Plasma membrane-associated vesicles in retinal capillaries of the rat

The structure and function of abluminal vesicles in endothelial cells of rat retinal capillaries was examined using glutaraldehyde-tannic acid fixation and the hemeproteins--horseradish peroxidase, microperoxidase, and lactoperoxidase--as tracers. Numerous vesicles, delimited by a tannic acid-positive membrane, were distributed along the abluminal front. Other vesicles were arranged in clusters and chains or tubule-like structures. Such vesicles were not found in the vicinity of the capillary lumen. When the retina was exposed to hemeproteins, either in vitro or after intravitreal injection, the abluminal vesicles became labeled with tracer reaction product. Apparently "free" vesicles and tubules seen in tangential sections through the basal lamina were also labeled, suggesting that they were in continuity with the plasma membrane in another plane of section. No enzyme reaction product was present in the capillary lumen. Peroxidase-positive multivesicular bodies were observed, suggesting that some protein was endocytosed and directed to lysosomes where it was presumably degraded. The results suggest that abluminal endothelial vesicles represent pits or invaginations of the plasma membrane and, as such, are not involved in the transendothelial transport of protein from the perivascular space to the capillary lumen. Tannic acid treatment revealed a population of similar vesicles associated with the plasma membrane of pericytes. After exposure to hemeproteins, enzyme reaction product was localized in these vesicles and in a few multivesicular bodies. The results suggest that the majority of these vesicles are in continuity with the plasma membrane and are not involved in endocytosis.     

Studies on the permeability of the blood-testis barrier in stainless steel-administered mice

Previous trials performed in our laboratory have demonstrated that stainless steel corrosion products induced morphological changes in mice seminiferous cells. In this study the functional nature of the blood-testis barrier was investigated, in view of the extesive use of this metallic alloy in orthopaedic surgery. The uptake of horseradish peroxidase by the seminiferous tubules was used as a tracer method being exposed in vitro for different periods of time and observed at the ultrastructural level. Both boundary cells as well as Sertoli cells of experimental group absorbed the tracer, as manifested by vesicles and vacuoles filled with the reaction product, evidencing its role within the testis. Germ cells were also able in the uptake of this macromolecule. However, the tracer did not spread into the junctional complexes between neighboring Sertoli cells, at any interval of time used in this study, demonstrating the functional integrity of this barrier.     

Distribution of calcitonin gene-related peptide-containing fibers in the urinary bladder of the rat and their origin

Summary Using horseradish peroxidase (HRP) as a tracer, we have investigated if the so-called apical tubules (AT) in the kidney proximal tubule cells are directly involved in the endocytic process by carrying the tracer into the cells, or if they are derived from the intracellular membrane compartments. Rat kidney was fixed by vascular perfusion at different time intervals after intravenous injection of HRP and prepared for electron microscopy. An analysis revealed that 0.5 min after injection, invaginations of the plasma membrane and small apical endocytic vesicles, including coated vesicles, were labelled with reaction product, whereas almost all large apical endocytic vacuoles and the AT were negative. The endocytic vacuoles and about 18% of the AT were labelled 1 min after injection. The reaction product in the large endocytic vacuoles was usually seen along the luminal surface of the vacuoles. The AT with reaction product appeared as a branched network, and were frequently connected with the labelled endocytic vacuoles. Three min after injection, reaction product was detected in about 38% of the AT, and thereafter, the percentage increased to about 74% after 7 min. No reaction product was detected in the Golgi complex at any time after HRP-injection. These findings indicate that the AT are probably formed by budding off from the large endocytic vacuoles, rather than being directly involved in the endocytic process.     

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.     

Absorption of intraarticularly injected horseradish peroxidase in synoviocytes of rat synovial membrane: an ultrastructural-cytochemical study

The ability of type A and type S synoviocytes to absorb horseradish peroxidase (HRP) and the intracellular fate of this tracer were studied by electron microscopic cytochemistry. Different concentrations of HRP (0.1-5 mg/ml) were injected into the left knee joint of rats and at intervals ranging from 1 min to 24 hr after injection the synovial membrane was fixed and incubated for HRP. Type A synoviocytes showed a striking ability to absorb HRP at low concentrations. At 1 and 5 min after injection reaction product was localized in coated pits and coated vesicles (110 nm) as well as in smooth-walled vesicles, vacuoles, and tubules. At 15 min to 4 hr postinjection the lysosomal system became increasingly loaded with reaction product. At 24 hr after injection reaction product had disappeared. At higher concentrations of HRP similar observations were made in the A cells, but reaction product was still apparent in lysosomes at 24 hr postinjection. With respect to type S synoviocytes no reaction product was detected within these cells at any time interval after injection of low concentrations of HRP. However, at 5 min after injection of higher concentrations of HRP reaction product was localized in smooth vesicles and vacuoles mainly restricted to the large cytoplasmic processes facing the joint cavity. At 30 min to 4 hr postinjection the lysosomal system became progressively more loaded with HRP reaction product. At 24 hr after injection reaction product still remained in the lysosomal system. The present findings that type A and type S synoviocytes showed major differences with respect to endocytic capacity and cellular structures involved in absorption of HRP support the interpretation that the A and S cells represent two distinct types of cells and further suggest that endocytosis in these two types of cells serve different functions.     

A comparison of the fine-structural localization of nucleoside phosphatase activity in large intracranial blood vessels and the thoracic aorta of rabbits

Summary Nucleoside phosphatase activity was localized in rabbit intracranial blood vessels, namely cerebral and basilar arteries and veins, and in the thoracic aorta using the electron microscope. In the intracranial vessels the same ultrastructural localization of reaction product was found when ATP or ADP was used as substrate in a modified Wachstein-Meisel procedure using Mg ions as the enzyme activator. No reaction product was seen when using AMP or -glycero-phosphate as substrates, or in controls without any substrate. Reaction product was sparsely localized within cell membrane invaginations on all sides of endothelial and, to a lesser extent, of smooth muscle cells. Pinocytotic vesicles occasionally contained reaction product. The greatest amount of lead phosphate (reaction product) precipitate was found in the basement membranes of both endothelial and smooth muscle cells, particularly intense in the former case. A diffuse precipitate of reaction product was observed in the cytoplasm of adventitial fibroblasts.The thoracic aorta demonstrated the same localization with the following exceptions: generally greater concentrations of reaction product were found using ATP as substrate, than in the corresponding intracranial vessels; interendothelial gaps and the cell membrane invaginations of these gaps were completely filled with reaction product; there was no specific localization of reaction product in the basement membranes; and reaction product could also be demonstrated using AMP and glycero-phosphate as substrates. Acid phosphatase activity was localized in lysosomes in endothelial and smooth muscle cells from both types of vessels. The differences in enzyme localization between aorta and intracranial vessels were discussed, particularly in light of the differences in nucleoside phosphatase activity and transport functions between brain and somatic capillaries.     

Access of horseradish peroxidase (HRP) to the extracellular spaces of the maturation zone of the rat incisor enamel organ

Adult rats received a single dose of HRP intravenously and were killed from 10 min to 6 hr after injection. Following fixation with glutaraldehyde, the enamel organs were treated with a Graham-Karnovsky-type procedure for peroxidase activity, post-osmicated, and embedded in plastic. Sections were studied with light and electron microscopes. Ten minutes after injection, reaction product was found in all extra-cellular spaces of the enamel organ, at the enamel-ameloblast interface over smooth-ended and intermediate ameloblasts, and in apical surface invaginations and vesicles of the latter cell types. The enamel-ameloblast interface over the ruffle-ended aemlo-blasts and the extracellular spaces within the ruffled border were free of reaction product and remained so for up to 6 hr. The apical terminal bars of the ruffle-ended ameloblasts functioned as a barrier to HRP. The basal terminal bars of the smooth-ended ameloblasts likewise seemed to prevent the passage of the HRP. Possibly, HRP flows in a lateral direction from groups of ruffle-ended into groups of smooth-ended ameloblasts. Between 10 min and 6 hr, HRP was cleared more rapidly from the extra-cellular spaces of the papillary layer than from those of the ameloblast layer, and there was little backflow of tracer from the ameloblast into the papillary layer. Eventually, tracer was cleared also from the extracellular spaces of the ameloblast layer, probably mainly through micropinocytosis by the ameloblasts. A working model is proposed regarding the handling of large molecules by the enamel organ in the maturation zone.     

THE USE OF BEEF LIVER CATALASE AS A PROTEIN TRACER FOR ELECTRON MICROSCOPY

Beef liver catalase was injected intravenously into mice, and its distribution in the kidney, myocardium, and liver was studied with the electron microscope. A specific and relatively sensitive method was developed for its ultrastructural localization, based on the peroxidatic activity of catalase and employing a modified Graham and Karnovsky incubation medium. The main features of the medium were a higher concentration of diaminobenzidine, barium peroxide as the source of peroxide, and pH of 8.5. Ultrastructurally, the enzyme was seen to permeate the endothelial fenestrae and basement membranes of tubular and glomerular capillaries of the kidney. The urinary space and tubular lumina contained no reaction product. In the myocardial capillaries, the tracer filled the pinocytotic vesicles but did not diffuse across the intercellular clefts of the endothelium. In liver, uptake of catalase was seen both in hepatocytes and in Kupffer cells.     

Superoxide- and nitric oxide-derived species mediate endothelial dysfunction, endothelial glycocalyx disruption, and enhanced neutrophil adhesion in the post-ischemic guinea-pig heart.

The study was aimed at testing the hypothesis that a toxic product of the reaction between superoxide (O(2)(-)) and nitric oxide (NO) mediates, not only endothelial dysfunction, but also endothelium-glycocalyx disruption, and increased neutrophil (PMN) accumulation in the heart subjected to ischemia/reperfusion (IR) injury. Accordingly, we studied if scavengers of either O(2)(-) or NO, or a compound that was reported to attenuate cardiac production of peroxynitrite, would prevent endothelial injury and subsequent PNM adhesion in IR heart. Langendorff-perfused guinea-pig hearts were subjected to 30 min ischemia/35 min reperfusion, and infusion of PMN between 15 and 25 min of the reperfusion. Coronary flow responses to acetylcholine (ACh) and sodium nitroprusside (SNP) were used as measures of endothelium-dependent and -independent vascular function, respectively. PMN adhesion and endothelium glycocalyx ultrastructure were assessed in histological preparations. IR impaired the ACh, but not SNP, response by approximately 60%, caused endothelium-glycocalyx disruption, and approximately nine-fold increase in PMN adhesion. These alterations were prevented by superoxide dismutase (150 U/ml), NO synthase inhibitor, L-NAME (10 microM), NO scavenger, oxyhemoglobin (25 microM), and NO donor, SNAP (1 microM), and were not affected by catalase (600 u/ml). The glycocalyx-protective effect of these interventions preceded their effect on PMN adhesion. The data imply that PMN adhesion in IR guinea-pig heart is a process secondary to functional and/or structural changes in coronary endothelium, and that a toxic product of the reaction between superoxide and NO mediates these endothelial changes.     

Papaverine effects on PGI2 and TXA2 release from the canine vascular wall.

Operative manipulation of blood vessels might lead to spasm, thereby destroying the endothelial cell function: the spasm can be prevented by the vasodilator papaverine. To study if this was mediated via the prostanoid pathway the following investigation was undertaken: canine jugular veins and carotid arteries were dissected with or without papaverine. Vessel segments were then perfused with Hank's balanced salt solution for five times 15 min. Prostacyclin was measured as the stable degradation product 6-keto-PGF1 alpha and thromboxane as TXB2, by radioimmunoassay. Control arterial segments' 6-keto-PGF1 alpha release was initially 129.5 + 20.1 pg/mm2/15 min, and 29.7 + 10.4 after 60 min (p less than 0.05 vs initial value) and responded to arachidonic acid (AA) with an increase to 139.2 +/- 23.1 pg/mm2/15 min (p less than 0.05). Segments treated with papaverine had the same release as the controls. In venous segments there was a lower initial release (p less than 0.05) from segments given papaverine than from controls, but this was more likely an effect of papaverine on the assay. There was no difference in release of prostacyclin from segments given papaverine in the perfusate compared to controls when using 125I tracer. When using 3H tracer including absorption of free antigen to dextran coated charcoal, papaverine displaced the free tracer giving artificially low values. There was no effect of papaverine given intraoperatively on the TXB2 release, neither from arteries nor from veins. In another experiment the vessel wall tension was examined and the cyclooxygenase inhibitor diclofenac did not inhibit the vasodilating effect of papaverine.(ABSTRACT TRUNCATED AT 250 WORDS)     

STUDIES ON INFLAMMATION : I. The Effect of Histamine and Serotonin on Vascular Permeability: An Electron Microscopic Study

The mechanism, whereby histamine and serotonin increase the permeability of blood vessels, was studied in the rat by means of the electron microscope. The drugs were injected subcutaneously into the scrotum, whence they diffused into the underlying (striated) cremaster muscle. An intravenous injection of colloidal HgS was also given, in order to facilitate the identification of leaks by means of visible tracer particles. After intervals varying from 1 minute to 57 days the animals were killed; the cremaster was fixed, embedded in methacrylate, and examined with the electron microscope. One to 12 minutes after the injection, the blood vessels of the smallest caliber (3 to 5 micra as measured on electron micrographs) appeared intact. Numerous endothelial openings were present in blood vessels with a diameter of 7 to 8 micra or more. These gaps were 0.1 to 0.8 micra in width; portions of intercellular junctions were often present in one or both of the margins. The underlying basement membrane was morphologically intact. An accumulation of tracer particles and chylomicra against the basement membrane indicated that the latter behaved as a filter, allowing fluid to escape but retaining and concentrating suspended particulate matter of the size used. Uptake of tracer particles by endothelial vesicles was minimal. Phagocytosis by endothelial cells became more prominent at 3 hours, but as a secondary occurrence; the pericytes were actively phagocytic at all stages. At the 3-hour stage no leaks were found. The changes induced by histamine and serotonin were indistinguishable, except that the latter was more potent on a mole-to-mole basis. In control animals only small accumulations of tracer particles were found in the wall of a number of blood vessels. With regard to the pathogenesis of the endothelial leaks, the electron microscopic findings suggested that the endothelial cells become partially disconnected along the intercellular junctions. Supporting evidence was provided at the level of the light microscope, by demonstrating—in the same preparation—the leaks with appropriate tracer particles¹, and the intercellular junctions by the silver nitrate method. The lipid nature of the chylomicron deposits observed in electron micrographs was also confirmed at the level of the light microscope, using cremasters fixed in formalin and stained in toto with sudan red.     

Erythrocyte-mediated microinjection of horseradish peroxidase into BHK cells

In order to establish the distribution with time of proteins microinjected into mammalian cells, horseradish peroxidase (HRP) was microinjected into baby hamster kidney (BHK) cells using chicken erythrocyte ghosts. At time intervals following initiation of fusion between ghosts and target cells, samples were fixed with aldehydes and the peroxidase visualized by reaction with diaminobenzidine and viewing by light and electron microscopy. At 10 min, the reaction product was observed within the cytoplasm of 60% of the microinjected cells, but was excluded from the nucleus and membranous organelles. In the other 40% of microinjected cells, the reaction product was also observed within the nucleus. At 30 min, the reaction product was observed to be evenly distributed throughout the cell, including the nucleus but excluded from organelles. By 6 h, the reaction product was present almost exclusively within the nucleus of 63% of microinjected cells. At all time points, 20–30% of the erythrocytes ghosts appear to have been taken up by cells by phagocytosis rather than fusion, as evidenced by the presence of peroxidase reaction product within intact and fragmented erythrocyte ghosts in the cytoplasm of target cells. Cells incubated with a lanthanum solution following fusion excluded this electron dense tracer, indicating that the cytoplasmic compartment is not opened during exposure to polyethylene glycol.     

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.     

Oxidation of cholesterol moiety of low density lipoprotein in the presence of human endothelial cells or Cu+2 ions: identification of major products and their effects

Oxidation of lipoproteins is believed to play a key role in atherogenesis. In this study, low density lipoproteins (LDL) was subjected to oxidation in the presence of either human umbilical vein endothelial cells or with Cu+2 ions and the major oxides formed were identified. While cholesterol-alpha-epoxide (C-alpha EP) was the major product of cholesterol peroxidation in the presence of endothelial cells, cholest-3,5-dien-7-one (CD) predominated in the presence of Cu+2 ion. Both steroids were identified by gas chromatography/mass spectrometry. HDL cholesterol was resistant to oxidation. When tested on human skin fibroblasts in culture C-alpha EP (10 micrograms/ml) caused marked stimulation of 14C-oleate incorporation into cholesterol esters, while CD stimulated cholesterol esterification only mildly. These studies show that a) C-alpha EP is the major peroxidation product of LDL cholesterol moiety in the presence of endothelial cells and b) it causes marked stimulation of cholesterol esterification in cells. C-alpha EP may play a key role in increasing cholesterol esterification noted in atherogenesis.     

Ultrastructural study of the permeability of the guinea-pig placenta to horseradish peroxidase

Summary Horseradish peroxidase (HRP) was used to study macromolecule permeation into the guinea-pig placenta perfused in situ. When tissue culture medium 199 (TC 199) was used as fetal-side perfusate, the tracer reaction product was found only lining the fetal endothelium. When a longer period of perfusion with HRP in TC 199 was used, a small amount of reaction product was found in the subendothelial space and syncytiotrophoblastic vesicles, but not in maternal lacunae. In similar experiments using a Krebs bicarbonate Ringer (KRBG) as perfusate the tracer was found (i) lining the fetal endothelium, (ii) in the lateral intercellular spaces of the endothelium, (iii) in the subendothelial space, and (iv) in the maternal lacunae.It is therefore evident that the vehicle influenced the permeability of the guinea-pig placenta to horseradish peroxidase. As other studies have shown that perfusion of the fetal side with salt solution increases pore size, the results with TC 199 are regarded as more representative of the situation in the intact animal. It is therefore suggested that the fetal endothelium of the guinea-pig placenta may be largely impermeable to molecules of the size of horseradish peroxidase (4 nm) or larger.     

Study on the origin of apical tubules in ileal absorptive cells of suckling rats using concanavalin-A as a membrane-bound tracer

Summary The ileal absorptive cells of suckling rats exhibit high levels of endocytic activity being engaged in nonselective uptake of macromolecules from the intestinal lumen. The apical cytoplasm usually contains an extensive network of small, membrane-limited tubules (apical tubules: AT), in addition to newly formed endocytic vesicles and large endocytic vacuoles. To determine whether the AT are directly involved in the endocytic process by carrying the tracer into the cell, we have analysed movements of the apical cell membrane of the ileal absorptive cells by using a membrane-bound tracer (horseradish peroxidase-labelled cancanavalin-A: Con-A HRP). The ileal absorptive cells were exposed in vitro to Con-A HRP for 10 min at 4° C, incubated for different times in Con-A free medium at 37° C, and prepared for electron microscopy. After 1 min incubation at 37° C, invaginations of the apical cell membrane, including coated pits, and endocytic vesicles were labelled with HRP-reaction product, whereas the AT and large endocytic vacuoles were negative. After 2.5 min, almost all the large endocytic vacuoles were labelled with reaction product, which was seen in their vacuolar lumen and along the luminal surface of their limiting membrane. A few AT with reaction product were seen in the apical cytoplasm; they were in frequent connection with the reaction-positive large endocytic vacuoles. With increasing incubation time, the number of the labelled AT increased. Thus, after 15 min at 37° C, the apical cytoplasm was fully occupied by the reaction-positive AT. The ends of these AT were often continuous with small spherical coated vesicles. No reaction product was detected in the Golgi complex at any time after incubation. These observations indicate that the AT located in the apical cytoplasm probably originate by budding off from the large endocytic vacuoles, rather than being involved in the process of endocytosis.     

Electron microscopic demonstration of neural connections using horseradish peroxidase: a comparison of the tetramethylbenzidine procedure with seven other histochemical methods

Eight methods for the electron microscopic demonstration of horseradish peroxidase (HRP) labeling have been compared in adjacent series of vibratome sections of mouse lumbar spinal cord. The tracer, a HRP-wheat germ agglutinin (WGA) conjugate, was injected into the gastrocnemius muscle complex. Following retrograde axonal transport to the lumbar motor neurons and transganglionic anterograde transport of the tracer to the dorsal horn, the HRP activity was demonstrated in eight series of adjacent sections of lumbar spinal cord using eight methods. These included procedures using tetramethylbenzidine (TMB), benzidine dihydrochloride (BDHC), o-tolidine, paraphenylenediamine-pyrocatechol (PPD-PC), and 4 methods using 3,3'-diaminobenzidine (DAB). All eight methods were able to demonstrate both retrograde labeling of motor neurons and transganglionic anterograde transport into the dorsal horn. However, there were differences in the appearance of the various reaction products under the electron microscope. In addition, differences in the distribution of the reaction products were observed by both light and electron microscopy. The largest distribution of reaction product was observed with TMB. BDHC and o-tolidine were next, followed by the DAB procedures and PPD-PC. The TMB, BDHC, and o-tolidine reaction products were all found to be suitable for electron microscopy. The TMB reaction product was electron dense and had a very distinctive crystalloid appearance that made identification of HRP-labeled neuronal profiles easy and unequivocal.     

The toxic effects of bis (tributyltin) oxide on the rat thoracic aorta.

The toxic effects of bis (tributyltin) oxide (TBTO) on the ultrastructure and permeability of rat thoracic aorta were studied electron microscopically and the accumulation sites of tin were determined with an X-ray microanalyzer. Male Wistar rats received 0.05ml/kg of TBTO as an emulsion in 1 ml of distilled water through a stomach tube. After time intervals of 2, 4, 6, 8, 10, 12 h after intubation, thoracic aortae were isolated and prepared for electron microscopy. Marked swelling of mitochondria in the aortic endothelial cells appeared at 4 h after TBTO treatment. By x-ray microanalysis, tin L-alpha peaks (3.44 keV) were obtained from these swollen mitochondria. Subendothelial edema progressed between 6 and 8 h after TBTO treatment. By tracer experiment, it was seen that large amounts of peroxidase reaction products filled the expanded subendothelial space. At 12 h after TBTO treatment, degenerative changes of the endothelial cells were prominent. These results indicated that orally administered TBTO accumulated in the mitochondria of the endothelial cells of thoracic aorta. The direct toxic effects of TBTO on mitochondria might induce severe damage to the endothelial cells and cause disturbance of the permeability barrier function of the endothelial layer and subendothelial edema.     

Structural modulation of the surface and cytoplasm of oocytes during vitellogenesis in the lobster,Homarus americanus. An electron microscope-protein tracer study

The present investigation describes the ultrastructural changes which occur at the surface and in the cytoplasm of developing oocytes of the lobster, Homarus americanus, during vitellogenesis. The immature oocytes showed no surface specializations of the oolemma and no pinocytotic activity was observed. Horseradish peroxidase (HRP) tracer studies showed penetration of the tracer into the perivitelline space, but no uptake by the oocytes. The surfaces of oocytes examined during vitellogenesis, when yolk protein accumulation was maximal, exhibited numerous microvilli that projected into the perivitelline space, often appearing to be embedded in the follicular cell mass. In addition, the plasma membrane of vitellogenic oocytes contained many pinocytotic pits frequently situated at the bases of microvilli. The perivitelline space was engorged with electrondense material which appeared similar to that contained in pinocytotic structures of the oocytes. Vitellogenic oocytes incubated in HRP showed uptake of tracer reaction product by the coated pits and vesicles of the oolemma. Aggregation and subsequent fusion of these vesicles into large multivesicular bodies of ingested material were also observed in vitellogenic oocytes. Animals artificially induced to undergo vitellogenesis exhibited modulations of oocyte ultrastructure similar to those of normal vitellogenesis, notably, pinocytotic incorporation of extra-oocytic material and hypertrophy of oocyte surface microvilli. This study supports the hypothesis for a dual source of yolk protein in the American lobster.     

Horseradish peroxidase: a reliable or a misleading tool for the investigations on the origin of the proteins of the aqueous humor?

The concept of the blood-aqueous barrier is largely based on the use of horseradish peroxidase (HRP). The present investigation was designed to check its reliability as a macromolecular tracer, especially with regard to the transport of plasma proteins. Rabbits were killed 5 min to 24 h after being intravenously injected with HRP. The tracer diffused rapidly, reaching the aqueous humor of the eye in 3 min or less and was detected at high concentration in the narrow space between the outer epithelial layer of the ciliary epithelium and the wall of the pervious capillaries in the stroma of the processes. HRP appeared to migrate from the blood to the posterior chamber, permeating the tight junctions, viz., the anatomical basis of the blood-aqueous barrier. It was detected at higher concentration at the anterior surface of the iris, at short time intervals; this was interpreted as penetration of the tracer from the aqueous humor of the anterior chamber. The choroid was also labeled in continuation with the reaction in the stroma of the pars plana of the ciliary body which, in turn, sometimes reached the iris root. Therefore, the pervious blood vessels of the choroid could be a source of macromolecules for the iris root. HRP also induced the formation of lysosomes in the ciliary epithelium. This can hardly be accepted as the way in which plasma proteins are physiologically transported to the aqueous humor. However, the pathway of HRP migration over short time intervals seems to be in agreement with previous research indicating that the entrance of serum albumin into the posterior chamber is the first step of its incorporation into the aqueous humor. Received: 7 June 1996 / Accepted: 15 January 1997