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PubMed1987

Scrape-loading and dye transfer. A rapid and simple technique to study gap junctional intercellular communication.

Gap junction-mediated intercellular communication has been recognized in cells from different tissues of various organisms and has been implicated in a variety of cellular functions and dysfunctions. Here we describe a new, direct and rapid technique with which to study this cellular phenomenon. It employs scrape-loading to introduce a low molecular weight (MW) fluorescent dye, Lucifer yellow CH (MW 457.2) into cells in culture and allows the monitoring of its transfer into contiguous cells. In communication-competent cells the dye transmission occurred within minutes after loading. The involvement of membrane junctions in Lucifer yellow transfer was verified by the concurrent loading of a high MW marker dye conjugate, rhodamine dextran (MW 10,000). Once introduced intracellularly the rhodamine dextran is unable to cross the relatively narrow membrane junctions. Chemicals of variable potency known to block junctional communication were tested in Chinese hamster V79 cells and other mammalian cells. The results showed effective blockage of the dye transfer at non-cytotoxic doses. This new technique can be applied to a wide variety of mammalian (including human) cells. In addition, it has the potential to be utilized as a rapid screening assay to detect chemicals that can modulate intercellular communication and to study their mechanism of action.

Animals↗

PubMed1985

Effects of 12-O-tetradecanoylphorbol-13-acetate and retinoids on intercellular junctional communication measured with a citrulline incorporation assay.

Inhibition of intercellular junctional communication by 12-O-tetradecanoylphorbol-13-acetate (TPA) and retinoids was investigated using a citrulline incorporation assay. This new assay uses metabolic co-operation between argininosuccinate lyase-deficient human fibroblasts and arginosuccinate synthetase-deficient cells as a measure of junctional communication. Short-term exposure to TPA resulted in virtually complete inhibition of metabolic co-operation when V79 cells were used as the synthetase-deficient type. When synthetase-deficient human fibroblasts were used, inhibition by TPA was only partial. Exposure to high concentrations of TPA for prolonged periods resulted in partial reversal of communication inhibition and a refractory state in which cells were unresponsive to TPA. Retinoic acid and other retinoids also inhibited metabolic co-operation, but did not cause desensitisation of the type seen with TPA after prolonged exposure. Cultures which had been made refractory to TPA remained sensitive to inhibition by retinoic acid and 1,1,1-trichloro-2,2-bis(p-chlorophenyl)ethane, indicating that these latter compounds inhibit junctional communication by a mechanism different from TPA. Simultaneous exposure of cultures to TPA and retinoic acid showed that the inhibitory effects on metabolic co-operation of these compounds were additive. Fluocinolone acetonide did not antagonise the effect of TPA. These results suggest that retinoic acid and fluocinolone acetonide exert their anti-tumor-promoting action by mechanisms which are not mediated by intercellular junctional communication.

Animals↗

PubMed1986

In vitro early changes in intercellular junctions by treatment with a chemical carcinogen.

To examine early intercellular junction changes caused by treatment with 9,10-dimethyl-1,2-benzanthracene (DMBA), rat lingual epithelium was cultivated in isolation and observed by electrophysiological, freeze-fracture and whole-mount electron microscopy. Electrophysiological measurements showed a transient decrease in membrane potential of -10.2 mV 6 h after the treatment. It returned to almost the same level as that of the control group 1 day later. Six hours after treatment, input resistance decreased rapidly to 5.3 M omega but increased to 18.0 M omega 12 h after treatment. Transient reduction of input resistance and membrane potential occurred prior to the decrease in the coupling ratio 6 h after treatment with DMBA. In freeze-fracture replicas, the number of gap junctions decreased by approximately 45% of the control value 6 h after treatment with DMBA. At 12 h and thereafter, the number and area of gap junctions subsequently decreased by 60-80% of the control value. Alterations in the number and area of desmosomes were similar to those of the gap junctions. The formation of epithelial cytoskeletons, partially devoid of the 2-4 and 5-8 nm filaments was also observed. A decrease in the density of filament networks beneath the plasma membranes was especially apparent. Treatment with a carcinogen brought about morphological cellular changes as early as 6 h after treatment, and such early changes might trigger metabolic cellular abnormalities. Affected cells appear to move away from normal cells in a process of repeated destruction and revision of intercellular junctions, and cytoskeletons.

9,10-Dimethyl-1,2-benzanthracene↗

PubMed1989

Freeze-etching study of intercellular junctions in the rat developing neural tube.

The intramembranous organization of the neuroepithelial intercellular junctions in the rat neural tube on days E13 and E15 was studied by using the freeze-etching technique. Tight junctions, frequently altered, are distinguished as the predominant type of intercellular junctions. Relatively rare gap junctions like intramembranous particle aggregations of varying sizes and particle packing occur too. The possible formation or breakdown of these probably temporary cell junction specializations is discussed.

Animals↗

PubMed1987

Effect of biological toxins on gap-junctional intercellular communication in Chinese hamster V79 cells.

Since chemical modulation of gap-junctional intercellular communication has been implicated in several toxicological endpoints, a study to examine the ability of several biological toxins to inhibit this process was undertaken. Eight biological toxins were tested for their ability to inhibit metabolic cooperation, a measure of gap-junctional intercellular communication, in the Chinese V79 cell system. Aplysiatoxin, anhydrodebromoaplysiatoxin and debromoaplysiatoxin showed the strongest ability to inhibit metabolic cooperation while T2-toxin and vomitoxin inhibited metabolic cooperation to a lesser degree. Aflatoxin B1, aflatoxin B2 and palytoxin were inactive in the Chinese V79 system. Palytoxin, which was extremely cytotoxic, might act as a tumor promoter if it induces compensatory hyperplasia in vivo.

Animals↗

PubMed1979

Freeze-fracture study of the epidermal cells of a teleost with particular reference to intercellular junctions and permeability to tracer.

The plasmatic membranes, the intercellular junctions and the intercellular spaces of the epidermis of the fish Pimelodus maculatus were studied by freeze-fracture and by lanthanum methods. The observations has confirmed the presence of desmosomes. Gap junctions were not found and the tight junctions can be seen very rarely, arranged to form small discrete maculae. The finger-print pattern due to the microridges of the apical plasma membrane of the superficial cells was studied by direct replicas. The tracer penetrates all the intercellular epidermal spaces but failed to penetrate the dermis, suggesting the presence of a barrier at the dermo-epidermal level.

Animals↗

PubMed1991

Intercellular junctions and tumor behavior in lung cancer.

We examined the ultrastructure of 76 lung cancers and counted the number of intercellular junctions per area of tissue section. The total number of desmosomes, intermediate junctions, and tight junctions ranged from zero to 300 per 0.157 cm2 of tissue section area, and each type of carcinoma showed a considerable range in concentration of intercellular junctions. Multivariate analysis showed that the number of junctions was significantly tied to the probability of the patient's having extensive stage and to survival time, even after accounting for the routine light microscopic diagnosis of small cell versus non-small cell carcinoma. These results suggest that a tumor's ability to spread and metastasize is inversely related to its number of intercellular junctions, and this conclusion appears to apply to both small cell and non-small cell carcinomas.

Adenocarcinoma↗

PubMed1988

The modulation of cell adhesion molecule expression and intercellular junction formation in the developing avian inner ear.

The cells that constitute the membranous labyrinth in the vertebrate inner ear are all derived from a single embryonic source, namely, the otocyst. The mature inner ear epithelia contain different regions with highly differentiated cells, displaying a highly specialized cytoarchitecture. The present study was designed to determine the presence of adherens-type intercellular junctions in this tissue and study the expression of cell adhesion molecules (CAMs) associated with these junctions, namely, A-CAM and L-CAM, in the developing avian inner ear epithelia. The results presented here show that throughout the early otocyst, A-CAM is coexpressed with L-CAM. The formation of asymmetries between sensory and nonsensory areas in the epithelium is accompanied by the modulation of CAMs expression and the assembly of intercellular junctional complexes. A-CAM and L-CAM display reciprocal expression patterns, the former being expressed mostly in the mosaic sensory epithelium, while L-CAM becomes conspicuous in the nonsensory areas but its expression in the sensory region is markedly reduced. Adherens-type junctions and numerous desmosomes are found in the junctional complexes of early otocyst cells. The former persist to maturity of the various inner ear epithelia, whereas desmosomes disappear from junctional complexes of hair cells but remain in the intercellular junctional complexes of all other cell types in the membranous labyrinth. Thus, adherens type intercellular junctions comprise the only defined cytoskeleton-bound junction in mature hair cells. A-CAM-positive cells are also found in the region of the acoustic ganglion in early developmental stages but not in the mature neural elements.

Actins↗

PubMed1986

Reversible inhibition of intercellular junctional communication by glycyrrhetinic acid.

Intercellular gap-junctional communication was measured using metabolic co-operation in co-cultures of argininosuccinate synthetase-deficient and argininosuccinate lyase-deficient human fibroblasts. 18-alpha-glycyrrhetinic acid (AGA) was found to inhibit communication by more than 95% at concentrations as low as 2 microM. Concentrations up to 100 microM were not cytotoxic over a period of 2 hours. Communication inhibition was of rapid onset and was readily reversible. Communication remained continuously yet reversibly blocked in cells cultured in the presence of AGA for 20 days. The related compounds 18-beta-glycyrrhetinic acid and carbenoxolone also caused communication inhibition. The effect is probably not mediated via mineralocorticoid or glucocorticoid receptors since aldosterone and glucocorticoids had no effect on communication. AGA thus has properties of a useful inhibitor in the study of intercellular junctional communication.

Carbenoxolone↗

PubMed1985

Inhibition of intercellular junctional communication in human fibroblasts by triphenylmethane, triphenylmethylchloride, tetraphenylboron and related compounds.

Intercellular junctional communication was measured using [14C]citrulline incorporation in co-cultures of argininosuccinate synthetase-deficient and argininosuccinate lyase-deficient human fibroblasts. Triphenylmethane, triphenylmethylchloride and tetraphenylboron inhibited communication at concentrations at least 12-fold lower than cytotoxic concentrations. This inhibition was of rapid onset and was rapidly reversible by washing the co-cultures. Refractoriness to inhibition did not develop after prolonged exposure. Several other compounds also induced communication inhibition, but only at concentrations slightly below cytotoxic concentrations. Treatment of co-cultures with calcium ionophore A23187 or cycloheximide did not cause communication inhibition. It is suggested that triphenylmethane, triphenylchloride and tetraphenylboron may be useful inhibitors for studying the roles of intercellular junctional communication in some biological systems.

Argininosuccinate Lyase↗

PubMed1979

Ultrastructure of intercellular junctions in the rat exocrine pancreas stimulated by pancreozymin.

Intercellular junctions in the exocrine pancreas, through which pancreatic enzymes may leak out from the lumen into the intercellular spaces, were studied in the rat by means of electron microscopy using tannic acid as a tracer and freeze-fracture methods as well as conventional thin sections. In thin sections and freeze-fracture replicas from the pancreas stimulated or not stimulated by exogenous pancreozymin, tight junctions were located between adjacent cells of the acinus or duct at the level just below the lumen. Observation of freeze-fracture replicas revealed that the number of strands or furrows in the tight junction did not differ significantly between the two groups but the width of the tight junction area was narrower and the distance between neighboring strands or furrows in the junction was smaller in the stimulated pancreas than in the control pancreas. Tannic acid, the molecular weight of which is smaller than that of pancreatic enzymes, could not pass beyond the tight junction area into the lumen. This study suggests that the pancreatic enzymes can not leak out from the glandular lumen into the intercellular spaces by dissociation of intercellular junctions even if the pancreas is stimulated by a high concentration of pancreozymin.

Animals↗

PubMed1980

Characterization of intercellular junctions in the caudal portion of the developing neural tube of the chick embryo.

The types of intercellular junctions present within caudal levels of the chick neural tube (i.e., future lower thoracic and lumbosacral regions of the spinal cord) were determined by freeze-fracture of stage 14 to 16 embryos. Two levels of the developing neural tube were examined: the region of the neurulation overlap zone--consisting of primary neural tube dorsally and secondary neural tube ventrally--and the portion of the primary neural tube located just cranial to the overlapping region. Gap junctions were the most numerous type of intercellular junction present within the primary neural tube. These junctions were located primarily in juxtaluminal areas, near the apices of neuroepithelial cells, and sometimes also at the bases of these same cells. In addition, focal, poorly defined tight junctions occasionally occupied juxtaluminal regions of the primary neural tube. The medullary cord (i.e., the immediate precursor of the secondary neural tube) and secondary neural tube contained gap junctions exclusively. Gap junctions were first found in these areas at the lateral borders of the medullary cord, concomitant with formation of this structure, and then at the interface between the elongated, peripheral cells of the cord and the irregularly shaped and loosely arranged central cells of this structure. Finally, gap junctions were distributed radially around secondary lumina formed by cavitation. The precise spatial and temporal correlation between the appearance of gap junctions and the specific changes occurring in cellular morphology and arrangement during secondary neurulation strongly suggest that gap junctions may have a role in coordinating cellular activities during formation of both the medullary cord and the secondary neural tube.

Animals↗

PubMed1989

[Electron microscopic study of intercellular junction in nasal mucosa of nasal allergy by lectin histochemistry].

To explain of the mechanism of the enhanced nasal epithelial permeability to HRP in patients with nasal allergy, the inferior turbinate mucosa was removed from 6 normal adults and 7 adults with nasal allergy. Difference of the fine structure of the intercellular junction was compared between normal mucosa and mucosa of nasal allergy by electron microscope. Staining pattern of four kinds of HRP-conjugated lectin (HRP-WGA, PNA, UEA-I and RCA-I) was also studied by electron microscope. There was no significant difference in the intercellular space of the mucosa between the normal mucosa and mucosa of nasal allergy. In the epithelial cell membrane, pattern of HRP-lectin staining was almost similar in both groups. In normal nasal epithelium, the intercellular junction consisted of junctional complex; adherent junction, desmosome and gap junction. The intercellular space was approximately 150-250 A in width. The tight junction was located beneath the luminal surface of the epithelium, and belt-like continuation connecting the adjacent cells. It was concluded that enhanced permeability to HRP in nasal allergy was not morphologic changes of the intercellular junction and component and distribution of the glycoconjugates in epithelial cellular membrane, but this may be based on functional changes.

Glycoconjugates↗

PubMed1989

Herbimycin A suppresses the reduction of gap-junctional intercellular communication induced by tumor-promoting phorbol ester in 3T3-L1 cells.

We have examined the suppressive effect of herbimycin A on the reduction of gap-junctional intercellular communication that is induced by a tumor-promoting phorbol ester in 3T3-L1 cells. Most cells in growth arrest participated in dye-coupling, as evaluated by the transfer between cells of a fluorescent dye (Lucifer Yellow CH). Treatment of cells with 0.25 microgram/ml herbimycin A slightly enhanced the dye-coupling. This enhancement required treatment for periods as long as 24 h. Addition of 100 ng/ml 12-O-tetradecanoylphorbol-13-acetate (TPA) caused a rapid reduction of dye-coupling. However, addition of TPA did not suppress dye-coupling in cells pretreated for more than 24 h with herbimycin A. Pretreatment of cells for less than 6 h with herbimycin A did not suppress the TPA-induced reduction of dye-coupling. These results suggest that herbimycin A suppresses the reduction of gap-junctional intercellular communication that is induced by TPA through enhancement of the ability of the cells to participate in gap-junctional intercellular communication.

Animals↗

PubMed1989

Inhibition of gap-junctional intercellular communication between epithelial cells transformed by the activated H-ras-1 oncogene.

In order to study the effects of an activated H-ras-1 oncogene on gap-junctional intercellular communication, we introduced the EJ/T24 H-ras-1 oncogene into cells of the epithelial Clone 9-3 cell line. Gap-junctional intercellular communication was significantly reduced in H-ras-1-transformed Clone 9-3 derivatives; this result shows that transformation by the activated H-ras-1 oncogene can inhibit gap-junctional intercellular communication. We postulate that the activated H-ras-1 oncogene product could mediate this effect through a change in the phosphorylation of the major gap-junction protein.

Animals↗

PubMed1975

Intercellular junctions of rat endocardium.

The ultrastructure of the intercellular junctions of rat endocardium has been characterized following lanthanum exposure in vitro and uranyl acetate staining en bloc. The interendothelial clefts of the endocardium run either a relatively straight or convoluted course and posses one or two loci where the plasma membranes are in close apposition or form punctate fusions. Elongate restrictions, that exhibit hexagonal arrays of subunits following lanthanum immersion (gap junctions), are also present in the intercellular endocardial clefts. The occurrence of interendothelial clefts of endocardium lacking occlusive foci can account for the permeability properties of ventricular endocardium, where the direction of diffusion of macromolecules has been attributed to pressure gradients between ventricular cavity and myocardium. The relationship of gap junctions to possible electrical phenomena within the endocardium is also discussed.

Animals↗

PubMedDate not supplied

Intercellular junctions in the organ of Corti.

The intercellular junctions between adjacent supporting cells and between apposed hair and supporting cells in the organ of Corti of cat and human were studied. At the endolymphatic surface, the intercellular space was closed by a series of tight junctions (zonula occludens), whereas there were no tight junctions at the basilar membrane surface of the neuroepithelium. Beneath the adlumenal zonula occludens between adjacent supporting cells, a zonula adherens (intermediate junction, "desmosone") was found. Many gap junctions joined apposed supporting cells both within apposed hair and supporting cells, membrane specialization sharing the morphological characterics of both the macula adherens and zonula adherens was found. Between cells, there were short areas of parallel limiting membranes separated by a 20A intercellular space. These areas were suggestive but not characteristic of gap junctional specialization. The functional significance of the junctional specialization between cells in the organ of Corti is discussed.

Animals↗

PubMed1984

A comparative study on the intercellular canalicular system and intercellular junctions in the pancreatic islets of some rodents.

Intercellular junctions were studied in pancreatic islets of some rodents by electron microscopy of thin sections and freeze-fracture-replicas. Junctions between islet cells can be classified into three types: macula occludens, a combination of the macula occludens and gap junction, and gap junction. The first and second types are mainly located on the cell membrane near the intercellular canaliculus, while the third type is present independently of it. The development of these types of junctions varies considerably among animal species: in mice, guinea pigs and hamsters, the second and third types are frequent, but the first type is rarely seen. In Mongolian gerbils, diminutive elements of the three types are infrequently present. In rats, only the third type is developed. In addition to intercellular communication through gap junctions, the first and second types of junctions may incompletely discriminate the intercellular canalicular lumen, and regulate the microenvironment of the islet cell.

Animals↗