Astrocytic gap junctional communication is reduced in amyloid-β-treated cultured astrocytes,but not in Alzheimer's disease transgenic mice

Alzheimer''s disease is characterized by accumulation of amyloid deposits in brain, progressive cognitive deficits and reduced glucose utilization. Many consequences of the disease are attributed to neuronal dysfunction, but roles of astrocytes in its pathogenesis are not well understood. Astrocytes are extensively coupled via gap junctions, and abnormal trafficking of metabolites and signalling molecules within astrocytic syncytia could alter functional interactions among cells comprising the neurovascular unit. To evaluate the influence of amyloid-β on astrocyte gap junctional communication, cultured astrocytes were treated with monomerized amyloid-β_1–40 (1 μmol/l) for intervals ranging from 2 h to 5 days, and the areas labelled by test compounds were determined by impaling a single astrocyte with a micropipette and diffusion of material into coupled cells. Amyloid-β-treated astrocytes had rapid, sustained 50–70% reductions in the area labelled by Lucifer Yellow, anionic Alexa Fluor® dyes and energy-related compounds, 6-NBDG (a fluorescent glucose analogue), NADH and NADPH. Amyloid-β treatment also caused a transient increase in oxidative stress. In striking contrast with these results, spreading of Lucifer Yellow within astrocytic networks in brain slices from three regions of 8.5–14-month-old control and transgenic Alzheimer''s model mice was variable, labelling 10–2000 cells; there were no statistically significant differences in the number of dye-labelled cells among the groups or with age. Thus amyloid-induced dysfunction of gap junctional communication in cultured astrocytes does not reflect the maintenance of dye transfer through astrocytic syncytial networks in transgenic mice; the pathophysiology of Alzheimer''s disease is not appropriately represented by the cell culture system.     

Lipopolysaccharide-induced inhibition of connexin43 gap junction communication in astrocytes is mediated by downregulation of caveolin-3

Astrocytes play a crucial role in maintaining the homeostasis of the brain. Changes to gap junctional intercellular communication (GJIC) in astrocytes and excessive inflammation may trigger brain damage and neurodegenerative diseases. In this study, we investigated the effect of lipopolysaccharide (LPS) on connexin43 (Cx43) gap junctions in rat primary astrocytes. Following LPS treatment, dose- and time-dependent inhibition of Cx43 expression was seen. Moreover, LPS induced a reduction in Cx43 immunoreactivity at cell–cell contacts and significantly inhibited GJIC, as revealed by the fluorescent dye scrape loading assay. Toll-like receptor 4 (TLR4) protein expression was increased 2–3-fold following LPS treatment. To study the pathways underlying these LPS-induced effects, we examined downstream effectors of TLR4 signaling and found that LPS induced a significant increase in phosphorylated extracellular signal-regulated kinase (pERK) levels up to 6 h, followed by signal attenuation and downregulation of caveolin-3 expression. Interestingly, LPS treatment also induced a dramatic increase in inducible nitric oxide synthase (iNOS) levels at 6 h, which were sustained up to 18–24 h. The LPS-induced downregulation of Cx43 and caveolin-3 was prevented by co-treatment of astrocytes with the iNOS cofactor inhibitor 1400W, but not the ERK inhibitor PD98059. Specific knockdown of caveolin-3 using siRNA had a significant inhibitory effect on GJIC and resulted in a downregulation of Cx43. Our results suggest that long-term LPS treatment of astrocytes leads to inhibition of Cx43 gap junction communication by the activation of iNOS and downregulation of caveolin-3 via a TLR4-mediated signaling pathway.     

Overexpression of the gap junction protein Cx43 as found in diabetic foot ulcers can retard fibroblast migration

Poor healing of DFUs (diabetic foot ulcers) is a major clinical problem that can be extremely debilitating and lead to lower limb amputation. In the normal acute wound, the Cx43 (connexin 43) gap junction protein is down-regulated at the wound edge as a precursor to cell migration and healing. In fibroblasts from the human chronic DFU wound edge there was a striking and significant 10-fold elevation of Cx43 protein, as well as a 6-fold increase in N-cadherin and a 2-fold increase in ZO-1 (zonular occludin-1), compared with unwounded skin. In streptozotocin diabetic rats, Cx43 was found to be up-regulated in intact dermal fibroblasts in direct proportion to blood glucose levels and increased 2-fold further in response to wounding of the skin. To mimic diabetes, NIH 3T3 fibroblasts were cultured under different concentrations of glucose or mannitol and Cx43 protein intercellular communication and migration rates were determined. Cultures of fibroblasts in very high (40 mM) glucose conditions showed significantly elevated Cx43 protein levels, as shown by immunostaining and Western blotting, and significantly increasing gap junctional communication, as shown by dye transfer. In scratch wound-healing assays, increased levels of Cx43 from high glucose resulted in repressed filopodial extensions and significantly slower migration rates than in either standard conditions (5.5 mM glucose) or the osmotic control of mannitol. Conversely, when glucose-induced Cx43 up-regulation was prevented with Cx43shRNA (Cx43 short-hairpin RNA) transduction, the fibroblasts extended long filopodia and migrated significantly faster. Cx43 protein was up-regulated in fibroblasts in DFUs as well as after high glucose exposure in culture which correlated with inhibition of fibroblast migration and is likely to contribute to impaired wound healing.     

A dominant connexin43 mutant does not have dominant effects on gap junction coupling in astrocytes

Dominant mutations in GJA1, the gene encoding the gap junction protein connexin43 (Cx43), cause oculodentodigital dysplasia (ODDD), a syndrome affecting multiple tissues, including the central nervous system (CNS). We investigated the effects of the G60S mutant, which causes a similar, dominant phenotype in mice (Gja1(Jrt/+)). Astrocytes in acute brain slices from Gja1(Jrt/+) mice transfer sulforhodamine-B comparably to that in their wild-type (WT) littermates. Further, astrocytes and cardiomyocytes cultured from Gja1(Jrt/+) mice showed a comparable transfer of lucifer yellow to those from WT mice. In transfected cells, the G60S mutant formed gap junction (GJ) plaques but not functional channels. In co-transfected cells, the G60S mutant co-immunoprecipitated with WT Cx43, but did not diminish GJ coupling as measured by dual patch clamp. Thus, whereas G60S has dominant effects, it did not appreciably reduce GJ coupling.     

The unfolded protein response to endoplasmic reticulum stress in cultured astrocytes and rat brain during experimental diabetes

Oxidative-nitrosative stress and inflammatory responses are associated with endoplasmic reticulum (ER) stress in diabetic retinopathy, raising the possibility that disturbances in ER protein processing may contribute to CNS dysfunction in diabetics. Upregulation of the unfolded protein response (UPR) is a homeostatic response to accumulation of abnormal proteins in the ER, and the present study tested the hypothesis that the UPR is upregulated in two models for diabetes, cultured astrocytes grown in 25 mmol/L glucose for up to 4 weeks and brain of streptozotocin (STZ)-treated rats with diabetes for 1–7 months. Markers associated with translational blockade (phospho-eIF2α and apoptosis (CHOP), inflammatory response (inducible nitric oxide synthase, iNOS), and nitrosative stress (nuclear translocation of glyceraldehyde-3-phosphate dehydrogenase, GAPDH) were not detected in either model. Nrf2 was present in nuclei of low- and high-glucose cultures, consistent with oxidative stress. Astrocytic ATF4 expression was not altered by culture glucose concentration, whereas phospho-IRE and ATF6 levels were higher in low- compared with high-glucose cultures. The glucose-regulated chaperones, GRP78 and GRP94, were also expressed at higher levels in low- than high-glucose cultures, probably due to recurrent glucose depletion between feeding cycles. In STZ-rat cerebral cortex, ATF4 level was transiently reduced at 4 months, and p-IRE levels were transiently elevated at 3 months. However, GRP78 and GRP94 expression was not upregulated, and iNOS, amyloid-β, and nuclear accumulation of GAPDH were not evident in STZ-diabetic brain. High-glucose cultured astrocytes and STZ-diabetic brain are relatively resistant to diabetes-induced ER stress, in sharp contrast with cultured retinal Müller cells and diabetic rodent retina.     

Astrocytic gap junctional networks suppress cellular damage in an in vitro model of ischemia

Astrocytes play pivotal roles in both the physiology and the pathophysiology of the brain. They communicate with each other via extracellular messengers as well as through gap junctions, which may exacerbate or protect against pathological processes in the brain. However, their roles during the acute phase of ischemia and the underlying cellular mechanisms remain largely unknown. To address this issue, we imaged changes in the intracellular calcium concentration ([Ca²⁺]_i) in astrocytes in mouse cortical slices under oxygen/glucose deprivation (OGD) condition using two-photon microscopy. Under OGD, astrocytes showed [Ca²⁺]_i oscillations followed by larger and sustained [Ca²⁺]_i increases. While the pharmacological blockades of astrocytic receptors for glutamate and ATP had no effect, the inhibitions of gap junctional intercellular coupling between astrocytes significantly advanced the onset of the sustained [Ca²⁺]_i increase after OGD exposure. Interestingly, the simultaneous recording of the neuronal membrane potential revealed that the onset of the sustained [Ca²⁺]_i increase in astrocytes was synchronized with the appearance of neuronal anoxic depolarization. Furthermore, the blockade of gap junctional coupling resulted in a concurrent faster appearance of neuronal depolarizations, which remain synchronized with the sustained [Ca²⁺]_i increase in astrocytes. These results indicate that astrocytes delay the appearance of the pathological responses of astrocytes and neurons through their gap junction-mediated intercellular network under OGD. Thus, astrocytic gap junctional networks provide protection against tissue damage during the acute phase of ischemia.     

Connexin43 Reduces Melanoma Growth within a Keratinocyte Microenvironment and during Tumorigenesis in Vivo

Connexins (Cx) have been identified as tumor suppressors or enhancers, a distinction that appears to be dependent on the type and stage of disease. However, the role of connexins in melanoma tumorigenesis and their status during cancer onset and progression remain controversial and unclear. Here, we show that the aggressive B16-BL6 mouse melanoma cell line expresses low basal levels of Cx26 and Cx43, rendering them gap junctional intercellular communication-deficient as elucidated by immunofluorescence, Western blotting, and dye transfer studies. Following ectopic expression of green fluorescent protein-tagged Cx26 and Cx43 in these connexin-deficient melanomas, punctate gap junction-like plaques were evident at sites of cell-cell apposition, and the incidence of dye transfer was significantly increased similar to connexin-rich keratinocytes. We found that the expression of Cx43, but not Cx26, significantly reduced cellular proliferation and anchorage-independent growth from control melanomas, whereas migration was unaffected. Additionally, melanomas expressing Cx43 displayed significantly reduced growth within the in situ-like microenvironment of keratinocytes, despite a lack of heterocellular gap junctional intercellular communication between the two cell types. Furthermore, when grown in vivo in the chicken chorioallantoic membrane, primary tumors derived from Cx43-expressing melanomas were significantly smaller than controls, whereas Cx26-expressing melanomas produced tumors similar to controls. Collectively, these results suggest that Cx43, and not Cx26, can act as a tumor suppressor during melanoma tumorigenesis.     

间隙连接蛋白Cx43在人胚肺和肺癌细胞表达的研究

细胞与细胞之间通过细胞膜上的间隙连接通道交换小分子和离子进行细胞间通讯,对细胞增殖分化调控和机体内环境稳定有重要作用。用间隙连接蛋白Ｃｘ４３ｃＤＮＡ探针Ｎｏｒｔｈｅｒｎ印迹杂交,Ｃｘ４３抗体免疫荧光染色和罗氏黄荧光染料传输方法检查,正常人胚肺细胞的Ｃｘ４３在ｍＲＮＡ和蛋白水平有高表达,Ｃｘ４３蛋白免疫荧光分布在间隙连接的部位,细胞间隙连接通讯功能明显。与正常相反,人肺癌ＰＧ系细胞Ｃｋ４３无论在ｍＲＮＡ或蛋白质水平都无表达,细胞通讯功能缺陷。结果表明Ｃｘ４３在培养的人胚肺细胞有功能性表达。人肺癌ＰＧ细胞通讯功能缺陷与Ｃｘ４３基因转录抑制有关。对Ｃｘ基因的抑癌基因性质进行讨论。     

ATP and glutamate released via astroglial connexin 43 hemichannels mediate neuronal death through activation of pannexin 1 hemichannels

Inflammation contributes to neurodegeneration in post-ischemic brain, diabetes, and Alzheimer's disease. Participants in this inflammatory response include activation of microglia and astrocytes. We studied the role of microglia treated with amyloid-β peptide (Aβ) on hemichannel activity of astrocytes subjected to hypoxia in high glucose. Reoxygenation after 3?h hypoxia in high glucose induced transient astroglial permeabilization via Cx43 hemichannels and reduction in intercellular communication via Cx43 cell-cell channels. Both responses were greater and longer lasting in astrocytes previously exposed for 24 h to conditioned medium from Aβ-treated microglia (CM-Aβ). The effects of CM-Aβ were mimicked by TNF-α and IL-1β and were abrogated by neutralizing TNF-α with soluble receptor and IL-1β with a receptor antagonist. Astrocytes under basal conditions protected neurons against hypoxia, but exposure to CM-Aβ made them toxic to neurons subjected to a sub-lethal hypoxia/reoxygenation episode, revealing the additive nature of the insults. Astrocytes exposed to CM-Aβ induced permeabilization of cortical neurons through activation of neuronal pannexin 1 (Panx1) hemichannels by ATP and glutamate released through astroglial Cx43 hemichannels. In agreement, inhibition of NMDA or P2X receptors only partially reduced the activation of neuronal Panx1 hemichannels and neuronal mortality, but simultaneous inhibition of both receptors completely prevented the neurotoxic response. Therefore, we suggest that responses to ATP and glutamate converge in activation of neuronal Panx1 hemichannels. Thus, we propose that blocking hemichannels expressed by astrocytes and/or neurons in the inflamed nervous system could represent a novel and alternative strategy to reduce neuronal loss in various pathological states including Alzheimer's disease, diabetes and ischemia.     

Microinjected anti-actin antibodies decrease gap junctional intercellular commmunication in cultured astrocytes

The purpose of the present study was to investigate the influence of the actin cytoskeleton on gap junctional intercellular communication (GJIC) in cultured astrocytes. This report describes an decrease of GJIC following microinjection of anti-actin antibodies or cytochalasin D treatment. Dye transfer of microinjected neurobiotin was used to assay gap junctional permeability in cultured astrocytes. Besides this translocation of connexins to the plasma membrane we investigated subsequent anti-actin antibody injection. While control cultures exhibited intensive dye spreading of microinjected neurobiotin, GJIC was impaired by microinjection of anti-actin antibodies. Additionally, impaired GJIC was observed after cytochalasin D treatment for 15 min. After the drug had been washed out, a recovery of GJIC was achieved. Cultured astrocytes exhibited a prominent actin cytoskeleton, with strong staining of actin filaments at the plasma membrane. Confocal laser scanning microscopy revealed an impaired translocation of Cx 43 from the Golgi apparatus to the cell membrane of cell processes following anti-actin antibody injection. These results suggest that the morphological integrity of microfilaments seems to be fundamental for GJIC, probably by means of associations among actin filaments, actin binding proteins, and Cx 43 at the plasma membrane or indirectly through the transport of connexins from the cytoplasm to the cell membrane.     

A Neuroprotective Role for Gap Junctions

Glial-neuronal interactions have been implicated in both normal information processing and neuroprotection. One pathway of cellular interactions involves gap junctional intercellular communication (GJIC). In astrocytes, gap junctions are composed primarily of the channel protein, connexin43 (Cx43), and provide a substrate for formation of a functional syncytium implicated in the process of spatial buffering in the CNS. Thus gap junctional communication may be neuroprotective following a CNS insult that entails glutamate cytotoxicity (i.e. ischemia). We have shown that blocking gap junctions during a glutamate insult to co-cultures of astrocytes and neurons results in increased neuronal injury. To assess the effect of reduced Cx43 and GJIC on neuroprotection, we examined brain infarct volume in wild type and Cx43 heterozygote null mice following focal ischemia. Cx43 heterozygous null mice exhibited a significantly larger infarct volume compared to wild type. At the cellular level, a significant increase in TUNEL positive cells was observed in the penumbral region of the Cx43 heterozygote mice. These results suggest that augmentation of GJIC in astrocytes may contribute to neuroprotection following ischemic injury. These findings support the hypothesis that gap junctions play a neuroprotective role against glutamate cytotoxicity.     

A neuroprotective role for gap junctions

Glial-neuronal interactions have been implicated in both normal information processing and neuroprotection. One pathway of cellular interactions involves gap junctional intercellular communication (GJIC). In astrocytes, gap junctions are composed primarily of the channel protein, connexin43 (Cx43), and provide a substrate for formation of a functional syncytium implicated in the process of spatial buffering in the CNS. Thus gap junctional communication may be neuroprotective following a CNS insult that entails glutamate cytotoxicity (i.e. ischemia). We have shown that blocking gap junctions during a glutamate insult to co-cultures of astrocytes and neurons results in increased neuronal injury. To assess the effect of reduced Cx43 and GJIC on neuroprotection, we examined brain infarct volume in wild type and Cx43 heterozygote null mice following focal ischemia. Cx43 heterozygous null mice exhibited a significantly larger infarct volume compared to wild type. At the cellular level, a significant increase in TUNEL positive cells was observed in the penumbral region of the Cx43 heterozygote mice. These results suggest that augmentation of GJIC in astrocytes may contribute to neuroprotection following ischemic injury. These findings support the hypothesis that gap junctions play a neuroprotective role against glutamate cytotoxicity.     

Reversible Inhibition of Gap Junctional Intercellular Communication, Synchronous Contraction, and Synchronism of Intracellular Ca Fluctuation in Cultured Neonatal Rat Cardiac Myocytes by Heptanol

We analyzed by Fotonic Sensor, a fiber-optic displacement measurement instrument, the effects of heptanol on synchronized contraction of primary neonatal rat cardiac myocytes cultured at confluent density. We also examined the effect of heptanol on the changes in gap junctional intercellular communication by using the microinjection dye transfer method, and on intercellular Ca²⁺ fluctuation by confocal laser scanning microscopy of myocytes loaded with the fluorescent Ca²⁺ indicator fluo 3. In addition, we studied expression, phosphorylation, and localization of the major cardiac gap junction protein connexin 43 (Cx43) using immunofluorescence and Western blotting. At Day 6 of culture, numerous myocytes exhibited spontaneous, synchronous contractions, excellent dye coupling, and synchronized intracellular Ca²⁺ fluctuations. We treated the cells with 1.5, 2.0, 2.5, and 3.0 mmol/liter heptanol. With 1.5 mmol/liter heptanol, we could not observe significant effects on spontaneous contraction of myocytes. At 3.0 mmol/liter, the highest concentration used in the current experiment, heptanol inhibited synchronous contractions and even after washing out of heptanol, synchronous contraction was not rapidly recovered. On the other hand, at the intermediate concentrations of 2.0 and 2.5 mmol/liter, heptanol reversely inhibited synchronized contraction, gap junctional intercellular communication, and synchronization of intracellular Ca²⁺ fluctuations in the myocytes without preventing contraction and changes of intracellular Ca²⁺ in individual cells. Brief exposure (5-20 min) to heptanol (2.0 mmol/liter) did not cause detectable changes in the expression, phosphorylation, or localization of Cx43, despite strong inhibition of gap junctional intercellular communication. These results suggest that gap junctional intercellular communication plays an important role in synchronous intracellular Ca²⁺ fluctuations, which facilitate synchronized contraction of cardiac myocytes.     

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.     

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.     

Role of astroglial Connexin 43 in pneumolysin cytotoxicity and during pneumococcal meningitis

Streptococcus pneumoniae or pneumococcus (PN) is a major causative agent of bacterial meningitis with high mortality in young infants and elderly people worldwide. The mechanism underlying PN crossing of the blood brain barrier (BBB) and specifically, the role of non-endothelial cells of the neurovascular unit that control the BBB function, remains poorly understood. Here, we show that the astroglial connexin 43 (aCx43), a major gap junctional component expressed in astrocytes, plays a predominant role during PN meningitis. Following intravenous PN challenge, mice deficient for aCx43 developed milder symptoms and showed severely reduced bacterial counts in the brain. Immunofluorescence analysis of brain slices indicated that PN induces the aCx43–dependent destruction of the network of glial fibrillary acid protein (GFAP), an intermediate filament protein specifically expressed in astrocytes and up-regulated in response to brain injury. PN also induced nuclear shrinkage in astrocytes associated with the loss of BBB integrity, bacterial translocation across endothelial vessels and replication in the brain cortex. We found that aCx4-dependent astrocyte damages could be recapitulated using in vitro cultured cells upon challenge with wild-type PN but not with a ply mutant deficient for the pore-forming toxin pneumolysin (Ply). Consistently, we showed that purified Ply requires Cx43 to promote host cell plasma membrane permeabilization in a process involving the Cx43-dependent release of extracellular ATP and prolonged increase of cytosolic Ca²⁺ in host cells. These results point to a critical role for astrocytes during PN meningitis and suggest that the cytolytic activity of the major virulence factor Ply at concentrations relevant to bacterial infection requires co-opting of connexin plasma membrane channels.     

Flow regulates intercellular communication in HAEC by assembling functional Cx40 and Cx37 gap junctional channels

Direct cell-to-cell transfer of ions and small signaling molecules via gap junctions plays a key role in vessel wall homeostasis. Vascular endothelial gap junctional channels are formed by the connexin (Cx) proteins Cx37, Cx40, and Cx43. The mechanisms regulating connexin expression and assembly into functional channels have not been fully identified. We investigated the dynamic regulation of endothelial gap junctional intercellular communication (GJIC) by fluid flow and the participation of each vascular connexin in functional human endothelial gap junctions in vitro. Human aortic endothelial cells (HAEC) were exposed for 5, 16, and 24 h to physiological flows in a parallel-plate flow chamber. Connexin protein expression and localization were evaluated by immunocytochemistry, and functional GJIC was evaluated by dye injection. Connexin-mimetic peptide inhibitors were used to assess the specific connexin composition of functional channels. HAEC monolayers in culture exhibited baseline functional communication at a striking low level despite abundant expression of Cx43 and Cx40 localized at cell-to-cell appositions. Upon exposure to flow, GJIC by dye spread demonstrated a significant time-dependent increase from baseline levels, reaching 7.5-fold in 24 h. Inhibition studies revealed that this response was mediated primarily by Cx40, with lesser contributions of the other two vascular connexins assembled into functional homotypic and/or heterotypic channels. This is the first study to demonstrate that flow simultaneously and differentially regulates expression of the Cx37, Cx40, and Cx43 proteins and their involvement in the augmentation of intercellular communication by dye transfer in human endothelial cells in vitro.     

Effects of streptozotocin-induced diabetes on connexin43 mRNA and protein expression in ventricular muscle

Abnormal QT prolongation with the associated arrhythmias is a significant predictor of mortality in diabetic patients. Gap junctional intercellular communication allows electrical coupling between heart muscle cells. The effects of streptozotocin (STZ)-induced diabetes mellitus on the expression and distribution of connexin 43 (Cx43) in ventricular muscle have been investigated. Cx43 mRNA expression was measured in ventricular muscle by quantitative PCR. The distribution of total Cx43, phosphorylated Cx43 (at serine 368) and non-phosphorylated Cx43 was measured in ventricular myocytes and ventricular muscle by immunocytochemistry and confocal microscopy. There was no significant difference in Cx43 mRNA between diabetic rat ventricle and controls. Total and phosphorylated Cx43 were significantly increased in ventricular myocytes and ventricular muscle and dephosphorylated Cx43 was not significantly altered in ventricular muscle from diabetic rat hearts compared to controls. Disturbances in gap junctional intercellular communication, which in turn may be attributed to alterations in balance between total, phosphorylated and dephosporylated Cx43, might partly underlie prolongation of QRS and QT intervals in diabetic heart.     

ZO-1 is required for protein kinase C gamma-driven disassembly of connexin 43

We have previously reported that protein kinase C gamma (PKC-gamma) is activated by phorbol-12-myristate-13-acetate (TPA) and that this causes PKC-gamma translocation to membranes and phosphorylation of the gap junction protein, connexin 43 (Cx43). This phosphorylation, on S368 of Cx43, causes disassembly of Cx43 out of cell junctional plaques resulting in the inhibition of dye transfer. The purpose of this study is to identify the specific role of zonula occludens protein-1 (ZO-1), a tight junction protein with recently established effects on gap junctions, in this PKC-gamma-driven Cx43 disassembly. For this purpose, ZO-1 levels in lens epithelial cells in culture were decreased by up to 70% using specific siRNA. The down-regulation of ZO-1 caused a stable interaction of PKC-gamma with Cx43 even without normal enzyme activation by TPA. However, after TPA activation of the PKC-gamma, the Cx43 did not disassemble out of plaques even though the PKC-gamma enzyme was activated and the Cx43 was phosphorylated on S368. Confocal microscopy demonstrated that the siRNA treatment caused a loss of ZO-1 from borders of large junctional Cx43 cell-to-cell plaques and resulted in the accumulation of Cx43 aggregates inside of cells. Loss of the specific "plaquetosome" arrangement of large Cx43 plaques surrounded by ZO-1 was accompanied by a complete loss of functional dye transfer. These results suggest that ZO-1 is required for Cx43 control, both for dye transfer, and, for the PKC-gamma-driven disassembly response.     

Selective astrocytic gap junctional trafficking of molecules involved in the glycolytic pathway: impact on cellular brain imaging

To assess the specificity of metabolite trafficking among gap junction-coupled astrocytes, we developed novel, real-time, single-cell enzymatic fluorescence assays to assay cell-to-cell transfer of unlabeled glycolytic intermediates and report (i) highly restricted transfer of glucose-6-phosphate (P) and two analogs, deoxyglucose (DG)-6-P, and 2-[ N -(7-nitrobenz-2-oxa-1,3-diazol-4-yl)amino]-2-DG-6-P, compared with DG and 2- and 6-[ N -(7-nitrobenz-2-oxa-1,3-diazol-4-yl)amino]-2-DG, (ii) extensive junctional diffusion of glyceraldehyde-3-P, NADH, and NADPH plus three anionic fluorescent dyes used as internal standards for transfer assays, and (iii) stimulation of gap junctional communication by increased intracellular Na⁺ that also evokes metabolic responses in nearby coupled astrocytes. Thus, dye transfer does not predict gap junctional permeability of endogenous metabolites. Intracellular retention of flux-regulating compounds (e.g. glucose-6-P) may be necessary for local metabolic control, whereas 'syncytial sharing' may dissipate the work load on peri-synaptic astrocytes. Imaging of brain functional activity depends on local accumulation of exogenous or endogenous signals, and DG-6-P is trapped in the cell where it is phosphorylated, whereas rapid dispersal of cytoplasmic NAD(P)H and labeled glucose metabolites throughout the astrocytic syncytium can interfere with cellular assessment of neuron–astrocyte relationships in autoradiographic, fluorescence microscopic, and magnetic resonance spectroscopic studies.