Cell surface "blanket" of apolipoprotein E on rat adrenocortical cells.

Apolipoprotein (apo) E is expressed at high levels by adrenocortical cells. In the present study, an affinity-purified antibody to rat apoE was used in combination with immunogold visualization at both the light and electron microscopic levels to determine the cellular and subcellular distribution of apoE within the rat adrenal cortex. At the light microscopic level, apoE was found primarily in z. fasciculata and z. reticularis with little or none detected in z. glomerulosa and medulla. Within the z. fasciculata and z. reticularis, apoE was present in the cytoplasm of all parenchymal cells. ApoE also was found on the cell surface both on the sinusoidal front and in regions well removed from the subendothelial space. Electron microscopic examination of the z. fasciculata showed that apoE on the sinusoidal front was on the parenchymal cell surface but not the endothelial cell. Cell surface apoE was prominent on microvilli as well as non-microvillar regions of plasma membrane in the subendothelial space. ApoE was also associated with the cell surface in intercellular spaces continuous with but well removed from the subendothelial space. These findings at the light and electron microscopic levels suggest that the z. fasciculata cell is encircled or covered with apoE on all faces of the cell. These results are consistent with the idea that this cell surface "blanket" of apoE participates in the uptake of lipoprotein cholesterol by either the endocytic or selective uptake pathways.

Light and electron microscope observations of the lymphatic drainage units of the peritoneal cavity of rodents.

Fluid, particles, and cells are taken up from the peritoneal cavity by lymphatic drainage units, which, in the mouse and rat, are located along the peritoneal surface of the muscular portion of the diaphragm. The drainage units are composed of three specifically differentiated components: a lymphatic lacuna, a covering of lacunar mesothelium, and intervening submesothelial connective tissue. The units are drained by connecting lymphatic vessels that cross the diaphragm to empty into collecting lymphatic vessels running along the pleural surface of the diaphragm. The collecting lymphatics empty into parasternal lymphatic trunks. In this report, we briefly review critical features of the drainage apparatus and describe new observations, summarized below, about their structure. Around the rim of stomata, the mesothelial openings that lead into the lymphatic lacunae, plasma membranes of lacunar mesothelial cells and of lacunar endothelial cells abut but are not linked to one another by recognizable junctional specializations. Lacunar endothelial cells often extend valve-like processes that bridge the distal end of the channel beneath the stoma. The configuration of the endothelial processes may be complex. Occasionally, processes from fibroblasts in the submesothelial connective tissue adjacent to stomata make contact with the interstitial surface of lacunar endothelial cells. A discontinuous elastic layer in the submesothelial connective tissue spans the roof of each lacuna. Connecting and collecting lymphatics, which drain lymphatic lacunae, possess endothelial valves. Possible functions for each of these newly described structural features are discussed.

Basement membrane of central nervous system capillaries lacks ruthenium red-staining sites.

We used the cationic dye ruthenium red to examine the distribution of anionic macro-molecules (presumably proteoglycans) in the basement membranes of the fenestrated capillaries and epithelium of the choroid plexus, and of the continuous capillaries forming the blood-brain barrier. Both the endothelial and epithelial basement membranes of choroid plexus displayed discrete, 10- to 20-nm-diameter, electron-dense sites after exposure to ruthenium red. These sites were similar in size, appearance, and distribution to those found in the vascular and epithelial basement membranes of a variety of tissues outside the central nervous system. In contrast, the basement membranes of continuous, blood-brain barrier capillaries did not display electron-dense sites following exposure to ruthenium red, even after measures had been taken to enhance penetration of the dye across the endothelial cells. The lack of discrete ruthenium red staining in the basement membrane of continuous blood-brain barrier capillaries could be due to a relative paucity of anionic macromolecules, or may be the result of the compact architecture of this particular basement membrane. Regardless of the final explanation, these findings suggest that the basement membrane of the blood-brain barrier, like its endothelium, is structurally (and perhaps functionally) unique.

Surgical exposure induces formation of an arteriovenous permeability gradient for macromolecules in the microcirculation of muscle.

Rous and his colleagues (P. Rous, H. P. Gilding, and F. Smith, 1930, J. Exp. Med. 51, 807-830, F. Smith and P. Rous, 1931, J. Exp. Med. 53, 195-217) uncovered evidence for an arteriovenous gradient of permeability in exchange vessels of muscle. After injecting vital dyes intravenously in laboratory animals, including mice and rats, they examined escape of the dyes from exchange vessels of abdominal muscles, prepared for observation by reflection of the overlying skin. They noted that a particular class of dyes, termed "poorly diffusible," escapes from venules but not from arterioles and capillaries a few minutes after injection. We now assume that the "poor" diffusibility of the dyes stems from their binding to plasma proteins. We repeated similar experiments in mice and rats, using Evans blue as tracer, and also observed leakage of Evans blue from venules but not from other exchange vessels. We made three additional observations. (1) Evans blue leaks from venules only after the skin overlying the abdominal muscles is reflected. (2) Reflection of the skin initiates degranulation of mast cells in the muscles. (3) Leakage of Evans blue from venules is inhibited by administration of promethazine, a histamine and serotonin antagonist, to the animals prior to reflection of the skin. On the basis of our observations, we conclude that the arteriovenous permeability gradient for poorly diffusible dyes in the microcirculation of muscle represents response to tissue injury resulting from reflection of overlying skin.

Anionic sites on the luminal surface of fenestrated and continuous capillaries of the CNS.

We injected intravenously cationic ferritin, pI 8.4, and studied patterns of labeling of the luminal surface of capillaries in the CNS of rats. Cationic ferritin consistently labeled the luminal aspect of the diaphragms of fenestrated capillaries in the choroid plexus, median eminence and pineal. No other feature of these endothelial cells was consistently labeled. Diaphragms spanning the mouths of vesicles open to the lumen were not labeled. Occasional ferritin molecules were seen in the BL of these fenestrated vessels. The luminal surfaces of endothelial cells of the continuous capillaries of the brain, and of reactive capillaries proliferating in a region of cold injury necrosis, were not labeled. Ferritin was not seen in cytoplasmic structures or the basal lamina of these vessels. The findings in the fenestrated capillaries of CNS are in agreement with those reported for other fenestrated endothelia. The absence of labeling of the continuous capillaries of the blood-brain barrier differs from findings reported for other continuous capillaries, with the exception of the blood-air barrier portions of lung capillaries.

Anionic sites in basement membranes. Differences in their electrostatic properties in continuous and fenestrated capillaries.

We have used ruthenium red, a cationic dye, to detect at the electron microscopic level the presence of anionic sites in various murine basement membranes, with particular emphasis on those of the microvasculature. We have observed anionic sites in all continuous and fenestrated capillaries examined. Terminal lymphatics, which have a discontinuous basement membrane, have sites only where the basement membrane is present. Anionic sites are not present beneath sinusoidal lining cells of the liver which lack a basement membrane. Basement membranes of epithelial cells and those surrounding striated and smooth muscle cells, pericytes, fat cells, and Schwann cells also exhibit anionic sites. We compared the electrostatic properties of anionic sites in basement membranes of continuous and fenestrated capillaries by determining the salt concentration (critical electrolyte concentration, Scott and Dorling, 1965) required to displace ruthenium red from the sites. A concentration of 0.5 M Na+ was required to displace ruthenium red from the basement membrane of continuous capillaries of muscle whereas 1.3 M Na+ was required to displace ruthenium red from the basement membrane of fenestrated peritubular capillaries of the renal cortex. Our results suggest that anionic sites in the basement membrane of fenestrated peritubular capillaries are more strongly negatively charged than those in the basement membrane of continuous capillaries of muscle. We conclude from this study, first, that anionic sites are a general property of vascular, epithelial, and pericellular basement membranes and, second, that the electrostatic properties of the sites differ in different vascular basement membranes. We speculate that the anionic sites in vascular basement membranes and the variation in their electrostatic properties in different types of capillaries may have important implications for exchange of substances across the capillary wall.

Abundant, uniquely oriented endoplasmic reticulum in capillaries of the CNS: demonstration using reduced-osmium and glucose-6-phosphatase cytochemistry.

In the endothelial cells of capillaries in the rat CNS, we have observed abundant, circumferentially oriented, smooth, membrane-bound profiles, found just beneath, and parallel to, the abluminal plasmalemma. These structures are seen particularly well in tissue exposed to potassium ferricyanide-reduced OsO4. We studied these structures cytochemically, using glucose-6-phosphatase as a marker for endoplasmic reticulum, and acid phosphatase as a marker for the Golgi-associated endoplasmic reticulum containing lysosomal enzymes (GERL). We found that they contained glucose-6-phosphate hydrolyzing activity but did not contain acid phosphatase activity. Comparable structures were not seen in the continuous capillaries of skeletal muscle. Based on their morphology and content of glucose-6-phosphate hydrolyzing activity, we conclude that these structures are uniquely oriented smooth endoplasmic reticulum, which is much more abundant in capillaries of the CNS than in other continuous capillaries. The function of this distinctive feature of the CNS capillary is not known.

Identification of the small pore in muscle capillaries.

Electron microscope studies have been carried out in order to identify the structural counterpart of the small pore of muscle capillaries. Experiments with horseradish peroxidase (40,000 MW) and microperoxidase (1,900 MW) indicate that the clefts between capillary endothelial cells, rather than endothelial vesicles or transendothelial channels, are the counterpart of the pore. The structural properties of the clefts that enable them to serve as the small pore are presumably related to the nature of the attachment between plasma membranes of neighboring endothelial cells.

Permeability of muscle capillaries to microperoxidase.

In this study we attempted to identify a morphologic counterpart of the small pore of muscle capillaries. The existence of such a pore has been postulated by physiologists to explain the permeability of muscle capillaries to small macromolecules. We injected mice intravenously with microperoxidase (MP) and fixed specimens of diaphragm at intervals of 0-250 s after the injection to localize the tracer by electron microscopy. The small size of MP (1,900 mol wt and 20 A molecular diameter [MD]) ensures its ready passage through the small pore since the latter is thought to be either a cylindrical channel 90 A in diameter or a slit 55 A wide. MP appears in the pericapillary interstitium within 30 s of initiation of its intravenous injection. The patterns of localization of MP observed within clefts between adjacent capillary endothelial cells indicate that some endothelial junctions are permeable to this tracer. Although small vesicles transfer MP across the endothelium, we do not believe that the vesicles transfer substantial amounts of MP into the pericapillary interstitium. We did not obtain evidence that MP crosses the endothelium of capillaries through channels formed either by a single vesicle or by a series of linked vesicles opening simultaneously at both surfaces of the endothelial cell. From our observations we conclude that some endothelial junctions of capillaries are permeable to MP, and that these permeable junctions are a plausible morphologic counterpart of the small pore.

Absorption from the peritoneal cavity: SEM study of the mesothelium covering the peritoneal surface of the muscular portion of the diaphragm.

Colored tracers, injected intraperitoneally in mice, are taken up by diaphragmatic lymphatics, outlining their large, terminal cisterns, the so-called lacunae. The lacunae occur exclusively on the muscular portion of the diaphragm. The mesothelium covering non-lacunar and lacunar areas of the muscular portion was examined with the SEM. Mesothelial cells overlying non-lacunar areas are extremely flat, and their boundaries are indistinct. Mesothelial cells overlying lacunae protrude towards the lumen of the peritoneal cavity and have distinct outlines. There are openings or stomata, 4-12 micron in diameter, between them. Some of the stomata overlie a deep pit; others overlie a shallower pit in which the surface of another cell can be seen beneath the opening. It seems likely that the bulk of the fluid draining from the peritoneal cavity passes through these stomata into underlying lymphatic lacunae.

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.

A biochemical and radioautographic analysis of protein secretion by thyroid lobes incubated in vitro.

In this study we analyzed several aspects of protein secretion by thyroid follicular cells. The study was carried out on intact thyroid lobes obtained from newborn rats and incubated in vitro. The fate of leucine-(3)H incorporated into protein within follicular cells of untreated and thyrotropic hormone (TSH)-treated lobes was traced by quantitative electron microscope radioautography. Our findings indicate that protein synthesized by the rough-surfaced endoplasmic reticulum during a pulse exposure to leucine-(3)H is released relatively slowly by this organelle. Approximately 1 hr after onset of the pulse, a peak of radioactive protein appears in the Golgi region. The significance of this peak is not clear. Newly synthesized secretory protein passes through the apex of follicular cells without being concentrated or temporarily stored there in the form of large secretory droplets. Passage probably takes place via small vesicles which are intermingled among diverse small vesicles at the apex of the cells as well as in the Golgi region. Exposure of the lobes to TSH in the incubation medium for 45 or 90 min does not stimulate incorporation of leucine-(3)H into protein. Acute stimulation with TSH does, however, modify the movement of secretory protein within the exocrine secretory apparatus of the follicular cell. It accelerates the arrival of the protein at the apex of follicular cells, and it accelerates the release of the protein into the follicular lumen.

Physiological and morphological effects of poly-L-lysine on the toad bladder.

Studies were carried out on the morphological and physiological effects of the binding of poly-L-lysine (polylysine; mol wt≊120,000) to the apical surface membrane of the toad bladder epithelium. Paired hemibladders were mounted in chambers and exposed to polylysine concentrations of 2, 8, or 80 µg/ml in the mucosal medium for periods of up to 2 hr. Radioautographs prepared after addition of(3)H-polylysine showed that the polymer was localized to the apical surface of the epithelium and in dense subapical masses in lysed cells. No significant morphological changes were seen in the epithelium by light or electron microscopy at polymer concentrations of 2 and 8 µg/ml. Exposure to 80 µg/ml lysed many epithelial cells, i.e., converted them to slightly swollen ghosts with pycnotic nuclei and empty cytoplasm, except for remnants of mitochondria and vesicular fragments of the endoplasmic reticulum. All of the superficial epithelial cells were lysed in stretched hemibladders. The plasma membranes of the lysed cells were uniformly thickened, and their intercellular attachments remained intact. In contracted hemibladders, lysed and normal-appearing cells were interspersed, and the number of lysed cells in the epithelium was proportional to the duration of exposure to high concentrations of the polycation. In parallel experiments, the effects of varying concentrations of polylysine on active Na(+) transport and osmotic flow of water were measured with and without vasopressin, aldosterone, or amphotericin B in the media. At a concentration of 2 µg/ml of polylysine in the mucosal bathing solutions, no change in the basal rate of Na(+) transport was seen, and the response to vasopressin was unimpaired. At a concentration of 8 µg/ml, there was a significant but small fall in electrical potential difference (PD) and in short-circuit current (SCC) and no interference with the response to vasopressin. At a concentration of 80 µg/ml, there was a rapid curvilinear fall in SCC to 54±4% of the baseline value and in PD to 21±3% of the baseline value in a 2-hr period. Simultaneous unidirectional isotope flux studies with(22)Na and(24)Na showed a more than twofold increase in the serosal to mucosal flux but no discrepancy between net flux and SCC. Despite the inhibitory action of the polymer, the stimulatory response in Na(+) transport to vasopressin, aldosterone, and amphotericin B was relatively preserved in that the percentage increase in SCC was the same in the polymer-treated and control hemibladders. The polycation produced a small but significant increase in osmotic water flow, and striking and irreversible inhibition of the water-flow response to vasopressin.

Membrane modifications in the apical endocytic complex of ileal epithelial cells.

Ileal lining cells of the suckling rat possess an "apical endocytic complex" capable of sequestering intact protein from the intestinal lumen. The complex consists of a network of invaginations of the apical plasma membrane, a number of subjacent small vesicles, and a giant supranuclear vacuole. The first two components initially incorporate material from the intestinal lumen and then transfer it to the giant vacuole where it is stored. Their limiting membrane displays striking structural modifications when viewed in various planes of section. Its lumenal dense leaflet appears discontinuous and consists of an ordered array of minute discrete plaques. A dense particle approximately 70 A in diameter is centered over each plaque. The particles are arranged in a two-dimensional square lattice with center-to-center spacing of approximately 120 A.