Inhibition of MAP kinase activity moderates changes in expression and function of Cx32 but not claudin-1 during DNA synthesis in primary cultures of rat hepatocytes

The signal transduction pathways and activation of the MAP kinase or PI3 kinase signaling cascade regulate a variety of cellular processes, including proliferation and differentiation in hepatocytes. To elucidate the mechanisms of signal transmission required for the regulation of gap and tight junctions during DNA synthesis in rat hepatocytes, we determined changes of expression and function of gap and tight junctions of cells grown in primary culture, using inhibitors of signaling pathways for MAP kinase (PD98059) and PI3 kinase (LY294002). During the stimulation of DNA synthesis induced by epidermal growth factor (EGF), immunoreactivity and mRNAs of gap junction protein Cx32 and of tight junction protein claudin-1 markedly decreased with reduction of gap junctional intercellular communication (GJIC) and the fence function of tight junctions. In Western blots, whole-cell lysate of claudin-1 protein decreased and phosphorylated Cx32 protein in the insoluble fraction of Triton X-100 increased during the stimulation of DNA synthesis. During reinhibition of DNA synthesis, the changes of Cx32 and claudin-1 returned to control levels, as did both functions. In treatment with the inhibitors before DNA synthesis, PD98059 inhibited the changes of expression and function of Cx32, but not claudin-1, without inhibition of cell growth, whereas LY294002 completely inhibited cell growth. These findings indicate that the PI3 kinase pathway rather than the MAP kinase pathway plays an important role for EGF-induced proliferation of rat hepatocytes, and that changes of Cx32 in hepatocytes during the stimulation of DNA synthesis may be in part controlled through MAP kinase. Furthermore, Cx32, but not claudin-1, protein may be a target of activated MAP kinase in hepatocytes.     

The gap effect and express saccades in the auditory modality

Latencies of eye movements to peripheral targets are reduced when there is a short delay (typically 200 ms) between the offset of a central visual fixation point and the target onset. This has been termed the gap effect. In addition, some subjects, usually with practice, exhibit a separate population of very short latency saccades, called express saccades. Both these phenomena have been attributed to disengagement of visual attention when the fixation point is extinguished. A competing theory of the gap effect attributes it to disengagement of oculomotor fixation during the temporal gap. It is known that auditory targets are effective in eliciting saccadic eye movements, and also that covert attention operates in the auditory modality. If the gap effect and express saccades are due to disengagement of spatial attention, both should persist in the auditory modality. However, fixation of gaze is largely under visual control. If the gap effect results from disengagement of fixation, then at least a reduced effect should be seen in the auditory modality. Human subjects performed the gap task and a control task in the dark, using auditory fixation points and saccadic targets, on five successive days. Despite this practice, express saccades were not observed. There was a reliable gap effect, but the reduction in saccadic latency was only 17 ms, compared with 32 ms for the same subjects in the visual modality. This suggests that about half the gap effect is due to disengagement of visual fixation. The remainder was not due to non-specific warning effects and could be attributed to offset of the auditory fixation stimulus. Received: 1 March 1996 / Accepted: 11 July 1997     

Effects of cGMP-dependent phosphorylation on rat and human connexin43 gap junction channels

The effects of 8-bromoguanosine 3:5-cyclic monophosphate (8Br-cGMP), a membrane-permeant activator of protein kinase G (PKG), were studied on rat and human connexin43 (Cx43), the most abundant gap junction protein in mammalian heart, which were exogenously expressed in SKHep1 cells. Under dual whole-cell voltage-clamp conditions, 8Br-cGMP decreased gap junctional conductance (g_j) in rat Cx43-transfected cells by 24.0±3.7% (mean±SEM, n=5), whereas g_j was not affected in human Cx43-transfected cells by the same treatment. The relaxation of g_j in response to steps in transjunctional voltage observed in rat Cx43 transfectants was best fitted with three exponentials. Time constants and amplitudes of the decay phases changed in the presence of 8Br-cGMP. Single rat and human Cx43 gap junction channels were resolved in the presence of halothane. Under control conditions, three single-channel conductance states (_j) of about 20, 40–45 and 70 pS were detected, the events of the intermediate size being most frequently observed. In the presence of 8Br-cGMP, the _j distribution shifted to the lower size in rat Cx43 but not in human Cx43 transfectants. Immunoblot analyses of Cx43 in subconfluent cultures of rat Cx43 or human Cx43 transfectants showed that 8Br-cGMP did not induce changes in the electrophoretic mobility of Cx43 in either species. However, the basal incorporation of [³²P] into rat Cx43 was significantly altered by 8Br-cGMP, whereas this incorporation of [³²P] into human Cx43 was not affected. We conclude that 8Br-cGMP modulates phosphorylation of rat Cx43 in SKHep1 cells, but not of human Cx43. This cGMP-dependent phosphorylation of rat Cx43 is associated with a decreased g_j, which results from both an increase in the relative frequency of the lowest conductance state and a change in the kinetics of these channels.     

Transmission electron microscopic demonstration of vimentin in rat osteoblast and osteocyte cell bodies and processes using the immunogold technique

Background: The immunogold labeling technique and transmission electron microscopy were used to demonstrate the expression and position of the intermediate filament vimentin in rat osteoblast and osteocyte cell bodies and cell processes. Conventional light and transmission electron microscopic studies of bone cells demonstrated adjacent cell linkage to be mediated by osteoblast and osteocyte processes present within the canalicular system traversing the bone matrix. The cell processes were filled with densely packed filaments, many of which have been shown previously to be actin microfilaments. The appearance, however, of 10 nm diameter filaments in some cell processes and the fact that the intermediate filament vimentin has been defined in many cells of mesenchymal origin raised the possibility that some of these filaments might be vimentin. The ultrastructural colloidal gold immunochemical technique allowed for demonstration in situ of the expression of vimentin filaments plus accurate definition of their position. Methods: The studies were performed in newborn rat femoral and tibial diaphyseal cortical bone and in 1-week-old repair bone from 2.4 mm diameter defects made through the lateral cortex in 6-week-old rat femurs and tibias. The bone tissues for the immunochemical study were fixed in 1% glutaraldehyde, 4% paraformaldehyde, and 0.1 M phosphate buffer (pH 7.4) for 2 days. Decalcification was performed in 6% EDTA for 2–3 days. Infiltration involved use of Lowicryl resin K4M, and the embedding and curing processes were performed in a cryostat with temperatures ?30°C. An antivimentin monoclonal antibody was used for labeling using the postembedding technique. Effective antibody dilutions ranged from 1:10 to 1:200, with the dilutions of 1:25 and 1:100 showing the best combination of filament labeling with the least matrix background. The grids were exposed to 10 nanometer gold colloid conjugated goat anti-mouse IgM for demonstration of binding. Results: Vimentin immunolabeling was defined clearly in relation to filaments within the osteoblast and osteocyte cell body cytoplasm, throughout the entire length of the osteoblast and osteocyte cell processes, and in close relationship to the intercellular gap junctions which were present within the cell processes both close to the cell bodies and within the canaliculi well away from them. Conclusions: Immunogold labeling demonstrates the presence of the intermediate filament vimentin in osteoblast and osteocyte cell bodies and processes of rat bone. Vimentin distribution is not concentrated to specific areas, is present throughout the extent of the bodies and processes, and is seen immediately adjacent to gap junctions. © 1995 Wiley-Liss, Inc.     

Stochastic and Coherence Resonance in an In Silico Neural Model

We show that it is possible for chaotic systems to display the main features of stochastic and coherence resonance. In particular, a model of coupled nonlinear oscillators which emulates the transmembrane voltage activities in CA3 neurons, operating in a chaotic regime and in the presence of noise, can exhibit coherence resonance and stochastic resonance. Certain firing frequencies become more "rhythmic" for some optimal values of noise intensity. The effect of noise in different coupling pathways is investigated. We found that the effect of coherence resonance and stochastic resonance are more prominent if noise is presented in either electric field or gap junction coupling pathways. Frequency sensitivity of the model is investigated as a preliminary step in illustrating the principles of possible epileptic seizure control strategies using "chaos control" concepts. Significant effects of stochastic resonance are observed in the 4-8 Hz range. Weaker effects can be found in the 1-4 Hz and 8-10 Hz ranges whereas 0.5 Hz does not exhibit any resonance phenomenon. Our results suggest that: (a) Stochastic resonance could enhance the intrinsic 4-8 Hz rhythms in CA3 neurons more prominently via field coupling pathways. It could also help explain why some reported seizure control strategies using pulse-trains would only be effective at 0.5 Hz. (b) Stochastic resonance-like behavior can occur in the gamma range only if noise is presented via chemical synaptic pathways.     

三磷酸腺苷对人横纹肌肉瘤细胞系诱导分化作用的研究

为了探讨三磷酸腺苷（ＡＴＰ０对人横纹肌肉瘤细胞增殖和分化的影响，用ＡＴＰ作用于人横纹肌肉瘤细胞亚系（ＲＤＬ６）细胞，观察到ＡＴＰ可抑制ＲＤＬ６细胞的增殖，使其生长速度明显减慢，作用第５ｄ时增殖抑制率为８１％，流式光度术检测；观察到ＡＴＰ ＲＤＬ６细胞Ｓ期的细胞数明显增多，说明细胞停滞在Ｓ期，用罗氏黄荧光染料传法实，ＡＴ家恢复ＲＤＬ６细胞间隙加接通讯功能的作用。用 光细胞化学方法观察到经ＡＴＰ处理后     

Linear gap and tight junctional assemblies between capillary endothelial cells in the eel rete mirabile

Background: Interendothelial tight junctions and gap junctions have been described in large blood vessels and in cultures of endothelium derived from large blood vessels. Transfer of microinjected smallmolecular weight tracers between adjacent endothelial cells also has been demonstrated indicating the presence of gap junctional interendothelial communication. Similar transfer of tracers is evident between microvessel endothelial cells in culture and in microvessels in situ. However, gap junctions have not been detectable by electron microscopy of intact capillary systems. This may be due to limited sampling available in diffuse capillary systems and a small area of overlap between adjacent endothelial membranes. Methods: Thin slices of the parallel, tightly packed capillary bed of the eel rete mirabile were cryofixed and prepared for conventional TEM by freeze substitution. Other samples were freeze-fractured and replicated for examination of endothelial junctional components. Results: A novel tight-gap junctional complex between rete capillary endothelial cells is described. In freeze-fracture replicas of the membrane P face, rows of gap junction subunits are flanked on either side by linear depressions representing grooves previously occupied by tight junctional strands that partition to the E face. In thin sections, the junctions appear in profile as short lengths of closely apposed membranes characteristic of gap junctions. Conclusions: The tight junctional components imply a barrier to paracellular transport across the capillary wall between the endothelial cells. The gap junctional component may provide a mechanism for communication between endothelial cells along the length of the vessel wall. © 1995 Wiley-Liss, Inc.     

The effect of transcranial magnetic stimulation on the latencies of vertical saccades

In this study, we investigated the effect of transcranial magnetic stimulation (TMS) over the right posterior parietal cortex (PPC) on the latency of two different types of visually-guided vertical saccades: reflexive saccades triggered by the sudden onset of a target, and saccades towards target locations known in advance. For this reason, we used two oculomotor tasks: a gap and a delay task, respectively. Nine normal subjects performed vertical saccades at ±7.5 and ±15°. TMS was applied at 80 and 100 ms after target onset in the gap task, and after fixation offset in the delay task. Without TMS, we confirmed a latency asymmetry in the gap task favouring upward saccades at the lower eccentricity (7.5°), and a latency symmetry in the delay task. TMS increased the latencies of all saccades in the delay task, when delivered at 100 ms. This effect was mostly pronounced for downward saccades at 7.5°. As a result, saccade latencies showed an asymmetry in this condition, similar to the one observed in the gap task without TMS. The gap task with TMS resulted in a variable latency distribution and no significant overall effect on saccade latency. Our results indicate that the right PPC is involved in the initiation of vertical saccades in the delay task, and that this involvement appears to be enhanced for downward saccades. A conclusion for the involvement of this area in the gap task could not be drawn from this study.     

Coordinated Movement of Bile Canalicular Networks Reconstructed by Rat Small Hepatocytes

Hepatocytes in vivo have a potential for liver regeneration, but it has been very difficult to reconstruct hepatic organoids in vitro. Recent studies have shown that small hepatocytes (SHs) can reconstruct hepatic organoids including functional bile canaliculi (BC). In the present study we analyzed the movement of BC formed in the hepatic organoids, focusing on the coordination of contraction and dilation among cells and the mechanism producing the coordination. Hepatic cells, including SHs, were isolated from an adult rat liver and cultured. Time-lapse images of BC movements were taken and analyzed in cells treated with or without cytochalasin B (CB). Time-lapse images revealed that all BC, regardless of region contracted in a coordinated manner. Actin filaments were observed along the BC even after the BC networks treated with CB dilated markedly. Microinjection of dye was also carried out to investigate the flow thorough BC. Secreted fluorescein from the injected cell flowed along BC, and gap junctional protein connexin 32 was expressed along BC networks, suggesting cell-to-cell communication. Thus, groups of hepatocytes in the hepatic organoids act in a coordinated manner through intercellular communication.     

Effect of resistive discontinuities on waveshape and velocity in a single cardiac fibre

A computer model of a one-dimensional cardiac fibre of resistively coupled cells is used to investigate the influence of the junction resistance on the nature of conduction. The results of the simulations are presented and indicate that the effect of the junction on both intracellular and extracellular waveshape and on the velocity of propagation depends on the size and frequency of the coupling resistance and the kinetics of the active membrane. Significant changes in these factors are not observed without the generation of prepotentials in the action potential upstroke. The absence of this ‘signature’ in microelectrode recordings of activity in ventricular muscle suggests that under normal conditions cardiac tissue behaves as a functional syncytium.