Gap junctions and particle aggregates in the tegumentary syncytium of a trematode

The tegumentary syncytium of a Trematode is studied by transmission EM and freeze-fracture with the following results. (1) Infoldings of the basal plasma membrane suggest that transport of water and solutes occur through the tegument. (2) Heterocellular gap junctions are found between the tegumentary cell bodies and the parenchymal cells. Gap junctional particles, 8 nm in diameter, are visible on the P face of membrane and form an irregular pattern. (3) Orthogonal arrays of small particles (6 nm in diameter) are abundant on the P face of the tegument basal plasma membrane and on the cell necks connecting tegumentary cell bodies to the tegument. (4) Hemidesmosomal particles are found on the E face of the tegument basal plasma membrane. The significance of these structures with respect to tegumentery permeability and exchanges with parenchyma are discussed.     

Cell junctions in the epithelium of Bowman's capsule

The intercellular connections between the epithelial cells of Bowman's capsule were investigated. It could be demonstrated that typical zonulae occludentes (tight junctions) are present in the species (rat, hamster, and Tupaia) studied. Freeze-fracturing shows a network of anastomizing strands; some species variations are described. In the rat two strands are common. In the golden hamster mostly two to four and occasionally five strands occur. In Tupaia regularly three tight junction strands are found and also gap junctions associated with the zonulae occludentes. In thin sections the goniometric analysis confirms the freeze-fracturing results and reveals attachment zones of macular shape, which are classified as intermediate junctions and desmosomes. The functional role of these cell junctions observed in the epithelium of Bowman's capsule is discussed.     

Regulation of connexin43 function by activated tyrosine protein kinases

Gap junctions are specialized membrane structures that are involved in the normal functioning of numerous mammalian tissues and implicated in several human disease processes. This mini-review focuses on the regulation of gap junctions through phosphorylation of connexin43 induced by the v-Src or epidermal growth factor receptor tyrosine kinases. These tyrosine kinases markedly disrupt gap junctional communication in mammalian cells. here, we describe work correlating the alteration of connexin43 function with the ability of the v-Src tyrosine kinase to phosphorylate connexin43 directly on two distinct tyrosine sites in mammalian cells (Y247 and Y265). We also present evidence that proline-rich regions and phosphotyrosine sites of connexin43 may mediate interactions with the SH3 and SH2 domains of v-Src. In contrast to v-Src, the activated epidermal growth factor receptor acts indirectly through activated MAP kinase which may stimulate phosphorylation of connexin43 exclusively on serine. This phosphorylation event is complex because MAP kinase phosphorylates three serine sites in connexin43 (S255, S279, and S282). These findings suggest novel interactions between connexin43, the v-Src tyrosine kinase, and activated MAP kinase that set the stage for future investigations into the regulation of gap junctions by protein phosphorylation.     

Lack of restriction at the blood-brain interface in Limulus despite atypical junctional arrangements

Tracer and freeze-fracture techniques are used to evaluate the capacity of the central and peripheral nervous system of the horseshoe crab, Limulus polyphemus to admit or exclude molecular or ionic constituents of the blood intercellularly. Both the peripheral and central nervous systems are contained within blood sinuses so there is intimate contact between the haemolymph and the neural lamella. No discrete perineurium exists so any protection afforded to the nerve cells must be provided by the ensheathing glial cells and any junctions between them. Using ionic lanthanum as a pre-fixation incubation medium the system is seen to be completely "open', with the tracer gaining access to all regions of the nervous tissue. Cellular association in the peripheral nervous system, as revealed by thin-section and freeze-fracture, consist only of small scattered gap junctions between glial cells which afford no restriction to tracer entry. Gap junctions are again present between glial cells in the C.N.S. but here they are far more numerous, sometimes forming extensive sheets of almost continuous gap junctional plaques. Between certain glial cells there also exists a junctional system of linear PF ridges and complementary EF grooves; these may associate with or surround, often discontinuous arrays, the gap junctional plaques. Given their characteristics and the freedom of tracer entry, they seem unlikely to represent either typical occluding tight junctions or septate junctions.     

Effects of antiarrhythmic agents on junctional resistance of guinea pig ventricular cell pairs

Modulation of intercellular coupling through gap junctions can lead to a decrease in conduction velocity and conduction block. Previous studies have suggested that antiarrhythmic agents alter the internal resistance (sum of cytoplasmic and gap junctions resistances) of cardiac fibers. The objective of this study was to directly assess the effect of antiarrhythmic agents on junctional resistance between two isolated cells using the double whole-cell patch-clamp technique. The experimental protocol consisted in holding the membrane potential of each guinea pig ventricular myocyte of a coupled cell pair at 0 mV. Then, a junctional voltage gradient was created by changing membrane potential in only one cell. Voltage gradients were varied between -50 to +50 mV in steps of 20 mV. The extracellular medium was set to minimize trans-sarcolemmal currents and the junctional current was recorded in the cell maintained at 0 mV. Drugs tested were quinidine, lidocaine, procainamide, flecainide, propranolol, sotalol, amiodarone and verapamil. Drugs were superfused after a control period of 5 min. during which junctional resistance was observed to be stable. None of the antiarrhythmic agents tested in this study directly affected junctional resistance, although procainamide slightly increased junctional resistance 110 +/- 8% after 10 min of exposure. In conclusion, drugs tested in this study, chosen among all classes of antiarrhythmic agents, did not affect junctional resistance of cardiac myocyte cell pairs. However, long-term modulation or indirect effects of antiarrhythmic agents on gap junctions under physiological conditions cannot be excluded.     

Controlled-release tablet matrices from carrageenans: compression and dissolution studies

The corpus luteum (CL) is an organ that exhibits extremely rapid growth, development, and regression during the course of each nonpregnant cycle. The CL consists of steroidogenic (parenchymal) and nonsteroidogenic (nonparenchymal) cells. The small and large parenchymal cells differ in numerous morphological and functional characteristics, and are thought to interact with each other to maintain normal luteal function. These steroidogenic luteal cells also interact with the nonsteroidogenic cells; for example, they produce factors that stimulate proliferation and migration of endothelial cells and proliferation of fibroblasts; they also may enhance or suppress immune cell function. Conversely, endothelial cells produce factors that modulate steroidogenesis, and immune cells produce cytokines that modify the secretory function of steroidogenic cells. Cellular interactions may be mediated by several mechanisms, including humoral (endocrine and paracrine) pathways as well as contact-dependent (gap junctional) pathways. Thus, hormones, growth factors and cytokines produced locally by steroidogenic or nonsteroidogenic cells may be transferred from cell to cell indirectly or directly to regulate luteal function. Gap junctions are present in luteal tissues of several species, and gap junctional intercellular communication is affected by the stage of luteal development and systemic and local regulators of luteal function. Such cellular interactions probably are important in luteal hormone production, signal transduction, angiogenesis, and luteolysis because of their role in coordinating function among the various luteal cell types.     

Gap junctions and growth control in liver regeneration and in isolated rat hepatocytes

The hepatocytes in the mature normal liver are tightly coupled through gap junctions, except during compensatory hyperplasia (regeneration) after partial hepatectomy when the gap junctions become down-regulated. The significance of this down-regulation has been a long-standing enigma. The present study of hepatocytes in primary culture and in the regenerating liver aimed at defining the relationship, if any, between hepatocyte gap junctional communication and proliferation. Gap junctional down-regulation in the regenerating liver appeared to be a specific phenomenon because desmosomes and the surface contact area between neighboring hepatocytes remained constant. All agents and conditions (dexamethasone in vivo; dexamethasone, cyclic adenosine monophosphate, serum, and high cell density in vitro) delaying gap junctional down-regulation also increased the lag before the cells reached competence to enter S phase. This raised the possibility that hepatocyte DNA replication was inhibited through preservation of gap junctions. However, we disproved this assumption by showing that the DNA replication (more specifically the G1/S transition rate constant) was inhibited even in hepatocytes completely devoid of gap junctional communication. The teleological advantage of linking gap junctional down-regulation to hepatocyte G1 progression therefore may not be to trigger DNA replication but to ensure that proliferating hepatocytes and hepatocytes responsible for liver-specific metabolic functions maintain separate pools of metabolites and signaling molecules.     

The distribution of connexin 43 is associated with the germ cell differentiation and with the modulation of the Sertoli cell junctional barrier in continual (guinea pig) and seasonal breeders' (mink) testes

To test whether the gap junction protein connexin 43 (Cx43) is associated with germ cell differentiation and with the Sertoli cell junctional blood barrier, we recorded the temporal changes in its distribution before birth, through the neonatal period, puberty, and adulthood in guinea pig, and throughout the annual seasonal reproductive cycle in the mink. We used the immunoperoxidase labeling technique on Bouin's perfused-fixed testes and with site-specific polyclonal affinity-purified antibodies against Cx43. Cx43 was localized between Leydig cells in fetal guinea pig testis. In this species, after birth, the appearance of Cx43 concurred with the onset of spermatogenesis. In the seminiferous epithelium, the distribution of Cx43 coincided with the gap junctions of the Sertoli cell junctional blood barrier. In guinea pig and mink, the distribution of the protein in the tubules changed in accordance with the germ cell differentiation in a stage-dependent manner and with the modulation, i.e., the assembly and disassembly of the junctional barrier accompanying the translocation of spermatocytes into the lumenal compartment. In the mink, the reaction product persisted during testicular regression but showed a similar distribution from one tubule to the next. In this paper, we documented the existence of a temporal correlation between the appearance of the gap junction protein Cx43 and both the germ cell differentiation and the modulation in the junctional barrier between Sertoli cells. The paper also discusses the possibility that cell-to-cell communications, generally attributed to gap junctions, may help changes in the barrier to take place in coordination with spermatogenesis.     

The postnatal development of the junctional complexes of the mouse Sertoli cells as revealed by freeze-fracture

Mouse testes of newborn to adult were examined by freeze-fracture. Between the newborn Sertoli cells, gap junctions consisting of aggregations of the intramembranous particles (about 8 nm in diameter) are frequently found. Some of the junctions are about 1 mum in diameter and show particle-free regions in the aggregation. Linear arrangements of a few particles, which appear to be the initial formation of the occluding junctions, are seen in the newborn sertoli cells. The occluding junctions are arranged in a meshwork, in which the gap junctions are situated between the stages of newborn to six days of age. The particles of the occluding junctions are predominantly located on the B face in the center of the groove instead of the A face of the ridge. The occluding junctions do not appear to surround the entire circumference of the Sertoli cell of the 6-day-old mouse. The gap junctions decrease in size. In later stages, many parallel occluding junctions (up to forty in number) are found over one Sertoli cell surface and are distributed circumferentially around the entire cell surface, indicating establishment of the blood-testis barrier. The occluding junctions dominate and the gap junctions diminish in number as development proceeds.     

Changes in cellular distribution of connexins 32 and 26 during formation of gap junctions in primary cultures of rat hepatocytes

In the adult rat hepatocyte, gap junction proteins consist of connexin 32 (Cx32) and connexin 26 (Cx26). Previously, we reported that both Cx32 and Cx26 were markedly induced and maintained in primary cultures of adult rat hepatocytes. The reappearing gap junctions were accompanied by increases in both the proteins and the mRNAs, and they were well maintained together with extensive gap junctional intercellular communication (GJIC) for more than 4 weeks. In the present study, we examined the cellular location of the gap junction proteins and the structures in the hepatocytes cultured in our system, using confocal laser microscopy and immunoelectron microscopy of cells processed for Cx32 and Cx26 immunocytochemistry and freeze-fracture analysis. In immunoelectron microscopy, the size of Cx32-immunoreactive gap junction structures on the plasma membrane increased with time of culture, and some of them were larger than those in liver sections in vivo. Freeze-fracture analysis also showed that the size of gap junction plaques increased and that the larger gap junction plaques were composed of densely packed particles. These results suggest that in this culture system, not only the synthesis of Cx proteins but also the size of the gap junction plaques was increased markedly. In the adluminal lateral membrane of the cells, Cx32-immunoreactive lines were observed and many small gap junction plaques were closely associated with a more developed tight junction network. In the basal region of the cells, small Cx32- and Cx26-immunoreactive dots were observed in the cytoplasm and several annular structures labeled with the antibody to Cx32 were observed in the cytoplasm. These results indicated the formation and degradation of gap junctions in the cultured hepatocytes.     

Child sexual abuse evaluations: the scientist-practitioner model

This review presents published data on various aspects of gap junction functioning during development. The process of formation of these junctions and their structure, along with relationships between molecular characteristics of connexins and properties of gap junctions channels are considered in detail. Some newly discovered functions of gap junctions in early embryonic development, their participation in the formation of various cellular compartments and in coordinated behavior are discussed; their relationship with other types of cell interactions is also reviewed. Promising problems for further research are outlined.     

Mechanisms of electrical coupling between pyramidal cells

Direct electrical coupling between neurons can be the result of both electrotonic current transfer through gap junctions and extracellular fields. Intracellular recordings from CA1 pyramidal neurons of rat hippocampal slices showed two different types of small-amplitude coupling potentials: short-duration (5 ms) biphasic spikelets, which resembled differentiated action potentials and long-duration (>20 ms) monophasic potentials. A three-dimensional morphological model of a pyramidal cell was employed to determine the extracellular field produced by a neuron and its effect on a nearby neuron resulting from both gap junctional and electric field coupling. Computations were performed with a novel formulation of the boundary element method that employs triangular elements to discretize the soma and cylindrical elements to discretize the dendrites. An analytic formula was derived to aid in computations involving cylindrical elements. Simulation results were compared with biological recordings of intracellular potentials and spikelets. Field effects produced waveforms resembling spikelets although of smaller magnitude than those recorded in vitro. Gap junctional electrotonic connections produced waveforms resembling small-amplitude excitatory postsynaptic potentials. Intracellular electrode measurements were found inadequate for ascertaining membrane events because of externally applied electric fields. The transmembrane voltage induced by the electric field was highly spatially dependent in polarity and wave shape, as well as being an order of magnitude larger than activity measured at the electrode. Membrane voltages because of electrotonic current injection across gap junctions were essentially constant over the cell and were accurately depicted by the electrode. The effects of several parameters were investigated: 1) decreasing the ratio of intra to extracellular conductivity reduced the field effects; 2) the tree structure had a major impact on the intracellular potential; 3) placing the gap junction in the dendrites introduced a time delay in the gap junctional mediated electrotonic potential, as well as deceasing the potential recorded by the somatic electrode; and 4) field effects decayed to one-half of their maximum strength at a cell separation of approximately 20 micron. Results indicate that the in vitro measured spikelets are unlikely to be mediated by gap junctions and that a spikelet produced by the electric field of a single source cell has the same waveshape as the measured spikelet but with a much smaller amplitude. It is hypothesized that spikelets are a manifestation of the simultaneous electric field effects from several local cells whose action potential firing is synchronized.     

Gap junctions mediate intercellular calcium signalling in cultured articular chondrocytes

Gap junction-mediated intercellular communication has been implicated in a variety of cellular functions. Among these, signal transduction can be coordinated among several cells due to gap junctional permeability to intracellular second messengers. Chondrocytes from articular cartilage in primary culture respond to extracellular ATP by rhythmically increasing their cytosolic Ca2+ concentration. Digital imaging fluorescence microscopy of Fura-2 loaded cells was used to monitor Ca2+ in confluent and semi-confluent cell layers. Under these conditions, Ca2+ spikes propagate from cell to cell giving rise to intercellular Ca2+ waves. The functional expression of gap junctions was assessed, in confluent chondrocyte cultures, by the intercellular transfer of Lucifer yellow dye in scrape-loading experiments. Intercellular dye transfer was blocked by the gap junction inhibitor 18 alpha-glycyrrhetinic acid. In imaging experiments, the inhibitor caused the loss of synchrony of ATP-induced Ca2+ oscillations, and blocked the intercellular Ca2+ propagation induced by mechanical stimulation of a single cell in a monolayer. It is concluded that gap junctions mediate intercellular signal transduction in cartilage cells and may provide a mechanism for co-ordinating their metabolic activity.     

Cell-junctional and cytoskeletal organization in mouse blastocysts lacking E-cadherin

Trophectoderm epithelium formation, the first visible differentiation process during mouse embryonic development, is affected in embryos lacking the cell adhesion molecule E-cadherin. Here we analyze the developmental potential of such E-cadherin-negative embryos, focusing on the organization of cell junctions and the cytoskeleton. To do this we used antibodies directed against alpha-, beta-, or gamma-(plakoglobin)-catenin and junctional and cytoskeletal proteins including ZO-1 and occludin (tight junctions), desmoglein1 (desmosomes), connexin43 (gap junctions), and EndoA (cytokeratin intermediate filaments). Membrane localization of alpha- and beta-catenin, and ZO-1, as well as cortical actin filament organization were abnormal in E-cadherin-negative embryos, and the expression levels of alpha- and beta-catenin were dramatically reduced, all suggesting a regulatory role for E-cadherin in forming the cadherin-catenin complex. In contrast, the membrane localization of plakoglobin, occludin, desmoglein1, connexin43, and cytokeratin filaments appeared unaltered. The unusual morphogenesis in E-cadherin-negative embryos apparently reflects defects in the molecular architecture of a supermolecular assembly involving zonulae adherens, tight junctions, and cortical actin filament organization, although the individual structures still appeared normal in electron microscopical analysis.     

Association between dendritic lamellar bodies and complex spike synchrony in the olivocerebellar system

Dendritic lamellar bodies have been reported to be associated with dendrodendritic gap junctions. In the present study we investigated this association at both the morphological and electrophysiological level in the olivocerebellar system. Because cerebellar GABAergic terminals are apposed to olivary dendrites coupled by gap junctions, and because lesions of cerebellar nuclei influence the coupling between neurons in the inferior olive, we postulated that if lamellar bodies and gap junctions are related, then the densities of both structures will change together when the cerebellar input is removed. Lesions of the cerebellar nuclei in rats and rabbits resulted in a reduction of the density of lamellar bodies, the number of lamellae per lamellar body, and the density of gap junctions in the inferior olive, whereas the number of olivary neurons was not significantly reduced. The association between lamellar bodies and electrotonic coupling was evaluated electrophysiologically in alert rabbits by comparing the occurrence of complex spike synchrony in different Purkinje cell zones of the flocculus that receive their climbing fibers from olivary subnuclei with different densities of lamellar bodies. The complex spike synchrony of Purkinje cell pairs, that receive their climbing fibers from an olivary subnucleus with a high density of lamellar bodies, was significantly higher than that of Purkinje cells, that receive their climbing fibers from a subnucleus with a low density of lamellar bodies. To investigate whether the complex spike synchrony is related to a possible synchrony between simple spikes, we recorded simultaneously the complex spike and simple spike responses of Purkinje cell pairs during natural visual stimulation. Synchronous simple spike responses did occur, and this synchrony tended to increase as the synchrony between the complex spikes increased. This relation raises the possibility that synchronously activated climbing fibers evoke their effects in part via the simple spike response of Purkinje cells. The present results indicate that dendritic lamellar bodies and dendrodendritic gap junctions can be downregulated concomitantly, and that the density of lamellar bodies in different olivary subdivisions is correlated with the degree of synchrony of their climbing fiber activity. Therefore these data support the hypothesis that dendritic lamellar bodies can be associated with dendrodendritic gap junctions. Considering that the density of dedritic lamellar bodies in the inferior olive is higher than in any other area of the brain, this conclusion implies that electrotonic coupling is important for the function of the olivocerebellar system.     

ATP- and gap junction-dependent intercellular calcium signaling in osteoblastic cells

Many cells coordinate their activities by transmitting rises in intracellular calcium from cell to cell. In nonexcitable cells, there are currently two models for intercellular calcium wave propagation, both of which involve release of inositol trisphosphate (IP3)- sensitive intracellular calcium stores. In one model, IP3 traverses gap junctions and initiates the release of intracellular calcium stores in neighboring cells. Alternatively, calcium waves may be mediated not by gap junctional communication, but rather by autocrine activity of secreted ATP on P2 purinergic receptors. We studied mechanically induced calcium waves in two rat osteosarcoma cell lines that differ in the gap junction proteins they express, in their ability to pass microinjected dye from cell to cell, and in their expression of P2Y2 (P2U) purinergic receptors. ROS 17/2.8 cells, which express the gap junction protein connexin43 (Cx43), are well dye coupled, and lack P2U receptors, transmitted slow gap junction-dependent calcium waves that did not require release of intracellular calcium stores. UMR 106-01 cells predominantly express the gap junction protein connexin 45 (Cx45), are poorly dye coupled, and express P2U receptors; they propagated fast calcium waves that required release of intracellular calcium stores and activation of P2U purinergic receptors, but not gap junctional communication. ROS/P2U transfectants and UMR/Cx43 transfectants expressed both types of calcium waves. Gap junction-independent, ATP-dependent intercellular calcium waves were also seen in hamster tracheal epithelia cells. These studies demonstrate that activation of P2U purinergic receptors can propagate intercellular calcium, and describe a novel Cx43-dependent mechanism for calcium wave propagation that does not require release of intracellular calcium stores by IP3. These studies suggest that gap junction communication mediated by either Cx43 or Cx45 does not allow passage of IP3 well enough to elicit release of intracellular calcium stores in neighboring cells.     

No junctional communication between epithelial cells in hydra

Diffusion gradients of morphogens have been inferred as a basis for the control of morphogenesis in hydra, and morphogenetic substances have been found which, on the basis of their molecular weight (MW), should be able to pass gap junctions. There have been several reports of the presence of gap junctions between epithelial cells of hydra. However, until now, there has been no report published on whether these junctions enable the epithelial cells to exchange molecules of small molecular weight, as has been described in other organisms. Therefore we decided to investigate the communicative properties of the junctional membranes by electrophysiological methods and by intracellular-dye iontophoresis. We report here that no electrotonic coupling is detectable between epithelial cells of Hydra attenuata in: (1) intact animals, (2) head-regenerating animals, (3) cell re-aggregates, and (4) hydra that have become nervefree. Furthermore we show that epithelial cells are unable to exchange low-molecular weight fluorescent dyes.     

Gap junctional communication between murine macrophages and intestinal epithelial cell lines

In intestinal inflammation, inflammatory cells infiltrate the submucosa and are found juxtaposed to intestinal epithelial cell (IEC) basolateral membranes and may directly regulate IEC function. In this study we determined whether macrophage (M phi), P388D1 and J774A.1, are coupled by gap junctions to IEC lines, Mode-K and IEC6. Using flow cytometric analysis, we show bi-directional transfer of the fluorescent dye, calcein (700 Da) between IEC and M phi resulting in a 3.5-20-fold increase in recipient cell fluorescence. Homocellular and heterocellular dye transfer between M phi and/or IEC was detected in cocultures of P388D1, J774A.1, Mode-K, IEC6 and CMT93. However, transfer between P388D1 and Mode-K was asymmetrical in that transfer from P388D1 to Mode-K was always more efficient than transfer from Mode-K to P388D1. Dye transfer was strictly dependent on IEC-M phi adhesion which in turn was dependent on the polarity of IEC adhesion molecule expression. Both calcein dye transfer and adhesion were inhibited by the addition of heptanol to cocultures. Furthermore we demonstrate both IEC homocellular, and M phi-IEC heterocellular propagation of calcium waves in response to mechanical stimulation, typical of gap junctional communication. Finally, areas of close membrane apposition were seen in electron micrographs of IEC-M phi cocultures, suggestive of gap junction formation. These data indicate that IEC and M phi are coupled by gap junctions suggesting that gap junctional communication may provide a means by which inflammatory cells might regulate IEC function.     

A pre-loading method of evaluating gap junctional communication by fluorescent dye transfer

We describe a simple method for evaluating gap junctional communication (GJC) between cells in culture. The procedure involves pre-loading cells with two fluorescent dyes: calcein and DiI. Calcein is able to pass through gap junctions, while DiI is not. These pre-loaded cells are then plated with unlabeled cells. The number of cells receiving calcein from each pre-loaded cell can then be quantified after the cells settle on the plate. Potent and reversible inhibitors of GJC can be used in this system to evaluate dye transfer within a given period of time.