Angiotensin II binding to tissues of the rainbow trout,Oncorhynchus mykiss,studied by autoradiography

Summary Tissue slices from seawater-adapted and freshwater-adapted rainbow trout, Oncorhynchus mykiss, were exposed to ¹²⁵I-angiotensin II (1.01·10^-9 M) and binding sites located by light-microscopic autoradiography. Binding/uptake was significantly inhibited by excess (10^-5 M) unlabelled angiotensin II, suggesting specific binding/uptake of angiotensin II to the ventral and dorsal aorta (smooth muscle), urinary bladder (smooth muscle and epithelial lining), glomeruli and proximal tubules, the gill (lamellae and central filament), skin (epithelium), intestine and oesophagus (mucosal epithelium), liver, heart (ventricular myocytes), adrenocortical tissue and brain (cerebellum and medulla oblongata). The specific binding/uptake of angiotensin II to tissues of freshwater- and seawater-adapted animals were generally similar. However, binding/uptake by the proximal tubules was significantly higher in freshwater-adapted trout than seawater-adapted trout. Specific binding/uptake of angiotensin II by the smooth muscle of the bladder was significantly higher in trout adapted to seawater than trout adapted to freshwater.     

Accessory cells in the gill epithelium of the freshwater rainbow trout Salmo gairdneri

Two types of mitochondria-rich cells were identified in the gill epithelium of the freshwater-adapted rainbow trout, Salmo gairdneri, after selective impregnation of their tubular system with reduced osmium. A first type consisted of large cells with a poorly developed and loosely anastomosed tubular system; thus, that resembled the chloride cells commonly encountered in the gill epithelium of freshwater-adapted euryhaline fishes. A second type comprised smaller cells with an extensively developed and tightly anastomosed tubular system. These never reached the basal lamina of the gill epithelium and were adjacent to chloride cells, to which they were linked by shallow apical junctions (100-200 nm); thus, they resembled accessory cells, which are currently found in the gill epithelium of seawater-adapted fishes but are usually lacking in freshwater living fishes. Transfer of the freshwater-adapted trout into seawater induced the proliferation of the tubular system in the chloride cells and the formation of lateral plasma membrane interdigitations between accessory cells and the apical portion of the chloride cells. The length of the apical junction sealing off this extended intercellular space was reduced to 20-50 nm. The tubular system of the accessory cells was not modified. The extension of the tubular system in the chloride cells of the seawater-adapted fishes indicated that, as in most euryhaline fishes, these cells have a role in the adaptation of the rainbow trout to seawater. In contrast, the function of the presumptive accessory cells in freshwater trout remains to be established.     

Studies on the relationship between osmotic or ionic regulation and thyroid gland activity in two salmonid fishes, Salmo gairdneri Richardson and Oncorhynchus kisutch Walbaum

In freshwater-acclimated rainbow trout a single intraperitoneal injection of ovine TSH significantly elevated plasma thyroxine (T₄) levels within 1 h after the injection. In seawater adapted trout the increase in T₄ after TSH-treatment was not evident until 6 h after the injection. TSH caused a transient fall in plasma Na⁺ and Cl^- between 3 h and 9 h after the injection in seawater-adapted fish and plasma Na⁺ was lowered in freshwater-adapted trout 24 h after the injection. Although there were clear histological changes in the thyroid gland of freshwater-adapted trout after TSH-injection, no such changes were evident in seawater-adapted fish.
Plasma thyroid hormone levels and thyroid histology in freshwater-adapted rainbow trout and coho salmon transferred to sea water, and seawater-adapted trout transferred to fresh water showed no consistent changes compared with controls.
The data are interpreted to indicate that although ambient salinity may have indirect effects on thyroid activity there is no direct involvement in ionic or osmotic regulation in the two species.     

Ultrastructural distribution of endogenous IgGs in the glomerular wall of control and diabetic rats

Summary Endogenous IgG molecules were revealed with high resolutionem over the glomerular wall in renal tissues sampled from short and longterm control and streptozotocin induced diabetic rats by applying the protein A-gold immunocytochemical approach. In tissues from control animals, IgG antigenic sites were revealed on the subendothelial side of the basement membrane, the epithelial side being only weakly labelled. In contrast, in longterm diabetic animals IgG antigenic sites were present throughout the entire thickness of the basement membrane, and in patches closely associated with the plasma membrane of the epithelial cells. Deposits of basement membrane-like material present in the mesangial area were also highly labelled for IgG. Numerous intensely labelled lysosome-like structures were present in the epithelial cells. Morphometrical evaluation of the distribution of the labelling over the basement membrane confirmed these observations. In control animals a peak of labelling was found at 30 nm from the endothelial cell region corresponding to the subendothelial side of the lamina densa. In longterm diabetic animals the labelling was more uniformly distributed throughout the entire thickness of the basement membrane. These data were correlated to biochemical determinations of proteinuria and IgG excretion in urine samples from the control and the diabetic animals. These results suggest that in normal conditions the lamina densa may represent the main barrier for the restriction of the passage of IgGs through the glomerular wall. Modifications at that level occur during diabetes leading to or participating in the loss of the selective permeability of the basement membrane.     

An ultrastructural study of connective tissue in mollusc integument: I. Bivalvia

The ultrastructure of the subepidermal connective tissue (SEC) in different areas of the integument of the bivalves Callista chione, Pecten jacobaeus, Mytilus galloprovincialis and Ostrea edulis was studied by transmission electron microscopy. The main organisation of the SEC was broadly similar in all species: the SEC was connected to the epidermis by a basement membrane and merged directly with the deeper connective tissue surrounding muscles. The SEC was not differentiated into layers like the papillary and reticular dermis of mammals, however, the architecture, thickness and shape of the basement membrane varied from species to species, as well as within species (in the foot, central or marginal zones of the mantle). The ultrastructure of the lamina densa was broadly similar to that in mammals: although basotubules and double pegs were absent, proteoglycans and rod-like units homologous to 'double tracks' were always abundant. A zone similar to the lamina lucida was irregularly present and was shot thorough with small protrusions of the lamina densa that connected with the epithelial hemidesmosomes or focal adhesions. Nevertheless zones were observed where the lamina densa fuse directly to the epithelial plasmamembrane. This variability of connection may be related to the various types of epidermal cell. A lamina fibroreticularis was not recognized since anchoring fibrils and microfibrils were not present; lamina densa protrusions into the extracellular matrix (ECM) of SEC characterize the connection between basement membrane and SEC. Collagen fibrils were small and of constant diameter and were never organised into fibres. Anchoring devices - similar to the anchoring plaques of mammalian dermis - were abundant and scattered between SEC collagen fibrils. The orange-pink pigmentation of C. chione seems due to electron-dense granules embedded within the connective ECM.     

Glomerular lysozyme binding in protein-overload proteinuria

Binding of the cationic molecule lysozyme to the glomerular basement membrane and to the glomerular epithelial cell coat was investigated in the glomerulus of normal female Wistar rats and in rats in which heavy proteinuria was induced by the daily administration of 1 g of bovine serum albumin. In normal rats the binding of lysozyme to the anionic groups in the glomerular basement membrane and the cell coat had no effect on the ultrastructure of the glomerular epithelial cell, in particular the foot processes were unchanged. In the proteinuric rats the lysozyme-binding to the glomerular basement membrane and the epithelial cell coat was completely lost in the damaged glomeruli. In the apparently normal glomeruli present in these proteinuric animals binding was similar to that seen in normal rats. These results suggest that in protein-overload proteinuria there is a loss of glomerular anion and hence a reduction in the glomerular charge barrier. This may account, at least in part, for the increased glomerular leak of negatively charged serum albumin in this experimental model of proteinuria.     

Direct effects of angiotensin II on glomerular ultrastructure in the rainbow trout,Salmo gairdneri

Summary Slices from the kidneys of the rainbow trout which were exposed to 10^-6 or 10^-5 M angiotensin II (AII) and isolated glomeruli exposed to 10^-7 or 10^-5 M AII showed ultrastructural changes compared to control tissues incubated without AII. The studies indicate that angiotensin II has a direct action on glomerular ultrastructure, flattening the epithelial podocytes and broadening the primary processes with fusion of pedicels in extreme cases. These changes suggest a probable effect of AII on water permeability of the trout glomerulus, an intrarenal action which is believed to form an essential part of the antidiuretic adaptation to increased environmental salinities.     

Morphological changes in the middle intestine of the rainbow trout,Salmo gairdneri,induced by a hyperosmotic environment

Summary The structural modifications in the middle intestine of the trout, Salmo gairdneri, induced by transfer to seawater have been studied. During the first two days in seawater, significant distensions of the intercellular spaces are observed between the apical tight junctions and the basement membrane. These dilations are more frequent in the apical part of the intestinal folds. At the basal part of the cell, numerous lamellar processes open in the intercellular spaces. They are closely associated with elongated mitochondria, and are often mixed with small clear vesicles. After seven days in seawater, intercellular spaces are less expanded. Numerous mitochondria are observed in the apical part of the cell, and numerous myelinic bodies with dense granules lie near the nucleus. After one month in seawater, the epithelium resembles that of the freshwater controls; mitochondria are more numerous and other organelles are well developed. The most important modifications of the ultrastructure of the intestine mucosa occur during the first two days in seawater, in correlation with important physiological changes following the abrupt increase of environmental salinity.     

Ontogenesis of glomerular basement membrane: structural and functional properties

Protein A-gold immunocytochemistry was applied in combination with morphometrical approaches to reveal the alpha 1(IV), alpha 2(IV), and alpha 3(IV) chains of type IV collagen as well as entactin on renal basement membranes, particularly on the glomerular one, during maturation. The results have indicated that a heterogeneity between renal basement membranes appears during the maturation process. In the glomerulus at the capillary loop stage, both the epithelial and endothelial cell basement membranes were labeled for the alpha 1(IV) and alpha 2(IV) chains of type IV collagen and entactin. After fusion, both proteins were present on the entire thickness of the typical glomerular basement membrane. At later stages, the labeling for alpha 1(IV) and alpha 2(IV) chains of type IV collagen decreased and drifted towards the endothelial side, whereas the labeling for the alpha 3(IV) chain increased and remained centrally located. Entactin remained on the entire thickness of the basement membrane during maturation and in adult stage. The distribution of endogenous serum albumin in the glomerular wall was studied during maturation, as a reference for the functional properties of the glomerular basement membrane. This distribution, dispersed through the entire thickness of the basement membrane at early stages, shifted towards the endothelial side of the lamina densa with maturation, demonstrating a progressive acquisition of the permselectivity. These results demonstrate that modifications in the content and organization of the different constituents of basement membranes occur with maturation and are required for the establishment of the filtration properties of the glomerular basement membrane.     

Alterations in the sialic acid content of the rat glomerular filter in aminonucleoside nephrosis

A daily injection protocol with puromycin aminonucleoside (PAN) causes loss of sialic acid from the glomerular filter. These changes have been studied previously by colloidal iron staining, but we have recently shown that phosphotungstic acid (PTA) at low pH allows the demonstration of sialic acid groups in the glomerular basement membrane in ultrathin sections of glycolmethacrylate(GMA)-embedded rat kidney. With this technique the slit diaphragm is seen as a continuation of the luminal cell coat and the method also gives an idea of the sialic acid distribution at the podocyte plasma membrane. The availability of this method made it possible to reevaluate the results obtained earlier in aminonucleoside (PAN) nephrosis indicating a decrease in the sialic acid content of the glomerulus. Although there are changes in the epithelial architecture, the ultrastructural appearances of the basement membrane are only slightly altered in PAN nephrosis. Detachment of epithelial cells was variable in different animals. Seven days after the first injection of PAN, staining with PTA revealed local defects in the lamina rara externa which later became more extensive. In PAN-treated animals the luminal cell coat showed reduced staining and large areas of the plasma membrane were completely devoid of a cell coat. These changes coincided with the onset of heavy proteinuria. The results indicate that both the basement membrane and the epithelial plasma membrane are affected in PAN nephrosis, as revealed by decreased staining for sialic acid-containing molecules in the basement membrane and by changes in the epithelial cell coat. The defects in the cell coat material point to functional alterations at the level of the slit pores and it is suggested that the decrease in sialic acid content of the lamina rara externa may be partly responsible for defects in the size-selective filtration barrier in PAN nephrosis.     

Ultrastructural organization of the glomerular basement membrane as revealed by a deep-etch replica method

Summary The fine structure of the glomerular basement membrane was re-evaluated by using a deep-etch replica method.The structure of the laminae rarae interna and externa of the rat glomerular basement membrane was basically identical in that 6 to 8 nm fibrils were interconnected to form a three-dimensional, polygonal network. The size of the mesh was quite variable but most often ranged from 20 to 25 nm in width. In addition, a zipper-like substructure of the epithelial slit diaphragm was observed. By contrast, the lamina densa was composed of closely packed particles.After exposure of the bovine glomerular basement membrane to ultrasonic waves or trypsin, the particles of the lamina densa were effectively removed. The underlying structure showed the fibrillar network closely resembled that seen in the laminae rarae of the rat glomerular basement membrane.The glomerular basement membrane thus revealed was as principally composed of a fibrillar network, which might be regularly arranged units of type-IV collagen. Numerous fine particles, most likely proper components of the glomerular basement membrane, were attached onto this basic fibrillar structure, giving rise to a morphologic appearance different from that of the laminae rarae.     

Fine structure of the glomerular basement membrane and immunolocalization of five basement membrane components to the lamina densa (basal lamina) and its extensions in both glomeruli and tubules of the rat kidney

Electron microscopic immunostaining was used to examine the localization of type IV collagen, laminin, entactin , heparan sulfate proteoglycan, and fibronectin within the basement membranes of the rat kidney. In preliminary experiments, various methods of processing formaldehyde-fixed kidney were compared using antilaminin antiserum and the indirect immunoperoxidase method. Little or no laminin immunostaining of the glomerular basement membrane was present in sections unless they had been frozen-thawed; and even in this case, the immunostaining was light in comparison to that of basement membranes in adjacent tubules. However, when frozen-thawed sections were treated with 0.5% sodium borohydride, immunostaining was then as strong in glomerular as in tubular basement membranes. Accordingly, this treatment was applied to frozen-thawed sections before immunostaining for any of the substances under study. Immunostaining of the glomerular basement membrane for each of the five substances was fairly uniform throughout the lamina densa (also called basal lamina), but uneven in the lamina lucida interna and externa (also called lamina rara interna and externa) in which stained bands extended from the lamina densa. Similarly in the basement membranes of tubules, immunostaining for the five substances was localized to the lamina densa and bands extending into the lamina lucida. When the ultrastructure of the glomerular basement membrane was examined, three structures were found: (1) a network of 4-nm-thick "cords," which seems to be the main component; the cords are closely packed in the lamina densa and more loosely arranged in the lamina lucida interna and externa; (2) straight, hollow 7-10-nm-thick structures referred to as " basotubules "; and (3) 3.5-nm elements composed of minute paired rods, referred to as "double pegs." The distribution of the cords, but not that of the other two structures, was related to the immunostaining pattern. It is concluded that (1) to fully reveal the antigenicity of the glomerular basement membrane, frozen-thawed sections must be treated with sodium borohydride prior to immunostaining, possibly because this basement membrane is more compact than the others; and (2) in both glomerular and tubular basement membranes, type IV collagen, laminin, entactin , heparan sulfate proteoglycan and fibronectin are colocalized in the lamina densa and its extensions to the laminae lucidae . Since the distribution of the cords corresponds to that of immunostaining, it is likely that the five substances are present within the cords.     

Effects of cortisol and salinity challenge on water balance in developing larvae of tilapia (Oreochromis mossambicus)

Effects of exogenous cortisol on drinking rate and water content in developing larvae of tilapia (Oreochromis mossambicus) were examined. Both freshwater- and seawater-adapted larvae showed increases in drinking rates with development. Drinking rates of seawater-adapted larvae were about four- to ninefold higher than those of freshwater-adapted larvae from day 2 to day 5 after hatching. Seawater-adapted larvae showed declines in drinking rate and water content at 4 and 14 h, respectively, after immersion in 10 mg L(-1) cortisol. In the case of freshwater-adapted larvae, the drinking rate decreased after 8 h of cortisol immersion, while the water content did not show a significant change even after 32 h of cortisol immersion. In a subsequent experiment of transfer from freshwater to 20 ppt (parts per thousand, salinity) seawater, immersion in 10 mg L(-1) cortisol for 8-24 h enhanced the drinking rate in larvae at 4 h after transfer, but no significant difference was found in water contents between cortisol-treated and control groups following transfer. These results suggest that cortisol is involved in the regulation of drinking activity in developing tilapia larvae.     

Ultrastructure of the basement membrane and its precursor in developing rat submandibular gland as shown by alcian blue staining

Summary The ultrastructure of the epithelial basement membrane and membrane precursor was studied in rat submandibular rudiment and a model system of the reconstructed basement membrane, by transmission electron microscopy following alcian blue staining. Directly beneath the epithelial plasma membrane, a meshwork layer was found to consist of anastomosing thin fibers arranged as a three-dimensional meshwork (100–400 nm in thickness). Straight strands (5–10 nm in diameter) could sometimes be seen to pass through the meshwork. Adjacent to this layer, a coarse network composed of threads (20–40 nm in diameter) connected the meshwork layer with collagen fibers of the underlying connective tissue. The earliest precursors recognized in the reconstruction-model system were part of the fine-meshwork structure, and showed this structure to be a fundamental component of the basement membrane.     

Glomerular extracellular matrices and anionic sites in aging ddY mice: a morphometric study

A morphometric study was undertaken to examine age-related changes in glomerular ultrastructure and anionic sites in ddY male mice at various ages. A progressive increase in glomerular extracellular matrices, including thickening of the glomerular basement membrane (GBM), formation of GBM nodules, and mesangial matrix increase, was found to be the primary age-related ultrastructural change in aging mice; there were also electron-dense deposits in mesangial and subepithelial regions. The extent of GBM thickening in mice was less than was reported in rats. Rather, the GBM nodules, which had the same electron density as the lamina densa (LD) and protruded on the subepithelial side of the GBM, were more striking. Quantitative evaluation showed that GBM thickness, number and size of GBM nodules, and the area of the mesangial matrix were significantly correlated with the age of the mice. The distribution of anionic sites in the glomeruli of aging animals was described for the first time. No statistically significant differences were noted between the number of glomerular anionic sites in the different age groups. These results indicate that the increase in glomerular extracellular matrices reported in aged rats was also present in aged mice, although the extent of various changes was different. The results also indicate that this increase in glomerular extracellular matrices with age was not accompanied by significant alteration in glomerular anionic sites.     

Fibronectin in basement membrane of Hertwig's epithelial sheath. Light and electron immunohistochemical localization

The distribution of fibronectin throughout the basement membrane of Hertwig's epithelial sheath was studied using specific antibodies with the immunoperoxidase technique in both light and electron microscopy. Our results demonstrate that, after collagenase digestion in situ, the basement membrane was strongly labelled by antifibronectin antibodies on the lamina lucida, the lamina densa and the lamina (pars) fibroreticularis which contained aperiodic fibrils of 5-10 nm in diameter.     

Ultrastructure of the kidney of a South American caecilian,Typhlonectes compressicaudus (Amphibia,Gymnophiona)

The ultrastructure of the renal corpuscle, the neck segment, the proximal tubule and the intermediate segment of the kidney of a South American caecilian, Typhlonectes compressicaudus (Amphibia, Gymnophiona) was examined by means of transmission electron microscopy (TEM), scanning electron microscopy (SEM) and freeze-fracture technique. The glomerular filter apparatus consists of the podocyte epithelium, a distinct basement membrane, a subendothelial space and the capillary endothelium. Emanating from the podocyte cell body, several long primary processes encircle neighboring capillaries. The short slender foot processes originating from the primary processes interdigitate with those from other primary processes, thereby forming the meandering filtration slit. Thick bundles of microfilaments are found in the primary processes, but absent in the foot processes. The basement membrane consists of a lamina rara externa and a rather thin lamina densa (50 nm thickness). The wide subendothelial space contains abundant microfibrils, a few collagen fibrils and many thin processes of mesangial cells. The endothelium is flat and fenestrated (compared to mammals displaying relatively few fenestrations); some of the fenestrations are bridged by a diaphragm. The glomerular mesangium is made up of the mesangial cells and a prominent mesangial matrix containing microfibrils and collagen fibrils. The cells of the neck and intermediate segments display numerous cilia with their microtubules arranged in the typical 9 + 2 pattern. The basal bodies of the cilia are attached to thick filaments with a clear crossbanding pattern of 65 nm periodicity. The proximal tubule is composed of cells typical for this segment (PT cells) and light cells lacking a brush border (bald-headed cells). The PT cells measure 10-25 micron in height and 15-30 micron in width and do not interdigitate at their lateral borders with each other. Their basolateral cell membrane is amplified by many folds projecting into lateral intercellular spaces and into basal recesses. The brush border is scarce and composed of loosely arranged short microvilli.     

Fibronectin in basement membrane of hertwig's epithelial sheath

Summary The distribution of fibronectin throughout the basement membrane of Hertwig's epithelial sheath was studied using specific antibodies with the immunoperoxidase technique in both light and electron microscopy.—Our results demonstrate that, after collagenase digestion in situ, the basement membrane was strongly labelled by antifibronectin antibodies on the lamina lucida, the lamina densa and the lamina (pars) fibroreticularis which contained aperiodic fibrils of 5–10 nm in diameter.     

Late ultrastructural effects of heavy ions and gamma irradiation in the gastrointestinal tract of the mouse

The irradiated gastrointestinal tract of LAF1 mice was examined one year following a single dose (1000 rad) of either 12C heavy ions or 60Co gamma rays. Qualitative ultrastructural analysis of the gastrointestinal tract of mice exposed to heavy ions or gamma irradiation did not show any discernible differences. In the stomach of irradiated mice, parietal cells contained numerous lysosomes; the gastric chief cells occasionally contained myelin figures. The epithelial cells of the small intestine, especially jejunum and ileum, showed several changes: (1) increased vacuolation was seen both inter- and intra-cellularly, (2) epithelial cell projections penetrated the basal lamina and were in contact with underlying mesenchymal cells, (3) occasional Paneth cells contained intracellular vacuoles consisting of fibrillar and granular material. In the large intestine occasional signs of degeneration were observed. Qualitative analysis of stromal elements of the gut in irradiated mice indicated the presence of damage to capillary endothelial cells, smooth muscle cells and some nerve processes. The amount of basement membrane (BM) around capillaries and small vessels was increased; the same phenomenon was observed to affect the nerve processes, but with less severity. Quantitative analysis of the basement membrane thickness around capillaries in irradiated vs. control mice showed significant differences. Basement membrane thickness around capillaries in the gastric mucosa and duodenum did not differ significantly in any of the treatment groups. In jejunum, the gamma treated animals exhibited significantly higher BM thickness when compared to unirradiated controls. In ileum, only 12C-heavy ion treated animals showed thicker BM when compared to their respective controls. In colon, both 12C- and 60Co-treated animals showed increased BM thickness when compared to controls.