Protein phosphatase modulation of the intercellular junctional communication: importance in cardiac myocytes

The rhythmic contraction of a four-chambered heart is a highly co-ordinated process, requiring the sequential activation of pacemaker cells and the propagation of activity throughout the whole myocardium. Gap-junctional channels, providing enclosed conduits for direct cell-to-cell transfer of ions and small molecules between adjacent cells, allow depolarising currents to flow from excited to non-excited regions of the network and a gradual spreading of the action potential. Gap-junctional channels are dodecamers of transmembrane proteins belonging in chordates to the connexin (Cx) family. In mammalian hearts, cardiomyocytes most prominently express junctional channels built of three Cxs: Cx40, Cx43 and Cx45. As with the great majority of Cx, they are phosphoproteins and exist under different phosphorylated levels. Phosphorylation, a widespread post-translational modification of proteins, is a primary means of mediating signal transduction events that control numerous cellular processes via a highly regulated dynamic interplay of protein kinases (PKs) and protein phosphatases (PPs). These processes appear implicated in the regulation of gap-junctional communication at several stages of the Cx lifecycle, including intracellular Cx trafficking, connexon assembly and disassembly, Cx degradation as well as the gating of gap-junction channels, but the underlying mechanisms remain poorly understood. Although PKs have an established role in this process, less is known about the involvement of PPs. The present review examines the roles played by protein dephosphorylation catalysers in the regulation of the gap-junctional communication in general, with a special focus on the junctional communication between cardiac cells.     

Effects of stochastic channel gating and distribution on the cardiac action potential

Ion channels exhibit stochastic conformational changes determining their gating behavior. In addition, the process of protein turnover leads to a natural variability of the number of membrane and gap junctional channels. Nevertheless, in computational models, these two aspects are scarcely considered and their impacts are largely unknown. We investigated the effects of stochastic current fluctuations and channel distributions on action potential duration (APD), intercellular conduction delays (ICDs) and conduction blocks using a modified ventricular cell model (Rudy et al.) with Markovian formulations of the principal ion currents (to simulate their stochastic open-close gating behavior) and with channel counts drawn from Poisson distributions (to simulate their natural variability). In single cells, APD variability (coefficient of variation: 1.6% at BCL=1000 ms) was essentially caused by stochastic channel gating of I_Ks, persistent I_Na and I_Ca,L. In cell strands, ICD variability induced by stochastic channel gating and Poissonian channel distributions was low under normal conditions. Nonetheless, at low intercellular coupling levels, Poissonian gap junctional channel distribution resulted in a large ICD variability (coefficient of variation >20%), highly heterogeneous conduction patterns and conduction blocks. Therefore, the stochastic behavior of current fluctuations and channel distributions can contribute to the heterogeneity of conduction patterns and to conduction block, as observed previously in experiments in cardiac tissue with altered intercellular coupling.     

Nontransformed cells can normalize gap junctional communication with transformed cells

We demonstrate that the Src kinase can augment gap junctional communication between cells derived from homozygous null Cx43 knockout mice. The total conductance between Src transformed cells was nearly twice that of nontransformed cells. In addition, the unitary conductance of the majority of single channel events between transformed cells was about 35% greater than that of nontransformed cells. Analysis showed that both nontransformed and transformed cells expressed at least two populations of channels, suggesting that Src increased junctional conductance by up-regulating one population and/or by increasing the unitary conductance of another population of channels. Interestingly, the conductance displayed by heterologous pairs of transformed and nontransformed cells resembled that of nontransformed cells. The majority of single channel events between heterologous pairs shifted back to lower conductances that were exhibited by nontransformed cells. Thus, nontransformed cells can effectively "normalize" the conductance of gap junction channels expressed by adjacent tumor cells.     

Effects of UV-C radiation on membrane potential and electric conductance in internodal cells of Nitellopsis obtusa

Effects of 253.7 nm ultraviolet radiation on membrane potential and conductance in internodal cells of Nitellopsis obtusa were studied. The radiation caused transient depolarization of plasmalemma and tonoplast and simultaneous increase in electric conductance. These effects were partly reversible and the degree of the recovery depended on the duration of the exposure. In cells with potential difference (between vacuole and external medium) more negative than – 140 mV, the radiation induced an action potential. The hyperpolarized state created by visible light and indole-3-acetic acid was fully suppressed by the radiation. The results are discussed taking into account the data for Chara corallina obtained by C. J. Doughty and A. B. Hope. It is suggested that 253.7 nm radiation inhibits electrogenic proton pumps in the plasmalemma and activates the Cl⁻ channels.     

Increased gap junctional area in the rat liver after administration of dibutyryl cAMP

Summary Since cAMP has recently been reported to be a possible physiological modulator of cell-to-cell communication, we performed a quantitative freeze-fracture investigation on the hepatocyte gap junctions after administration of a membrane-permeant derivative of this cyclic nucleotide. For this purpose, male rats received two intraperitoneal injections of 100 mg dibutyryl cAMP/kg body weight with a time interval of 2.5 h. Litter mates were injected with saline only. Five hours after the start of the treatment, tissue blocks of the left lateral liver lobe were fixed by immersion and processed for freeze-fracture. By point counting on negatives projected on a square double-lattice test system the relative gap junctional area on contiguous hepatocyte membranes was determined. As compared to control animals, the proportion of the membrane area occupied by gap junctions in dibutyryl cAMP-treated liver parenchyma significantly increased from 4.9% to 6.1%. Within the gap junctions no changes in shape, particle density or packing pattern were observed. Possibly, the enlarged gap junctional area provides structural pathways for the integration of the response of hepatocytes to messages mediated by cAMP.This investigation was supported by grant nr. 3.0059.81 (to D.W.S.) from the Fund for Medical Scientific Research (Belgium).Research assistant of the National Fund for Scientific Research (Belgium).     

Simulating patterns of excitation, repolarization and action potential duration with cardiac Bidomain and Monodomain models

Parallel numerical simulations of excitation and recovery in three-dimensional myocardial domains are presented. The simulations are based on the anisotropic Bidomain and Monodomain models, including intramural fiber rotation and orthotropic or axisymmetric anisotropy of the intra- and extra-cellular conductivity tensors. The Bidomain model consist of a system of two reaction-diffusion equations, while the Monodomain model consists of one reaction-diffusion equation. Both models are coupled with the phase I Luo-Rudy membrane model describing the ionic currents. Simulations of excitation and repolarization sequences on myocardial slabs of different sizes show how the distribution of the action potential durations (APD) is influenced by both the anisotropic electrical conduction and the fiber rotation. This influence occurs in spite of the homogeneous intrinsic properties of the cell membrane. The APD dispersion patterns are closely correlated to the anisotropic curvature of the excitation wavefront.     

Effects of transmural electrical heterogeneities and electrotonic interactions on the dispersion of cardiac repolarization and action potential duration: A simulation study

It has been shown in the literature that myocytes isolated from the ventricular walls at various intramural depths have different action potential durations (APDs). When these myocytes are embedded in the ventricular wall, their inhomogeneous properties affect the sequence of repolarization and the actual distribution of the APDs in the entire wall. In this article, we implement a mathematical model to simulate the combined effect of (a) the non-homogeneous intrinsic membrane properties (in particular the non-homogeneous APDs) and (b) the electrotonic currents that modulate the APDs when the myocytes are embedded in the ventricular myocardium. In particular, we study the effect of (a) and (b) on the excitation and repolarization sequences and on the distribution of APDs in the ventricles. We implement a Monodomain tissue representation that includes orthotropic anisotropy, transmural fiber rotation and homogeneous or heterogeneous transmural intrinsic membrane properties, modeled according to the phase I Luo-Rudy membrane ionic model. Three-dimensional simulations are performed in a cartesian slab with a parallel finite element solver employing structured isoparametric trilinear finite elements in space and a semi-implicit adaptive method in time. Simulations of excitation and repolarization sequences elicited by epicardial or endocardial pacing show that in a homogeneous slab the repolarization pathways approximately follow the activation sequence. Conversely, in the heterogeneous cases considered in this study, we observed two repolarization wavefronts that started from the epi and the endocardial faces respectively and collided in the thickness of the wall and in one case an additional repolarization wave starting from an intramural site. Introducing the heterogeneities along the transmural epi-endocardial direction affected both the repolarization sequence and the APD dispersion, but these effects were clearly discernible only in transmural planes. By contrast, in planes parallel to epi- and endocardium the APD distribution remained remarkably similar to that observed in the homogeneous model. Therefore, the patterns of the repolarization sequence and APD dispersion on the epicardial surface (or any other intramural surface parallel to it) do not reveal the uniform transmural heterogeneity.     

Regulation of gap junctional coupling in isolated pancreatic acinar cell pairs by cholecystokinin-octapeptide,vasoactive intestinal peptide (VIP) and a VIP-antagonist

Cholecystokinin-octapeptide (CCK-OP) induces a time- and dose-dependent decrease of gap Junctional conductance in isolated pairs of pancreatic acinar cells. In double whole-cell experiments, the time course could be described by the latency and the half-life time (t _1/2 ) of cell-to-cell uncoupling. The latency shows a biphasic dependence on [CCK-OP] with a minimum of about 50 sec at 10^–9 m CCK-OP. In the presence of vasoactive intestinal peptide (VIP), the biphasic relationship is shifted to lower CCK-OP concentrations. The increase of latency at high concentrations of CCK-OP (> 10⁰⁹ m) was blocked by addition of a VIP-antagonist. t _1/2 decreases monophasically with increasing [CCKOP]. Addition of GTPS to the pipette solution suppresses the [CCK-OP] dependence of the latency and potentiates the uncoupling phase. The kinetic data are discussed in terms of CCK binding to receptors of high and low affinity. Evidence is presented that secretion and cell-to-cell coupling are not related by an all-ornone process, but that for physiological CCK-OP concentrations, gap junctional uncoupling follows secretion.The authors would like to thank Dipl. Biol. F. Mendez for his support in software development for analysis of gap junctional conductance. The work was supported by the Graduiertenkolleg Biochemische Pharmakologie, the Herrmann und Lilly Schilling Stiftung and the Sonderforschungsbereich 156 of the Deutsche Forschungsgemeinschaft.     

Closing and opening of gap junction pores between two- and threedimensionally cultured tumor cells

Intercellular signal transfer via gap junction pores in cultured multicell spheroids of BICR/M1R-K cells decreases with increasing spheroid age. In two days old spheroids the pores allow passage of Lucifer yellow molecules. Two days later, this fluorescent dye is retained in the injected cell even though the cells are still electrically coupled. Gap junction plaques of considerable size are still found in 9 days old spheroids, when the cells are completely uncoupled. The same cells growing as monolayer cultures do not exhibit such a gradual closing of their gap junction pores: Their coupling is established at first cell contact, probably by a gradual opening of the pores, which remain open even up to 9 days in culture.Based on material presented at the Symposium Intercellular Communication Stuttgart, September 16–17, 1982     

ATP activates P2X receptors to mediate gap junctional coupling in the cochlea

ATP is an important extracellular signaling molecule and can activate both ionotropic (P2X) and metabotropic purinergic (P2Y) receptors to influence cellular function in many aspects. Gap junction is an intercellular channel and plays a critical role in hearing. Here, we report that stimulation of ATP reduced gap junctional coupling between cochlear supporting cells. This uncoupling effect could be evoked by nanomolar physiological levels of ATP. A P2X receptor agonist benzoylbenzoyl-ATP (BzATP) but not a P2Y receptor agonist UTP stimulated this uncoupling effect. Application of P2X receptor antagonists pyridoxalphosphate-6-azophenyl-2′,4′-disulfonic acid (PPADS, 50 μM) or oxidized ATP (oATP, 0.1 mM) eliminated this uncoupling effect. We further found that ATP activated P2X receptors in the cochlear supporting cells allowing Ca²⁺ influxing, thereby increasing intracellular Ca²⁺ concentration to mediate gap junctions. These data suggest that ATP can mediate cochlear gap junctions at the physiological level by the activation of P2X receptors rather than P2Y receptors. This P2X receptor-mediated purinergic control on the cochlear gap junctions may play an important role in the regulation of K⁺-recycling for ionic homeostasis in the cochlea and the reduction of hearing sensitivity under noise stress for protection.     

Changes in the fluctuation of the contraction rhythm of spontaneously beating cardiac myocytes in cultures with and without cardiac fibroblasts

The heart functions as a syncytium of cardiac myocytes and surrounding supportive non-myocytes such as fibroblasts. There is a possibility that a variety of non-myocyte-derived factors affect the maturation of cardiac myocytes in the development of the heart. Cultured neonatal cardiac myocytes contract spontaneously and cyclically. The fluctuation of beating rhythm varies depending on the strength of coupling through gap junctions among cardiac myocytes, indicating that the development of intercellular communication via gap junctions is crucial to the stability of contraction rhythm in cardiac myocytes. In this study, we aimed at elucidating whether and how cardiac fibroblasts affect the development of cardiac myocytes from the point of view of the changes in the fluctuation of the contraction rhythm of cardiac myocytes in cardiac myocyte–fibroblast co-cultures. The present study suggested that cardiac fibroblasts co-cultured with cardiac myocytes enhanced the intercellular communication among myocytes via gap junctions, thereby stabilizing the spontaneous contraction rhythm of cultured cardiac myocytes.     

Junctions between interstitial cells of the renal medulla: A freeze-fracture study

Summary Cell junctions between interstitial cells of the renal medulla were studied in freeze-fracture replicas of kidneys from rat, rabbit, hamster and the tree-shrew Tupaia belangeri. In all species studied a composite type of intercellular junction was found comprising elements of tight junctions and irregular gap junctions of highly variable size and shape. The number of these junctions increased towards the tip of the papilla.Our findings suggest that the composite junctions observed play a role in the maintainance of the ladder-like arrangement of the interstitial cells in the inner zone. The existence of irregular gap junctions raises the possibility that the functions of the interstitial cells are coordinated, especially during alterations of the functional state of the kidney.Supported by the Deutsche Forschungsgemeinschaft     

Passage through vertebrate gap junctions of 17/18 kDa molecules is primarily dependent upon molecular configuration

In fish, amphibians and mammals, gap junctions of some cells allow passage of elongate molecules as large as 18 kDa, while excluding smaller, less elongate molecules. Fluorescently labeled Calmodulin (17 kDa) and fluorescently labeled Troponin-C (18 kDa), when microinjected into oocytes of Danio rerio, Xenopus laevis or Mus domestica, were able to transit the gap junctions between these oocytes and the granulosa cells which surrounded them. Co-microinjected with these Ca²⁺-binding proteins, Texas-red-labeled dextran (10 kDa) remained in the microinjected cell. Osteocalcin (6 kDa), also a Ca²⁺-binding protein, but with a wide “V” shape proved unable to transit these gap junctions. Calmodulin, but not Troponin-C, was able to transit gap junctions of gonadotropin treated WB cells in culture. We show evidence that molecules as large as 18 kDa can pass through some vertebrate gap junctions, both homologous and heterologous, and that it is primarily molecular configuration which governs gap junctional permeability.     

Voltage noise influences action potential duration in cardiac myocytes

Stochastic gating of ion channels introduces noise to membrane currents in cardiac muscle cells (myocytes). Since membrane currents drive membrane potential, noise thereby influences action potential duration (APD) in myocytes. To assess the influence of noise on APD, membrane potential is in this study formulated as a stochastic process known as a diffusion process, which describes both the current-voltage relationship and voltage noise. In this framework, the response of APD voltage noise and the dependence of response on the shape of the current-voltage relationship can be characterized analytically. We find that in response to an increase in noise level, action potential in a canine ventricular myocytes is typically prolonged and that distribution of APDs becomes more skewed towards long APDs, which may lead to an increased frequency of early after-depolarization formation. This is a novel mechanism by which voltage noise may influence APD. The results are in good agreement with those obtained from more biophysically-detailed mathematical models, and increased voltage noise (due to gating noise) may partially underlie an increased incidence of early after-depolarizations in heart failure.     

Temperature dependence of embryonic cardiac gap junction conductance and channel kinetics

We have investigated the effects of temperature on the conductance and voltage-dependent kinetics of cardiac gap junction channels between pairs of seven-day embryonic chick ventricle myocytes over the range of 14–26°C. Records of junctional conductance (G _j ) and steady-state unit junctional channel activity were made using the whole-cell double patch-clamp technique while the bath temperature was steadily changed at a rate of about 4°C/min. The decrease inG _j upon cooling was biphasic with a distinct break at 21°C. In 12 cell pairs,Q ₁₀ was 2.2 from 26 to 21°C, while between 21 and 14°C it was 6.5. The meanG _j at 22°C (G _j22 ) was 3.0±2.1 nS, ranging in different preparations from 0.24 to 6.4 nS. At room temperature, embryonic cardiac gap junctions contain channels with conductance states near 240, 200, 160, 120, 80 and 40 pS. In the present study, we demonstrate that cooling decreases the frequency of channel openings at all conductance levels, and at temperatures below 20°C shifts the prevalence of openings from higher to lower conductance states: all 240 pS openings disappear below 20°C; 200 pS openings are suppressed at 17°C; below 16°C 160 and 120 pS events disappear and only 80 and 40 pS states are seen. Temperature also affected the voltage-dependent kinetics of the channels. Application of a 6 sec, 80 mV voltage step across the junction (V _j80 ) caused a biexponential decay in junctional conductance. Decay was faster at lower temperatures, whereas the rate of recovery ofG _j after returning toV _j0 was slowed. Cooling reduced the fast decay time constant, increased both recovery time constants, and decreased the magnitude of GitG_j decay, thus leaving a 10–16% larger residual conductance (G _ss/G _init,±80 mVV _j ) at 18 than at 22°C. From these results we propose that embryonic chick cardiac gap junctions contain at least two classes of channels with different conductances and temperature sensitivities.     

Loss and recovery of the blood–nerve barrier in the rat sciatic nerve after crush injury are associated with expression of intercellular junctional proteins

The blood–nerve barrier in peripheral nerves is important for maintaining the environment for axons. Breakdown of the barrier by nerve injury causes various pathologies. We hypothesized that the breakdown and recovery of the blood–nerve barrier after injury are associated with the changes in the expression of intercellular junctional proteins. To test this hypothesis, we induced crush injuries in the rat sciatic nerve by ligation and analyzed spatiotemporal changes of claudin-1, claudin-5, occludin, VE-cadherin, and connexin43 by immunoconfocal microscopy and morphometry and compared them with changes in the permeability of the blood–nerve barrier by intravenous and local administration of Evans blue–albumin (EBA). On day 1 after removal of the ligature EBA leaked into the connective tissue in the endoneurium and then the leakage gradually decreased and disappeared on day 7. On day 1 claudin-1, claudin-5, occludin, VE-cadherin, and connexin43 had totally disappeared from the perineurium and endoneurium. Thereafter, claudin-1, claudin-5, occludin, and VE-cadherin recovered from day 2, whereas connexin43 was redetected on day 5. These results indicate that the breakdown and following recovery of the blood–nerve barrier are closely associated with changes in the expression of claudins, occludin, VE-cadherin, and connexin43 and that the recovery time course is similar but nonidentical.     

Role of the p38 MAP-Kinase Signaling Pathway for Cx32 and Claudin-1 in the Rat Liver

Liver regeneration and cholestasis are associated with adaptive changes in expression of gap and tight junctions through signal transduction. The roles of stress responsitive MAP-kinase, p38 MAP-kinase, in the signaling pathway for gap junction protein, Cx32, and tight junction protein, claudin-1, were examined in rat liver in vivoand in vitro, including regeneration following partial hepatectomy and cholestasis after common bile duct ligation. Changes in the expression and function of Cx32 and claudin-1 in hepatocytes in vivowere studied using the p38 MAP-kinase inhibitor SB203580. Following partial hepatectomy and common bile duct ligation, down-regulation of Cx32 protein was inhibited by SB203580 treatment. Up-regulation of claudin-1 protein was enhanced by SB203580 treatment after partial hepatectomy but not common bile duct ligation. However, no change of the Ki-67 labeling index (which is a marker for cell proliferation) in the livers treated with SB203580, was observed compared to that without SB203580 treatment. In primary cultures of rat hepatocytes, however, treatment with a p38 MAP-kinase activator, anisomycin, decreased Cx32 and claudin-1 protein levels. p38 MAP-kinase may be an important signaling pathway for regulation of gap and tight junctions in hepatocytes. Changes of gap and tight junctions during liver regeneration and cholestasis are shown to be in part controlled via the p38 MAP-kinase signaling pathway and are independent of cell growth.     

An ephaptic transmission model of CA3 pyramidal cells: an investigation into electric field effects

Extracellular electric fields existing throughout the living brain affect the neural coding and information processing via ephaptic transmission, independent of synapses. A two-compartment whole field effect model (WFEM) of pyramidal neurons embedded within a resistive array which simulates the extracellular medium i.e. ephapse is developed to study the effects of electric field on neuronal behaviors. We derive the two linearized filed effect models (LFEM-1 and LFEM-2) from WFEM at the stable resting state. Through matching these simplified models to the subthreshold membrane response in experiments of the resting pyramidal cells exposed to applied electric fields, we not only verify our proposed model’s validity but also found the key parameters which dominate subthreshold frequency response characteristic. Moreover, we find and give its underlying biophysical mechanism that the unsymmetrical properties of active ion channels results in the very different low-frequency response of somatic and dendritic compartments. Following, WFEM is used to investigate both direct-current (DC) and alternating-current field effect on the neural firing patterns by bifurcation analyses. We present that DC electric field could modulate neuronal excitability, with the positive field improving the excitability, the modest negative field suppressing the excitability, but interestingly, the larger negative field re-exciting the neuron back into spiking behavior. The neuron exposed to the sinusoidal electric field exhibits abundant firing patterns sensitive to the input frequency and intensity. In addition, the electrical properties of ephapse can modulate the efficacy of field effect. Our simulated results are qualitatively in line with the relevant experimental results and can explain some experimental phenomena. Furthermore, they are helpful to provide the predictions which can be tested in future experiments.