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PubMed1982

Intramembrane changes in retinal pigment epithelial cell junctions of the dystrophic rat retina.

A progressive failure in phagocytosis by the retinal pigment epithelium (RPE) occurs in the Royal College of Surgeons rat with inherited retinal degeneration. Another change that can be attributed to a defect in the RPE is a breakdown in the blood-retinal barrier. RPE cell junctions, which form a part of this barrier, become permeable to extracellular tracer during the dystrophic process. We have used the freeze-fracture technique to study the structure of RPE cell junctions in normal and dystrophic retinas. In normal retinas, tight junctions between RPE cells consisted of 8 to 16 anastomosing strands on the cytoplasmic membrane leaflet (P-face) and a complementary pattern of grooves on the external membrane leaflet (E-face). Gap-junctional aggregates of hexagonally packed P-face particles and complementary E-face pits were enclosed within the tight junctional strands. In dystrophic retinas changes were first seen at postnatal day 21. Subtle breaks in P-face tight-junctional strands became more pronounced with time. Eventually the tight junctions appeared to unravel from the gap junctional aggregates, which became isolated and appeared to break off into patches of particle aggregates. The increased density of background particles in the membranes adjacent to disassembling junctions suggested that junctional elements were being removed by dispersal. Endocytosis of junctional elements was observed in both dystrophic and control retinas but may be accelerated in the dystrophic retina. In the late stages of the dystrophy some RPE cell junctions appeared to have proliferated and occupied extensive areas of the RPE membrane.

Animals↗
PubMed1984

Cell relationships during aggregation between preimplantation embryos and teratocarcinoma-derived cells.

Cleavage-stage mouse embryos aggregate and form chimaeric blastocysts with embryonal carcinoma (EC) cells. We used scanning and transmission electron microscopy to study cell relationships during aggregate formation between 8-cell-stage embryos and F9 EC cells. Relations between heterotypic cells were similarly studied in aggregation experiments with embryos and teratocarcinoma-derived visceral (PSA5-E) and parietal (PYS-2) endoderm cells and in experiments with EC cells and endoderm cells. The embryos and F9 cells always adhered to each other and rapidly formed compacted aggregates. Numerous microvilli and cell processes, originating from both embryo and EC cells, extended between the two cell types during adhesion and early phases of aggregation. The aggregation process involved spreading of the blastomeres on the EC cells. Frequent adherent junctions and close contacts, including possible focal gap or tight junctions were observed between the embryo and F9 cells after 3 h of culture. Apparent gap or tight junctions were infrequent during the early phases of aggregation but during further culture, extensive typical gap junctions were also seen between embryo and EC cells. The embryos adhered only irregularly and loosely to PSA5-E and PYS-2 cells; this interaction never led to aggregate formation comparable to that seen in the experiments with embryos and EC cells. Close contacts but no gap or tight junctions could be observed between the embryo and endoderm cells. On the other hand, both PSA5-E and PYS-2 cells readily adhered to and aggregated with EC cells. The present results suggest that microvilli and cell processes mediate membrane interactions during adhesion and early phases of aggregation between embryos and EC cells. During aggregation, blastomeres spread over the EC cells, and rapid formation of adherent junctions and close contacts, including possible focal gap or tight junctions is involved during the early phases of this process. After this initial phase, typical gap junctions are also seen between the embryo and EC cells. Interestingly, adhesive properties of embryo and EC cells differ: the former aggregate only with EC cells, whereas the latter do so also with teratocarcinoma-derived visceral and parietal endoderm cells. Mechanisms operating in the morphogenetic movement of cells in this experimental setup may be involved also in the development of the blastocyst in vivo.

Animals↗
PubMed1985

Lanthanum and freeze-fracture studies of retinal pigment epithelial cell junctions in the streptozotocin diabetic rat.

The blood-retinal barrier breaks down early in diabetes and previous morphological studies suggest that the retinal pigment epithelium (RPE) is the site of this defect. In the present study the electron microscope lanthanum nitrate tracer technique has been used to study RPE cell permeability in the streptozotocin diabetic rat retina. The freeze-fracture technique has been used to study RPE cell tight junction structure as permeability increases. In the lanthanum experiments, RPE cell permeability is normal in control rats and in diabetic rats 3 weeks after the injections. After 8 or 16 weeks of diabetes, however, the RPE cell layer no longer forms a barrier to the tracer and electron dense material is present in the subretinal space, in the apical and basal regions of the RPE cell junctions and intracellularly within the RPE. Freeze-fracture studies of tight junctions during this period show (1) an increase in the complexity of the tight junction network due to an increase in anastomoses between the tight junctions; (2) a change in membrane fracturing properties such that the tight junctional intra-membrane particles adhering to the E-face grooves are more numerous than in the control junctions; (3) no change in the number or size of the tight and gap junctional elements. These results suggest that the blood-retinal barrier breakdown in the diabetic RPE is due to alteration of plasma membrane permeability rather than to a loss of tight junctions.

Animals↗
PubMedDate not supplied

Maturation of junctional complexes during embryonic and early postnatal development of inner ear secretory epithelia.

The development and maturation of tight junctions in secretory epithelia of the inner ear, i.e., the stria vascularis in the cochlea and the dark cell epithelium around the vestibular organs, have been analyzed with the freeze-fracture technique. Inner ears were followed from the otocyst stage to the mature stage (mouse; gestational age 20 to 21 days). Before epithelial cells differentiate into specific tissues, tight junctions have a loose network of sealing elements with zero to four sealing strands. During and after cytodifferentiation, the numbers of strands increase both in the marginal cells of the stria vascularis and in the dark cells around the vestibular organs. Large intra- and intercellular variations in the numbers of sealing strands of the tight junction occur prior to birth in both types of secretory epithelia. In the dark cells the mature structure of the tight junction with five to eight sealing elements is reached at birth. Maturation of marginal cell junctions occurs during the first few days postnatally. Thus, the tight junctions are morphologically mature before the development of the high potassium concentration in the endolymph.

Animals↗
PubMed1981

Structure and function of the junctional complement of spontaneous and transplanted murine mammary carcinomas.

In this study the morphology of intercellular junctions in a murine mammary adenocarcinoma and in a solid carcinoma which resulted from continuous transplantations of this spontaneous tumor are described employing the techniques of ultrathin-sectioning after conventional fixation and tannic acid fixation and freeze-fracturing. The acini of the original adenocarcinoma are separated from the intercellular spaces by tight junctions which form a narrow belt-like zonula around the adluminal parts of all epithelial cells. The apical-to-basal width of the tight junctions varies from 0.4 to 0.8 micrometer. Desmosomes and gap junctions are located close to the zonulae occludentes. The size of gap junctions ranges from small spots 0.2 micrometer in diameter to large areas approximately 1.5 micrometer in diameter. In the solid carcinoma these cellular junctions appear without acinar organization randomly between the cells. In addition, special contact zones can be observed. The contact zones seem to be the precursors of a formation plaque for gap or tight junction formation. The sizes of the gap junctions show a wide range of variation, from as small as 5 particles to as large as 0.5 micrometer in diameter. Tight junctions do not form continuous belt-like zonulae indicating that they have lost their significance as a permeability barrier. They are interpreted as mechanical links. In addition to isolated gap junctions and tight junctions there exist gap junctions adjacent to tight junction fibrils suggesting a probable biogenetic relationship of these two structures.

Adenocarcinoma↗
PubMed2007

In utero exposure to tributyltin alters the expression of E-cadherin and localization of claudin-1 in intercellular junctions of the rat ventral prostate.

Tributyltin (TBT) is an environmental contaminant, exhibiting well-established toxicity to reproductive systems in aquatic organisms. Little information exists regarding the effects of TBT on mammalian reproduction. Cellular junctions are crucial for sperm development and maturation. Intercellular tight junctions are formed by transmembrane proteins such as claudins (Cldns), while the formation of tight junctions involves signaling components of adhering junctions, comprised of cadherins. The objectives of this study were to determine the effects of in utero exposure to TBT on the rat ventral prostate. Pregnant Sprague-Dawley rats were given doses of TBT (2.5, 10, or 20 mg/kg) throughout gestation and sacrificed at Day 91. Ventral prostate weights of TBT-treated rats were decreased in all treatment groups. Results of gene expression macro-array analysis indicated that numerous genes related to cellular adhesion and cell polarity were affected. Cldn-1 mRNA levels decreased after exposure to TBT. Cldn-1 was immunolocalized to the apical lateral margins of adjacent prostatic epithelial cells in controls, but was increasingly dispersed along the lateral plasma membrane with increasing TBT dose, suggesting that the targeting of Cldn-1 or its localization to tight junctions was altered as a result of fetal TBT exposure. E-cadherin mRNA levels and immunolocalization were decreased in a dose-dependent manner. These data indicate that in utero TBT exposure results in permanent alterations in ventral prostate and that these are associated with alterations in the expression and distribution of cell adhesion and tight junctional proteins.

Animals↗
PubMed2000

Expression from the human occludin promoter is affected by tumor necrosis factor alpha and interferon gamma.

The 65 kDa protein occludin is a membrane-spanning part of the epithelial tight junction, which is the main barrier of the paracellular pathway. The function of occludin as part of tight junctions is still poorly understood and even less is known about the regulatory mechanisms that influence occludin gene expression. This study aimed to identify the sequences essential in cis for genomic regulation of tight junction formation and to investigate their funcional role in cytokine-dependent tight junction regulation. Using genome walking cloning of occludin-specific human genomic DNA sequences, a 1853 bp DNA fragment containing the transcription start point of occludin cDNA sequences was amplified and sequenced. Subcloning of this fragment in front of the luciferase reporter gene revealed strong expression of enzymatic activity after transfection of the human intestinal cell line HT-29/B6. With subsequent deletions of parts of the promoter fragment, its size was reduced to 280 bp that are necessary and sufficient to mediate promoter activity. Tumor necrosis factor alpha and another cytokine involved in inflammation, interferon gamma, reduced transepithelial resistance in HT-29/B6 cells, which was preceded by a decrease in occludin mRNA expression as revealed by northern blot analysis. Tumor necrosis factor alpha and interferon gamma diminished occludin promoter activity alone and even synergistically, suggesting a genomic regulation of alterations of the paracellular barrier. In conclusion, proinflammatory cytokines such as tumor necrosis factor alpha and interferon gamma can downregulate the expression of the transmembrane tight junction strand protein occludin, paralleling the barrier disturbance detected electrophysiologically. This could be an important mechanism in gastrointestinal diseases accompanied by barrier defects, for example inflammatory bowel diseases.

Base Sequence↗
PubMed1989

Application of a fiber-matrix model to transport in renal tubules.

The effects of tight junction structure on water and solute fluxes across proximal tubular epithelium were examined with fiber-matrix equations previously derived by Curry and Michel (1980. Microvascular Research. 20:96-99). Using plausible estimates of tight junction fiber length and width the model predicts solute (Ps) and water permeability (Lp) coefficients that agree with the measured values. When fiber-matrix and pore models were compared for physiologically relevant ranges of matrix void fraction (80-98%) and pore radii (0-20 A), the fiber-matrix model predicted a 10-fold higher Lp/Ps ratio. Lp/Ps was most sensitive to small changes in tight junction structure when void fractions exceeded 90%. Void fractions of 96.5% and 97.1% predicted previously measured values for Lp and solute permeabilities in rat and rabbit proximal tubules. These values are consistent with void fractions and permeabilities of artificial membranes. The fiber-matrix tight junction model was incorporated into a model of reabsorption from the rat proximal tubule developed by Weinstein (1984). American Journal of Physiology. 247:F848-F862.) A void fraction of 98% predicted the experimental results for isosmotic reabsorption driven by active transport. Changing void fraction over the range of 97-99% produced a 50-75% change in predicted volume reabsorption with active transport. According to the fiber-matrix model: (a) solute permeabilities alone cannot be used to predict Lp, (b) previously measured solute permeabilities in the proximal tubule are compatible with significant water reabsorption through a water-permeable tight junction, and (c) hydraulic and solute permeabilities may be sensitive to small changes in tight junction fiber length and diameter or ionic strength within the tight junction.

Absorption↗
PubMed1984

Intercellular junctions in upper airway submucosal glands of the rat: a tracer and freeze fracture study.

The structure of intercellular tight junctions of rat airway submucosal glands was examined by freeze fracture techniques and their permeability assessed by the use of colloidal lanthanum. The submucosal glands were organized into three distinct regions: a) serous tubules and b) mucous tubules lined, respectively, by serous and mucous cells, and c) ducts lined by cuboidal epithelial cells, containing few secretory granules, and some ciliated cells. The mean number of parallel fibrils constituting the tight junctions between serous cells was 3.6 +/- 0.4, which was significantly smaller than those between any of the other cell types. Colloidal lanthanum permeated the tight junctions between serous cells up to the level of the acinar lumen. There was a progressive increase in the mean number of parallel fibrils of tight junctions between mucous (5.1 +/- 0.6), ductal (5.4 +/- 0.5), and ciliated cells (8.5 +/- 0.7); none of these junctions was permeated by colloidal lanthanum. These results imply that tight junctions between serous cells are more permeable to small water-soluble solutes than those present in the more proximal portions of the gland. Gap junctions were observed between serous cells and between mucous cells, suggesting that these secretory cells may be electotronically and metabolically coupled.

Animals↗
PubMed2004

Connexin 26-mediated gap junctional intercellular communication suppresses paracellular permeability of human intestinal epithelial cell monolayers.

In some cell types, gap junctional intercellular communication (GJIC) is associated with tight junctions. The present study was performed to determine the roles of GJIC in regulation of the barrier function of tight junctions. Caco-2 human colonic cells were used as a monolayer model, and barrier function was monitored by measuring mannitol permeability and transepithelial electrical resistance (TER). The monolayers were chemically disrupted by treatment with oleic acid and taurocholic acid. Western blotting analyses were performed to evaluate the protein levels of connexins, which are components of gap junctional intercellular channels. Cx26 expression was detected in preconfluent Caco-2 cells, and its level increased gradually after the monolayer reached confluency. These results prompted us to examine whether overexpression of Cx26 affects barrier function. Monolayers of Caco-2 cells stably expressing Cx26 showed significantly lower mannitol permeability and higher TER than mock transfectants when the monolayers were chemically disrupted. The levels of claudin-4, an important component of tight junctions, were significantly increased in the stable Cx26 transfectant. These results suggest that Cx26-mediated GJIC may play a crucial role in enhancing the barrier function of Caco-2 cell monolayers.

Caco-2 Cells↗
PubMed2004

Tyrosine phosphatase inhibition induces loss of blood-brain barrier integrity by matrix metalloproteinase-dependent and -independent pathways.

Tight junctions between endothelial cells of brain capillaries form the structural basis of the blood-brain barrier (BBB), which controls the exchange of molecules between blood and CNS. Regulation of cellular barrier permeability is a vital and complex process involving intracellular signalling and rearrangement of tight junction proteins. We have analysed the impact of tyrosine phosphatase inhibition on tight junction proteins and endothelial barrier integrity in a primary cell culture model based on porcine brain capillary endothelial cells (PBCEC) that closely mimics the BBB in vitro. The tyrosine phosphatase inhibitor phenylarsine oxide (PAO) induced increased matrix metalloproteinase (MMP) activity, which was paralleled by severe disruption of cell-cell contacts and proteolysis of the tight junction protein occludin. ZO-1 and claudin-5 were not affected. Under these conditions, the transendothelial electrical resistance (TEER) was markedly reduced. PAO-induced occludin proteolysis could be prevented by different MMP inhibitors. Pervanadate (PV) reduced the TEER similar to PAO, but did not increase MMP activity. Cell-cell contacts of PV-treated cells appeared unaffected, and occludin proteolysis did not occur. Our results suggest that tyrosine phosphatase inhibition can influence barrier properties independent of, but also correlated to MMPs. Evidence is given for a role of MMPs in endothelial tight junction regulation at the BBB in particular and probably at tight junctions (TJs) in general.

Animals↗
PubMed1988

The effect of heat shock on primary cultures of brain capillary endothelium: inhibition of assembly of zonulae occludentes and the synthesis of heat-shock proteins.

Subjecting primary cultures of bovine brain microvessel endothelial cells to thermal stress (heat shock) results in: (1) an inhibition of further tight junction assembly, (2) the disappearance and/or disassembly of tight junctions, (3) a 30-fold increase in the number of plasmic fracture (PF)-face intramembrane particles, and (4) the new and/or enhanced synthesis of at least three heat-shock polypeptides (HSPs) with molecular masses of approximately 100,000, 90,000 and 70,000. Endothelial cells which are heat-shocked and allowed to recover at 37 degrees C exhibit, within the first 2 h, a marked depression in the synthesis of HSPs and the new and/or enhanced synthesis of a 47,000 dalton "recovery" polypeptide. In later periods of recovery (2-4 h), the synthesis of this polypeptide is even more pronounced and is accompanied by the new and/or enhanced synthesis of a polypeptide(s) with a molecular mass of 35 to 37,000. The appearance of these "recovery protein(s)" in the endothelial cells is concomitant with a decrease in the number of PF-face intramembrane particles and the resumption of tight junction assembly. Results of this study suggest that some of the HSPs synthesized by thermally-stressed cultures of brain endothelial cells may activate or be directly involved in a mechanism(s) to ensure survival of these cells by decreasing membrane fluidity and stabilizing the plasma membrane of these cells. Moreover, our results also suggest that the recovery of these cells from the stress of heat shock is accompanied by the synthesis of "recovery" proteins which, in some manner, may be directly involved in, or necessary for, rapidly reversing the membrane-stabilizing effect of heat shock by promoting membrane fluidity and the apparent amplified synthesis and assembly and/or reassembly of tight junctions.

Animals↗
PubMed1982

Intercellular junctions and transfer of small molecules in primary vascular endothelial cultures.

The ultrastructure of gap and tight junctions and the cell-to-cell transfer of small molecules were studied in primary cultures and freshly isolated sheets of endothelial cells from calf aortae and umbilical veins. In thin sections and in freeze-fracture replicas, the gap and tight junctions in the freshly isolated cells from both sources appeared similar to those found in the intimal endothelium. Most of the interfaces in replicas had complex arrays of multiple gap junctions either intercalated within tight junction networks or interconnected by linear particle strands. The particle density in the center of most gap junctions was noticeably reduced. In confluent monolayers, after 3-5 days in culture, gap and tight junctions were present, although reduced in complexity and apparent extent. Despite the relative simplicity of the junctions, the cell-to-cell transfer of potential changes, dye (Lucifer Yellow CH), and nucleotides was readily detectable in cultures of both endothelial cell types. The extent and rapidity of dye transfer in culture was only slightly less than that in sheets of freshly isolated cells, perhaps reflecting a reduced gap junctional area combined with an increase in cell size in vitro.

Animals↗
PubMed1984

Fracture faces of cell junctions in cerebral endothelium during normal and hyperosmotic conditions.

Infusion of hyperosmolar solutions into the internal carotid artery causes opening of blood-brain barrier to macromolecules. Ultrastructural tracer studies indicate that extravasation of macromolecules takes place primarily in segments of large penetrating cortical blood vessels. The purpose of the present study was to examine fracture faces of cerebral endothelium in normal and hyperosmolar mannitol-treated rat brains in an attempt to elucidate: (a) the organization of endothelial cell junctions in various segments of the cerebral vascular bed and (b) the structural basis of blood-brain barrier opening in hyperosmotic conditions. We found that in control rat brains: (a) capillary endothelium is provided with complex networks of continuous multistranded tight junctions; (b) continuous capillary-type tight junctions extend, although in a simpler beltlike fashion, into the endothelium of postcapillary venules; (c) the endothelium of collecting veins possess widely discontinuous single- or double-stranded tight junctions associated with gap junctions; (d) arteries have endothelial tight junctions containing focal discontinuities associated with gap junctions. In hyperosmolar mannitol-treated rat brains, there appeared focal distensions of compartments but no definitive structural discontinuities in capillary-type tight junctions. Our data suggest that the blood-brain barrier consists of: (a) an extended tight region (comprising both capillaries and postcapillary venules) and (b) focal, potentially leaky regions (restricted to collecting veins and possibly arteries). Our studies furnished no direct evidence for the structural basis of blood-brain barrier opening in hyperosmolar mannitol-treated rat brains.

Animals↗
PubMed1990

[An ultrastructural investigation of changes in hypoxia in the subependymal layer of the newborn rabbit].

Fetal and neonatal hypoxia is suggested as a noticeable risk factor related to the occurrence of subependymal hemorrhages. To investigate the cause of newborn subependymal hemorrhages, a 0 day old newborn rabbit was exposed to either 5 or 10 minutes hypoxia by N2 box. After exposure, the brain was observed by optical and electric microscope using horseradish peroxidase (HRP) as the protein tracer to study the change in the capillary permeability. The result obtained were as follows: 1) In the newborn rabbit blood gas analysis, PO2 (mmHg) decreased more in the 5 and 10 min hypoxia groups than the control group which used the N2 box. pH and BE changes were similar to PO2 (mmHg). On the other hand, PCO2 (mmHg) increased proportionately. These results indicated that this hypoxic method changed blood gas and caused acidosis. 2) In the optical microscopic examination, the 0 day old newborn rabbit brain was found to have a thin subependymal layer. We could not find obvious subependymal hemorrhage using optical microscope. 3) In the electric microscopic examination, HRP was found in the cavity of the brain capillary at the subependymal layer and was slightly incorporated into the pinocytic vesicle of the lumen on the control. Since the tight junction filled the roll out, HRP was not found outside the capillary. 4) The 5 min hypoxia group caused astrocyte foot swelling, edema and slight opening of the tight opening of the tight junction. HRP was passed through the tight junction, but not outside the capillary. 5) The 10 min hypoxia group caused destruction of astrocyte and edema around the capillary. The tight junction was more opened than in the 5 min hypoxia group and HRP leaked out of the capillary through the tight junction. From the findings mentioned above, it is suggested that our hypoxic model increased permeability of the capillary in subependymal layers and that this change might be the first ultrastructural change before the onset of subependymal hemorrhages.

Animals↗
PubMed2005

Claudin-1 is a strong prognostic indicator in stage II colonic cancer: a tissue microarray study.

Tight junction associated proteins are key molecular components governing cellular adhesion, polarity and glandular differentiation. Tight junction proteins also play critical roles in cellular proliferation and neoplastic pathways via their functions as couplers of the extracellular milieu to intracellular signaling pathways and the cytoskeleton. Neoplastic cells frequently exhibit structural and functional deficiencies in the tight junction. The purpose of this study was to determine the pattern of expression and prognostic value of four tight junction associated proteins, claudin-1, claudin-4, occludin and ZO-1 in a cohort of TNM stage II colon cancer using tissue microarray technology. In this study, we retrospectively analyzed, resected and otherwise untreated paraffin embedded specimens from 129 consecutive patients with TNM stage II colonic carcinomas for claudin-1, claudin-4, occludin and ZO-1 protein expression by immunohistochemistry. Seventy-five, 58, 56 and 44% of the tumors exhibited normal to elevated expression levels (+2 and +3 immunopositivity) of claudin-1, claudin-4, occludin and ZO-1 respectively. Low expression levels of claudin-1 and ZO-1 were directly associated with higher tumor grade (P=0.05 and 0.03 respectively). Multivariate analysis indicated that lymphovascular invasion (P=0.01) and low levels of claudin-1 (P=0.0001) expression were independent predictors of recurrence and that reduced claudin-1 expression (P=0.0001) was associated with poor survival. This study is the first to comprehensively examine the expression of several tight junction associated proteins in colonic neoplasms and to correlate their expression with disease progression. Loss of claudin-1 expression proved to be a strong predictor of disease recurrence and poor patient survival in stage II colon cancer.

Adult↗
PubMed1983

Ionic lanthanum passage across cerebral endothelium exposed to hyperosmotic arabinose.

Hyperosmotic media infused into the cerebral circulation open the blood-brain barrier to protein and colloid. The mechanism whereby such substances cross the affected vessels is still disputed. We describe here the transendothelial route taken by ionic lanthanum (La3+), a small electron-dense tracer which, unlike colloidal lanthanum, can be administered to the living animal. In adult rats, 2.9 ml of hyperosmotic (1.4 M) arabinose was infused into the internal carotid artery as a 30-s bolus, followed by 5 mM LaCl3. To find the extravasated La3+, which is invisible by light microscopy, horseradish peroxidase (HRP) was injected simultaneously into the femoral vein. The hyperosmotic treatment resulted in exudation of both HRP and La3+ primarily around cerebral arterioles. The La3+ crossed arterioles through successive tight junctions between endothelial cells. Although the tight junctions were not discernibly opened, they must have become permeable because the extracellular pools between successive tight junctions were penetrated by the La3+. These pools are normally inaccessible to La3+. Luminal and abluminal pits and cytoplasmic vesicles, some of them containing La3+, formed intraendothelial clusters. Their role, if any, in the transfer of ion remains uncertain.

Animals↗
PubMed1982

Freeze-fracture elucidation of hepatocyte membrane alterations following beta-pyridylcarbinol application.

The effect of beta-pyridylcarbinol on mice was investigated in long-term studies (21 days, daily dose of 0.1 mg) making use of thin-sectioning and freeze-fracture techniques. Thin sectioning merely revealed only subtle pathological changes including dilatation of the intercellular space and foundation of hepatocytic vacuoles. Only upon investigation using freeze-fracture was it possible to demonstrate more profound alterations, primarily involving the cell contacts, i.e. the gap and tight junctions. The organized structure of the tight junctions appeared dissipated. The meshwork was disorganized in some cases and reduced in size. Large, proliferative tight junctional maculae were frequently observed on the plasmalemma. In some cases the gap junctions had lost their round to oval shape and had increased in size to form large plaques protruding at numerous points. Moreover, even the bile canaliculi, which presented no pathological alterations on ultrathin section, exhibited damage as seen in freeze-fracture preparations. The lumen was uneven in contour and processes were visible extending into the adjacent cytoplasm. The present investigation shows that hepatocytic damage primarily affecting the membranes was first made evident using freeze-fracture technique. The issue is addressed as to the extent to which freeze-fracture technique should be employed in routine investigations of pharmaceuticals.

Animals↗