Intercellular interactions through gap junctions in embryonic stem cells

The minireview considers the structure and function of gap junctions, their role in embryonic stem cell persistence and differentiation, and in this context, the first data of the last research project launched by Levon M. Chailakhyan.     

Intercellular Ca2+ signaling in alveolar epithelial cells through gap junctions and by extracellular ATP

Inter- and extracellular-mediated changes in intracellular Ca2+ concentration ([Ca2+]i) can ensure coordinated tissue function in the lung. Cultured rat alveolar epithelial cells (AECs) have been shown to respond to secretagogues with increases in [Ca2+]i and have been shown to be gap junctionally coupled. However, communication of [Ca2+]i changes in AECs is not well defined. Monolayers of AECs were mechanically perturbed and monitored for [Ca2+]i changes. Perturbation of AECs was administered by a glass probe to either mechanically stimulate or mechanically wound individual cells. Both approaches induced a change in [Ca2+]i in the stimulated cell that was propagated to neighboring cells (Ca2+ waves). A connexin mimetic peptide shown to uncouple gap junctions eliminated Ca2+ waves in mechanically stimulated cells but had no effect on mechanically wounded cells. In contrast, apyrase, an enzyme that effectively removes ATP from the extracellular milieu, had no effect on mechanically stimulated cells but severely restricted mechanically wounded Ca2+ wave propagation. We conclude that AECs have the ability to communicate coordinated Ca2+ changes using both gap junctions and extracellular ATP.     

Intercellular communication via gap junctions affected by mechanical load in the bovine annulus fibrosus

Cells in the intervertebral disc, as in other connective tissues including tendon, ligament and bone, form interconnected cellular networks that are linked via functional gap junctions. These cellular networks may be necessary to affect a coordinated response to mechanical and environmental stimuli. Using confocal microscopy with fluorescence recovery after photobleaching methods, we explored the in situ strain environment of the outer annulus of an intact bovine disc and the effect of high-level flexion on gap junction signalling. The in situ strain environment in the extracellular matrix of the outer annulus under high flexion load was observed to be non-uniform with the extensive cellular processes remaining crimped sometimes at flexion angles greater than 25°. A significant transient disruption of intercellular communication via functional gap junctions was measured after 10 and 20 min under high flexion load. This study illustrates that in healthy annulus fibrosus tissue, high mechanical loads can impede the functioning of the gap junctions. Future studies will explore more complex loading conditions to determine whether losses in intercellular communication can be permanent and whether gap junctions in aged and degenerated tissues become more susceptible to load. The current research suggests that cellular structures such as gap junctions and intercellular networks, as well as other cell–cell and cell–matrix interconnections, need to be considered in computational models in order to fully understand how macroscale mechanical signals are transmitted across scales to the microscale and ultimately into a cellular biosynthetic response in collagenous tissues.     

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.     

Intercellular calcium signaling in astrocytes via ATP release through connexin hemichannels.

Astrocytes are capable of widespread intercellular communication via propagated increases in intracellular Ca(2+) concentration. We have used patch clamp, dye flux, ATP assay, and Ca(2+) imaging techniques to show that one mechanism for this intercellular Ca(2+) signaling in astrocytes is the release of ATP through connexin channels ("hemichannels") in individual cells. Astrocytes showed low Ca(2+)-activated whole-cell currents consistent with connexin hemichannel currents that were inhibited by the connexin channel inhibitor flufenamic acid (FFA). Astrocytes also showed molecular weight-specific influx and release of dyes, consistent with flux through connexin hemichannels. Transmembrane dye flux evoked by mechanical stimulation was potentiated by low Ca(2+) and was inhibited by FFA and Gd(3+). Mechanical stimulation also evoked release of ATP that was potentiated by low Ca(2+) and inhibited by FFA and Gd(3+). Similar whole-cell currents, transmembrane dye flux, and ATP release were observed in C6 glioma cells expressing connexin43 but were not observed in parent C6 cells. The connexin hemichannel activator quinine evoked ATP release and Ca(2+) signaling in astrocytes and in C6 cells expressing connexin43. The propagation of intercellular Ca(2+) waves in astrocytes was also potentiated by quinine and inhibited by FFA and Gd(3+). Release of ATP through connexin hemichannels represents a novel signaling pathway for intercellular communication in astrocytes and other non-excitable cells.     

Intercellular communication via intracellular calcium oscillations

In this letter, we present the results of a simple model for intercellular communication via calcium oscillations, motivated in part by a recent experimental study. The model describes two cells (a "donor" and "sensor") whose intracellular dynamics involve a calcium-induced, calcium release process. The cells are coupled by assuming that the input of the sensor cell is proportional to the output of the donor cell. As one varies the frequency of calcium oscillations of the donor cell, the sensor cell passes through a sequence of N : M phase-locked regimes and exhibits a "Devil's staircase" behavior. Such a phase-locked response has been seen experimentally in pulsatile stimulation of single cells. We also study a stochastic version of the coupled two-cell model. We find that phase locking holds for realistic choices for the cell volume.     

Intercellular junctions in myriapods

Tissue from the intestinal tract of myriapods, including millipedes, centipedes and pauropods were examined in tracer-impregnated sections and freeze-fracture replicas. The foregut and hindgut of all three classes exhibit pleated septate junctions; these display undulating intercellular ribbons in thin sections. In replicas they show discrete intramembranous particle (IMP) arrays aligned in rows in parallel; with one another. The tissues of the hindgut also possess scalariform junctions, characterized by cross-striated intercellular clefts in sections and IMP-enriched membranes in replicas. Gap junctions occur in all groups, but they are atypical in replicas in that their component IMPs do not always fracture onto the E face, as is characteristic of other arthropods; some IMPs cleave to the P face and others to the E face. The midgut of these organisms exhibits smooth septate junctions with conventional straight septal ribbons and occasional interseptal columns. However the intramembranous appearance in replicas is variable, particularly in centipedes, in that the rows of IMPs in chemically-unfixed propanecryofixed tissues, are prominent and adhere preferentially to the E face, with complementary P face grooves, while in fixed tissues the IMPs are much less distinct and fracture to either P face or E face. They tend not to protrude far beyond the mid-plane of the membrane bilayer and lie in rows which commonly take on the form of a network. Individual rows of the network sometimes curve to run beside a second row, over a short distance, before bending away into another part of the network. The aligned particle rows, which are much more prominent in millipedes, where they frequently lie in close parallel appositions, do not fuse into ridges as often occurs in insect tissues. The myriapod junctions, therefore, are of the same general kind as are found in the gut tract of other arthropod groups, but differ with respect to the subtleties of their intramembranous organization and disposition.     

Intercellular junctions between Novikoff hepatoma cells and hepatic cells

In the course of an ultrastructural study of liver invasion by Novikoff hepatoma transplanted in rat liver, several instances of intercellular junctions between tumor cells and non-neoplastic hepatic cells were noted. Such junctions were of the macula adherens type. This finding is interpreted as evidence of differentiation of the tumor cells.     

Intercellular junctions in nerve-free hydra

Summary Epithelial cells of nerve-free hydra contain septate and gap junctions. In thin sections the gap junctions are characterized by a gap of 3–4 nm. Freeze-fracture demonstrates the presence of septate junctions and two further types of structures: (i) the E-type or inverted gap junctions with particles in an en plaque conformation appearing as a raised plateau on the E-face or as a depression on the P-face; (ii) structures morphologically similar to gap junctions in rat liver, containing particles on the P-face and corresponding pits on the E-face, both having hexagonal packing with a lattice constant of 8 nm. We propose that these structures are also gap junctions.     

Isolation and characterization of Chinese hamster cells defective in cell-cell coupling via gap junctions

Chinese hamster Wg3-h-o cells which were descended from DON cells have been mutagenized and selected for derivatives defective in metabolic cooperation via gap junctions (i.e., mec-). The selection protocol included four consecutive cycles of cocultivating mutagenized cells, deficient in hypoxanthine phosphoribosyltransferase (HPRT) and wild-type cells in the presence of thioguanine (cf Slack, C, Morgan, R H M & Hooper, M L, Exp cell res 117 (1978) 195-205) [8]. We carried out the last two selection cycles in the presence of 1 mM dibutyryl cyclic adenosine monophosphate (db-cAMP). The isolated Chinese hamster CI-4 cells which expressed the mec- phenotype most stringently showed the following characteristics: 1. In standard culture medium no cell-cell coupling was detected among CI-4 cells when assayed by injections of the fluorescent dye Lucifer yellow or by electrical measurements. Between 73 and 100% of the mec+ parental cells were coupled under these conditions. Up to 14% positive contacts were found between CI-4 cells and Chinese hamster Don cells (mec+). Confluent CI-4 cells grown in the presence of 1 mM db-cAMP showed 9% coupled cells. 2. No gap junction plaques were found on electron micrographs of freeze-fractured, confluent CI-4 cells. The mec+ parental cells showed small gap junction plaques (0.013% of the total cell surface analyzed). 3. CI-4 cells exhibited 16% positive contacts and the parental Wg3-h-o cells showed 92% positive contacts in autoradiographic measurements of metabolic cooperation with DON cells. On an extracellular matrix, prepared from normal embryonic fibroblasts, metabolic cooperation between CI-4 and DON cells was autoradiographically measured to be 68%. Other cells of spontaneous mec- phenotype (for example mouse L cells or human fibrosarcoma HT1080 cells) also appeared to exhibit increased metabolic cooperation when grown on an extracellular matrix and assayed by autoradiographic measurements. When tested by Lucifer yellow injections, however, only very few positive contacts were found for CI-4/DON cell pairs and no positive contacts were found among mouse L cells grown on an extracellular matrix. 4. The mec- defect in the genome of CI-4 cells was cured in somatic cell hybrids with mouse embryonic fibroblasts or with mouse embryonal carcinoma cells. The results of isozyme and karyotype studies of mec-, as well as mec+ somatic cell hybrids suggest that mouse chromosome 16 may be involved in complementation of the mec- defect.     

Activation of L-type calcium channels is required for gap junction-mediated intercellular calcium signaling in osteoblastic cells

The propagation of mechanically induced intercellular calcium waves (ICW) among osteoblastic cells occurs both by activation of P2Y (purinergic) receptors by extracellular nucleotides, resulting in "fast" ICW, and by gap junctional communication in cells that express connexin43 (Cx43), resulting in "slow" ICW. Human osteoblastic cells transmit intercellular calcium signals by both of these mechanisms. In the current studies we have examined the mechanism of slow gap junction-dependent ICW in osteoblastic cells. In ROS rat osteoblastic cells, gap junction-dependent ICW were inhibited by removal of extracellular calcium, plasma membrane depolarization by high extracellular potassium, and the L-type voltage-operated calcium channel inhibitor, nifedipine. In contrast, all these treatments enhanced the spread of P2 receptor-mediated ICW in UMR rat osteoblastic cells. Using UMR cells transfected to express Cx43 (UMR/Cx43) we confirmed that nifedipine sensitivity of ICW required Cx43 expression. In human osteoblastic cells, gap junction-dependent ICW also required activation of L-type calcium channels and influx of extracellular calcium.     

Early responses to mechanical load in tendon: role for calcium signaling, gap junctions and intercellular communication

Tendon and other connective tissue cells are subjected to diverse mechanical loads during daily activities. Thus, fluid flow, strain, shear and combinations of these stimuli activate mechanotransduction pathways that modulate tissue maintenance, repair and pathology. Early mechanotransduction events include calcium (Ca2+) signaling and intercellular communication. These responses are mediated through multiple mechanisms involving stretch-activated channels, voltage-activated channels such as Ca(v)1, purinoceptors, adrenoceptors, ryanodine receptor-mediated Ca2+ release, gap junctions and connexin hemichannels. Calcium, diacylglycerol, inositol (1,4,5)-trisphosphate, nucleotides and nucleosides play intracellular and/or extracellular signaling roles in these pathways. In addition, responses to mechanical loads in tendon cells vary among species, tendon type, anatomic location, loading conditions and other factors. This review includes a synopsis of the immediate responses to mechanical loading in connective tissue cells, particularly tenocytes. These responses involve Ca2+ signaling, gap junctions and intercellular communication.     

Evidence for signaling via gap junctions from smooth muscle to endothelial cells in rat mesenteric arteries: possible implication of a second messenger

We investigated heterocellular communication in rat mesenteric arterial strips at the cellular level using confocal microscopy. To visualize Ca(2+) changes in different cell populations, smooth muscle cells (SMCs) were loaded with Fluo-4 and endothelial cells (ECs) with Fura red. SMC contraction was stimulated using high K(+) solution and Phenylephrine. Depending on vasoconstrictor concentration, intracellular Ca(2+) concentration ([Ca(2+)](i)) increased in a subpopulation of ECs 5-11s after a [Ca(2+)](i) rise was observed in adjacent SMCs. This time interval suggests chemical coupling between SMCs and ECs via gap junctions. As potential chemical mediators we investigated Ca(2+) or inositol 1,4,5-trisphosphate (IP(3)). First, phospholipase C inhibitor U-73122 was added to prevent IP(3) production in response to the [Ca(2+)](i) increase in SMCs. In high K(+) solution, all SMCs presented global and synchronous [Ca(2+)](i) increase, but no [Ca(2+)](i) variations were detected in ECs. Second, 2-aminoethoxydiphenylborate, an inhibitor of IP(3)-induced Ca(2+) release, reduced the number of flashing ECs by 75+/-3% (n = 6). The number of flashing ECs was similarly reduced by adding the gap junction uncoupler palmitoleic acid. Thus, our results suggest a heterocellular communication through gap junctions from SMCs to ECs by diffusion, probably of IP(3).     

Connexin mimetic peptides reversibly inhibit Ca(2+) signaling through gap junctions in airway cells

The effect of peptides with sequences derived from connexins, the constituent proteins of gap junctions, on mechanically stimulated intercellular Ca(2+) signaling in tracheal airway epithelial cells was studied. Three peptides with sequences corresponding to connexin extracellular loop regions reversibly restricted propagation of Ca(2+) waves to neighboring cells. Recovery of communication began within 10 min of removal of the peptides, with inhibition totally reversed by 20-40 min. The peptides were shown to be more effective in inhibiting Ca(2+) waves than glycyrrhetinic acid or oleamide. Inhibition of intercellular Ca(2+) waves by connexin mimetic peptides did not affect the Ca(2+) response to extracellular ATP. Although the intracellular Ca(2+) response of tracheal epithelial cells to ATP was greatly reduced by either pretreatment with high doses of ATP or application of apyrase, mechanically stimulated intercellular Ca(2+) signaling was not affected by these agents. We conclude that connexin mimetic peptides are effective and reversible inhibitors of gap junctional communication of physiologically significant molecules that underlie Ca(2+) wave propagation in tracheal epithelial cells and propose a potential mechanism for the mode of action of mimetic peptides.     

Freezing monolayers of cells without gap junctions

Cells in monolayers have been reported to be more susceptible to freezing injury than the same cell type frozen in dispersed suspensions. There appears to be an enhanced susceptibility to intracellular freezing in the monolayers, which is thought to be facilitated by the presence of gap junctions allowing the spread of ice between neighbouring cells. MDCK Type II cells do not form gap junctions in monolayer culture. When frozen at rates of 0.2 to 10 degrees C/min, monolayers in 10% (v/v) propane-1,2-diol or dimethyl sulphoxide showed little influence of cooling rate on survival. This suggested that, in the absence of gap junctions, cells in monolayers did not display enhanced susceptibility to intracellular freezing. In contrast, however, monolayers frozen in glycerol showed a marked increase in cell damage when cooled at rates higher than 0.5 degrees C/min. This does not necessarily counter the suggestion that lack of gap junctions mitigates intracellular freezing as there is evidence that glycerol may itself promote intracellular freezing.     

Cultured myometrial cells establish communicating gap junctions

Myometrial cells were isolated and cultured from term rat uterus. The myometrial origin of the cultures was verified by antibody staining of cellular desmin and alpha-smooth muscle actin. The presence of functional gap junctions was indicated by transfer of radiolabeled nucleotide and microinjected Lucifer yellow dye. The cultured cells expressed mRNA recognized by a connexin43 gap junction cDNA probe. To our knowledge, this is the first report that isolated myometrial cells form gap junctions in culture.     

Mechanically stimulated osteocytes regulate osteoblastic activity via gap junctions

The strong correlation between a bone's architectural properties and the mechanical forces that it experiences has long been attributed to the existence of a cell that not only detects mechanical load but also structurally adapts the bone matrix to counter it. One of the most likely cellular candidates for such a "mechanostat" is the osteocyte, which resides within the mineralized bone matrix and is perfectly situated to detect mechanically induced signals. However, as osteocytes can neither form nor resorb bone, it has been hypothesized that they orchestrate mechanically induced bone remodeling by coordinating the actions of cells residing on the bone surface, such as osteoblasts. To investigate this hypothesis, we developed a novel osteocyte-osteoblast coculture model that mimics in vivo systems by permitting us to expose osteocytes to physiological levels of fluid shear while shielding osteoblasts from it. Our results show that osteocytes exposed to a fluid shear rate of 4.4 dyn/cm² rapidly increase the alkaline phosphatase activity of the shielded osteoblasts and that osteocytic-osteoblastic physical contact is a prerequisite. Furthermore, both functional gap junctional intercellular communication and the mitogen-activated protein kinase, extracellular signal-regulated kinase 1/2 signaling pathway are essential components in the osteoblastic response to osteocyte communicated mechanical signals. By utilizing other nonosteocytic coculture models, we also show that the ability to mediate osteoblastic alkaline phosphatase levels in response to the application of fluid shear is a phenomena unique to osteocytes and is not reproduced by other mesenchymal cell types. osteocyte; osteoblast; fluid-flow; coculture; mechanical stimulation; gap junction; intercellular communication