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.     

Differentiated microdomains on the luminal surface of capillary endothelium: distribution of lectin receptors

Lectins conjugated with either peroxidase or ferritin were used to detect specific monosaccharide residues on the luminal front of he fenestrated endothelium in the capillaries of murine pancreas and intestinal mucosa. The lectins tested recognize, if accessible, the following residues: alpha-N-acetylgalactosaminyl (soybean lectin), beta- D-galactosyl (peanut agglutinin [PA] and Ricinus communis agglutinin- 120 [RCA]), beta-N-acetylglucosaminyl and sialyl residues (wheat germ agglutinin [WGA]), alpha-L-fucosyl (lotus tetragonolobus lectin), and alpha-D-glucosyl and beta-D-mannosyl (concanavalin A [ConA]). Thi labeled lectins were introduced by perfusion in situ after thoroughly flushing with phosphate-buffered saline the microvascular beds under investigation. Specimens were fixed by perfusion, and subsequently processed for peroxidase detection and electron microscopy. Control experiments included perfusion with: (a) unlabeled lectin before lectin conjugate; (b) labeled lectin together with the cognate hapten sugar, and (c) horseradish peroxidase or ferritin alone. Binding sites were found to be relatively homogeneously distributed on the plasmalemma proper, except for Lotus tetragonolobus lectin and Con A, which frequently bound in patches. Plasmalemmal vesicles, transendothelial channels, and their associated diaphragms were particularly rich in residues recognized by RCA and PA (beta-D-galactosyl residues) and by WGA (beta-N-acetylglucosaminyl residues). Receptors for all lectins tested appeared to be absent or considerably less concentrated on fenestral diaphragms. The results reported here extend and complement previous findings on the existence of microdomains generated by the preferential distribution of chemically different anionic sites (Simionescu et al., 1981, J. Cell Biol., 9:605-613 and 614-621).     

Redistribution of surface anionic sites on the luminal front of blood vessel endothelium after interaction with polycationic ligand

The ability of anionic groups on the luminal surface of blood vessels to redistribute by lateral migration under the influence of multivalent ligands was analyzed by electron microscopy, using cationized ferritin (CF). In vitro interaction of blood vessel segments with CF results in rapid aggregation of most anionic sites on the luminal fromt of the endothelium, followed by internalization or detachment of the CF patches, leaving most of the luminal surface devoid of anionic sites. Further incubation of such endothelial cells without CF results in regeneration of binding capacity for the polycationic label. Transport of CF, but not of native ferritin, across the endothelium by vesicle transport, followed by exocytosis of the interiorized CF clusters on the tissue front of the endothelium, was also observed. The possibility that such activities in the blood vessels in vivo may be associated with local changes in the normal distribution of the surface anionic sites as well as in accumulation of debris in the subendothelial layers of the vessels is suggested.     

The distribution of negative charges on the luminal surface of Descemet's endothelium

The localization of anionic sites on the surface of corneal endothelium was investigated using colloidal nickel. The distribution of the binding sites of colloidal nickel along the cell surface was examined with the scanning electron microscope. The application a special detector of high sensitivity reflection allows to register a composed contrast determined by colloidal nickel. Colloidal nickel is bound with the surface of endothelium in areas of cell junctions only. A 1 X 10(-4)% sol. adrenaline injection into the anterior rabbit eye chamber in vivo changed the distribution of colloidal nickel. These findings demonstrate that colloidal nickel allows to mark the anionic sites on the cell surface for scanning electron microscopic studies.     

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.     

Isolation and partial characterization of the luminal plasmalemma of microvascular endothelium from rat lungs.

This paper describes a procedure for isolating in high yield and at a high degree of purity the endothelial luminal plasmalemma from the microvasculature of the rat lung. The procedure relies on the modification of the density of the luminal plasmalemma obtained by coating it by perfusion in situ first, with cationized colloidal silica and then with Na polyacrylate. These steps generate a strongly adhering coat to the luminal plasmalemma that resists tissue homogenization to yield, upon repeated centrifugation through Nycodenz density gradients, a nearly homogeneous fraction of coated luminal plasmalemmal fragments still carrying their associated plasmalemmal vesicles. The fraction is enriched in the luminal plasmalemmal antigen, angiotensin converting enzyme, contains gp60, an antigen expected to occur on both plasmalemmal domains, is not enriched in either alkaline phosphatase or 5'-nucleotidase activity and is free of the mitochondrial and endoplasmic reticulum antigens so far tested. This procedure, that can be extended--in principle--to any vascular bed, obviates the use of cultured cells for studying the biochemistry of the endothelium, at least as far as the luminal endothelial plasmalemma is concerned.     

Prediction of LDL concentration at the luminal surface of a vascular endothelium

To find out whether concentration polarization of low-density lipoprotein (LDL) occurs at the surface of a vascular endothelium or not, transport of LDL in flowing blood to an water-permeable endothelium was studied theoretically by means of CFD. Calculations were carried out for an endothelium exposed to a Couette flow by assuming that the surface geometry of the endothelium could be expressed by a cosine function. Two typical cases were considered for the permeability of endothelium to water; one was uniform permeability everywhere in the endothelium, and the other was uneven permeability which was augmented at the intercellular junction. It was found that, in both cases, the surface concentration of LDL increased in going distally from the entrance, taking locally high and low values at the valleys and hills of the endothelium, respectively, and the variation was larger in the case of endothelium with uneven permeability. These results clearly showed that concentration polarization of LDL which might affect the uptake of LDL by the arterial wall certainly occurs at the surface of the endothelium even if the flow is disturbed microscopically by the uneven surface of the endothelium.     

Demonstration of anionic sites on the luminal and abluminal fronts of endothelial cells with poly-L-lysine-gold complex

An attempt was made to demonstrate the anionic sites on the endothelial cell (EC) surfaces of mouse brain micro-blood vessels (MBVs) after embedding of tissue samples in hydrophilic media: Lowicryl K4M, LR White, and Polyamph-10. As a cationic probe, poly-L-lysine-gold complex (PLG), prepared according to the procedure of Skutelsky and Roth (J Histochem Cytochem 34:693, 1986), was used. In ultra-thin sections of brain samples embedded in Lowicryl K4M and LR White, the anionic sites were demonstrated in the entire cross-section of the vessel wall. After embedding in Polyamph-10, however, the anionic sites could not be detected. Brain capillaries, representing blood-brain barrier type MBVs, showed polar distribution of anionic sites, evidenced by more intense labeling of luminal than of abluminal plasma membrane of the EC. Some differences in labeling of ECs and of basement membrane in arterioles and venules were also noted. The use of cationic gold and the ultra-thin sections of tissue samples embedded in hydrophilic media (Lowicryl K4M and LR White) seems to be a promising new method for detection of anionic constituents located on both luminal and abluminal surfaces of the EC, in the basement membrane, and in other components of the vessel wall.     

The distribution of anionic sites on the surface of the Golgi complex

The distribution of anionic binding sites has been investigated in the isolated Golgi complex using cationic ferritin. The greatest density of anionic sites occurs on the tubular network and small vesicles, and this binding is accompanied by increased levels of galactosyltransferase activity. The density of anionic sites on the cisternae is less than on the tubules and shows anisotropic distribution, with higher density on the convex surface and lower density on the concave surface. The distribution of anionic sites may reflect the functional activity of the Golgi complex and possibly the interaction or cohesion between cisternae in this organelle.     

Partial characterization of immunosuppressive proteins from bovine uterine luminal secretions

Unfractionated and fractionated uterine luminal protein (ULP) secretions collected from nonpregnant and pregnant beef cows on Day 17 post-breeding were tested in vitro for suppression of lymphocyte blastogenesis to the mitogen phytohemagglutinin (PHA). In replicated experiments, ULP from nonpregnant and pregnant cows was separated into five molecular weight (M_r) fractions with Sephacryl S-200. Unfractionated (25 to 400 μg/ml) and fractionated (25 to 100 μg/ml) ULP was added to cultures containing 5 × 10⁵ bovine lymphocytes and 0.4 μg of PHA in a complete culture medium. At 48 hr, 0.5 μCi of ³H-thymidine was added to cultures, and cells were harvested at 60 h by automation. Thymidine incorporation data were expressed as percentage of control (no ULP) values. Unfractionated and all S-200 ULP fractions from nonpregnant and pregnant cows suppressed (P<0.05 to 0.001) lymphocyte blastogenesis to PHA, but to varying degrees of suppression. Unfractionated ULP was more suppressive (P<0.05) for pregnant than nonpregnant cows, which was likely due to the greater (P<0.05) immunosuppressive activity of S-200 fractions I (>219,000 M_r) and V (∼14,000 M_r) from the pregnant cows. At 25 μg ULP/ml, mean (± SEM) % of control values for fraction I from pregnant and nonpregnant cows were 9.1 ± 3.3 and 36.6 ± 8.5%, respectively (P<0.05). Values for fraction V were 15.7 ± 6.5 and 46.6 ± 6.1%, respectively (P<0.01). Within each reproductive class, fractions I and V were more suppressive (P<0.05) than fractions II, III and IV. Immunosuppression was not mediated by lymphocyte cytotoxicity.     

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.     

Concanavalin A binding sites on the luminal surface of ependymal cells of third ventricle

Summary Detection of the Concanavalin A binding sites within the luminal surface of ependymal cells of the third ventricle has been accomplished using perfusion techniques as a way of exposing the cells to reaction solutions. Parameters for this procedure were established. It was found that electron dense reaction products form a layer ranging from 25–40 nm in thickness on the luminal surface of investigated cells. The microvilli exhibited a slightly thiner layer, the reaction products formed small patches on cilia.     

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.     

Electrical resistance of muscle capillary endothelium.

A recently developed technique for in vivo determination of the electrical resistance of vascular endothelium in microvessels was applied to the vessels in a thin frog muscle, m. cutaneus pectoris. The technique consists of injection of current via a glass micropipette into a capillary and measurement of the resulting intra- and extravascular potential profiles with another micropipette placed at various distances from the current source. The theory of Peskoff and Eisenberg (1974) was used to handle the problems arising from distributed extravascular resistances and was experimentally shown to describe the external field satisfactorily. With this extension of one-dimensional cable theory the specific electrical resistance of arterial microvessels was 33 omega cm2 and of venous capillaries 23 omega cm2. The "length constants" were 135 and 112 micrometers, respectively. If results from arterial and venous vessels are taken together, the ionic permeabilities at 20 degrees C were PNa = 3.9 X 10(-5) cm X s-1, PK = 5.7 X 10(-5) cm X s-1, PCl = 5.9 X 10(-5) cm X s-1 and PHCO3 = 3.4 X 10(-5) cm X s-1. These figures agree with figures for capillary permeability obtained in tracer experiments on whole muscle. The study bridges a gap between single capillary and whole organ techniques with the conclusion that the two different approaches lead to similar results in muscle capillaries.