Propagation Of Intercellular Ca2 Research Articles

Ca2+ Dynamics of Gap Junction Coupled and Uncoupled Deiters' Cells in the Organ of Corti in Hearing BALB/c Mice.

ATP, as a paracrine signalling molecule, induces intracellular Ca2+ elevation via the activation of purinergic receptors on the surface of glia-like cochlear supporting cells. These cells, including the Deiters' cells (DCs), are also coupled by gap junctions that allow the propagation of intercellular Ca2+ waves via diffusion of Ca2+ mobilising second messenger IP3 between neighbouring cells. We have compared the ATP-evoked Ca2+ transients and the effect of two different gap junction (GJ) blockers (octanol and carbenoxolone, CBX) on the Ca2+ transients in DCs located in the apical and middle turns of the hemicochlea preparation of BALB/c mice (P14-19). Octanol had no effect on Ca2+ signalling, while CBX inhibited the ATP response, more prominently in the middle turn. Based on astrocyte models and using our experimental results, we successfully simulated the Ca2+ dynamics in DCs in different cochlear regions. The mathematical model reliably described the Ca2+ transients in the DCs and suggested that the tonotopical differences could originate from differences in purinoceptor and Ca2+ pump expressions and in IP3-Ca2+ release mechanisms. The cochlear turn-dependent effect of CBX might be the result of the differing connexin isoform composition of GJs along the tonotopic axis. The contribution of IP3-mediated Ca2+ signalling inhibition by CBX cannot be excluded.

Smooth muscle gap-junctions allow propagation of intercellular Ca2+ waves and vasoconstriction due to Ca2+ based action potentials in rat mesenteric resistance arteries

The role of vascular gap junctions in the conduction of intercellular Ca2+ and vasoconstriction along small resistance arteries is not entirely understood. Some depolarizing agents trigger conducted vasoconstriction while others only evoke a local depolarization. Here we use a novel technique to investigate the temporal and spatial relationship between intercellular Ca2+ signals generated by smooth muscle action potentials (APs) and vasoconstriction in mesenteric resistance arteries (MA). Pulses of exogenous KCl to depolarize the downstream end (T1) of a 3 mm long artery increased intracellular Ca2+ associated with vasoconstriction. The spatial spread and amplitude of both depended on the duration of the pulse, with only a restricted non-conducting vasoconstriction to a 1 s pulse. While blocking smooth muscle cell (SMC) K+ channels with TEA and activating L-type voltage-gated Ca2+ channels (VGCCs) with BayK 8644 spread was dramatically facilitated, so the 1 s pulse evoked intercellular Ca2+ waves and vasoconstriction that spread along an entire artery segment 3000 μm long. Ca2+ waves spread as nifedipine-sensitive Ca2+ spikes due to SMC action potentials, and evoked vasoconstriction. Both intercellular Ca2+ and vasoconstriction spread at circa 3 mm s−1 and were independent of the endothelium. The spread but not the generation of Ca2+ spikes was reversibly blocked by the gap junction inhibitor 18β-GA. Thus, smooth muscle gap junctions enable depolarization to spread along resistance arteries, and once regenerative Ca2+-based APs occur, spread along the entire length of an artery followed by widespread vasoconstriction.

Dedifferentiation, Proliferation, and Redifferentiation of Adult Mammalian Cardiomyocytes After Ischemic Injury.

Adult mammalian hearts have a limited ability to generate new cardiomyocytes. Proliferation of existing adult cardiomyocytes (ACMs) is a potential source of new cardiomyocytes. Understanding the fundamental biology of ACM proliferation could be of great clinical significance for treating myocardial infarction (MI). We aim to understand the process and regulation of ACM proliferation and its role in new cardiomyocyte formation of post-MI mouse hearts. β-Actin-green fluorescent protein transgenic mice and fate-mapping Myh6-MerCreMer-tdTomato/lacZ mice were used to trace the fate of ACMs. In a coculture system with neonatal rat ventricular myocytes, ACM proliferation was documented with clear evidence of cytokinesis observed with time-lapse imaging. Cardiomyocyte proliferation in the adult mouse post-MI heart was detected by cell cycle markers and 5-ethynyl-2-deoxyuridine incorporation analysis. Echocardiography was used to measure cardiac function, and histology was performed to determine infarction size. In vitro, mononucleated and bi/multinucleated ACMs were able to proliferate at a similar rate (7.0%) in the coculture. Dedifferentiation proceeded ACM proliferation, which was followed by redifferentiation. Redifferentiation was essential to endow the daughter cells with cardiomyocyte contractile function. Intercellular propagation of Ca2+ from contracting neonatal rat ventricular myocytes into ACM daughter cells was required to activate the Ca2+-dependent calcineurin-nuclear factor of activated T-cell signaling pathway to induce ACM redifferentiation. The properties of neonatal rat ventricular myocyte Ca2+ transients influenced the rate of ACM redifferentiation. Hypoxia impaired the function of gap junctions by dephosphorylating its component protein connexin 43, the major mediator of intercellular Ca2+ propagation between cardiomyocytes, thereby impairing ACM redifferentiation. In vivo, ACM proliferation was found primarily in the MI border zone. An ischemia-resistant connexin 43 mutant enhanced the redifferentiation of ACM-derived new cardiomyocytes after MI and improved cardiac function. Mature ACMs can reenter the cell cycle and form new cardiomyocytes through a 3-step process: dedifferentiation, proliferation, and redifferentiation. Intercellular Ca2+ signal from neighboring functioning cardiomyocytes through gap junctions induces the redifferentiation process. This novel mechanism contributes to new cardiomyocyte formation in post-MI hearts in mammals.

Linear Channel Modeling and Error Analysis for Intra/Inter-Cellular Ca2+ Molecular Communication.

The use of intra/inter-cellular calcium ion (Ca2+) signaling for molecular communication (MC) is investigated in this paper. In particular, the elevation of the intracellular Ca2+ concentration upon the external excitation, i.e., Ca2+ wave generation, and the intercellular propagation of Ca2+ wave over consecutive cells are studied for information transmission. The main objective of this paper is to develop a linear channel model for intra/inter-cellular Ca2+ MC. In this context, the end-to-end Ca2+ MC system is studied under three blocks: the wave generation, the gap junctional (intercellular) propagation, and the intracellular propagation. The wave generation block captures the intracellular Ca2+ signaling pathway including the release of Ca2+ from the organelles and the buffers inside a cell, and the intake from the extracellular space. The gap junctional (intercellular) propagation block captures the Ca2+ transition through the gap junctions between the touching cells. The intracellular propagation block defines the effect of the cytoplasmic diffusion. Using the developed blocks for the different biophysical phenomena, the end-to-end channel gain and delay formulas are derived. Furthermore, the bit error probability is studied to reveal the impact of the detection threshold. This work provides the basis for the modeling, analysis and the design of Ca2+ MC systems.

Hemichannels: new roles in astroglial function.

The role of astrocytes in brain function has evolved over the last decade, from support cells to active participants in the neuronal synapse through the release of “gliotransmitters.”Astrocytes express receptors for most neurotransmitters and respond to them through Ca2+ intracellular oscillations and propagation of intercellular Ca2+ waves. While such waves are able to propagate among neighboring astrocytes through gap junctions, thereby activating several astrocytes simultaneously, they can also trigger the release of gliotransmitters, including glutamate, d-serine, glycine, ATP, adenosine, or GABA. There are several mechanisms by which gliotransmitter release occurs, including functional hemichannels. These gliotransmitters can activate neighboring astrocytes and participate in the propagation of intercellular Ca2+ waves, or activate pre- and post-synaptic receptors, including NMDA, AMPA, and purinergic receptors. In consequence, hemichannels could play a pivotal role in astrocyte-to-astrocyte communication and astrocyte-to-neuron cross-talk. Recent evidence suggests that astroglial hemichannels are involved in higher brain functions including memory and glucose sensing. The present review will focus on the role of hemichannels in astrocyte-to-astrocyte and astrocyte-to neuron communication and in brain physiology.

Human Articular Chondrocytes Express Multiple Gap Junction Proteins: Differential Expression of Connexins in Normal and Osteoarthritic Cartilage

Human Articular Chondrocytes Express Multiple Gap Junction Proteins: Differential Expression of Connexins in Normal and Osteoarthritic Cartilage

Can triggered arrhythmias arise from propagation of calcium waves between cardiac myocytes

Intracellular Ca2+ overload can induce regenerative Ca2+ waves that activate inward current in cardiac myocytes, allowing the cell membrane to achieve threshold. The result is a triggered extrasystole that can, under the right conditions, lead to sustained triggered arrhythmias. In this review, we consider the issue of whether or not Ca2+ waves can travel between neighboring myocytes and if this intercellular Ca2+ diffusion can involve enough cells over a short enough period of time to actually induce triggered activity in the heart. This review is not intended to serve as an exhaustive review of the literature summarizing Ca2+ flux through cardiac gap junctions or of how Ca2+ waves move from cell to cell. Rather, it summarizes many of the pertinent experimental studies and considers their results in the theoretical context of whether or not the intercellular propagation of Ca2+ overload can contribute to triggered beats and arrhythmias in the intact heart.

EGF activates intracellular and intercellular calcium signaling by distinct pathways in tumor cells

Epidermal growth factor (EGF)-mediated Ca2+ signaling in multiple cell lines derived from human gliomas and in the A431 epidermoid carcinoma cell line was observed using fluorescence videomicroscopy. Bath application of EGF evoked an oscillatory increase in [Ca2+]i in 4 different human glioma cell lines as well as the A431 cell line. This effect was blocked by the EGF receptor tyrosine kinase inhibitors gefitinib and erlotinib, as well as by the EGFR antibody cetuximab. In addition to this acute Ca2+ signaling response, transient exposure to EGF also potentiated subsequent Ca2+ signaling responses to other stimuli. Tumor cells transiently exposed to EGF (5 minutes), showed a sustained increase in propagation of intercellular Ca2+ waves, which have been previously shown to involve release of ATP and activation of purinergic receptors. Cells transiently exposed to EGF also showed a sustained potentiation of the Ca2+ signaling response to ATP. In contrast to the acute Ca2+ signaling response to EGF, this sustained potentiation of purinergic intercellular signaling was not blocked by gefitinib or erlotinib, while it was blocked by cetuximab. These results indicate that while the acute Ca2+ signaling response requires tyrosine kinase activation, the sustained potentiation of intercellular signaling occurs via a distinct pathway. Distinct intra- and intercellular Ca2+ signaling pathways may be mechanisms by which EGF modulates the growth and migration of tumor cells.

Intercellular Ca2+-transient propagation in normal and high glucose solutions in rat retinal epithelial (RPE-J) cells during mechanical stimulation

We investigated the effect of high glucose and modulation of protein kinase C (PKC) on the intercellular propagation of Ca2+-waves in a rat retinal pigment epithelial cell line (RPE-J cells) in order to compare its properties with the properties previously investigated in primary LE-RPE cells. The intercellular propagation of the Ca2+-waves in RPE-J cells was analyzed by fluorescence imaging confocal microscopy and fluorescence recovery after photobleaching (FRAP). In control conditions the maximal normalized fluorescence in the mechanically stimulated (MS) cell and the propagation towards the neighboring RPE-J cells were similar to LE-RPE cells. As in LE-RPE cells, the propagation was reduced by the gap junction (GJ) blocker halothane, and FRAP experiments demonstrated the presence of functional GJ coupling. Similar to the effect in LE-RPE cells, the propagation of the Ca2+-transient was reduced by 25mM glucose. However, unlike LE-RPE cells, the neighboring RPE-J cells presented a Ca2+-rise of amplitude similar to that in normal glucose levels. PKC activation with 1μM PMA for 30min resulted in inhibition of the Ca2+-wave propagation, which could be overcome by PKC downregulation as in LE-RPE cells. Cells grown for 72h in a high glucose solution in which PKC activity was downregulated, did not develop the inhibitory effect on Ca2+-wave propagation that was induced by elevated glucose levels. However, the effects were not as pronounced as in LE-RPE cells. We concluded that despite marked similarities, the transduction and the modulation of intercellular propagation of the Ca2+-transients in RPE-J cells are not identical to the mechanisms in primary LE-RPE cells.

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.

A Fundamental Role for the Nitric Oxide-G-Kinase Signaling Pathway in Mediating Intercellular Ca2+Waves in Glia

In this study, we highlight a role for the nitric oxide-cGMP-dependent protein kinase (NO-G-kinase) signaling pathway in glial intercellular Ca(2+) wave initiation and propagation. Addition of the NO donor molsidomine (100-500 microM) or puffing aqueous NO onto primary glial cell cultures evoked an increase in [Ca(2+)](i) in individual cells and also local intercellular Ca(2+) waves, which persisted after removal of extracellular Ca(2+). High concentrations of ryanodine (100-200 microM) and antagonists of the NO-G-kinase signaling pathway essentially abrogated the NO-induced increase in [Ca(2+)](i), indicating that NO mobilizes Ca(2+) from a ryanodine receptor-linked store, via the NO-G-kinase signaling pathway. Addition of 10 microM nicardipine to cells resulted in a slowing of the molsidomine-induced rise in [Ca(2+)](i), and inhibition of Mn(2+) quench of cytosolic fura-2 fluorescence mediated by a bolus application of 2 microM aqueous NO to cells, indicating that NO also induces Ca(2+) influx in glia. Mechanical stress of individual glial cells resulted in an increase in intracellular NO in target and neighboring cells and intercellular Ca(2+) waves, which were NO, cGMP, and G-kinase dependent, because incubating cells with nitric oxide synthase, guanylate cyclase, and G-kinase inhibitors, or NO scavengers, reduced Delta[Ca(2+)](i) and the rate of Ca(2+) wave propagation in these cultures. Results from this study suggest that NO-G-kinase signaling is coupled to Ca(2+) mobilization and influx in glial cells and that this pathway plays a fundamental role in the generation and propagation of intercellular Ca(2+) waves in glia.

Heterotypic coupling between glial cells of the mammalian central nervous system

Heterotypic coupling, defined as gap-junctional coupling between cells of different classes, may be common among the different types of non-neuronal cells in the central nervous system. Since gap junctions provide a route for the intercellular exchange of signaling molecules, heterotypic coupling may serve to coordinate the activities of many types of "support cells" in the brain. The evidence for heterotypic coupling between astrocytes and oligodendrocytes, astrocytes and retinal Müller glial cells, and astrocytes and ependymal cells is reviewed. The finding that some heterotypic gap junctions are chemically rectifying implies that there is asymmetry between the two sides of these gap junctions, and the connexin composition of heterotypic gap junctions is discussed. Finally, I speculate about the functions of heterotypic gap junctions, including their proposed roles in K+ spatial buffering around axons and in the propagation of intercellular Ca2+ waves between astrocytes and other glial cells.

Heterotypic coupling between glial cells of the mammalian central nervous system

Heterotypic coupling, defined as gap-junctional coupling between cells of different classes, may be common among the different types of non-neuronal cells in the central nervous system. Since gap junctions provide a route for the intercellular exchange of signaling molecules, heterotypic coupling may serve to coordinate the activities of many types of “support cells” in the brain. The evidence for heterotypic coupling between astrocytes and oligodendrocytes, astrocytes and retinal Müller glial cells, and astrocytes and ependymal cells is reviewed. The finding that some heterotypic gap junctions are chemically rectifying implies that there is asymmetry between the two sides of these gap junctions, and the connexin composition of heterotypic gap junctions is discussed. Finally, I speculate about the functions of heterotypic gap junctions, including their proposed roles in K+ spatial buffering around axons and in the propagation of intercellular Ca2+ waves between astrocytes and other glial cells. GLIA 24:85–96, 1998. © 1998 Wiley-Liss, Inc.

Synchronized Ca2+ signaling by intercellular propagation of Ca2+ action potentials in NRK fibroblasts.

The intercellular propagation of Ca2+ waves by diffusion of inositol trisphosphate has been shown to be a general mechanism by which nonexcitable cells communicate. Here, we show that monolayers of normal rat kidney (NRK) fibroblasts behave like a typical excitable tissue. In confluent monolayers of these cells, Ca2+ action potentials can be generated by local depolarization of the monolayer on treatment with either bradykinin or an elevation of the extracellular K+ concentration. These electronically propagating action potentials travel intercellularly over long distances in an all-or-none fashion at a speed of approximately 6.1 mm/s and can be blocked by L-type Ca2+ channel blockers. The action potentials are generated by depolarizations beyond the threshold value for L-type Ca2+ channels of about -15 mV. The result of these locally induced, propagating Ca2+ action potentials is an almost synchronous, transient increase in the intracellular Ca2+ concentration in large numbers of cells. These data show that electrically coupled fibroblasts can form an excitable syncytium, and they elucidate a novel mechanism of intercellular Ca2+ signaling in these cells that may coordinate synchronized multicellular responses to local stimuli.

Paracrine mediation of calcium signaling in human SK-N-MCIXC neuroepithelioma cells.

Paracrine-mediated Ca2+ signaling in SK-N-MCIXC neuroepithelioma cells was evaluated by means of two experimental paradigms. In the first, single SK-N-MCIXC cells were microinjected with inositol 1,4,5-trisphosphate [Ins(1,4,5)P3] and cytoplasmic Ca2+ was monitored by fura 2 digital-imaging microfluorometry. In response to Ins(1,4,5)P3 or CaCl2, but not inositol 1,3,4-trisphosphate, an increase in cytoplasmic Ca2+ concentration ([Ca2+]i) was observed in injected cells and also in neighboring cells. The direction of intercellular propagation of Ca2+ signals was influenced by the presence of a flow in the extracellular medium and occurred in the absence of any detectable gap-junctional communication. The P2 purinoceptor antagonist suramin, but not antagonists of other phosphoinositide-linked receptors, blocked cell-to-cell Ca2+ signaling initiated by microinjections of Ins(1,4,5)P3. In the second paradigm, conditioned medium (CM) obtained from monolayers of SK-N-MCIXC cells elicited increases in [Ca2+]i when reapplied to cells on coverslips. The Ca(2+)-mobilizing activity of CM was reversibly antagonized by suramin and abolished by pretreatment with apyrase. The presence of nucleotide di- and triphosphates in CM was confirmed by high-performance liquid chromatography. We conclude that SK-N-MCIXC cells release nucleotides that then activate specific receptors on neighboring cells. A rise in [Ca2+]i in these cells, and subsequent additional release of nucleotides, serves to further the propagation of intercellular Ca2+ signals.

Coordination of Ca2+ Signaling by Intercellular Propagation of Ca2+ Waves in the Intact Liver

Activation of the inositol lipid signaling system results in cytosolic Ca2+ oscillations and intra- and intercellular Ca2+ waves in many isolated cell preparations. However, this form of temporal and spatial organization of signaling has not been demonstrated in intact tissues. Digital imaging fluorescence microscopy was used to monitor Ca2+ at the cellular and subcellular level in intact perfused rat liver loaded with fluorescent Ca2+ indicators. Perfusion with low doses of vasopressin induced oscillations of hepatocyte Ca2+ that were coordinated across entire lobules of the liver by propagation of Ca2+ waves along the hepatic plates. At the subcellular level these periodic Ca2+ waves initiated from the sinusoidal domain of cells within the periportal region and propagated radially across cell-cell contacts into the pericentral region, or until terminated by annihilation collision with other Ca2+ wave fronts. With increasing agonist dose, the frequency but not the amplitude of the Ca2+ waves increased. Intracellular Ca2+ wave rates were constant, but transcellular signal propagation was determined by agonist dose, giving rise to a dose-dependent increase in the rate at which Ca2+ waves spread through the liver. At high vasopressin doses, a single Ca2+ wave was observed and the direction of Ca2+ wave propagation was reversed, initiating in the pericentral region and spreading to the periportal region. It is concluded that intercellular Ca2+ waves may provide a mechanism to coordinate responses across the functional units of the liver.