The psychostimulant modafinil enhances gap junctional communication in cortical astrocytes.

Sleep-wake cycle is characterized by changes in neuronal network activity. However, for the last decade there is increasing evidence that neuroglial interaction may play a role in the modulation of sleep homeostasis and that astrocytes have a critical impact in this process. Interestingly, astrocytes are organized into communicating networks based on their high expression of connexins, which are the molecular constituents of gap junction channels. Thus, neuroglial interactions should also be considered as the result of the interplay between neuronal and astroglial networks. Here, we investigate the effect of modafinil, a wakefulness-promoting agent, on astrocyte gap junctional communication. We report that in the cortex modafinil injection increases the expression of mRNA and protein of connexin 30 but not those of connexin 43, the other major astroglial connexin. These increases are correlated with an enhancement of intercellular dye coupling in cortical astrocytes, which is abolished when neuronal activity is silenced by tetrodotoxin. Moreover, gamma-hydroxybutyric acid, which at a millimolar concentration induces sleep, has an opposite effect on astroglial gap junctions in an activity-independent manner. These results support the proposition that astroglia may play an important role in complex physiological brain functions, such as sleep regulation, and that neuroglial networking interaction is modified during sleep-wake cycle. This article is part of the Special Issue Section entitled 'Current Pharmacology of Gap Junction Channels and Hemichannels'.

Gap Junctional Communication Modulates Gene Expression in Osteoblastic Cells

Bone-forming cells are organized in a multicellular network interconnected by gap junctions. In these cells, gap junctions are formed by connexin43 (Cx43) and connexin45 (Cx45). Cx43 gap junctions form pores that are more permeable to negatively charged dyes such as Lucifer yellow and calcein than are Cx45 pores. We studied whether altering gap junctional communication by manipulating the relative expression of Cx43 and Cx45 affects the osteoblast phenotype. Transfection of Cx45 in cells that express primarily Cx43 (ROS 17/2.8 and MC3T3-E1) decreased both dye transfer and expression of osteocalcin (OC) and bone sialoprotein (BSP), genes pivotal to bone matrix formation and calcification. Conversely, transfection of Cx43 into cells that express predominantly Cx45 (UMR 106–01) increased both cell coupling and expression of OC and BSP. Transient cotransfection of promoter–luciferase constructs and connexin expression vectors demonstrated that OC and BSP gene transcription was down-regulated by Cx45 cotransfection in ROS 17/2.8 and MC3T3-E1 cells, in association with a decrease in dye coupling. Conversely, cotransfection of Cx43 in UMR 106–01 cells up-regulated OC and BSP gene transcription. Activity of other less specific osteoblast promoters, such as osteopontin and osteonectin, was less sensitive to changes in gap junctional communication. Thus, altering gap junctional permeability by manipulating the expression of Cx43 and Cx45 in osteoblastic cells alters transcriptional activity of osteoblast-specific promoters, presumably via modulation of signals that can diffuse from cell to cell. A communicating intercellular network is required for the full elaboration of a differentiated osteoblastic phenotype.

Effects of cAMP modulators during in vitro prematuration of bovine oocytes on gap junctional communication and embryo development potential.

2016

Transient disruption of liver gap junctional intercellular communication and induction of apoptosis after administration of 1,4-bis[2-(3,5 dichloropyridyloxy)]benzene in mice

Gap junctional intercellular communication (GJIC) and connexin expression (Cx26 and Cx32) in mouse liver were studied after administration of 4-bis[2-(3,5 dichloropyridyloxy)]benzene (TCPOBOP), a phenobarbital-like enzyme inducer. Female C57BI/6 mice were administered TCPOBOP (5.8 mg/kg BW) and euthanized 0, 24, 48 and 72 hours later. Liver samples were snap frozen, or fixed in formalin, or submitted to GJIC analysis. The proliferating cell nuclear antigen (PCNA) immunohistochemistry and the Western blotting for Cx26 and Cx32 were performed. After 48 and 72 h of drug administration the liver-to-body weight ratio was increased 70% and 117% (p < 0.0001), respectively. There were temporal-dependent alterations in liver histopathology and a significant increase in cell proliferation was noted after 48h and sustained after 72h, though to a lesser extent (p < 0.0001). In addition. TCPOBOP administration induced apoptosis, which appeared to be time-dependent showing statistical significance only after 72h (p < 0.0001). Interestingly, a transient disruption by nearly 50% of GJIC capacity was detected after 48 h of drug ingestion, which recovered after 72 h (p = 0.003). These GJIC changes were due to altered levels of Cx26 and Cx32 in the livers of TCPOBOP-treated mice. We concluded that a single administration of TCPOBOP transiently disrupted the levels of GJIC due to decreased expression of connexins and increased apoptotic cell death in mouse liver. (C) 2009 Elsevier GmbH. All rights reserved.

Involvement of gap junctional intercellular communication in the pathophysiology of inflammation

SUMMARY Inflammation has evolved as a mechanism to defend the body against invading microorganisms and to respond to injury. It requires the coordinated response of a large number of cell types from the whole organism in a time- and space-dependent fashion. This coordination involves several cell-cell communication mechanisms. Exchange of humoral mediators such as cytokines is a major one. Moreover, direct contact between cells happens and plays a primordial role, for example when macrophages present antigens to lymphocytes. Contact between endothelial cells and leucocytes occurs when the latter cross the blood vessel barrier and transmigrate to the inflammatory site. A particular way by which cells communicate with each other in the course of inflammation, which at this time starts to gain attention, is the intercellular communication mediated by gap junctions. Gap junctions are channels providing a direct pathway (i.e. without transit through the extracellular space) for the diffusion of small molecules between adjacent cells. This process is known as gap junctional intercellular communication (GJIC). The general aim of this thesis was to study a possible involvement of GJIC in the pathophysiology of inflammation. A first part of the work was dedicated to study the implication of GJIC in the modification of vascular endothelial function by inflammation. In a second part, we were interested in the possible role of GJIC in the transmigration of neutrophil polymorphonuclear leucocytes through the endothelium. The main positive finding of this work is that acute inflammation preferentially modulates the expression of connexin 40 (Cx40), a gap junction protein specifically expressed in vascular endothelium. The modulation could be towards overexpression (aortic endothelium of septic rats) or towards downregulation (acutely inflamed mouse lung). We put a lot of efforts in search of possible functions of these modulations, in two directions: a potential protective role of Cx40 increased expression against sepsis-induced endothelial dysfunction, and a facilitating role of Cx40 decreased expression in neutrophil transmigration. To pursue both directions, it seemed logical to study the impact of Cx40 deletion using knock-out mice. Concerning the potential protective role of Cx40 overexpression we encountered a roadblock as we observed, in the aorta, a Cx40 downregulation in wild type mouse whereas Cx40 was upregulated in the rat. Regarding the second direction and using an in vivo approach, we observed that pulmonary neutrophil transmigration was not affected by the genetic deletion of Cx40. In spite of their negative nature, these results are the very first ones regarding the potential implication of GJIC concerning leucocyte transmigration in vivo. Because this process involves such tight cell-cell physical contacts, the hypothesis for a role of GJIC remains attractive.

Ca2+/calmodulin-dependent kinase II mediates simultaneous enhancement of gap-junctional conductance and glutamatergic transmission

While chemical synapses are very plastic and modifiable by defined activity patterns, gap junctions, which mediate electrical transmission, have been classically perceived as passive intercellular channels. Excitatory transmission between auditory afferents and the goldfish Mauthner cell is mediated by coexisting gap junctions and glutamatergic synapses. Although an increased intracellular Ca2+ concentration is expected to reduce gap junctional conductance, both components of the synaptic response were instead enhanced by postsynaptic increases in Ca2+ concentration, produced by patterned synaptic activity or intradendritic Ca2+ injections. The synaptically induced potentiations were blocked by intradendritic injection of KN-93, a Ca2+/calmodulin-dependent kinase (CaM-K) inhibitor, or CaM-KIINtide, a potent and specific peptide inhibitor of CaM-KII, whereas the responses were potentiated by injection of an activated form of CaM-KII. The striking similarities of the mechanisms reported here with those proposed for long-term potentiation of mammalian glutamatergic synapses suggest that gap junctions are also similarly regulated and indicate a primary role for CaM-KII in shaping and regulating interneuronal communication, regardless of its modality.

Contribution of the gap junction proteins Connexin40 and Connexin43 to the control of blood pressure

Summary Cells in tissues and organs coordinate their activities by communicating with each other through intercellular channels named gap junctions. These channels are conduits between the cytoplasmic compartments of adjacent cells, allowing the exchange of small molecules which may be crucial for hormone secretion. Renin is normally secreted in a regulated manner by specific cells of the juxtaglomerular apparatus located within the renal cortex. Gap junctional communication may be requisite to maintain an accurate functioning in coordination of renin-producing cells, more especially as renin is of paramount importance for the control of blood pressure. Connexin43 (Cx43) and Cx40 form gap junctions that link in vivo the cells of the juxtaglomerular apparatus. Cx43 links the endothelial cells, whereas gap junctions made of Cx40 connect the endothelial cells, the renin secreting cells, as well as the endothelial cells of to the renin-secreting cells of the afferent arteriole. The observation that loss of Cx40 results in chronic hypertension associated with altered vasomotion and signal conduction along arterioles, has lead us to suggest that connexins may contribute to control blood pressure by participating to the integration of various mechanical, osmotic and electrochemical stimuli involved in the control of renin secretion and by mediating the adaptive changes of the vascular wall induced by elevated blood pressure and mechanical stress. We therefore postulated that the absence of Cx40 could have deleterious effects on the coordinated functioning of the renin-containing cells, hence accounting for hypertension. In the first part of my thesis, we reported that Cx40-deficient mice (Cx40) are hypertensive due to increased plasma renin levels and numbers of renin-producing cells. Besides, we demonstrated that prostaglandins and nitric oxide, which are possible mediators in the regulation of renin secretion by the macula densa, exert a critical role in the mechanisms controlling blood pressure ín Cx40 knockout hypertensive mice. In view of previous studies that stated avessel-specifc increase in the expression of Cx43 during renin-dependent hypertension, we hypothesized that Cx43 channels are particularly well-matched to integrate the response of cells constituting the vascular wall to hypertensive conditions. Using transgenic mice in which Cx43 was replaced by Cx32, we revealed that the replacement of Cx43 by Cx32 is associated with decreased expression and secretion of renin and prevent the renin-dependent hypertension which is normally induced in the 2K1C model. To gain insights into the regulation of connexins in two separate tissues exposed to the same fluid pressure, the second part of my thesis work was dedicated to the study of the impact of chronic hypertension and related hypertrophy on the expression of the cardiovascular connexins (Cx40, Cx37, Cx43 and Cx45) in mouse aorta and heart. Our results documented that the expression of connexins is differentially regulated in mouse aorta. according to the models of hypertension. Thus, blood pressure induces mechanical forces that differentially alter the expression of vascular connexins in order to respond to an adaptation of the aortic wall observed under pathological conditions. Altogether these data provide the first evidences that intercellular communication mediated by gap junctions is required for a proper renin secretion from the juxtaglomerular apparatus in order to control blood pressure.

Regulation of gap junctions by protein phosphorylation

Gap junctions are constituted by intercellular channels and provide a pathway for transfer of ions and small molecules between adjacent cells of most tissues. The degree of intercellular coupling mediated by gap junctions depends on the number of gap junction channels and their activity may be a function of the state of phosphorylation of connexins, the structural subunit of gap junction channels. Protein phosphorylation has been proposed to control intercellular gap junctional communication at several steps from gene expression to protein degradation, including translational and post-translational modification of connexins (i.e., phosphorylation of the assembled channel acting as a gating mechanism) and assembly into and removal from the plasma membrane. Several connexins contain sites for phosphorylation for more than one protein kinase. These consensus sites vary between connexins and have been preferentially identified in the C-terminus. Changes in intercellular communication mediated by protein phosphorylation are believed to control various physiological tissue and cell functions as well as to be altered under pathological conditions.

SIGNALING MECHANISMS THAT CONTROL GAP JUNCTIONAL COUPLING BETWEEN RETINAL NEURONS

Gap junctions between neurons form the structural substrate for electrical synapses. Connexin 36 (Cx36, and its non-mammalian ortholog connexin 35) is the major neuronal gap junction protein in the central nervous system (CNS), and contributes to several important neuronal functions including neuronal synchronization, signal averaging, network oscillations, and motor learning. Connexin 36 is strongly expressed in the retina, where it is an obligatory component of the high-sensitivity rod photoreceptor pathway. A fundamental requirement of the retina is to adapt to broadly varying inputs in order to maintain a dynamic range of signaling output. Modulation of the strength of electrical coupling between networks of retinal neurons, including the Cx36-coupled AII amacrine cell in the primary rod circuit, is a hallmark of retinal luminance adaptation. However, very little is known about the mechanisms regulating dynamic modulation of Cx36-mediated coupling. The primary goal of this work was to understand how cellular signaling mechanisms regulate coupling through Cx36 gap junctions. We began by developing and characterizing phospho-specific antibodies against key regulatory phosphorylation sites on Cx36. Using these tools we showed that phosphorylation of Cx35 in fish models varies with light adaptation state, and is modulated by acute changes in background illumination. We next turned our focus to the well-studied and readily identifiable AII amacrine cell in mammalian retina. Using this model we showed that increased phosphorylation of Cx36 is directly related to increased coupling through these gap junctions, and that the dopamine-stimulated uncoupling of the AII network is mediated by dephosphorylation of Cx36 via protein kinase A-stimulated protein phosphatase 2A activity. We then showed that increased phosphorylation of Cx36 on the AII amacrine network is driven by depolarization of presynaptic ON-type bipolar cells as well as background light increments. This increase in phosphorylation is mediated by activation of extrasynaptic NMDA receptors associated with Cx36 gap junctions on AII amacrine cells and by Ca2+-calmodulin-dependent protein kinase II activation. Finally, these studies indicated that coupling is regulated locally at individual gap junction plaques. This work provides a framework for future study of regulation of Cx36-mediated coupling, in which increased phosphorylation of Cx36 indicates increased neuronal coupling.

Gap junctional coupling in lenses lacking α3 connexin

Fiber cells of the lens are interconnected by an extensive network of gap junctions containing α3 (Cx46) and α8 (Cx50) connexins. A specific role for these connexins in lens homeostasis is not known. To determine the contribution of these connexins to lens function, we used impedance techniques to study cell-to-cell coupling in lenses from homozygous α3 knockout (−/−), heterozygous (+/−), and wild-type (+/+) mice. Western blots and immunofluorescence data indicated that α8 remained at similar levels in the three classes of lenses, whereas α3 was approximately 50% of the normal level in the +/− lenses, and it was absent from the −/− lenses. Moreover, the data from +/+ lenses suggest that a cleavage of connexins occurs abruptly between the peripheral shell of differentiating fibers (DF) and the inner core of mature fibers (MF). The appearance of the cleaved connexins was correlated to a change in the coupling conductance. In −/− lenses the coupling conductance of MF was zero, and these fibers were depolarized by about 30 mV from normal (≈−65 mV). The DF remained coupled, but the conductance was reduced to 30–35% of normal. However, the gap junctions in the DF of α3 −/− lenses remained sensitive to pH. We conclude that α3 connexin is necessary for the coupling of central fibers to peripheral cells, and that this coupling is essential for fiber cell homeostasis because uncoupled MF depolarize and subsequently become opaque.

Microglia at brain stab wounds express connexin 43 and in vitro form functional gap junctions after treatment with interferon-γ and tumor necrosis factor-α

Gap junctional communication between microglia was investigated at rat brain stab wounds and in primary cultures of rat and mouse cells. Under resting conditions, rat microglia (FITC-isolectin-B4-reactive cells) were sparsely distributed in the neocortex, and most (95%) were not immunoreactive for Cx43, a gap junction protein subunit. At brain stab wounds, microglia progressively accumulated over several days and formed aggregates that frequently showed Cx43 immunoreactivity at interfaces between cells. In primary culture, microglia showed low levels of Cx43 determined by Western blotting, diffuse intracellular Cx43 immunoreactivity, and a low incidence of dye coupling. Treatment with the immunostimulant bacterial lipopolysaccharide (LPS) or the cytokines interferon-γ (INF-γ) or tumor necrosis factor-α (TNF-α) one at a time did not increase the incidence of dye coupling. However, microglia treated with INF-γ plus LPS showed a dramatic increase in dye coupling that was prevented by coapplication of an anti-TNF-α antibody, suggesting the release and autocrine action of TNF-α. Treatment with INF-γ plus TNF-α also greatly increased the incidence of dye coupling and the Cx43 levels with translocation of Cx43 to cell–cell contacts. The cytokine-induced dye coupling was reversibly inhibited by 18α-glycyrrhetinic acid, a gap junction blocker. Cultured mouse microglia also expressed Cx43 and developed dye coupling upon treatment with cytokines, but microglia from homozygous Cx43-deficient mice did not develop significant dye coupling after treatment with either INF-γ plus LPS or INF-γ plus TNF-α. This report demonstrates that microglia can communicate with each other through gap junctions that are induced by inflammatory cytokines, a process that may be important in the elaboration of the inflammatory response.

Leukocytes express connexin 43 after activation with lipopolysaccharide and appear to form gap junctions with endothelial cells after ischemia-reperfusion.

Levels and subcellular distribution of connexin 43 (Cx43), a gap junction protein, were studied in hamster leukocytes before and after activation with endotoxin (lipopolysaccharide, LPS) both in vitro and in vivo. Untreated leukocytes did not express Cx43. However, Cx43 was clearly detectable by indirect immunofluorescence in cells treated in vitro with LPS (1 micrograms/ml, 3 hr). Cx43 was also detected in leukocytes obtained from the peritoneal cavity 5-7 days after LPS-induced inflammation. In some leukocytes that formed clusters Cx43 immunoreactivity was present at appositional membranes, suggesting formation of homotypic gap junctions. In cell homogenates of activated peritoneal macrophages, Cx43, detected by Western blot analysis, was mostly unphosphorylated. A second in vivo inflammatory condition studied was that induced by ischemia-reperfusion of the hamster cheek pouch. In this system, leukocytes that adhered to venular endothelial cells after 1 hr of ischemia, followed by 1 hr of reperfusion, expressed Cx43. Electron microscope observations revealed small close appositions, putative gap junctions, at leukocyte-endothelial cell and leukocyte-leukocyte contacts. These results indicate that the expression of Cx43 can be induced in leukocytes during an inflammatory response which might allow for heterotypic or homotypic intercellular gap junctional communication. Gap junctions may play a role in leukocyte extravasation.

Gap-junction communication between subtypes of direction-selective ganglion cells in the developing retina

The On-Off direction-selective ganglion cells (DSGCs) in the rabbit retina comprise four distinct subtypes that respond preferentially to image motion in four orthogonal directions; each subtype forms a regular territorial array, which is overlapped by the other three arrays. In this study, ganglion cells in the developing retina were injected with Neurobiotin, a gap-junction-permeable tracer, and the DSGCs were identified by their characteristic type 1 bistratified (BiS1) morphology. The complex patterns of tracer coupling shown by the BiSl ganglion cells changed systematically during the course of postnatal development. BiSl cells appear to be coupled together around the time of birth, but, over the next 10 days, BiSl cells decouple from each other, leading to the mature pattern in which only one subtype is coupled. At about postnatal day 5, before the ganglion cells become visually responsive, each of the BiSl cells commonly showed tracer coupling both to a regular array of neighboring BiSl cells, presumably destined to be DSGCs of the same subtype, and to a regular array of overlapping BiSl cells, presumably destined to be DSGCs of a different subtype. The gap-junction intercellular communication between subtypes of DSGCs with different preferred directions may play an important role in the differentiation of their synaptic connectivity, with respect to either the inputs that DSGCs receive from retinal interneurons or the outputs that DSGCs make to geniculate neurons. (C) 2004 Wiley-Liss, Inc.

Blood pressure variability increases connexin expression in the vascular smooth muscle of rats

Aims Following sinoaortic denervation (SAD), isolated rat aortas present oscillatory contractions and demonstrate a heightened contraction for alpha-adrenergic agonists. Our aim was to verify the effects of SAD on connexin43 (Cx43) expression and phenylephrine-induced contraction in isolated aortas. Methods and results Three days after surgery (SAD or sham operation), isolated aortic rings were exposed to phenylephrine and acetylcholine (0.1-10 mu M) in the presence or absence of the gap junction blocker 18 beta-glycyrrhetinic acid (18 beta-GA, 100 mu M). Vascular reactivity to potassium chloride (KCl, 4.7-120 mM) was also examined. The incidence of rats presenting oscillatory contractions was measured. Effects of SAD on the vascular smooth muscle expression of the Cx43 mRNA by RT-PCR and western blotting for Cx43 protein were examined. Phenylephrine-induced contraction was higher in SAD rat aortas compared with the control. In the presence of 18 beta-GA, the response to phenylephrine was similar in both groups. Oscillatory contractions were observed in 10/10 SAD rat aortas vs. 2/10 controls. Relaxing response to acetylcholine was similar in both groups, but in the presence of 18 beta-GA, the response to acetylcholine decreased significantly in the sham-operated group (82.7 +/- 7.6% reduction of relaxation), whereas a half-maximal relaxation (reduction of 46.2 +/- 5.3%) took place in SAD rat aortas. KCl-induced contraction was similar in both groups. Following SAD, RT-PCR revealed significantly increased levels of Cx43 mRNA (9.85 fold, P < 0.01). Western blot analysis revealed greater levels of Cx43 protein (P < 0.05). Conclusion Blood pressure variability evoked by SAD leads to increased expression of Cx43, which could contribute to enhanced phenylephrine-induced contraction and oscillatory activity in isolated aortas.

A new nitrosyl ruthenium complex nitric oxide donor presents higher efficacy than sodium nitroprusside on relaxation of airway smooth muscle

Nitric oxide (NO) has been demonstrated to be the primary agent in relaxing airways in humans and animals. We investigated the mechanisms involved in the relaxation induced by NO-donors, ruthenium complex [Ru(terpy)(bdq)NO(+)](3+) (TERPY) and sodium nitroprusside (SNP) in isolated trachea of rats contracted with carbachol in an isolated organs chamber. For instance, we verified the contribution of K(+) channels, the importance of sGC/cGMP pathway, the influence of the extra and intracellular Ca(2+) sources and the contribution of the epithelium on the relaxing response. Additionally, we have used confocal microscopy in order to analyze the action of the NO-donors on cytosolic Ca(2+) concentration. The results demonstrated that both compounds led to the relaxation of trachea in a dependent-concentration way. However, the maximum effect (E(max)) of TERPY is higher than the SNP. The relaxation induced by SNP (but not TERPY) was significantly reduced by pretreatment with ODQ (sGC inhibitor). Only TERPY-induced relaxation was reduced by tetraethylammonium (K(+) channels blocker) and by pre-contraction with 75 mM KCl (membrane depolarization). The response to both NO-donors was not altered by the presence of thapsigargin (sarcoplasmic reticulum Ca(2+)-ATPase inhibitor). The epithelium removal has reduced the relaxation only to SNP, and it has no effect on TERPY. The both NO-donors reduced the contraction evoked by Ca(2+) influx, while TERPY have shown a higher inhibitory effect on contraction. Moreover, the TERPY was more effective than SNP in reducing the cytosolic Ca(2+) concentration measured by confocal microscopy. In conclusion, these results show that TERPY induces airway smooth muscle relaxation by cGMP-independent mechanisms, it involves the fluxes of Ca(2+) and K(+) across the membrane, it is more effective in reducing cytosolic Ca(2+) concentration and inducing relaxation in the rat trachea than the standard drug, SNP. (C) 2011 Elsevier B.V. All rights reserved.