Intercellular calcium waves in glial cells with bistable dynamics

A two-dimensional model is proposed for intercellular calcium (Ca(2 +)) waves with Ca(2 +)-induced IP(3) regeneration and the diffusion of IP(3) through gap junctions. Many experimental observations in glial cells, i.e. responding to local mechanical stimulation, glutamate application, mechanical stimulation followed by ACh application, and glutamate followed by mechanical stimulation, are reproduced and classified by the model. We show that a glial cell model with bistable dynamics, i.e. a Ca(2 +) oscillation state coexisting with a fixed point, can cause a prolonged plateau of Ca(2 +) signals in the cells nearby the stimulated cell when the cell network responds to the local mechanical stimulation.     

Intercellular calcium signaling via gap junctions in glioma cells

Calcium signaling in C6 glioma cells in culture was examined with digital fluorescence video microscopy. C6 cells express low levels of the gap junction protein connexin43 and have correspondingly weak gap junctional communication as evidenced by dye coupling (Naus, C. C. G., J. F. Bechberger, S. Caveney, and J. X. Wilson. 1991. Neurosci. Lett. 126:33-36). Transfection of C6 cells with the cDNA encoding connexin43 resulted in clones with increased expression of connexin43 mRNA and protein and increased dye coupling, as well as markedly reduced rates of proliferation (Zhu, D., S. Caveney, G. M. Kidder, and C. C. Naus. 1991. Proc. Natl. Acad. Sci. USA. 88:1883-1887; Naus, C. C. G., D. Zhu, S. Todd, and G. M. Kidder. 1992. Cell Mol. Neurobiol. 12:163-175). Mechanical stimulation of a single cell in a culture of non-transfected C6 cells induced a wave of increased intracellular calcium concentration ([Ca2+]i) that showed little or no communication to adjacent cells. By contrast, mechanical stimulation of a single cell in cultures of C6 clones expressing transfected connexin43 cDNA induced a Ca2+ wave that was communicated to multiple surrounding cells, and the extent of communication was proportional to the level of expression of the connexin43 cDNA. These results provide direct evidence that intercellular Ca2+ signaling occurs via gap junctions. Ca2+ signaling through gap junctions may provide a means for the coordinated regulation of cellular function, including cell growth and differentiation.     

Metabotropic glutamate receptor 5 and calcium signaling in retinal amacrine cells

To begin to understand the modulatory role of glutamate in the inner retina, we examined the mechanisms underlying metabotropic glutamate receptor 5 (mGluR5)-dependent Ca(2+) elevations in cultured GABAergic amacrine cells. A partial sequence of chicken retinal mGluR5 encompassing intracellular loops 2 and 3 suggests that it can couple to both G(q) and G(s). Selective activation of mGluR5 stimulated Ca(2+) elevations that varied in waveform from cell to cell. Experiments using high external K(+) revealed that the mGluR5-dependent Ca(2+) elevations are distinctive in amplitude and time course from those engendered by depolarization. Experiments with a Ca(2+) -free external solution demonstrated that the variability in the time course of mGluR5-dependent Ca(2+) elevations is largely due to the influx of extracellular Ca(2+). The sensitivity of the initial phase of the Ca(2+) elevation to thapsigargin indicates that this phase of the response is due to the release of Ca(2+) from the endoplasmic reticulum. Pharmacological evidence indicates that mGluR5-mediated Ca(2+) elevations are dependent upon the activation of phospholipase C. We rule out a role for L-type Ca(2+) channels and cAMP-gated channels as pathways for Ca(2+) entry, but provide evidence of transient receptor potential (TRP) channel-like immunoreactivity, suggesting that Ca(2+) influx may occur through TRP channels. These results indicate that GABAergic amacrine cells express an avian version of mGluR5 that is linked to phospholipase C-dependent Ca(2+) release and Ca(2+) influx, possibly through TRP channels.     

Switching from simple to complex oscillations in calcium signaling

We present a new model for calcium oscillations based on experiments in hepatocytes. The model considers feedback inhibition on the initial agonist receptor complex by calcium and activated phospholipase C, as well as receptor type-dependent self-enhanced behavior of the activated G(alpha) subunit. It is able to show simple periodic oscillations and periodic bursting, and it is the first model to display chaotic bursting in response to agonist stimulations. Moreover, our model offers a possible explanation for the differences in dynamic behavior observed in response to different agonists in hepatocytes.     

Effects of calcium buffers and calbindin-D28k upon histamine-induced calcium oscillations and calcium waves in HeLa cells

The effects of the artificial Ca(2+) buffers EGTA and BAPTA upon histamine-induced Ca(2+) oscillations and calcium waves were studied in HeLa cells. These events were also examined in HeLa cell lines transfected with the intracellular calcium-binding protein calbindin-D28k (CaBP; HeLa-CaBP) or the pCINeo vector alone (HeLa-pCINeo). High concentrations of the Ca(2+) indicators fluo-3 and fura-2 significantly influenced the oscillatory pattern of intracellular Ca(2+) in HeLa-pCINeo cells exposed to 1 microM histamine. Loading cells with low concentrations of the cell-permeant esters of the artificial Ca(2+)-buffers EGTA or BAPTA, resulted in fewer cells with a distinct "baseline" oscillatory pattern, and loading with higher concentrations of BAPTA almost completely abolished them. In HeLa-CaBP cells, stimulation with 1 microM histamine resulted in individual Ca(2+) spikes that had a flattened profile when compared to control cells; peak [Ca(2+)](i) was lowered, the rate of increase in [Ca(2+)](i) was slower and transients were prolonged. When compared to HeLa-pCINeo cells, loading with EGTA or BAPTA, or transfection of CaBP, significantly reduced the propagation velocity (by up to 60%) of Ca(2+) waves induced by exposure to 100 microM histamine. We conclude that intracellular Ca(2+) buffering exerts a significant influence on global Ca(2+) responses in HeLa cells and the propagation of Ca(2+) waves that underlie them. The relative effectiveness of different Ca(2+) buffers, including CaBP, appears to be particularly dependent upon the rapidity of their binding kinetics, with BAPTA being the most effective.     

Neuronal and glial calcium signaling in Alzheimer's disease

Cognitive impairment and emotional disturbances in Alzheimer's disease (AD) result from the degeneration of synapses and death of neurons in the limbic system and associated regions of the cerebral cortex. An alteration in the proteolytic processing of the amyloid precursor protein (APP) results in increased production and accumulation of amyloid beta-peptide (Abeta) in the brain. Abeta has been shown to cause synaptic dysfunction and can render neurons vulnerable to excitotoxicity and apoptosis by a mechanism involving disruption of cellular calcium homeostasis. By inducing membrane lipid peroxidation and generation of the aldehyde 4-hydroxynonenal, Abeta impairs the function of membrane ion-motive ATPases and glucose and glutamate transporters, and can enhance calcium influx through voltage-dependent and ligand-gated calcium channels. Reduced levels of a secreted form of APP which normally regulates synaptic plasticity and cell survival may also promote disruption of synaptic calcium homeostasis in AD. Some cases of inherited AD are caused by mutations in presenilins 1 and 2 which perturb endoplasmic reticulum (ER) calcium homeostasis such that greater amounts of calcium are released upon stimulation, possibly as the result of alterations in IP(3) and ryanodine receptor channels, Ca(2+)-ATPases and the ER stress protein Herp. Abnormalities in calcium regulation in astrocytes, oligodendrocytes, and microglia have also been documented in studies of experimental models of AD, suggesting contributions of these alterations to neuronal dysfunction and cell death in AD. Collectively, the available data show that perturbed cellular calcium homeostasis plays a prominent role in the pathogenesis of AD, suggesting potential benefits of preventative and therapeutic strategies that stabilize cellular calcium homeostasis.     

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.     

Orientation response of arterial smooth muscle cells to mechanical stimulation

Arterial smooth muscle cells from rabbit aortic media in primary culture and subculture were grown on hydrophilized and collagen-coated silicone membranes which were then subjected to cyclic and directional stretches and relaxations at a frequency of 60 times/min. The membranes were stretched with various amplitudes ranging from 2% to 20%. Smooth muscle cells on unstretched membranes in the same incubation chamber served as controls. In long-term experiments the stretching and relaxing of the membranes was continued for several days. While the smooth muscle cells grown on unstretched membranes remained in random orientation in all experiments, the cells which underwent mechanical stimulation showed a high degree of orientation. The angle of cell orientation varied in direct relation to the stretching amplitude and became steeper in correlation to the intensity of the mechanical stimulus. The angle of cell orientation was reversible, as preoriented cells changed their orientation when another stretching amplitude was applied. To study the role of cytoskeleton in the process of cell orientation, we examined the behaviour of the intracellular actin filament system. In short-term experiments the smooth muscle cells were exposed for 3 to 12 h to cyclic and directional stretches and relaxations with an amplitude of 10%. We observed a rearrangement of the intracellular actin filament system prior to the orientation of the whole cell bodies. The present study provides evidence that stretching the artery wall by blood pulsation may result in an orientation response of the intracellular actin cytoskeleton and in the orientation of the smooth muscle cells within the media of artery walls.     

Spontaneous and glutamate evoked calcium oscillations in rat hippocampal astrocytes

In this work, we researched spontaneous and glutamate evoked Ca²⁺ oscillations in rat hippocampal astrocytes using confocal laser scanning microscopy and bulk-loading of the Ca²⁺-sensitive dye Oregon Green Bapta 1-AM.     

The latest waves in calcium signaling

Ca2+ is a universal second messenger that is a key component of myriad processes in all cell types. Perturbations in normal intracellular Ca2+ concentrations underlie many common pathological conditions, ranging from cardiac hypertrophy to ischemic death of neurons. A recent meeting addressed the contributions of Ca2+ and Ca2+ binding proteins to health and disease. Insights gleaned from mechanistic studies offered the potential for new therapeutic approaches to combat a variety of diseases resulting from alterations in Ca2+ homeostasis.     

Intercellular communication upon mechanical stimulation of CPAE- endothelial cells is mediated by nucleotides

Intercellular Ca(2+)-signaling, after mechanical stimulation of calf pulmonary artery endothelial cells (CPAE), was investigated with fluorescence video imaging. Mechanical stimulation evoked an intracellular Ca(2+)-response in the mechanically stimulated (MS) cell, proceeding to the neighboring (NB) cells as a Ca(2+)-wave. The intercellular propagation of the Ca(2+)-wave was unaffected by the gap junction blockers halothane or heptanol. Therefore the intercellular communication (IC) pathway of the Ca(2+)-wave in CPAE cells does not depend on gap junctional communication but is most likely mediated by release of an extracellular mediator. Continuous unilateral flow experiments confirmed the presence of a diffusible mediator: the Ca(2+)-rise in upstream NB cells is significantly lower than in control experiments. After desensitization of purinergic receptors by pretreatment of CPAE cells with ATP (100mM), UTP (100 microM), 2MeSATP (100microM) or ADPbS (100 microM), the propagation of the intercellular Ca(2+)-wave upon mechanical stimulation was significantly inhibited. Also suramin (200 and 400 microM), a non-specific purinergic receptor blocker, reduced the IC. Application of the nucleotidase apyrase VI (10U/ml), which has a high ATPase/ADPase ratio, enhanced Ca(2+)-signaling and IC. In contrast, apyrase VII (10U/ml), which has a high ADPase/ATPase ratio, significantly depressed the propagation of the intercellular Ca(2+)-wave upon mechanical stimulation. Our experiments therefore demonstrate that the IC, evoked by a mechanical stimulus of CPAE cells, is mediated via release of nucleotides in the extracellular space. The data indicate that the diffusible messenger, responsible for the propagation of a Ca(2+)-wave, is mainly ADP or a combination of ADP/ATP.     

Intercellular calcium waves in HeLa cells expressing GFP-labeled connexin 43, 32, or 26

This study was undertaken to obtain direct evidence for the involvement of gap junctions in the propagation of intercellular Ca(2+) waves. Gap junction-deficient HeLa cells were transfected with plasmids encoding for green fluorescent protein (GFP) fused to the cytoplasmic carboxyl termini of connexin 43 (Cx43), 32 (Cx32), or 26 (Cx26). The subsequently expressed GFP-labeled gap junctions rendered the cells dye- and electrically coupled and were detected at the plasma membranes at points of contact between adjacent cells. To correlate the distribution of gap junctions with the changes in [Ca(2+)](i) associated with Ca(2+) waves and the distribution of the endoplasmic reticulum (ER), cells were loaded with fluorescent Ca(2+)-sensitive (fluo-3 and fura-2) and ER membrane (ER-Tracker) dyes. Digital high-speed microscopy was used to collect a series of image slices from which the three-dimensional distribution of the gap junctions and ER were reconstructed. Subsequently, intercellular Ca(2+) waves were induced in these cells by mechanical stimulation with or without extracellular apyrase, an ATP-degrading enzyme. In untransfected HeLa cells and in the absence of apyrase, cell-to-cell propagating [Ca(2+)](i) changes were characterized by initiating Ca(2+) puffs associated with the perinuclear ER. By contrast, in Cx-GFP-transfected cells and in the presence of apyrase, [Ca(2+)](i) changes were propagated without initiating perinuclear Ca(2+) puffs and were communicated between cells at the sites of the Cx-GFP gap junctions. The efficiency of Cx expression determined the extent of Ca(2+) wave propagation. These results demonstrate that intercellular Ca(2+) waves may be propagated simultaneously via an extracellular pathway and an intracellular pathway through gap junctions and that one form of communication may mask the other.     

Ultrasound-induced calcium oscillations and waves in Chinese hamster ovary cells in the presence of microbubbles

This study investigated the effects of ultrasound on the intracellular [Ca(2+)] of Chinese hamster ovary cells in the presence of albumin-encapsulated Optison microbubbles. Cells were exposed to 1 MHz ultrasound (tone burst of 0.2 s duration, 0.45 MPa peak pressure) while immersed in solution of 0.9 mM Ca(2+). Calcium imaging of the cells was performed using digital video fluorescence microscopy and Ca(2+)-indicator dye fura-2AM. Experimental evidence indicated that ultrasound caused a direct microbubble-cell interaction resulting in the breaking and eventual dissolution of the microbubble and concomitant permeabilization of the cells to Ca(2+). These cells exhibited a large influx of Ca(2+) over 3-4 s and did not return to their equilibrium levels. Subsequently, some cells exhibited one or more Ca(2+) oscillations with the onset of oscillations delayed by 10-80 s after the ultrasound pulse. A variety of oscillations were observed including decaying oscillations returning to the baseline value over 35-100 s, oscillations superimposed on a more gradual recovery over 150-200 s, and oscillations continued with increased amplitude caused by a second ultrasound tone burst. The delays in onset appeared to result from calcium waves that propagated across the cells after the application of the ultrasound pulse.     

Cytoplasmic compartmental response to local mechanical stimulation of internal tissue cells

A convenient experimental system was established to test how cells derived from higher-plant internal tissues respond to mechanical stimulation. Short-term culture of tobacco ovules in vitro led to the generation of bar-shaped cells from the parenchyma tissue of the ovule funicle. These cells are still connected to the mother tissue and are almost undifferentiated. The cells are translucent, and one end protrudes from the funicle, making them easy to manipulate and observe. Mechanical stimulation tests performed on these cells indicated that the cells are less sensitive to mechanical stimulation than epidermal hair cells but still possess the ability to respond to stimulation. Interestingly, the cells showed a cytoplasmic compartmental response to the stimulation. The nucleus, some plastids, and mitochondria were organized into a responsive unit that moved in unison to the stimulated sites, whereas most of the other organelles were not notably influenced by the stimulation. This suggests that the cytoplasm is highly organized and functionally divided in response to environmental stimulation.     

Intercellular calcium waves in primary cultured rat mesenteric smooth muscle cells are mediated by connexin43

Intercellular Ca(2+) wave propagation between vascular smooth muscle cells (SMCs) is associated with the propagation of contraction along the vessel. Here, we characterize the involvement of gap junctions (GJs) in Ca(2+) wave propagation between SMCs at the cellular level. Gap junctional communication was assessed by the propagation of intercellular Ca(2+) waves and the transfer of Lucifer Yellow in A7r5 cells, primary rat mesenteric SMCs (pSMCs), and 6B5N cells, a clone of A7r5 cells expressing higher connexin43 (Cx43) to Cx40 ratio. Mechanical stimulation induced an intracellular Ca(2+) wave in pSMC and 6B5N cells that propagated to neighboring cells, whereas Ca(2+) waves in A7r5 cells failed to progress to neighboring cells. We demonstrate that Cx43 forms the functional GJs that are involved in mediating intercellular Ca(2+) waves and that co-expression of Cx40 with Cx43, depending on their expression ratio, may interfere with Cx43 GJ formation, thus altering junctional communication.     

Intercellular calcium waves in primary cultured rat mesenteric smooth muscle cells are mediated by Connexin43

Abstract

Intercellular Ca^{2 +} wave propagation between vascular smooth muscle cells (SMCs) is associated with the propagation of contraction along the vessel. Here, we characterize the involvement of gap junctions (GJs) in Ca^{2 +} wave propagation between SMCs at the cellular level. Gap junctional communication was assessed by the propagation of intercellular Ca^{2 +} waves and the transfer of Lucifer Yellow in A7r5 cells, primary rat mesenteric SMCs (pSMCs), and 6B5N cells, a clone of A7r5 cells expressing higher connexin43 (Cx43) to Cx40 ratio. Mechanical stimulation induced an intracellular Ca^{2 +} wave in pSMC and 6B5N cells that propagated to neighboring cells, whereas Ca^{2 +} waves in A7r5 cells failed to progress to neighboring cells. We demonstrate that Cx43 forms the functional GJs that are involved in mediating intercellular Ca^{2 +} waves and that co-expression of Cx40 with Cx43, depending on their expression ratio, may interfere with Cx43 GJ formation, thus altering junctional communication.     

Alpha 1-adrenergic modulation of metabotropic glutamate receptor-induced calcium oscillations and glutamate release in astrocytes

Astrocytic responses to activation of metabotropic glutamate receptors group I (mGluRs I) and alpha(1)-adrenoreceptors in cultured cells have been assessed using spectral analyzes and calcium imaging. Concentration-dependent changes were observed after stimulation with the mGluR I agonist (S)-3,5-dihydroxyphenylglycine (DHPG). These responses changed from a regular low frequency signal with sharp peaks at 1 microm to a pronounced stage of irregularity at 10 microm. After stimulation with 100 microm the signal was again homogenous in shape and regularity but occurred at a higher frequency. In contrast, the spectral properties after stimulation with the alpha(1)-adrenoreceptor agonist phenylephrine, exhibited considerable variation for all investigated concentrations. DHPG-induced increases in [Ca(2+)](i) were also associated with astroglial glutamate release, whereas no release was observed after noradrenergic stimulation. Both DHPG-mediated calcium signaling and glutamate release were inhibited by preincubation with 10 or 100 microm phenylephrine. Collectively, the present investigation provides new information about the spatial-temporal characteristics of astroglial intracellular calcium responses and demonstrates distinct differences between noradrenergic and glutamatergic receptors regarding intracellular calcium signaling and coupling to glutamate release. The noradrenergic modulation of DHPG-induced responses indicates that intracellular astroglial processes can be regulated in a bi-directional feedback loop between closely connected astrocytes and neurons in the central nervous system.     

Metabolic signaling between neurons and glial cells: a short review.

There is convincing evidence that astrocytes transform blood-born glucose to lactate, alpha-Keto-glutarate and alanine and supply the neurons. There is a tight regulation of this metabolic coupling by means of chemical signals released by functioning neurons. Previous, pioneer, studies have explored several signals-candidates the major being K(+), Ca(++) and several neuromodulators. However, recent results of numerous studies identify glutamate as the major signal that traffics between excited neurons and astrocytes. The excited neurons also produce and release NH(4)(+) in the extracellular space. Both glutamate and ammonium are taken up preferentially by astrocytes and form glutamine. Ammonia fixation by glutamine synthase controls the amount of lactate, glutamine and alanine produced and released by Muller cells in the extracellular space and then taken up by neurons. Thus, there is a tight coupling between function and metabolism in the central neurons system.