Inhibition Of Intercellular Communication Research Articles

Regulation of cell‐cell communication in rat bone cells: The effect of phorbol esters

The skin tumor promoter 12-O-tetradecanoylphorbol-13-acetate (TPA) is a potent inhibitor of gap junctional intercellular communication. In the present study, the inhibition of cell-cell communication by TPA has been investigated in primary bone cells from newborn rat calvaria, with an emphasis on the involvement of intracellular pH (pHi) and cytosolic calcium ([Ca+2]i) in this process. The results show that TPA (5 × 10−8 M) caused a complete inhibition of intercellular communication within 40–60 min. The intercellular communication was fully restored after overnight incubation in the presence of TPA. This effect was found to be associated with an elevation of pHi. However, neither an increase of pHi alone nor exposure to TPA, under conditions preventing pHi-shift, were found to affect intercellular communication. It is suggested that the inhibition of intercellular communication, in the presence of TPA, depends on the pHi-shift itself rather than on the absolute value of pHi. In addition, elevation of cytosolic calcium by ionomycin led to the termination of intercellular communication after 30 min. This inhibitory effect was abolished when the cells were incubated for overnight with TPA and then intracellular calcium was elevated by the addition of ionomycin. These results indicate that shift of pHi and the increase of intracellular calcium are involved in repression of intercellular communication by TPA. J. Cell. Biochem. 74:349–356, 1999. © 1999 Wiley-Liss, Inc.

Regulation of cell-cell communication in rat bone cells: The effect of phorbol esters

The skin tumor promoter 12-O-tetradecanoylphorbol-13-acetate (TPA) is a potent inhibitor of gap junctional intercellular communication. In the present study, the inhibition of cell-cell communication by TPA has been investigated in primary bone cells from newborn rat calvaria, with an emphasis on the involvement of intracellular pH (pH(i)) and cytosolic calcium ([Ca(+2)](i)) in this process. The results show that TPA (5 x 10(-)(8) M) caused a complete inhibition of intercellular communication within 40-60 min. The intercellular communication was fully restored after overnight incubation in the presence of TPA. This effect was found to be associated with an elevation of pH(i). However, neither an increase of pH(i) alone nor exposure to TPA, under conditions preventing pH(i)-shift, were found to affect intercellular communication. It is suggested that the inhibition of intercellular communication, in the presence of TPA, depends on the pH(i)-shift itself rather than on the absolute value of pH(i). In addition, elevation of cytosolic calcium by ionomycin led to the termination of intercellular communication after 30 min. This inhibitory effect was abolished when the cells were incubated for overnight with TPA and then intracellular calcium was elevated by the addition of ionomycin. These results indicate that shift of pH(i) and the increase of intracellular calcium are involved in repression of intercellular communication by TPA.

Spatial and temporal regulation of gap junction connexin43 in vascular endothelial cells exposed to controlled disturbed flows in vitro.

Hemodynamic regulation of the endothelial gap junction protein connexin43 (Cx43) was studied in a model of controlled disturbed flows in vitro. Cx43 mRNA, protein expression, and intercellular communication were mapped to spatial variations in fluid forces. Hemodynamic features of atherosclerotic lesion-prone regions of the vasculature (flow separation and recirculation) were created for periods of 5, 16, and 30 h, with laminar shear stresses ranging between 0 and 13.5 dynes/cm2. Within 5 h, endothelial Cx43 mRNA expression was increased in all cells when compared with no-flow controls, with highest levels (up to 6- to 8-fold) expressed in regions of flow recirculation corresponding to high shear stress gradients. At 16 h, Cx43 mRNA expression remained elevated in regions of flow disturbance, whereas in areas of fully developed, undisturbed laminar flow, Cx43 expression returned to control levels. In all flow regions, typical punctate Cx43 immunofluorescence at cell borders was disrupted by 5 h. After 30 h of flow, disruption of gap junctions persisted in cells subjected to flow separation and recirculation, whereas regions of undisturbed flow were substantially restored to normal. These expression differences were reflected in sustained inhibition of intercellular communication (dye transfer) throughout the zone of disturbed flow (84.2 and 68.4% inhibition at 5 and 30 h, respectively); in contrast, communication was fully reestablished by 30 h in cells exposed to undisturbed flow. Up-regulation of Cx43 transcripts, sustained disorganization of Cx43 protein, and impaired communication suggest that shear stress gradients in regions of disturbed flow regulate intercellular communication through the expression and function of Cx43.

Inhibitory effects of organochlorine pesticides on intercellular transfer of lucifer yellow in cultured bovine oviductal cells

The present study investigated the effects of dichlorodiphenyltrichloroethane (DDT), methoxychlor (MXC), and γ-hexachlorocyclohexane (γHCH, lindane) on gap junctional intercellular communication (GJIC) in cultured bovine oviductal cells. GJIC was evaluated by microinjecting fluorescent dye Lucifer Yellow and observing the inhibition of the spreading of dye into adjacent cells. After incubation for 1 h at 37°C, a dose-dependent inhibition of GJIC was observed over a concentration range of 16 to 128 μ m DDT, MXC, or γHCH compared with nonexposed controls. A significant inhibition began at 32 μ m DDT, MXC, or γHCH. After incubation for 5 h, a dose-dependent inhibition of GJIC was obtained in the concentration range from 8 to 64 μ m of the pesticides. The first significant inhibitory effect on GJIC was caused by 8 μ m DDT, 16 μ m MXC, and 32 μ m γHCH. The 128 μ m concentration of the pesticides was toxic. At pesticide concentration of 64 μ m, the decrease in dye-coupling observed was not due to lethal cell injury, as is indicated by the use of trypan blue dye exclusion. After removal of 64 μ m DDT from the culture medium, intercellular communication was reestablished within 3 h. Measurement of cytosolic free Ca 2+ concentration [Ca 2+] i in fura-2/AM-loaded oviductal cells showed that the inhibition of GJIC by addition of DDT, MXC, or γHCH was not associated with a detectable increase in [Ca 2+] i. Coincubation of the DDT with dibutyryl-cAMP prevented the 64 μ m DDT-induced inhibition of intercellular communication in adherent oviduct cells. It is suggested that organochlorine pesticides can influence cells responsible for reproduction.

Involvement of oxidative stress in motorcycle exhaust particle-induced DNA damage and inhibition of intercellular communication

In this study, we investigated the involvement of reactive oxygen species (ROS) in the motorcycle exhaust particle (MEP)-induced genotoxic and non-genotoxic activity in mammalian cell systems. Initially, the capability of MEP to induce ROS was evaluated by using 2′,7′-dichlorofluorescin diacetate (DCFH-DA) to detect hydrogen peroxide (H 2O 2). A five-fold increase in H 2O 2 was observed in Chinese hamster lung V79 and human lung carcinoma Calu-1 cells treated with 100 μg/ml MEP for 2 h. Under the same experimental conditions, only a two-fold elevation in H 2O 2 was detected in hepatic cell systems such as BNL.Cl.2, HepG2, and Hep3B. Treatment of the V79 cells with varying concentrations of MEP caused a dose-dependent increase in sister chromatid exchanges (SCEs), which are effectively inhibited by addition of antioxidants, N-acetyl- l-cysteine (NAC) and ascorbic acid. Furthermore, we determined the oxidized bases in the V79 cells after exposure to MEP. The result showed that 500 μg/ml MEP induced a 3.7-fold increase in thymine glycol (TG) and a seven-fold increase in 8-hydroxy-guanosine (8-OHGua) as compared to untreated cells. We subsequently examined whether MEP would affect gap junctional intercellular communication (GJIC), a tumor promotion process, in V79 cells. We found that MEP inhibited GJIC in a dose–response fashion. Maximal inhibition occurred at 500 μg/ml. The concentration that inhibited at 0.5 of the fraction of the control was 200 μg/ml. Interestingly, when cells were pretreated with NAC or ascorbic acid, they could abolish the MEP-mediated inhibition of GJIC. In addition, a moderate decrease of glutathione was observed in the V79 cells during exposure to MEP. Taken together, our findings suggest that MEP can induce oxidative stress in a broad range of cell lines, especially in lung cell systems. The MEP-induced oxidative stress was critically involved in both genotoxic and non-genotoxic activity.

Antioxidant prevention of tumour promoter induced inhibition of intercellular communication in renal cell lines.

Antioxidant prevention of tumour promoter induced inhibition of intercellular communication in renal cell lines.

P XIX.15 - P XIX.15 Morphological transformation and inhibition of intercellular communication of Syrian hamster embryo cells by hepatic peroxisome proliferators

P XIX.15 - P XIX.15 Morphological transformation and inhibition of intercellular communication of Syrian hamster embryo cells by hepatic peroxisome proliferators

Oleic acid inhibits gap junction permeability and increases glucose uptake in cultured rat astrocytes.

The role of oleic acid in the modulation of gap junction permeability was studied in cultured rat astrocytes by the scrape-loading/Lucifer yellow transfer technique. Incubation with oleic acid caused a dose-dependent inhibition of gap junction permeability by 79.5% at 50 microM, and no further inhibition was observed by increasing the oleic acid concentration to 100 microM. The oleic acid-mediated inhibition of gap junction permeability was reversible and was prevented by bovine serum albumin. The potency of oleic acid-related compounds in inhibiting gap junction permeability was arachidonic acid > oleic acid > oleyl alcohol > palmitoleic acid > stearic acid > octanol > caprylic acid > palmitic acid > methyloleyl ester. Oleic acid and arachidonic acid, but not methyloleyl ester, increased glucose uptake by astrocytes. Neither oleic acid nor arachidonic acid increased glucose uptake in the poorly coupled glioma C6 cells. These results support that the inhibition of gap junction permeability is associated with the increase in glucose uptake. We suggest that oleic acid may be a physiological mediator of the transduction pathway leading to the inhibition of intercellular communication.

Flavonoids (apigenin, tangeretin) counteract tumor promoter-induced inhibition of intercellular communication of rat liver epithelial cells.

We have shown previously that two flavonoids, apigenin and tangeretin, enhance gap junctional intercellular communication (GJIC) in rat liver epithelial cells, named REL cells. Here, we show that these two flavones also antagonize the inhibition of GJIC induced by tumor promoters like 12-O-tetradecanoyl-phorbol-acetate (TPA) and 3,5,di-tertio-butyl-4-hydroxytoluene (BHT). Their preventive effect is rapid. It does not seem to involve any change of the amount of the connexin expressed in REL cells, connexin 43 (Cx 43), and in its phosphorylation state. Other flavonoids tested including naringenin, myricetin, catechin and chrysin did not enhance GJIC nor counteract TPA-induced inhibition of GJIC.

Loss of gap junction plaques and inhibition of intercellular communication in ilimaquinone-treated BICR-M1Rk and NRK cells.

To examine the mechanism(s) and pathways of gap junction formation and removal a novel and reversible inhibitor of protein secretion, ilimaquinone (IQ), was employed. IQ has been reported to cause the vesiculation of Golgi membranes, block protein transport at the cis-Golgi and depolymerize cytoplasmic microtubules. Connexin43 (Cx43) immunolabeling and dye microinjection experiments revealed that gap junction plaques were lost and intercellular communication was inhibited following IQ treatment for 1 hr in BICR-M1Rk rat mammary tumor cells and for 2 hr in normal rat kidney (NRK) cells. Gap junction plaques and intercellular communication recovered within 2 hr when IQ was removed. IQ, however, did not affect the distribution of zonula occludens-1, a protein associated with tight junctions. Western blot analysis revealed that the IQ-induced loss of gap junction plaques was accompanied by a limited reduction in the highly phosphorylated form of Cx43, previously shown to be correlated with gap junction plaques. The presence of IQ inhibited the formation of new gap junction plaques in BICR-M1Rk cells under conditions where preexisting gap junctions were downregulated by brefeldin A treatment. Treatment of BICR-M1Rk and NRK cells with other microtubule depolymerization agents did not inhibit plaque formation or promote rapid gap junction removal. These findings suggest that IQ disrupts intercellular communication by inhibiting the events that are involved in plaque formation and/or retention at the cell surface independent of its effects on microtubules. Our results also suggest that additional factors other than phosphorylation are necessary for Cx43 assembly into gap junction plaques.

Stimulation of cell proliferation and inhibition of gap junctional intercellular communication by linoleic acid

The effect of linoleic acid (LA) on gap-junction permeability, connexin 43 mRNA level, protein level, and phosphorylation, and the numbers of gap-junctional membrane plaques were studied in the rat liver epithelial cell line WB-F344 to determine whether changes in these parameters correlated with the enhanced cell growth and the inhibition of gap-junction function. When cultured in a medium with low serum (1%), these cells exhibited a slower growth rate than in the high serum medium (7%). Addition of linoleic acid (0.01–3 mg/ml) to the low serum medium increased the growth rate and inhibited gap junctional intercellular communication (GJIC) in a dose-dependent manner. In a comparison of short-term and long-term treatments with LA, GJIC in short-term treated (1 h) WB cells was inhibited at 3 mg/ml LA but readily recovered by washing and removing LA from cells, whereas GJIC in long-term treated (6 days) WB cells did not recover by washing and removing LA from WB cells. Western blot analysis of connexin 43 showed that a short-term incubation with linoleic acid increased the relative amount of unphosphorylated connexin 43 protein, but a long-term incubation with linoleic acid decreased the amount of unphosphorylated connexin 43 protein and increased the relative amount of hyperphosphorylated connexin 43 protein. Connexin 43 and p53 mRNA levels decreased in a time- and dose-dependent manner in linoleic acid-treated cells. These results suggest that growth stimulation and gap junctional intercellular communication inhibition of rat liver epithelial cells by linoleic acid may be mediated in part through modulation of p53 expression and function.

Alteration of intercellular communication in a human urothelial carcinoma cell-line by tumor-promoting agents.

Most of the findings that support a good association between inhibition of intercellular communication and tumor promotion have been demonstrated by studies of animal cells, while it has been accepted that most tumor-promoting agents are specific to species and organs. Therefore, the influence of tumor-promoting agents on intercellular communication was investigated using a human urothelial cell-line. The effects of six tumor-promoting agents, including dichlorodiphenyltrichloroethane (DDT), butylated hydroxyanisole (BHA), saccharin, cyclamic acid, phenobarbital, and DL-tryptophan, on intercellular communication in a human urothelial cell-line (JTC-30) were investigated by using a dye-transfer assay after 48- and 96-hour exposure to each agent. Cytotoxicity was determined using the colony-forming method after 48-hour and 7-day exposure to each agent. DDT, BHA, saccharin, and cyclamic acid inhibited intercellular communication after either 48- or 96-hour exposure at concentrations that were nontoxic to the cells after 7-day exposure. Phenobarbital and DL-tryptophan inhibited intercellular communication only at toxic concentrations. Since the former four agents are known to be direct tumor promoters, and the metabolites of the later two agents have been regarded as tumor promoters, the results suggest that current methodology using the JTC-30 cell-line may be applicable to the screening to detect direct tumor promoters of human urothelium.

Ultraviolet absorption characteristics and calculated semi-empirical parameters as chemical descriptors in multivariate modelling of polychlorinated biphenyls

The structural variation within the polychlorinated biphenyls (PCBs) was characterized by using principal component analysis (PCA). A multivariate model was evolved from 52 physicochemical descriptors including measured ultraviolet (UV) absorption spectra, calculated semiempirical parameters (AM1) and properties captured from the literature. Parameters calculated by using the AM1-Hamiltonian were e.g. heat of formation, dipole moments, ionization potential and the barrier of internal rotation. The UV spectra were measured and digitized in the range 200–300 nm. The multivariate model revealed that most of the information within the set of physicochemical parameters was related to molecular size. Descriptors depending on size were e.g. GC retention times, partition coefficients and a subset of semiempirically derived energy terms. Important also were parameters reflecting differences in substitution patterns and related to electronic and steric properties, such as UV absorption in the wavelength region 245–300 nm, the barrier of internal rotation and the ionization potential. The developed model describes the large variation in physicochemical characteristics within the PCBs. The importance of a broad chemical characterization is illustrated by a quantitative structure-activity relationship (QSAR) for the potency of inhibition of intercellular communication for 27 structurally diverse tetra- to heptachlorinated PCBs.

Inhibition of gap junction intercellular communications of cultured rat hepatocytes by ethanol: role of ethanol metabolism

Background/Aims: In a previous study, we reported that in cultured rat hepatocytes, ethanol inhibits intercellular communication which is known to play a central role in the regulation of cell growth and differentiation. This work was designed to find out if ethanol exerts a direct action on cell membranes, comparable to other long-chain (C6–C9) alcohols, or an indirect action. Methods: Intercellular communication was measured on short-term cultured rat hepatocytes by the fluorescent Lucifer-Yellow CH transfer method. Intracellular pH was measured by spectrofluorimetry and membrane expression of connexin 32 by indirect immunofluorescence. Results: Under our conditions, ethanol (20 mM) inhibited intercellular communication of hepatocytes to the same extent as did octanol at 1 mM. Immunofluorescence semi-quantitative studies of connexin 32 suggested that the observed inhibition was not related to a decrease in the number of gap junction plaques. In contrast with those of octanol, the inhibitory effects of ethanol appeared to be indirect because the inhibition of ethanol metabolism by 4-methyl pyrazole abolished its effects on intercellular communication, while 4-methyl pyrazole did not influence the effects of octanol. Acetaldehyde, the main metabolite of ethanol was without effect on gap junctions. Conclusions: This suggests that the inhibition of intercellular communication induced by ethanol may be included among the consequences of intermediary cell metabolism disturbances indirectly due to ethanol oxidation. This may be one of the mechanisms by which ethanol metabolism exerts a hepatotoxic and possibly carcinogenic action.

Inhibition of intercellular communication and induction of ethoxyresorufin- O-deethylase activity by polychlorobiphenyls, -dibenzo- p-dioxins and -dibenzofurans in mouse hepa1c1c7 cells

Inhibition of intercellular communication (IC) between hepa1c1c7 cells was used as a possible bioassay to predict tumor promoting potency of polyhalogenated aromatic hydrocarbons (PAHs). Relative potencies with regard to 2,3,7,8-TCDD to inhibit IC and to induce cytochrome P450IA1/2 (EROD) in these hepa1c1c7 cells were compared in order to investigate the possible role of the Ah receptor (AhR). For the PCDD/F and the co-planar PCB congeners relative potencies of both responses were within the same range. However, the mono-ortho PCBs, 2,3,3′,4,4′-PeCB, 2,3,4,4′,5-PeCB, 2,3′,4,4′,5-PeCB and 2,3,3′,4,4′,5,5′-HxCB showed a 30–1300 times higher potency to inhibit IC compared to EROD induction activity. These potency differences were even more pronounced for the di-ortho PCBs, 2,2′,5,5′-TeCB and 2,2′,3,3′,4,4′-HxCB. The data presented here indicate that for IC inhibition by these non-planar PCBs a non-AhR mediated mechanism, with a different structure-activity relationship may be responsible. Given the high IC inhibition potency of mono- and di-ortho PCBs and their abundancy in environmental mixtures, the mono- and di-ortho PCBs may contribute for a major part to the total tumor promoting potency of complex mixtures relevant to human exposure. Using the traditional TEF values, these compounds do not account for much toxic potency in a mixture, which may imply that the tumor promotion potential is not covered by the commonly derived TEF values.

Mechanistic Studies of the Inhibition of Intercellular Communication by Organochlorine Compounds

Mechanistic Studies of the Inhibition of Intercellular Communication by Organochlorine Compounds

Mechanistic studies of the inhibition of intercellular communication by organochlorine compounds

Mechanistic studies of the inhibition of intercellular communication by organochlorine compounds

Inhibition of intercellular communication by condensates of high and low tar cigarettes

Inhibition of gap junctional intercellular communication (GJIC) is a predictive short term test for tumor promoting activity. A new metabolic cooperation assay has been developed, which takes the cytochrome P-450 metabolism into account. In this assay the inhibitory activity of tobacco smoke condensates (CSC) and CSC fractions from high and low tar cigarettes was tested. CSC of both high and low tar cigarettes and fractions thereof contained tumor promoting activity. The tar yield of the cigarettes did not closely reflect the effects in the GJIC assay and the major constituent nicotine had no effect. The effect was only marginally greater in cells expressing different cytochrome P-450 enzymes, indicating that the active substances are not metabolized by these enzymes. The activities of CSC fractions were considerably lower than the activities in the unfractionated CSC. This may indicate that compounds in the CSC act strongly synergistically. Furthermore, CSC and CSC fractions synergistically inhibit GJIC with the tumor promoter 12-O-tetradecanoylphorbol-13-acetate, indicating different mechanisms of action.

In vitro inhibition of gap junctional intercellular communication by chemical carcinogens

This study was conducted to assess the effects of chemical carcinogens on the gap junction-mediated intercellular communication in cultured mammalian cells. The method of scrape-loading dye transfer of lucifer yellow was adapted as a measure of gap junctional communication. Clone 9 cells derived from rat liver were treated with a model chemical carcinogen, N-methyl- N′-nitro- N-nitrosoguanidine (MNNG) and the gap junctional communication was assessed by measuring the transfer of scrape-loaded lucifer yellow dye. When cells were treated with the carcinogen at 0.3 mg/ml, the fluorescent dye transfer was inhibited by 90% in 60 min. Other chemical agents, which include direct or indirect carcinogens and antitumor drugs, were also examined for their effects on the gap junctional communication. Direct carcinogens, such as MNNG, hydroxylamine and ethidium bromide, exhibited strong inhibition of intercellular communication, while indirect carcinogens, such as aflatoxin B 1 and ethionine, exerted minor effects. Effects of test chemicals on the cell communication through gap junctions were readily quantitated by counting the number of cells stained with the fluorescent dye.

Inhibition of Intercellular Communication via Gap Junction in Cultured Aortic Endothelial Cells by Elevated Glucose and Phorbol Ester

Gap junctional intercellular communication (GJIC) is important in coordinating the cells in maintaining tissue homeostasis and in regulating signal transmission. We examined the effect of elevated glucose on GJIC activity in cultured bovine aortic endothelial cells. GJIC activity was assessed by quantitating the transfer from cell to cell of directly microinjected fluorescent dye molecules. GJIC was activated in the subconfluent monolayer. In this condition, exposing the cells to elevated glucose (400 mg/dl) for 24 hrs significantly inhibited GJIC activity, as compared with low glucose (100mg/dl). This inhibition of GJIC activity induced by elevated glucose was mimicked by addition of 12-O-tetradecanoylphorbol-13-acetate and was restored by addition of staurosporin (10−8 M), a PKC inhibitor. These results suggest that inhibition of GJIC activity induced by elevated glucose probably through activation of PKC may be involved in the vascular endothelial cell dysfunction associated with diabetes.