Sinusoidal Face Research Articles

Liver in guinea pig: Scanning and transmission electron microscopic studies

The liver performs many functions, which support metabolism, immunity, digestion, detoxification, vitamin storage, and so on. The liver is the storage organ for fat-soluble vitamins, iron and copper. Six adult healthy guinea pigs of 16-32 weeks of age (Irrespective of sex) were procured from the Department of Laboratory Animal Medicine, TANUVAS as per ethical committee approval. Animals were dissected as per CPCSEA norms and liver pieces were utilized for scanning electron microscopy (SEM) and transmission electron microscopy (TEM) study. The study was performed to document the ultrastructural details of liver of guinea pigs by SEM and TEM. By SEM, the liver parenchyma appeared as lobular with anastomosed one cell thick hepatic cell plates, which appeared as continuous sheets with small to large holes representing sinusoids. The sinusoidal lumen was lined by two types of cells namely endothelial cells and Kupffer cells. Endothelial cells had numerous large and small cytoplasmic fenestrations. Presence of Kupffer cells along with Ito cells were observed in the sinusoids. By TEM, hepatocytes had angular shape with sinusoidal face and bile canalicular face. Hepatocytes had oval nucleus with peripheral heterochromatin and central electron lucent granules and a prominent nucleolus. Bile canaliculi were formed by two adjacent hepatocytes. Fenestrated endothelial cells with flat and elongated shape were found to be in the formation of hepatic sinusoidal wall. A narrow space of Disse was found between the sinusoidal endothelium and hepatocytes. Kupffer cells and pit cells were found within the sinusoidal lumen. Ito cells were found in the space of Disse. RESEARCH HIGHLIGHTS: By scanning electron microscopy, the liver parenchyma appeared as lobular with anastomosed one cell thick hepatic cell plates, which appeared as continuous sheets with small to large holes representing sinusoids. By transmission electron microscopy, hepatocytes had angular shape with sinusoidal face and bile canalicular face. Hepatocytes had oval nucleus with peripheral heterochromatin and central electron lucent granules and a prominent nucleolus.

Adherence Patterns of Histoplasma capsulatum Yeasts to Bat Tissue Sections

The adherence of Histoplasma capsulatum yeasts to lung, spleen, liver, gut, and trachea cryosections of Artibeus hirsutus bats and inbred BALB/c mice (control) was studied after in vitro yeast-tissue incubations. Candida albicans yeasts were used as a well-known adherent fungal model in the mice host, and latex beads were used as a negative adherence control. Adhered yeast cells were identified by using crystal violet staining and the immunoperoxidase method with specific antibodies. H. capsulatum yeasts adhered to all tissues tested, mainly in the lung. Moreover, H. capsulatum yeasts adhered preferentially to white and red spleen pulp, in contrast to the dispersed distribution of C. albicans yeasts. H. capsulatum yeasts were mostly found on the sinusoidal face of hepatocytes. In general, the gut showed a higher number of adhered H. capsulatum yeasts than the trachea in both bats and mice. H. capsulatum and C. albicans yeasts developed high selectivity for the lamina propria of the gut. In addition, H. capsulatum yeasts interacted better with the lamina propria and adventitia of the trachea. The number of H. capsulatum yeast cells that adhered to each tissue section type was always greater than the corresponding number of C. albicans yeast cells, and latex beads never adhered to the tissue sections. Controls with anti-H. capsulatum and normal rabbit sera showed a significant blockage of H. capsulatum yeast adherence to lung tissue. This is the first study describing the patterns of H. capsulatum yeast adherence to different bat and mouse tissues.

H2, the Minor Subunit of the Human Asialoglycoprotein Receptor, Trafficks Intracellularly and Forms Homo-oligomers, but Does Not Bind Asialo-orosomucoid

The functional human hepatic asialoglycoprotein receptor (ASGP-R) is a hetero-oligomer composed of two subunits, designated H1 and H2, which are highly homologous. Despite their extensive homology, the major H1 subunit is stably expressed by itself, whereas in the absence of H1 most of the H2 subunits are degraded in the ER. In this study, we were able to investigate the capability of the minor ASGP-R subunit, H2, to function independently of H1, because it was apparently stabilized by fusing its NH(2) terminus with an epitope tag. We could thus create stable cell lines in hepatoma-derived SK-Hep-1 cells that expressed the H2 subunit alone. H2 was expressed on the cell surface and was internalized, predominantly through the clathrin-coated pit pathway. Since the internal pool of H2 was also able to traffick to the cell surface, we conclude that H2 recycles between the surface and intracellular compartments, similar to the constitutive recycling of hetero-oligomeric ASGP-R complexes. However, the rate of H2 recycling and internalization was approximately 25-33% that of H1. Similar to H1, the H2 polypeptides were also able to self-associate to form homo-oligomers, including trimers and tetramers. However, unlike H1, which can bind the ligand asialo-orosomucoid (ASOR) when overexpressed in COS-7 cells, H2 failed to bind or endocytose ASOR. In summary, the H2 subunit of the human ASGP-R contains functional, although weak, signal(s) for endocytosis and recycling and has the ability to oligomerize. H2 homo-oligomers, however, do not create binding sites for desialylated glycoproteins, such as ASOR, that contain tri- and tetra-antennary N-linked oligosaccharides. Nonetheless, these results raise the intriguing possibility that naturally occurring H2 homo-oligomers may exist in human hepatocytes and have an as yet undiscovered function.

Analysis of gap junction formation in rat hepatocytes by intravenous injection of an anti-connexin32 monoclonal antibody (HAM8).

HAM8 monoclonal antibody was used to examine the mechanism of connexin32 (Cx32) formation in the rat model in vivo by immunofluorescence and immunoelectron microscopy. After a single intravenous injection of HAM8 IgG, a number of HAM8 signals were observed at the sinusoidal face, between adjacent hepatocytes as well as in the cytoplasm stained with only 2nd fluorescent antibody. Moreover, the in vivo localization of the HAM8 antigen appeared to change with time. At 5 min after the antibody injection, Cx32 signals from liver sections were clearly detected at the sinusoidal face. Fifteen minutes later, numerous linear and dotted fluorescent signals were observed between hepatocytes; in addition, much punctate staining was found at the sinusoidal face and in hepatocytes. These findings were identified by immunoelectron microscopy. Interestingly, 1 hr later, much punctate staining considered to be similar to those seen in the normal rat liver tissues was observed between adjacent hepatocytes, suggesting that a great deal of Cx32 combined with HAM8 have been assembled into identifiable gap junction plaques. Five hours later, intercellular and intracellular Cx32 signals were infrequently detected. When staining was performed with HAM8 and 2nd antibody, however, numerous Cx32 signals were again observed between neighboring hepatocytes, as punctate staining appearing in a pattern approximately the same as that seen in the normal liver tissue. Based on these results, we assumed that a precursor gap was present during Cx32 formation, and discussed the pathways of Cx32 formation and the degradation of Cx32 as well as that of HAM8.

Three-dimensional thermoelasticity problems for nonuniformly heated laminar transversally isotropic plates

Three-dimensional heat-conduction and thermoelasticity boundary-value problems are considered for laminar transversally isotropic rectilinear plates under the action of a nonuniform temperature field. Both ideal and nonideal thermal and mechanical contact conditions at the interfaces are fulfilled. Cases where the roots of the basic characteristic equation are different and include duplicate values are discussed for transversally isotropic bodies (rectangular anisotropy). In the numerical calculation of thermoelastic deformation for a two-layer plate, a sinusoidal face temperature is specified. Characteristic thermomechanical effects are discovered.

Immunoelectron Microscopic Localization of Cell Surface Antigens on Rat Hepatocytes Detected with Monoclonal Antibodies (HAM2 and HAM4).

The localization of surface antigens and the binding activity of two monoclonal antibodies, HAM2 and HAM4, which recognize the rat major histocompatibility complex (MHC) antigen class I and the rat hepato-renal antigen respectively, on dissociated (free) hepatocytes was examined by light (LM) and electron microscopy (EM), and by radioimmunoassay (RIA). Fixed hepatocytes, fixed before dissociation, and fresh hepatocytes, dissociated by collagenase, were treated by direct staining with HAM2- or HAM4-immunogold complexes (HAM2-gold and HAM4-gold). Some of the directly stained hepatocytes were further mixed with antimouse IgG-gold complex (IgG-gold) to supplement the direct staining. The polarity of the sinusoidal and contiguous faces and the bile canaliculus, i.e. the in situ morphology, was well preserved in the fixed hepatocytes, while the fresh cells had lost the polarity and were round. On the fixed hepatocytes HAM2-gold particles were distributed predominantly on the sinusoidal face, while HAM4-gold particles were localized on both the bile canalicular and sinusoidal faces. No different antigen distribution on the fresh cells was detected with the two antibodies. Supplementation by IgG-gold was noticeable in most cases. The extent of binding activity in both the immunogold and RIA experiments was lower in the fixed cells than in fresh cells. These results suggest that HAM2 and HAM4 are useful monoclonal antibodies for detecting the localization of the MHC class I antigen and the hepato-renal antigen on the hepatocytes, respectively.

Microcystin-LR-induced Ultrastructural Changes in Rats

The ultrastructure of hepatic, pulmonary, and renal lesions was evaluated in rats injected intraperitoneally with a lethal dose of microcystin-LR (MCLR, 160 micrograms/kg), a cyclic heptapeptide hepatotoxin produced by the blue-green algae, Microcystis aeruginosa. Hepatic lesions were first seen at 10 minutes post-dosing and consisted of mild widening of hepatocyte intercellular spaces centrilobularly. At 20 minutes post-dosing, hepatocyte plasma membrane alterations were more pronounced, consisting of plasma membrane invagination with formation of variably sized and shaped intracytoplasmic vacuoles, loss of microvilli along the sinusoidal face, and widespread, pronounced hepatocyte separation. By 30 minutes, the space of Disse was markedly widened. At 60 minutes post-dosing, centrilobular areas contained necrotic cells and apparently intact, isolated, organelles intermingled with erythrocytes and platelets. In less severely affected regions there was prominent hepatocyte rounding, and erythrocytes and platelets were present in the widened space of Disse. Large amounts of hepatocellular debris and intact hepatocytes were present in the pulmonary vasculature, while smaller amounts of debris were also seen in the glomerular and peritubular capillaries of the renal cortex. This study shows that initial lesions are confined to shape changes in the plasma membrane of hepatocytes. These changes are consistent with the hypothesis that microcystin-LR induces alterations in the hepatocyte cytoskeleton. Later changes consist of hepatocyte disassociation and necrosis, as well as endothelial damage, which allow release of hepatocytes and debris into the circulation with microembolism in lungs and kidneys.

A characterization of γ-glutamyltranspeptidase in normal, perinatal, premalignant and malignant rat liver

1. 1. γ-Glutamyltranspeptidase displays the following order of activity in tissues of the Fischer 344 rat: kidney ⪢ small intestine ⪢ cerebral cortex = testis > lung ⪢ liver = heart. 2. 2. The activity of the hepatic enzyme in rats is: 4-fold higher in females than males; 4-fold higher in male Wistar, Sprague-Dawley and Zucker rats than male Fischer 344 rats; increased 10-fold in very old vs young male Fischer 344. 3. 3. The hepatic enzyme displays significant species variation: the mouse and rat liver enzymes are similar and low in activity, while duck, dog, pig and beef enzymes are 7, 13, 86 and 92-fold higher, respectively, in activity than the male Fischer rat liver enzyme. 4. 4. A liver plasma membrane isolation procedure has been devised which selects for the sinusoidal face of the liver parenchymal cell as assessed by marker enzyme analysis: for these plasma membranes the purification of γ-glutamyltranspeptidase is 21.5 and the recovery is 42% and the recovery indicating that this is the cellular and subcellular locus of the enzyme in rat liver. 5. 5. The characteristics of the liver plasma membrane from female rats are: pH optimum of 8.0; classical Michaelis-Menten kinetics; K m of 1.43 mM and V max of 33.3 nmol·mg −1·min −1. 6. 6. In Fischer 344 rats, γ-glutamyltranspeptidase activities are elevated over adult levels in perinatal liver: in fetal liver homogenates and plasma membranes the activities are increased 179 and 109-fold, respectively. The activity peaks just after birth and declines rapidly over the first 15 postnatal days. 7. 7. The activity of the liver enzyme in the male Fischer 344 rat exhibits a progressive increase throughout diethylnitrosamine-induced hepatocarcinogenesis: it is increased 7.8-fold in homogenates and 5.4-fold in plasma membranes at the early premalignant stage; 74-fold in homogenates and 31-fold in plasma membranes at the later hyperplastic nodular premalignant stage; and 174-fold in homogenates and 61-fold in plasma membranes at the hepatoma stage. The gradual drop in purification during hepatocarcinogenesis is associated with the appearance of the enzyme in the blood.

Characterization of plasma-membrane glycoproteins from functional domains of the rat hepatocyte.

Plasma-membrane glycoproteins from the three different functional domains of the rat hepatocyte were radioactively labelled by oxidation with NaIO4, followed by reduction with NaB3H4. Analysis of the radioactively labelled glycoproteins by polyacrylamide-gel electrophoresis revealed the presence of at least 12 major sialoglycoproteins in each different region of the hepatocyte surface. The Mr-110 000 component was homogeneously distributed over the plasma membrane, whereas the Mr-90 000 polypeptide was only located at the sinusoidal face. These radiolabelled glycoproteins were solubilized in 1% Triton X-100, and the soluble fraction was subjected to affinity chromatography on Sepharose-conjugated wheat-germ agglutinin (WGA). The labelled glycoproteins were poorly bound to WGA. Membrane glycoproteins were also labelled by the galactose oxidase/NaB3H4 method. The results show that the polypeptides with apparent Mr 170 000 from the sinusoidal, 230 000 from the canalicular and 170 000 from the lateral membranes were specifically labelled. When the membranes were treated with neuraminidase and galactose oxidase/NaB3H4, the electrophoretic patterns showed changes in the apparent Mr values of the glycoproteins, owing to loss of sialic acid, and a clear increase in labelling in the sinusoidal and canalicular membranes compared with the lateral membranes. When these labelled membranes were solubilized in 1% Triton X-100 and subjected to affinity chromatography on Sepharose-conjugated Ricinus communis agglutinin and/or Lens culinaris agglutinin, the results showed that the former columns efficiently bound the radiolabelled glycoproteins, whereas the latter columns bound poorly. The results show that there is a differential distribution of glycoproteins along the hepatocyte's surface.

Centrilobular liver necrosis induced by hypoxia in chronic ethanol-fed rats.

Rats fed ethanol from 21 to 130 days were subjected to one or more episodes of hypoxia (6% O2) in order to determine if ethanol predisposed to centrilobular liver necrosis induced by hypoxia. Pair-fed control rats were fed the diet regimen in parallel with the ethanol-fed rats through an indwelling gastric cannula. The diet and ethanol were fed continuously 24 hr per day so as to maintain high blood alcohol levels in the ethanol-fed rats. Serum enzyme levels, SGOT and SGPT were measured before and after the hypoxic episodes as an indicator of centrilobular necrosis. Animal livers were studied for centrilobular necrosis by light and electron microscopy. Necrosis was documented to be present when flocculent densities were found in hepatocytic mitochondria or the plasma membrane permitted lanthanum entrance into the cell. The results showed that ethanol feeding to maintain high blood alcohol levels did increase the propensity of the liver to undergo centrilobular necrosis when the rats were subjected to hypoxia (1 hr 45 min to 5 hr 30 min). Centrilobular necrosis was observed in the ethanol-fed rats only. Serum enzyme levels (SGPT and SGOT) rose to very high levels in these rats when they were permitted to die of hypoxia. Serum sediment from the ethanol-fed rats contained numerous cell fragments and free organelles. Since the plasma membranes were missing along the sinusoidal face of centrilobular hepatocytes and microbodies were present, it was concluded that the cell fragments in the blood had originated from necrotic hepatocytes.

Abnormal secretion of proteins into bile from colchicine-treated isolated perfused rat livers.

The microtubule poison, colchicine, caused an abnormal output of a variety of proteins into rat bile. After 3 h of exposure to the drug, livers were isolated and perfused with media of defined protein composition. There was no essential change in permeability of the hepatobiliary system to proteins (e.g. bovine serum albumin) entering bile from the perfusion fluid. The rat (serum) albumin and fibrinogen that were secreted into bile from colchicine-treated livers were probably derived from the hepatocytes. Disruption of the microtubular system reduces the secretion of proteins at the sinusoidal face of the hepatocyte and results in an accumulation of secretory vesicles in the cytoplasm. It is suggested that under these conditions some of the vesicles discharge their contents into the bile canaliculus.

Distribution of an asialoglycoprotein receptor on rat hepatocyte cell surface.

Direct ferritin immunoelectron microscopy was applied to visualize the distribution of the hepatocyte cell surface of the asialoglycoprotein receptor which is responsible for the rapid clearance of serum glycoproteins and lysosomal catabolism. For this purpose, rabbit antibody against the purified hepatic binding protein specific for asialoglycoproteins was prepared and coupled to ferritin by glutaraldehyde. The specific antibody conjugates were incubated with the hepatocytes, which were isolated from rat liver homogenate after fixation by glutaraldehyde perfusion. These cells preserved well the original polygonal shape and polarity, and it was easy to identify the sinusoidal, lateral, and bile canalicular faces. The surface density of the ferritin particles bound to the sinusoidal face was about four times higher than that of particles bound to the lateral face, while the bile canalicular face was hardly labeled and almost at the control level. Using the surface area of hepatocyte measured by morphometrical analyses, it was estimated that approximately 90% of bound ferritin particles were at the sinusoidal face, approximately 10% at the lateral face, and approximately 1% at the bile canalicular face. Nonhepatic cells such as endothelial and Kupffer cells had no receptor specific for asialoglycoproteins.

Localization of paternal H-2K antigens on murine trophoblast cells in vivo.

We have previously shown the presence of H-2K and D antigens of both parental haplotypes on dispersed murine trophoblast cells. The question still remained whether such antigens are sequestered away from the sinusoidal face of these cells making them inert as allografts. The in vivo expression of H-2 antigens on these cells was therefore examined radioautographically after perfusion of 125I-labeled monoclonal and anti-H-2Kk (anti-paternal) antibody directly into individual placental branches of the uterine artery suppling 15-d-old (C57BL/6J female) X CBA/J male) placentae. Syngeneic C57BL/6J placentae served as negative controls. A radioautographic examination of 0.5-micrometer-thick sections revealed specific labeling of labyrinthine trophoblasts lining the sinusoids of allogeneic placentae. Most of this labeling was localized to the sinusoidal face of the cells as opposed to a weak labeling of the intracellular aspect. Spongiotrophoblasts and trophoblast giant cells did not label, but specific labeling of fetal capillary endothelium and some macrophages was also noted.

Distribution of secretory component in hepatocytes and its mode of transfer into bile.

Immunoglobin A in bile and other external secretions is mostly bound to a glycoprotein known as secretory component. This glycoprotein is not synthesized by the same cells as immunoglobulin A and is not found in blood. We now report the mechanism by which secretory component reaches the bile and describe its function in immunoglobulin A transport across the hepatocyte. Fractionation of rat liver homogenates by zonal centrifugation was followed by measurement of the amounts of secretory component in the various fractions by rocket immunoelectrophoresis. Secretory component was found in two fractions. One of these was identified as containing Golgi vesicles from its isopycnic density and appearance in the electron microscope; the other contained principally fragments of the plasma membrane of the sinusoidal face of the hepatocyte, as shown by its particle size and content of marker enzymes. Only the latter fraction bound (125)I-labelled immunoglobulin A added in vitro. At 5min after intravenous injection of [(14)C]fucose, the secretory component in the Golgi fraction was labelled, but not that in the plasma membrane. The secretory component in the sinusoidal plasma membrane did, however, become labelled before the first labelled secretory component appeared in bile, about 30min after injection. We suggest that fucose is added to the newly synthesized secretory component in the Golgi apparatus. The secretory component then passes, with the other newly secreted glycoproteins, to the sinusoidal plasma membrane. There it remains bound but exposed to the blood and able to bind any polymeric immunoglobulin A present in serum. The secretory component then moves across the hepatocyte to the bile-canalicular face in association with the endocytic-shuttle vesicles which carry immunoglobulin A. Hence there is a lag before newly synthesized secretory component appears in bile.

Scanning electron microscopy of normal rat liver: the surface structure of its cells and tissue components.

Scanning electron microscopy (SEM) allows the surface ultrastructure of intrahepatic cells and other tissue components of liver to be delineated. Excellent depth of focus of the SEM makes it possible to visualize surfaces of intact cells in their native configurations. This report details the surface characteristics and inter-relationships of hepatocytes and hepatic plates, sinusoidal endothelial cells and sinusoids, presumed Kupffer cells, vessels, bile ducts, connective tissue, and the capsule of rat liver. Hepatocytes present three structurally distinctive faces--the intercellular face containing flat surfaces and bile canaliculus, the sinusoidal face, and the connective tissue face which abuts portal tracts and hepatic veins. Sinusoidal endothelium is penetrated by large (1 to 3 mum) and small (0.1 mum) fenestrae, the latter occurring in clusters of up to 50. The width of bile canaliculi and distribution of large fenestrae vary proximodistally along hepatic plate or sinusoid. The cells of portal bile ductules contain microvilli located in linear rows and sparse cilia. Endothelium of hepatic artery and of portal vein is sparsely fenestrated.