Increased Gap Junctional Communication Research Articles

Single and fractionated ionizing radiation induce alterations in endothelial connexin expression and channel function

Radiotherapy is an effective treatment for most tumor types. However, emerging evidence indicates an increased risk for atherosclerosis after ionizing radiation exposure, initiated by endothelial cell dysfunction. Interestingly, endothelial cells express connexin (Cx) proteins that are reported to exert proatherogenic as well as atheroprotective effects. Furthermore, Cxs form channels, gap junctions and hemichannels, that are involved in bystander signaling that leads to indirect radiation effects in non-exposed cells. We here aimed to investigate the consequences of endothelial cell irradiation on Cx expression and channel function. Telomerase immortalized human Coronary Artery/Microvascular Endothelial cells were exposed to single and fractionated X-rays. Several biological endpoints were investigated at different time points after exposure: Cx gene and protein expression, gap junctional dye coupling and hemichannel function. We demonstrate that single and fractionated irradiation induce upregulation of proatherogenic Cx43 and downregulation of atheroprotective Cx40 gene and protein levels in a dose-dependent manner. Single and fractionated irradiation furthermore increased gap junctional communication and induced hemichannel opening. Our findings indicate alterations in Cx expression that are typically observed in endothelial cells covering atherosclerotic plaques. The observed radiation-induced increase in Cx channel function may promote bystander signaling thereby exacerbating endothelial cell damage and atherogenesis.

Follicle-Stimulating Hormone Increases Gap Junctional Communication Between Somatic and Germ-Line Follicular Compartments During Murine Oogenesis.

Germ cells develop in intimate contact and communication with somatic cells of the gonad. In female mammals, oocyte development depends crucially on gap junctions that couple it to the surrounding somatic granulosa cells of the follicle, yet the mechanisms that regulate this essential intercellular communication remain incompletely understood. Follicle-stimulating hormone (FSH) drives the terminal stage of follicular development. We found that FSH increases the steady-state levels of mRNAs encoding the principal connexins that constitute gap junctions and cadherins that mediate cell attachment. This increase occurs both in granulosa cells, which express the FSH-receptor, and in oocytes, which do not. FSH also increased the number of transzonal projections that provide the sites of granulosa cell-oocyte contact. Consistent with increased connexin expression, FSH increased gap junctional communication between granulosa cells and between the oocyte and granulosa cells, and it accelerated oocyte development. These results demonstrate that FSH regulates communication between the female germ cell and its somatic microenvironment. We propose that FSH-regulated gap junctional communication ensures that differentiation processes occurring in distinct cellular compartments within the follicle are precisely coordinated to ensure production of a fertilizable egg.

Activation of 5' adenosine monophosphate-activated protein kinase blocks cumulus cell expansion through inhibition of protein synthesis during in vitro maturation in Swine.

The serine/threonine kinase 5' adenosine monophosphate-activated protein kinase (AMPK), a heterotrimeric protein known as a metabolic switch, is involved in oocyte nuclear maturation in mice, cattle, and swine. The present study analyzed AMPK activation in cumulus cell expansion during in vitro maturation (IVM) of porcine cumulus-oocyte complexes (COC). 5-Aminoimidazole-4-carboxamide-1-beta-d-ribofuranoside (AICAR) is a well-known activator of AMPK. It inhibited oocyte meiotic resumption in COC. Moreover, cumulus cell expansion did not occur in the presence of AICAR, demonstrating its marked impact on cumulus cells. Activation of AMPK was supported by AICAR-mediated phosphorylation of alpha AMPK subunits. Furthermore, the presence of AICAR increased glucose uptake, a classical response to activation of this metabolic switch in response to depleted cellular energy levels. Neither nuclear maturation nor cumulus expansion was reversed by glucosamine, an alternative substrate in hyaluronic acid synthesis, through the hexosamine biosynthetic pathway, which ruled out possible depletion of substrates. Both increased gap junction communication and phosphodiesterase activity in COC are dependent on protein synthesis during the initial hours of IVM; however, both were inhibited in the presence of AICAR, which supports the finding that activation of AMPK by AICAR mediated inhibition of protein synthesis. Moreover, this protein synthesis inhibition was equivalent to that of the well-known protein synthesis inhibitor cycloheximide, as observed on cumulus expansion and protein concentration. Finally, the phosphorylation level of selected kinases was investigated. The pattern of raptor phosphorylation is supportive of activation of AMPK-mediated inhibition of protein synthesis. In conclusion, AICAR-mediated AMPK activation in porcine COC inhibited cumulus cell expansion and protein synthesis. These results bring new considerations to the importance of this kinase in ovarian physiology and to the development of new oocyte culture medium.

Ouabain Increases Gap Junctional Communication in Epithelial Cells

Background/Aims: The finding that endogenous ouabain acts as a hormone prompted efforts to elucidate its physiological function. In previous studies, we have shown that 10 nM ouabain (i.e., a concentration within the physiological range) modulates cell-cell contacts such as tight junctions and apical/basolateral polarity. In this study, we examined whether 10 nM ouabain affects another important cell-cell feature: gap junction communication (GJC). Methods: We employed two different approaches: 1) analysis of the cell-to-cell diffusion of neurobiotin injected into a particular MDCK cell (epithelial cells from dog kidneys) in a confluent monolayer by counting the number of neighboring cells reached by the probe and 2) measurement of the electrical capacitance. Results: We found that 10 nM ouabain increase GJC by 475% within 1 hour. The Na+-K+-ATPase acts as a receptor of ouabain. In previous works we have shown that ouabain activates c-Src and ERK1/2 in 1 hour; in the present study we show that the inhibition of these proteins block the effect of ouabain on GJC. This increase in GJC does not require synthesis of new protein components, because the inhibitors cycloheximide and actinomycin D did not affect this phenomenon. Using silencing assays we also demonstrate that this ouabain-induced enhancement of GJC involves connexins 32 and 43. Conclusion: Ouabain 10 nM increases GJC in MDCK cells.

Injury-triggered Akt phosphorylation of Cx43: a ZO-1-driven molecular switch that regulates gap junction size

The proteins that form vertebrate gap junctions, the connexins, are highly regulated and have short (<2 hour) half-lives. Phosphorylation of connexin43 (Cx43) affects gap junction assembly, channel gating and turnover. After finding dramatic effects on gap junctions with Akt inhibitors, we created an antibody specific for Cx43 phosphorylated on S373, a potential Akt substrate. We found S373 phosphorylation in cells and skin or heart almost exclusively in larger gap-junctional structures that increased dramatically after wounding or hypoxia. We were able to mechanistically show that Akt-dependent phosphorylation of S373 increases gap junction size and communication by completely eliminating the interaction between Cx43 and ZO-1. Thus, phosphorylation on S373 acts as a molecular 'switch' to rapidly increase gap-junctional communication, potentially leading to initiation of activation and migration of keratinocytes or ischemic injury response in the skin and the heart, respectively.

WNT3A induces a contractile and secretory phenotype in cultured vascular smooth muscle cells that is associated with increased gap junction communication

Evidence suggests a role for Wnt signaling in vascular wound repair and remodeling events. Despite this, very little is known about the effect of Wnt ligands on the structure and function of vascular cells. In this study, we treated vascular smooth muscle cells with 250 ng/ml of recombinant Wnt3a for 72 h and observed changes in the cell phenotype. Our data suggest Wnt3a completely alters the phenotype of vascular smooth muscle cells. The Wnt3a-treated cells appeared larger and had increased formation of stress fibers. These cells also had increased expression of the smooth muscle contractile proteins, calponin and smooth muscle α-actin, and contracted a collagen lattice faster than control cells. The Wnt3a-treated smooth muscle cells displayed increased extracellular matrix synthesis, as measured by collagen I and III mRNA expression, along with increased expression of MMP2 and MMP9, but decreased TIMP2 levels. The Wnt3a-induced change in cell phenotype was associated with increased expression of the gap junction protein connexin 43. Consistent with this, Wnt3a-treated smooth muscle cells displayed enhanced intercellular communication, as measured by the scrape-loading dye transfer technique. The canonical Wnt antagonist, dickkopf-related protein 1, completely reversed the contractile protein and connexin 43 expression seen in the Wnt3a-treated cells, suggesting these changes were dependent on canonical Wnt signaling. Collectively, this data suggest Wnt3a promotes a contractile and secretory phenotype in vascular smooth muscle cells that is associated with increased gap junction communication.

P38 MAPK activation, JNK inhibition, neoplastic growth inhibition, and increased gap junction communication in human lung carcinoma and Ras‐transformed cells by 4‐Phenyl‐3‐butenoic acid

Human lung neoplasms frequently express mutations that down-regulate expression of various tumor suppressor molecules, including mitogen-activated protein kinases such as p38 MAPK. Conversely, activation of p38 MAPK in tumor cells results in cancer cell cycle inhibition or apoptosis initiated by chemotherapeutic agents such as retinoids or cisplatin, and is therefore an attractive approach for experimental anti-tumor therapies. We now report that 4-phenyl-3-butenoic acid (PBA), an experimental compound that reverses the transformed phenotype at non-cytotoxic concentrations, activates p38 MAPK in tumorigenic cells at concentrations and treatment times that correlate with decreased cell growth and increased cell-cell communication. H2009 human lung carcinoma cells and ras-transformed rat liver epithelial cells treated with PBA showed increased activation of p38 MAPK and its downstream effectors which occurred after 4 h and lasted beyond 48 h. Untransformed plasmid control cells showed low activation of p38 MAPK compared to ras-transformed and H2009 carcinoma cells, which correlates with the reduced effect of PBA on untransformed cell growth. The p38 MAPK inhibitor, SB203580, negated PBA's activation of p38 MAPK downstream effectors. PBA also increased cell-cell communication and connexin 43 phosphorylation in ras-transformed cells, which were prevented by SB203580. In addition, PBA decreased activation of JNK, which is upregulated in many cancers. Taken together, these results suggest that PBA exerts its growth regulatory effect in tumorigenic cells by concomitant up-regulation of p38 MAPK activity, altered connexin 43 expression, and down-regulation of JNK activity. PBA may therefore be an effective therapeutic agent in human cancers that exhibit down-regulated p38 MAPK activity and/or activated JNK and altered cell-cell communication.

Pharmacological modulation of connexin-formed channels in cardiac pathophysiology.

Coordinated electrical activity in the heart is supported by gap junction channels located at the intercalated discs of cardiomyocytes. Impaired gap junctional communication between neighbouring cardiomyocytes contributes to the development of re-entry arrhythmias after myocardial ischaemia. Current antiarrhythmic therapy is hampered by a lack of efficiency and side effects, creating the need for a new generation of drugs. In this review, we focus on compounds that increase gap junctional communication, thereby increasing the conduction velocity and decreasing the risk of arrhythmias. Some of these compounds also inhibit connexin 43 (Cx43) hemichannels, thereby limiting adenosine triphosphate loss and volume overload following ischaemia/reperfusion, thus potentially increasing the survival of cardiomyocytes. The compounds discussed in this review are: (i) antiarrythmic peptide (AAP), AAP10, ZP123; (ii) GAP-134; (iii) RXP-E; and (vi) the Cx mimetic peptides Gap 26 and Gap 27. None of these compounds have effects on Na(+) , Ca(2+) and K(+) channels, and therefore have no proarrhythmic activity associated with currently available antiarrhythmic drugs. GAP-134, RXP-E, Gap 26 and Gap 27 are pharmalogical agents with a favorable clinical safety profile, as already confirmed in phase I clinical trials for GAP-134. These agents show an excellent promise for treatment of arrhythmias in patients with ischaemic cardiomyopathy.

Interleukin‐1 β increases gap junctional communication among synovial fibroblasts via the extracellular‐signal‐regulated kinase pathway

The gap junction protein, Cx43 (connexin 43), has been implicated in the aetiology of osteoarthritis. Studies have revealed that the size and number of gap junctions increase in synovial biopsies from patients with osteoarthritis. Furthermore, pharmacological inhibition of Cx43 function has been shown to reduce IL-1beta (interleukin-1beta)-induced metalloproteinase production by synovial fibroblasts in vitro. In the present study, we examined the link between IL-1beta and Cx43 function. We demonstrated that treatment of a rabbit synovial fibroblast cell line with IL-1beta markedly increased the level of the Cx43 protein in a concentration- and time-dependent manner. The impact on Cx43 protein levels appeared to occur post-transcriptionally, as mRNA levels are unaffected by IL-1beta administration. Additionally, we showed by fluorescence microscopy that IL-1beta alters the cellular distribution of Cx43 to cell-cell junctions and is concomitant with a striking increase in gap junction communication. Furthermore, we demonstrated that the increase in Cx43 protein, and the associated change in protein localization and gap junction communication following IL-1beta treatment, are dependent upon activation of the ERK (extracellular-signal-regulated kinase) signalling cascade. These data show that IL-1beta acts through the ERK signalling cascade to alter the expression and function of Cx43 in synovial fibroblasts.

Complementary effects of HDAC inhibitor 4-PB on gap junction communication and cellular export mechanisms support restoration of chemosensitivity of PDAC cells

Pancreatic ductal adenocarcinoma (PDAC) is a fatal disease and one of the cancer entities with the lowest life expectancy. Beside surgical therapy, no effective therapeutic options are available yet. Here, we show that 4-phenylbutyrate (4-PB), a known and well-tolerable inhibitor of histone deacetylases (HDAC), induces up to 70% apoptosis in all cell lines tested (Panc 1, T4M-4, COLO 357, BxPc3). In contrast, it leads to cell cycle arrest in only half of the cell lines tested. This drug increases gap junction communication between adjacent T3M-4 cells in a concentration-dependent manner and efficiently inhibits cellular export mechanisms in Panc 1, T4M-4, COLO 357 and BxPc3 cells. Consequently, in combination with gemcitabine 4-PB shows an overadditive effect on induction of apoptosis in BxPc3 and T3M-4 cells (up to 4.5-fold compared to single drug treatment) with accompanied activation of Caspase 8, BH3 interacting domain death agonist (Bid) and poly (ADP-ribose) polymerase family, member 1 (PARP) cleavage. Although the inhibition of the mitogen-activated protein kinase-pathway has no influence on fulminant induction of apoptosis, the inhibition of the JNK-pathway by SP600125 completely abolishes the overadditive effect induced by the combined application of both drugs, firstly reported by this study.

A potential novel mechanism involving connexin 43 gap junction for control of sertoli cell proliferation by thyroid hormones

There is strong evidence that thyroid hormones through triiodothyronine (T3) regulate Sertoli cell proliferation and differentiation in the neonatal testis. However, the mechanism(s) by which they are able to control Sertoli cell proliferation is unclear. In the present study in vivo approaches (PTU-induced neonatal hypothyroidism known to affect Sertoli cell proliferation) associated with in vitro experiments on a Sertoli cell line were developed to investigate this question. We demonstrated that the inhibitory effect of T3 on Sertoli cell growth, analyzed by evaluating DNA-incorporated [3H] thymidine, was associated with a time and dose-dependent increase in the levels of Cx43, a constitutive protein of gap junctions, known to participate in the control of cell proliferation and the most predominant Cx in the testis. These Cx43 changes were associated with increased gap junction communication measured by gap FRAP. Consistent with these results two specific inhibitors of gap junction coupling, AGA and oleamide, were able to significantly reverse the T3 inhibitory effect on Sertoli cell proliferation. The present data also revealed a nongenomic effect of T3 on Cx43 Sertoli cells that was evidenced by a rapid up-regulation of gap junction plaque number as identified in Cx43-GFP transfected cells exposed to the hormone. This process appears mediated through actin cytoskeleton since incubation of the cells with cytochalasin D totally reversed the T3 stimulatory effect on Cx43-GFP gap junction plaques. Based on these data, we propose a working hypothesis in which Cx43 could be an intermediate target for T3 inhibition of neonatal Sertoli cell proliferation.

The Antiarrhythmic Peptide Analog Rotigaptide (ZP123) Stimulates Gap Junction Intercellular Communication in Human Osteoblasts and Prevents Decrease in Femoral Trabecular Bone Strength in Ovariectomized Rats

Gap junctions play an important role in bone development and function, but the lack of pharmacological tools has hampered the gap junction research. The antiarrhythmic peptides stimulate gap junction communication between cardiomyocytes, but effects in noncardiac tissue are unknown. The purpose of this study was to examine whether antiarrhythmic peptides, which are small peptides increasing gap junctional conductivity, show specific binding to osteoblasts and investigate the effect of the stable analog rotigaptide (ZP123) on gap junctional intercellular communication in vitro and on bone mass and strength in vivo. Cell coupling and calcium signaling were assessed in vitro on human, primary, osteoblastic cells. In vivo effects of rotigaptide on bone strength and density were determined 4 wk after ovariectomy in rats treated with either vehicle, sc injection twice daily (300 nmol per kilogram body weight) or by continuous ip infusion (158 nmol per kilogram body weight per day). During metabolic stress, a high affinity-binding site (KD=0.1 nM) with low density (15 fmol/mg protein) for [125I]di-I-AAP10 was demonstrated. During physiological conditions, specific binding sites for [125I]AAP10 could not be shown. Studies of the effects of rotigaptide on propagation of intercellular calcium waves and cell-to-cell coupling demonstrated that 10 nM rotigaptide produced a small increase in intercellular communication during physiological conditions (+4.5+/-1.6% vs. vehicle; P<0.05). During conditions with metabolic stress, 10 nM rotigaptide produced an increase in coupling measured by both methods. Four weeks after ovariectomy, bone strength of the femoral head was reduced by 20% in vehicle-treated ovariectomized rats, which was completely prevented in both rotigaptide-treated groups. Rotigaptide also prevented decreases in bone mineral. We conclude that the stable analog rotigaptide increases gap junctional communication in osteoblasts in vitro and preferably during conditions with metabolic stress. Rotigaptide further prevents ovariectomy-induced bone loss in vivo. Thus, gap junction modulation may be a promising new target for osteoporosis therapy.

Transforming Growth Factor-β3 Increases Gap-Junctional Communication among Folliculostellate Cells to Release Basic Fibroblast Growth Factor

Folliculostellate (FS) cells are known to communicate with each other and with endocrine cells via gap junctions in the anterior pituitary. We investigated whether TGFbeta3 and estradiol, known to regulate FS cell production and secretion of basic fibroblast growth factor (bFGF), increases gap junctional communication to alter bFGF secretion from FS cells. FS cells in monolayer cultures were treated with TGFbeta3 or vehicle alone for 24 h and then microinjected with Lucifer Yellow and high-molecular-weight Texas Red dextran. Ten minutes later the transfer of dye among adjacent cells was recorded with a digital microscope. TGFbeta3 increased the transfer of dye. The TGFbeta3-neutralizing antibody and the gap junction inhibitor octanol reduced the effect of TGFbeta3 on the transfer of dye. The TGFbeta3-induced transfer of dye was unaltered by simultaneous treatment with estradiol. The steroid alone also had no effect. TGFbeta3 increased total and phosphorylated levels of connexin 43. Estradiol treatment did not produce any significant changes on basal or TGFbeta3-induced increases in connexin 43 levels. The gap-junction inhibitor octanol reduced TGFbeta3-increased levels of bFGF in FS cells. Taken together, these results suggest that TGFbeta3 may act on FS cells to increase gap-junctional communication to maximize its effect on bFGF secretion.

The increase in gap junctional communication decreases the rate of glucose uptake in C6 glioma cells by releasing hexokinase from mitochondria

We have previously shown that the enhancement of glucose uptake caused by the inhibition of gap junctional communication is a consequence of the increase in astrocyte proliferation. Since C6 glioma cells are highly proliferative and are poorly coupled through gap junctions, we used these cells to investigate the effect of increasing gap junctional communication on the rate of glucose uptake. Previous work by us had shown that tolbutamide increases gap junctional communication in C6 glioma cells, as does dbcAMP, a classical activator of gap junctional communication. In this work, our results show that both tolbutamide and dbcAMP reduce the rate of glucose uptake in C6 glioma cells and that their effects are additive. The main glucose transporters expressed in C6 glioma cells are GLUT-1 and GLUT-3. Neither the expression nor the cellular localization of either GLUT-1 or GLUT-3 were modified by increasing gap junctional communication. The estimation of glucose uptake with 2-deoxyglucose includes not only glucose transport but also glucose phosphorylation, which in C6 glioma cells is mainly catalyzed by type I and type II hexokinase. Our results reveal that the increase in gap junctional communication caused by tolbutamide and dbcAMP is associated with a decrease in the activity of hexokinase. In agreement with this, tolbutamide and dbcAMP caused a rapid change in the localization of both type I and type II hexokinase, which were detached from the mitochondria to the cytosol.

Gap junctions in hematopoietic stroma control proliferation and differentiation of blood cell precursors

We examined gap junction communication in an in vitro model of hematopoiesis, using the murine bone marrow stroma cell line S-17, and primary cultures of murine marrow-derived blood cell precursors. S-17 cells express several connexins, the major one being connexin 43. Connexin expression and formation of functional gap junctions is modulated by stroma cell density. Transfection of S-17 cells with a vector containing connexin 43 sense or anti-sense sequences increased or decreased, respectively, connexin 43 synthesis and intercellular dye coupling. Under these conditions, modulation of gap junction-mediated communication modified the growth pattern of stroma itself, as well as the ability of the stroma to sustain hematopoiesis. Increased connexin 43 expression was associated with a delay in differentiation of blood cells, resulting in increased production of hematopoietic precursors, while decreased connexin 43 expression elicited an accelerated differentiation of myeloid blood cell precursor cells. These results suggest that connexin-mediated coupling in the stroma modulates the ratio between proliferation and differentiation of hematopoietic precursors. We therefore propose that increased gap junction communication in the stroma elicits an enhanced production of immature bone marrow cells through the delay in their terminal differentiation, inducing consequently an extended proliferation period of blood cell precursors.

Upregulation of connexin 43 protein expression and increased gap junctional communication by water soluble disodium disuccinate astaxanthin derivatives

Carotenoids are plant pigments whose consumption is associated with lower cancer rates in humans. Studies in experimental animal and cell systems have confirmed the cancer chemopreventive activity of these compounds. However, their extremely hydrophobic nature makes these compounds biologically unavailable unless delivered in organic solution to model systems. We have synthesized novel disodium salt disuccinate astaxanthin derivatives that possess high aqueous dispersibility. When delivered to mouse embryonic fibroblast C3H/10T1/2 cell cultures, either in aqueous or aqueous/ethanol solutions, these derivatives are biologically active. Biological activity was demonstrated by (1) upregulated expression of connexin 43 (Cx43) protein; (2) increased formation of Cx43 immunoreactive plaques in regions of the plasma membrane consistent with localization of gap junctions; (3) significantly upregulated gap junctional intercellular communication (GJIC) as demonstrated by Lucifer Yellow dye transfer after microinjection ( P<0.03; Fisher's Exact test). Enhanced expression of Cx43 and increased GJIC have been previously demonstrated to result in inhibition of in vitro neoplastic transformation of 10T1/2 cells as well as growth reduction of human tumors in xenografts. These novel derivatives possess increased utility as water soluble and water dispersible agents, allowing for aqueous delivery both in vitro and in vivo, properties that could enhance their potential clinical utility as potent cancer chemopreventive agents.

Hydrogen peroxide increases gap junctional communication and induces astrocyte toxicity: regulation by brain macrophages.

Cultured astrocytes are highly coupled by gap junction channels mainly constituted by connexin 43. We have previously shown that gap junctional communication (GJC) represents a functional property of astrocytes that is a target for their interaction with other brain cell types, including neurons and brain macrophages. In pathological situations, neurons as well as brain macrophages produce superoxide ions leading to the formation of hydrogen peroxide (H2O2) that can be cytotoxic. We report here that 10-min exposure to 100 microM H2O2 increases GJC in astrocytes. Moreover, 30-min exposure to 100 microM H2O2 induces, 24 h later, an astrocyte cell death by both apoptosis and necrosis. This H2O2-induced astrocyte cell death is not affected when gap junctions are inhibited by several uncoupling agents, including 18alpha-glycyrrhetinic acid, halothane, heptanol, and endothelin-1, indicating that the proportion of cell death is not related to the level of GJC. The effect of H2O2 on gap junction channels does not result from the production of free radicals but is rather linked to modification of the redox equilibrium in astrocytes. Indeed, an oxidative agent reproduces the H2O2-evoked response while reducing agents prevent the effect of H2O2. Finally, when astrocytes are cocultured with brain macrophages, the effects of H2O2 on both GJC and toxicity are not observed, revealing a new protective role of brain macrophages during oxidative stress.

Epileptiform activity in hippocampal slice cultures exposed chronically to bicuculline: increased gap junctional function and expression.

Chronic (18 h) exposure of cultured hippocampal slices to the type-A GABA receptor blocker, bicuculline methiodide (BMI) 10 micro m increased the levels of connexin 43 (Cx43) and connexin 32 (Cx32) mRNAs, but not connexin 26 and connexin 36, as demonstrated by RNase protection assays. The levels of Cx43 and Cx32 proteins in membrane fractions detected by western blotting were also significantly increased. Immunoblotting indicated that BMI also promoted a significant expression of the transcription protein c-fos. The rate of fluorescence recovery after photobleaching, an index of gap junctional coupling, was also significantly increased, whereas it was blocked by the gap junctional blocker, carbenoxolone (100 micro m). Extracellular recordings in CA1 stratum pyramidale, performed in BMI-free solution, demonstrated that BMI-exposed cultures possessed synaptic responses characteristic of epileptiform discharges: (i) significantly greater frequency of spontaneous epileptiform discharges, (ii) post-synaptic potentials with multiple population spikes, and (iii) significantly longer duration of primary afterdischarges. Carbenoxolone (100 micro m), but not its inactive analog, oleanolic acid (100 micro m), reversibly inhibited spontaneous and evoked epileptiform discharges. The findings of BMI-induced parallel increases in levels of gap junction expression and function, and the increase in epileptiform discharges, which were sensitive to gap junctional blockers, are consistent with the hypothesis that increased gap junctional communication plays an intrinsic role in the epileptogenic process.

In vivo electrophysiological evidences for cortical neuron–glia interactions during slow (

The cortical activity results from complex interactions within networks of neurons and glial cells. The dialogue signals consist of neurotransmitters and various ions, which cross through the extracellular space. Slow (<1 Hz) sleep oscillations were first disclosed and investigated at the neuronal level where they consist of an alternation of the membrane potential between a depolarized and a hyperpolarized state. However, neuronal properties alone could not account for the mechanisms underlying the oscillatory nature of the sleeping cortex. Here I will show the behavior of glial cells during the slow sleep oscillation and its relationship with the variation of the neuronal membrane potential (pairs of neurons and glia recorded simultaneously and intracellularly) suggesting that, in contrast with previous assumptions, glial cells are not idle followers of neuronal activity. I will equally present measurements of the extracellular concentration of K + and Ca 2+, ions known to modulate the neuronal excitability. They are also part of the ionic flux that is spatially buffered by glial cells. The timing of the spatial buffering during the slow oscillation suggests that, during normal oscillatory activity, K + ions are cleared from active spots and released in the near vicinity, where they modulate the excitability of the neuronal membrane and contribute to maintain the depolarizing phase of the oscillation. Ca 2+ ions undergo a periodic variation of their extracellular concentration, which modulates the synaptic efficacy. The depolarizing phase of the slow oscillation is associated with a gradual depletion of the extracellular Ca 2+ promoting a progressive disfacilitation in the network. This functional synaptic neuronal disconnection is responsible for the ending of the depolarizing phase of the slow oscillation and the onset of a phasic hyperpolarization during which the neuronal network is silent and the intra- and extracellular ionic concentrations return to normal values. Spike-wave seizures often develop during sleep from the slow oscillation. Here I will show how the increased gap junction communication substantiates the facility of the glial syncytium to spatially buffer K + ions that were uptaken during the spike-wave seizures, and therefore contributing to the long-range recruitment of cortical territories. Similar mechanisms as those described during the slow oscillation promote the periodic (2–3 Hz) recurrence of spike-wave complexes.

Proliferation of C6 glioma cells is blunted by the increase in gap junction communication caused by tolbutamide

We have previously reported that tolbutamide prevents the inhibition of gap junction communication in astrocytes. Here, we show that tolbutamide increases gap junction communication and connexin 43 expression in poorly coupled C6 glioma cells. The increase in communication is concurrent with the inhibition of the rate of proliferation due to a block of the progression of C6 glioma cells through the S phase of the cell cycle. The effects of tolbutamide were quantitatively similar to that found after the elevation of intracellular cAMP. Furthermore, the effects of tolbutamide and cAMP were additive. The possible beneficial effect of tolbutamide on gene therapy for gliomas is discussed.