Binding and transcytosis of glycoalbumin by the microvascular endothelium of the murine myocardium: evidence that glycoalbumin behaves as a bifunctional ligand

The binding and transport of glycoalbumin (gA) by the endothelium of murine myocardial microvessels were studied by perfusing in situ 125I-gA or gA-gold complexes (gA-Au) and examining the specimens by radioassays and EM, respectively. After a 3-min perfusion, the uptake of radioiodinated gA is 2.2-fold higher than that of native albumin; it is partially (approximately 55%) competed by either albumin or D-glucose, and almost completely abolished by the concomitant administration of both competitors or by gA. D-mannose and D-galactose are not effective competitors. Unlike albumin-gold complexes that bind restrictively to plasmalemmal vesicles, gA-Au labels the plasma-lemma proper, plasmalemmal vesicles open on the lumen, and most coated pits. Competing albumin prevents gA-Au binding to the membrane of plasmalemmal vesicles, while glucose significantly reduces the ligand binding to plasmalemma proper. Competition with albumin and glucose gives additive effects. Transcytosis of gA-Au, already detected at 3 min, becomes substantial by 30 min. No tracer exit via intercellular junctions was detected. gA-Au progressively accumulates in multivesicular bodies. The results of the binding and competition experiments indicate that the gA behaves as a bifunctional ligand which is recognized by two distinct binding sites: one, located on the plasma membrane, binds as a lectin the glucose residues of gA; whereas the other, confined to plasmalemmal vesicles, recognizes presumably specific domains of the albumin molecule.     

Identification of albumin-binding proteins in capillary endothelial cells

Isolated fat tissue microvessels and lung, whose capillary endothelia express in situ specific binding sites for albumin, were homogenized and subjected to SDS-gel electrophoresis and electroblotting. The nitrocellulose strips were incubated with either albumin-gold (Alb-Au) and directly visualized, or with [125I]albumin (monomeric or polymeric) and autoradiographed. The extracts of both microvascular endothelium and the lung express albumin-binding proteins (ABPs) represented by two pairs of polypeptides with major components of molecular mass 31 and 18 kD. The ABP peptides have pIs 8.05 to 8.75. Rabbit aortic endothelium, used as control, does not express detectable amounts of ABPs. The ABPs subjected to electrophoresis bind specifically and with high affinity (Kd = approximately 60 X 10(-9)M) both monomeric and polymeric albumin: the binding is saturable at approximately 80 nM concentration and 50% inhibition is reached at 5.5 micrograms/ml albumin concentration. Sulfhydryl-reducing agents beta-mercaptoethanol and dithiothreitol do not markedly affect the ABPs electrophoretic mobility and binding properties. As indicated by cell surface iodination of isolated capillary endothelium followed by electroblotting, autoradiography, and incubation with Alb-Au, the bands specifically stained by this ligand are also labeled with radioiodine.     

Fatty acids binding to albumin increases its uptake and transcytosis by the lung capillary endothelium

To determine whether uptake and transcytosis of albumin (A) in continuous capillary endothelia are modified when this protein carries fatty acids, the transport of albumin-oleic acid and albumin-palmitic acid complexes was compared with that of defatted albumin. The probes, either radioiodinated or tagged with 5-nm gold particles (Au), or both, were perfused in situ or injected in vivo; after 3 or 30 min lung fragments were radioassayed or examined by electron microscopy. Both in situ and in vivo, the uptake of fatty acid-carrying albumin (A-FA) was consistently 2 to 3 times higher than that of defatted A. Electron microscopy revealed that A-FA complexes tagged with gold were taken up and transported across the endothelium by plasmalemmal vesicles. Morphometric analysis showed that as compared with A-Au, at 3 min the density of (A-FA)Au bound to plasmalemmal vesicles was 2 to 3 times higher, and the extent of transcytosis was increased. Uptake of the iodinated albumin was more effectively competed by A-FA complexes than by defatted A, suggesting a higher affinity of the former for the albumin binding sites of the endothelium. The results indicate that when carrying fatty acids, albumin is taken up specifically and with high affinity by the capillary endothelium, a process that may play a role in the transport of fatty acids from the plasma to the cells where they are metabolized.     

Quantitative immunocytochemical studies of endogenous albumin in rat aortic endothelial and mesothelial cells

Endogenous albumin was revealed over cellular structures of rat ascendent aorta endothelia and mesothelium, with high resolution and specificity, by applying the protein A-gold immunocytochemical approach. This approach allows albumin distribution to be studied under steady-state conditions. The cellular layers evaluated were the aortic endothelium, the capillary endothelium (vasa vasorum), and the mesothelium externally lining the aorta at this level. Gold particles, revealing albumin antigenic sites, were preferentially located over plasmalemmal vesicles and intercellular clefts of endothelial and mesothelial cells, though with different labeling intensities. The interstitial space was also labeled. Morphometrical evaluation of plasmalemmal vesicles demonstrated a higher surface density for these structures in capillary endothelial cells (12%) compared with those in aortic endothelial (5%) and mesothelial cells (2%). Quantitation of gold labeling intensities over these structures revealed a higher labeling over plasmalemmal vesicles of capillary endothelium than over those of aortic endothelium and mesothelium. This result, together with the higher surface density of plasmalemmal vesicles found in capillary endothelium, suggest an important role of these structures in the transendothelial passage of endogenous albumin, particularly for capillary endothelium. On the other hand, labeling densities over mesothelial clefts were found to be higher than those of capillary and aortic endothelia. Results from this study concur with the proposal of a differential passage of albumin according to the cell lining considered, and suggest to a role for mesothelial intercellular clefts in contributing to the presence of albumin in interstitial spaces.     

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.     

Rings of membrane sterols surround the openings of vesicles and fenestrae, in capillary endothelium

We investigated the distribution of sterols in the cell membrane of microvascular endothelium (mouse pancreas, diaphragm, brain, heart, lung, kidney, thyroid, adrenal, and liver) with the polyene antibiotic filipin, which reportedly has binding specificity for free 3-beta- hydroxysterols. In some experiments, concomitantly, cell-surface anionic sites were detected with cationized ferritin. Vessels were perfused in situ with PBS, followed by light fixation and filipin administration for 10 to 60 min. Tissues were further processed for thin-section and freeze-fracture electron microscopy. Short exposure (10 min) to filipin-glutaraldehyde solution resulted in the initial appearance, on many areas, of rings of characteristic filipin-sterol complexes within the rim surrounding stomata of most plasmalemmal vesicles, transendothelial channels, and fenestrae. Such rings were absent from the rims of the large openings of the sinusoid endothelium (liver, adrenal), coated pits and phagocytic vacuoles. After longer exposure (30-60 min), filipin-sterol complexes labeled randomly the rest of plasma membrane (except for coated pits, and partially the interstrand areas of junctions), and also marked most plasmalemmal vesicles. These peristomal rings of sterols were displayed mostly on the P face, and, at their full development, consisted of 6-8 units around a vesicle stoma, and 10-12 units around a fenestra. At their level, the intramembranous particles and the cell surface anionic sites were virtually excluded. Peristomal rings of sterols were also detected on the plasma membrane of pericytes and smooth muscle cells of the microvascular wall, which otherwise were poorly labeled with filipin- sterol complexes as compared to endothelial plasmalemma. It is presumed that the peristomal rings of cholesterol may represent important contributors to the local transient stabilization of plasma membrane and to the phase separation between cell membrane and vesicle membrane at a certain stage of their fusion/fission process.     

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.     

Albumin-binding proteins of endothelial cells: immunocytochemical detection of the 18 kDa peptide.

Extracts of isolated microvascular endothelial cells (MEC) and cultured bovine aortic endothelial cells (BAEC) were subjected to sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE), electrotransfer and incubation with albumin either radioiodinated or adsorbed to 5-nm gold particles. Both ligands reacted exclusively with two peptides of 18 and 31 kDa. To the 18 kDa peptide (excised from preparative SDS-PAGE), an antibody was raised in rabbits and purified by affinity on 18 kDa obtained from two-dimensional gel electrophoresis and immobilized on nitrocellulose paper. The specificity of the anti-18 kDa was assessed by immunoblotting and immunoprecipitation of endothelial cell extracts. To check whether the 18 kDa peptide is exposed on the endothelial cell surface and/or its components (uncoated pits, open plasmalemmal vesicles), the apical membrane of BAEC was radioiodinated, the solubilized proteins incubated with the anti-18 kDa, and the immune complexes formed were precipitated with protein A-Sepharose CL-4B. The ensuing SDS-PAGE and autoradiography revealed that from all radioiodinatable surface proteins, the 18 kDa was the only polypeptide immunoprecipitated by the anti-18 kDa antibody. To localize the 18 kDa peptide, we applied indirect immunofluorescence technique on cultured MEC and BAEC and immunoelectron microscopy (EM) on ultrathin cryosections of mouse heart. Nonpermeabilized whole MEC and BAEC incubated with anti-18 kDa followed by rhodamine-conjugated second antibody showed a relatively intense surface fluorescence often appearing as small dots. At the EM level, heart ultrathin cryosections exposed anti-18 kDa followed by gold-conjugated second antibody revealed that 18 kDa was primarily associated with the membrane of plasmalemmal vesicles of capillary endothelia.(ABSTRACT TRUNCATED AT 250 WORDS)     

Anionized and cationized hemeundecapeptides as probes for cell surface charge and permeability studies: differentiated labeling of endothelial plasmalemmal vesicles

To obtain small membrane markers easily accessible to the charged groups of the cell surface, we prepared, from hemeundecapeptide (HUP), three derivatives that maintain the peroxidatic activity: the anionized hemeundecapeptide, Mr 1,963, estimated diameter 1.68 nm, pl 3.5, for the detection of basic groups; and both a cationized hemeundecapeptide containing predominantly tertiary amino groups, Mr 2,215, estimated diameter 1.75 nm, pl 9.0, and a cationized hemeundecapeptide containing only primary amino groups, Mr 2,271, estimated diameter 1.75 nm, pl 10.6, for labeling acidic residues. The markers were perfused in situ in mice to label the luminal surface of fenestrated endothelium of pancreatic capillaries. Specimens were processed through the cytochemical reaction for peroxidatic activity and examined by electron microscopy. The anionized HUP and HUP (pl 4.85) marked the plasmalemma proper, the coated pits, and the membrane and diaphragms of plasmalemmal vesicles and transendothelial channels. The cationized HUP containing predominantly tertiary amino groups (pl 9.0) decorated all cell surface components with the exception of plasmalemmal vesicles and channels; the latter were, however, labeled by the cationized HUP containing only primary groups (pl 10.6), which suggests that these structures contain on their luminal surface very weak acidic residues of high pKa values. The fact that the membrane of plasmalemmal vesicles can discriminate against permeant cationic macromolecules only up to a pl of approximately 9.0 indicates that in the electrostatic restriction there is a charge limit. In the case of fenestrated capillary endothelium, the upper charge limit seems to be a pl of approximately 9.0. In these vessels, the charge discrimination is effective for molecules as small as 2 nm.     

Plasminogen activator: Morphological evidence of binding, internalization and delivery to lysosomes in 3T3 mouse fibroblasts

Summary Using a direct conjugate of urokinase and ferritin, the binding has been followed at the plasma membrane and the internalization of urokinase into BALB/C-3T3 fibroblasts, cultured in plasminogen-free conditions. At 0° C, the conjugate was observed bound on both coated and uncoated cell surface regions as singlets, and small and large clusters. No binding was observed in the presence of excess native urokinase. The binding was impaired by preincubation of the conjugate with a competitive inhibitor of the catalytic site, suggesting an interaction between the receptor and the catalytic site of the enzyme.Within 1 min at 37° C, urokinase clustered on coated regions of the plasma membrane. At 5 min after warming, ferritin was found on deeply indented coated pits and in both coated and uncoated vesicles close to the cell surface. By 10 min at 37° C, ferritin particles were present in uncoated endosomes and in multivesicular bodies in the Golgi area. Within 10 min, the receptors on the surface strongly decreased. New receptors were observed on the membrane after 20 min at 37° C. At this time, ferritin was observed both in endosomes or multivesicular bodies and in vesicles close to the plasma membrane.     

Surface charges associated with fenestrated brain capillaries. II. In vivo studies on the role of molecular charge in endothelial permeability

We report on the effect of the net charge of a tracer (ferritin) on its permeability in fenestrated capillaries of the brain. Our experiments show that the charge of this tracer actually influences its interaction with the endothelium. Three phases of tracer-endothelial interaction could be discriminated. Anionic and slightly cationic derivatives (pH 4.5-7.8) do not show any affinity to the luminal endothelial membrane. Ferritin derivatives with a pI value between 7.8 and 9.3 result in the labeling of the fenestrae without coating additional luminal plasmalemmal structures (i.e., coated pits and plasmalemmal vesicles). Tracers with a high positive net charge (pI greater than 9.3) led to their endocytotic uptake and extravasation by some transcytotic mechanism. Extravasated cationic ferritin accumulates in the endothelial basement membrane and binds to striated collagen fibrils. It is suggested that the pericapillary collagen fibrils of fenestrated brain capillaries act as a charge filter with respect to macromolecules.     

Transcytosis of albumin in capillary endothelium

We have used a variety of immunocytochemical procedures to localize albumin in transit through the capillary endothelium of the murine myocardium and thereby identify endothelial cell structures involved in albumin efflux. The most informative results were obtained with a protocol that included (a) removal of endogenous albumin by perfusion of the heart with PBS supplemented with 14 mM glucose, (b) perfusion of the heart vasculature with exogenous (bovine) albumin for various short time periods, (c) fixation of the vessels by formaldehyde- glutaraldehyde mixtures, (d) processing of fixed myocardium specimens through L. R. White embedding followed by sectioning, or direct thin frozen sectioning, and (e) reacting the surface of such specimens with antialbumin antibodies followed by gold-labeled secondary antibodies. The results indicate that (a) monomeric albumin binds (with low affinity) to the luminal surface of the capillary endothelium, (b) it is restricted in transit through the endothelium to plasmalemmal vesicles, and (c) it appears in the pericapillary spaces less than 15 s after the beginning of its perfusion. No albumin concentration gradients, centered with their maxima on the exits from intercellular spaces, were detected at any time points, including the shortest ones (15 and 30 s) investigated. Additional information comparing monomeric vs. polymeric albumin transcytosis was obtained using albumin-gold complexes. The results are discussed in terms of vesicular transport of albumin across the endothelium and the relations of this type of transport to the postulated pore systems of the physiological literature.     

Filipin-sensitive caveolae-mediated transport in endothelium: reduced transcytosis, scavenger endocytosis, and capillary permeability of select macromolecules

Caveolae or noncoated plasmalemmal vesicles found in a variety of cells have been implicated in a number of important cellular functions including endocytosis, transcytosis, and potocytosis. Their function in transport across endothelium has been especially controversial, at least in part because there has not been any way to selectively inhibit this putative pathway. We now show that the ability of sterol binding agents such as filipin to disassemble endothelial noncoated but not coated plasmalemmal vesicles selectively inhibits caveolae-mediated intracellular and transcellular transport of select macromolecules in endothelium. Filipin significantly reduces the transcellular transport of insulin and albumin across cultured endothelial cell monolayers. Rat lung microvascular permeability to albumin in situ is significantly decreased after filipin perfusion. Conversely, paracellular transport of the small solute inulin is not inhibited in vitro or in situ. In addition, we show that caveolae mediate the scavenger endocytosis of conformationally modified albumins for delivery to endosomes and lysosomes for degradation. This intracellular transport is inhibited by filipin both in vitro and in situ. Other sterol binding agents including nystatin and digitonin also inhibit this degradative process. Conversely, the endocytosis and degradation of activated alpha 2- macroglobulin, a known ligand of the clathrin-dependent pathway, is not affected. Interestingly, filipin appears to inhibit insulin uptake by endothelium for transcytosis, a caveolae-mediated process, but not endocytosis for degradation, apparently mediated by the clathrin-coated pathway. Such selective inhibition of caveolae not only provides critical evidence for the role of caveolae in the intracellular and transcellular transport of select macromolecules in endothelium but also may be useful for distinguishing transport mediated by coated versus noncoated vesicles.     

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.     

PERMEABILITY OF INTESTINAL CAPILLARIES : Pathway followed by Dextrans and Glycogens

The pathway followed by macromolecules across the wall of visceral capillaries has been studied by using a set of tracers of graded sizes, ranging in diameter from 100 A (ferritin) to 300 A (glycogen). Polysaccharide particles, i.e. dextran 75 (mol wt ~75,000; diam ~125 A), dextran 250 (mol wt 250,000; diam ~225 A), shellfish glycogen (diam ~200 A) and rabbit liver glycogen (diam ~300 A), are well tolerated by Wistar-Furth rats and give no vascular reactions ascribable to histamine release. Good definition and high contrast of the tracer particles were obtained in a one-step fixation—in block staining of the tissues by a mixture containing aldehydes, OsO₄ and lead citrate in phosphate or arsenate buffer, pH 7.4, followed by lead staining of sections. The glycogens and dextrans used move out of the plasma through the fenestrae and channels of the endothelium relatively fast (3–7 min) and create in the pericapillary spaces transient (2–5 min) concentration gradients centered on the fenestrated sectors of the capillary walls. The tracers also gained access to the plasmalemmal vesicles, first on the blood front and subsequently on the tissue front of the endothelium. The particles are temporarily retained by the basement membrane. No probe moved through the intercellular junctions. It is concluded that, in visceral capillaries, the fenestrae, channels, and plasmalemmal vesicles, viewed as related parts in a system of dynamic structures, are the structural equivalent of the large pore system.     

Maturation of myocardial capillaries in the fetal and neonatal rat: an ultrastructural study with a morphometric analysis of the vesicle populations

The ultrastructural morphology of the cellular and extracellular components of the developing myocardial capillary wall--from the 16-day-gestation fetus of the rat to the 21-day neonate--was examined. A morphometric analysis of plasmalemmal vesicles and of coated vesicles and pits of capillary endothelial cells was performed during the same developmental period. As the lateral extensions of the capillary endothelial cells change from irregular to regular in their thickness during development, there is an increase in the number of plasmalemmal vesicles and a progression from clusters of plasmalemmal vesicles to a uniform distribution in the endothelial cell. The ratio of vesicles which are open to the luminal front, which are "free" in the cytoplasm, or which are open to the abluminal front of the endothelial cell was consistent throughout development. The numerical density of plasmalemmal vesicles demonstrates a gradual and significant increase. In contrast, the numbers of coated vesicles and pits are variable within a very narrow range, and no pattern of increase or decrease is discernible during development. Similarly, there is no change in interendothelial cell junctions, which consist of occluding and primitive adhesive junctional types, during development. The lamina densa of the basal lamina gradually develops from discontinuous, patchy densities along the abluminal surface of the endothelial cells to a continuous and distinct layer by 21 days gestation. The presence of the proteoglycan species in the developing basal lamina was assessed with the cationic dye ruthenium red (RR), and the appearance of RR-marked proteoglycans was found to parallel the appearance of lamina densa material. found to parallel the appearance of lamina densa material. The RR sites appear discontinuously in patches; and later, the RR sites appear in a continuous and regular planar lattice in the lamina rara interna and externa at 21 days gestation. A complete array of RR-stainable anionic sites outside a continuous lamina densa near birth indicates that the basal laminae of developing capillaries in the heart are morphologically, and in part biochemically, mature by the end of the first neonatal week. Our results show that the endothelial cells and the subtending basal lamina of myocardial capillaries gradually mature morphologically during the final days of gestation and the first neonatal week.(ABSTRACT TRUNCATED AT 400 WORDS)     

Endocytic vesicles and surface invaginations in cultured vascular endothelium: a morphometric comparison

Ruthenium red staining of plasma membrane glycoproteins of confluent cultured arterial endothelial cells revealed that the limiting membrane of many apparently discrete cytoplasmic vesicles was continuous with the plasmalemma. Surface invaginations accessible to ruthenium red appeared as vesicles when sectioned out of the plane of attachment to the cell surface, Morphometric analysis of ruthenium red-positive (RR+) and ruthenium red-negative vesicles (RR-) indicated that 47.2% of the total apparent vesicle population was RR+ and that those infoldings accounted for 19.6 +/- 1.4% of the cell surface in transverse sections. Whereas 14.9% of the true vesicles (ruthenium red-negative) were coated vesicles, only 1.1% of RR+ "vesicles" were coated pits. These studies show that although many deep infoldings of the cell surface may be misinterpreted as vesicles, almost all are uncoated. The existence of discrete coated vesicles (independent of coated pits) in vascular endothelium in vitro is readily apparent.     

Vesicular transport in capillary endothelium: does it occur?

A revised picture of the organization of endothelial plasmalemmal vesicles is presented. Three-dimensional reconstructions of endothelial segments from frog mesenteric capillaries and rat heart capillaries based on ultrathin serial sectioning have shown that plasmalemmal vesicles are not true vesicles but parts of an elaborate system of invaginations of the surface membrane. The revised picture probably applies to capillary endothelia in general. The absence of free cytoplasmic vesicles implies that vesicular transport is unlikely to occur. A reinterpretation of previous studies of vesicular transport shows that they are equally compatible with the present view that plasmalemmal vesicles are static elements of invaginations of the endothelial surface membrane.     

The membrane composition of coated pits, microvilli, endosomes, and lysosomes is distinctive in the rat kidney proximal tubule cell

The distribution of a number of membrane proteins on plasmalemmal microdomains (microvilli, coated pits) and in endosomes and lysosomes of the proximal tubule epithelial cell was determined in normal rat kidneys by immunofluorescence and immunoelectron microscopy. Two major brush border proteins, 130 and 94 kD, and gamma-glutamyl transpeptidase were detected on the membranes of the microvilli but were not found on membranes of coated pits. Gp330, the Heymann nephritis antigen, and clathrin were localized in coated pits. The lysosomal membrane glycoprotein, lgp120 (Lewis, V., S. A. Green, M. Marsh, P. Vihko, A. Helenius, and I. Mellman, 1985, J. Cell Biol., 100: 1839-1847) was restricted to lysosomes where it co-localized with beta-glucuronidase. Endosomes, identified by preloading with HRP injected 5-15 min before rats were killed, did not contain detectable amounts of any antigen tested. The distribution of the same proteins was also determined in rats given sodium maleate, which is known to slow or reduce protein absorption by the proximal tubule and to cause vacuolation of the endocytic apparatus. After maleate treatment the distribution of microvillar and lysosomal markers was unchanged, but the coated pit markers were redistributed--gp330 was concentrated in newly formed apical vacuoles, and clathrin was diffusely distributed in the apical cytoplasm or on apical coated vesicles. These findings indicate that the membrane composition of microvilli, coated pits, endosomes, and lysosomes is distinctive in the proximal tubule cell; and that gp330, unlike other known coated pit membrane components, is not transferred to endosomes during endocytosis. After maleate treatment, the coated pits lose their clathrin coats, and the corresponding membrane is internalized.     

Receptor-mediated endocytosis of immunoglobulin-coated colloidal gold particles in cultured mouse peritoneal macrophages

Endocytosis of immunoglobulin G (IgG)-coated colloidal gold particles in cultured mouse peritoneal macrophages was studied by scanning and transmission electron microscopy. At 4 degrees C, the tracers adhered to the plasma membrane and accumulated in coated pits located in the bottom of furrows or deep invaginations on the cell surface. In the presence of an excess of unlabeled mouse IgG, cellular binding of the tracer was reduced by 80 to 90%. After warming to 37 degrees C, surface-bound tracer particles were rapidly ingested and transported to small and large vesicles lacking membrane coat. From here, they were then passed over to multivesicular bodies and lysosomes characterized by their content of myelin-like figures and other inclusions. Double-labeling experiments with IgG-coated colloidal gold particles of two different sizes (20 and 5 nm diameter) indicated that the plasma membrane was depleted of binding sites after uptake of a polyvalent ligand. The restoration of the binding capacity was a slow process requiring ongoing protein synthesis. On the basis of these observations, a model for endocytosis of immune complexes in macrophages is presented. It includes the following four steps: IgG-containing macromolecular aggregates bind to specific receptors in the plasma membrane. These appear to be preclustered in coated pits or able to move laterally within the membrane even at 4 degrees C. The receptor-ligand complexes are internalized and transferred sequentially to larger uncoated vesicles or endosomes, multivesicular bodies, and lysosomes with inclusions of varying appearance. Receptors and ligands are degraded within the lysosomes.(ABSTRACT TRUNCATED AT 250 WORDS)