The ultrastructural basis of the permeability of arterial endothelium to horseradish peroxidase

The thoracic aorta and basilar artery, in which the incidence of atherosclerosis is known to be different, were examined to elucidate the correlation between the structure of the intercellular cleft junction between adjacent endothelial cells and its permeability to HRP. Tannic acid or HRP in the vessel lumen passed through the intercellular clefts of the thoracic aorta into the subendothelial space, whereas in the basilar artery they were unable to penetrate beyond the tight junction of the intercellular clefts. Freeze-fracture replicas revealed that the tight junctions of the thoracic aorta consisted of one to two junctional strands in most areas of the cleaved planes, with discontinuities in some places, whereas those of the basilar artery consisted of a continuous belt-like meshwork of six anastomosing junctional strands on average. These observations confirm that the structure of endothelial junctions in arteries has a close correlation with the permeability of the intercellular clefts to HRP.     

Horseradish peroxidase as a tracer for capillary permeability studies

The permeability of capillaries was investigated utilizing an in vivo injection of horseradish peroxidase (HRP) and an in situ perfusion of a balanced salt solution containing HRP and lanthanum chloride. In the continuous capillaries of heart and muscle, HRP diffuses mainly through intercellular junctions, while in testicular capillaries, the transport is via micropinocytotic vesicles. The diffusion and micropinocytotic transport of HRP was demonstrated in both directions, i.e. from the capillary lumen to the interstitium and vice versa. Lanthanum can be used as a bidirectional inhibitor of micropinocytosis. The transport of HRP is then almost completely hindered in testicular capillaries. In heart muscle, the effect on HRP transport is not significant, due to second transport pathway, i.e. intercellular cleft passage.     

The permeability of muscle capillaries to horseradish peroxidase

The main objective of this study was to determine the pathways by which horseradish peroxidase (HRP) can cross the endothelium of muscle capillaries. Specimens of mouse diaphragm were fixed for cytochemical analysis at various intervals after intervenous injection of 0.5 mg HRP, at 4 min after intervenous injection of varied amounts of HRP, and at 4 min after intervenous injections in various volumes of isotonic NaCl. Our findings indicate that endothelial junctions serve as a barrier which may allow passage of very limited amounts of HRP. They also suggest that endothelial vesicles transfer HRP from the capillary lumen to the pericapillary interstitium as well as in the reverse direction. Increasing the volume of solution injected to approximately 30% of total blood volume did not increase the amount of HRP that left the capillary lumen. Our results with HRP do not provide clearcut evidence that endothelial junctions are the site of the small pore.     

Vascular permeability and hyperpermeability in a murine adenocarcinoma after fractionated radiotherapy: an ultrastructural tracer study

Large radiation doses cause postradiation vascular hyperpermeability by disrupting endothelia. The cumulative sequences of small doses (fractionated radiotherapy) standard in clinical practice cause it too, but not by endothelial disruption: the mechanisms are unknown. In this study, correlated fluorescent and ultrastructural localisation of a tracer revealed the architecture, fine structure and function of microvessels in mouse AT17 tumours, before and after 42 Gy fractionated radiation. Before irradiation, tumour vascular permeability lay in the normophysiological range defined by the gut and cerebral cortex. A double barrier regulated permeability: vesicular transport through the endothelial wall required approximately 2 h and then the basement membrane charge barrier trapped tracer for 2 h longer. Irradiation abolished the double barrier: tracer passed instantly through both endothelial wall and underlying basement membrane, forming diffusion haloes around microvessels within 2-5 min. Structurally, irradiated tumour microvessels were lined by a continuous and vital endothelium with closed interendothelial junctions; endothelial basement membranes were intact, though loosened. Irradiated endothelia exhibited extremely active membrane motility and intracellular vesicle trafficking. Radiation treatment raised vascular permeability by enhancing transendothelial transcytosis, and by altering the passive filter properties of the subendothelial basement membrane. This type of vascular hyperpermeability should be susceptible to pharmacological modulation.     

Mechanism of action of collagenase on the blood-brain barrier permeability. Increase of endothelial cell pinocytotic activity as shown with horse-radish peroxidase as a tracer.

The ultrastructural mechanism of the protease induced blood-brain barrier permeability-increase was studied with horse-radish peroxidase as a tracer. After intravenous injection of collagenase or pronase, a significantly increased number of pinocytotic vesicles was found in brain capillary endothelial cells. alpha-Chymotrypsine did not exert such an action.     

Molecular basis of bacterial outer membrane permeability revisited.

Gram-negative bacteria characteristically are surrounded by an additional membrane layer, the outer membrane. Although outer membrane components often play important roles in the interaction of symbiotic or pathogenic bacteria with their host organisms, the major role of this membrane must usually be to serve as a permeability barrier to prevent the entry of noxious compounds and at the same time to allow the influx of nutrient molecules. This review summarizes the development in the field since our previous review (H. Nikaido and M. Vaara, Microbiol. Rev. 49:1-32, 1985) was published. With the discovery of protein channels, structural knowledge enables us to understand in molecular detail how porins, specific channels, TonB-linked receptors, and other proteins function. We are now beginning to see how the export of large proteins occurs across the outer membrane. With our knowledge of the lipopolysaccharide-phospholipid asymmetric bilayer of the outer membrane, we are finally beginning to understand how this bilayer can retard the entry of lipophilic compounds, owing to our increasing knowledge about the chemistry of lipopolysaccharide from diverse organisms and the way in which lipopolysaccharide structure is modified by environmental conditions.     

The permeability of the primary decidual zone in the rat uterus: an ultrastructural tracer and freeze-fracture study

The rat primary decidual zone (PDZ) is a transitory avascular region of transformed fibroblasts surrounding the implanting embryo. Studies using fluorescein-labeled tracers have shown that the PDZ is selectively permeable to macromolecules, permeability decreasing with increasing molecular weight. In the present study we investigated the morphologic basis of the permeability barrier. Horseradish peroxidase (HRP) or HRP-labeled immunoglobulin G (IgG-HRP) was administered i.v. to rats on Day 7 of pregnancy, and the animals were killed 30 min to 2 h later. The reaction product of HRP was the same density in uterine blood vessels as in the intercellular spaces of the endometrium and PDZ at 30 min and 1 h after administration. Two hours after administration, the reaction product of IgG-HRP was dense in uterine blood vessels, much less dense in the interstitial spaces of the endometrium, and was not detected in the PDZ. There was an abrupt change in the density of the IgG-HRP reaction product at the intercellular clefts between endothelial cells, where cellular junctions were observed in control tissue. This suggests that the passage of large macromolecules from blood to the implantation chamber is limited initially by cellular junctions between capillary endothelial cells. The exclusion of IgG-HRP from the PDZ indicates that an additional barrier(s) to macromolecules in this region. Lanthanum nitrate tracer was uniformly present throughout the intercellular spaces of the PDZ except at tight junctions between decidual cells. Freeze-fracture replicas of the PDZ showed tight junctions that varied from single strands to interconnected networks of strands oriented mainly parallel to the long axis of the PDZ. Some strands were discontinuous. The tight junctions between decidual cells appear to be functionally discontinuous because HRP readily penetrated the PDZ, but such junctions may retard larger macromolecules such as IgG-HRP. The biological significance of the permeability barrier of the PDZ is discussed.     

The ultrastructural basis of transcapillary exchanges

A brief survey is given of current views correlating the ultrastructural and permeability characteristics of capillaries. Observations based on the use of peroxidase (mol wt 40,000), as an in vivo, and colloidal lanthanum, as an in vitro, ultrastructural tracer, are presented. In capillaries with "continuous" endothelium, the endothelial intercellular junctions are thought to be permeable to the tracers, and are regarded as maculae occludentes rather than zonulae occludentes, with a gap of about 40 A in width between the maculae. Some evidence for vesicular transport is also presented. It is inferred that the cell junctions are the morphological equivalent of the small-pore system, and the vesicles the equivalent of the large-pore system. Peroxidase does not apparently cross brain capillaries: the endothelial cell junctions are regarded as zonulae occludentes, and vesicles do not appear to transport across the endothelium. This is regarded as the morphological equivalent of the blood-brain barrier for relatively large molecules. The tracers appear to permeate the fenestrae of fenestrated capillaries, and the high permeability of these capillaries to large molecules is attributed to the fenestrae. Capillaries with discontinuous endothelium readily allow passage of the tracers through the intercellular gaps. A continuous basement membrane may act as a relatively coarse filter for large molecules. In general, the morphology of capillaries correlates well with physiological observations.     

Non-electrolyte permeability as a tool for studying membrane fluidity

1. The reflection coefficient for the permeation of thiourea through bilayers of phosphatidylcholine is a function of the fatty-acid composition of the lipid molecules. By means of these reflection coefficients an index for membrane fluidity has been given to each of those lipids, relative to that of egg phosphatidylcholine. 2. The maximum number of water molecules that can copermeate with each molecule of solute by means of solute-solvent interaction is a function of the packing of the lipid molecules in the bilayer. This parameter has been used in this paper for characterizing the fluidity of cholesterol-containing membranes and for membranes with their lipids in the gel state.     

Protamine-induced permeability changes in the neutrophil plasma membrane as the basis of activation of exocytosis.

Protamine induces a gradual change in plasma membrane permeability in rabbit neutrophils, which is evident from the increase of indol fluorescence, and the leakage of quin2 from quin2-loaded neutrophils. The influx of extracellular Ca2+ into the neutrophil provides an explanation for exocytosis which occurs in the presence of Ca2+ and protamine. The dependence of exocytosis on Ca2+ concentration follows the same pattern as is observed in neutrophils permeabilized by other means. In the absence of Ca2+, and in the presence of protamine, La3+ has an activating effect on exocytosis. At higher concentrations La3+ inhibits exocytosis that occurs in the presence of Ca2+ and protamine, as do some other metal ions. The resemblance between the membrane effects of a number of toxins, as reported in literature, and protamine-induced membrane damage suggests that they occur via the same mechanism.     

The permeability of the guinea pig cochlear capillaries to horseradish peroxidase

Summary Guinea pigs were given horseradish peroxidase intracardially. Examination of the cochlear capillaries 2 to 10 min after the injection revealed that the capillaries of the vascular stria are permeable to the peroxidase whereas the capillaries of the basilar membrane, the spiral ligament, and the spiral prominence are impermeable.

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Zusammenfassung Meerrettich-Peroxydase wurde Meerschweinchen intracardial verabfolgt. Die Untersuchung der Kapillaren der Schnecke 2–10 min nach der Injektion zeigte, daß die Kapillaren der Stria vascularis für die Peroxydase permeabel waren, jene der Membrana basilaris, des Ligamentum spirale und der Prominentia spiralis dagegen impermeabel sind.

     

Fusion of phospholipid vesicles with a planar membrane depends on the membrane permeability of the solute used to create the osmotic pressure

Phospholipid vesicles fuse with a planar membrane when they are osmotically swollen. Channels in the vesicle membrane are required for swelling to occur when the vesicle-containing compartment is made hyperosmotic by adding a solute (termed an osmoticant). We have studied fusion using two different channels, porin, a highly permeable channel, and nystatin, a much less permeable channel. We report that an osmoticant's ability to support fusion (defined as the magnitude of osmotic gradient necessary to obtain sustained fusion) depends on both its permeability through lipid bilayer as well as its permeability through the channel by which it enters the vesicle interior. With porin as the channel, formamide requires an osmotic gradient about ten times that required with urea, which is approximately 1/40th as permeant as formamide through bare lipid membrane. When nystatin is the channel, however, fusion rates sustained by osmotic gradients of formamide are within a factor of two of those obtained with urea. Vesicles containing a porin-impermeant solute can be induced to swell and fuse with a planar membrane when the impermeant bathing the vesicles is replaced by an isosmotic quantity of a porin-permeant solute. With this method of swelling, formamide is as effective as urea in obtaining fusion. In addition, we report that binding of vesicles to the planar membrane does not make the contact region more permeable to the osmoticant than is bare lipid bilayer. In the companion paper, we quantitatively account for the observation that the ability of a solute to promote fusion depends on its permeability properties and the method of swelling. We show that the intravesicular pressure developed drives fusion.