Permeability of junctions between animal cells. Intercellular transfer of nucleotides but not of macromolecules.
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Biological Transport↗
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Permeability of junctions between animal cells. Intercellular exchange of various metabolites and a vitamin-derived cofactor.
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Animals↗
Modulation of the tight junctions of the Caco-2 cell monolayers by H2-antagonists.
PURPOSE: The tight junctions in the intestinal epithelium represent highly specialized intercellular junctions. Ranitidine, an H2-antagonist, causes a tightening of the tight junctions. Hence, we have investigated the effect of ranitidine and other H2-antagonists on the function of the intestinal tight junctions. METHODS: Effect of the H2-antagonists on the tight junctions has been investigated using the transepithelial electrical resistance (TEER) and the transport of mannitol across the Caco-2 cell monolayers. RESULTS: Four different H2-antagonists caused an increase in the TEER across the Caco-2 cell monolayers, accompanied by a decrease in the permeability for mannitol. The effect was concentration-dependent and saturable. Ranitidine and famotidine, caused a decrease in their own transport rate across the Caco-2 cells. Ranitidine competitively inhibited the increase in TEER caused by famotidine, whereas compounds which represent molecular fragments of ranitidine had no effect. The relative potency of the four H2-antagonists in causing an increase in the TEER correlated inversely with the oral bioavailability of these compounds in humans. CONCLUSIONS: We hypothesize that the H2-antagonists exert their effect on the tight junctions of Caco-2 cells by modulation of interactions among proteins associated with the tight junctional complex.
Biological Availability↗
Differentiation of a colon cancer cell line on a reconstituted basement membrane in vitro.
Basement membrane, a thin extracellular matrix, functions as a tissue stabilizer that promotes tissue integrity and differentiated phenotype. We studied a human colon cancer cell line, SNU 61, to evaluate its ability to differentiate on basement membrane. Cells were cultured on plastic, reconstituted basement membrane (Matrigel) or polyhydroxyethyl methacrylate (poly HEMA) for 72 h and evaluated by light and electron microscopy. On Matrigel, the cells showed gland formation with highly polarized cells containing basal nuclei and well developed brush border microvilli on the luminal surface. Apoptosis was noted mainly at the luminal side. On electron microscopic examination, numerous long microvilli, abundant cytoplasmic organelles and intercellular junctions were noted in the Matrigel-cultured cells. Intermediate cytoskeletons were scattered in the cytoplasm and existed on the axes of microvilli. Junctional complexes and desmosomes were frequently formed along intercellular spaces. The cells cultured on poly HEMA, on the other hand, were poorly differentiated and contained a few glandular structures with small lumens. Brush border microvilli, characteristic of enterocytic differentiation, were few in number and were developed on the basal surface. Intermediate filaments and microtubules were fewer than in the Matrigel-cultured cells. Carcinoembryonic antigen was expressed on the luminal surface of the Matrigel-cultured cells and in the cytoplasm of the poly HEMA cultured cells. CD44 stained the basolateral surface in the Matrigel-cultured cells, but the basal side was not stained in the poly HEMA cultured cells. These results are consistent with the different localization of microvilli in the Matrigel and in the poly HEMA cultured cells. Our observations suggest that human colon cancer cells on basement membrane can undergo glandular differentiation and that extracellular matrix is an important factor in morphogenesis.
Basement Membrane↗
Regulation of connexin43-protein binding in astrocytes in response to chemical ischemia/hypoxia.
Connexin-protein interactions are believed to be critical for the regulation of gap junctional intercellular communication and for the function of gap junctions formed by these complexes. We have primarily used immunoprecipitation strategies to investigate whether connexin43 binds to selected signaling and cytoskeletal proteins and whether connexin43-protein binding is altered in cultured astrocytes exposed to chemical ischemia/hypoxia, a treatment that resembles ischemia in vivo. Chemical ischemia/hypoxia induced marked dephosphorylation of connexin43, which was accompanied by increased association of connexin43 with c-Src, ERK1/2, and mitogen-activated protein kinase phosphatase-1 and by decreased association between connexin43 and beta-actin. Moreover, we found that endogenous c-Src in normal astrocytes exists primarily in the Triton X-100-soluble membrane fraction, distinct from the Triton-insoluble fraction, which contains gap junctions. After chemical ischemia/hypoxia, c-Src appeared in the Triton-insoluble fraction and was co-immunoprecipitated with connexin43, suggesting that chemical ischemia/hypoxia induced translocation of c-Src to the Triton-insoluble fraction and association with connexin43. Furthermore, the "dephosphorylated" form of connexin43 was immunoprecipitated by a phosphotyrosine antibody, suggesting tyrosine phosphorylation of connexin43 by c-Src. In addition, the association between connexin43 and c-Src was blocked by inhibition of connexin43 dephosphorylation, suggesting that the interaction between connexin43 and c-Src can be regulated by alterations in the phosphorylation state of connexin43. These results identify new binding partners for connexin43 and demonstrate that interactions between connexin43 and protein kinases and phosphatases are dynamically altered as a consequence of connexin43 phosphorylation.
Animals↗
Enhancement of liver cell gap junction protein expression by glucocorticoids.
Gap junctional intercellular communication (GJIC) and the expression of gap junction proteins (connexins) may be involved in growth regulation and neoplastic transformation. The mechanisms of connexin gene regulation in normal and neoplastic tissues are poorly understood. In this study, the glucocorticoids, dexamethasone and hydrocortisone, enhanced fluorescent dye-coupling in primary cultured rat hepatocytes and MH1C1 rat hepatoma cells. Other types of steroids (beta-estradiol, testosterone, aldosterone and progesterone) had no effect. Northern blot, Western blot, nuclear run-on and immunohistochemical analyses showed that glucocorticoids enhanced the expression of connexin32 in these cells in a dose- and time-dependent fashion. Connexin26 expression was also enhanced slightly by dexamethasone in hepatocytes, but not MH1C1 cells. Connexin43 expression in these cells was not affected by steroids. In WB-F344 rat liver epithelial cells, which were highly coupled and expressed high levels of connexin43 and no detectable connexin32 or connexin26, dexamethasone had no effect on coupling or connexin expression. These results indicate that dye-coupling and the expression of connexin32 and connexin26, but not connexin43, were upregulated by glucocorticoids in a cell-specific manner. These effects on GJIC and connexin expression may be involved in the induction of hepatic differentiation and inhibition of growth.
Animals↗
Expression and localization of N- and E-cadherin in the human testis and epididymis.
Cellular interactions in the testis and epididymis are an important prerequisite for spermatogenesis and sperm maturation, and involve a well-developed complex of intercellular junctions. Cadherins are cell surface proteins which mediate intercellular Ca(2+)-dependent adhesion and are believed to be fundamentally important for maintaining multicellular structures. In the present study we report the expression of a 135 kDa N-cadherin polypeptide in the human seminiferous epithelium by immunoblotting. The presence of N-cadherin was demonstrated by immunohistochemistry on the surface of spermatogonia and primary spermatocytes, and possibly also around some early spermatids, whereas late spermatids were always negative. Endothelial cells also stained for N-cadherin, whereas peritubular cells and Leydig cells did not. No expression of E-cadherin could be demonstrated in the human testis. In the human epididymis E-cadherin, but not N-cadherin, was expressed and localized to the surface of the principal epithelial cells as shown by immunohistochemistry. These observations indicate that cadherins play an important role in the organization of the seminiferous and epididymal epithelium.
Adult↗
Normal cells control the growth of neighboring transformed cells independent of gap junctional communication and SRC activity.
The growth of many types of cancer cells can be controlled by surrounding normal cells. However, mechanisms underlying this phenomenon have not been defined. We used a layered culture system to investigate how nontransformed cells suppress the growth of neighboring transformed cells. Direct physical contact between transformed and nontransformed cells was required for growth suppression of transformed cells in this system; communication by diffusible factors was not sufficient. However, significant gap junctional communication was not required, indicating that other intercellular junctions mediated this growth regulatory response. We also report that the Src kinase activity in transformed cells was not directly inhibited by contact with nontransformed cells. Instead, nontransformed cells increased the expression of serum deprivation-response protein and the transcription factor four and a half LIM domain 1 in tumor cells. In addition, these results suggest mechanisms by which normal cells may block Wnt signaling, inhibit insulin-like growth factor activity, and promote host recognition of neighboring tumor cells.
Animals↗
Localization of a novel 210 kDa protein in Xenopus tight junctions.
The tight junction is the most apical member of the intercellular junctional complex. It functions as a permeability barrier between epithelial cells and maintains the integrity of the apical and basolateral membrane domains. In order to study tight junctions in Xenopus laevis, a polyclonal antibody was raised which recognized Xenopus ZO-1. Monoclonal antibody 19B1 (mAb 19B1) was generated in rats using a crude membrane preparation from Xenopus lung as antigen. mAb 19B1 gave immunofluorescent staining patterns identical to those seen with anti-ZO-1 on monolayers of Xenopus A6 kidney epithelial cells and on frozen sections of Xenopus kidney, liver, and embryos. Electron microscopy showed that the 19B1 antigen colocalized with ZO-1 at the tight junction. Western blotting and immunoprecipitation demonstrated that ZO-1 is an approximately 220 kDa protein in Xenopus, while mAb 19B1 identified an approximately 210 kDa antigen on immunoblots. Immunoprecipitates of ZO-1 were not recognized by mAb 19B1 by western analysis. The solubility properties of the 19B1 antigen suggested that it is a peripheral membrane protein. Thus, the antigen recognized by the new monoclonal antibody 19B1 is not ZO-1 and represents a different Xenopus tight junction associated protein.
Animals↗
Adaptation of the heart to hypertension is associated with maladaptive gap junction connexin-43 remodeling.
We hypothesized that hypertension-related myocardial remodeling characterized by hypertrophy and fibrosis might be accompanied by cell-to-cell gap junction alterations that may account for increased arrhythmogenesis. Intercellular junctions and expression of gap junction protein connexin-43 were analyzed in rat heart tissues from both spontaneous (SHR) and L-NAME model of hypertension. Isolated heart preparation was used to examine susceptibility of the heart to lethal ventricular fibrillation induced by low potassium perfusion. Ultrastructure observation revealed enhanced neoformation of side-to-side type while internalization of end-to-end type (intercalated disc-related) of gap junctions prevailed in the myocardium of rats suffering from either spontaneous or L-NAME-induced hypertension. In parallel, immunolabeling showed increased number of connexin-43 positive gap junctions in lateral cell membrane surfaces, particularly in SHR. Besides, focal loss of immunopositive signal was observed more frequently in hearts of rats treated with L-NAME. There was a significantly higher incidence of hypokalemia-induced ventricular fibrillation in hypertensive compared to normotensive rat hearts. We conclude that adaptation of the heart to hypertension-induced mechanical overload results in maladaptive gap junction remodeling that consequently promotes development of fatal arrhythmias.
Adaptation, Physiological↗
[Inhibition of in vivo growth of lung carcinoma cells after transfection with gap junction gene Cx43].
OBJECTIVE: To explore the effects of expression of gap junction gene and cell-cell communication on tumor growth. METHODS: A highly metastatic human lung carcinoma cell line PG was used. PG cells were defective of gap junctional intercellular communication(GJIC) and lacking expression of gap junction gene Cx43. By transfection, Cx43 cDNA was introduced into PG cells and blank vector cDNA was used as mock control. By using Northern-blot, dye-transfer methods and examinations of in vitro/in vivo growth, stable Cx43 transfectant cells were studied. RESULTS: The mock control cells resembled untransfected PG cells in lacking expression of Cx43 and GJIC function. They grew fast in soft agar(colony formation rate 11.6%) and in nude mice (average tumor weight 3.47 g in 28 days). The Cx43 transfectant cells showed increased level of Cx43 mRNA and increased function of GJIC. Cell growth in soft agar and in nude mice was markedly retarded. The inhibition rate was 90% and 75%, respectively. CONCLUSION: Increased expression of gap junction gene Cx43 induced tumor-suppressing effects in human lung carcinoma cells.
Animals↗
Gap junctions, electrotonic coupling, and intercellular communication.
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Animals↗
The pathological shape variability of the junctional complexes along the intercellular spaces of the liver.
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Biopsy↗
On the mechanisms of cell uncoupling induced by a tumor promoter phorbol ester in clone 9 cells, a rat liver epithelial cell line.
It is known that in Clone 9 (C9) cells, intercellular gap junctional communication (IGJC) is rapidly blocked by the tumor promoter phorbol ester, 12-O-tetradecanoylphorbol-13-acetate (TPA), but it recovers spontaneously a few hours later and becomes refractory to TPA (Yada et al., J. Membr. Biol. 88, 217-232 (1985)). We now report that gap junctions between C9 cells contain at least two junctional proteins, connexin26 (Cx26) and connexin43 (Cx43), and that the TPA-induced changes in IGJC correlate temporally to changes in the state of phosphorylation of Cx43. The latter changes were prevented by inhibition of protein kinase C. Phosphoamino acid analysis and two-dimensional tryptic peptide maps of 32P-labeled Cx43 showed that during the TPA-induced phosphorylation at least two of the phosphorylated forms of Cx43 were differentially phosphorylated in seryl residues as compared to control. TPA induced a drastic reduction in junctional conductance as well as a redistribution of unitary gap junction channel event sizes seen in control cells. These changes were associated with retrieval of Cxs from the plasma membrane. Reappearance of gap junctions formed by Cx43 but not by Cx26 accounted for the spontaneous recovery in IGJC. It is proposed that gap junctions between C9 cells contain two types of channels each formed by Cx43 or Cx26 and that they are differentially affected during the action of TPA.
Animals↗
Thrombin-induced increase in endothelial permeability is associated with changes in cell-to-cell junction organization.
Thrombin increases endothelial permeability in a rapid and reversible way. This effect requires the catalytic activity of the enzyme and thrombin receptor engagement. Endothelial cell permeability is mostly regulated by intercellular junction organization. In the present study, we investigated whether opening of intercellular gaps after thrombin treatment could be related to changes in adherence-junction molecular organization. By immunofluorescence analysis, we found that thrombin stimulation of endothelial cells caused a marked alteration of the distribution of vascular endothelial (VE)-cadherin and of the associated catenins. These molecules, which are strictly localized at intercellular boundaries in confluent resting cells, were absent in the areas of intercellular retraction. Immunoprecipitation analysis indicated that thrombin disrupted the VE-cadherin/catenin complex. This effect was reversible and correlated with the increase in endothelial permeability. The use of a protein kinase C inhibitor (calphostin C) blocked both thrombin-induced permeability and disassembly of adherence-junction components. We propose that thrombin's effect on endothelial cell junction organization is an important determinant in the increase in endothelial permeability induced by this agent.
Amino Acid Sequence↗
Multiple connexins contribute to intercellular communication in the Xenopus embryo.
To explore the role of gap junctional intercellular communication (GJIC) during Xenopus embryogenesis, we utilized the host-transfer and antisense techniques to specifically deplete Cx38, the only known maternally expressed connexin. Cx38-depleted embryos developed normally but displayed robust GJIC between blastomeres at 32-128 cell stages, suggesting the existence of other maternal connexins. Analysis of embryonic cDNA revealed maternal expression of two novel connexins, Cx31 and Cx43.4, and a third, Cx43, that had been previously identified as a product of zygotic transcription. Thus, the early Xenopus embryo contains at least four maternal connexins. Unlike Cx38, expression of Cx31, Cx43 and Cx43.4 continue zygotically. Of these, Cx43.4 is the most abundant, accumulating significantly in neural structures including the brain, the eyes and the spinal cord.
Animals↗
Gap junction assembly: multiple connexin fluorophores identify complex trafficking pathways.
The assembly of gap junction channels was studied using mammalian cells expressing connexin (Cx) 26, 32 and 43 in which the carboxyl terminus was fused to green, yellow or cyan fluorescent proteins (GFP, YFP, CFP). Intracellular targeting of Cx32-CFP and 43-GFP to gap junctions was disrupted by brefeldin A treatment and resulted in a severe loss of gap junctional intercellular communication reflected by low intercellular dye transfer. Cells expressing Cx43-GFP exposed to nocodazole showed normal targeting to gap junctions and dye transfer. Cx32 and 43 thus appear to be transported and assembled into gap junctions via the classical secretory pathway. In contrast, we found that assembly of Cx26-GFP into functional gap junctions was relatively unaffected by treatment of cells with brefeldin A, but was extremely sensitive to nocodazole treatment. Coexpression of Cx26-YFP and Cx32-CFP indicated a different intracellular distribution that was accentuated in the presence of brefeldin A, with the gap junctions in these cells constructed predominantly of Cx26-YFP. A site specific mutation in the first transmembrane domain that distinguished Cx32 from Cx26 (Cx32128L) resulted in the adoption of the trafficking properties of Cx26 as well as its unusual post-translational membrane integration characteristics. The results indicate that multiple intracellular connexin trafficking routes exist and provide a further mechanism for regulating the connexin composition of gap junctions and thus specificity in intercellular signalling.
Animals↗
Intercellular communication in primary cultures of putative preneoplastic and 'normal' hepatocytes.
Gap junctional intercellular communication (IC) was studied in gamma-glutamyltranspeptidase (gamma-GT)-positive and -negative hepatocytes isolated from carcinogen-treated rats. Putative preneoplastic gamma-GT-positive hepatocytes were visualized in monolayer cultures by indirect immunofluorescence using anti-gamma-GT-antibodies. IC was evaluated by studying dye coupling of the cells. gamma-GT-positive hepatocytes showed a significantly lower dye coupling than did gamma-GT-negative liver cells. Spread of the dye Lucifer Yellow CH to neighboring cells was decreased further by the tumor-promoting chemical phenobarbital in both cell types in vitro. Also treatment in vivo with the barbiturate significantly reduced dye coupling of hepatocytes. The findings suggest that as a result of their decreased ability to communicate, preneoplastic hepatocytes may escape from growth control and differentiation signals given out by surrounding 'normal' cells.
2-Acetylaminofluorene↗