The role of sialated glycoproteins in endocytosis, permeability and transmural passage in the myeloid endothelium.

Changes in the anionic charge distribution at the luminal face of the endothelium of the sinusoids of the bone marrow have been studied at sites of endocytosis by large bristle coated vesicles and at the sites of molecular permeability through diaphragmed fenestrae. The anionic charge distribution has also been studied at the abluminal aspect of these vessels at sites of transmural blood cell passage. Cationic surface markers such as colloidal iron, native ferritin and polycationic ferritin used at low pH, 1.8, and the use of neuraminidase show that the nonmodified endothelial cell surface has exposed sialic acid groups, which are absent at the sites of these functional specializations. Polycationic ferritin binding over a range of pH levels indicates the prsence of another species of anionic materials present at both the nonmodified cell surface and at the sites of the cell surface modifications. This second group of anionic compounds is neuraminidase resistant and has a pKa higher than that of sialic acid (pKa:2.6).     

Interaction of plasma proteins with negatively charged sites on the pulmonary capillary endothelium of the rat

Summary The endothelial glycocalyx, a polyanionic structure which may regulate the passage of solutes and water through the endothelium, readily binds cationic ferritin (CF). In normal, nonexchange-transfused rats, however, only 7.5% and 6.0% of the luminal plasma membrane and 7.5% and 5.0% of vesicle diaphragms on the thick and thin side of pulmonary capillaries, respectively, bound cationic ferritin. With the graded removal of circulating proteins by exchange transfusion with fluorocarbon emulsion, up to 89 and 82% of the luminal surface, and 76 and 73% of vesicle diaphragms on the thick and thin sides, respectively, bound CF. Although the extent of binding on the thin side was consistently less than on the thick side, the difference was not statistically significant. The extensive binding of CF to the glycocalyx in totally exchange-transfused rats was completely reversible upon addition of lyophilized rat serum protein to the perfusate. These data suggest that in vivo anionic sites of the endothelial glycocalyx are partially masked by adsorbed plasma proteins.     

Differentiated microdomains of the luminal plasmalemma of murine muscle capillaries: segmental variations in young and old animals

We investigated the luminal surface of the continuous endothelium of the microvasculature of the murine heart and diaphragm to find out whether it has differentiated microdomains. The probes were ferritin molecules, cationized to pI's 6.8, 7.15, 7.6, 8.0 and 8.4, which were introduced by retrograde or anterograde perfusion through the aorta or vena cava after the blood was removed from the vasculature. The pattern of labeling was analyzed by electron microscopy and assessed quantitatively by morphometry in arterioles, capillaries, and venules identified in bipolar microvascular fields in the diaphragm. The results showed that the plasmalemma proper was heavily but discontinuously labeled by all cationized ferritins (CF) used, the labeling being less extensive on the venular endothelium. CF had access as individual molecules to a fraction of the vesicular population opened on the luminal front of the endothelium. Plasmalemmal vesicle labeling increased from approximately 10 to approximately 25% as the pI decreased from 8.4 to 6.8. Vesicle labeling also increased with CF concentration in the perfusate. All CF binding sites were removed by pronase and papain. Heparinase and heparitinase caused only a slight reduction in CF labeling. Neuraminidase decreased the extent and density of labeling, especially on the plasmalemma proper of the venular endothelium; this decrease was particularly pronounced in old animals.     

The cell surface of a restrictive fenestrated endothelium

Summary The location and chemical composition of anionic sites on the endothelium of the choriocapillaris was investigated with cationic ferritin and enzyme digestion techniques. Cationic ferritin administered intravenously initially labeled essentially all fenestral diaphragms. Within 30 min after injection, no diaphrams remained labeled, but they could be relabeled by a second cationic ferritin injection. Following perfusion of cationic ferritin, the entire luminal front of the endothelium was labeled: the plasmalemma and fenestral, vesicle, and channel diaphragms. Perfusion of neuraminidase or chondroitinase did not affect subsequent cationic ferritin binding. In contrast, heparitinase removed anionic sites on all structures except fenestral diaphragms. Cationic ferritin did not mark the endothelium following heparinase digestion. All sites were cleaved with pronase E. These results indicate that heparin is the anionic moiety on fenestral diaphragms while the glycocalcyces of the plasmalemma and vesicle and channel diaphragms are rich in a heparan sulfate proteoglycan. Furthermore, since the heparan sulfate localized to these structures was digested by both heparinase and heparitinase, it is in a form similar to heparin. These findings demonstrate that the endothelium of the choriocapillaris bears cell-surface anionic components that are different than those described for fenestrated endothelia lining other vascular beds.Supported by NIH EY 03776     

Nonrandom distribution of sialic acid over the cell surface of bristle- coated endocytic vesicles of the sinusoidal endothelium cells

Previous studies with protein tracers have shown that the luminal surface of the vascular endothelium of the bone marrow is endocytic. The endocytosis occurs through the formation of large bristle-coated vesicles (LCV). The anionic charge distribution in this process was examined at the luminal surface of the endothelial cell, At pH 1.8, colloidal iron (CI), native ferritin, and polycationic ferritin (PCF) are bound by the luminal surface of the endothelial cell, but not at the sites of LCV formation. PCF used over a pH range of 1.8--7.2 (CI is unstable at higher pH levels) revealed LCV binding of this agent in increasing manner from pH 3.5 upwards. PCF binding at low pH (1.8) at the endothelial cell surface was markedly reduced by neuraminidase. Neuraminidase did not reduce PCF binding by the endothelial cell surface nor by the LCV at higher pH levels. It is concluded that the luminal surface of the endothelial cell has exposed sialic acid groups which are absent or significantly diminished at endocytic sites. The free surface of the endothelial cells as well as the sites of endocytosis have, in addition, anionic material with a pKa higher than that of sialic acid (pKa 2.6). These anionic materials may be different at the sites of endocytosis as compared to those present at the free cell surface.     

A simple two-step labeling procedure for ultrastructural localization of cell surface anionic sites

We propose a new method for ultrastructural localization of cell surface anionic sites. The method consists of sequential interaction of aldehyde-fixed cells with a polycationic reagent, poly-L-lysine (PL), followed by secondary interaction with a negatively charged marker, ferritin. By use of PL of low molecular weight (4000) on aldehyde-pre-fixed red blood cells and macrophages, the reaction resulted in binding of ferritin particles to cell surface anionic sites with a density distribution resembling that of cationized ferritin (CF). The density of the attached ferritin molecules increased in direct correlation with the MW of PL used. The primary PL interaction can be carried out at low pH (less than 2), thus restricting the labeling mainly to membrane-bound sialyl residues.     

Anionic surface properties of aortic and mitral valve endothelium from New Zealand white rabbits

The luminal surfaces of the endothelium lining the two surfaces of the aortic arterial (AAR) and ventricular (AVT), and mitral ventricular (MVT) and atrial (MAT), valve cusps were studied with cationic ferritin (CF) and ferritin (Fer)-conjugated lectins (WGA, RCA, SBA). The arterial (AAR) and ventricular (MVT) surfaces of the aortic and mitral cusps, which are exposed to more turbulent fluid mechanical forces and lower wall shear stresses, had the greatest density of CF labeling. The endothelia of the four surfaces displayed a gradient of decreasing density from the nuclear region to the periphery. Neuraminidase, chondroitinase ABC and AC, heparinase, heparitinase, hyaluronidase (testicular), and pronase E digestions suggested that a significant number of the anionic sites labeled by CF are associated with sialoglycoproteins and glycosaminoglycans such as chondroitin 4/6 sulfates, dermatan sulfates, and heparan sulfates. The localization of WGA receptors on the endothelium of AAR and MVT demonstrated a greater density of sialyl moieties than on the AVT and MAT. There was no binding of Fer-RCA with specificity for D-galactopyranosides or Fer-SBA with affinity for N-acetylglucosamine and D-galactose to the endothelium unless it was first treated with neuraminidase. Hence, sialic acids are shown to be among the more superficial components of this glycocalyx and to be largely responsible for the greater densities over the endothelium of AAR and MVT.     

Regeneration of plasmalemma and surface properties in macrophages

The regeneration of surface anionic groups in mouse peritoneal macrophages was investigated by electron microscopy, using cationized ferritin (CF) as a tool for the localization and evaluation of negative charge density on the cell surface. In vitro interaction of living macrophages with CF resulted in removal of most anionic groups, either by concentration of their receptor sites to a part of the membrane which is subsequently internalized, or by detachment of the aggregated label from the surface. After incubation of macrophages lacking surface anionic groups in tissue culture medium without the ligand, regeneration of the binding capacity for CF took place within 3 h. The first regenerated parts of the membrane can be visualized within 1 h on the upper part of the adherent cells; there is a discontinuous coating of ferritin, with the lateral regions of the plasmalemma free of label. The attached CF particles on the regenerated membrane are closer to the membrane and their density is considerably higher than on the normal control macrophages. The results indicate that the turnover of the plasmalemma is regional and not dispersed; the mechanism involved is insertion of membrane patches into the pre-existing plasma membrane.     

CHANGES IN CHARGED GROUPS ON THE CELL SURFACE DURING DEVELOPMENT OF THE CELLULAR SLIME MOLD DICTYOSTELIUM DISCOIDEUM: AN ELECTRON MICROSCOPIC STUDY

The distribution of charged groups on the surface of Dictyostelium cells and their change during development were examined by electronmicroscopy using cationic and anionic ferritins. The number of anionic sites on the cell surface decreased greatly during the course of development. The whole surface of vegetative cells stained strongly with cationic ferritin (CF). On the other hand, the surface of aggregation-competent cells had fewer negative charges and these were unequally distributed, the surface of the advancing area (lobopodial region) being devoid of anionic sites. The number of anionic sites on the cell surface decreased progressively during further development, and the suface of slug cells did not stain at all with CF. The cell surface did not stain with anionic ferritin at any developmental stage, indicating the absence of detectable cationic sites. The biological significance of these findings is discussed in connection with cell adhesiveness and movement.     

The endothelial pocket

Summary A new endothelial cell structure, named the endothelial pocket, has been found by combined transmission and scanning electron microscopic studies of renal peritubular capillaries. Transmission EM observations made on these and other fenestrated capillaries demonstrated that each pocket consists of an attenuated fold of fenestrated endothelium that projects 200 nm into the lumen above the rest of the endothelial surface. Beneath this luminal fold, there is a space and then another layer of fenestrated endothelium which abuts the basal lamina. The linear density of endothelial pockets was measured in the capillaries of the kidney cortex, intestinal mucosa and exocrine pancreas in mice and determined to be 0.067, 0.017 and 0.007 pockets·m^-1 respectively. Cationic ferritin decoration of the anionic sites on the luminal surface of the endothelium in these capillary beds revealed that both unlabelled and labelled diaphragms are clustered. In such specimens, the majority of the luminal diaphragms on endothelial pockets did not have cationic ferritin binding sites detectable by either scanning or transmission EM. On this account as well as on account of their general morphology, endothelial pockets appear to be multifold versions of the simple transendothelial channel.     

Immunoelectron microscopic study of PECAM-1 expression on lymphatic endothelium of the human tongue

The expression of platelet-endothelial cell adhesion molecule-1 (PECAM-1) on lymphatic and blood vessels of the human tongue was examined with fluorescence and transmission electron microscopy (TEM). The study used anti-desmoplakins antiserum for light microscopic identification of the lymphatic vessels, plus a pre-embedding immunogold electron microscopic technique for TEM observations. Before making TEM observations, cryostat serial sections were immunostained with anti-desmoplakins or anti-PECAM-1 and then embedded. Semithin sections from each cryostat section were photographed under a light microscope and compared in order to identify the lymphatic vessels expressing PECAM-1. In fluorescence microscopy, PECAM-1 expression on lymphatic vessels was weaker than that on blood vessels. TEM observations showed that PECAM-1 expression on the blood vessels was observed only on the luminal surface of the endothelium. In lymphatic vessels, PECAM-1 expression was found both on the luminal and abluminal surfaces of the endothelium. The density of the PECAM-1 reaction products was lower in lymphatic vessels than in blood vessels. The density of PECAM-1 reaction products on the luminal surface of lymphatic vessels was higher than on the abluminal surfaces. The results suggest that blood vessels are more active than lymphatic vessels in leukocyte migration. The expression of PECAM-1 on the abluminal surface of lymphatic endothelium may allow leukocytes to adhere to the endothelium and interact in their migration from tissue into lymphatic vessels.     

Internalization of cationized ferritin receptors by rat hepatoma ascites cells.

Cationized ferritin (CF) was used to label the cell surface anionic sites of Chang rat hepatoma ascites cells. If the hepatoma cells were fixed with glutaraldehyde and treated with CF, the label was distributed evenly over the external surface of the plasma membrane. Treatment of unfixed ascites cells with CF yielded clusters of ferritin particles separated by label-free areas of the plasma membrane. Some unfixed ascites cells were treated firstly with CF, then incubated in veronal buffered saline at 37 °C for 10, 20, 30 and 45 min, subsequently fixed in glutaraldehyde and re-exposed to CF. After 10 min of incubation, the label was arranged into large clusters with the remaining areas of the plasma membrane lightly labelled with CF. At 20 min, only clusters of ferritin were present on the plasma membrane; the remaining area of the cell surface was totally free of label. The ability of the plasma membrane to bind additional CF was completely restored after 45 min of incubation. These results suggest that for some period of time after internalization of CF label on cell surface the plasma membrane is devoid of any detectable negative charge.     

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.     

Membrane retrieval in epithelial cells of isolated thyroid follicles.

Follicles from rat and pig thyroid glands were isolated by digestion with collagenase. The epithelial cells of isolated follicles maintain their structural and functional polarity as shown by incorporation of 3H-leucine and autoradiography. To trace the fate of surface membrane, isolated follicles were opened, stimulated with thyrotropin and incubated for various time intervals with cationized ferritin (CF), uncharged dextran, native ferritin (NF), and latex spheres (0.5 mum in diameter) which were either pre-coated with CF or added together with CF. Uncharged dextran and native ferritin did not bind to the luminal cell membrane, were taken up in small amounts and accumulated in lysosomes; anionic NF was not found in Golgi cisternae in contrast to uncharged dextran which occassionally reached a few Golgi stacks. CF bound rapidly and in clusters to the luminal plasmalemma, preferentially to coated pits, was taken up by endocytosis, accumulated in lysosomes after 5 min and reached the Golgi cisternae after 30 min. Latex spheres were taken up by engulfment through fusion of microvilli and reached the lysosomes. CF particles coating the latex spheres may detach at this station and reach the Golgi cisternae. The findings show that the route of small tracers depends on the charge of the tracer, in agreement with results obtained by Farquhar [8]. Vesicles carrying NF can be traced to lysosomes only, whereas vesicles containing uncharged dextran or - more conspicuously -CF also fuse with Golgi membranes. Large tracers (latex beads) reach only the lysosomes, but CF taken up with them may move to Golgi cisternae.     

Leaky intra-acinar arteries in rat lungs perfused with hydrogen peroxide

We have investigated the effects of H2O2 (150 or 300 microM) on the ultrastructure and permeability of the pulmonary endothelium in rat lungs perfused for 60 min with buffered Hanks' bovine serum albumin medium. In one group of experiments, we examined the effect of H2O2 on the uptake and transport of cationized ferritin (CF) by endothelial cells in intra-acinar arteries, alveolar capillaries, and interlobular veins. The influence of the oxidant on endothelial adsorptive endocytic processes was assessed by measuring the density of ferritin particles in luminal vesicles, multivesicular bodies, and basal lamina. In a second group of experiments, we examined the effects of H2O2 on the fine structure and permeability to electron-dense macromolecules of arterial, microvascular, and venous endothelium. For this purpose, at the end of the 60-min perfusion with H2O2, CF was perfused to identify leaky vessels. We found that H2O2 caused a dose-dependent inhibition of transcytosis of CF in all vascular segments. At the lower dose of H2O2, inhibition of transcytotic activity was not associated with structural injury to the vascular endothelium or with elevation of wet-to-dry ratios. At the higher oxidant dose, inhibition of transcytosis was associated with leaky arterial endothelium and elevation of wet-to-dry ratios (6.44 +/- 0.12 vs. 5.64 +/- 0.16, P less than 0.02). The effects of H2)2 were prevented by adding catalase to the perfusate. The selective loss of structural integrity and leakiness of the arterial endothelium were diminished but not completely abolished by perfusing the oxidant retrograde from the venous side.(ABSTRACT TRUNCATED AT 250 WORDS)     

Reduction of anionic sites on the rabbit uterine surface during the preimplantation period

Studies have been carried out to analyze distribution of anionic sites on the uterine epithelium of the rabbit, using cationized ferritin as a label. A negatively charged glycocalyx was demonstrated by transmission electron microscopy on the luminal cell surface during estrus and days 5–7 of pregnancy. There was a general reduction of labeling from estrus and day 5 to 7 of pregnancy. At estrus and on day 5 and 6 of pregnancy, the results were similar on the meso- and antimesometrial sides of uterine horns and at or between egg recovery sites. At day 7, anionic sites were no longer detected antimesometrially facing the eggs. These results suggested that the progressive loss of anionic sites during the preimplantation period was due to the combined actions of uterus and egg and that this loss might play a role in blastocyst antimesometrial implantation.     

Prevention by retinoic acid of anionic site redistribution on the surface of cultured human sarcoma cells

The distribution of cell surface negatively-charged macromolecules was determined electron microscopically on untreated and on retinoic acid (RA)-treated cultured human osteosarcoma Hs791 and chondrosarcoma Hs705 cells using cationized ferritin (CF), an electron-dense marker of anionic sites. Labeling on the surface of prefixed cells was continuous and uniform whether they were grown in the absence or presence of RA. In contrast, CF distribution on unfixed cells was markedly affected by RA; CF labeling of untreated cells occurred in patches and clusters whereas the label on RA-treated cells was continuous, as on prefixed cells. CF labeling of unfixed cells decreased considerably after incubation of the cells either with hyaluronidase or neuraminidase. There was also a reduction in patching and clustering. Changes induced by RA in the apparent membrane microviscosity, in neuraminidase-releasable sialic acid, or in transglutaminase activity could not be related to the effect of RA on CF-induced anionic site redistribution since these characteristics were modulated differently in the two cell lines. In contrast, RA increased the sialylation of specific cell surface membrane glycoproteins on both cell types. These results suggest that RA prevents redistribution of cell surface sialoglycoconjugates and glycosaminoglycans by CF. This effect may be the result of increased sialylation of specific surface components and may be related causally to the suppression of the transformed phenotype in the sarcoma cells.     

Lateral mobility of negative charge sites at the surface of sheep erythrocytes

Neuraminidase-sensitive negative charge sites on sheep red blood cells (SRBCs) are uniformly distributed over the cell surface, as in other species. Unlike most other species in which charge sites are immobile, however, in intact RBCs interaction of SRBCs with cationized ferritin (CF) induces clustering (i.e. patching) of CF-binding sites. This clustering is both time- and temperature-dependent. The clustering of negative charge sites results in the formation of domains up to 1 000 Å across which are totally free of CF binding sites. The unusually high degree of lateral mobility of ionogenic groups at the SRBC surface may contribute to the species specificity of E rosetting.     

The distribution and mobility of anionic sites on the surfaces of baby hamster kidney cells

The distribution and mobility of anionic sites on the surfaces of baby hamster kidney cells were studied by utilizing the multivalent ligand, polycationic ferritin, as a visual probe. Our observations revealed that anionic sites are distributed over the entire cell surface, with the highest density of sites being located on cell surface microextensions. Following the initial binding of polycationic ferritin to the surface of unfixed cells, the ligand-bound anionic sites redistributed by migrating from the surface of microextensions to the surface of the cell body. In 20 min, this migration resulted in a total clearing of anionic sites from the surface of microextensions concomitant with the formation of patches of anionic sites on the surface of the cell body. Polycationic ferritin-induced migration and patch formation of anionic sites was not prevented by 2,4- dinitrophenol, N-ethylmaleimide, colchicine, or cytochalasin B. However, the ligand-induced redistribution of cell surface anionic sites was prevented by prefixation of cells with glutaraldehyde.     

The distribution of cationized ferritin receptors on ciliated epithelial cells of rat trachea

The interaction of tracheal cilia with the biphasic mucus layer covering the surface of the mammalian respiratory tract may be influenced by many cell surface coat components including those having an overall negative charge. In order to assess the distribution of ciliary anionic sites, cationized ferritin (CF) was used to label the surface of rat tracheal epithelium. If pieces of trachea were fixed with 3% glutaraldehyde and treated with CF at low (L) (0.08 mg/ml), medium (M) (0.32 mg/ml PBS), or high (H) (0.64 mg/ml PBS) concentrations, the label was distributed evenly over the entire external surface of the ciliary membrane at all concentrations. Unfixed tracheal tissue was also treated with L, M, and H CF for 1 or 5 min at 4 degrees C in order to minimize lateral redistribution of CF receptors. To ensure accessibility of the cell surface to CF the samples were agitated thoroughly during exposure. Exposure for 1 min to L, M, and H CF resulted in a light binding of ferritin particles on all portions of the ciliary membrane with occasional areas of multilayered binding distributed randomly on the ciliary shaft. When unfixed trachea was treated with CF for 5 min at 4 degrees C, CF binding was similar except heavier and more uniform. In no instance was there any preferential binding of CF to the ciliary tips at any of the concentrations used. Moreover, as indicated by the CF binding pattern at L concentrations, high density negative charges are present over almost the entire surface of the cilium. These results suggest that, unlike the ciliary membrane of other organs such as oviduct, negatively charged cell surface coat molecules are present on all areas of the ciliary membrane of rat tracheal epithelia.