Role Of Gap Junctional Communication Research Articles

L-Proline improves the cytoplasmic maturation of mouse oocyte by regulating glutathione-related redox homeostasis

Recent studies have shown that l-proline (proline) is an antioxidant to protect cells from oxidative stress in vivo and in vitro. Glutathione (GSH) is a major cellular redox regulator involved in controlling redox balance and is regarded as one of the key indices to predict the cytoplasmic maturation of oocytes. The objectives of this study are to investigate the effect of proline on the developmental potential of mouse oocytes and to determine the role of gap junctional communication (GJC) on intraoocyte GSH concentration during in vitro maturation (IVM). Compared with control (0 mmol/L), 0.5 mmol/L proline supplementation enhanced rates of activated oocytes, 2-cell and 4-cell embryos, and blastocysts. Furthermore, 0.25 and 0.5 mmol/L proline supplementation markedly upregulated mRNA expression of glutamate-cysteine ligase catalytic subunit (GCLC) and glutamate-cysteine ligase modifier subunit (GCLM) in oocytes and cumulus cells, enhanced GSH concentration in oocytes, and reduced reactive oxygen species (ROS) level in oocytes. Interestingly, carbenoxolone disodium salt (CBX) treatment reduced GSH concentration in oocytes and the rate of early embryo development without proline incubation. Notably, CBX-triggered reduction in the rates of the number of 2-cell and 4-cell embryos and blastocysts were rescued by 0.5 mmol/L proline supplementation. Collectively, these results indicate a novel functional role of proline in oocyte cytoplasmic maturation and regulation of glutathione-related redox homeostasis.

Dynamic intercellular transport modulates the spatial patterning of differentiation during early neural commitment

The initiation of heterogeneity within a population of phenotypically identical progenitors is a critical event for the onset of morphogenesis and differentiation patterning. Gap junction communication within multicellular systems produces complex networks of intercellular connectivity that result in heterogeneous distributions of intracellular signaling molecules. In this study, we investigate emergent systems-level behavior of the intercellular network within embryonic stem cell (ESC) populations and corresponding spatial organization during early neural differentiation. An agent-based model incorporates experimentally-determined parameters to yield complex transport networks for delivery of pro-differentiation cues between neighboring cells, reproducing the morphogenic trajectories during retinoic acid–accelerated mouse ESC differentiation. Furthermore, the model correctly predicts the delayed differentiation and preserved spatial features of the morphogenic trajectory that occurs in response to intercellular perturbation. These findings suggest an integral role of gap junction communication in the temporal coordination of emergent patterning during early differentiation and neural commitment of pluripotent stem cells.

Gap junctional communication is involved in differentiation of osteoclasts from bone marrow and peripheral blood monocytes

AimsThe aim of the study was to compare the influence of gap junctional communication (GJC) in osteoclastogenesis from bone marrow (BM) and peripheral blood (PB) monocytes. These widely used sources differ in purity, since BM cultures contain a significant number of stromal cells. We studied whether stimulation of GJC in BM monocyte/stromal cell cultures differs from the effect in pure PB monocyte cultures. We compared the differentiation also in acidosis, which is a known inducer of bone resorption. Main methodsHuman BM and PB monocytes were isolated from BM aspirates or whole blood samples. The cells were cultured on human bone slices with osteoclastogenic growth factors and a GJC modulator, antiarrhythmic peptide AAP10, at physiological and acidic pH. Key findingsBoth BM and PB monocytes differentiated into osteoclasts. Acidosis increased resorption in both cultures but stimulated cell fusion only in BM cultures, which demonstrates the role of stromal cells in osteoclastogenesis. At physiological pH, AAP10 increased the number of multinuclear cells and bone resorption in both BM and PB cultures indicating that GJC is involved in differentiation in both of these osteoclastogenesis assays. Interestingly, in PB cultures at pH 6.5 the stimulation of GJC with AAP10 inhibited both osteoclastogenesis and bone resorption suggesting a different role of GJC in BM and PB monocytes at stressed environment. SignificanceThe study is conducted with primary human tissue samples and adds new knowledge on factors affecting osteoclastogenesis from different monocyte sources.

Abstract 1815: Bystander effect and genomic instability in human cells and their progeny after irradiation with X rays, protons or carbon ions: role of gap junction communication

Abstract Purpose: Ionizing Radiation (IR)-induced bystander effects and genomic instability have important implication in radiotherapy and radioprotection. Their persistence in the progeny of normal cells may contribute to risk of long-term radiation-related health effect in human, including cancer. Hence, this study investigates the role of gap junction intercellular communication (GJIC) and the quality of radiation in the propagation of stressful effects in the unirradiated bystander cells and their progeny. Material and methods: Human skin fibroblasts in the confluent state were exposed to microbeam irradiations with different linear energy transfer (LET) at mean absorbed dose of 0.4 Gy, in the presence or absence of GJIC inhibitor (AGA) by which 0.036-0.4% of cells were directly targeted by IR. After 4 h irradiation or following 20 population doublings, the cells were harvested and assayed for micronucleus (MN) formation, gene mutation and protein oxidation. Results: Our results showed that high-LET carbon microbeams (LET ∼103 keV/μm) and high-LET proton microbeams (LET ∼11 keV/μm) were more effective than low-LET X ray microbeam (LET ∼6 keV/μm) in the induction of DNA damage (MN formation) in bystander cells. Interestingly, significant attenuation of MN formation occurred in bystander cells in the presence of AGA after proton and carbon microbeams. In contrast, incubation of the cells with AGA did not significantly affect the induction of MN formation in bystander cells during confluent holding after X irradiation. Further, the progeny of bystander cells exposed to X rays or protons showed persistent oxidative stress which correlated MN formation and mutation frequency. Such effects were not observed after irradiation by carbon ions. Importantly, the progeny of bystander cells from cultures exposed to protons or carbon ions under conditions where GJIC was inhibited harbored reduced oxidative and genetic damage. This mitigating effect was not detected when the cultured were exposed to X rays. Taken together, the overall results show the expression of stressful effects in the bystander cells and their progeny are dependent on the radiation quality or LET. Conclusions: Our findings suggest that the involvement of GJIC-dependent of radiation quality in the propagation of radiation induces stressful effects to bystander cells and their progeny. In addition, this work provides a strong support to the fact that carbon ions can significantly reduce the risk of cancer and have potential implications in the therapeutic outcome of radiotherapy compared to X rays or protons. Citation Format: Narongchai Autsavapromporn, Ianik Plante, Cuihua Liu, Teruaki Konishi, Noriko Usami, Tomoo Funayama, Yukio Uchihori, Tom K. Hei, Edouard I. Azzam, Sirikan Yamada, Takeshi Murakami, Masao Suzuki. Bystander effect and genomic instability in human cells and their progeny after irradiation with X rays, protons or carbon ions: role of gap junction communication. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 1815. doi:10.1158/1538-7445.AM2015-1815

Role of gap junction communication in hepatocyte/fibroblast co-cultures: Implications for hepatic tissue engineering

The success of cell-based therapies for treatment of liver disease hinges on the phenotypic stability of isolated hepatocytes; however, primary hepatocytes rapidly lose many liver-specific functions following enzymatic isolation from the liver. Previous studies have demonstrated that liver-specific functions can be stabilized by cocultivation with non-parenchymal cells. Although the precise mechanisms underlying these effects have not yet been elucidated, it is believed that gap junctional intercellular communication plays an important role in cell-cell communication. In this study, indirect immunofluorescence was used to identify connexin 32 and 26 as homotypic gap junction proteins that were present between hepatocytes during co-culture with 3T3 fibroblasts, but not in pure hepatocyte cultures. Additionally, dye transfer of Lucifer yellow revealed that these gap junctions were functionally coupled. Although previous studies reported abundant connexin 43 expression in 3T3 fibroblasts and functional coupling by dye transfer, we did not observe heterotypic gap junction (connexin 43) expression or its functional coupling by either immunofluorescence or dye transfer. Incubation with a gap junction inhibitor, 18β-glycyrrhetinic acid, in hepatocyte co-culture diminished the level of liverspecific markers such as albumin production and P450 activity relative to control co-cultures. Overall, these findings suggest that homotypic gap junctions are present in hepatocytes co-cultured with fibroblasts and might play a functional role in homotypic hepatocyte communication. In conclusion, homotypic gap junctional communication may be important to phenotypic stability of isolated hepatocytes in co-culture and crucial to the design of tissue-engineered therapies for treatment of liver failure.

Critical role of gap junction communication, calcium and nitric oxide signaling in bystander responses to focal photodynamic injury.

Ionizing and nonionizing radiation affect not only directly targeted cells but also surrounding "bystander" cells. The underlying mechanisms and therapeutic role of bystander responses remain incompletely defined. Here we show that photosentizer activation in a single cell triggers apoptosis in bystander cancer cells, which are electrically coupled by gap junction channels and support the propagation of a Ca2+ wave initiated in the irradiated cell. The latter also acts as source of nitric oxide (NO) that diffuses to bystander cells, in which NO levels are further increased by a mechanism compatible with Ca(2+)-dependent enzymatic production. We detected similar signals in tumors grown in dorsal skinfold chambers applied to live mice. Pharmacological blockade of connexin channels significantly reduced the extent of apoptosis in bystander cells, consistent with a critical role played by intercellular communication, Ca2+ and NO in the bystander effects triggered by photodynamic therapy.

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.

Connexins and Cap-independent translation: Role of internal ribosome entry sites

Cap-independent translation using an internal ribosome entry site instead of the 5'-Cap structure has been discovered in positive-sense RNA viruses and eukaryotic genomes including a subset of gap junction forming connexins genes. With a growing number of mutations found in human connexin genes and studies on genetically modified mouse models mechanisms highlighting the important role of gap junctional communication in multicellular organism it is obvious that mechanism need to be in place to preserve this critical property even under conditions when Cap-mediated translation is scrutinized. To ensure sustained gap junctional communication, rapid initiation of translation of preexisting connexin mRNAs is one possibility, and the presence of internal ribosome entry sites in gap junction genes comply with such a requirement. In this review, we will summarize past and recent findings to build a case for IRES mediated translation as an alternative regulatory pathway facilitating gap junctional communication. This article is part of a Special Issue entitled Electrical Synapses.

The role of gap junction communication and oxidative stress in the propagation of toxic effects among high-dose α-particle-irradiated human cells.

We investigated the roles of gap junction communication and oxidative stress in modulating potentially lethal damage repair in human fibroblast cultures exposed to doses of α particles or γ rays that targeted all cells in the cultures. As expected, α particles were more effective than γ rays at inducing cell killing; further, holding γ-irradiated cells in the confluent state for several hours after irradiation promoted increased survival and decreased chromosomal damage. However, maintaining α-particle-irradiated cells in the confluent state for various times prior to subculture resulted in increased rather than decreased lethality and was associated with persistent DNA damage and increased protein oxidation and lipid peroxidation. Inhibiting gap junction communication with 18-α-glycyrrhetinic acid or by knockdown of connexin43, a constitutive protein of junctional channels in these cells, protected against the toxic effects in α-particle-irradiated cell cultures during confluent holding. Upregulation of antioxidant defense by ectopic overexpression of glutathione peroxidase protected against cell killing by α particles when cells were analyzed shortly after exposure. However, it did not attenuate the decrease in survival during confluent holding. Together, these findings indicate that the damaging effect of α particles results in oxidative stress, and the toxic effects in the hours after irradiation are amplified by intercellular communication, but the communicated molecule(s) is unlikely to be a substrate of glutathione peroxidase.

489 New animal model in colorectal cancer

489 New animal model in colorectal cancer

Effects of gap junctional blockers on cerebral vasospasm after subarachnoid hemorrhage in rabbits

The role of gap junctional communication in the coordination of vascular behavior has been well established experimentally. The aim of this study was to investigate whether the gap junctional blockers would inhibit cerebral vasospasm after experimental subarachnoid hemorrhage (SAH). We used the double-hemorrhage model of SAH with injection of autologous arterial blood into the cisterna magna on days 1 and 2. Octanol or carbenoxolone was administered intracisternally on day 3, and repeated on days 4 and 5. Angiography was performed before (day 0) and 7 days after (day 7) SAH, and the diameter of the basilar artery was measured. Paraffin blocks of brain tissues were prepared and sectioned for hematoxylin and eosin staining for morphologic analysis. Arterial narrowing in SAH + octanol group and SAH + carbenoxolone group at day 7 was significantly less than that in SAH-only group and each SAH + vehicle group, respectively. Less morphologic changes in SAH + octanol group and SAH + carbenoxolone group were observed when compared to that in SAH-only group and each SAH + vehicle group. Western blotting showed that carbenoxolone down-regulated C×43 protein expression in the BA, which in the SAH-only group was significantly higher than that of the normal group. Gap junction blockers attenuate the experimental cerebral vasospasm and down-regulate the increase in expression of C×43 protein after SAH in rabbits. These data suggest that gap junctions play an important role in the development of cerebral vasospasm.

Role of Gap Junctional Intercellular Communication and NO Signaling in Low-Dose Radiation Hypersensitivity

In the cells irradiated at low density, U87 cell line has shown a highly distinguishable HRS/IRR response (Chandna et al, Radiat Res, 157, 516-525, 2002), which was mitigated at higher density. In order to understand the basis of increased cellular lethality at low doses as well as the density-mediated suppression of HRS/IRR response, effect of low gamma radiation doses was studied using additional parameters including micronucleation, apoptosis induction and cell cycle progression delay in the absence and presence of inhibitors of gap junctional intercellular communication (GJIC) and NO signaling. Cells plated/ seeded at high cell multiplicity or density were pre-treated with phorbol ester PMA (phorbyl myristate acetate) and/or the NOS inhibitor LNMMA from 2h pre-irradiation, and treatment continued till 1h post-irradiation, after which cells were incubated in fresh growth medium. The low dose of 20cGy induced significant micronucleation in cells, and inhibition of GJIC by PMA (5nM) significantly enhanced this radiation-induced micronucleation in high density cultures. On the other hand, the NOS inhibitor LNMMA reduced micronucleation induced at 20cGy, and also significantly mitigated the PMA-induced enhancement of radiation-induced micronucleation. Interestingly, PMA could induce relatively larger number of binucleated cells, which indicates abrogation of G2/M checkpoint and might explain the higher induction of micronuclei in irradiated cells pre-treated with this agent. In contrast to PMA that enhanced micronucleation and reduced irradiated cell survival at low doses, LNMMA inhibited the micronucleation and enhanced survival in the irradiated cells, indicating the role of nitrosative stress in low-dose sensitivity of cells. A small but significant proportion of cells also showed nuclear fragmentation typical of apoptosis when pre-treated with PMA prior to irradiation at 20cGy. Further, a significant proportion of micronuclei induced at 20cGy were relatively larger in size, possibly indicating the induction of chromosome laggards due to spindle disturbances during the cell division. In this study, inhibition of GJIC significantly enhanced low dose lethality in the high density tumour cell cultures, whereas NOS inhibition reversed this effect. Indeed, the cell density mediated abrogation of HRS/IRR was significantly reversed in the presence of PMA, indicating protective role of gap junctional communication in HRS/IRR response. The study further indicated that PMA induced enhancement of radiation lethality could be effected through abrogation of G2/M cell cycle checkpoint.

Connexin 43 expression in human and mouse testes with impaired spermatogenesis.

Connexin 43 (Cx43) belongs to a family of proteins that form gap junction channels. The aim of this study was to examine the expression of Cx43 in the testis of a patient with Klinefelter's syndrome and of mice with the mosaic mutation and a partial deletion in the long arm of the Y chromosome. These genetic disorders are characterized by the presence of numerous degenerated seminiferous tubules and impaired spermatogenesis. In mouse testes, the expression and presence of Cx43 were detected by means of immunohistochemistry and Western blot analysis, respectively. In testes of Klinefelter's patient only immunoexpression of Cx43 was detected. Regardless of the species Cx43 protein was ubiquitously distributed in testes of reproductively normal males, whereas in those with testicular disorders either a weak intensity of staining or no staining within the seminiferous tubules was observed. Moderate to strong or very strong staining was confined to the interstitial tissue. In an immunoblot analysis of testicular homogenates Cx43 appeared as one major band of approximately 43 kDa. Our study adds three more examples of pathological gonads in which the absence or apparent decrease of Cx43 expression within the seminiferous tubules was found. A positive correlation between severe spermatogenic impairment and loss of Cx43 immunoreactivity observed in this study supports previous data that gap junctions play a crucial role in spermatogenesis. Strong Cx43 expression detected mostly in the interstitial tissue of the Klinefelter's patient may presumably be of importance in sustaining Leydig cell metabolic activity. However, the role of gap junction communication in the control of Leydig cell function seems to be more complex than originally thought.

The use of antibodies to gap junction protein to explore the role of gap junctional communication during development.

Antibodies raised against the major 27 kDa protein electrophoretically eluted from isolated gap junctions and affinity purified against the antigen have been used to explore the role of communication through gap junctions in the early amphibian and mouse embryos. In both species, injection of the antibodies into one cell completely blocks both dye transfer and electrical coupling between cells connected by gap junctions. In the amphibian embryo the generation of a communication-incompetent clone of cells leads to patterning defects in the region derived from the antibody-injected cell. In the mouse embryo, blocking cell-to-cell communication leads to decompaction of the communication-incompetent cells. The possible significance of these findings in relation to development in general and to the organization of the first transporting epithelia to appear during development is discussed.

Gap junctional intercellular communication in the mouse ovarian follicle.

A targeted disruption of the gene encoding the gap junction protein connexin37 (Cx37; alpha 4) results in female infertility. Mutant follicles are not observed to develop beyond early antral stages, and there is a lack of both observable mature Graafian follicles and ovulation. The oocytes are unable to acquire meiotic competence. Following oocyte failure, the residual follicular cells do not undergo atresia but rather transdifferentiate into luteal cells, resulting in a mutant ovary populated with numerous, inappropriate corpora lutea. These results indicate that the Cx37-containing gap junctions formed between oocyte and follicular cells permit bidirectional signalling between the two cell types. These junctions are required for oocyte growth and development during preantral stages of the follicle, and for the inhibition of follicle cell luteinization. An additional role for these junctions may be to permit transfer of cytoplasmic signals required to hold oocytes in meiotic arrest. Since the mutant follicles never acquire meiotic competence, this latter role for gap junctional communication cannot be tested in this model.

Connexin 43 expression of foreign body giant cells after implantation of nanoparticulate hydroxyapatite

In bone a role of connexin 43 has been implicated with the fusion of mononuclear precursors of the monocyte/macrophage lineage into multinucleated cells. In order to investigate the putative role of connexin 43 in formation of bone osteoclast-like foreign body giant cells which are formed in response to implantation of biomaterials, nanoparticulate hydroxyapatite had been implanted into defects of minipig femura. After 20 days the defect areas were harvested and connexin 43 expression and synthesis were investigated by using immunohistochemistry, Western Blot, and in situ hybridization within macrophages and osteoclast-like foreign body giant cells. Morphological analysis of gap junctions is performed ultrastructurally. As shown on protein and mRNA level numerous connexin 43 positive macrophages and foreign body giant cells (FBGC) were localized within the granulation tissue and along the surfaces of the implanted hydroxyapatite (HA). Besides, the formation of FBGC by fusion of macrophages could be shown ultrastructurally. Connexin 43 labeling observed on the protein and mRNA level could be attributed to gap junctions identified ultrastructurally between macrophages, between FBGC, and between FBGC and macrophages. Annular gap junctions in the cytoplasm of FBGC pointed to degradation of the channels, and the ubiquination that had occurred in the course of degradation was confirmed by Western blot analysis. All in all, the presently observed pattern of connexin 43 labeling refers to an functional role of gap junctional communication in the formation of osteoclast-like foreign body giant cells formed in response to implantation of the nanoparticulate HA.

Gap‐junction blocker carbenoxolone differentially enhances NMDA‐induced cell death in hippocampal neurons and astrocytes in co‐culture

The beneficial or detrimental role of gap junction communication in the pathophysiology of brain injury is still controversial. We used co-cultures of hippocampal astrocytes and neurons, where we identified homocellular astrocyte-astrocyte and heterocellular astrocyte-neuron coupling by fluorescence recovery after photobleaching, which was decreased by the gap junction blocker carbenoxolone (CBX). In these cultures, we determined the cell type-specific effects of CBX on the excitotoxic damage caused by N-methyl-D-aspartate (NMDA). We determined in both astrocytes and neurons the influence of CBX, alone or together with NMDA challenge, on cytotoxicity using propidium iodide labeling. CBX alone was not cytotoxic, but CBX treatment differentially accelerated the NMDA-induced cell death in both astrocytes and neurons. In addition, we measured mitochondrial potential using rhodamine 123, membrane potential using the oxonol dye bis(1,3-diethylthiobarbituric acid)trimethine oxonol, cytosolic Ca(2+) level using fura-2, and formation of reactive oxygen species (ROS) using dihydroethidium. CBX alone induced neither an intracellular Ca(2+) rise nor a membrane depolarization. However, CBX elicited a mitochondrial depolarization in both astrocytes and neurons and increased the ROS formation in neurons. In contrast, NMDA caused a membrane depolarization in neurons, coinciding with intracellular Ca(2+) rise, but neither mitochondrial depolarization nor ROS production seem to be involved in NMDA-mediated cytotoxicity. Pre-treatment with CBX accelerated the NMDA-induced membrane depolarization and prevented the repolarization of neurons after the NMDA challenge. We hypothesize that these effects are possibly mediated via blockage of gap junctions, and might be involved in the mechanism of CBX-induced acceleration of excitotoxic cell death, whereas the CBX-induced mitochondrial depolarization and ROS formation are not responsible for the increase in cytotoxicity. We conclude that both in astrocytes and neurons gap junctions provide protection against NMDA-induced cytotoxicity.

Vascular Gap Junctions in Hypertension

The proteins that form the gap junctions (connexins) are widely expressed in organs that are central to the development of hypertension: endocrine organs, kidney, brain, heart, and vasculature (Figure 1). Surprisingly, there is little information on the modification of connexins in hypertension in any of these organs except the vasculature, the subject of our review, but it would be hoped that this lack of information might spur research on these organs. Figure 1. Possible roles for the connexins in the control of blood pressure. A, Electron micrograph of a longitudinal view of an arteriole. Vascular connexins can play multiple roles in regulating function and vasomotor tone. Cell–cell communication, both radial and longitudinal, links the cells of the arteriolar wall. B, Most every organ involved in the control of blood pressure abundantly expresses ≥1 of the connexins. Essentially no experimental evidence exists regarding a possible link between connexins and hypertension in nonvascular tissues. Augmented vasomotor tone typically plays a key role in the development of hypertension, and tone depends on cell–cell communication established by paracrine molecules and, in addition, gap junctions. Paracrine-based linkages between cells of the vasculature are well known, and the possible roles of such linkages in the genesis of hypertension have been extensively explored. Much less is known of the roles of gap junctional communication in establishing vasomotor tone and of the effects of modification of gap junctions on hypertension. The gap junctions are formed by joining 2 hexameric assemblies of connexin protein monomers, that is, hemichannels (1 in Figure 2A). In the vasculature, assays of message and protein show that the hemichannels can be assembled from combinations of 4 connexins, named according to their apparent molecular weight: Cx37, Cx40, Cx43, and Cx45. Two hemichannels are linked to form a gap junction, which can provide both homocellular …

Glutamate pretreatment affects Ca2+ signaling in processes of astrocyte pairs

Simultaneous somatic patch-pipette recording of a single astrocyte to evoke voltage-gated calcium currents, and Ca(2+) imaging, were used to study the spatial and temporal profiles of depolarization-induced changes in intracellular Ca(2+) ([Ca(2+)](i)) in the processes of cultured rat cortical astrocytes existing as pairs. Transient Ca(2+) changes locked to depolarization were observed as microdomains in the processes of the astrocyte pairs, and the responses were more pronounced in the adjoining astrocyte. Considering the functional significance of higher concentrations of glutamate observed in certain pathological conditions, Ca(2+) transients were recorded following pretreatment of cells with glutamate (500 microM for 20 min). This showed distance-dependent incremental scaling and attenuation in the presence of the metabotropic glutamate receptor (mGluR) antagonist, alpha-methyl(4-carboxy-phenyl) glycine (MCPG). Estimation of local Ca(2+) diffusion coefficients in the astrocytic processes indicated higher values in the adjoining astrocyte of the glutamate pretreated group. Intracellular heparin introduced into the depolarized astrocyte did not affect the Ca(2+) transients in the heparin-loaded astrocyte but attenuated the [Ca(2+)](i) responses in the adjoining astrocyte, suggesting that inositol 1,4,5 triphosphate (IP(3)) may be the transfer signal. The uncoupling agent, 1-octanol, attenuated the [Ca(2+)](i) responses in both the control and glutamate pretreated astrocytes, indicating the role of gap junctional communication. Our studies indicate that individual astrocytes have distinct functional domains, and that the glutamate-induced alterations in Ca(2+) signaling involve a sequence of intra- and intercellular steps in which phospholipase C (PLC), IP(3), internal Ca(2+) stores, VGCC and gap junction channels appear to play an important role.

Regulation of osteoclastogenesis by gap junction communication

Receptor activator of NF-kappaB ligand (RANKL) is crucial in osteoclastogenesis but signaling events involved in osteoclast differentiation are far from complete and other signals may play a role in osteoclastogenesis. A more direct pathway for cellular crosstalk is provided by gap junction intercellular channel, which allows adjacent cells to exchange second messengers, ions, and cellular metabolites. Here we have investigated the role of gap junction communication in osteoclastogenesis in mouse bone marrow cultures. Immunoreactive sites for the gap junction protein connexin 43 (Cx43) were detected in the marrow stromal cells and in mature osteoclasts. Carbenoxolone (CBX) functionally blocked gap junction communication as demonstrated by a scrape loading Lucifer Yellow dye transfer technique. CBX caused a dose-dependent inhibition (significant > or = 90 microM) of the number of tartrate-resistant acid phosphatase (TRAP)-positive multinucleated cells formed in 7- to 8-day marrow cultures stimulated by parathyroid hormone (PTH; 10 nM) or forskolin (FSK; 1 microM). Furthermore, CBX (100 microM) significantly inhibited prostaglandin E2 (PGE2; 10 microM) and 1,25(OH)2-vitamin D3 stimulated osteoclast differentiation in the mouse bone marrow cultures. Consequently, quantitative real-time polymerase chain reaction (PCR) analysis demonstrated that CBX downregulated the expression of osteoclast phenotypic markers, but without having any significant effects on RANK, RANKL, and osteoprotegerin (OPG) mRNA expression. However, the results demonstrated that CBX significantly inhibits RANKL-stimulated (100 ng/ml) osteoclastogenesis in the mouse bone marrow cultures. Taken together, our results suggests that gap junctional diffusion of messenger molecules interacts with signaling pathways downstream RANKL in osteoclast differentiation. Further studies are required to define the precise mechanisms and molecular targets involved.