Surface charge distribution on the endothelial cell of liver sinusoids.

The topography of the charged residues on the endothelial cell surface of liver sinusoid capillaries was investigated by using electron microscopic tracers of different size and charge. The tracers used were native ferritin (pl 4.2-4.7) and its cationized (pl 8.4) and anionized (pl 3.7) derivatives, BSA coupled to colloidal gold (pl of the complex 5.1), hemeundecapeptide (pl 4.85), and alcian blue (pl greater than 10). The tracers were either injected in vivo or perfused in situ through the portal vein of the mouse liver. In some experiments, two tracers of opposite charge were sequentially perfused with extensive washing in between. The liver was processed for electron microscopy and the binding pattern of the injected markers was recorded. The electrostatic nature of the tracer binding was assessed by perfusion with high ionic strength solutions, by aldehyde quenching of the plasma membrane basic residues, and by substituting the cell surface acidic moieties with positively charged groups. Results indicate that the endothelial cells of the liver sinusoids expose on their surface both cationic and anionic residues. The density distribution of these charged groups on the cell surface is different. While the negative charge is randomly and patchily scattered all over the membrane, the cationic residues seem to be accumulated in coated pits. The charged groups co-exist in the same coated pit and bind the opposite charged macromolecule. It appears that the fixed positive and negative charges of the coated pit glycocalyx are mainly segregated in space. The layer of basic residues is located at 20-30-nm distance of the membrane, while most of the negative charges lie close to the external leaflet of the plasmalemma.

Specific binding sites for albumin restricted to plasmalemmal vesicles of continuous capillary endothelium: receptor-mediated transcytosis.

The interaction of homologous and heterologous albumin-gold complex (Alb-Au) with capillary endothelium was investigated in the mouse lung, heart, and diaphragm. Perfusion of the tracer in situ for from 3 to 35 min was followed by washing with phosphate-buffered saline, fixation by perfusion, and processing for electron microscopy. From the earliest time examined, one and sometimes two rows of densely packed particles bound to some restricted plasma membrane microdomains that appeared as uncoated pits, and to plasmalemmal vesicles open on the luminal front. Morphometric analysis, using various albumin-gold concentrations, showed that the binding is saturable at a very low concentration of the ligand and short exposure. After 5 min, tracer-carrying vesicles appeared on the abluminal front, discharging their content into the subendothelial space. As a function of tracer concentration 1-10% of plasmalemmal vesicles contained Alb-Au particles in fluid phase; from 5 min on, multivesicular bodies were labeled by the tracer. Plasma membrane, coated pits, and coated vesicles were not significantly marked at any time interval. Heparin or high ionic strength did not displace the bound Alb-Au from vesicle membrane. No binding was obtained when Alb-Au was competed in situ with albumin or was injected in vivo. Gold complexes with fibrinogen, fibronectin, glucose oxidase, or polyethyleneglycol did not give a labeling comparable to that of albumin. These results suggest that on the capillary endothelia examined, the Alb-Au is adsorbed on specific binding sites restricted to uncoated pits and plasmalemmal vesicles. The tracer is transported in transcytotic vesicles across endothelium by receptor-mediated transcytosis, and to a lesser extent is taken up by pinocytotic vesicles. The existence of albumin receptors on these continuous capillary endothelia may provide a specific mechanism for the transport of albumin and other molecules carried by this protein.

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.

Alterations of phospholipid asymmetry in the membrane of spontaneously aggregated platelets in diabetes.

The changes of asymmetric distribution of anionic phospholipids of human platelets in diabetic patients were studied by fluorescent and freeze fracture cytochemistry, using merocyanine 540 (MC 540) and polymyxin B (PxB) as specific markers. The membrane anionic phospholipids were detected with PxB, a membrane nonpermeant probe, used either in native form for freeze fracture electron microscopy or as dansylated or iodinated derivative for fluorescence microscopy or gamma counting, respectively. MC 540 is a naturally fluorescent probe which reportedly inserts into less packed bilayer domains. Both in platelet rich plasma and in washed platelets obtained from diabetic patients, some small platelet aggregates were observed, their number being generally dependent on the level of hyperglycemia. In contrast with single platelets, the aggregated ones bind PxB as revealed by all assay methods. The fluorescence microscopic studies with dansyl PxB and MC 540 displayed a strong binding of the fluorescent markers to aggregated platelets. The electron microscopic examination of freeze fracture replicas showed the appearance of characteristic PxB-induced deformations in the plasmalemma of aggregated platelets. The gamma counting of 125I-PxB incubated samples indicates significant differences on the platelets of diabetic patients as compared to those obtained from healthy subjects. Our data provide evidence that in diabetic patients, the spontaneous aggregated platelets are a result of the appearance of the anionic phospholipids in the outer half of plasmalemma. These changes may enhance the procoagulant activity and should represent a determinant of activated platelet recognition and their removal from circulation by splenic macrophages.

Albumin binding sites are expressed on the abluminal plasma membrane of capillary endothelium.

The presence of albumin binding sites on the abluminal front of vascular endothelium was examined on the capillaries of the adipose tissue. The experimental procedure consisted in injecting interstitially within the rat epididymal fat and epicardial fat, albumin (alone or bearing oleic acid) either conjugated with gold particles (Alb-Au or Alb-OA-Au) or radioiodinated [( 125I]-Alb). In controls, polyethyleneglycol-gold complex (PEG-Au) and [125I]-IgG were used as tracers. The results revealed that: a) albumin binding sites are expressed on the abluminal front of endothelium especially concentrated on plasmalemmal vesicles; b) the retrotranscytosis of albumin conjugates from the interstitium to the capillary lumen is a poorly represented process; c) the binding of the tracers used appears to be time and concentration dependent; d) albumin conjugates do not bind significantly to plasmalemmal vesicles of adipocytes, pericytes and smooth muscle cells; e) PEG-Au and [125I]-IgG do not show a binding pattern similar to that of albumin conjugates.

The cerebral microvasculature of the rat: structure and luminal surface properties during early development.

The development of the cerebral microvasculature of the rat was studied during three successive postnatal periods, namely: 1) neonatal period, i.e., 1 to 9 days after birth (capillary sprouting period); 2) myelinization period, i.e., 10 to 20 days; and 3) young adult period, i.e., 2 to 3 months. The survey covered structural aspects and distribution of binding sites for anionic or cationic probes and for albumin-gold complexes on the luminal surface of the microvascular endothelium. The salient results are: a) an extensive development of the endoplasmic reticulum of endothelial cells during the first period (presumably in relation with the production of basement membrane components); b) the high surface density of coated pits and coated vesicles that peaks during the myelinization period; c) the paucity of plasmalemmal vesicle and their differential distribution (their volume density is higher in the endothelium of arterioles than in that of capillaries and venules); d) the existence of an extensive smooth surface tubular system in the cytoplasm of endothelial cells, whose structural connections and functional significance remains to be established; and e) the presence of pericytes with elaborate interactions with endothelia in the early developmental periods. Labeling by perfused tracers indicates an uneven patchy distribution of binding sites for cationic ferritin (generally limited to the plasmalemma proper) and a more even distribution of binding sites for cationic and anionic hemeundecapeptides. Binding patterns did not change during the developmental periods studied. No binding sites were detected for albumin-gold complexes.