Inhibition of gap junctional intercellular communication by tumor promoters in connexin43 and connexin32-expressing liver cells: cell specificity and role of protein kinase C

Nur77 is a member of the steroid receptor superfamily and is known to be expressed in animals under stress. We studied the role of nur77 in the regulation of the hypothalamic-pituitary-adrenal (HPA) axis during the stress response using a murine pituitary corticotrope cell line, AtT-20. Corticotropin-releasing hormone (CRH), a stress mediator in the HPA axis, induced the expression of nur77 transiently in AtT-20 cells. Gel shift assay showed that nur77 bound to negative glucocorticoid responsive element (nGRE) in the promoter of the human proopiomelanocortin (POMC) gene and the formation of the nur77-nGRE complex increased after treatment of the cells with CRH. Negative GRE is known to be necessary for the negative regulation by glucocorticoid of the POMC gene expression. In stable transformants of AtT-20 cells expressing a human homolog of nur77, NAK-1, at a high level, glucocorticoid-mediated inhibition of both POMC mRNA induction and ACTH secretion was significantly lower than that in the NAK-1-non-expressing cells (P < 0.001). These results strongly suggest that nur77 antagonizes the negative feedback effect of glucocorticoid on the synthesis and secretion of ACTH in pituitary corticotropes. This suggests that nur77 plays an important role in the pituitary gland in the biological adaptation to overcome stress.     

Inhibition of gap junctional intercellular communication by barbiturates in long-term primary cultured rat hepatocytes is correlated with liver tumour promoting activity

Rodent liver tumor formation can be promoted by certain barbiturates and this may involve their ability to inhibit hepatocyte gap junctional intercellular communication (GJIC). In order to address the mechanisms and specificity of action of barbiturates on hepatocyte gap junctions, we have compared the effects of liver tumor-promoting barbiturates (phenobarbital, sodium barbital and amobarbital: PB, SB and AB, respectively) and a non-liver tumor-promoting barbiturate (barbituric acid: BA) on primary cultured rat hepatocyte GJIC and connexin32 (Cx32) expression after short (1-24 h) and long (2-14 days) treatment. GJIC was evaluated by fluorescent dye microinjection (dye-coupling); Cx32 expression was monitored by Northern blot, Western blot and immunohistochemistry. Both parameters were maintained at high levels over 14 days by coculture of the cells with WB-F344 rat liver epithelial cells in the presence of dexamethasone. Treatment with PB (2 mM) for 1 h sharply reduced dye-coupling from approximately 90-30%, but the cells fully recovered by 24 h. No inhibition was seen with the other barbiturates over this 1-day treatment period. Longer treatments (2-14 days) with the promoters PB, SB and AB, however, gradually reduced hepatocyte dye-coupling to approximately 30-50%. The non-promoter, BA, did not affect hepatocyte GJIC. These decreases in hepatocyte dye-coupling occurred without changes in Cx32 or gap junction expression. Dye-coupling of WB-F344 cells and expression of their predominant gap junction protein, connexin43 (Cx43), were also not affected. Thus, the inhibition of GJIC was specific to liver tumor promoting barbiturates in hepatocytes, was time-dependent and was not due to altered Cx32 expression.     

A characterization of permolybdate and its effect on cellular tyrosine phosphorylation, gap junctional intercellular communication and phosphorylation status of the gap junction protein, connexin43

Biological and analytical characterizations of permolybdate (a mixture of H2O2 and molybdate) were done. Molybdate (10 mM) and molybdenum(V) chloride (3 mM) did not affect gap junctional intercellular communication (GJIC), phosphorylation status of connexin43 (Cx43) or cellular tyrosine phosphorylation in early passage hamster embryonic cells (mainly fibroblast-like). High concentrations of H2O2 (3-10 mM) affected some of the parameters. Acidified permolybdate was clearly more stable than the unadjusted permolybdate. The maximum biological potency of acidified permolybdate was found at a molar ratio of 2:1 (H2O2:molybdate). The mixtures of molybdenum(V) chloride and H2O2 gave a maximum effect at 4:1 molar ratio (H2O2:molybdenum(V)). This can be explained by decomposition of H2O2 and by the generation of less biologically active compounds. Spectrophotometric analyses of the mixtures corroborated the biological results. The Mo(V) electron spin resonance spectrum disappeared upon addition of H2O2 to Mo(V) solutions, and no spectrum appeared when H2O2 was mixed with Mo(VI). Thus, permolybdate is probably diperoxomolybdate, a Mo(VI) compound. Regardless of the parent metal salt, the H2O2/metal salt mixtures showed concentration-dependent biphasic responses with an initial decrease in GJIC followed by an increase. A dissociation between alteration in Cx43 phosphorylation status and GJIC was obtained under certain conditions. The biological activities of permolybdate were only partially mimicked by phenylarsine oxide, an alternative protein tyrosine phosphatase inhibitor.     

Possible molecular mechanism of loss of homologous and heterologous gap junctional intercellular communication in rat liver epithelial cell lines

We have identified beta-galactosidase activity in purified bovine rod outer segments (ROS), using rho-nitrophenyl-beta-D-galactopyranoside (PNPG) and chlorophenol red-beta-D-galactopyranoside (CPRG) as substrates. This glycosylhydrolase activity did not appear to represent contamination from other retinal subcellular fractions, based upon the relative specific activities of beta-galactosidase vs. other hydrolases (N-acetyl-beta-glucosaminidase, alpha- and beta-mannosidase, alpha-fucosidase, and acid phosphatase) in bovine retina and ROS homogenates. Using PNPG as a substrate, two pH optima were observed (at 3.5 and 5.5), while the hydrolysis of CPRG exhibited a single, broad pH optimum centered at 5.5. In contrast, hydrolysis of PNPG and CPRG by retinal homogenates exhibited single pH optima, at 3.5 and 5.5., respectively. ROS beta-galactosidase activity increased linearly with time, temperature, and protein concentration, and obeyed Michaelis-Menten kinetics with both substrates. For PNPG, Vmax approximately 88 nmol/h/mg protein and the apparent Km approximately 147 microM. For CPRG, Vmax approximately 33 nmol/h/mg protein and the apparent Km approximately 50 microM. ROS beta-galactosidase activity was affected by carbohydrates and their derivatives: glucose, fucose, sucrose, maltose and N-acetyl-galactosamine were found to stimulate the activity, while D-galactono-gamma-lactone and, to a lesser extent, D-galactose were inhibitory. The enzyme activity also was slightly stimulated by [Cl-] and markedly by dithiothreitol (DTT), while rho-chloro-mercuribenzoic acid (PCMB) and rho-hydroxymercuribenzoic acid (PHMB) inactivated the enzyme. In addition, the enzymatic activity was also found to be differentially sensitive to various anionic and nonionic detergents. However, n-octyl-beta-D-glucoside was slightly stimulatory.(ABSTRACT TRUNCATED AT 250 WORDS)     

Changing patterns of gap junctional intercellular communication and connexin distribution in mouse epidermis and hair follicles during embryonic development

This paper is concerned with whether transport accident risk tends to peak at particular times, in relation to both time of day and time on task, and with the underlying causes of such peaks. Macro-analyses confirmed the presence of a clear circadian (ca 24 hour) rhythm in road accident risk with a major peak at ca 03:00 but suggested that this rhythm could not be entirely accounted for in terms of drivers falling asleep at the wheel. Sleep propensity clearly shows a pronounced circadian rhythm and performance efficiency in wakeful subjects shows a similar trend implying that the 03:00 road accident peak may simply reflect lowered performance capabilities. However, there are 'residual' peaks in accidents at certain times of day that are difficult to account for in terms of circadian rhythmicity. It is suggested that these may reflect a time on task effect which shows a pronounced, but transient, 2-4 hour peak in risk. Only when individuals had been on duty for 12 hours or more did the risk exceed that found during the 2-4 hour peak. While an explanation for this transient peak is offered, the underlying reason for it is, as yet, uncertain and clearly warrants investigation in view of its practical implications. It is concluded that there are 'black times' when accidents are far more likely and that there is a strong need to investigate possible countermeasures.     

Differential dose-dependent effects of alpha-, beta-carotenes and lycopene on gap-junctional intercellular communication in rat liver in vivo

In order to examine the relevance of alteration of gap-junctional intercellular communication (GJIC) to chemopreventive activity against carcinogenesis, the effects of alpha- and beta-carotene as well as lycopene, typical chemopreventive carotenoids, on cell coupling via gap junctions in rat liver in vivo were studied using a direct functional dye-transfer technique. We found that all three test compounds given at a dose of 50 mg/kg-body weight (b.w.) daily, 5 times by gavage, inhibited GJIC, while similar treatment with 5 mg/kg b.w. caused enhancement, especially in the beta-carotene- and lycopene-treated groups. At the dose level of 0.5 mg/kg b.w., the three compounds had no effect. The findings show that all three agents differentially modulate GJIC depending on the dose, with beneficial effects on cell communication only detected at the one dose. The result suggests that determination of the dose of chemicals to be used is crucial for human intervention studies.     

Coordinated intercellular calcium waves induced by noradrenaline in rat hepatocytes: dual control by gap junction permeability and agonist

Calcium-mobilizing agonists induce intracellular Ca2+ concentration ([Ca2+]i) changes thought to trigger cellular responses. In connected cells, rises in [Ca2+]i can propagate from cell to cell as intercellular Ca2+ waves, the mechanisms of which are not elucidated. Using fura2-loaded rat hepatocytes, we studied the mechanisms controlling coordination and intercellular propagation of noradrenaline-induced Ca2+ signals. Gap junction blockade with 18 alpha-glycyrrhetinic acid resulted in a loss of coordination between connected cells. We found that second messengers and [Ca2+]i rises in one hepatocyte cannot trigger Ca2+ responses in connected cells, suggesting that diffusion across gap junctions, while required for coordination, is not sufficient by itself for the propagation of intercellular Ca2+ waves. In addition, our experiments revealed functional differences between noradrenaline-induced Ca2+ signals in connected hepatocytes. These results demonstrate that intercellular Ca2+ signals in multicellular systems of rat hepatocytes are propagated and highly organized through complex mechanisms involving at least three factors. First, gap junction coupling ensures coordination of [Ca2+]i oscillations between the different cells; second, the presence of hormone at each hepatocyte is required for cell-cell Ca2+ signal propagation; and third, functional differences between adjacent connected hepatocytes could allow a 'pacemaker-like' intercellular spread of Ca2+ waves.     

Inhibition by anandamide of gap junctions and intercellular calcium signalling in striatal astrocytes

Anandamide, an endogenous arachidonic acid derivative that is released from neurons and activates cannabinoid receptors, may act as a transcellular cannabimimetic messenger in the central nervous system. The biological actions of anandamide and the identity of its target cells are, however, still poorly documented. Here we show that anandamide is a potent inhibitor of gap-junction conductance and dye permeability in striatal astrocytes. This inhibitory effect is specific for anandamide as compared to co-released congeners or structural analogues, is sensitive to pertussis toxin and to protein-alkylating agents, and is neither mimicked by cannabinoid-receptor agonists nor prevented by a cannabinoid-receptor antagonist. Glutamate released from neurons evokes calcium waves in astrocytes that propagate via gap junctions, and may, in turn, activate neurons distant from their initiation sites in astrocytes. We find that anandamide blocks the propagation of astrocyte calcium waves generated by either mechanical stimulation or local glutamate application. Thus, by regulating gap-junction permeability, anandamide may control intercellular communication in astrocytes and therefore neuron-glial interactions.     

Processing of the gap junction protein connexin50 in the ocular lens is accomplished by calpain

Scorpion venom can induce in dogs severe haemodynamic changes leading to rapid rise in systemic blood pressure and cardiac output, followed by reduction of cardiac output and blood pressure within 1 h. The decrease in cardiac output is not related to myocardial dysfunction (Tarasiuk et al. 1994). We hypothesized that scorpion venom affects cardiac output by reducing venous return to the heart. Venous return was studied by steady-state measurements of cardiac output, the pressure gradient and resistance to venous return, in 16 dogs following injection of 0.05 mg kg-1 venom obtained from the scorpion species Leiurus quinquestriatus. In eight of the 16 dogs, atropine (0.1 mg kg-1) was given 15 min prior to venom injection (n = 4) or 85 min (n = 4) after venom administration. In five additional dogs, the stability of the preparation over time was evaluated following the same protocol without the injection of the venom. At 15 min, the venom induced an increase in blood pressure (80%) and cardiac output (250%) (P < 0.001) with little effect on heart rate. At 90 min, cardiac output and heart rate declined considerably below baseline (P < 0.001). Atropine prevented the decrease in heart rate, but did not affect the reduction of cardiac output. Five minutes after venom injection, mean circulatory pressure increased by 300% (P < 0.001), which was accompanied by a rightward shift of the venous return curve with no effect on resistance to venous return. At 120 min, mean circulatory pressure recovered and resistance to venous return remained at 40% (P < 0.01) above baseline. This study indicates that, in dogs, scorpion venom affects cardiac output by modifying the determinants of venous return. The initial increase in cardiac output is related to increased mean circulatory pressure since resistance to venous return did not change. The later fall in cardiac output is related to the reduction of mean circulatory pressure and increased resistance to venous return.     

Effects of angiotensin II on expression of the gap junction channel protein connexin43 in neonatal rat ventricular myocytes

OBJECTIVES: To elucidate signal transduction pathways regulating expression of myocardial gap junction channel proteins (connexins) and to determine whether mediators of cardiac hypertrophy might promote remodeling of gap junctions, we characterized the effects of angiotensin II on expression of the major cardiac gap junction protein connexin43 (Cx43) in cultured neonatal rat ventricular myocytes. BACKGROUND: Remodeling of the distribution of myocardial gap junctions appears to be an important feature of anatomic substrates of ventricular arrhythmias in patients with heart disease. Remodeling of intercellular connections may be initiated by changes in connexin expression caused by chemical mediators of the hypertrophic response. METHODS: Cultures were exposed to 0.1 micromol/liter angiotensin II for 6 or 24 h, and Cx43 expression was characterized by immunoblotting, confocal microscopy and electron microscopy. RESULTS: Immunoblot analysis revealed a twofold increase in Cx43 content in cells treated for 24 h with angiotensin II (n=4, p < 0.05). This response was inhibited by the presence of 1.0 micromol/liter losartan, an AT1-receptor blocker. Confocal and electron microscopy demonstrated enhanced Cx43 immunoreactivity and increases in the number and size of gap junction profiles in cells exposed to angiotensin II for 24 h. These effects were also blocked by losartan. Immunoprecipitation of Cx43 from cells metabolically labeled with [35S]methionine demonstrated 2.4- and 2.9-fold increases in Cx43 radioactivity after 6 and 24 h exposure to angiotensin II, respectively (p < 0.03 at each time point). CONCLUSIONS: Angiotensin II up-regulates gap junctions in cultured neonatal rat ventricular myocytes by increasing Cx43 synthesis. Signal transduction pathways activated by angiotensin II under pathophysiologic conditions could initiate remodeling of conduction pathways, leading to the development of anatomic substrates of arrhythmias.     

Stimulation of gap junctional intercellular communication by thalidomide and thalidomide analogs in human skin fibroblasts

It has been speculated that gap junctional intercellular communication (GJIC), an intercellular signalling pathway, is involved in embryogenesis by coupling compartments of the same developmental potential. We found that thalidomide induces GJIC in human fibroblasts after activation by liver microsomes in concentrations as low as 10(-7) M. Treatment of cells with the thalidomide analog EM-12 increased GJIC without prior activation. No alteration of GJIC was detected with phthalimide and glutamate, the components of thalidomide. However, 2-phthalimido glutaric acid (PGA), a hydrolysis product of thalidomide, stimulated GJIC without activation at concentrations between 10(-10) M and 10(-5) M. We suggest modification of GJIC as a biochemical mechanism responsible for pharmacological and toxicological properties of thalidomide and related compounds.     

Partial saturation of rat liver Cd-binding proteins by Cd2+ in vivo

Liver Cd-binding proteins (Cd-BP) were isolated from rats chronically treated with 109Cd-labeled CdCl2 for ten days. Fractions purified using Sephadex G-75 and DEAE-Sephadex were characterized and found to be similar to those isolated by other investigators. Cd-binding was not saturated in any of the preparations and significant amounts of Cu and Zn were also found bound to the proteins. The percentage of saturation of Cd-BP1, and Cd-BP2 was independently determined by atomic absorption spectrometry and spectroscopy at 254 nm. These results indicate that the fraction of binding sites unoccupied by Cd on Cd-BP approaches 20% in vivo.     

Effect of perchloroethylene and its metabolites on intercellular communication in clone 9 rat liver cells

Gap junction intercellular communication (IC) is thought to be important in chemical carcinogenesis as abnormalities in IC have been found in cancer cells. Perchloroethylene (PERC) is metabolized in rodent liver to dichloroacetic acid (DCA) and trichloroacetic acid (TCA), which are rodent liver carcinogens. Chloral hydrate (CH) and trichloroethanol (TCEth) are kidney metabolites. We used Lucifer yellow scrape-load dye transfer as a measure of IC to look at the effect of PERC, DCA, TCA, CH, and TCEth on Clone 9 cell cultures (normal rat liver cells). Four independent experiments were performed for each chemical using exposure times of 1, 4, 6, 24, 48, and 168 h. Concentrations for each chemical varied and were based on preliminary data on effect and cytotoxicity. To compare the relative effectiveness of each chemical to cause biological change, we identified the lowest concentration and shortest time to significantly reduce dye transfer. DCA caused a significant change at 10 mM at 6 h; TCA, 1 mM at 1 h; CH and TCEth, 1 mM at 24 h; and PERC, 0.01 mM at 48 h. Over a 24-h treatment period, the relative efficiencies, as defined by the concentration needed to produce 50% reduction in IC, were PERC (0.3 mM) > TCA (3.8 mM) > TCEth (6.6 mM) = CH (7.0 mM) > DCA (41 mM). Time-course data indicated that PERC, DCA, and TCA produced reduction in IC in a similar fashion, but 5 mM CH or TCEth exhibited variances from these results and may indicate specific cell responses to these chemicals. The mechanism(s) responsible for inhibition of IC by these structurally related chemicals needs to be established.     

Propagation of mechanically induced intercellular calcium waves via gap junctions and ATP receptors in rat liver epithelial cells

Mechanical stimulation was used to initiate Ca2+ waves in rat liver epithelial cells in order to ascertain the degree to which gap junctional intercellular communication (GJIC) is involved in communication of Ca2+ to adjacent cells and to assess alternative Ca2+ signaling pathways that may be present between these cells. In both WB-F344 cells, which show a high degree of GJIC, and WB-aB1 cells, which are GJIC deficient, mechanical stimulation of a single cell induced a Ca2+ wave which propagated away from the point of stimulation, across cell borders, to neighboring cells directly or indirectly in contact with the stimulated cell. In addition, the Ca2+ wave was transmitted to nearby isolated cells that exhibited no direct or indirect contact with the stimulated cell. Treatment of cells with 18beta-glycyrrhetinic acid, a compound that has been shown to block GJIC, did not significantly affect propagation of the Ca2+ wave. In contrast, treatment with suramin, a P2-purinergic receptor inhibitor, significantly reduced both the rate and the extent of Ca2+ wave propagation in WB-F344 cells and completely blocked its propagation in WB-aB1 cells. Cotreatment with suramin and glycyrrhetinic acid was found to completely block the mechanically induced Ca2+ wave in both cell lines. These studies indicate that mechanically induced cell injury in rat liver epithelial cells initiates signaling through at least two pathways, involving intercellular communication via gap junctions and extracellular communication via ATP activation of purinergic receptors.     

Inhibition of intercellular adhesion molecule-1 with antisense deoxynucleotides prolongs renal isograft survival in the rat

BACKGROUND: Delayed graft function from ischemia-reperfusion injury has a negative impact on long-term renal graft survival. We tested the utility of antisense oligodeoxynucleotide (ODN) against intercellular adhesion molecule-1 (ICAM-1) in the pretransplant treatment of renal isografts in improving long-term graft survival. METHODS: Three groups of 16 inbred Lewis rats each underwent unilateral nephrectomy and were then transplanted with a kidney from a Lewis donor rat, which had received antisense ODN, reverse sense ODN, or saline vehicle six hours prior to nephrectomy. The kidneys were subjected to one hour of warm ischemia and 30 minutes of cold ischemia, which when untreated results in delayed graft function. The remaining native kidney was removed 10 days later. Serum creatinine and urinary protein excretion were measured in surviving rats at weeks 2, 4, 6, 8, 12, 16, and 20 after native nephrectomy. RESULTS: A Kaplan-Meier analysis revealed that by week 6 one half of the animals receiving reverse sense ODN and saline vehicle treatment had died, while all but 2 rats in the antisense ODN-treatment group survived to 20 weeks. Serum creatinine concentrations and urine protein excretion of surviving reverse sense and saline vehicle-treated rats were significantly higher than antisense treated rats at every time point. Histology at week 20 revealed marked interstitial fibrosis, focal glomerular sclerosis, vascular intimal and medial thickening and tubular atrophy in reverse sense and saline vehicle-treated kidneys, while antisense ODN-treated kidneys showed only modest changes. Immunohistochemistry showed macrophage and lymphocyte infiltration, as well as substantial up-regulation of MHC class II, in reverse sense and saline vehicle-treated kidneys compared to antisense ODN-treated kidneys. CONCLUSIONS: These results suggest that by ameliorating acute nonimmunological renal isograft injury, the long-term chronic nonimmunologic processes are improved as well. Furthermore, the data suggest that an antisense ODN strategy directed against ICAM-1 may have utility in human kidney transplantation.     

Inhibition of DNA methylation by S-adenosylethionine with the production of methyl-deficient DNA in regenerating rat liver

Ethionine, a liver carcinogen, was administered p.o. (300 mg/kg) to rats 17 hr after partial hepatectomy. At 6 hr after administration of the ethionine, hepatic S-adenosylethionine levels were 30- to 40-fold greater than the hepatic level of S-adenosylmethionine. A 10-fold ratio of S-adenosylethionine to S-adenosylmethionine still persited at 24 hr after ethionine administration. When given at 17 hr after partial hepatectomy, ethionine produced a 30% inhibition of DNA synthesis, measured by the incorporation of [methyl-3H]thymidine at 23 to 24 hr after partial hepatectomy (6 to 7 hr after ethionine administration). DNA synthesized during this interval was methyl deficient as judged by the reduced incorporation of radioactivity from L-[methyl-3H]methionine into 5-methylcytosine residues of DNA. In an assay for DNA methylation in vitro using whole nuclei, the methyl-deficient DNA was methylated by S-adenosylmethionine 8 times more than was control DNA; the DNA methylation was competitively inhibited by S-adenosylethionine. These data suggest that S-adenosylethionine, formed in vivo from ethionine, competitively inhibits the methylation of DNA in vivo by S-adenosylmethionine, resulting in the production of methyl-deficient DNA.     

Inhibition by warfarin of prothrombin synthesis and of lipid-saccharide synthesis in rat liver

In vivo and in vitro studies using [3H]glucosamine incorporation into prothrombin and into glycolipids were conducted in rat liver to determine the role of lipid-saccharides in the biosynthesis of prothrombin. In vivo studies demonstrated that 10 mg warfarin/kg inhibited the incorporation of radiolabeled glucosamine into liver prothrombin and glycolipids. This inhibition was similar to the kinetics of inhibition of prothrombin synthesis in the liver. In vitro studies demonstrated a time-dependent increase in the incorporation of radiolabeled glucosamine into lipid-saccharides and prothrombin. This incorporation was inhibited 50% by 5 . 10(-4) M warfarin. Warfarin also inhibited the incorporation of radiolabeled glucosamine into glycolipids in a dose-related manner. In all studies, vitamin K-1 reversed the inhibition of glucosamine incorporation into glycolipids and into prothrombin.     

Emergence of undifferentiated rat tracheal cell carcinomas, but not squamous cell carcinomas, is associated with a loss of expression of E-cadherin and of gap junction communication

A series of cells representing normal, non-tumorigenic cell lines, as well as differentiating neoplastic and undifferentiated neoplastic rat tracheal epithelial cell populations were evaluated for their ability to establish homologous and/or heterologous cell-cell gap junction communication in culture. Gap junction communication was evaluated by flow cytometric quantitation of the transfer of the fluorescent dye calcein from a donor to a recipient cell population via gap junctions. The data indicate that normal primary cultures of rat tracheal epithelial cells, as well as non-tumorigenic cell lines and squamous cell carcinomas cell populations, retain the ability to establish both homologous and heterologous gap junction communication. In all cases an average of >48% of recipient cells had acquired calcein label during a 5-h interval of co-culture of donor and recipient cells at confluent densities. Cells harvested directly from squamous cell carcinoma tumors exhibited similar levels of cell-cell communication. In contrast, cells giving rise to undifferentiated carcinomas, as well as cells harvested from undifferentiated carcinomas, exhibited very low levels or no homologous or heterologous cell-cell communication. Cell populations exhibiting distinctly different communication phenotypes were evaluated by Northern blot analysis for expression of connexins (Cx 26, 32 and 43) and E-cadherin. Neither communicating nor non-communicating cells expressed connexin 32. Those cell populations, which established functional gap junctions, expressed E-cadherin as well as connexin 26 and/or 43. In contrast, those cell populations that lacked the ability to communicate universally lacked expression of E-cadherin, and a quarter also lacked expression of detectable levels of connexin.