or dextran sulfate have no effect on native ferritin uptake and transport in either pulse-chase or continuous perfusions. Since adsorptive or receptor-mediated mechanisms play a role in the transendothelial movement of plasma proteins, and other plasma constituents, our findings suggest a general role of the endothelial acidic compartments in modulating microvascular permeability to macromolecules.

Gap junctions may provide the structural basis for communication between arterial and endothelial cells and modulate their response to injury. We have utilized autoradiography and freeze fracture techniques to correlate proliferative activity with changes in the organization, density, and surface area of gap junctions in healing arterial endothelium. A well-circumscribed, reproducible area of endothelial loss was produced in the common carotid artery of the rabbit by desiccation of the intima. The lesion healed in 15 days by centripetal proliferation and migration of endothelial cells from adjacent uninjured areas. As controls, we used the uninjured contralateral artery. In parallel experiments, the vascular endothelium was sampled either for autoradiography after administration of 3H-thymidine or for freeze fracturing. Proliferative activity was measured by calculating the labeling index in autoradiographs of endothelium. The surface areas of gap junctions and that of the lateral endothelial membranes was measured by planimetry in electron micrographs of freeze fracture replicas. Two days after injury, cells at the growing edge of the endothelium revealed a marked decrease in the density and surface area of gap junctions, loss of tight junctions, and high labeling index (232 +/- 55 S.E.M.). At seven days, the labeling index was lower (61 +/- 31 S.E.M.), gap junctions were small but numerous, and short segments of tight junctions were present. At fifteen days, the endothelial integrity was restored, the labeling index was at control levels (3.32 +/- 3 S.E.M.), and both gap and tight junctions were well developed and indistinguishable from controls. The study shows that loss of junctions between endothelial cells is associated with high proliferative activity. As a corollary, well-developed gap junctions may prevent the response of the uninjured endothelium to circulating mitogens.