ARTICLE |
Correspondence to: Moïse Bendayan, Dept. of Anatomy, Université de Montréal, CP 6128 Succ. Centre Ville, Montreal, Quebec, Canada H3C 3J7.
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Summary |
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The arterial endothelial cells of the rete capillaries of the eel were examined by transmission electron microscopy on thin sections, on freeze-fracture replicas, by scanning electron microscopy, after cytochemical osmium impregnation and perfusion with peroxidase. The study revealed the existence of membrane-bound tubules and vesicles that open at both the luminal and abluminal poles of the cell and at the level of the intercellular space. The tubules are straight or present successive dilations and constrictions. They branch in various directions and intrude deeply into the cell cytoplasm, forming a complex tubular network within the cell. Immunocytochemical techniques were applied on immersion-fixed tissues and on perfusion of the capillaries with albumin and insulin. These demonstrated that the tubular-vesicular system is involved in the transport of circulating proteins. Furthermore, protein A-gold immunocytochemistry has revealed the association of actin with the membranes of this system. On the basis of these results, we suggest that the transendothelial transport of serum proteins takes place by a transcytotic process through a membrane-bound tubular-vesicular system and is equivalent to the large pore system presumed from functional studies. (J Histochem Cytochem 45:1365-1378, 1997)
Key Words: capillary, transcytosis, tubular system, vascular permeability, actin
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Introduction |
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Vascular permeability and identification of pathways for transendothelial transport of serum proteins have been matters of intense investigation because of their importance for the diagnosis and treatment of a variety of human diseases. Although vascular permeability can be assessed by techniques using radiolabeled tracers in vivo and in vitro, the recognition of cellular transport pathways can be achieved only by morphological means. Physiological data have conceptually defined a system of pores on the endothelial wall to account for the vascular permeability for both small solutes and larger circulating proteins (
The rete mirabile on the eel swimbladder wall constitutes a unique model for the study of the morphofunctional properties of blood capillaries (
Although the morphological characteristics of the capillary tissue isolated from the eel swimbladder rete mirabile have already been reported (
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Materials and Methods |
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The morphological characteristics of the rete capillaries and their involvement in transcapillary transport were investigated in electron microscopy by applying various morphological and cytochemical techniques.
Tissue Preparation
Large eels of the genus Anguilla anguilla, weighing 1-1.5 kg, were caught in the St. Lawrence river and kept in running tapwater. After anesthesia with tricainemethane sulfonate, MS222 (Sandoz Pharmaceutical; East Hanover, NJ) added to the water at the concentration of 0.3 g/liter and abdominal incision, the two retia on the swimbladder wall were exposed and fixed by perfusion through the pre-retal artery with 1% glutaraldehyde in 0.1 M phosphate buffer with or without 2% acrolein and 5% DMSO (
For transmission electron microscopy, the tissues were postfixed with 1% osmium tetroxide and embedded in Epon according to standard techniques. In a different set of experiments, osmium impregnation was carried out after glutaraldehyde fixation according to the techniques of Friend (
In a different series of experiments, the capillary tissue was fixed by perfusion with 1% glutaraldehyde for 15 min. After perfused washing with phosphate buffer, the capillaries were perfused with a 1% solution of horseradish peroxidase (Sigma Chemical; St Louis, MO). This was carried out for 30 min, after which the tissue was immersion-fixed for 2 hr in 1% glutaraldehyde. After rinsing, the capillary tissue was embedded in gelatin and thick sections were obtained with a Smith-Farquhar tissue chopper. These sections were then reacted with DAB (
For scanning electron microscopy, thick segments of the tissue were sectioned using a Smith-Farquhar tissue chopper and digested with 2% diastase in Ringer's lactate solution for 15 min. After critical point-drying, the segments were mounted on supports and coated with gold before examination.
For the freeze-etching technique, platinum-carbon replicas were generated according to standard techniques after fixation of the tissue with buffered 1% glutaraldehyde.
For the immunocytochemical investigation of vascular permeability, the rete was perfused through the pre-retal artery with a Krebs-Ringers' bicarbonate buffer supplemented with glucose (5 mM) and bovine serum albumin (4 g/100 ml) and equilibrated with a gas mixture of 95% O2-5% CO2 (
For immunodetection of endogenous serum albumin, we used the capillary tissue that was fixed by immersion. The thin sections were pretreated with sodium metaperiodate as mentioned above and then incubated with the anti-BSA antibody diluted 1:25 overnight at 4C. The protein A-gold complex was then applied for 30 min at RT. The crossreactivity of the anti-BSA with eel serum albumin was previously assessed by an immunodot experiment.
For immunodetection of actin, the tissue was fixed with 1% glutaraldehyde and processed for embedding in Lowicryl K4M at -20C without postfixation with osmium tetroxide (
Several control experiments were performed to assess the specificity of the different immunolabelings. These included omission of the specific antibodies in the labeling protocol or adsorption of the specific antibodies with their respective antigens before the immunolabelings (
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Results |
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The rete mirabile of the eel swimbladder is composed of a rich pure network of blood capillaries arranged in parallel circuits. Arterial and venous capillaries alternate in regular fashion and carry blood in opposite directions. The arterial capillaries are connected at the poles of the rete by pre-retal and post-retal arteries, and the venous capillaries are connected by pre-retal and post-retal veins. In transmission electron microscopy, the arterial capillaries are formed by a high continuous endothelium, whereas the venous capillaries are thinner and fenestrated (Figure 1). Both endothelia rest on well-defined basement membranes and some pericytes surrounded by bundles of collagen fibers are found in the interstitial space (Figure 1). Tight intercellular junctions between endothelial cells seal the blood vessel lumina (Figure 2). At higher magnification (Figure 2 Figure 3 Figure 4 Figure 5 Figure 6), the endothelial cells display a tubular-vesicular system, composed of plasma membrane invaginations, which is particularly developed in the arterial capillaries. This system consists of vesicular and tubular membrane profiles that penetrate the endothelial cell cytoplasm and open at the luminal and abluminal spaces as well as at the level of the intercellular junctional space (Figure 2). Some of the tubules intrude deeply into the cell cytoplasm, forming channel-like structures (Figure 2). Observations made on tilting the tissue sections (20-30°) confirm the existence of true continuous membrane-bound tubular structures. In relatively thicker sections (Figure 3), these tubules can be followed over a path reaching several micrometers in length. The tubules can be straight or branched in various directions (Figure 2 Figure 3 Figure 4 Figure 5 Figure 6). Some of these branching structures form bottle-neck figures (Figure 4 and Figure 6) because their narrow tubular connections have been cut out of the tissue section. The tubules either appear linear or exhibit a series of dilations and constrictions (Figure 2 and Figure 6). In some instances, narrow tubular extensions are present, either arising from vesicular structures or extending between dilated vesicular profiles (Figure 6). In addition, narrower tubular structures are found close to the plasmalemmal membranes (Figure 5). Continuity of membranes is observed between the tubular structures and smooth membranes that could be assigned to the smooth endoplasmic reticulum (Figure 5). Occasionally, part of the membrane of this tubular-vesicular organelle appears coated (Figure 2). The continuity of membrane between the tubular system and the plasmalemmal membrane on both the luminal and abluminal fronts is evident. As reported previously (
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When prefixed tissues were perfused with peroxidase, the tracer was detected in association with the luminal plasma membrane, in the intracellular junctions as well as within the basement membrane (Figure 8). Extravasation of the tracer had therefore occurred, probably related to rupture of the interendothelial junctions as a consequence of the perfusion-fixation process. Nevertheless, vesicular and tubular profiles within the endothelial cells were labeled with the peroxidase, demonstrating that after fixation these structures remained open to the luminal space (Figure 8). Most of these labeled structures were located at or close to the luminal plasma membrane. Occasionally, some vesicular profiles deep within the endothelial cells were also labeled, demonstrating that they are indeed connected to the luminal membrane. On the other hand, no peroxidase-labeled tubular profile was found connecting directly the luminal and abluminal fronts of the cells (Figure 8). Scanning electron microscopy at high magnification (Figure 9) revealed the existence of an endothelial intracellular network of interconnected tubules with dilations. The size of such structures falls within that of the tubulo-vesicular system observed by transmission electron microscopy. On replicas generated by freeze-fracture (Figure 10), long membrane-delineated intracellular tubules as well as vesicles are seen intruding deeply into the cell cytoplasm from the plasmalemmal membrane. The leaflets of these plasmalemmal membranes display their typical intramembrane particles. Replicas of lateral membranes also revealed the existence of tight occluding junctional elements composed of a few characteristic ridges or strands, as demonstrated previously (
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The particular arrangement of the rete capillaries allows countercurrent perfusion and accurate measurement of the transcapillary permeability of tracers present in the perfusate (
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In a second series of experiments we used bovine serum albumin and porcine insulin as exogenous tracers. These tissues were perfused for 30 min as described in Materials and Methods. The specific antibodies were combined with the protein A-gold complex to reveal the location of their corresponding antigens in the capillary tissue sections with high resolution. Aside from variations in intensities of labeling due to differences in concentrations of the tracers and antibody titers, the pattern of labeling was similar for both proteins. Because the perfusions were carried out until steady-state conditions were reached, the labeling by gold particles reflecting the location of the tracers was found in both the arterial and the venous capillary lumina, in the endothelial cells, and in the interstitial space. In the arterial endothelial cells, the labeling was mainly located over the tubular-vesicular structures, in those opened at both the blood and tissue fronts of the cells as well as in those present inside the cell cytoplasm (Figure 11C). Very few gold particles were seen over mitochondria or nuclei. Notably, the intercellular junctions were devoid of any labeling. When labeling was found close to the junctions, it was confined to either the luminal or the basal side of the junctions but not at the level of the tight occluding complex. In the intercellular space, the labeling was located within the basement membranes and between the collagen fibers. Little labeling was detected in pericytes and, when present, it was associated with vesicular profiles (data not shown).
For the study of actin, a different set of experiments was performed in which actin antigenic sites were detected on immersion-fixed nonosmicated tissues embedded in Lowicryl. Because of these conditions of tissue preparation, the ultrastructural preservation was suboptimal. Strong labeling for actin was found on pericytes, particularly over filament-rich regions (data not shown). The labeling was also present in endothelial cells, but to a lesser extent. It was concentrated along the plasmalemmal membrane (Figure 12A), the intercellular junctional complex, and associated with the membranes of the vesicular and tubular profiles (Figure 12B).
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Control experiments for the immunocytochemical studies resulted in an absence of specific labeling, very few gold particles being present in the capillary lumina and endothelial cells (data not shown).
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Discussion |
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The rete mirabile of the eel swimbladder consists of a rich network of blood capillaries, the function of which is the build-up of the partial pressure of oxygen, allowing its diffusion into the swimbladder. This increase in oxygen pressure is achieved by a hairpin countercurrent multiplication mechanism that pumps lactic acid from the venous capillaries into the arterial ones. The resulting acidification of the arterial blood at the bladder pole of the rete raises the partial pressure of oxygen, which causes the gas to enter the bladder (
The presence of tubules in the rete capillaries in such a significant number might be attributed to the large size of these endothelial cells and to the very active countercurrent transport taking place in these capillaries for adaptation of fish buoyancy to water depth (
With respect to the functional properties of the tubular system in endothelial cells, we have shown the presence of circulating endogenous and exogenous albumin, insulin, and large serum proteins of various sizes (
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Acknowledgments |
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Supported by research grants from the Medical Research Council of Canada.
We are grateful to Dr. L. Ghitescu for his interest and constructive discussion. We acknowledge the technical assistance of G. Mayer, D. Gingras, M.P. Dea, and J. Léveillé.
Received for publication October 15, 1996; accepted May 15, 1997.
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