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Featured researches published by Horst Robenek.
Journal of Ultrastructure Research | 1982
Horst Robenek; Wilhelm Jung; Rolf Gebhardt
The present study, employing freeze-fracture and filipin as a cytochemical marker for cholesterol or cholesterol-like components in cell membranes, shows that there is an apparent heterogeneity in the distribution of filipin-cholesterol complexes in the plasma membranes of cultured hepatocytes, thus indicating differences in their cholesterol content. A high number of filipin-cholesterol complexes are uniformly and randomly distributed over the entire plasma membrane of isolated hepatocytes. A similar pattern was observed in the upper and lower plasma membranes of cultured hepatocytes, whereas the plasma membrane areas between adjoining hepatocytes contained far fewer filipin-cholesterol complexes. Tight junction and gap junction formation zones show a nearly complete absence of filipin-cholesterol complexes. It is suggested that changes in membrane fluidity due to the low cholesterol content may play a role in the recognition and adhesion of cultured hepatocytes. The localized deficiency of cholesterol in specialized domains of the plasma membranes recognized as early junction formation zones may facilitate the essential adhesion between cell surfaces and may alter lipid-protein interactions which further affect assembly of tight or gap junctions.
Cell and Tissue Research | 1982
Jürgen Rassat; Horst Robenek; Hermann Themann
SummaryFollowing the administration of colchicine at a dosage (1 mg/mouse) known to cause an antimicrotubular effect, membranes as well as tight and gap junctions of hepatocytes were studied using the thin-sectioning and freeze-fracturing technique.As early as l h after administration of colchicine the intercellular spaces were dilated and vacuoles were visible within the cytoplasm. The bile canaliculi became enlarged, and after lanthanum perfusion the tracer was found in the canalicular lumen, i.e., the tight junctions became permeable to the tracer. These findings correlated with a disorganized arrangement of the tight junctional strands of the zonula occludens. In some regions the strands showed interruptions and frequently ended freely in a diffuse pattern on the plasma membrane. Proliferation of tight junctions could be observed at various locations on the plasma membrane.The gap junctions also exhibited alterations. They showed an irregular outline with outpouchings, in addition to an enlargement in their total area from approximately 0.5 μm2 in controls up to approximately 2 μm2 in treated mice. The surface area occupied by these junctions increased from 4 % (controls) to 20% (treated) of the hepatic plasma membrane. In the cytoplasm of hepatocytes from colchicine-treated mice gap-junctional vesicles were frequently observed.In view of the antimicrotubular effect of colchicine it is tentatively suggested that the intact microtubular system of the cell may play a decisive role in the regular formation of gap and tight junctions, either directly or indirectly via microfilaments.
Journal of Ultrastructure Research | 1980
Horst Robenek; Margarete Döldissen; Hermann Themann
The freeze-fracture technique was used to examine the morphology of the tight junctions between rat hepatocytes after 2, 5, 7, and 10 weeks of chronic administration of N -nitrosomorpholine (NNM). NNM produced an extensive proliferation and rearrangement of the tight junctions in the rat liver. The junctional meshwork became loosened and the strands were oriented in a perpendicular direction with respect to the lumen of the bile canaliculus. The ridges were interrupted and fragmented into many pieces which could be found devoid of tight junctions as loosely branched strands in the lateral surface of the plasmalemma. After prolonged treatment many tight junctions became completely disorganized and occupied broad areas of the plasma membrane along the bile canaliculus. The formation of large discrete macular tight junctional complexes dispersed in the lateral region of the plasmalemma was frequently observed. These findings indicate that the tight junctions of the rat liver appear to be a mobile structure due to the fluid mosaic model of biological membranes which is not restricted to the bile canaliculus and which can be modified in various conditions by different stimuli. The results of this investigation are discussed in relation to those obtained in other systems.
Virchows Archiv B Cell Pathology Including Molecular Pathology | 1981
Horst Robenek; Jürgen Rassat; Hermann Themann
SummaryThe gap and tight junctions in normal human liver and in cholestasis following bile duct obstruction have been studied by freeze-fracture to analyze quantitatively the changes in these membrane specializations.The length of tight junctions/1 μm of bile canaliculus lies within a wide range both in normal (from 4 to 14 μm:mean 7.4 μm) and cholestatic livers (from 2 to 20 μm:mean 6.5 μm; = −12%). The mean width of the zonulae occludentes is 0.32 μm in the normal liver, consisting on the average of 5.4 strands. In cholestatic livers they exhibit a mean width of 0.26 μm (= −19%) and consist of 4.4 strands (= −19%). 0.5% of all measurements at every 0.25 μm of zonula occludens in the cholestatic liver revealed no junctions at all, i.e., an interruption of the permeability barrier.The packing density of gap junctions is 3.6÷100 μm2 of plasmalemma in normal livers and 3.1÷100 μm2 (= −14%) in cholestatic livers. They occupy approximately 1% and 0.3% (= −60%) of the cell surface in normal and cholestatic livers, respectively.The main effects of cholestasis was to a cause a decrease in gap junction area and, to a lesser extent, in the number of gap junctions. Measurements of the center-to-center spacing of gap junctional particles lay in a wide range of values (6–15 nm in the normal liver, 4–23 nm in the cholestatic liver). This distribution probably indicates open and closed configurations of gap junctional particles. Furthermore, this distribution shows that the diminution in gap junctional area in the cholestatic liver may depend partly on either a dispersion or clumping of particles. The structural and quantitative parameters of these liver cell junctions have been correlated with known or proposed physiological functions under normal and pathological conditions.
Virchows Archiv B Cell Pathology Including Molecular Pathology | 1985
Wilhelm Jung; Rolf Gebhardt; Horst Robenek
SummaryIn the present paper rat hepatocytes in primary monolayer culture were used to investigate the adverse effects of chlorpromazine (CPZ) at the cellular level. As revealed by thin sectioning many of the ultrastructural alterations were comparable to those described for the isolated perfused rat liver under the influence of CPZ. Alterations comprised short-term effects, such as dilation of the rough endoplasmic reticulum and the nuclear envelope, and long-term effects including huge accumulations of myeloid bodies within the cytoplasm as well as dilation and diverticulation of bile canaliculi. Freeze-fracturing revealed the dislocation of intramembrane particles in the sinusoidal plasma membrane which could be detected as early as 30 min after exposure to CPZ. As judged from filipin cytochemistry, alterations in the cholesterol content seems to play a minor role in the process of membrane damage except at the sinusoidal surface where a reduction of cholesterol content may contribute to the impairment of membrane functions.It is concluded that CPZ exerts its cholestatic effect primarily by a rapid disturbance of the membrane architecture of the sinusoidal surface and secondarily by other interactions with the bile secretory apparatus.
Journal of Ultrastructure Research | 1983
Horst Robenek; Arpad Hesz; Jürgen Rassat
In this investigation the membrane-perturbing effect of filipin, a polyene antibiotic which reacts specifically with cholesterol or cholesterol-like compounds in cell membranes, has been exploited to study the distribution of coated pits in cultured human skin fibroblasts. The coated pits, showing no filipin-cholesterol complexes, occurred singly or in clusters without apparent localization of either type to a particular region of the fibroblast membrane. Colloidal gold, conjugated to low-density lipoprotein, has proven to be an excellent marker, allowing the localization of low-density lipoprotein receptors on the surface of cultured cells. A pattern similar to that for the coated pits in the plasma membrane fracture faces was observed in the distribution of gold-low-density lipoprotein conjugates in surface replicas, indicating that the low-density lipoprotein receptors are associated with these coated pits. It was shown that there is an apparent heterogeneity in the distribution of low-density lipoprotein receptors, from cell to cell and even among different areas of the same cell membrane. The binding capacity for gold-low-density lipoprotein complexes, as represented by the extent of surface labeling, was directly related to the cell variety within the culture or to the particular experimental procedure. The observation of differences in the distribution of gold-low-density lipoprotein conjugates, even among adjacent coated pits, provides evidence for various stages of activity of the low-density lipoprotein receptors corresponding to incorporation, mobility, and internalization.
Virchows Archiv B Cell Pathology Including Molecular Pathology | 1982
Horst Robenek; Jürgen Rassat; V. Grosser; Hermann Themann
SummaryOver a period of 20 weeks estradiol valerate (1.5 mg/kg body weight/week) was administered subcutaneously to male Wistar rats from which the livers were examined at four week intervals employing a freeze-fracture technique and colloidal lanthanum tracer studies.In connection with intrahepatic cholestasis, distinct alterations in the tight junctions were observed, consisting of disorganization, rarification and proliferation. Disruption of the tight junctions was not seen and colloidal lanthanum did not penetrate into the bile canalicular lumen. Holding the view that the term “leakiness” of tight junctions should be defined with reference to the tracer employed, we conclude that in the liver one tight junctional strand is sufficient to prevent the escape of larger bile constituents such as bile acids and that a back diffusion of bile acids over the tight junctional barrier does not play a role in the pathogenesis of the estrogen-induced cholestasis. Interruptions of tight junctions, as described by other authors, are interpreted as a secondary mechanical effect. On the other hand, we consider an increased permeability of the tight junctions to water and small solute molecules as probable; possibly this increased permeability is brought about by alterations in the microfilaments. A model for the pathogenesis of the estrogen-induced intrahepatic cholestasis is proposed.
Virchows Archiv B Cell Pathology Including Molecular Pathology | 1980
Horst Robenek; Hermann Themann
SummaryThe technique of freeze-fracture electron microscopy was used to examine the alterations in the rat liver after chronic administration of Thioacetamide (TAA) after two and ten weeks. The results of these studies indicate that TAA altered the morphological appearance of the bile canaliculi and induced a progressive disorganization of the zonulae occludentes. During the treatment the bile canaliculi dilatated, showing a loss of microvilli, irregularities, and side branches extending into the hepatocytes. The tight junctions limited to a narrow band of hepatocytic membrane on each side of the bile canaliculus in the normal adult liver proliferated and anastomosed frequently. The junctional meshwork loosened and the predominantly parallel strands positioned themselves perpendicularly to the lumen of the bile canaliculus. Sometimes the complexity of the branching pattern and the number of strands varied extensively at very short intervals along the zonula occludens. Only one or two parallel strands were observed, whereas in other instances up to ten rows of interconnecting strands were present in one fracture face. In the ablumenal side of the tight junction, extended loops and loose ends were frequently observed. Although most strands appeared as continuous ridges in the protoplasmic face, regions in which strands had a fragmented or even a particulate appearance were common. As a consequence, the lateral and the lumenal regions of the plasmalemma become directly continuous.After five weeks of treatment individual strands were occasionally observed on the lateral surface of the plasmalemma away from their usual pericanalicular location. After ten weeks, junctional strands had proliferated in the lateral plasmalemma yielding large arrays with a very complex appearance and complicated configuration. The results of this investigation are discussed in relation to those obtained in other experimental systems.
Pathobiology | 1980
Horst Robenek; Rüdiger Meiss; Hermann Themann; Sabine Himmels
The ultrastructural changes in the liver cells of male Wistar rats induced by oral and subcutaneous application of o-phenylphenol were studied electron microscopically using the thin section and freeze-fracture technique. The rats were given the o-phenylphenol once at a dose of 2,500 mg/kg b.w. and were sacrificed 60 h after treatment. In the orally treated animals, alterations in the nuclei and nucleoli of the hepatocytes were the most prominent alterations. Furthermore, an increase in smooth endoplasmic reticulum, lysosomes and microbodies were found. After the subcutaneous injection of o-phenylphenol enlargements of the bile canaliculi, intercellular space and pathological alterations in the mitochondria were seen. A proliferation of the smooth endoplasmic reticulum, an increase of microbodies and a decrease in the rough endoplasmic reticulum could be found. Both groups showed an increase in small intracellular fat droplets in the hepatocytes. In general, the effects of the subcutaneous application on the liver were more pronounced than the effects of the oral application. Freeze-fracture replicas exhibited a disorganization of the zonulae occludentes and an apparent increase in the number and size of gap junctions. These alterations were interpreted as an attempt of the liver cells to counteract the intracanalicular pressure and to increase the mechanical stability of the liver tissue.
Cell and Tissue Research | 1982
Hartmut Greven; Horst Robenek
SummaryThe apical portion of the uterine lining of the ovoviviparous fire salamander, Salamandra salamandra, was studied by the freeze-fracture technique in conjunction with the polyene antibiotic filipin. Filipin-sterol complexes were found in the luminal plasmalemma and in the membranes limiting the mucous secretory granules typical of this epithelium. In all females, but particularly in non-pregnant females, more or less discrete clusters of filipinsterol complexes were occasionally found overlying heavily affected secretory granules. The findings are discussed with regard to comparable results (Orci et al. 1980) based on the examination of collapsed and stretched urinary bladders of toads.ZusammenfassungDas apikale Plasmalemm und die Membranen der Sekretgrana im Uterusepithel des Feuersalamanders, Salamandra salamandra, sind nach Behandlung mit Filipin im Gefrierbruch unterschiedlich stark markiert. Bei trächtigen und nichtträchtigen Weibchen —hier wahrscheinlich vermehrt —finden sich gelegentlich Felder von Filipin-Sterol Komplexen im apikalen Plasmalemm unmittelbar unter ebenfalls meist stark markierten Sekretgrana. Die Befunde werden im Hinblick auf im wesentlichen vergleichbare Beobachtungen an kollabierten und gedehnten Harnblasen von Kröten (Orci et al. 1980) diskutiert.