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PubMed1995

Functional analysis of amino acid sequences in connexin43 involved in intercellular communication through gap junctions.

Gap junctions allow direct communication between cells without recourse to the extracellular space and have been widely implicated as important mediators of cell-cell signalling. They are constructed from the connexin proteins, which form a large family, and individual connexins show complex spatial and temporal variations in their expression patterns. Understanding how this variation contributes to the control of intercellular signalling, both in the adult and during embryonic development, is an important problem that would be aided by reagents that interfere with gap junctional communication through specific connexins. We have begun to address this issue by raising antibodies to peptides derived from connexin43 and connexin32. Connexin43 peptides were located in the amino terminus, cytoplasmic loop and carboxytail. Connexin32 peptides came from the cytoplasmic loop and the first extracellular loop. Immunoblotting and immunostaining properties of purified IgGs were characterized on mouse heart, liver and the 8- to 16-cell mouse embryo. Effects on transfer through gap junctions were assessed in the fully compacted 8-cell mouse embryo by co-injection with Lucifer Yellow or Cascade Blue. Embryos were maintained in culture to assess the developmental consequences of injection. Peptide competition was used to confirm the specificity of immunostaining and inhibition of dye transfer. All connexin specific antibodies recognized their parent connexin on immunoblots and showed no 43/32 cross-reactivity. The connexin32 extracellular loop antibody recognized both connexin 32 and 43 on immunoblots, as predicted by the amino acid sequence homology in this region, but did not immunostain intact gap junctions. Connexin specific antibodies that immuno-stained showed the predicted connexin specificity. Antibodies to either connexin43 amino acids (AA) 1-16 (amino terminus) or AA 101-112 (cytoplasmic loop) neither immunostained nor prevented functional communication through 8-cell embryo gap junctions. Antibodies to AA 123-136 and AA 131-142 in the cytoplasmic loop immunostained heart and 8-cell embryo gap junctions and blocked transfer through them with high efficiency. Fab' fragments were equally effective. Peptide competition showed that both antibodies contained epitopes within AA 131-136 of connexin43. Antibodies against AA 313-324 in the carboxytail immunostained heart and the 8-cell embryo and, as IgGs, prevented dye transfer. Fab' fragments were ineffective. All connexin43 antibodies that blocked gap junctional communication between cells of the 8-cell mouse embryo induced non-communicating cells subsequently to withdraw from compaction.(ABSTRACT TRUNCATED AT 400 WORDS)

Amino Acid Sequence↗
PubMed1996

Theoretical analysis of intercellular communication between the vestibular type I hair cell and its calyx ending.

1. The vestibular type I hair cell and its calyx ending can communicate in three ways. 1) In conventional synaptic transmission an excitatory neurotransmitter is released in multimolecular packets from the hair cell and depolarizes the ending. 2) Ephaptic transmission occurs because currents originating in one structure change the membrane potential of the other structure. 3) Potassium is released from the hair cell during transduction, accumulates in the intercellular space, and can depolarize both the hair cell and the ending. 2. A system of steady-state cable equations was used to analyze conventional and ephaptic transmission. The equations describe the membrane potentials of the hair cell's basolateral surface and of the ending's inner and outer faces. A terminal resistor represents the apical pole of the hair cell and a second terminal resistor represents the parent fiber and other processes connected to the ending. Transducer currents are delivered to the hair cell at its apex and synaptic currents are delivered to the inner or outer faces of the ending at its base. 3. The presence of a calyx ending can reduce conventional synaptic transmission by lowering the postsynaptic input impedance and by introducing an intercellular component into the postsynaptic depolarization of the inner face. Transmission is expressed as a percentage of the synaptic depolarization that would occur in the absence of the ending. Increasing the specific resistance of the inner and outer faces from 150 to 15,000 omega.cm2 improves transmission almost tenfold, from 10% to > 90%. Selectively increasing the impedance of one or the other face results in only a twofold improvement. Under all conditions, transmission is similar for inner- and outer-face synaptic inputs. 4. Hair cell transducer currents cause an ephaptic hyperpolarization of the calyx inner face and a depolarization of the outer face. Excitatory postsynaptic currents originating in the calyx inner face hyperpolarize the apical part of the hair cell and depolarize its base. Outer-face excitatory currents depolarize the hair cell apex and hyperpolarize the base. On the basis of plausible assumptions about the magnitudes of input currents and about the electrical properties of the elements, it was estimated that ephaptic transmission to or from the inner face results in voltages of 0.5-2 mV in the target structure. Transmission to or from the outer face is 10-25 times less effective. 5. During transduction, K+ ions leave the hair cell's basolateral surface and accumulate in the intercellular space separating it from the calyx inner face. The accumulation was analyzed by combining cable and electrodiffusion theories. To make the theories consistent, the resistance of the intercellular space was derived from the Nernst-Planck equation. It was found that the steady-state intercellular current consisted of a diffusional and an electrical component. The diffusional component was 50-100 times larger than the electrical component. 6. Delta x[K+], the steady-state increase in intercellular K+ concentration from a baseline concentration of 4 mM, is proportional to IA, the magnitude of the transducer current, and is only slightly affected by the basolateral impedance. For a uniform distribution of basolateral currents, the proportionality constant, delta x[K+]/IA, is 0.07 mM/pA at the base and declines to 0 at the apex. A transducer current of 100 pA can result in a delta x[K+] of 7 mM at the base and a possible 25-mV depolarization of the hair cell and the ending. 7. Intercellular K+ accumulation has kinetics with a dominant rate constant of 12 s-1, corresponding to a first-order low-pass filter with a corner frequency of 2 Hz. Kinetics is sufficiently fast for accumulation to participate in the transduction of normally occurring head movements. (ABSTRACT TRUNCATED)

Animals↗
PubMed1997

Intercellular communication in rapidly proliferating and differentiated C6 glioma cells in culture.

Glial cells in the brain are known to provide structural and functional supports to neurons. To sustain such a supportive role, they have developed cell-to-cell communicating gap junctional channels. The authors studied the effect of dbcAMP on gap junctional channels mediated communication in C6 cells, a rat glioma cell line. Quantitative assessment of coupled cells under microscope after microinjection of a fluorescent dye was taken as a measure of junctional permeability. An enhanced coupling between cells was observed following dbcAMP treatment and this elevated coupling was found to be dependent on the duration of exposure of cells to dbcAMP. The studies have focused on a subtle shift in the spatial organization of the functional channels to the processes of dbcAMP induced differentiated cells from the cell cytoplasms and membranes of dbcAMP untreated cells. Immunofluorescence study with affinity purified antibody against gap junction further confirmed the spatial distribution of gap junctional protein(s) in the processes and also showed an increase in the density of the protein at the intercellular spaces in dbcAMP induced differentiated C6 glioma cells.

Animals↗
PubMed1992

The crystalline lens. A system networked by gap junctional intercellular communication.

The vertebrate eye lens is a solid cyst of cells which grows throughout life by addition of new cells at the surface. The older cells, buried by the newer generations, differentiate into long, prismatic fibers, losing their cellular organelles and filling their cytoplasms with high concentrations of soluble proteins, the crystallins. The long-lived lens fibers are interconnected by gap junctions, both with themselves and with an anterior layer of simple cuboidal epithelial cells at the lens surface. This network of gap junctions joins the lens cells into a syncytium with respect to small molecules, permitting metabolic co-operation: intercellular diffusion of ions, metabolites, and water. In contact with nutrients at the lens surface, the epithelial cells retain their cellular organelles, and are able to provide the metabolic energy to maintain correct ion and metabolite concentrations within the lens fiber cytoplasms, such that the crystallins remain in solution and do not aggregate (cataract). Gap junctions are formed by a family of integral membrane channel-forming proteins called connexins. Gap junctions between lens epithelial cells are composed of a connexin which is common between many different cell types, notably myocardial cells and connective tissue fibroblasts. The gap junctions between epithelial cells and lens fibers have not yet been biochemically characterized. The gap junctions formed between lens fibers are composed of at least two different connexins, one of which has not been detected between other cell types. The unusual physiology and longevity of the lens fibers may require the special set of connexins which are found joining these cells.

Animals↗
PubMed1997

Intercellular communication between dorsal root ganglion cells and colonic smooth muscle cells in vitro.

The mechanism(s) by which intestinal smooth muscle tension is signaled to extrinsic primary afferent neurons is poorly understood. In order to characterize myocyte-neuron communication, we developed a coculture system using rat dorsal root ganglion (DRG) neurons and myocytes obtained from the circular muscle layer of the rat distal colon. Both cell types maintained their phenotype in culture, as demonstrated by positive immunocytochemical staining for neuron-specific enolase and smooth muscle actin. Myocytes showed mechanosensitivity in the form of increases in [Ca2+]i in response to light mechanical touch of the plasma membrane. This increase in [Ca2+]i was independent of extracellular Ca2+ and passed as a propagated wave from muscle cells into adjacent DRG neurites. The inhibitory effect of octanol on this intercellular propagation suggests propagation of [Ca2+]i gradients via heterologous gap junctions. This preparation may serve a useful model system for the study of the interaction of visceral afferents and their target cells.

1-Octanol↗
PubMed1999

Morphology and intercellular communication in glial cells of intramural ganglia from the guinea-pig urinary bladder.

Neurons in most peripheral ganglia are surrounded by satellite glial cells (SCs), but these cells have so far received little attention. We used immunohistochemistry and intracellular injections of tracers to characterize SCs in the intramural ganglia of the guinea-pig urinary bladder, which are part of the parasympathetic system. Intracellular injections of horseradish peroxidase (HRP) revealed two morphological types: cells that surrounded neurons and are SCs proper, and bipolar cells with processes that projected into the nerve fiber bundles connecting the ganglia. SCs were immunopositive for glutamine synthetase (GS) and S100beta and immunonegative for glial fibrillary acidic protein (GFAP). Injections of Lucifer yellow (LY) or biocytin (molecules known to cross gap junctions) into single SCs showed that these cells have a very low degree of intercellular coupling. A mean of 0.31 and 0.71 cells were coupled to the injected cells, using LY and biocytin, respectively. It appears that SCs in the bladder ganglia are distinct from central and enteric glial cells in the small degree of their coupling and in the absence of GFAP immunostaining.

Animals↗
PubMed2003

Application of two-photon flash photolysis to reveal intercellular communication and intracellular Ca2+ movements.

Two-photon excitation makes it possible to excite molecules in volumes of much less than 1 fl. In two-photon flash photolysis (TPFP) this property is used to release effector molecules from caged precursors with high three-dimensional resolution. We describe and examine the benefits of using TPFP in model solutions and in a number of cell systems to study their spatial and temporal properties. Using TPFP of caged fluorescein, we determined the free diffusion coefficient of fluorescein (D=4 x 0(-6) cm(2)/s at 20 degrees C, which is in close agreement with published values). TPFP of caged fluorescein in lens tissue in situ revealed spatial nonuniformities in intercellular fiber cell coupling by gap junctions. At the lens periphery, intercellular transport was predominantly directed along rows of cells, but was nearly isotropic further from the periphery. To test an algorithm aiming to reconstruct the Ca(2+) release flux underlying physiological Ca(2+) signals in heart muscle cells, TPFP of DM-Nitrophen was utilized to generate artificial microscopic Ca(2+) signals with known underlying Ca(2+) release flux. In an experiment with mouse oocytes, the recently developed Ca(2+) cage dimethoxynitrophenyl-ethyleneglycol-bis-(beta-aminoethylether)-N,N,N('),N(') tetraacetic acid-4 (DMNPE-4) was released in the oocyte cytosol and inside a nucleolus. Analysis of the resulting fluorescence changes suggested that the effective diffusion coefficient within the nucleolus was half of that in the cytosol. These experiments demonstrate the utility of TPFP as a novel tool for the optical study of biomedical systems.

Action Potentials↗
PubMed1979

Pancreatic acinar cells: the effect of carbon dioxide, ammonium chloride and acetylcholine on intercellular communication.

1. Segments of mouse pancreatic or exorbital lacrimal gland were superfused with saline solutions. Under visual control two micro-electrodes were inserted into neighbouring cells within the same acinus or into neighbouring acini. Cell to cell electrical coupling was assessed by injecting rectangular current pulses through one electrode and measuring the electrotonic potential change in the same cell (V(1)) and in the neighbouring cell (V(2)). Acetylcholine (ACh) was added locally to impaled acini by micro-ionophoresis from an extracellular micropipette.2. Exposure of the tissues to a Krebs solution equilibrated with 100% CO(2) caused a rapid increase in the size of electrotonic potential changes in the current injection cell and disappearance of the electrotonic potential changes in a neighbouring acinus or cell. This electrical uncoupling of previously coupled cells was rapidly reversible upon return to a solution equilibrated with 95% O(2) and 5% CO(2).3. Reduction of electrical intercellular coupling was also obtained using smaller CO(2) concentrations (50, 20 or 10%). In these cases the effects developed more slowly and were less dramatic. Reducing the extracellular HCO(3) concentration enhanced the uncoupling effect of 10 or 20% CO(2). However, weak uncoupling effects were still observed using 10 or 20% CO(2) in combination with a high bicarbonate concentration maintaining a constant extracellular pH (7.4).4. Reductions in extracellular pH (down to 5.5) achieved by varying combinations of Tris base and Tris HCl had no effect on electrical coupling. Brief periods of anoxia (100% N(2)) also had no effect.5. Exposure to 20% CO(2) markedly enhanced the uncoupling effect of a brief ionophoretic pulse of ACh.6. Exposure of the tissue to 10 mM-NH(4)Cl, a procedure expected to increase the intracellular pH, counteracted the uncoupling effect of ACh. During sustained uncoupling caused by a sustained ACh stimulation a brief period of exposure to NH(4)Cl caused an immediate and fully reversible recoupling.7. It is concluded that variations in intracellular pH have marked effects on the electrical coupling between neighbouring cells in the pancreatic and lacrimal acinar tissue.

Acetylcholine↗
PubMed1996

[Role of the Notch receptors in intercellular communication].

The Notch gene was discovered in Drosophila at the beginning of the century and is currently the subject of intensive investigation, not only in invertebrates but also in vertebrates where remarkably well conserved homologues have been recently found. Notch encodes a new kind of cellular receptor whose functioning is still unclear and plays a role in a large number of cell interactions throughout development and in tissue renewal in the adult. Detailed study in invertebrates of some of these interactions has led to the identification of other genes required for transduction of the signal initiated by the receptor. Notch is always involved in processes where cells have the potential to choose between several different programmes of differentiation. Cells adopt a specific developmental pathway as a result of the inhibition of some programmes through Notch signalling. In this review we discuss the contribution of different experimental models to an understanding of the role of Notch in intercellular signalling.

Animals↗
PubMed1975

Intercellular communication in a positional field. Ultrastructural correlates and tracer analysis of communication between insect epidermal cells.

The junctional membrane in the epidermal cells of the larval beetle (Tenebrio molitor L.) is comprised of macular gap junctions embedded in septate junctions. Ultrastructural and morphometric analysis of the distribution of gap junctions within the segmental epidermis suggests that this junction alone could account for the high electrotonic coupling recorded for the epidermal sheet. Analysis of the lanthanum-impregnated septate junction makes it doubtful that this junction serves as a communicating channel between beetle cells. A new model for the septate junction is presented in which pleated septa, less than 30 A thick, connect adjacent plasma membranes; the septa themselves are interconnected by two interseptal platforms that are coplanar with the plasma membranes. Iontophoretic injection of organic tracers into single epidermal cells suggests that only molecules of less than MW 1000 can transfer between cells through low-resistance junctions.

Animals↗
PubMed2001

Intercellular communication via connexin43 gap junctions is required for ovarian folliculogenesis in the mouse.

The ovarian follicle in mammals is a functional syncytium, with the oocyte being coupled with the surrounding cumulus granulosa cells, and the cumulus cells being coupled with each other and with the mural granulosa cells, via gap junctions. The gap junctions coupling granulosa cells in mature follicles contain several different connexins (gap junction channel proteins), including connexins 32, 43, and 45. Connexin43 immunoreactivity can be detected from the onset of folliculogenesis just after birth and persists through ovulation. In order to assess the importance of connexin43 gap junctions for postnatal folliculogenesis, we grafted ovaries from late gestation mouse fetuses or newborn pups lacking connexin43 (Gja1(-)/Gja1(-)) into the kidney capsules of adult females and allowed them to develop for up to 3 weeks (this was necessitated by the neonatal lethality caused by the mutation). By the end of the graft period, tertiary (antral) follicles had developed in grafted normal (wild-type or heterozygote) ovaries. Most follicles in Gja1(-)/Gja1(-) ovaries, however, failed to become multilaminar, with the severity of the effect depending on strain background. Dye transfer experiments indicated that intercellular coupling between granulosa cells is reduced, but not abolished, in the absence of connexin43, consistent with the presence of additional connexins. These results suggest that coupling between granulosa cells mediated specifically by connexin43 channels is required for continued follicular growth. Measurements of oocyte diameters revealed that oocyte growth in mutant follicles is retarded, but not arrested, despite the arrest of folliculogenesis. The mutant follicles are morphologically abnormal: the zona pellucida is poorly developed, the cytoplasm of both granulosa cells and oocytes is vacuolated, and cortical granules are absent from the oocytes. Correspondingly, the mutant oocytes obtained from 3-week grafts failed to undergo meiotic maturation and could not be fertilized, although half of the wild-type oocytes from 3-week grafted ovaries could be fertilized. We conclude that connexin43-containing gap junction channels are required for expansion of the granulosa cell population during the early stages of follicular development and that failure of the granulosa cell layers to develop properly has severe consequences for the oocyte.

Animals↗
PubMed1975

A morphological basis for intercellular communication between alpha- and beta-cells in the endocrine pancreas.

By degranulating beta-cells in the islets of Langerhans of the rat with sulfonylurea, it has been possible to distinguish unambiguously alpha-cells from beta-cells in freeze-fracture replicas. In such preparations, we found morphologically typical tight and gap junctions occurring between alpha- and beta-cells. The presence of gap junctions offers indirect evidence that these cells are coupled with one another; coupling may influence the secretory behavior of alpha- and beta-cells maintaining glucose homeostasis within tightly constricted limits.

Animals↗
PubMed1977

Intercellular communication and tissue growth: IX. Junctional membrane structure of hybrids between communication-competent and communication-incompetent cells.

The structure of the membrane junctions of the hybrid cell system, examined in the companion paper in respect to competence for communication through cell-to-cell membrane channels, is here examined by freeze-fracture electron microscopy. The junctions of the channel-competent parent cell and of the channel-competent hybrid cells present aggregates of intramembranous particles typical of "gap junction"; those of the channel-incompetent parent cell and channel-incompetent segregant hybrid cells do not. Competence for junctional communication and for gap junction formation are genetically related. The junctions of the intermediate hybrid cells with incomplete channel-competence (characterized by cell-to-cell transfer of small inorganic ions but not of fluorescein), present special intramembranous fibrillar structures instead of discrete gap-junctional particles. The possibility that these structures may constitute coupling elements with subnormal permeability is discussed in terms of incomplete dominance of the genetic determinants of gap junction.

Animals↗
PubMed1976

Intercellular communication and tissue growth: VII. A cancer cell strain with retarded formation of permeable membrane junction and reduced exchange of a 330-dalton molecule.

A cancer (hepatoma) cell strain is described in which the formation of junctional membrane channels is abnormally slow. The development of electrical junctional coupling following the establishment of contact between these (reaggregated) cells is at least 15 times slower than that between their normal counterparts; and junctional transfer of fluorescein eventually develops, but only in about 5 per cent of the contacts (as against 100 per cent normally). This deviant membrane behavior is interpreted as a retardation in the process of accretion of junctional membrane channels. Its possible etiological role in defective growth regulation is discussed.

Carcinoma, Hepatocellular↗
PubMed2001

Multiple pathways in the trafficking and assembly of connexin 26, 32 and 43 into gap junction intercellular communication channels.

The assembly of gap junctions was investigated in mammalian cells expressing connexin (Cx) 26, 32 and 43 fused to green, yellow or cyan fluorescent proteins (GFP, YFP, CFP). Targeting of Cx32-CFP and 43-GFP to gap junctions and gap junctional communication was inhibited in cells treated with Brefeldin A, a drug that disassembles the Golgi. However gap junctions constructed of Cx26-GFP were only minimally affected by Brefeldin A. Nocodazole, a microtubule disruptor, had little effect on the assembly of Cx43-GFP gap junctions, but perturbed assembly of Cx26-GFP gap junctions. Co-expression of Cx26-YFP and Cx32-CFP in cells treated with Brefeldin A resulted in assembly of gap junctions constructed of Cx26-YFP. Two amino acids that distinguish Cx26 from Cx32 in transmembrane domains were mutated in Cx32 to investigate underlying mechanisms determining trafficking routes to gap junctions. One mutation, Cx32I28L, conferred on it partial Cx26-like trafficking properties as well the post-translational membrane insertion characteristics of Cx26, suggesting that a key determinant regulating trafficking was present in the first transmembrane domain. The results provide a protein trafficking basis for specifying and regulating connexin composition of gap junctions and thus selectivity of intercellular signaling, with Cx32 and 43 trafficking through the secretory pathway and Cx26 also following an alternative pathway.

Animals↗
PubMed1976

New membrane formation and intercellular communication in the early Xenopus embryo.

The ionic permeability of the nonjunctional and newly formed junctional membranes was investigated in embryos of Xenopus laevis up to the onset of the fifth cleavage. Continuous measurements were made of the equivalent nonjunctional (R'o) and junctional resistances (R'i) in different pairs of adjacent cells separated by one of the four cleavage membranes formed in that period. The specific resistance of the nonjunctional membranes (ro) and of each cleavage membrane (ri) as a function of time were derived using a generally applicable computer simulation model. ro decreased from about 40 komega cm2 in the in the uncleaved egg to about 10 komega cm2 at the 16-cell stage, due to the insertion of a small fraction of the relatively permeable newly formed cleavage membranes into the outer surface. Superimposed on this overall decline, a transient decrease of ro was observed during each cycle, caused by a temporary partial separation of the peripheral parts of adjacent blastomeres. The changes in followed the same pattern. R'1 increased stepwise during each cleavage cycle. At the onset of each cleavage there were no significant differences in R'i as measured between different pairs of cells. After an initial phase of membrane formation ri of all cleavage membranes remained constant at about 400 omega cm2. In the states investigated the coupling ratio ranged from 0.8 to 1. It is argued that this close coupling could be the result of the highly impermeable outer surface even in the absence of specialized junctions in the intercellular membranes.

Animals↗
PubMed2005

Intercellular communication: the Drosophila innexin multiprotein family of gap junction proteins.

Gap junctions belong to the most conserved cellular structures in multicellular organisms, from Hydra to man. They contain tightly packed clusters of hydrophilic membrane channels connecting the cytoplasms of adjacent cells, thus allowing direct communication of cells and tissues through the diffusion of ions, metabolites, and cyclic nucleotides. Recent evidence suggests that gap junctions are constructed by three different families of four transmembrane proteins: the Connexins and the Innexins found in vertebrates and in invertebrates, respectively, and the Innexin-like Pannexins, which were recently discovered in humans. This article focuses on the Drosophila Innexin multiprotein family, which is comprised of eight members. We highlight common structural features and discuss recent findings that suggest close similarities in cellular distribution, function, and regulation of Drosophila Innexins and vertebrate gap junction proteins.

Adherens Junctions↗