Binding and endocytosis of heparin-gold conjugates by the fenestrated endothelium of the rat choriocapillaris

Summary Heparin-gold probes were used to localize regions of heparin binding on the luminal surface of the diaphragmed-fenestrated endothelium of the rat choriocapillaris. Structures of endothelial cells were unlabeled when rats were kept on ice and perfused with solutions at 4° C. When the heparin-gold quantity was doubled, only a few heparin-gold particles marked some diaphragms spanning fenestrae, vesicles and channels, parajunctional regions of the plasmalemma, and coated pits. With solutions at 4° C, but the animals kept at room temperature, all of these structures in the endothelial cells were labeled. This binding was not altered by the perfusion of low levels of unlabeled heparin, but was eliminated following high levels of unlabeled heparin, and by digestion with trypsin and pronase. At 37° C, heparin was localized to the above structures and, in addition, was internalized into coated vesicles, endosomes, and multivesicular bodies, but not other types of lysosomes. Some particles were found in tubules adjacent to the Golgi stacks. Heparin-gold was also transported to the abluminal front of the endothelium by vesicles. A desulfated, non-anticoagulant, fraction of heparin bound to gold was localized to the endothelium in the same manner. These results demonstrate receptors for heparin on the surface of a fenestrated endothelium. Furthermore, they show the pathway of endocytosis and transport of heparin. The possible roles of heparin in the function of the endothelium is discussed.     

Transendothelial transport (transcytosis) of iron-transferrin complex in the rat liver

To determine the transport pathway of iron-transferrin complex (Fe-TF) into the hepatocyte, we labeled Fe-TF with colloidal gold and perfused rat liver through the portal vein with this probe under different conditions. The tissue was then studied by transmission electron microscopy. At a cold temperature (approximately 4 degrees C), the probe bound to the luminal surface of sinusoidal endothelium without internalization. The binding was limited to the endothelium and there was no binding to Kupffer cells or hepatocytes. The binding was inhibitable in the presence of excess soluble Fe-TF, indicating the specificity of the bindings. At 37 degrees C the probe was internalized via a system of coated pits and vesicles. Morphometric analyses of the temporal sequence of events suggested that the probe was transported, in part, across the endothelium via a system of tubules and vesicles and externalized on the abluminal side via a system of coated pits. The probe was then taken up by hepatocytes. Only minimal uptake was noted when gold-labeled bovine serum albumin was used either at the low temperature or at 37 degrees C. The findings suggest that the liver uptake of Fe-TF complex by hepatocytes is a transendothelial phenomenon (transcytosis).     

Plasma membrane-associated vesicles in retinal capillaries of the rat

The structure and function of abluminal vesicles in endothelial cells of rat retinal capillaries was examined using glutaraldehyde-tannic acid fixation and the hemeproteins--horseradish peroxidase, microperoxidase, and lactoperoxidase--as tracers. Numerous vesicles, delimited by a tannic acid-positive membrane, were distributed along the abluminal front. Other vesicles were arranged in clusters and chains or tubule-like structures. Such vesicles were not found in the vicinity of the capillary lumen. When the retina was exposed to hemeproteins, either in vitro or after intravitreal injection, the abluminal vesicles became labeled with tracer reaction product. Apparently "free" vesicles and tubules seen in tangential sections through the basal lamina were also labeled, suggesting that they were in continuity with the plasma membrane in another plane of section. No enzyme reaction product was present in the capillary lumen. Peroxidase-positive multivesicular bodies were observed, suggesting that some protein was endocytosed and directed to lysosomes where it was presumably degraded. The results suggest that abluminal endothelial vesicles represent pits or invaginations of the plasma membrane and, as such, are not involved in the transendothelial transport of protein from the perivascular space to the capillary lumen. Tannic acid treatment revealed a population of similar vesicles associated with the plasma membrane of pericytes. After exposure to hemeproteins, enzyme reaction product was localized in these vesicles and in a few multivesicular bodies. The results suggest that the majority of these vesicles are in continuity with the plasma membrane and are not involved in endocytosis.     

The role of liver endothelium in the binding and uptake of ceruloplasmin: studies with colloidal gold probe

To determine the mode of uptake of ceruloplasmin (CP) by liver, the protein was labeled with colloidal gold and infused into the portal vein. In cold almost all probes bound to the sinusoidal endothelium, and at 37 degrees C internalization via a system of coated pits and vesicles occurred. Only rarely did the probe appear to bypass the endothelium, moving to the abluminal side through the gaps between endothelial cells. In the endothelial cytoplasm, the probe was seen in coated vesicles, endosomes, tubules, and large vesicles which may have formed by fusion of endosomes and tubules. Moreover, externalization of the probe to the abluminal side was noted, and this also occurred via a system of coated vesicles. The findings suggest that the uptake of CP in the liver may be primarily a transendothelial phenomenon (transcytosis).     

Distribution of sialoglycoconjugates on the luminal surface of the endothelial cell in the fenestrated capillaries of the pancreas

Sialic acid-bearing molecules on the luminal surface of the vascular endothelium in mouse and rat pancreatic capillaries were detected electron microscopically by using a procedure with ferritin hydrazide (FH), after preferential oxidation of sialyl residues with sodium periodate. The distribution of FH on the endothelial surface demonstrated the existence of microdomains with various densities of sialoglycoconjugates oxidizable by sodium periodate and accessible to the tracer. On the plasmalemma proper, FH binding sites were heterogeneously distributed. Their concentration on various microdomains decreased as follows: plasmalemma proper greater than coated pits greater than stomal diaphragms of plasmalemmal vesicles and transendothelial channels, and fenestral diaphragms. The membrane of plasmalemmal vesicles and transendothelial channels was not labeled by FH. Nonspecific binding of FH to the nonoxidized endothelial surface or that oxidized after neuraminidase treatment was relatively low.     

Transendothelial transport (transcytosis) of iron-transferrin complex in the bone marrow

To determine the transport pathway of iron-transferrin complex (Fe-TF) across the marrow-blood barrier, we labeled Fe-TF with colloidal gold and perfused rat femoral marrow with this probe. At 4 degrees C, the probe bound to the luminal surface of marrow sinus endothelium. The binding was inhibitable in the presence of excess native Fe-TF indicating the specificity of the binding. At 37 degrees C, the probe was internalized largely via a system of coated pits and vesicles and transported across the endothelium via a system of tubules and endosomal vesicles. It could not be ascertained if all Fe-TF was still associated with the colloidal gold probe within the endothelium, but the probe appeared to be externalized on the abluminal side into the interstitium where it subsequently bound to the surface of marrow erythroblasts and was internalized. Endothelium appeared to store part of the probe within a large vesicular system. No transport of Fe-TF was noted through diaphragmed fenestrations, diaphragmed vesicles, or interendothelial junctions. No endothelial uptake of this magnitude was noted when native gold particles or gold-labeled bovine serum albumin was used. Our findings indicate that in the bone marrow, gold-labeled Fe-TF is first taken up by sinus endothelium through a receptor-mediated mechanism and is possibly transported transendothelially via a vesicular system (transcytosis).     

Retinol-binding protein is synthesized in the mammalian eye

As the chromophoric component of the visual pigment, retinol plays an essential role in vision. In the plasma, retinol is transported by retinol-binding protein (RBP) in complex with transthyretin (TTR, prealbumin). In previous work we demonstrated intraocular synthesis of TTR. To determine whether RBP is also synthesized in the eye, we performed Northern and Western blot analysis of rat eye, and detected both RBP mRNA and immunoreactive RBP. Regional Northern analysis of bovine eye localized RBP mRNA to ciliary body/iris and retina/RPE. Preliminary immunohistochemical studies revealed a widespread but heterogeneous distribution of RBP in rat eye. We postulate that ocular RBP and TTR are involved in the intraocular translocation of retinol.     

Temperature dependence of protein transport across lymphatic endothelium in vitro

The purpose of the work was to develop an in vitro model for the study of lymphatic endothelium and to determine, using this model, whether or not a cytoplasmic process may be involved in transendothelial transport. Segments of canine renal hilar lymphatics were dissected clean, cannulated at both ends, and transferred to a perfusion chamber for measurement of transendothelial protein transport and for ultrastructural tracer studies. The segments were subsequently processed for light and electron microscopy. By both structural and functional criteria the lymphatics were judged to have retained their integrity. At 37 degrees C, 36 lymphatics showed a mean rate of protein transport of 3.51 +/- 0.45 (SEM) micrograms/min per cm2 of lymphatic endothelium. The rate was influenced by the temperature of the system, being significantly reduced by 49% +/- 4.8, 31% +/- 5.3, and 29% +/- 3.9 when the temperature was lowered to 4 degrees, 24 degrees, and 30 degrees C, respectively. When the temperature was raised to 40 degrees C, the rate was significantly increased by 48% +/- 12.2. The vesicular system and the intercellular regions in vessels with increased or reduced rates of transport were analyzed quantitatively to ascertain whether the rate changes could be correlated with ultrastructurally demonstrable changes in either of these postulated pathways. No significant changes in junctional or vesicular parameters were found between the control lymphatics and those perfused at 24 degrees, 30 degrees, and 40 degrees C. At 4 degrees C, the temperature at which the rate of protein transport was maximally reduced, vesicular size decreased, and the number of free cytoplasmic vesicles increased, whereas the number associated with the abluminal and luminal surfaces decreased. We concluded that isolated perfused lymphatic segments transport protein at a relatively constant rate under control conditions, and that this transendothelial transport comprises both temperature-dependent and temperature-independent mechanisms. The findings were considered in terms of the different theories of lymph formation and were interpreted as providing support for the vesicular theory.     

Impermeability of hagfish cerebral capillaries to horseradish peroxidase

Summary Brain capillaries and their permeability to intravenously injected horseradish peroxidase, HRP, (MW: 40,000) were examined electron-microscopically in an attempt to find a structural explanation for the poorly developed blood-brain barrier in the hagfish, Myxine glutinosa. In particular, it was the aim of this study to examine the role of the numerous endothelial vesicles and tubules in the transport of this tracer between blood and brain. Many of the vesicles and tubules were found to be in continuity with the luminal or abluminal surfaces, but tubules generating channels through the endothelial cells were never observed. The cleft between adjacent endothelial cells was obliterated by punctate junctions. HRP, which was allowed to circulate for up to 35 min, was not found in the basal lamina or in the surrounding brain parenchyma. Few of the luminal vesicles and tubules were marked by the tracer. In the intercellular cleft HRP was stopped by the junctions. It is concluded that the hagfish like other vertebrates has a blood-brain barrier to HRP, and the numerous vesicles and tubules occurring in hagfish brain endothelium are not involved in the transendothelial transport of this macromolecule.     

Interactions of retinol with binding proteins: studies with retinol-binding protein and with transthyretin.

The interactions within the molecular complex in which retinol circulates in blood were studied. To monitor binding between retinol-binding protein (RBP) and transthyretin (TTR), TTR was labeled with a long-lived fluorescence probe (pyrene). Changes in the rotational volume of TTR following its association with RBP were monitored by fluorescence anisotropy of the probe. Titration of TTR with holo-RBP revealed the presence of 1.5 binding sites characterized by a dissociation constant Kd = 0.07 microM. At 0.15 M NaCl, binding of RBP to TTR showed an absolute requirement for the native ligand, retinol. At higher ionic strength (0.5 M NaCl), RBP complexed with retinal also bound to TTR with high affinity (Kd = 0.134 microM). RBP containing retinoic acid did not bind to TTR even at the high salt concentration. The data suggest that the TTR binding site on RBP is in close proximity to the retinoid binding site and that the head group of retinoic acid, when bound to RBP, presents steric hindrance for the interactions with TTR. The implications of the data for selectivity in retinoid transport in the circulation are discussed. The kinetics of the steps leading to complete dissociation of the retinol-RBP-TTR complex was also studied. The first step of this process was dissociation of retinol, which had a rate constant of 0.06/min. Following loss of retinol, the two proteins dissociate. The rate of dissociation is slow (k = 0.055/h), however, indicating that the complex apo-RBP-TTR will be an important factor in regulating serum levels of retinol.     

Differentiated microdomains on the luminal surface of the capillary endothelium. II. Partial characterization of their anionic sites

To investigate the chemical nature of the cationic ferritin (CF)- binding sites of the differentiated microdomains of the capillary endothelium, the vasculature of the mouse pancreas and intestinal mucosa was perfused in situ with neuraminidase, hyaluronidase, chondroitinase ABC, heparinase, and three proteases: trypsin, papain, and pronase. Proteases of broad specificity removed all anionic sites, suggesting that the latter are contributed by acid glycoproteins or proteoglycans. Neuraminidase, hyaluronidase, and chondroitinase ABC reduced the density of CF-binding sites on the plasmalemma proper, but had no effect on either coated pits or fenestral diaphragms. Heparinase removed CF-binding sites from fenestral diaphragms and had no effect on coated pits. Taken together, these results indicate that the anionic sites of the fenestral diaphragms are contributed primarily by heparan sulfate and/or heparin, whereas those of the plasmalemma proper are of mixed chemical nature. The membranes and diaphragms of plasmalemmal vesicles and transendothelial channels do not bind CF in control specimens; this condition is not affected by the enzymic treatments mentioned above.     

The cell surface of a restrictive fenestrated endothelium

The choriocapillaris is one example of a capillary bed lined by a fenestrated endothelium that is restrictive to exogenous tracers and endogenous plasma proteins. In this study we have examined the distribution of cell-surface monosaccharides utilizing biotinylated lectin-avidin ferritin cytochemistry. Receptors for wheat germ agglutinin were localized to the plasmalemma and diaphragms of some fenestrae, vesicles, and channels at the luminal endothelial front in amounts greater than seen for the other lectins employed. The absence of labeling following inhibition with N-acetylglucosamine and after tissue digestion with N-acetylhexosaminidase, but not after neuraminidase indicated that this lectin marked N-acetylglucosamine residues and not sialic acid. Wheat germ agglutinin receptors were not affected by pronase E or trypsin digestion, but were partially removed by proteinase K. The latter also removed many fenestral diaphragms. Wheat germ agglutinin receptors were cleaved with endoglycosidase D. The combined results indicate that the wheat germ agglutinin receptor is of the low-mannose type and part of a protein with hydrophobic properties. Receptors for concanavalin A (mannose) and Ricinus communis agglutinin (galactose) were also localized to the plasmalemma and endothelial diaphragms. The examination of sections at different tilt angles revealed that these lectins bound to the endothelium in a non-random distribution, encircling diaphragms of fenestrae and channels. Soybean agglutinin (N-acetylgalactosamine) marked endothelial structures sparsely. Following digestion with pronase E or trypsin, receptor sugars for the latter three lectins were completely removed, indicating their presence on protease susceptible glycoproteins. These findings demonstrate that the endothelium of the choriocapillaris bears carbohydrate moieties that are different than those described for permeable fenestrated endothelia.     

Study on the mechanism of interference of 3,4,3',4'-tetrachlorobiphenyl with the plasma retinol-binding proteins in rodents

The mechanism of plasma retinol reduction in rodents by 3,4,3',4'-tetrachlorobiphenyl (TCB) was investigated by radioimmunochemical analysis of the amounts of circulating and hepatic retinol-binding protein (RBP) and transthyretin (TTR) in exposed and control animals. Plasma RBP concentrations were markedly reduced in C57BL/Rij mice (50%) at 4 days, in DBA/2 mice (37-41%) at 4 and 8 days, and in Sprague-Dawley rats (58%) at 2 days after exposure to TCB. These reductions paralleled the time course of reduction of plasma retinol after exposure to TCB. Hepatic RBP concentrations were somewhat increased in TCB-treated animals, especially in the C57BL/Rij mouse and Sprague-Dawley rat. However, the release of hepatic RBP into the circulation was not blocked by TCB treatment, as analysed in vitamin A deficient rats. In addition, the amount of plasma TTR was in the normal range in TCB-treated rats. The dissociation constants of the RBP-TTR complex as analysed by polarization of fluorescence appeared to be significantly increased (from 0.5 x 10(-7) M-1 to 2.4 x 10(-7) M-1) in the presence of a TCB metabolite, isolated from plasma of TCB-treated rats. In addition, the estimated number of binding sites for RBP on the TTR molecule was reduced (from 2.8 to 1.7 sites) upon treatment of TTR with the TCB metabolite. These data support the hypothesis that plasma retinol reduction by TCB might result from a weakening of the RBP-TTR complex, in the presence of the TCB metabolite bound to the TTR.     

Restriction of exogenous transthyretin (prealbumin) by the endothelium of the rat choriocapillaris

The endothelium of the choriocapillaris has been shown to restrict molecules with Einstein-Stokes radii greater than or equal to 3.2 nm which correspond to minimal molecular weights of approximately 64,000-68,000 daltons. The present study was undertaken to determine if the endothelium restricts exogenous transthyretin (prealbumin), a 55,000-dalton carrier of retinol-binding protein. Rats were injected with human 125I-transthyretin which was allowed to circulate for 15 and 30 min. Chromatographic analysis demonstrated that the human transthyretin did not bind to rat blood proteins. Eye tissue from injected rats was prepared for light and ultrastructural autoradiographic analysis. Autoradiographic grains were confined to areas over the lumen of the choriocapillaris with few present on Bruch's membrane, the basement membrane common to the endothelium of the choriocapillaris and the retinal pigment epithelium. These findings demonstrate that the choriocapillaris can restrict transthyretin and suggest a possible role of its endothelium in the metabolism of retinol-carrier molecules.     

Immunocytochemical studies on the localization of plasma and of cellular retinol-binding proteins and of transthyretin (prealbumin) in rat liver and kidney

The immunocytochemical localization of cellular retinol-binding protein (CRBP), of plasma retinol-binding protein (RBP), and of plasma transthyretin (TTR) was studied in rat liver and kidney. The studies employed normal rats, retinol-deficient rats, and rats fed excess retinol. Antisera were prepared in rabbits against purified rat CRBP, RBP, and TTR. The primary antibodies and goat anti-rabbit IgG were purified by immunosorbent affinity chromatography, using the respective pure antigen coupled to Sepharose as the immunosorbent. This procedure effectively removed cross-reactive and heterophile antibodies, which permitted the specific staining and localization of each antigen by the unlabeled peroxidase-antiperoxidase method. CRBP was found to be localized in two cell types in the liver, the parenchymal cells and the fat-storing cells. Diffuse cytoplasmic staining for CRBP was seen in all the parenchymal cells. Much more intense staining for CRBP was seen in the fat-storing cells. The prominence of the CRBP-positive fat- storing cells changed markedly with vitamin A status. Thus, these cells were most prominent, and appeared most numerous, in liver from rats fed excess retinol. Both RBP and TTR were localized within liver parenchymal cells. The intensity of RBP staining increased markedly in retinol-deficient rat liver, consistent with previous biochemical observations. With the methods employed, specific staining for RBP or TTR was not seen in cells other than the parenchymal cells. In the kidney, all three proteins (CRBP, RBP, and TTR) were localized in the proximal convoluted tubules of the renal cortex. Staining for RBP was much more intense in normal kidney than in kidney from retinol- deficient rats. These findings reflect the fact that RBP in the tubules represents filtered and reabsorbed RBP. The pattern of specific staining for CRBP among the various tubules was very similar to that seen for RBP on adjacent, serial sections of kidney. The function of CRBP in the kidney is not known.     

Differentiated microdomains on the luminal surface of the capillary endothelium. I. Preferential distribution of anionic sites

Cationized ferritin (CF), introduced systemically in vivo or by perfusion in situ, binds preferentially to certain microdomains of the luminal plasmalemma of fenestrated capillaries (mouse pancreas and jejunum). The density and affinity of binding decrease in the following order: fenestral diaphragms greater than coated pits greater than plasmalemma proper. CF binds neither to the membrane of plasmalemmal vesicles and transendothelial channels nor to the corresponding stomatal diaphragms. The distribution pattern is the same when glutaraldehyde fixation precedes the administration of the tracer by perfusion, provided fixation is followed by quenching of residual free aldehyde groups. A much smaller cationic probe (alcian blue) perfused together with the fixative reveals a similar distribution pattern. The functional implications of the association of these microdomains with structures involved in capillary permeability are discussed.     

Production of recombinant human transthyretin with biological activities toward the understanding of the molecular basis of familial amyloidotic polyneuropathy (FAP).

Transthyretin (TTR) is a plasma protein interacting with thyroxine T4 and retinol binding protein (RBP). Several variants of TTR with single amino acid substitutions have been identified as the major components of the amyloid fibrils of familial amyloidotic polyneuropathy (FAP), a fetal, autosomal dominant genetic disease. The elucidation of the molecular nature of the variants distinct from that of the wild-type TTR is crucial for understanding the amyloidogenesis in FAP, but our understanding is very poor mainly because of the unavailability of pure variant TTRs. In the present study, we used an Escherichia coli OmpA secretion vector (Ghrayeb et al., 1984) and achieved an effective production of the variant TTRs related to FAP including Met-30, Ile-33, Ala-60, Tyr-77, Met-111, and Ile-122 types. The variant TTRs produced in this system were efficiently secreted to the culture media. The chemical analysis showed that the secreted TTR (Met-30 type) has the same N-terminus as the native one. IEF analyses also indicated that the secreted product is properly processed as assessed by its pI. Furthermore, the secreted TTR was shown to have biological activities, namely, the thyroxin binding activity and the ability to associate with retinol binding protein, indicating that the secreted TTR polypeptide is properly folded. The present work also demonstrated that the processing/secretion of the recombinant TTR molecules in E. coli was strongly affected by single amino acid substitutions.     

Application of an allosteric model to describe the interactions among retinol binding protein 4, transthyretin, and small molecule retinol binding protein 4 ligands

Retinol binding protein 4 (RBP4) is a serum protein that serves as the major transport protein for retinol (vitamin A). Recent reports suggest that elevated levels of RBP4 are associated with insulin resistance and that insulin sensitivity may be improved by reducing serum RBP4 levels. This can be accomplished by administration of small molecules, such as fenretinide, that compete with retinol for binding to RBP4 and disrupt the protein-protein interaction between RBP4 and transthyretin (TTR), another serum protein that protects RBP4 from renal clearance. We developed a fluorescence resonance energy transfer (FRET) assay that measures the interaction between RBP4 and TTR and can be used to determine the binding affinities of RBP4 ligands. We present an allosteric model that describes the pharmacology of interaction among RBP4, TTR, retinol, and fenretinide, and we show data that support the model. We show that retinol increases the affinity of RBP4 for TTR by a factor of 4 and determine the affinity constants of fenretinide and retinyl acetate. The assay may be useful for characterizing small molecule ligands that bind to RBP4 and disrupt its interaction with TTR. In addition, such a model could be used to describe other protein-protein interactions that are modulated by small molecules.     

Immunocytochemical and labelled tracer approaches to uptake and intracellular routing of Immunoglobulin-G (IgG) in the human placenta

Summary Isolated lobules of freshly delivered human term placenta were (a) subjected to an indirect immunoelectron ultracryo method in which the immunoreactivity of endogenous Immunoglobulin-G (IgG) to rabbit anti-human IgG antibody was localized with protein-A-colloidal gold and (b) extracorporeally perfused and human IgG molecules complexed to horseradish peroxidase (HRP) added to the maternal perfusate and the uptake of IgG-HRP over different perfusion durations visualized ultrastructurally by using diaminobenzidine cytochemistry. Immunoreactivity to anti-human IgG antibody was localized all along the apical plasmalemma, in apical coated and uncoated vesicles, in apical and juxtanuclear multivesicular bodies, and in basal vesicles of the syncytiotrophoblast layer of the placenta. The stroma separating the syncytiotrophoblast from the foetal endothelium as well as vesicles within the endothelium were immunoreactive. No immunoreactivity was localized in paracellular clefts of endothelia. A similar distribution of exogenous IgG-HRP was observed for the perfused placentae. When bovine IgG-HRP or HRP alone were used as control tracers no uptake was seen for the former whilst the latter was observed only in early endosomal vesicles of the syncytiotrophoblast. The pattern of localization visualized in both studies is consistent with receptor-mediated uptake of IgG by the syncytiotrophoblast and a vesicular transport of IgG across the foetal endothelium.     

Transendothelial transport of serum albumin: a quantitative immunocytochemical study

The steady-state distribution of endogenous albumin in mouse diaphragm was determined by quantitative postembedding protein A-gold immunocytochemistry using a specific anti-mouse albumin antibody. Labeling density was recorded over vascular lumen, endothelium, junctions, and subendothelial space. At equilibrium, the volume density of interstitial albumin was 18% of that in circulation. Despite this large difference in albumin concentration between capillary lumen and interstitium, plasmalemmal vesicles labeling was uniformly distributed across the endothelial profile. 68% of the junctions displayed labeling for albumin, which was however low and confined to the luminal and abluminal sides. The scarce labeling of the endothelial cell surface did not confirm the fiber matrix theory. The kinetics of albumin transcytosis was evaluated by injecting radioiodinated and DNP-tagged BSA. At 3, 10, 30, and 60 min, and 3, 5, and 24 h circulation time, blood radioactivity was measured and diaphragms were fixed and embedded. Anti-DNP antibodies were used to map the tracer in aforementioned compartments. A linear relationship between blood radioactivity and vascular labeling density was found, with a detection sensitivity approaching 1 gold particle per DNP-BSA molecule. Tracer presence over endothelial vesicles reached rapidly (10 min) a saturation value; initially localized near the luminal front, it evolved towards a uniform distribution across endothelium during the first hour. An hour was also needed to reach the saturation limit within the subendothelial space. Labeling of the junctions increased slowly, out of phase with the inferred transendothelial albumin fluxes. This suggests that they play little, if any, role in albumin transcytosis, which rather seems to proceed through the vesicular way.