Non-excitable Cell Types Research Articles

Ionotropic glycine receptor in nonexcitable cells in regenerative medicine: a narrative review

The vast majority of ionotropic glycine receptor research has historically concentrated on receptor function in neurons and, to a somewhat lesser extent, in myocytes. However, an ever-increasing volume of experimental data indicates the important role of glycine receptor in cytoprotective and modulatory effects in nonexcitable cells and tissues. The key mechanisms underlying these glycine receptor effects are connected to the regulation of the electrical potential of the plasmalemma, Ca2+ influx from the extracellular media, and Ca2+ efflux from the endoplasmic reticulum. These mechanisms trigger a complex network of biochemical signaling pathways that, in turn, partially regulate glycine receptor activity. In addition, glycine effects in several cell types suggest the existence of modulatory signaling chains of a yet unknown nature. Taken together, these findings clearly demonstrate the important role of the glycine receptor in understudied (although widespread) molecular and cellular mechanisms of potentially high significance. Therefore, we provide a brief review of glycine receptor effects on different types of nonexcitable cells, and we believe that targeting glycine receptor in nonexcitable cells is a potential strategy for regenerative medicine in the clinic.

An Ultrasensitive Genetically Encoded Voltage Indicator Uncovers the Electrical Activity of Non-Excitable Cells.

Most animal cell types are classified as non-excitable because they do not generate action potentials observed in excitable cells, such as neurons and muscle cells. Thus, resolving voltage signals in non-excitable cells demands sensors with exceptionally high voltage sensitivity. In this study, the ultrabright, ultrasensitive, and calibratable genetically encoded voltage sensor rEstus is developed using structure-guided engineering. rEstus is most sensitive in the resting voltage range of non-excitable cells and offers a 3.6-fold improvement in brightness change for fast voltage spikes over its precursor ASAP3. Using rEstus, it is uncovered that the membrane voltage in several non-excitable cell lines (A375, HEK293T, MCF7) undergoes spontaneous endogenous alterations on a second to millisecond timescale. Correlation analysis of these optically recorded voltage alterations provides a direct, real-time readout of electrical cell-cell coupling, showing that visually connected A375 and HEK293T cells are also largely electrically connected, while MCF7 cells are only weakly coupled. The presented work provides enhanced tools and methods for non-invasive voltage imaging in living cells and demonstrates that spontaneous endogenous membrane voltage alterations are not limited to excitable cells but also occur in a variety of non-excitable cell types.

Voltage-Gated T-Type Calcium Channel Modulation by Kinases and Phosphatases: The Old Ones, the New Ones, and the Missing Ones.

Calcium (Ca2+) can regulate a wide variety of cellular fates, such as proliferation, apoptosis, and autophagy. More importantly, changes in the intracellular Ca2+ level can modulate signaling pathways that control a broad range of physiological as well as pathological cellular events, including those important to cellular excitability, cell cycle, gene-transcription, contraction, cancer progression, etc. Not only intracellular Ca2+ level but the distribution of Ca2+ in the intracellular compartments is also a highly regulated process. For this Ca2+ homeostasis, numerous Ca2+ chelating, storage, and transport mechanisms are required. There are also specialized proteins that are responsible for buffering and transport of Ca2+. T-type Ca2+ channels (TTCCs) are one of those specialized proteins which play a key role in the signal transduction of many excitable and non-excitable cell types. TTCCs are low-voltage activated channels that belong to the family of voltage-gated Ca2+ channels. Over decades, multiple kinases and phosphatases have been shown to modulate the activity of TTCCs, thus playing an indirect role in maintaining cellular physiology. In this review, we provide information on the kinase and phosphatase modulation of TTCC isoforms Cav3.1, Cav3.2, and Cav3.3, which are mostly described for roles unrelated to cellular excitability. We also describe possible potential modulations that are yet to be explored. For example, both mitogen-activated protein kinase and citron kinase show affinity for different TTCC isoforms; however, the effect of such interaction on TTCC current/kinetics has not been studied yet.

Structural Insights into Ca2+ Permeation through Orai Channels.

Orai channels belong to the calcium release-activated calcium (CRAC) channel family. Orai channels are responsible for the influx of extracellular Ca2+ that is triggered by Ca2+ depletion from the endoplasmic reticulum (ER); this function is essential for many types of non-excitable cells. Extensive structural and functional studies have advanced the knowledge of the molecular mechanism by which Orai channels are activated. However, the gating mechanism that allows Ca2+ permeation through Orai channels is less well explained. Here, we reviewed and summarized the existing structural studies of Orai channels. We detailed the structural features of Orai channels, described structural comparisons of their closed and open states, and finally proposed a “push–pull” model of Ca2+ permeation.

Neuromodulation of Glial Function During Neurodegeneration.

Glia, a non-excitable cell type once considered merely as the connective tissue between neurons, is nowadays acknowledged for its essential contribution to multiple physiological processes including learning, memory formation, excitability, synaptic plasticity, ion homeostasis, and energy metabolism. Moreover, as glia are key players in the brain immune system and provide structural and nutritional support for neurons, they are intimately involved in multiple neurological disorders. Recent advances have demonstrated that glial cells, specifically microglia and astroglia, are involved in several neurodegenerative diseases including Amyotrophic lateral sclerosis (ALS), Epilepsy, Parkinson’s disease (PD), Alzheimer’s disease (AD), and frontotemporal dementia (FTD). While there is compelling evidence for glial modulation of synaptic formation and regulation that affect neuronal signal processing and activity, in this manuscript we will review recent findings on neuronal activity that affect glial function, specifically during neurodegenerative disorders. We will discuss the nature of each glial malfunction, its specificity to each disorder, overall contribution to the disease progression and assess its potential as a future therapeutic target.

KCNQs: Ligand- and Voltage-Gated Potassium Channels.

Voltage-gated potassium (Kv) channels in the KCNQ (Kv7) family are essential features of a broad range of excitable and non-excitable cell types and are found in organisms ranging from Hydra vulgaris to Homo sapiens. Although they are firmly in the superfamily of S4 domain-bearing voltage-sensing ion channels, KCNQ channels are highly sensitive to a range of endogenous and exogenous small molecules that act directly on the pore, the voltage-sensing domain, or the interface between the two. The focus of this review is regulation of KCNQs by direct binding of neurotransmitters and metabolites from both animals and plants and the role of the latter in the effects of plants consumed for food and as traditional folk medicines. The conceptual question arises: Are KCNQs voltage-gated channels that are also sensitive to ligands or ligand-gated channels that are also sensitive to voltage?

Pathophysiological Significance of Store-Operated Calcium Entry in Cardiovascular and Skeletal Muscle Disorders and Angiogenesis.

Store-Operated Ca2+ Entry (SOCE) is an important Ca2+ influx pathway expressed by several excitable and non-excitable cell types. SOCE is recognized as relevant signaling pathway not only for physiological process, but also for its involvement in different pathologies. In fact, independent studies demonstrated the implication of essential protein regulating SOCE, such as STIM, Orai and TRPCs, in different pathogenesis and cell disorders, including cardiovascular disease, muscular dystrophies and angiogenesis. Compelling evidence showed that dysregulation in the function and/or expression of isoforms of STIM, Orai or TRPC play pivotal roles in cardiac hypertrophy and heart failure, vascular remodeling and hypertension, skeletal myopathies, and angiogenesis. In this chapter, we summarized the current knowledge concerning the mechanisms underlying abnormal SOCE and its involvement in some diseases, as well as, we discussed the significance of STIM, Orai and TRPC isoforms as possible therapeutic targets for the treatment of angiogenesis, cardiovascular and skeletal muscle diseases.

An Allosteric Inhibitory Site Conserved in the Ectodomain of P2X Receptor Channels.

P2X receptors constitute a gene family of cation channels gated by extracellular ATP. They mediate fast ionotropic purinergic signaling in neurons and non-excitable cell types in vertebrates. The highly calcium-permeable P2X4 subtype has been shown to play a significant role in cardiovascular physiology, inflammatory responses and neuro-immune communication. We previously reported the discovery of a P2X4-selective antagonist, the small organic compound BX430, with submicromolar potency for human P2X4 receptors and marked species-dependence (Ase et al., 2015). The present study investigates the molecular basis of P2X4 inhibition by the non-competitive blocker BX430 using a structural and functional approach relying on mutagenesis and electrophysiology. We provide evidence for the critical contribution of a single hydrophobic residue located in the ectodomain of P2X4 channel subunits, Ile312 in human P2X4, which determines blockade by BX430. We also show that the nature of this extracellular residue in various vertebrate P2X4 orthologs underlies their specific sensitivity or resistance to the inhibitory effects of BX430. Taking advantage of high-resolution crystallographic data available on zebrafish P2X4, we used molecular dynamics simulation to model the docking of BX430 on an allosteric binding site around Ile315 (zebrafish numbering) in the ectodomain of P2X4. We also observed that the only substitution I312D (human numbering) that renders P2X4 silent by itself has also a profound silencing effect on all other P2X subtypes tested when introduced at homologous positions. The generic impact of this aspartate mutation on P2X function indicates that the pre-TM2 subregion involved is conserved functionally and defines a novel allosteric inhibitory site present in all P2X receptor channels. This conserved structure-channel activity relationship might be exploited for the rational design of potent P2X subtype-selective antagonists of therapeutic value.

Measurement of Store-Operated Calcium Entry in Human Neural Cells: From Precursors to Differentiated Neurons.

Calcium imaging in an ex-vivosetup is used to understand the calcium status of isolated cells or tissue. In this chapter we explain the use of the ratiometric chemical indicator Fura-2 which can be loaded into isolated cells in the form of lipophilic acetomethyl (AM) esters. Fura-2 is a combination of calcium chelator and fluorophore, and can be used with dual wavelength excitation (340/380nm) for quantitative calcium concentrations. The cells can then be viewed using a fluorescence microscope and captured by a CCD camera. We specifically discuss the technique involved in understanding the endoplasmic reticulum (ER)-driven store-operated calcium entry (SOCE) in human neural precursors (NPCs)and spontaneously differentiated neurons derived from a pluripotent humanembryonic stem cell(hESC) line. The derivation of neural precursors from stem cells and their subsequent spontaneous neural differentiation is also explained. The method can be used for various non-excitable and excitable cell types including neurons, be it freshly isolated, from frozen vials, or derived from different stem cell lines.

External K+ dependence of strong inward rectifier K+ channel conductance is caused not by K+ but by competitive pore blockade by external Na+

Strong inward rectifier K+ (sKir) channels determine the membrane potentials of many types of excitable and nonexcitable cells, most notably the resting potentials of cardiac myocytes. They show little outward current during membrane depolarization (i.e., strong inward rectification) because of the channel blockade by cytoplasmic polyamines, which depends on the deviation of the membrane potential from the K+ equilibrium potential (V - EK) when the extracellular K+ concentration ([K+]out) is changed. Because their open-channel conductance is apparently proportional to the "square root" of [K+]out, increases/decreases in [K+]out enhance/diminish outward currents through sKir channels at membrane potentials near their reversal potential, which also affects, for example, the repolarization and action-potential duration of cardiac myocytes. Despite its importance, however, the mechanism underlying the [K+]out dependence of the open sKir channel conductance has remained elusive. By studying Kir2.1, the canonical member of the sKir channel family, we first show that the outward currents of Kir2.1 are observed under the external K+-free condition when its inward rectification is reduced and that the complete inhibition of the currents at 0 [K+]out results solely from pore blockade caused by the polyamines. Moreover, the noted square-root proportionality of the open sKir channel conductance to [K+]out is mediated by the pore blockade by the external Na+, which is competitive with the external K+ Our results show that external K+ itself does not activate or facilitate K+ permeation through the open sKir channel to mediate the apparent external K+ dependence of its open channel conductance. The paradoxical increase/decrease in outward sKir channel currents during alternations in [K+]out, which is physiologically relevant, is caused by competition from impermeant extracellular Na.

Hearing Loss Controlled by Optogenetic Stimulation of Nonexcitable Nonglial Cells in the Cochlea of the Inner Ear.

Light-gated ion channels and transporters have been applied to a broad array of excitable cells including neurons, cardiac myocytes, skeletal muscle cells and pancreatic β-cells in an organism to clarify their physiological and pathological roles. Nonetheless, among nonexcitable cells, only glial cells have been studied in vivo by this approach. Here, by optogenetic stimulation of a different nonexcitable cell type in the cochlea of the inner ear, we induce and control hearing loss. To our knowledge, deafness animal models using optogenetics have not yet been established. Analysis of transgenic mice expressing channelrhodopsin-2 (ChR2) induced by an oligodendrocyte-specific promoter identified this channel in nonglial cells—melanocytes—of an epithelial-like tissue in the cochlea. The membrane potential of these cells underlies a highly positive potential in a K+-rich extracellular solution, endolymph; this electrical property is essential for hearing. Illumination of the cochlea to activate ChR2 and depolarize the melanocytes significantly impaired hearing within a few minutes, accompanied by a reduction in the endolymphatic potential. After cessation of the illumination, the hearing thresholds and potential returned to baseline during several minutes. These responses were replicable multiple times. ChR2 was also expressed in cochlear glial cells surrounding the neuronal components, but slight neural activation caused by the optical stimulation was unlikely to be involved in the hearing impairment. The acute-onset, reversible and repeatable phenotype, which is inaccessible to conventional gene-targeting and pharmacological approaches, seems to at least partially resemble the symptom in a population of patients with sensorineural hearing loss. Taken together, this mouse line may not only broaden applications of optogenetics but also contribute to the progress of translational research on deafness.

Infrared neural stimulation induces intracellular Ca2+ release mediated by phospholipase C.

The influence of infrared laser pulses on intracellular Ca2+ signaling was investigated in neural cell lines with fluorescent live cell imaging. The probe Fluo-4 was used to measure Ca2+ in HT22 mouse hippocampal neurons and nonelectrically excitable U87 human glioblastoma cells exposed to 50 to 500 ms infrared pulses at 1470 nm. Fluorescence recordings of Fluo-4 demonstrated that infrared stimulation induced an instantaneous intracellular Ca2+ transient with similar dose-response characteristics in hippocampal neurons and glioblastoma cells (half-maximal effective energy density EC50 of around 58 J.cm-2 ). For both type of cells, the source of the infrared-induced Ca2+ transients was found to originate from intracellular stores and to be mediated by phospholipase C and IP3 -induced Ca2+ release from the endoplasmic reticulum. The activation of phosphoinositide signaling by IR light is a new mechanism of interaction relevant to infrared neural stimulation that will also be widely applicable to nonexcitable cell types. The prospect of infrared optostimulation of the PLC/IP3 cell signaling cascade has many potential applications including the development of optoceutical therapeutics.

Cardiovascular and Hemostatic Disorders: SOCE in Cardiovascular Cells: Emerging Targets for Therapeutic Intervention.

The discovery of the store-operated Ca2+ entry (SOCE) phenomenon is tightly associated with its recognition as a pathway of high (patho)physiological significance in the cardiovascular system. Early on, SOCE has been investigated primarily in non-excitable cell types, and the vascular endothelium received particular attention, while a role of SOCE in excitable cells, specifically cardiac myocytes and pacemakers, was initially ignored and remains largely enigmatic even to date. With the recent gain in knowledge on the molecular components of SOCE as well as their cellular organization within nanodomains, potential tissue/cell type-dependent heterogeneity of the SOCE machinery along with high specificity of linkage to downstream signaling pathways emerged for cardiovascular cells. The basis of precise decoding of cellular Ca2+ signals was recently uncovered to involve correct spatiotemporal organization of signaling components, and even minor disturbances in these assemblies trigger cardiovascular pathologies. With this chapter, we wish to provide an overview on current concepts of cellular organization of SOCE signaling complexes in cardiovascular cells with particular focus on the spatiotemporal aspects of coupling to downstream signaling and the potential disturbance of these mechanisms by pathogenic factors. The significance of these mechanistic concepts for the development of novel therapeutic strategies will be discussed.

TRPM4-dependent post-synaptic depolarization is essential for the induction of NMDA receptor-dependent LTP in CA1 hippocampal neurons

TRPM4 is a calcium-activated but calcium-impermeable non-selective cation (CAN) channel. Previous studies have shown that TRPM4 is an important regulator of Ca2+-dependent changes in membrane potential in excitable and non-excitable cell types. However, its physiological significance in neurons of the central nervous system remained unclear. Here, we report that TRPM4 proteins form a CAN channel in CA1 neurons of the hippocampus and we show that TRPM4 is an essential co-activator of N-methyl-d-aspartate (NMDA) receptors (NMDAR) during the induction of long-term potentiation (LTP). Disrupting the Trpm4 gene in mice specifically eliminates NMDAR-dependent LTP, while basal synaptic transmission, short-term plasticity, and NMDAR-dependent long-term depression are unchanged. The induction of LTP in Trpm4−/− neurons was rescued by facilitating NMDA receptor activation or post-synaptic membrane depolarization. Accordingly, we obtained normal LTP in Trpm4−/− neurons in a pairing protocol, where post-synaptic depolarization was applied in parallel to pre-synaptic stimulation. Taken together, our data are consistent with a novel model of LTP induction in CA1 hippocampal neurons, in which TRPM4 is an essential player in a feed-forward loop that generates the post-synaptic membrane depolarization which is necessary to fully activate NMDA receptors during the induction of LTP but which is dispensable for the induction of long-term depression (LTD). These results have important implications for the understanding of the induction process of LTP and the development of nootropic medication.

Comparative End-to-End Analysis of Ca2+-Signaling-Based Molecular Communication in Biological Tissues

Calcium (Ca2+)-signaling-based molecular communication is a short-range communication process that diffuses and propagates ions between the cells of a tissue. The communication process is initiated via stimulation and amplification of the production of Ca2+ ions within a cell; these ions then diffuse through a physical connection between cells called a gap junction. Ca2+ signaling can be found in different classes of cell. In excitable cells, initiation of the Ca2+-signaling process is accompanied by an electrical component; for nonexcitable cell types, the electrical component is absent; while hybrid cells exhibit both behaviors. This paper provides a comparison and analysis of the communication behavior in tissues comprised three specific cell types that utilize Ca2+ signaling: epithelium cells (nonexcitable), smooth muscle cells (excitable), and astrocytes (hybrid). The analysis focuses on spatiotemporal Ca2+ concentration dynamics and how they are influenced by the intracellular signaling process, the molecular diffusion delay, the gain and capacity of the communication channel, as well as intracellular signaling interference. This analysis of the communication behavior in the context of tissues provides insights useful for, inter alia , the design of nanomachines that are situated within tissues and that use analysis of the communication channel to infer tissue health.

Sodium channel β subunits: emerging targets in channelopathies.

Voltage-gated sodium channels (VGSCs) are responsible for the initiation and propagation of action potentials in excitable cells. VGSCs in mammalian brain are heterotrimeric complexes of α and β subunits. Although β subunits were originally termed auxiliary, we now know that they are multifunctional signaling molecules that play roles in both excitable and nonexcitable cell types and with or without the pore-forming α subunit present. β subunits function in VGSC and potassium channel modulation, cell adhesion, and gene regulation, with particularly important roles in brain development. Mutations in the genes encoding β subunits are linked to a number of diseases, including epilepsy, sudden death syndromes like SUDEP and SIDS, and cardiac arrhythmia. Although VGSC β subunit-specific drugs have not yet been developed, this protein family is an emerging therapeutic target.

Impaired Bone Formation in TRIC-B-Knockout Mice

Osteogenesis imperfect (OI) is a heritable bone dysplasia characterized by bone fragility, but has divergent phenotypic manifestations, suggesting heterogeneous OI-responsible genes in the human genome. Most of OI cases result from defected type I collagen; structural mutations and altered post-translational modifications lead to its insufficient content, folding and trafficking errors, and also compromised matrix incorporation. Recently, a homozygous deletion mutation in the TRIC-B (also referred to as TMEM38B) locus has been identified in Saudi Arabic and Bedouin Israeli OI pedigrees. The OI mutant gene encodes a truncated TRIC-B protein lacking its C-terminal half, and is likely associated with severe impairment of TRIC-B channel activity in various cell types. However, pathological mechanism is still unknown in the OI pedigrees.The TRIC (trimeric intracellular cation) channel subtypes, namely TRIC-A and TRIC-B, form homotrimeric complexes to function as intracellular monovalent cation-specific channels. TRIC-A channels are predominantly expressed in muscle and brain, while TRIC-B channels are ubiquitously detected throughout excitable and non-excitable cell types. Based on our observations in knockout mice, TRIC channels seem to mediate, in part, counterion movements to support efficient Ca2+ release from the sarco/endoplasmic reticulum. Tric-b-knockout mice develop respiratory failure at birth, and the mutant alveolar epithelial cells exhibit compromised IP3R-mediated Ca2+ release and insufficient handling of surfactant lipids. In skeleton preparations from Tric-b-knockout neonates, semi-translucent parts were frequently detected in major bones, indicating impaired bone formation. Indeed, insufficient bone mineralization was confirmed by computed tomography imaging and histochemical analysis. RT-PCR experiments detected decreased expression of osteocalcin and RanklmRNAs as osteoblast makers in Tric-b-knockout femoral bones, suggesting the possibility that Tric-b deficiency may affect osteoblast functions. Therefore, Tric-b-knockout neonates provide a useful model for OI bearing the mutant TRIC-B gene.

Application of femtosecond‐pulsed lasers for direct optical manipulation of biological functions

Absorption of photon energy by cells or tissue can evoke photothermal, photomechanical, and photochemical effects, depending on the density of the deposited energy. Photochemical effects require a low energy density and can be used for reversible modulation of biological functions. Ultrashort‐pulsed lasers have a high intensity due to the short pulse duration, despite its low average energy. Through nonlinear absorption, these lasers can deliver very high peak energy into the submicrometer focus area without causing collateral damage. Absorbed energy delivered by ultrashort‐pulsed laser irradiation induces free electrons, which can be readily converted to reactive oxygen species (ROS) and related free radicals in the localized region. Free radicals are best known to induce irreversible biological effects via oxidative modification; however, they have also been proposed to modulate biological functions by releasing calcium ions from intracellular organelles. Calcium can evoke variable biological effects in both excitable and nonexcitable cell types. Controlled stimulation by ultrashort laser pulses generate intracellular calcium waves that can modulate many biological functions, such as cardiomyocyte beat rate, muscle contractility, and blood–brain barrier (BBB) permeability. This article presents optical methods that are useful therapeutic and research tools in the biomedical field and discuss the possible mechanisms responsible for biological modulation by ultrashort‐pulsed lasers, especially femtosecond‐pulsed lasers.

Deletion of Orai1 alters expression of multiple genes during osteoclast and osteoblast maturation

Store-operated Ca2+ entry (SOCE) is a major Ca2+ influx pathway in most non-excitable cell types and Orai1 was recently identified as an essential pore-subunit of SOCE channels. Here we investigate the physiological role of Orai1 in bone homeostasis using Orai1-deficient mice (Orai1−/−). Orai1−/− mice developed osteopenia with decreased bone mineral density and trabecular bone volume. To identify the nature and origin of the bone defect, bone-resorbing osteoclasts and bone-forming osteoblasts from Orai1−/− mice were examined. Orai1-mediated SOCE was completely abolished in Orai1−/− osteoclast precursor cells and osteoclastogenesis in vitro from Orai1−/− mice was impaired due to a defect in cell fusion of pre-osteoclasts. Also, resorption activity in vitro was comparable but the size of pits formed by Orai1−/− osteoclasts was smaller. We next assessed the role of Orai1 in osteoblast differentiation and function by using a pre-osteoblast cell line, as well as primary osteoblasts from wild-type and Orai1−/− mice. SOCE in MC3T3-E1 pre-osteoblastic cells was inactivated by lentiviral overexpression of a pore-dead Orai1 mutant. Lack of SOCE in MC3T3-E1 had no effect on alkaline phosphatase staining and expression but substantially inhibited mineralized nodule formation. Consistent with this finding, Orai1-mediated SOCE was markedly reduced in Orai1−/− osteoblast precursor cells and osteoblastogenesis in vitro from Orai1−/− stromal cells showed impaired mineral deposition but no change in differentiation. This indicates that Orai1 is involved in the function but not in the differentiation of osteoblasts. Together, these results suggest that Orai1 plays a critical role in bone homeostasis by regulating both osteoblasts and osteoclasts.

Real-time electrical measurement of L929 cellular spontaneous and synchronous oscillation.

Nonexcitable cell types, fibroblasts of heart muscle or astrocytes, are well known for their spontaneous Ca2+ oscillations. On the other hand, murine fibroblast (L929) cells are known to be deficient in cell–cell adhesive proteins and therefore lack gap junctions for cellular communication. However, these cells exhibit a unique property of collectively synchronized and spontaneous oscillation, as revealed by real-time monitoring of cells cultured on a 250-μm diameter microelectrode for more than 3 days using an electrical cell-substrate impedance-sensing system (ECIS). Live-cell imaging is a widely used technique for oscillation detection, but it has limitations relating to cellular physiological environment maintenance for microscopic analysis and for prolonged periods of study. The present research emphasizes an electrical-sensing technique (ECIS) capable of overcoming the most important issues inherent in live-cell imaging systems for the detection of L929 cellular spontaneous and synchronized oscillation in real-time for longer periods. Possible mechanisms involved in L929 oscillation were elucidated to be periodic extension/contraction of lamellipodia continued as blebbing, which is produced by signals from the actomyosin complex initiated by connexin hemichannel opening and adenosine triphosphate (ATP) release. By applying the connexin hemichannel inhibitor, flufenamic acid, the hindrance of ATP release and calcium transients were analyzed to elucidate this hypothesis.