Block Gap Junctional Communication Research Articles

Analysis of carbenoxolone by ultra‐high‐performance liquid chromatography tandem mass spectrometry in mouse brain and blood after systemic administration

Carbenoxolone is a derivative of glycyrrhetinic acid found in the root of Glycyrrhiza glabra, colloquially known as licorice. It has been used as a treatment for peptic and oral ulcers. In recent years, carbenoxolone has been utilized in basic research for its ability to block gap junctional communication. Better understanding the distribution of carbenoxolone after systemic administration can lead to a better understanding of its potential sites of action. Presented is an ultra high-performance liquid chromatography tandem mass spectrometer (UHPLC-MS/MS) method for the identification and quantification of carbenoxolone in mouse blood and brain tissue. Twenty mice were injected intraperitoneally with 25 mg/kg carbenoxolone and brain tissue and blood were collected for analysis. Blood concentrations (mean ± SD) at 15, 30, 60 and 120 min were determined to be (n = 5) 5394 ± 778, 2636 ± 836, 1564 ± 541 and 846 ± 252 ng/mL, respectively. Brain concentrations (mean ± SD) at 15, 30, 60 and 120 mins were determined to be (n = 5) 171 ± 62, 102 ± 35, 55 ± 10 and 27 ± 9 ng/g, respectively. The analysis of these specimens at the four different time points resulted in blood and brain half-lives in mice of ~43 and 41 min, respectively. The UHPLC-MS/MS method was determined to be sensitive and robust for quantification of carbenoxolone.

GABA and Gap Junctions in the Development of Synchronized Activity in Human Pluripotent Stem Cell-Derived Neural Networks

The electrical activity of the brain arises from single neurons communicating with each other. However, how single neurons interact during early development to give rise to neural network activity remains poorly understood. We studied the emergence of synchronous neural activity in human pluripotent stem cell (hPSC)-derived neural networks simultaneously on a single-neuron level and network level. The contribution of gamma-aminobutyric acid (GABA) and gap junctions to the development of synchronous activity in hPSC-derived neural networks was studied with GABA agonist and antagonist and by blocking gap junctional communication, respectively. We characterized the dynamics of the network-wide synchrony in hPSC-derived neural networks with high spatial resolution (calcium imaging) and temporal resolution microelectrode array (MEA). We found that the emergence of synchrony correlates with a decrease in very strong GABA excitation. However, the synchronous network was found to consist of a heterogeneous mixture of synchronously active cells with variable responses to GABA, GABA agonists and gap junction blockers. Furthermore, we show how single-cell distributions give rise to the network effect of GABA, GABA agonists and gap junction blockers. Finally, based on our observations, we suggest that the earliest form of synchronous neuronal activity depends on gap junctions and a decrease in GABA induced depolarization but not on GABAA mediated signaling.

Effective protection of rabbits’ explosive brain injury through blocking gap junction communication

The gap junction plays an important role in spreading of apoptotic and necrotic signals from injured and stressed cells to the neighboring viable cells. The present study was performed to investigate the important role of gap junction communication on rabbits' explosive brain injury. Explosion of paper detonators was used to create explosive brain injury model in 60 rabbits, which was randomly divided into control group and experimental group. Octanol, an efficient blocker of gap junction, was injected in the left ventricle to block gap junction communication in the experimental group 2 hours before injury, while the same volume of saline was utilized in the control group. Penumbra volume around the brain contusion in the experimental group was significantly less than that in the control group at 1d and 3d after brain damage. RT-PCR and Western blotting analysis indicated that the expression of connexin-43 (Cx43) and caspase-3 was significantly lower in the experimental group than that in the control group at all time points. Rabbits' explosive brain injury can be efficiently attenuated through blocking the gap junction communication, which benefit for deeper understanding the mechanism of brain injury.

Curious and contradictory roles of glial connexins and pannexins in epilepsy

Glia play an under-recognized role in epilepsy. This review examines the involvement of glial connexins (Cxs) and pannexins (Panxs), proteins which form gap junctions and membrane hemichannels (connexins) and hemichannels (pannexins), in epilepsy. These proteins, particularly glial Cx43, have been shown to be upregulated in epileptic brain tissue. In a cobalt model of in vitro seizures, seizures increased Panxs1 and 2 and Cx43 expression, and remarkably reorganized the interrelationships between their mRNA levels (transcriptome) which then became statistically significant. Gap junctions are highly implicated in synchronous seizure activity. Blocking gap junctional communication (GJC) is often anticonvulsant, and assumed to be due to blocking gap junctionally-medicated electrotonic coupling between neurons. However, in organotypic hippocampal slice cultures, connexin43 specific peptides, which attenuate GJC possibly by blocking connexon docking, diminished spontaneous seizures. Glia have many functions including extracellular potassium redistribution, in part via gap junctions, which if blocked, can be seizuregenic. Glial gap junctions are critical for the delivery of nutrients to neurons, which if interrupted, can depress seizure activity. Other functions of glia possibly related to epileptogenesis are mentioned including anatomic reorganization in chronic seizure models greatly increasing the overlapping domains of glial processes, changes in neurotransmitter re-uptake, and possible glial generation of currents and fields during seizure activity. Finally there is recent evidence for Cx43 hemichannels and Panx1 channels in glial membranes which could play a role in brain damage and seizure activity. Although glial Cxs and Panxs are increasingly recognized as contributing to fundamental mechanisms of epilepsy, the data are often contradictory and controversial, requiring much more research. This article is part of a Special Issue entitled Electrical Synapses.

Glial gap junctional communication involvement in hippocampal damage after middle cerebral artery occlusion

Most patients with stroke caused by middle cerebral artery occlusion (MCAO) show cognitive deficit that is generally regarded as resulting from damage to the cerebral cortex rather than the hippocampus. Whether MCAO induces hippocampal damage and whether this contributes to the cognitive defects remains unclear. Here we investigate the hippocampal damage and its correlation to cognitive defects after exclusively unilateral MCAO and the underlying mechanism for that damage. Patients were assessed for hippocampal damage by magnetic resonance imaging (MRI) and magnetic resonance angiography (MRA), and the Mini Mental-Status Evaluation (MMSE) and Rey Auditory Verbal Learning Test (RAVLT) were used to assess for cognitive defects. We provide the first evidence that patients with exclusively unilateral MCAO showed hippocampal damage characterized by an infarct-size-independent atrophy and alterations in neuronal and glial metabolites in the ipsilateral hippocampus, in parallel with cognitive impairment. Rodent MCAO also induced delayed shrinkage and pyramidal neuronal death in the ipsilateral hippocampus and an impairment of hippocampal-dependent spatial memory. Blocking Gap junctional communication (GJC) with carbenoxolone or downregulation of connexin43 (Cx43) significantly increased the survival of the pyramidal neurons in the ipsilateral hippocampus and improved behavioral scores. Furthermore, Cx43 heterozygous mice showed reduced shrinkage and metabolite abnormality in ipsilateral hippocampus after MCAO. Astroglial GJC plays a significant role in MCAO-induced remote hippocampal damage and cognitive impairment. It might be possible to improve the cognition in patients with MCAO by manipulating interastrocytic communication via the gap junction channels.

Src Utilizes Cas to Block Gap Junctional Communication Mediated by Connexin43

The Src tyrosine kinase phosphorylates Cas (Crk-associated substrate) to confer anchorage independence and invasive growth potential to transformed cells. Gap junctional communication is often lower between aggressive tumor cells compared with normal or benign precursors. The gap junction protein connexin43 (Cx43) is a tumor suppressor that can inhibit tumor cell growth. Src can phosphorylate Cx43 to block gap junctional communication between transformed cells. However, mechanisms by which this event actually closes intercellular channels have not been clearly defined. Here, we report that Src and Cas associate with each other at intercellular junctions. In addition, Cas is required for Src to reduce dye transfer and electrical coupling between cells expressing Cx43. Thus, Src utilizes Cas to inhibit gap junctional communication mediated by Cx43. This finding introduces a novel role of the Cas focal adhesion linker protein in the gap junction complex. This observation may help explain how gap junctional communication can be suppressed between malignant and metastatic tumor cells.

Connexin‐Mimetic Peptides Bind Initially to Cx43 Hemichannels Before Binding to Pericellular Cx43 Gap Junctions

Connexin mimetic peptides (e.g., “gap27”) have been used to block gap junctional communication. However, inconsistent reports of efficacy of peptides have raised questions as to their usefulness. Previous reports have identified gap2737,43 as a more rapid blocker of connexin hemichannels (<15 minutes; e.g., Leybaert et al, Cell Comm Adh, 2003) than gap junctions (>45 minutes; e.g., Boitano and Evans, AJP, 2000). We hypothesized this reflects greater access of the peptides to hemichannels than to fully formed gap junctions. To monitor the binding of the peptide to the cells, we conjugated gap2737,43 with a lissamine-rhodamine tag (GLR). After 45 minute incubations with GLR, HeLa cells expressing Cx43 were stained with the lipophillic dye FM 1-43FX to visualize membranes. Examination of these cells revealed extensive co-localization of FM 1-43FX and peptides and thus junctional localization of the peptide. GLR was also bath applied to HeLa cells transfected with Cx43GFP. XYZT scans using confocal microscopy demonstrated GLR co-localization with Cx43GFP in apical sections within 12 minutes and GLR binding to pericellular Cx43GFP only after a 48 minute incubation. This correlated with functional analyses of Cx43GFP gap junctions in HeLa cells. Microinjected dye diffused between Cx43GFP HeLa cells and this was significantly inhibited after a >45 minute incubation with the GLR peptide.

Anticonvulsant actions of gap junctional blockers in an in vitro seizure model.

Gap junctions (gjs) are increasingly recognized as playing a significant role in seizures. We demonstrate that different types of gap junctional blocking agents reduce the duration of evoked seizure-like primary afterdischarges (PADs) in the rat in vitro CA1 hippocampal pyramidal region, following repetitive tetanization of the Schaffer collaterals. Intracellular acidosis, which is known to block gap junctional communication, decreased the PADs, whereas alkalinization increased the PADs. Cellular excitability was not significantly depressed as determined by input/output relations recorded before and during perfusion of the gj blockers blockers carbenoxolone and sodium propionate. There was a small decrease following 1-octanol perfusion and a large decrease following NH(4)Cl application. Carbenoxolone diminished PAD duration, but increased neuronal excitability in whole-cell recordings. After robust PADs were established, the expression of several gj proteins including connexins (Cxs) 26, 32, 36, and 43, as measured by Western blotting, was unchanged, although the level of nonphosphorylated Cx43 was decreased. Our data support the concept that blocking gap junctional communication is an anticonvulsant mechanism.

Gap junction intercellular communication during lymphocyte transendothelial migration.

Migration of lymphocytes across the endothelium of central or peripheral tissues, a process occurring following activation or differentiation, involves cell to cell interactions featuring adhesion and heterotypic signalling 'cross-talk'. Since lymphocytes and endothelial cells express connexins, the subunit proteins of gap junction intercellular channels, we investigated whether these channels feature in heterotypic signalling during transendothelial migration of lymphocytes. We show, using FACS analysis, that calcein, a gap junction permeant fluorescent dye, was transferred from endothelial cell layers to lymphocytes. The gap junction involvement in intercellular dye transfer was reinforced by studies showing that the process was inhibited by connexin mimetic peptides, a new class of reagents shown to block gap junction communication. Further evidence for the involvement of lymphocyte gap junctions in intercellular communication during transendothelial migration was obtained by two-photon laser scanning microscopy. Although gap junctional communication was inhibited by connexin mimetic peptides, they had little influence on the transmigration process.

Blockade of connexin 43 expression by stable transfection of antisense cDNA in cultured vascular smooth muscle cells.

Gap junctional communication is involved in embryogenesis, cell growth control, and coordinated contraction of cardiac myocytes. It has been hypothesized that gap junctions coordinate responses of vascular cells to constrictor or dilator stimulation. Three connexin (Cx) proteins, 37, 40, and 43, are found in the vasculature. Cx43 gap junctions are widely distributed along the vascular tree, although a precise physiologic role in vascular function is unknown because of a lack of specific functional inhibitors and of suitable animal models. To investigate the role of Cx43 in intercellular communication among vascular smooth muscle (VSM) cells, we selectively modified the expression of the Cx43 gene using antisense cDNA stable transfections in culture. Results show that in cells stably transfected with antisense Cx43 cDNA, gene expression of Cx43 could be reduced to 20% of that observed in vector-transfected cells. In spite of the mRNA and protein reduction, the antisense Cx43 cDNA-transfected cells did not show a significant reduction in dye transfer or a difference in cell growth rate as compared with control. These results suggest either that the residual amount of Cx43 protein is sufficient for dye transfer and growth control or that the dye transfer in these cells can be mediated by Cx40 or other connexin proteins. Therefore, more potent approaches, such as dominant negative and gene knockout, are required to fully block gap junctional communication in VSM cells.

Expression of connexin 30 in Xenopus embryos and its involvement in hatching gland function.

Connexins are a family of proteins that assemble to form gap junction channels. Cell-cell communication through gap junctions mediates many important events in embryogenesis, including limb patterning, lens physiology, neuronal function, left-right asymmetry, and secretion from gland tissue. We studied the expression of connexin 30 (Cx30) in the Xenopus embryo and find that it is expressed in the developing hatching gland and pronephros. To determine whether its expression plays a functional role in the activity of the hatching gland, we exposed pre-hatching embryos to drugs that block gap junctional communication. This resulted in a continuation of normal growth and development but specifically abolished hatching. The treatment did not affect Cx30 or Xenopus hatching enzyme transcription, suggesting a post-transcriptional effect on Cx30 gap junctions. We conclude that junctional communication, possibly mediated by Cx30, is involved in secretion of hatching enzyme in Xenopus.

Chemical requirements for inhibition of gap junction communication by the biologically active lipid oleamide.

Oleamide is an endogenous fatty acid primary amide that possesses sleep-inducing properties in animals and has been shown to effect serotonergic systems and block gap junction communication in a structurally specific manner. Herein, the structural features of oleamide required for inhibition of the gap junction-mediated chemical and electrical transmission in rat glial cells are defined. The effective inhibitors fall into two classes of fatty acid primary amides of which oleamide and arachidonamide are the prototypical members. Of these two, oleamide constitutes the most effective, and its structural requirements for inhibition of the gap junction are well defined. It requires a chain length of 16-24 carbons of which 16-18 carbons appears optimal, a polarized terminal carbonyl group capable of accepting but not necessarily donating a hydrogen bond, a Delta9 cis double bond, and a hydrophobic methyl terminus. Within these constraints, a range of modifications are possible, many of which may be expected to improve in vivo properties. A select set of agents has been identified that serves both as oleamide agonists and as inhibitors of fatty acid amide hydrolase, which is responsible for the rapid inactivation of oleamide.

Structural requirements for 5-HT2A and 5-HT1A serotonin receptor potentiation by the biologically active lipid oleamide.

Oleamide is an endogenous fatty acid primary amide that possesses sleep-inducing properties in animals and that has been shown to effect serotonergic receptor responses and block gap junction communication. Herein, the potentiation of the 5-HT1A receptor response is disclosed, and a study of the structural features of oleamide required for potentiation of the 5-HT2A and 5-HT1A response to serotonin (5-HT) is described. Of the naturally occurring fatty acids, the primary amide of oleic acid (oleamide) is the most effective at potentiating the 5-HT2A receptor response. The structural features required for activity were found to be highly selective. The presence, position, and stereochemistry of the delta9-cis double bond is required, and even subtle structural variations reduce or eliminate activity. Secondary or tertiary amides may replace the primary amide but follow a well defined relationship requiring small amide substituents, suggesting that the carboxamide serves as a hydrogen bond acceptor but not donor. Alternative modifications at the carboxamide as well as modifications of the methyl terminus or the hydrocarbon region spanning the carboxamide and double bond typically eliminate activity. A less extensive study of the 5-HT1A potentiation revealed that it is more tolerant and accommodates a wider range of structural modifications. An interesting set of analogs was identified that inhibit rather than potentiate the 5-HT2A, but not the 5-HT1A, receptor response, further suggesting that such analogs may permit the selective modulation of serotonin receptor subtypes and even have opposing effects on the different subtypes.

Blocking gap junctional intercellular communication in myoblasts inhibits myogenin and MRF4 expression.

Cells rely heavily on cues from their extracellular environment and other cells to coordinate normal physiological processes, and the exchange of molecules via gap junctions has been suggested as on important avenue for cell-cell communication. Gap junctions are found in virtually all mammalian tissues with the notable exception of adult skeletal muscle. However, since functional gap junctions have been detected during the early stages of muscle development, gap junctional intercellular communication (GJIC) may play on important role in myogenesis. In this study, GJIC in normal 16 myoblasts was inhibited using the known blockers l-octanol and beta-glycyrrhetinic acid (beta-GA). Under differentiation promoting conditions, 16 cells fused to form multinucleated myotubes, but when treated with either octanol or beta-GA, no fusion was observed. The expression of two muscle regulatory factors (MRFs), myogenin and MRF4, was examined in both the blocked and control cells. As expected, the activation of both the myogenin and MRF4 genes coincided with the onset of differentiation in the control 16 cells. Neither of these genes were turned on in the blocked cells, even when grown under low serum conditions. This inhibition of differentiation by octanol and beta-GA was reversible, since the activation of both MRF genes as well as myoblast fusion were observed when the blocking medium was replaced with normal differentiating medium. These results suggest that intercellular communication via gap junctions plays an important role in skeletal muscle development and perhaps in the cell signaling events that trigger the activation of muscle-specific MRF genes.

Evidence That Disruption of Connexon Particle Arrangements in Gap Junction Plaques Is Associated with Inhibition of Gap Junctional Communication by a Glycyrrhetinic Acid Derivative

Glycyrrhetinic acid exhibits many pharmacological activities, including the ability to block gap junctional communication. However, the mechanism of glycyrrhetinic action is not clear. Others have shown that glycyrrhetinic acid apparently binds to a single proteinatious binding site in the plasma membrane. We present evidence that while exposure to glycyrrhetinic acid derivatives may not affect protein synthesis or location, it does seem to alter connexon particle packing in gap junction plaques.

Role of gap junctions in the development of the preimplantation mouse embryo.

We have taken several approaches to study the role of gap junctional communication during preimplantation mouse development. Firstly, the normal expression pattern of gap junctions has been characterized using immunostaining in conjunction with laser scanning confocal microscopy. Changes in junctional distribution have been correlated with developmental events. We have gone on to study development and junctional organization in mice which naturally exhibit reduced cell to cell communication (DDK syndrome), and in normal mice in which gap junction permeability has been artificially manipulated. Furthermore, anti-peptide antibodies have been tested for their ability to block gap junction communication and for the effects of such a block on subsequent development. Collectively, the results demonstrate that gap junctional communication plays an important role in the maintenance of compaction and the differentiation of an organized epithelium within an embryo, features which are vital for preimplantation development to progress successfully.

Manipulation of Gap Junctional Communication Between the Yolk Syncytial Layer and Blastoderm Cells and Its Effect On Embryonic Development of Cyprinus Carpio

In an attempt to block gap junctional communication during early development antibodies against connexins were injected into the YSL of carp embryos at the 30% epiboly stage. No effect on development in terms of survival or specific aberrations was observed. It appeared that no antibodies could be demonstrated in the YSL bordering the blastoderm. Connexin translation was aimed to be interrupted by antisense connexin RNA injection into the yolk just beneath the blastoderm cells of the 2- to 16-cell stage carp embryos. Preliminary results indicate a relatively small body volume in a number of treated embryos, which might indicate a disturbance of cell convergence during early gastrulation. The overexpression of connexin RNA by the injection of sense connexin RNA formed another approach to study gap junctional communication. This treatment resulted in aberrant development, which most seriously affected embryos after injection at the 2- to 8-cell stage.

Functional gap junctions are not required for muscle gene activation by induction in Xenopus embryos.

Muscle gene expression is known to be induced in animal pole cells of a Xenopus blastula after 2-3 h of close contact with vegetal pole cells. We tested whether this induction requires functional gap junctions between vegetal and animal portions of an animal-vegetal conjugate. Muscle gene transcription was assayed with a muscle-specific actin gene probe and the presence or absence of communication through gap junctions was determined electrophysiologically. Antibodies to gap junction protein were shown to block gap junction communication for the whole of the induction time, but did not prevent successful induction of muscle gene activation. The outcome was the same whether communication between inducing vegetal cells and responding animal cells was blocked by introducing antibodies into vegetal cells alone or into animal cells alone. We conclude that gap junctions are not required for this example of embryonic induction.