神经组织缝隙连接

近年来神经组织中缝隙连接的分布和功能研究取得了一些显著进展。分子生物学方法的应用促进了ＧＪ结构,亚型及生物物理特性的揭示,染料偶联实验和Ｃａ＾２＋成像技术为ＧＪ的功能研究提供了直观有效的手段。ＧＪ的调控涉及ＧＪ的表达,导通性的改变等环节。ＧＪ胞间通讯的基本形式是交换第二信使和电偶联。     

Detecting and estimating rectification of gap junction conductance based on simulations of dual-cell recordings from a pair and a network of coupled cells

Gap junctions can exhibit rectification of conductance. Some reports use inequality of coupling coefficients as the first sign of the possible existence of rectification ( [Devor and Yarom, 2002], [Fan et al., 2005], [31], [Mann-Metzer and Yarom, 1999], [Nolan et al., 1999] and [Szabadics et al., 2001]). However, mathematical modeling and simulations of electrotonic coupling between an isolated pair of neurons showed conditions where the coupling coefficients were unreliable indicators of rectification. On the other hand, the transfer resistances were found to be reliable indicators of junctional rectification. The existing mathematical model of cell coupling ( [Bennett, 1966], [Devor and Yarom, 2002] and [Verselis and Veenstra, 2000]) was extended in order to measure rectification of the junctional conductances directly between dual-recorded neurons whether isolated or surrounded by a simulated 3-dimensional network of heterogeneous cells whose gap junctions offered parallel paths for current flow between the recorded neurons. The results showed that the transfer resistances could still detect rectification of the gap junction linking the dual-recorded neurons when embedded in a coupled cell network and that a mathematical model could estimate the conductances in both directions through this gap junction using only data that would be available from real dual-intracellular penetrations which allow electrophysiological recordings and intracellular staining. Rectification of gap junctions in unrecorded cells of a biologically realistic coupled cell network had negligible effects on the voltage responses of the dual-recorded neurons because of minimal current passing through these surrounding cells.     

Bistable dynamics of cardiac cell models coupled by dynamic gap junctions linked to Cardiac Memory

In an earlier study, we suggested that adaptive gap junctions (GJs) might be a basis of cardiac memory, a phenomenon which refers to persistent electrophysiological response of the heart to external pacing. Later, it was also shown that the proposed mechanism of adaptation of GJs is consistent with known electrophysiology of GJs. In the present article, we show that a pair of cardiac cell models coupled by dynamic, voltage-sensitive GJs exhibits bistable behavior under certain conditions. Three kinds of cell pairs are considered: (1) a Noble–Noble cell pair that represents adjacent cells in Purkinje network, (2) a pair of DiFranceso–Noble cells that represents adjacent SA nodal cells, and (3) a model of Noble cell coupled to Luo–Rudy cell model, which represents an interacting pair of a Purkinje fiber and a ventricular myocyte. Bistability is demonstrated in all the three cases. We suggest that this bistability might be an underlying factor behind cardiac memory. Focused analysis of a pair of Noble cell models showed that bistability is obtained only when the properties of GJs “match” with the properties of the pair of cells that is coupled by the GJs. This novel notion of match between GJs and cardiac cell types might give an insight into specialized distributions of various connexin proteins in cardiac tissue.     

Development changes in regulation of embryonic chick heart gap junctions

Summary Embryonic chick myocyte pairs were isolated from ventricular tissue of 4-day, 14-day, and 18-day heart for the purpose of examining the relationship between macroscopic junctional conductance and transjunctional voltage during cardiac development. The double whole-cell patch-clamp technique was employed to directly measure junctional conductance over a transjunctional voltage range of ±100 mV. At all ages, the instantaneous junctional current (or conductance=current/voltage) varied linearly with respect to transjunctional voltage. This initial response was followed by a time- and voltage-dependent decline in junctional current to new steady-state values. For every experiment, the steady-state junctional conductance was normalized to the instantaneous value obtained at each potential and the data was pooled according to developmental age. The mean steadystate junctional conductance-voltage relationship for each age group was fit using a two-state Boltzmann distribution described previously for other voltage-dependent gap junctions. From this model, it was revealed that half-inactivation voltage for the transjunctional voltage-sensitive conductance shifted towards larger potentials by 10 mV, the equivalent gating charge increased by approximately 1 electron, and the minimal voltage-insensitive conductance exactly doubled (increased from 18 to 36%) between 4 and 18 days of development. Decay time constants were similar at all ages examined as rate increased with increasing transjunctional potential. This data provides the first direct experimental evidence for developmental changes in the regulation of intercellular communication within a given tissue. This information is consistent with the hypothesis that developmental expression of multiple gap junction proteins (connexins) may confer different regulatory mechanisms on intercellular communication pathways within a given cell or tissue.     

Conditional Gene Targeting of Connexin43: Exploring the Consequences of Gap Junction Remodeling in the Heart

Abnormalities in cardiac gap junction expression have been postulated to contribute to arrhythmias and ventricular dysfunction. We investigated the role of cardiac gap junctions by generating a heart-specific conditional knock-out (CKO) of connexin43 (Cx43), the major cardiac gap junction protein. While the Cx43 CKO mice have normal heart structure and contractile function, they die suddenly from spontaneous ventricular arrhythmias. Because abnormalities in gap junction expression in the diseased heart can be focal, we also generated chimeric mice formed from Cx43-null embryonic stem (ES) cells and wildtype recipient blastocysts. Heterogeneous Cx43 expression in the chimeric mice resulted in conduction defects and depressed contractile function. These novel genetic murine models of Cx43 loss of function in the adult mouse heart define gap junctional abnormalities as a key molecular feature of the arrhythmogenic substrate and an important factor in heart dysfunction.     

Implication of connexins 40 and 43 in functional coupling between mouse cardiac fibroblasts in primary culture

Cardiac fibroblasts contribute to the structure and function of the myocardium. However their involvement in electrophysiological processes remains unclear; particularly in pathological situations when they proliferate and develop fibrosis. We have identified the connexins involved in gap junction channels between fibroblasts from adult mouse heart and characterized their functional coupling. RT-PCR and Western blotting results show that mRNA and proteins of connexin40 and connexin43 are expressed in cultured cardiac fibroblasts, while Cx45 is not detected. Analysis of gap junctional communications established by these connexins with the gap-FRAP technique demonstrates that fibroblasts are functionally coupled. The time constant of permeability, k, calculated from the fluorescence recovery curves between cell pairs is 0.066 ± 0.005 min^− 1 (n = 65). Diffusion analysis of Lucifer Yellow through gap junction channels with the scrape-loading method demonstrates that when they are completely confluent, a majority of fibroblasts are coupled forming an interconnecting network over a distance of several hundred micrometers. These data show that cardiac fibroblasts express connexin40 and connexin43 which are able to establish functional communications through homo and/or heterotypic junctions to form an extensive coupled cell network. It should then be interesting to study the conditions to improve efficiency of this coupling in pathological conditions.     

Gap junctions modulate apoptosis and colony growth of human embryonic stem cells maintained in a serum-free system

We investigated the gap junctional properties of human embryonic stem cells (hESC) cultivated in a serum-free system using sphingosine-1-phosphate and platelet-derived growth factor (S1P/PDGF). We compared this condition to hESC grown on Matrigel in mouse embryonic fibroblast conditioned medium (MEF-CM) or unconditioned medium (UM). We show that in all culture systems, hESC express connexins 43 and 45. hESC maintained in S1P/PDGF conditions and hESC grown in presence of MEF-CM are coupled through gap junctions while hESC maintained on Matrigel in UM do not exhibit gap junctional intercellular communication. In this latter condition, coupling was retrieved by addition of noggin, suggesting that BMP-like activity in UM inhibits gap junctional communication. Last, our data indicate that the closure of gap junctions by the decoupling agent alpha-glycyrrhetinic acid increases cell apoptosis and inhibits hESC colony growth. Altogether, these results suggest that gap junctions play an important role in hESC maintenance.     

Light-dependent dynamics of gap junctions between horizontal cells in the retina of the crucian carp

Summary The dynamics of gap junctions between outer horizontal cells or their axon terminals in the retina of the crucian carp were investigated during light and dark adaptation by use of ultrathin-section and freeze-fracture electron microscopy. Light adaptation was induced by red light, while dark adaptation took place under ambient dark conditions. The two principal findings were: (1) The density of connexons within an observed gap junction is high in dark-adapted retina, and low in light-adapted retina. This, respectively, may reflect the coupled and uncoupled state of the gap junction. (2) The size of individual gap junctions is larger in light-than in dark-adapted retinae. Whereas the overall area occupied by gap junctions is reduced with dark adaptation, the percentage of small and very small gap junctions increases dramatically. A lateral shift of connexons in the gap junctional membrane is strongly suggested by these reversible processes of densification and dispersion. Two additional possibilities of gap junction modulation are discussed: (1) the de novo formation of very small gap junctions outside the large ones in the first few minutes of dark adaptation, and (2) the rearrangement of a portion of the very large gap junctions. The idea that the cytoskeleton is involved in such modulatory processes is corroborated by thin-section observations.Dedicated to Professor J. Peiffer on the occasion of his 65th birthday     

Learning Theories Reveal Loss of Pancreatic Electrical Connectivity in Diabetes as an Adaptive Response

Cells of almost all solid tissues are connected with gap junctions which permit the direct transfer of ions and small molecules, integral to regulating coordinated function in the tissue. The pancreatic islets of Langerhans are responsible for secreting the hormone insulin in response to glucose stimulation. Gap junctions are the only electrical contacts between the beta-cells in the tissue of these excitable islets. It is generally believed that they are responsible for synchrony of the membrane voltage oscillations among beta-cells, and thereby pulsatility of insulin secretion. Most attempts to understand connectivity in islets are often interpreted, bottom-up, in terms of measurements of gap junctional conductance. This does not, however, explain systematic changes, such as a diminished junctional conductance in type 2 diabetes. We attempt to address this deficit via the model presented here, which is a learning theory of gap junctional adaptation derived with analogy to neural systems. Here, gap junctions are modelled as bonds in a beta-cell network, that are altered according to homeostatic rules of plasticity. Our analysis reveals that it is nearly impossible to view gap junctions as homogeneous across a tissue. A modified view that accommodates heterogeneity of junction strengths in the islet can explain why, for example, a loss of gap junction conductance in diabetes is necessary for an increase in plasma insulin levels following hyperglycemia.     

The fine structure of identified electrotonic synapses following increased coupling resistance

Summary Gap junctions exist in the septa between the segments of the lateral giant axons in the ventral nerve cord of the crayfish Procambarus. A large increase in the resistance (uncoupling) of these gap junctions was brought about by mechanical injury to the axonal segments. Both thin sections and freeze-fracture preparations were used to monitor the morphological changes which occurred up to 45 min after injury.There was no apparent change in the organization (a loose polygonal array) of the intramembrane particles which make up the junctional complex up to 45 min after injury. In some instances, however, the intramembrane particles appeared to have moved away from the junctional area. Other junctional regions were internalized and appeared similar to what have been called annular gap junctions. Also at this time (20–25 min after injury), a dense cytoplasmic plug formed in uninjured axon near the junctional region. It is concluded that the gap junctions that exhibit a loose polygonal organization of the intramembrane particles may be either in a state of low resistance (coupled) or a state of high resistance (uncoupled).     

Remodeling of Gap Junctions in Ischemic and Nonischemic Forms of Heart Disease

Electrical activation of the myocardium to produce effective pumping of blood depends on the orderly coordinated spatial and temporal transfer of current from one cell to another via gap junctions. Normal ventricular myocytes are extensively coupled by gap junctions and have the capacity to rapidly increase the amount of connexin within gap junction plaques to meet physiological demands for enhanced cell-cell communication. However, myocytes can also rapidly uncouple in response to injury or disease. In general, both acute and chronic forms of heart disease caused by diverse etiologies are associated with changes in the expression of connexins and remodeling of gap junctions. Such remodeling may have both adaptive and maladaptive consequences and contribute to major clinical processes such as heart failure and sudden cardiac death. Our laboratory has investigated mechanisms regulating cell-cell electrical coupling in the heart under physiological and pathophysiological conditions. This review is focused on selected aspects of this work pertaining to changes in coupling in response to acute and chronic ischemic heart disease and in familial cardiomyopathies caused by mutations in genes encoding desmosomal proteins.     

Estimating conductances of dual-recorded neurons within a network of coupled cells

Simultaneous pre- and postsynaptic cell recordings are used to calculate gap junction conductance based on an equivalent electrical circuit of an electrically coupled pair of cells. This calculation is imprecise when recording from a cell pair that is coupled to neighboring cells providing indirect conductance paths between the recorded cells. Despite this imprecision, junctional conductance has been calculated for coupled cell networks during the past 40 years since a more accurate method was lacking. The present study simulated a three-dimensional network of electrically coupled heterogeneous neurons and used mathematical modeling to reduce the complexity to the simplest equations that could more accurately estimate the electrical properties of dual-recorded cells in the network. Analyses of the simulations showed that knowledge of the number of unrecorded cells directly linked to the recorded cells and of the voltage responses of these recorded cells were largely sufficient to accurately predict the direct junctional resistance linking the recorded cells as well as the input resistance of the recorded cells that would exist in the absence of junctional coupling. All model parameters could be obtained from real dual-intracellular penetrations which allow electrophysiological recordings and intracellular staining.     

Molecular characterization and embryonic expression of innexins in the leech Hirudo medicinalis

Gap junctions are direct intercellular channels that permit the passage of ions and small signaling molecules. The temporal and spatial regulation of gap junctional communication is, thus, one mechanism by which cell interactions, and hence cell properties and cell fate, may be regulated during development. The nervous system of the leech, Hirudo medicinalis, is a particularly advantageous system in which to study developmental mechanisms involving gap junctions because interactions between identified cells may be studied in vivo in both the embryo and the adult. As in most invertebrates, gap junctions in the leech are composed of innexin proteins, which are distantly related to the vertebrate pannexins and are encoded by a multi-gene family. We have cloned ten novel leech innexins and describe the expression of these, plus two other previously reported members of this gene family, in the leech embryo between embryonic days 6 and 12, a period during which the main features of the central nervous system are established. Four innexins are expressed in neurons and two in glia, while several innexins are expressed in the excretory, circulatory, and reproductive organs. Of particular interest is Hm-inx6, whose expression appears to be restricted to the characterized S cell and two other neurons putatively identified as presynaptic to this cell. Two other innexins also show highly restricted expressions in neurons and may be developmentally regulated. Electronic Supplementary Material Supplementary material is available for this article at .     

心肌细胞缝隙连接重塑与心律失常

缝隙连接是相邻心肌细胞间电、化学偶联的通道,亦是心室肌成为功能性合胞体的重要结构.心肌有缝隙连接蛋白(connexin,CX) 40、43与45的表达,心室肌主要表达CX43.CX43形成的缝隙连接大部分呈点状分布于闰盘部位,心肌细胞膜侧面分布极少.心肌缺血-再灌注、肥厚、衰竭、高胆同醇与糖尿病条件下,心肌细胞缝隙连接...     

Hyperglycaemia and diabetes impair gap junctional communication among astrocytes

Sensory and cognitive impairments have been documented in diabetic humans and animals, but the pathophysiology of diabetes in the central nervous system is poorly understood. Because a high glucose level disrupts gap junctional communication in various cell types and astrocytes are extensively coupled by gap junctions to form large syncytia, the influence of experimental diabetes on gap junction channel-mediated dye transfer was assessed in astrocytes in tissue culture and in brain slices from diabetic rats. Astrocytes grown in 15–25 mmol/l glucose had a slow-onset, poorly reversible decrement in gap junctional communication compared with those grown in 5.5 mmol/l glucose. Astrocytes in brain slices from adult STZ (streptozotocin)-treated rats at 20–24 weeks after the onset of diabetes also exhibited reduced dye transfer. In cultured astrocytes grown in high glucose, increased oxidative stress preceded the decrement in dye transfer by several days, and gap junctional impairment was prevented, but not rescued, after its manifestation by compounds that can block or reduce oxidative stress. In sharp contrast with these findings, chaperone molecules known to facilitate protein folding could prevent and rescue gap junctional impairment, even in the presence of elevated glucose level and oxidative stress. Immunostaining of Cx (connexin) 43 and 30, but not Cx26, was altered by growth in high glucose. Disruption of astrocytic trafficking of metabolites and signalling molecules may alter interactions among astrocytes, neurons and endothelial cells and contribute to changes in brain function in diabetes. Involvement of the microvasculature may contribute to diabetic complications in the brain, the cardiovascular system and other organs.     

Prevention of organochlorine-induced inhibition of gap junctional communication by chaetoglobosin K in astrocytes

Innumerable toxic substances present in the environment inhibit gap junctions, intercellular membrane channels that play fundamental roles in coordinated function of cells and tissues. Included are persistent organochlorine compounds, which pose health risks to humans and animals owing to their widespread use, bioaccumulation, and ability to inhibit gap junction channel-mediated intercellular communication in liver, lung, skin, heart, and brain cells. In this study, the organochlorine xenobiotics dieldrin and endosulfan, at micromolar concentrations, were found to inhibit gap junction-mediated intercellular communication and induce hypophosphorylation of connexin 43 in cultured rat astrocytes, the predominant cell type in the brain coupled through gap junctions. This inhibition of gap junctional communication was substantially reduced by preincubation with chaetoglobosin K (ChK), a bioactive natural produce previously shown to have ras tumor suppressor activity. Chaetoglobosin K also prevented dieldrin and endosulfan-induced hypophosphorylation of connexin 43 and prevented dieldrin-induced connexin 43 plaque dissolution in both astrocytes and cultured liver epithelial cells. The results suggest that stabilization of the native, phosphorylated form of connexin 43 by ChK may contribute to its ability to prevent organochlorine-induced inhibition of gap junction-mediated communication and dissolution of gap junction plaques within the plasma membrane. This revised version was published online in July 2006 with corrections to the Cover Date.     

Biochemical and ultrastructural evidence for the association of basic fibroblast growth factor with cardiac gap junctions

Basic fibroblast growth factor (bFGF) is a ubiquitous and multifunctional polypeptide that is believed to have a role in tissue repair and to act as a morphogen in embryonic development. Here, we have used immunohistochemical and biochemical methods with antibodies directed against the amino-terminal domain of bFGF, designated IS2, which recognize native and denatured bFGF, to demonstrate that in addition to its known intracellular and extracellular localization in heart, bFGF is also associated with cardiomyocyte gap junctions. In tissue sections, IS2 labeled regions of intercalated discs, producing an immunofluorescence pattern virtually indistinguishable from that obtained with antibodies against the heart gap junction protein connexin-43. By electron microscopy, gap junctions but not other regions of plasma membrane were heavily immunolabeled with this antibody. By solid phase immunoassay, bFGF was found to be more concentrated in a fraction enriched in cardiac gap junctions than in whole sarcolemmal preparations. Finally, an 18-kDa protein was recognized by several different antibodies specific for bFGF on Western blots of heart subcellular fractions enriched in gap junctions. We suggest that bFGF-like peptides are either an integral part of, or exist in close association with, cardiac gap junctions and thus may play a role in modulating gap junctional intercellular communication.     

Functional analysis of connexin-26 mutants associated with hereditary recessive deafness

The physiological importance of connexin-26 (Cx26) gap junctions in regulating auditory function is indicated by the finding that autosomal recessive DFNB1 deafness is associated with mutations of the Cx26 gene. To investigate the pathogenic role of Cx26 mutation in recessive hearing loss, four putative DFNB1 Cx26 mutants (V84L, V95M, R127H, and R143W) were stably expressed in N2A cells, a communication-deficient cell line. In N2A cells expressing (R127H) Cx26 gap junctions, macroscopic junctional conductance and ability of transferring neurobiotin between transfected cells were greatly reduced. Despite the formation of defective junctional channels, immunoreactivity of (R127H) Cx26 was mainly localized in the cell membrane and prominent in the region of cell-cell contact. Mutant (V84L), (V95M), or (R143W) Cx26 protein formed gap junctions with a junctional conductance similar to that of wild-type Cx26 junctional channels. (V84L), (V95M), or (R143W) Cx26 gap junctions also permitted neurobiotin transfer between pairs of transfected N2A cells. The present study suggests that (R127H) mutation associated with hereditary sensorineural deafness results in the formation of defective Cx26 gap junctions, which may lead to the malfunction of cochlear gap junctions and hearing loss. Further studies are required to determine the exact mechanism by which mutant (V84L), (V95M), and (R143W) Cx26 proteins, which are capable of forming functional homotypic junctional channels in N2A cells, cause the cochlear dysfunction and sensorineural deafness.     

Endothelial adherens and tight junctions in vascular homeostasis, inflammation and angiogenesis

Endothelial cells lining the vessel wall are connected by adherens, tight and gap junctions. These junctional complexes are related to those found at epithelial junctions but with notable changes in terms of specific molecules and organization. Endothelial junctional proteins play important roles in tissue integrity but also in vascular permeability, leukocyte extravasation and angiogenesis. In this review, we will focus on specific mechanisms of endothelial tight and adherens junctions.     

Gap junctions in the cardiac muscle cells of the lamprey

Summary Electron microscopy of both thin sections and freeze-fracture replicas has demonstrated the occurrence of gap junctions (nexuses) in the cardiac muscle cells of the lamprey. These gap junctions are identical in basic structure with those found in the mammalian heart. However, they are much smaller (less than 0.5 in diameter), and more irregularly distributed than the typical gap junction in the mammalian heart. These small gap junctions seem to provide a structural basis for the electrical coupling between cardiac muscle cells in the lower vertebrates.In addition, the well developed sarcoplasmic reticulum and subsurface cisternae, which contain an electron dense spheroidal cast, are frequently observed in the cardiac muscles of the lamprey.This work is supported by a research grant from the Ministry of Education, Japan