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PubMed1997

Effects of melatonin on proliferation, oxidative stress and Cx32 gap junction protein expression in primary cultures of adult rat hepatocytes.

Melatonin has antiproliferative and antioxidant effects on cells in vivo and in vitro. Gap junctions mediate the communication between adjacent cells and are closely related to cellular growth and oxidative stress. We previously reported that 2% dimethylsulfoxide (DMSO) which has antiproliferative and antioxidative effects on hepatocytes, induces connexin 32 (Cx32) gap junction protein in primary cultures of adult rat hepatocytes. In the present study, we have examined the effects of melatonin on proliferation, oxidant stress and Cx32 gap junction protein expression in the cultured rat hepatocytes as compared to 2% DMSO treatment. 10(-2) M melatonin significantly inhibited the proliferation and the oxidative stress of the cells, and markedly induced Cx32 gap junction protein expression and gap junctional intercellular communication (GJIC). These effects of 10(-2) M melatonin treatment were not due to cytotoxicity to the cultured rat hepatocytes and they were as strong as those of 2% DMSO treatment. These results suggested that melatonin might be a useful substance to maintain the functions of the hepatocytes in vitro by modulating the levels of proliferation, oxidative stress and gap junction expression.

Animals↗
PubMed1982

Disrupted communication between late-stage premalignant human colon epithelial cells by 12-O tetradecanoylphorbol-13-acetate.

Premalignant epithelial cells derived from benign tumors of the human colon fall into three classes based on the properties of the cultured cells and the pathology and clinical prognosis of the tumors themselves. Cellular communication was compared by injection of fluorescein into single cells in primary cultures of late- and early-stage premalignant cells, in primary cultures of colon adenocarcinomas, and in cultures of a tumorigenic established human colon carcinoma cell line. Many cells in both types of premalignant cultures appeared highly coupled inasmuch as the dye was often detected in several cells adjacent to the injection site. Thus, most premalignant cells within a colony were in communication with the surrounding cells. In contrast, many malignant cells were uncoupled and passed dye to few, if any, neighboring cells. The tumor promoter 12-O-tetradecanoyl-phorbol-13-acetate caused extensive uncoupling of cells in late-stage premalignant cultures, while not eliminating the extensively coupled areas found in early-stage premalignant cultures. 12-O-Tetradecanoyl-phorbol-13-acetate had no effect on coupling between tumorigenic cells from an established carcinoma cell line which were already extensively uncoupled. Tumor promoters may play an important role in the transition of late-stage premalignant cells to malignancy by decreasing intercellular communication, between premalignant cells at this specific stage in tumor development.

Adenoma↗
PubMed1999

Cell-to-cell communication via plant endomembranes.

Cell-to-cell communication was investigated in epidermal cells cut from stem internodal tissue of Nicotiana tabacum and Torenia fournieri. Fluorescently labelled peptides and dextrans were microinjected using iontophoresis into the cytoplasm andcortical endomembrane network of these cells. The microinjected endomembrane network was similar in location and structure to the endoplasmic reticulum (ER) as revealed by staining with 3, 3'-dihexyloxacarbocyanine iodide (DiOC(6)). No cell-to-cell movement of dextrans was observed following cytoplasmic injections but injection of dextrans into the endomembrane network resulted in rapid diffusion of the probes to neighbouring cells. It is proposed that the ER acts as a pathway for intercellular communication via the desmotubule through plasmodesmata.

Carbocyanines↗
PubMed1996

Effect of tumor-promoting and anti-promoting chemicals on the viability and junctional coupling of human HeLa cells transfected with DNAs coding for various murine connexin proteins.

Gap-junctional intercellular communication is thought to be essential for maintaining cellular homeostasis and growth control. Its perturbation entails toxicological implications and it has been correlated with the in vivo tumor-promoting potential of chemicals. Little is known about the mechanism(s) responsible for the tumor promoters interference with the cellular coupling. Moreover, nongenotoxic carcinogens, as well as connexins (gap-junctional protein subunits), are known to be organ-/tissue-specific; this implies that the effect of different agents should be evaluated on their specific target, that is, connexin. To investigate the role of different connexins in regulating gap-junctional gating and to compare the properties of homotypic junctional channels, we evaluated the effects of tissue-specific tumor promoters and anti-promoters on the viability and intercellular coupling (dye-transfer) of HeLa cells stably transfected with cDNAs coding for connexin(cx)43, cx40, cx26 and cx32. The results demonstrate that the transfectants possess individual junctional permeabilities, differentially affected by the chemicals, they also show different sensitivities to the cytotoxic effect of the compounds. These findings confirm that connexin diversity may be responsible for the different gating properties of gap-junctional channels, being also suggestive for their separate functions and independent regulatory mechanisms.

Animals↗
PubMed2001

Regulation of gap junctional communication in CFTR-expressing pancreatic epithelial cells.

Gap junction channels provide a pathway for coordinating multicellular activity. To evaluate the contribution of cell-to-cell communication in the function of epithelial cells, we studied the strength of gap junctional coupling in pancreatic acinar and duct cells exposed to agents known to elevate the intracellular concentration of Ca(2+) or cAMP. In acinar cells, we observed that maximal concentrations of acetylcholine evoked a biphasic increase in cytosolic Ca(2+) mobilization. The second sustained phase, which depends on Ca(2+) influx into the cell, was associated with the rapid closure of gap junction channels. In duct cells, stimulation of CFTR-dependent Cl(-) currents with cAMP analogs markedly increased gap junctional conductance in pairs of cells. Interestingly, cAMP had no effect on intercellular communication between cells harboring the DeltaF508 mutation of CFTR. An abnormal pattern of gap junctional coupling may contribute to the altered functions of tissues affected in cystic fibrosis.

Acetylcholine↗
PubMed2005

The role of human adult stem cells and cell-cell communication in cancer chemoprevention and chemotherapy strategies.

Since carcinogenesis is a multi-stage, multi-mechanism process, involving mutagenic, cell death and epigenetic mechanisms, during the "initiation/promotion/and progression" phases, chemoprevention must be based on understanding the underlying mechanism(s) of each phase, In principle, prevention of each of these phases could reduce the risk to cancer. However, because reducing the mutagenic/initiation phase to a zero level is impossible, the most efficacious intervention would be at the promotion phase that requires a sustained exposure to promoting conditions/agents. In addition, assuming the "target" cells for carcinogenesis are the pluri-potent stem cells and their early progenitor or transit cells, chemoprevention strategies for inhibiting the promotion of these two types of pre-malignant "initiated" cells will require different kinds of agents. A hypothesis will be proposed that involves adult stem cells, which express Oct-4 gene and lack gap junctional intercellular communication (GJIC-) or the early progenitor cells which express GJIC+ and are partially-differentiated, if initiated, will be promoted by agents that either inhibit secreted negative growth regulators or by inhibitors of GJIC. Consequently, anti-tumor promoting chemopreventing agents to each of these two types of initiated cells must have different mechanisms of action and work on different target cells. Assuming stem cells are target cells for carcinogenesis, an alternative method of chemoprevention would be to reduce the stem cell pool. Many classes of anti-tumor promoter chemopreventive agents, such as green tea components, resveratrol, caffeic acid phenethylene ester, either up-regulate GJIC in stem cells or prevent the down regulation of GJIC by tumor promoters in early progenitor cells.

Adult↗
PubMed1989

cAMP delays disappearance of gap junctions between pairs of rat hepatocytes in primary culture.

Freshly isolated adult rat hepatocytes were found to be coupled through gap junctions, but coupling decreased abruptly 5-8 h after plating the cells on plastic culture dishes in physiological saline containing insulin and fetal calf serum. Loss of intercellular coupling was associated with disappearance of 27-kDa gap junction protein and of gap junctions seen by electron microscopy or immunocytochemistry. This disappearance of coupling was delayed approximately 8 h by treatment of the cultures with membrane permeant adenosine 3',5'-cyclic monophosphate (cAMP) [but not guanosine 3',5'-cyclic monophosphate (cGMP)] derivatives. Levels of gap junction protein and anatomically identified junctions were also maintained by 8-bromoadenosine 3',5'-cyclic monophosphate (8-BrcAMP). Level of mRNA encoding the gap junction protein was maintained longer in cells treated with 8-BrcAMP than in untreated cells, but 8-BrcAMP did not detectably increase the transcription rate. Thus prolongation of coupling must result at least partially from extension of the lifetime of gap junction mRNA, allowing translation of message and assembly of channels for a longer period after plating. Treatment of cells with mRNA or protein synthesis inhibitors (alpha-amanitin and cycloheximide) prolonged coupling to a similar extent as did treatment with 8-BrcAMP. alpha-Amanitin blocked transcription of gap junction mRNA, but levels of cytoplasmic mRNA encoding the 27-kDa gap junction protein were maintained, presumably by block of transcription of an mRNA degrading factor. The factor is probably a protein, since a similar effect on mRNA level was produced in cycloheximide-treated cells. Cells cultured in control medium were also observed to flatten as they became uncoupled, whereas cells cultured for as long as 16 h in elevated 8-BrcAMP remained round and well coupled. The correlation between shape and coupling strength was not obtained after treatment with the microtubule stabilizing agent, taxol, which maintained the spherical shape of the cells but did not delay the disappearance of dye coupling. Nocodazole, which blocks microtubule formation, also maintained the spherical shape of the cells but delayed the disappearance of dye coupling. In addition to gating by covalent modification or other mechanisms, hormones and drugs that alter the intracellular cAMP concentration may affect intercellular communication by changing the lifetime of the mRNA encoding the main gap junction protein, thereby decreasing or increasing its synthesis. In addition, cAMP may act by decreasing removal of junctions from appositional membranes.

8-Bromo Cyclic Adenosine Monophosphate↗
PubMed2000

The epsilon subtype of protein kinase C is required for cardiomyocyte connexin-43 phosphorylation.

Gap junctions (GJs), composed of connexins, are intercellular channels ensuring electric and metabolic coupling between cardiomyocytes. We have shown previously that an endogenous mitogenic and cardioprotective protein, fibroblast growth factor-2 (FGF-2), decreases cardiomyocyte GJ permeability by stimulating phosphorylation of connexin-43 (Cx43). Identifying the kinase(s) phosphorylating cardiac Cx43 may thus provide a way of modulating cardiac intercellular communication. Because FGF-2 activates receptors linked to protein kinase C (PKC) and mitogen-activated protein kinase, we first investigated participation of these enzymatic systems in Cx43 phosphorylation. The inhibitor PD98059 blocked activation of mitogen-activated protein kinase, but it did not prevent the FGF-2 effects on GJs. In contrast, the PKC inhibitor chelerythrine blocked the effects of FGF-2 on Cx43 phosphorylation and permeability. Because the epsilon-isoform of PKC localizes to plasma membrane sites, we examined whether it is directly involved in the FGF-2-induced Cx43 phosphorylation. In nonstimulated myocytes, PKCepsilon displayed a discontinuous pattern of localization at intercellular contact sites and partial colocalization with Cx43. Treatment with FGF-2 or phorbol 12-myristate 13-acetate induced a more continuous pattern of PKCepsilon distribution, whereas the anti-Cx43 staining appeared to overlap extensively with that of PKCepsilon. In immunoprecipitation experiments using specific anti-Cx43 antibodies, PKCepsilon but not PKCalpha coprecipitated with Cx43. FGF-2 increased levels of coprecipitated PKCepsilon, suggesting increased association between PKCepsilon and Cx43 on stimulation. Transient gene transfer and overexpression of cDNAs coding for truncated or mutated dominant-negative forms of PKCepsilon decreased cardiomyocyte Cx43 phosphorylation significantly. We conclude that PKC mediates the FGF-2-induced effects on cardiac GJs and that PKCepsilon likely interacts with and phosphorylates cardiac Cx43 at sites of intercellular contact.

Alkaloids↗
PubMed2000

Phosphorylation of connexin43 on serine368 by protein kinase C regulates gap junctional communication.

Phorbol esters (e.g., TPA) activate protein kinase C (PKC), increase connexin43 (Cx43) phosphorylation, and decrease cell-cell communication via gap junctions in many cell types. We asked whether PKC directly phosphorylates and regulates Cx43. Rat epithelial T51B cells metabolically labeled with (32)P(i) yielded two-dimensional phosphotryptic maps of Cx43 with several phosphopeptides that increased in intensity upon TPA treatment. One of these peptides comigrated with the major phosphopeptide observed after PKC phosphorylation of immunoaffinity-purified Cx43. Purification of this comigrating peptide and subsequent sequencing indicated that the phosphorylated serine was residue 368. To pursue the functional importance of phosphorylation at this site, fibroblasts from Cx43(-/-) mice were transfected with either wild-type (Cx43wt) or mutant Cx43 (Cx43-S368A). Intercellular dye transfer studies revealed different responses to TPA and were followed by single channel analyses. TPA stimulation of T51B cells or Cx43wt-transfected fibroblasts caused a large increase in the relative frequency of approximately 50-pS channel events and a concomitant loss of approximately 100-pS channel events. This change to approximately 50-pS events was absent when cells transfected with Cx43-S368A were treated with TPA. These data strongly suggest that PKC directly phosphorylates Cx43 on S368 in vivo, which results in a change in single channel behavior that contributes to a decrease in intercellular communication.

Animals↗
PubMed2001

Gap junctional communication underpins EDHF-type relaxations evoked by ACh in the rat hepatic artery.

Synthetic peptides homologous to the Gap 26 and Gap 27 domains of the first and second extracellular loops of the major vascular connexins (Cx37, Cx40, and Cx43) have been used to investigate the role of gap junctions in endothelium-derived hyperpolarizing factor (EDHF)-type relaxations of the rat hepatic artery. These peptides were designated 37,40Gap 26, 43Gap 26, 37,43Gap 27, and 40Gap 27, according to connexin specificity. When administered at 600 microM, none of the peptides individually affected maximal EDHF-type relaxations to ACh. By contrast, at 300 microM each, paired peptide combinations targeting more than one connexin subtype attenuated relaxation by up to 50%, and responses were abolished by the triple peptide combination 43Gap 26 + 40Gap 27 + 37,43Gap 27. In parallel experiments with A7r5 cells expressing Cx40 and Cx43, neither 43Gap 26 nor 40Gap 27 affected intercellular diffusion of Lucifer yellow individually but, in combination, significantly attenuated dye transfer. The findings confirm that functional cell-cell coupling may depend on more than one connexin subtype and demonstrate that direct intercellular communication via gap junctions constructed from Cx37, Cx40, and Cx43 underpins EDHF-type responses in the rat hepatic artery.

Acetylcholine↗
PubMed2006

The gap junction cellular internet: connexin hemichannels enter the signalling limelight.

Cxs (connexins), the protein subunits forming gap junction intercellular communication channels, are transported to the plasma membrane after oligomerizing into hexameric assemblies called connexin hemichannels (CxHcs) or connexons, which dock head-to-head with partner hexameric channels positioned on neighbouring cells. The double membrane channel or gap junction generated directly couples the cytoplasms of interacting cells and underpins the integration and co-ordination of cellular metabolism, signalling and functions, such as secretion or contraction in cell assemblies. In contrast, CxHcs prior to forming gap junctions provide a pathway for the release from cells of ATP, glutamate, NAD+ and prostaglandin E2, which act as paracrine messengers. ATP activates purinergic receptors on neighbouring cells and forms the basis of intercellular Ca2+ signal propagation, complementing that occuring more directly via gap junctions. CxHcs open in response to various types of external changes, including mechanical, shear, ionic and ischaemic stress. In addition, CxHcs are influenced by intracellular signals, such as membrane potential, phosphorylation and redox status, which translate external stresses to CxHc responses. Also, recent studies demonstrate that cytoplasmic Ca2+ changes in the physiological range act to trigger CxHc opening, indicating their involvement under normal non-pathological conditions. CxHcs not only respond to cytoplasmic Ca2+, but also determine cytoplasmic Ca2+, as they are large conductance channels, suggesting a prominent role in cellular Ca2+ homoeostasis and signalling. The functions of gap-junction channels and CxHcs have been difficult to separate, but synthetic peptides that mimic short sequences in the Cx subunit are emerging as promising tools to determine the role of CxHcs in physiology and pathology.

Animals↗
PubMed1985

Intercellular junctions in human tooth-pulp cells in culture in vitro revealed by freeze-fracture, lanthanum impregnation and filipin treatment.

Three kinds of intercellular junctions were detected between human dental pulp cells in explant culture with electron microscopy included filipin detection for cholesterol; desmosome-like junctions observed on ultrathin sections probably contribute to the cohesiveness between cells in culture. Gap junctions, responsible for intercellular communication, exhibited two morphologies on freeze-fracture replicas: a conventional arrangement of their intramembranous particles and a crystalline array corresponding to the formation stage of junctions. Primitive tight junctions were detected on freeze-fracture replicas but not on ultrathin sections. It is likely that they contribute to the cell-to-cell adhesion under culture conditions.

Cells, Cultured↗
PubMed1981

Fine structure of the fully keratinized hair cuticle in the head hair of the human.

The cuticle is composed of five cell layers in the cross section of the hair at a level of 100 mm above the scalp. Intercellular junctions between two cuticular cells are zonulae occludentes (tight junction); they consist of five layers. The gap junctions of the intercellular communications occur near the cortex. The trichohyalin granule appears in the cuticular cells of completely cornified head hair. The granule is generally smaller than that of the keratohyalin granule, and spherical in shape. The granule is not visible in the area near the surface of the hair. The keratin in the cytoplasm of the cuticular cell may originate from the trichohyalin granule.

Adult↗
PubMed2006

Cell deformation and micromechanical environment in the intervertebral disc.

The purpose of this research was to explore the in situ anatomic and mechanical environment of disc cells. Laser scanning confocal microscopy was used to characterize three-dimensional morphology of intervertebral disc cells, micromechanical deformation and interaction with extracellular matrix, and functional intercellular communication. Bovine coccygeal discs were used for both the anatomic and micromechanical investigations. Anulus fibrosus cells had a complex morphology with sinuous processes woven into the extracellular matrix, particularly in the outer aspect of the anulus where they were also interconnected via functional gap junctions. They were also found in an extensive pericellular matrix of type-VI collagen, joining as many as ten cells into linear cell arrays that could be extracted from the matrix as functional units. Mechanically, collagen fibril sliding was demonstrated to govern cell mechanics and strain transfer in the anulus fibrosus during loading activities. Lamellar cells were largely protected from direct tensile strains in the matrix, with minimal intercellular strains. However, intercellular strains between lamellar cells in adjacent arrays were large, illustrating shearing between linear cell arrays. Appreciable shear was observed across the lamellar cell bodies as well as to the cellular processes woven into the matrix. These findings demonstrated the morphologic and micromechanical complexity of anulus fibrosus cells. The knowledge of the in situ environment of disc cells will provide a base to investigate the mechanical implications of disc degeneration on the cellular environment and to better understand how mechanical and genetic risk factors can impact the cells that are essential to maintaining the intervertebral disc.

Animals↗
PubMed2003

Zebrafish Cx35: cloning and characterization of a gap junction gene highly expressed in the retina.

The vertebrate connexin gene family encodes protein subunits of gap junction channels, which provide a route for direct intercellular communication. Consequently, gap junctions play a vital role in many developmental and homeostatic processes. Aberrant functioning of gap junctions is implicated in many human diseases. Zebrafish are an ideal vertebrate model to study development of the visual system as they produce transparent embryos that develop rapidly, thereby facilitating morphological and behavioral testing. In this study, zebrafish connexin35 has been cloned from a P1 artificial chromosome (PAC) library. Sequence analysis shows a high degree of similarity to the Cx35/36 orthologous group, which are expressed primarily in nervous tissue, including the retina. The gene encodes a 304-amino acid protein with a predicted molecular weight of approximately 35 kDa. Injection of zebrafish Cx35 RNA into paired Xenopus oocytes elicited intercellular electrical coupling with weak voltage sensitivity. In development, Cx35 is first detectable by Northern analysis and RT-PCR, at 2 days post-fertilization (2 dpf), and in the adult it is expressed in the brain and retina. Immunohistochemical analysis revealed that the Cx35 protein is expressed in two sublaminae of the inner plexiform layer of the adult retina. A similar pattern was seen in the 4 and 5 dpf retina, but no labeling was detected in the retina of earlier embryos.

Animals↗
PubMed1994

Regulation of cell-cell communication mediated by connexin 43 in rabbit myometrial cells.

The aim of this study was to evaluate the rapid regulation of cell-cell communication by using the microinjection of purified cAMP-dependent protein kinase (protein kinase A), the Ca(2+)-phospholipid-dependent protein kinase (protein kinase C), or the inhibitor proteins (PKI and CKI) that are, respectively, specific for each of these enzymes. Gap junction phenotypes of myometrial tissue and cells were studied by means of immunocytochemistry with antibody to connexin 43 (alpha 1; Cx43). Cells were enzymatically disaggregated from myometrium of nonpregnant, mid-pregnant (Day 14), and late-pregnant (Day 29) rabbit uteri (n = 8 per group) and seeded at high density such that after 4 days, cultures had the appearance of a cross-sectioned myometrium. Purified proteins and their subunits were microinjected, and intercellular communication was evaluated by monitoring Lucifer Yellow dye transfer. Cultures were treated with 0.5 mM 8Br-cAMP (8-bromo adenosine 3',5' cyclic monophosphate) or 10 microM OAG (1-oleoyl-2-acetyl-sn-glycerol), which, respectively, activate protein kinase A and protein kinase C. Immunoreactive Cx43 and cell-cell communication were examined 5 min to 2 h later. Cx43 was detected in myometrial cryosections and cultured cells by indirect immunofluorescence, and its expression increased with gestation. Exposure to 8Br-cAMP increased the amount of immunoreactive Cx43. Basal dye transfer was minimal in nonpregnant cells, increased in cells of mid-pregnant uteri, and was maximal in late-pregnant cells. Treatment with 8Br-cAMP enhanced transfer in mid- and late-pregnant cells but had no obvious effect on cells from nonpregnant animals. OAG treatment inhibited dye transfer in greater than 95% of the cells tested irrespective of pregnancy status. PKI inhibited cell-cell communication within 2 min and up to 40 min. Injection of free catalytic subunit of protein kinase A following PKI inhibition restored communication within 2-3 min, with maximal transfer in 4-5 min. Protein kinase C inhibited communication, which resumed in < 3 min after injection of CKI. We conclude that rabbit myometrial cells engage in Cx43-mediated cell-cell communication and that this process increases during pregnancy. Further, activators of protein kinase A or injected free catalytic subunit rapidly enhances cell-cell communication, whereas activators of protein kinase C or the enzyme itself diminishes this process.

8-Bromo Cyclic Adenosine Monophosphate↗
PubMed2002

Phospholipase-mediated inhibition of spontaneous oscillatory uterine contractions by lindane in vitro.

Although regulation of uterine contractility is fundamental for parturition, mechanisms by which toxicants modify uterine muscle contractions remain poorly understood. In a previous cumulative concentration-response study, 10 microM lindane (gamma-hexachlorocyclohexane) reduced contraction force and 30 microM lindane abolished contractions in Gestation Day 10 rat uterine strips when lindane was added to muscle baths at 10-min intervals. Other studies showed that brief (<10 min) exposures to 10-100 microM lindane inhibit gap junctions and activate phospholipase pathways in rat myometrial cells in culture. Consequently, lindane was used as a prototype toxicant with known uterine activity to investigate the hypothesis that activation of a specific phospholipase pathway provides a mechanistic link between inhibition of uterine contraction and inhibition of myometrial gap junctions. Uterine tissue and cells were pretreated with phospholipase pathway inhibitors to evaluate the role of phospholipase pathways in lindane's actions in the uterus. Concentrations of inhibitors were selected based on previous reports of effective concentrations for the enzyme activity and on pilot toxicity studies of the inhibitors on uterine contraction and gap junction communication. To monitor uterine contractions, longitudinal uterine strips were excised from Gestation Day 10 rats and suspended in isometric muscle baths, consistent with previous experiments. Exposure in vitro for 60 min to 10-50 microM lindane, an effective concentration range for the uterine responses of interest, revealed that 30 microM lindane rapidly abolished contractions. Subsequently, uterine strips were pretreated with phospholipase pathway inhibitors and then challenged with 30 microM lindane, the lindane concentration that elicited maximal inhibition of uterine contraction. Pretreatment with 20-50 microM of the phosphatidylinositol-specific phospholipase C inhibitor 1-O-octadecyl-2-O-methyl-sn-glycerol-3-phosphorylcholine (ET-18-OCH(3)) reversed lindane-induced inhibition of spontaneous uterine contractions. Gap junction intercellular communication was monitored by injecting the fluorescent dye Lucifer yellow into rat myometrial cells grown in culture and assessing dye transfer to adjacent cells using epifluorescence microscopy. Similar to uterine contraction, pretreatment of cell cultures with phospholipase C inhibitors (30 microM ET-18-OCH(3), 50 microM tricyclodecan-p-yl-xanthogenate.K [D609] or 50 microM tricyclodecan-p-yl-xanthogenate.K or 2-nitro-4-carboxyphenyl-N,N-dophenylcarbamate [NCDC]) partially reversed inhibition of dye transfer by 100 microM lindane, a lindane concentration previously shown to abolish myometrial Lucifer yellow dye transfer under similar culture conditions. In contrast, pretreatment with 20 microM of bromoenol lactone (BEL) to inhibit the calcium-independent phospholipase A(2) or 100 mM ethanol to interrupt the phospholipase D pathway failed to prevent inhibition of spontaneous uterine contractions and inhibition of Lucifer yellow dye transfer by lindane (100 microM). These data suggest that lindane inhibits myometrial gap junctions and spontaneous oscillatory contractions by a phospholipase C-mediated pathway.

Animals↗
PubMed2001

Phosphorylation of connexin43 and inhibition of gap junctional communication in 12-O-tetradecanoylphorbol-13-acetate-exposed R6 fibroblasts: minor role of protein kinase C beta I and mu.

12-O:-tetradecanoylphorbol-13-acetate (TPA) inhibits gap junctional communication in many cell culture systems, but TPA-induced phosphorylation of the gap junction protein connexin43 (Cx43) varies much between systems. We have here studied whether these responses and their sensitivities can be correlated with total protein kinase C (PKC) enzyme activity and if specific PKC isoenzymes are involved. Rat R6 fibroblasts transfected with the cDNA sequence encoding PKC beta I (R6-PKC3) had a total PKC activity 7- to 16-fold higher than the corresponding control cells (R6-C1), depending on the selection pressure (G418 concentration). Still, R6-PKC3 cells were no more sensitive than R6-C1 cells to TPA-induced down-regulation of communication, except at the highest selection pressure (500 micrograms/ml G418). Thus, total PKC activity does not indicate absolute sensitivity of a cell system to TPA-induced suppression of communication, but within a certain cell system increasing PKC activity may enhance the sensitivity to TPA in this respect. The results also suggest that PKC beta I is of minor importance for TPA-induced regulation of communication. Experiments with the Lilly compound 379196, a PKC beta-specific inhibitor, further supported this conclusion. Except for PKC beta I in R6-PKC3 cells, both cell lines contained the TPA-responsive PKC isoenzymes alpha, delta, epsilon and mu. Long-term treatment with TPA caused strong down-regulation of PKC alpha, delta and epsilon, but little down-regulation of PKC mu. Concurrently, the cells became refractory to repeated exposure to TPA, indicating that PKC mu is of minor importance. Experiments with the general PKC inhibitor GF109203X and the PKC alpha (and beta/gamma) inhibitor Gö6976 suggested that both classical (alpha) and novel PKCs (delta and epsilon) might be involved in TPA-induced suppression of intercellular communication, while phosphorylation of Cx43 may mainly be mediated by PKC alpha in the present systems.

Animals↗