Modulation Of Gap Junctional Communication Research Articles

Connexin43 in Gap Junctional Intercellular Communication in Astrocytes and Glioma Cells

Gap junctional intercellular communication built by hemichannels on cell membrane is a functional syncytium which allows rapid transfer of ions and molecules between cells. Recent findings have shown that gap junction proteins, and specifically Cx43, can play a significant role in cell migration, tissue formation and organ development, impacting adhesion and cytoskeletal rearrangements. Some literatures have shown that Cx43 was able to contribute to the treatment of gliomas by modulation of gap junctional communication. However, the exact cellular mechanism behind those pharmaceutical efficacies on Cx43 still remains unknown. Hereby, targeting Cx43 on astrocytes in gliomas is mainly discussed.

Gap junctional communication in health and disease.

Gap junctional communication in health and disease.

Cyclic dinucleotides mediate gap junction-dependent spread of DNA-induced IRF3 activation. (IRM9P.729)

Abstract Cell death and sterile inflammation are central to the pathogenesis of human diseases such as myocardial infarction and acute liver failure. Since patients often present after injury has begun, a primary therapeutic goal is to limit the extent and duration of injury. Therefore, we investigated the molecular mechanisms by which innate immune responses to dying cells are spread. When a cell dies, it releases danger signals such as DNA, which are sensed by neighboring cells and spread by undefined mechanisms, ultimately defining the extent and duration of injury. To study this process, we created stable monoclonal fluorescent reporter cell lines for key transcriptional regulators of danger signaling (IRF3 and NFkB), exposed them to various danger signals, and monitored their spatiotemporal activation by quantitative fluorescence microscopy. We found IRF3 to be strongly activated not only in cells sensing DNA, but also in their unexposed neighbors. Using pharmacologic inhibitors and genetically modified co-cultures, we found that spread of IRF3 activity is contact-dependent and requires gap junctions. We also provide evidence that the recently described cyclic dinucleotide cGAMP is a gap junction permeable mediator of IRF3 activation spread. In vivo experiments show that genetic or pharmacologic modulation of gap junction communication is protective against acute organ injury, demonstrating the importance of injury spread, and its promise as a therapeutic target.

Wound healing of cutaneous nitrogen mustard injuries by dynamic modulation of gap junction communication using connexin43 antisense oligodeoxynucleotides (216.6)

Gap junction communication is tightly regulated in skin wound healing. Studies showed that targeting of connexin43 (Cx43) improves skin wound repair in diabetes. Vesicant induced skin injury results in dermal‐epidermal junction disruption and delayed wound repair. We treated Cx43 antisense oligodeoxynucleotides (asODN) on NM exposed SKH‐1 mouse dorsal skin to evaluate the wound healing response. We examined markers of basal keratinocytes, keratin5 (K5); epidermal differentiation, keratin10 (K10); proliferation, Ki67; and basement membrane, laminin 332 (LM332). Immunofluorescent (IF) studies showed reduced K5 expression, loss of K10 and Ki67, and an interrupted LM332 pattern of expression 1 day after NM exposure. By day 3, K10 reappeared in a diffuse pattern. By day 7, the LM332 appearance was thick and continuous. By day 10, there was intense expression of Ki67, accompanied by extreme hyperplasia. Animals treated with Cx43AsODN after NM exposure had an accelerated wound rate and improved reepithelialization so that the skin marker patterns above appeared at earlier timepoints. RT‐PCR and Western blot analysis showed significant reduction of Cx43 at days 3 and 7. IF studies also showed that Cx43 asODN treatment successfully reduced Cx43 expression and modulated higher Cx26 expression in the wound margin by day 3. The use of Cx43asODN may alter Cx expression and accelerate vesicant wound repair.Grant Funding Source: Supported by ES005022, EY09056, and NIAMS U54AR055073

Osteoclastogenesis is Influenced by Modulation of Gap Junctional Communication with Antiarrhythmic Peptides

Osteoclasts are formed by the fusion of mononuclear precursor cells of the monocyte-macrophage lineage. Among several putative mechanisms, gap-junctional intercellular communication (GJC) has been proposed to have a role in osteoclast fusion and bone resorption. We examined the role of GJC in osteoclastogenesis and in vitro bone resorption with mouse bone marrow hematopoietic stem cells and RAW 264.7 cells. Blocking of gap junctions with 18-α-glycyrrhetinic acid (18GA) led to inhibition of osteoclastogenesis and in vitro bone resorption. Similarly, the GJC inhibitor GAP27 inhibited osteoclast formation. GJC modulation with the antiarrhythmic peptides (AAPs) led to increased amounts of multinuclear RAW 264.7 osteoclasts as well as increased number of nuclei per multinuclear cell. In the culture of bone marrow hematopoietic stem cells in the presence of bone marrow stromal cells AAP reduced the number of osteoclasts, and coculture of MC3T3-E1 preosteoblasts with RAW 264.7 macrophages prevented the action of AAPs to promote osteoclastogenesis. The present data indicate that AAPs modulate the fusion of the pure culture of cells of the monocyte-macrophage lineage. However, the fusion is influenced by GJC in cells of the osteoblast lineage.

Improved Corneal Wound Healing through Modulation of Gap Junction Communication Using Connexin43-Specific Antisense Oligodeoxynucleotides

Gap junctions play a major role in corneal wound healing. This study used reproducible models of corneal wound healing to evaluate the effect of a gap junction channel modulator, connexin43 (Cx43) antisense oligodeoxynucleotides (AsODN), on corneal healing dynamics. A mechanical scrape wound model was used to evaluate Cx43 AsODN penetration and initial wound reepithelialization 12 hours postsurgery. Thereafter, detailed analyses of corneal edema, inflammation, and healing were performed in an excimer laser surface ablation model. In vivo confocal microscopy determined clinical parameters (edema, haze) and cellular changes (stromal hypercellularity, reepithelialization), whereas histology and immunohistochemistry were used to quantify stromal edema, inflammation, and reepithelialization. Cx43 AsODN penetrated through the hydrophilic stroma where the epithelium had been removed and accumulated in the basal epithelium close to the wound edge. Twelve hours after scrape wounding, Cx43 AsODN-treated eyes showed a significant reduction in wound area compared with the vehicle alone (1.59±0.37 and 2.29±0.58 mm2, respectively, P<0.01). After excimer laser ablation, stromal edema and inflammation were reduced, with endothelial structures being clearly visible, and reepithelialization rates were again increased in Cx43 AsODN-treated eyes. Histologic analysis confirmed reduced edema in the central wound site and at the periphery of treated corneas (P<0.05), whereas immunohistochemistry showed lower Cx43 levels (P<0.05), reduced myofibroblast activation, and improved epithelial basal lamina deposition in antisense-treated wounds (P<0.01). Application of Cx43 AsODN to the cornea reduces stromal edema and inflammation, promoting faster wound closure and a more uniform repair of the epithelial basal lamina after laser ablation.

Role of Lycopene in the Control of ROS-Mediated Cell Growth: Implications in Cancer Prevention

Dietary intakes of tomatoes and tomato products containing lycopene have been shown to be associated with decreased risk of chronic diseases, such as cancer. Although several mechanisms, including modulation of gap junction communication and enhancement of immune system, are thought to be implicated in its beneficial activities, evidence is accumulating to suggest that lycopene may act as a modulator of intracellular reactive oxygen species (ROS) and, therefore, control ROS-mediated cell growth. According with this, at high concentration, ROS have been reported to be hazardous for living organisms, whereas at moderate concentrations, they play an important role as regulatory mediators in signaling processes regulating cell growth. In this review, we report the available evidence on a role of lycopene as a redox agent in cell proliferation, differentiation and apoptosis. In particular, we focused our attention on lycopene prevention of cell oxidative damage and its influence in cell growth as well as on lycopene modulation of redox-sensitive molecular targets in cell signalling: growth factors and growth factor receptors, antioxidant response elements, MAPKs, transcription factors, such as NF-kB and AP-1, and cytokine expression. Moreover, we speculate on the possible influence that lycopene may have as a redox agent in cancer.

E-cadherin contributes to the bystander effect of TK/GCV suicide therapy and enhances its antitumoral activity in pancreatic cancer models.

The thymidine kinase/ganciclovir (TK/GCV) cancer gene therapy approach is based on inducing GCV metabolite cytotoxicity in tumor cells expressing the herpes simplex virus TK gene and exposed to GCV. A bystander effect, mediated by gap junctions, accounts for the transfer of toxic metabolites from TK-expressing cells to neighboring cells. It has been proposed that E-cadherin participates in the formation and function of such gap junctions. In this study we investigate the influence of E-cadherin on TK/GCV suicide therapy with a panel of cellular and in vivo models of pancreatic ductal adenocarcinoma. We observed a strong correlation of E-cadherin expression and the TK/GCV bystander effect, associated with the modulation of gap junction communication and connexin expression or localization. Importantly, the co-expression of TK and E-cadherin genes in the adenoviral vector AdTat8TKIE improved TK/GCV cytotoxicity and triggered a potent antitumoral effect, superior to standard AdTat8TK/GCV in MIAPaCa-2 xenografts. The increased expression of E-cadherin resulted in the reduction of the bcl-2 content. Interestingly, the knockdown of bcl-2 sensitized cells to TK/GCV. Thus, we propose that by restoring E-cadherin in pancreatic tumor cells we will improve TK/GCV therapy, both by enhancing the bystander effect and by facilitating the induction of apoptosis.

Role of the gap junction in ischemic preconditioning in the heart

The gap junction plays roles not only in electrical coupling of cardiomyocytes but also in intercellular transport of biologically active substances. Furthermore, the gap junction participates in decision making on cell survival versus cell death in various types of cells, and a part of reperfusion injury in the heart has been indicated to be gap junction mediated. The contribution of gap junction communication (GJC) and/or mitochondrial "hemichannels" to protective signaling during the trigger phase of ischemic preconditioning (IPC) is suggested by observations that IPC failed to protect the heart when GJC was blocked during IPC. Although ischemia suppresses both electrical and chemical GJC, chemical GJC persists for a considerable time after electrical GJC is lost. IPC facilitates the ischemia-induced suppression of chemical GJC, whereas IPC delays the reduction of electrical GJC after ischemia. The inhibition of GJC during sustained ischemia and reperfusion by GJC blockers mimics the effect of IPC on myocardial necrosis. IPC induces distinct effects on the interaction of connexin-43 with protein kinases, and the phosphorylation of connexin-43 at Ser368 by PKCepsilon is a primary mechanism of inhibition of chemical GJC by IPC. Several lines of evidence support the notion that the modulation of GJC is a part of the mechanism of IPC-induced protection against myocardial necrosis and arrhythmias, though what percentage of IPC protection is attributable to the inhibition of GJC during ischemia-reperfusion still remains unclear.

TGF‐β induces connexin43 gene expression in normal murine mammary gland epithelial cells via activation of p38 and PI3K/AKT signaling pathways

One of the shared physiological roles between TGF-beta and connexin family members is to inhibit epithelial cell cycle progression and consequently, to provide protection against malignant transformation. Herein, we demonstrated that TGF-beta1 induces the expression of connexin43 (Cx43) in normal murine mammary gland (NMuMG) cell lines at the protein and mRNA levels, and transcriptionally. Using overexpression of a truncated dominant-negative form of Cx43, we determined that the modulation of gap junctional communication by TGF-beta1 plays a key role in the control of NMuMG cells proliferation by TGF-beta1. In addition, using overexpression of truncated dominant-negative forms of either Smad2 or Smad3, and MDA-MB-468 human breast carcinoma cells deficient for Smad4, we determined that the Smad cascade is not implicated in TGF-beta1 effect on Cx43 expression. Using specific pharmacologic inhibitors for JNK, ERK, p38, and PI3K/AKT signaling pathways, we demonstrated the cooperative role of p38 and PI3K/AKT signaling in TGF-beta1-induced Cx43 expression and gap junctional communication. Furthermore, transfection of a c-jun antisense expression vector significantly prevented TGF-beta1-induced Cx43 gene expression demonstrating the involvement of c-Jun/AP-1 pathway together with p38 and PI3K/AKT pathways in mediating TGF-beta1-induced Cx43 gene expression.

Connexin-43 upregulation in micrometastases and tumor vasculature and its role in tumor cell attachment to pulmonary endothelium

BackgroundThe modulation of gap junctional communication between tumor cells and between tumor and vascular endothelial cells during tumorigenesis and metastasis is complex. The notion of a role for loss of gap junctional intercellular communication in tumorigenesis and metastasis has been controversial. While some of the stages of tumorigenesis and metastasis, such as uncontrolled cell division and cellular detachment, would necessitate the loss of intercellular junctions, other stages, such as intravasation, endothelial attachment, and vascularization, likely require increased cell-cell contact. We hypothesized that, in this multi-stage scheme, connexin-43 is centrally involved as a cell adhesion molecule mediating metastatic tumor attachment to the pulmonary endothelium.MethodsTumor cell attachment to pulmonary vasculature, tumor growth, and connexin-43 expression was studied in metastatic lung tumor sections obtained after tail-vein injection into nude mice of syngeneic breast cancer cell lines, overexpressing wild type connexin-43 or dominant-negatively mutated connexin-43 proteins. High-resolution immunofluorescence microscopy and Western blot analysis was performed using a connexin-43 monoclonal antibody. Calcein Orange Red AM dye transfer by fluorescence imaging was used to evaluate the gap junction function.ResultsAdhesion of breast cancer cells to the pulmonary endothelium increased with cancer cells overexpressing connexin-43 and markedly decreased with cells expressing dominant-negative connexin-43. Upregulation of connexin-43 was observed in tumor cell-endothelial cell contact areas in vitro and in vivo, and in areas of intratumor blood vessels and in micrometastatic foci.ConclusionConnexin-43 facilitates metastatic 'homing' by increasing adhesion of cancer cells to the lung endothelial cells. The marked upregulation of connexin-43 in tumor cell-endothelial cell contact areas, whether in preexisting 'homing' vessels or in newly formed tumor vessels, suggests that connexin-43 can serve as a potential marker of micrometastases and tumor vasculature and that it may play a role in the early incorporation of endothelial cells into small tumors as seeds for vasculogenesis.

Different mechanisms of modulation of gap junction communication by non-genotoxic carcinogens in rat liver in vivo

This is a comparative study of the mechanisms by which three different rodent non-genotoxic carcinogens modulate connexin-mediated gap junction intercellular communication in male rat liver in vivo. In the case of the peroxisome proliferating agent Wy-14,643, a non-hepatotoxic dose of 50 mg/kg led to a marked loss of inter-hepatocyte dye transfer associated with a loss of both Cx32 and Cx26 protein expression. In contrast, p, p′-dichlorodiphenyltrichloroethane (DDT) at a non-hepatotoxic dose (25 mg/kg) was not found to alter Cx32 or Cx26 expression or to produce a measurable Cx32 serine phosphorylation but did give a small, significant reduction of cell communication. Carbon tetrachloride (CCl 4) did not affect cell communication (despite a small significant reduction of Cx32 content) at a non-hepatotoxic dose. Both loss of communication and Cx32 expression was observed only at a dose that caused hepatocyte toxicity as evidenced by increased serum alanine aminotransferase activity. Overall, the findings emphasise that loss of gap junctional communication in vivo can contribute to carcinogenesis by non-genotoxic carcinogens through different primary mechanism. In contrast to Wy-14,643 and DDT, the results with CCl 4 are consistent with a requirement for hepatotoxicity in its carcinogenic action.

Closing the Gap at Hot Spots

See related article, pages 1026–1035 Coordination of cellular behavior is indispensable for vascular function. Because of the structural design of the vascular tree only an orchestrated diameter change along the vessel length increases conductance significantly and ensures optimal tissue perfusion. Mechanisms that suffice these needs include locally generated signals at upstream sites which reflect downstream requirements (flow-induced dilation)1 and longitudinal signal transmission through communication channels residing in the vessel wall itself which enable the synchronisation of cellular behavior. This latter is achieved by homocellular gap junctions which are composed of connexins that connect adjacent cells by intercellular low-resistance channels establishing a functional syncitium.2 Electrical communication through these channels is experimentally studied by locally initiated vasomotor responses which conduct along the vessel wall and promote synchronized dilations or constrictions of arteriolar segments reflecting the coordination of cellular behavior.3 Longitudinal communication through gap junctions is also required for the synchronisation of intracellular Ca2+ ([Ca2+]i) changes in smooth muscle and subsequent vasomotion, ie, rhythmic oscillations of vascular diameter. Although the physiological significance of vasomotion is still to be clearly defined it depends critically on Ca2+ release from intracellular stores which occurs in an oscillating fashion and the synchronization of [Ca2+]i transients through gap junctional communication.4,5 In addition, endothelial and smooth muscle cells are coupled heterocellularly (myoendothelial junctions) allowing the direct spread of current from the endothelium to the smooth muscle6 or other signaling molecules.7 The importance of gap junctional communication in vascular function contrasts with the poor knowledge of its regulation in vessels which is complicated by the existence of functional and structural different types of gap junctions. Moreover, longterm regulation because of degradation or upregulation of connexins has to be separated from shortterm effects which reflect an …

Differential roles of 2, 6, and 8 carbon ceramides on the modulation of gap junctional communication and apoptosis during carcinogenesis

The inhibition of apoptosis and gap junctional intercellular communication (GJIC) has been implicated in tumor promotion. Ionizing radiation and oxidative toxicants activate sphingomyelinases resulting in the release of ceramides that control cell proliferation and apoptosis. A rat liver epithelial cell line treated with ceramides containing a 6 (C6) or 8 (C8) carbon acyl-group were potent inhibitors of GJIC and apoptosis, whereas a C2-ceramide was only a weak inhibitor of GJIC and strong inducer of apoptosis. Apoptosis induced by either serum deprivation or C2-ceramide was inhibited by the GJIC inhibitory C8-ceramide. In conclusion, these results suggest that a chronic release of ceramides with acyl groups larger than C6 might act as tumor promoters.

Gap junction modulation by extracellular signaling molecules: the thymus model.

Gap junctions are intercellular channels which connect adjacent cells and allow direct exchange of molecules of low molecular weight between them. Such a communication has been described as fundamental in many systems due to its importance in coordination, proliferation and differentiation. Recently, it has been shown that gap junctional intercellular communication (GJIC) can be modulated by several extracellular soluble factors such as classical hormones, neurotransmitters, interleukins, growth factors and some paracrine substances. Herein, we discuss some aspects of the general modulation of GJIC by extracellular messenger molecules and more particularly the regulation of such communication in the thymus gland. Additionally, we discuss recent data concerning the study of different neuropeptides and hormones in the modulation of GJIC in thymic epithelial cells. We also suggest that the thymus may be viewed as a model to study the modulation of gap junction communication by different extracellular messengers involved in non-classical circuits, since this organ is under bidirectional neuroimmunoendocrine control.

GABAergic modulation of gap junction communication in slice cultures of the rat suprachiasmatic nucleus

We employed morphological and electrophysiological methods in order to elucidate mechanisms which are responsible for communication between cellular oscillators in the cultured rat suprachiasmatic nucleus, the site of the endogenous biological clock that regulates circadian rhythms in mammals. When a gap junction-permeable dye, Lucifer Yellow, was injected into single neurons in the suprachiasmatic nucleus culture, the dye was transferred to neighboring cells in a gap junction blocker-sensitive manner. Optical imaging of neural activity evoked by electrical stimulation in the culture revealed that the spread of depolarization was inhibited by gap junction blockers but not by a blocker of voltage-dependent Na + channels. Depolarization propagation was inhibited by muscimol, a GABA A receptor agonist, in a dose-dependent manner and the inhibition was reversed by bicuculline, a GABA A receptor antagonist. Furthermore, muscimol inhibited dye-transfer between neurons in the suprachiasmatic nucleus culture in a dose-dependent fashion. These independent lines of evidence suggest that the gap junction communication is involved in interneuronal communication in the suprachiasmatic nucleus slice culture and that the coupling state between neurons is not static but dynamically regulated via GABA A receptor systems.

Mitogen-activated protein kinase and phosphorylation of connexin43 are not sufficient for the disruption of gap junctional communication by platelet-derived growth factor and tetradecanoylphorbol acetate.

Disruption of gap junctional communication (GJC) by various compounds, including growth factors and tumor promoters, is believed to be modulated by the phosphorylation of a gap junctional protein, connexin43 (Cx43). We have previously demonstrated a platelet-derived growth factor (PDGF)-induced blockade of GJC and phosphorylation of Cx43 in T51B rat liver epithelial cells expressing wild-type PDGF receptor beta (PDGFr beta). Both of these actions of PDGF required participation of protein kinase C (PKC) and mitogen-activated protein kinase (MAPK). Similar requirements of MAPK were suggested in the modulation of GJC by other agents, including epidermal growth factor (EGF) and lysophosphatidic acid (LPA). Since many of these agents activate additional protein kinases, our present study examined whether activation of MAPK was sufficient for Cx43 phosphorylation and GJC blockade. By utilizing a variety of MAPK activators, we now show that activation of MAPK is not always associated with either Cx43 phosphorylation or disruption of GJC, which suggests a requirement for additional factors. Furthermore, pretreatment with hydrogen peroxide (H2O2), a potent MAPK activator but inefficient GJC/Cx43 modulator, abrogated PDGF- or TPA-induced disruption of GJC. While a 5 min H2O2 pretreatment abolished both PDGF- and TPA-induced Cx43 phosphorylation and GJC blockade, a simultaneous H2O2 treatment interfered only with GJC closure but not with the phosphorylation of Cx43 induced by PDGF and TPA. This finding indicates that, in addition to the Cx43 phosphorylation step, inhibition of GJC requires interaction with other components. H2O2-mediated abrogation of PDGF/TPA signaling can be neutralized by the antioxidant N-acetylcysteine (NAC) or by the tyrosine kinase inhibitor genistein. Taken together, our results suggest that disruption of GJC is not solely mediated by either activated MAPK or Cx43 phosphorylation but requires the participation of additional kinases and regulatory components. This complex mode of regulation is perhaps essential for the proposed functional role of GJC.

Rapid disruption of gap junctional communication and phosphorylation of connexin43 by platelet-derived growth factor in T51B rat liver epithelial cells expressing platelet-derived growth factor receptor.

Gap junctional communication (GJC) between contacting cells has been postulated to be involved in the regulation of cell proliferation. This suggestion stems from numerous studies showing modulation of GJC by agents that influence cellular proliferation. Platelet-derived growth factor (PDGF), a strong mitogen, inhibits GJC in many cell types. To understand the molecular nature of the signal transduction pathway responsible for the GJC blockade, T51B rat liver epithelial cells, which lack endogenous PDGF receptor (PDGFr), were infected with a retrovirus containing either wild-type full-length cDNA of human PDGFr beta (Kin+) or a mutant PDGFr beta lacking receptor tyrosine kinase activity (Kin-). PDGF caused a complete but transient interruption of cell communication in Kin+ cells within 15-20 min of addition. This interruption of GJC was not associated with a gross destabilization of gap junction plaques but with the phosphorylation of connexin43 (Cx43), the only known gap junction protein expressed in these cells. These effects were exhibited in either control T51B cells or in Kin- cells, indicating a requirement of the receptor tyrosine kinase activity. Further examination revealed that the newly phosphorylated Cx43 then undergoes a rapid degradation utilizing the lysosomal pathway resulting in a decreased total Cx43 protein level. The re-establishment of GJC following PDGF treatment was dependent on protein synthesis. This report describes a suitable cell system which is currently being utilized for the characterization of the PDGF signaling pathway responsible for the inhibition of GJC.

Neurotransmitter Modulation of Gap Junctional Communication in the Rat Hippocampus

Increasing experimental evidence indicates that gap junctions can be modulated by neurotransmitters, in particular dopamine. To examine possible modulation of gap junctional communication in the rat hippocampus by neurotransmitters, we studied dye coupling and electrotonic transmission in the CA1 area in the presence of carbachol, a cholinergic agonist, and dopamine agonists. Carbachol markedly reduced dye coupling and the frequency of electrotonic potentials (spikelets). Spikelet amplitudes were decreased in the presence of carbachol. These effects were reversed by the cholinergic antagonist atropine, suggesting a muscarinic action of carbachol on gap junctional function. The non-specific dopamine agonist apomorphine, and the specific D1 receptor agonist SKF 38393, reduced dye coupling between pyramidal cells. Spikelet frequency was also decreased in the presence of dopamine agonists, but less than with carbachol. The specific D1 receptor antagonist, SCH 23390, reversed the effects of both dopamine agonists. These observations indicate that cholinergic and dopaminergic transmission can affect electrical and chemical (dye coupling) communication through gap junctions, and could therefore alter properties of neuronal assemblies, in addition to their effects on intrinsic membrane properties.

Inhibition of gap junctional communication by polyunsaturated fatty acids in WB cells: evidence that connexin 43 is not hyperphosphorylated.

Polyunsaturated fatty acids have attracted much interest due to their wide spectrum of biological activities which include the modulation of gap junctional communication (GJC). Since gap junctions play critical roles in maintaining the functional integrity of organs and tissues, and loss of intercellular communication is associated with a number of pathological conditions, we investigated the effects of the n-6 and n-3 series of polyunsaturated fatty acids and their derivatives on GJC in WB cells as determined by the ability of Lucifer Yellow-loaded cells to transfer the dye to neighbouring recipient cells. Studies were also conducted to investigate the possible mechanisms of action of the fatty acids. Treatment of cells with 10 microM arachidonic acid (20:4 n-6) resulted in a rapid and transient loss of communication competence. The response to 20 microM 20:4 (n-6) was prolonged (> 210 min) but was readily reversible by washing the cells with fatty acid-free bovine serum albumin. Cells which had regained their communication competence responded to further additions of 20:4 (n-6). The fatty acids, 18:3 (n-6), 20:5 (n-3), 22:6 (n-3) and the 15-hydroxy- and the 15-hydroperoxy-derivatives of 20:4 (n-6) were also powerful inhibitors of GJC, while 23:4 (n-6) was a relatively weak inhibitor. The saturated 20 carbon fatty acid, 20:0, and the methyl ester of 20:4 (n-6) were without effect. This illustrates the importance of unsaturation and the carboxyl group as structural requirements for activity. 20:4 (n-6)-induced inhibition of dye transfer was not attenuated by pretreating the cells with either phorbol-12-myristate-13-acetate (PMA) or indomethacin, suggesting that regulation of gap junctional permeability by 20:4 (n-6) in WB cells was neither dependent on PMA-responsive isozymes of protein kinase C nor required the metabolism of the fatty acids by cyclo-oxygenase. However, the effect of 20:4 (n-6) was antagonized by preincubating WB cells with either nordihydroguaiaretic acid or (+/-)-isoproterenol and isobutylmethyl-xanthine. Western blot analysis of connexin 43 (Cx43), the major gap junctional protein expressed in these cells, revealed no detectable changes to the electrophoretic mobility of Cx43 even after 60 min of incubation in the presence of 20:4 (n-6). As expected, other inhibitors of gap junctional permeability including epidermal growth factor, phorbol ester or lysophosphatidic acid induced a retardation in the mobility of Cx43, indicating an enhancement in the phosphorylation of Cx43 protein.(ABSTRACT TRUNCATED AT 400 WORDS)