Intercellular Calcium Research Articles

Intrinsic Electromagnetic Noise from Cortical Neurons and its Measurement Ex Vivo

An overview of measurements of intrinsic electromagnetic noise from rat cortical neurons ex vivo is reported here. A series of experiments was conceived around a modified Kerr brain chamber setup with a special vector microelectrode array to measure electromagnetic noise intrinsic to living cells, beyond the Johnson noise limit. Cortical cell sections (mainly neurons) from Wistar rats were obtained and kept in specially developed brain chambers wherein recordings were made under physiological conditions. There were spectra of conspicuous electromagnetic noise measured from living cells distinguishable from Johnson and 1/f noise and from the noise measured from dead cells or medium alone. An exhaustive overview of the experiments and the suggested technique, involving measurements reported here, is presented. A preliminary model is outlined to explain this noise and its origin as well as possible underlying connections with neuronal physiology, especially with intercellular calcium ion flux and waves. This study may have some significance as it may provide possibility of electromagnetic mechanisms playing possible roles in higher functions of the cerebral cortex. In addition, the technique offers promise as a useful new biophysical method in analysis of electromagnetic signals from living cells in vitro or ex vivo.

A positive feedback cell signaling nucleation model of astrocyte dynamics

We constructed a model of calcium signaling in astrocyte neural glial cells that incorporates a positive feedback nucleation mechanism, whereby small microdomain increases in local calcium can stochastically produce global cellular and intercellular network scale dynamics. The model is able to simultaneously capture dynamic spatial and temporal heterogeneities associated with intracellular calcium transients in individual cells and intercellular calcium waves (ICW) in spatially realistic networks of astrocytes, i.e., networks where the positions of cells were taken from real in vitro experimental data of spontaneously forming sparse networks, as opposed to artificially constructed grid networks or other non-realistic geometries. This is the first work we are aware of where an intracellular model of calcium signaling that reproduces intracellular dynamics inherently accounts for intercellular network dynamics. These results suggest that a nucleation type mechanism should be further investigated experimentally in order to test its contribution to calcium signaling in astrocytes and in other cells more broadly. It may also be of interest in engineered neuromimetic network systems that attempt to emulate biological signaling and information processing properties in synthetic hardwired neuromorphometric circuits or coded algorithms.

Mechanically induced intercellular calcium communication in confined endothelial structures

Calcium signaling in the diverse vascular structures is regulated by a wide range of mechanical and biochemical factors to maintain essential physiological functions of the vasculature. To properly transmit information, the intercellular calcium communication mechanism must be robust against various conditions in the cellular microenvironment. Using plasma lithography geometric confinement, we investigate mechanically induced calcium wave propagation in networks of human umbilical vein endothelial cells organized. Endothelial cell networks with confined architectures were stimulated at the single cell level, including using capacitive force probes. Calcium wave propagation in the network was observed using fluorescence calcium imaging. We show that mechanically induced calcium signaling in the endothelial networks is dynamically regulated against a wide range of probing forces and repeated stimulations. The calcium wave is able to propagate consistently in various dimensions from monolayers to individual cell chains, and in different topologies from linear patterns to cell junctions. Our results reveal that calcium signaling provides a robust mechanism for cell–cell communication in networks of endothelial cells despite the diversity of the microenvironmental inputs and complexity of vascular structures.

Multiscale vision model highlights spontaneous glial calcium waves recorded by 2-photon imaging in brain tissue

Intercellular glial calcium waves (GCW) constitute a signaling pathway which can be visualized by fluorescence imaging of cytosolic Ca(2+) changes. Reliable detection of calcium waves in multiphoton imaging data is challenging because of low signal-to-noise ratio. We modified the multiscale vision model (MVM), originally employed to detect faint objects in astronomy data to process stacks of fluorescent images. We demonstrate that the MVM identified and characterized GCWs with much higher sensitivity and detail than pixel thresholding. Origins of GCWs were often associated with prolonged secondary Ca(2+) elevations. The GCWs had variable shapes, and secondary GCWs were observed to bud from the primary, larger GCW. GCWs evaded areas shortly before occupied by a preceding GCW instead circulating around the refractory area. Blood vessels uniquely reshaped GCWs and were associated with secondary GCW events. We conclude that the MVM provides unique possibilities to study spatiotemporally correlated Ca(2+) signaling in brain tissue.

Intercellular calcium signaling in a gap junction-coupled cell network establishes asymmetric neuronal fates in C. elegans

The C. elegans left and right AWC olfactory neurons specify asymmetric subtypes, one default AWC(OFF) and one induced AWC(ON), through a stochastic, coordinated cell signaling event. Intercellular communication between AWCs and non-AWC neurons via a NSY-5 gap junction network coordinates AWC asymmetry. However, the nature of intercellular signaling across the network and how individual non-AWC cells in the network influence AWC asymmetry is not known. Here, we demonstrate that intercellular calcium signaling through the NSY-5 gap junction neural network coordinates a precise 1AWC(ON)/1AWC(OFF) decision. We show that NSY-5 gap junctions in C. elegans cells mediate small molecule passage. We expressed vertebrate calcium-buffer proteins in groups of cells in the network to reduce intracellular calcium levels, thereby disrupting intercellular communication. We find that calcium in non-AWC cells of the network promotes the AWC(ON) fate, in contrast to the autonomous role of calcium in AWCs to promote the AWC(OFF) fate. In addition, calcium in specific non-AWCs promotes AWC(ON) side biases through NSY-5 gap junctions. Our results suggest a novel model in which calcium has dual roles within the NSY-5 network: autonomously promoting AWC(OFF) and non-autonomously promoting AWC(ON).

Calcium signaling: overview and research directions of a molecular communication paradigm

In the ongoing effort to build micro- and nanoscale machines, one of the key approaches is the bio-hybrid approach, which focuses on the use of biological constructs and engineered cells. As a natural extension of this concept to nanoscale communication, molecular communication is an umbrella term encompassing various communication systems that are built based on biological intra- and intercellular communication methods, most of which use molecules and molecular concentration as the information carrier. Compared to other proposed molecular communication systems such as diffusion-based communication and microtubular networks, calcium signaling is expected to provide a faster and more controllable system that is suitable for information dissemination and group behavior in nanoscale sensor networks. In this article, we give a general overview of calcium signaling, a novel communication paradigm that uses intercellular calcium waves in biology as a baseline, explain its capabilities, limitations, and some possible deployment scenarios. We also describe various open issues of this novel communication system and elaborate on some research directions for calcium signaling.

Preparation of Acute Subventricular Zone Slices for Calcium Imaging

The subventricular zone (SVZ) is one of the two neurogenic zones in the postnatal brain. The SVZ contains densely packed cells, including neural progenitor cells with astrocytic features (called SVZ astrocytes), neuroblasts, and intermediate progenitor cells. Neuroblasts born in the SVZ tangentially migrate a great distance to the olfactory bulb, where they differentiate into interneurons. Intercellular signaling through adhesion molecules and diffusible signals play important roles in controlling neurogenesis. Many of these signals trigger intercellular calcium activity that transmits information inside and between cells. Calcium activity is thus reflective of the activity of extracellular signals and is an optimal way to understand functional intercellular signaling among SVZ cells. Calcium activity has been studied in many other regions and cell types, including mature astrocytes and neurons. However, the traditional method to load cells with calcium indicator dye (i.e. bath loading) was not efficient at loading all SVZ cell types. Indeed, the cellular density in the SVZ precludes dye diffusion inside the tissue. In addition, preparing sagittal slices will better preserve the three-dimensional arrangement of SVZ cells, particularly the stream of neuroblast migration on the rostral-caudal axis. Here, we describe methods to prepare sagittal sections containing the SVZ, the loading of SVZ cells with calcium indicator dye, and the acquisition of calcium activity with time-lapse movies. We used Fluo-4 AM dye for loading SVZ astrocytes using pressure application inside the tissue. Calcium activity was recorded using a scanning confocal microscope allowing a precise resolution for distinguishing individual cells. Our approach is applicable to other neurogenic zones including the adult hippocampal subgranular zone and embryonic neurogenic zones. In addition, other types of dyes can be applied using the described method.

Slow Calcium-Depolarization-Calcium waves may initiate fast local depolarization waves in ventricular tissue

Intercellular calcium waves in cardiac myocytes are a well-recognized, if incompletely understood, phenomenon. In a variety of preparations, investigators have reported multi-cellular calcium waves or triggered propagated contractions, but the mechanisms of propagation and pathological importance of these events remain unclear. Here, we review existing experimental data and present a computational approach to investigate the mechanisms of multi-cellular calcium wave propagation.Over the past 50 years, the standard modeling paradigm for excitable cardiac tissue has seen increasingly detailed models of the dynamics of individual cells coupled in tissue solely by intercellular and interstitial current flow. Although very successful, this modeling regime has been unable to capture two important phenomena: 1) the slow intercellular calcium waves observed experimentally, and 2) how intercellular calcium events resulting in delayed after depolarizations at the cellular level could overcome a source-sink mismatch to initiate depolarization waves in tissue. In this paper, we introduce a mathematical model with subcellular spatial resolution, in which we allow both inter- and intracellular current flow and calcium diffusion. In simulations of coupled cells employing this model, we observe: a) slow inter-cellular calcium waves propagating at about 0.1 mm/s, b) faster Calcium-Depolarization-Calcium (CDC) waves, traveling at about 1 mm/s, and c) CDC-waves that can set off fast depolarization-waves (50 cm/s) in tissue with varying gap-junction conductivity.

Intercellular Ca2+Waves: Mechanisms and Function

Intercellular calcium (Ca(2+)) waves (ICWs) represent the propagation of increases in intracellular Ca(2+) through a syncytium of cells and appear to be a fundamental mechanism for coordinating multicellular responses. ICWs occur in a wide diversity of cells and have been extensively studied in vitro. More recent studies focus on ICWs in vivo. ICWs are triggered by a variety of stimuli and involve the release of Ca(2+) from internal stores. The propagation of ICWs predominately involves cell communication with internal messengers moving via gap junctions or extracellular messengers mediating paracrine signaling. ICWs appear to be important in both normal physiology as well as pathophysiological processes in a variety of organs and tissues including brain, liver, retina, cochlea, and vascular tissue. We review here the mechanisms of initiation and propagation of ICWs, the key intra- and extracellular messengers (inositol 1,4,5-trisphosphate and ATP) mediating ICWs, and the proposed physiological functions of ICWs.

ATP signaling is deficient in cultured pannexin1‐null mouse astrocytes

Pannexins (Panx1, 2, and 3) comprise a group of proteins expressed in vertebrates that share weak yet significant sequence homology with the invertebrate gap junction proteins, the innexins. In contrast to the other vertebrate gap junction protein family (connexin), pannexins do not form intercellular channels, but at least Panx1 forms nonjunctional plasma membrane channels. Panx1 is ubiquitously expressed and has been shown to form large conductance (500 pS) channels that are voltage dependent, mechanosensitive, and permeable to relatively large molecules such as ATP. Pharmacological and knockdown approaches have indicated that Panx1 is the molecular substrate for the so-called "hemichannel" originally attributed to connexin43 and that Panx1 is the pore-forming unit of the P2X(7) receptor. Here, we describe, for the first time, conductance and permeability properties of Panx1-null astrocytes. The electrophysiological and fluorescence imaging analyses performed on these cells fully support our previous pharmacological and Panx1 knockdown studies that showed profoundly lower dye uptake and ATP release than wild-type untreated astrocytes. As a consequence of decreased ATP paracrine signaling, intercellular calcium wave spread is altered in Panx1-null astrocytes. Moreover, we found that in astrocytes as in Panx1-expressing oocytes, elevated extracellular K(+) activates Panx1 channels independently of membrane potential. Thus, on the basis of our present findings and our previous report, we propose that Panx1 channels serve as K(+) sensors for changes in the extracellular milieu such as those occurring under pathological conditions.

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.

Analysis of intercellular calcium signaling using microfluidic adjustable laminar flow for localized chemical stimulation

The propagation of intercellular calcium signals provides a mechanism to coordinate cell population activity, which is essential for regulating cell behavior and organ development. However, existing analytical methods are difficult to realize localized chemical stimulation of a single cell among a population of cells that are in close contact with one another for studying the propagation of calcium wave. In this work, a microfluidic method is presented for the analysis of contact-dependent propagation of intercellular calcium wave induced by extracellular ATP using multiple laminar flows. Adjacent cells were seeded ∼300μm downstream the intersection of a Y-shaped microchannel with negative pressure pulses. Consequently, the lateral diffusion distance of the chemical at cell locations was limited to ∼26μm with a total flow rate of 20μLmin−1, which prevented the interference of diffusion-induced cellular responses. Localized stimulation of the target cell with ATP induced the propagation of intercellular calcium wave among the cell population. In addition, studies on the spread of intercellular calcium wave under octanol inhibition allowed us to characterize the gap junction mediated cell–cell communication. Thus, this novel device will provide a versatile platform for intercellular signal transduction studies and high throughput drug screening.

Intercellular Calcium Waves in Vascular Smooth Muscle Cells

Muscular arteries are able to actively modify their diameter by modulating the tone of the smooth muscle cells (SMCs) located within the arterial wall. The contractile state of the SMCs is regulated by cytosolic calcium transients and propagates as a wave over a significant distance along the vessel.We studied the intercellular calcium wave propagation in primary cultured SMCs from rat mesenteric arteries. By using photolithography technique, two types of in vitro cells networks were developed: lines and loop patterns. We have recorded and analyzed calcium response and membrane potential variations for each cell, induced by single cell mechanical stimulus. The loop network was more efficient in transmitting the calcium signals from cell to cell. The calcium wave propagation through gap junctions was spatially limited in line patterns. Longer distance in calcium propagation was obtained in the presence of angiotensin II.This study provides new experimental data supporting the idea that mechanical stimulation evokes a membrane potential depolarization which propagates to neighboring cells. The electrical depolarization is followed by a fast calcium entry that triggers calcium release from intracellular stores. In addition, these results suggest that increasing the expression of the gap junction protein connexin 43 by angiotensin II treatment, facilitates longer distance propagation of calcium waves in primary cultured SMCs.

Identification of Additional Calmodulin Binding Regions in Connexin43

Gap junction, allowing the intercellular transmission of molecules through its specialized cell membrane channels, plays a major role in intercellular calcium signaling between adjacent cells. Connexin43 (Cx43), the most ubiquitous connexin, belongs to α family of gap junction proteins expressed in heart where are essential for normal heart development. Calmodulin (CaM) has been implicated in mediating the Ca2+-dependent regulation of gap junctions. We have reported CaM binding site in the second half of intracellular loop of Cx43. In this study, two additional CaM binding regions in cytosol loop and C-termini of Cx43 have been identified by biophysical studies. Our results indicate that in the presence of Ca2+, synthesized Cx peptide fragments encompassing predicted CaM binding regions are able to bind with high affinity to CaM using NMR spectroscopy. Our results elucidate the molecular level of regulation of Cx43 by multiple CaM targeting regions and provide insight into molecular basis of gap junction gating mechanism.

Dysferlin Interacts with Histone Deacetylase 6 and Increases alpha-Tubulin Acetylation

Dysferlin is a multi-C2 domain transmembrane protein involved in a plethora of cellular functions, most notably in skeletal muscle membrane repair, but also in myogenesis, cellular adhesion and intercellular calcium signaling. We previously showed that dysferlin interacts with alpha-tubulin and microtubules in muscle cells. Microtubules are heavily reorganized during myogenesis to sustain growth and elongation of the nascent muscle fiber. Microtubule function is regulated by post-translational modifications, such as acetylation of its alpha-tubulin subunit, which is modulated by the histone deacetylase 6 (HDAC6) enzyme. In this study, we identified HDAC6 as a novel dysferlin-binding partner. Dysferlin prevents HDAC6 from deacetylating alpha-tubulin by physically binding to both the enzyme, via its C2D domain, and to the substrate, alpha-tubulin, via its C2A and C2B domains. We further show that dysferlin expression promotes alpha-tubulin acetylation, as well as increased microtubule resistance to, and recovery from, Nocodazole- and cold-induced depolymerization. By selectively inhibiting HDAC6 using Tubastatin A, we demonstrate that myotube formation was impaired when alpha-tubulin was hyperacetylated early in the myogenic process; however, myotube elongation occurred when alpha-tubulin was hyperacetylated in myotubes. This study suggests a novel role for dysferlin in myogenesis and identifies HDAC6 as a novel dysferlin-interacting protein.

Brief review on systematic hypothermia for the protection of central nervous system during aortic arch surgery: a double-sword tool?

Antegrade selective cerebral perfusion in conjunction with hypothermia attenuate postoperative neurological injury, which in turn still remains the main cause of mortality and morbidity following aortic arch surgery. Hypothermic circulatory arrest however could be a useful tool during arch surgery, surgery for chronic thromboembolic disease, air on the arterial line during CPB, during cavotomy for extraction of renal cell carcinoma with level IV extension, or when dealing with difficult trauma to the SVC or IVC. Cerebral protective effects with hypothermic procedures including inhibition of neuron excitation, and discharge of excitable amino acids, and thereby, prevention of an increase in intercellular calcium ions, hyperoxidation of lipids in cell membranes, and free radical production.The authors are briefly discussing the fundamental principles of using hypothermia as an adjunct tool of the cardiothoracic surgeon's practice. The relationship between temperature, flow, metabolic requirements and adverse effects is addressed.

Gap junction‐mediated calcium waves define communication networks among murine postnatal neural progenitor cells

In the postnatal neurogenic niche, two populations of astrocyte-like cells (B cells) persist, one acting as neural progenitor cells (NPCs, B1 cells) and one forming a structural boundary between the neurogenic niche and the striatum (B2 cells, niche astrocytes). Despite being viewed as two distinct entities, we found that B1 and B2 cells express the gap junction protein connexin 43 and display functional coupling involving 50-60 cells. Using neonatal electroporation to label slowly cycling radial glia-derived B1 cells, which send a basal process onto blood vessels, we further confirmed dye coupling between NPCs. To assess the functionality of the coupling, we used calcium imaging in a preparation preserving the three-dimensional architecture of the subventricular zone. Intercellular calcium waves were observed among B cells. These waves travelled bidirectionally between B1 and B2 cells and propagated on blood vessels. Inter-B-cell calcium waves were absent in the presence of a gap junction blocker but persisted with purinergic receptor blockers. These findings show that privileged microdomains of communication networks exist among NPCs and niche astrocytes. Such functional coupling between these two cell types suggests that niche astrocytes do not merely have a structural role, but may play an active role in shaping the behavior of NPCs.

Preeclampsia activates 15-lipoxygenase and its metabolite 15-hydroxyeicosatetraenoic acid enhances constriction in umbilical arteries

Objective To compare the differential expression of 15-lipoxygenase (15-LO) isoenzymes, 15-LO-1 and 15-LO-2 in preeclampsia (PE), and normal pregnancy and its metabolite 15-hydroxyeicosatetraenoic acid (15-HETE) on the vasoconstriction of human umbilical artery (HUA) rings. Study design We performed western blotting and isometric tension studies and t-test analysis on data from 6 women with normal pregnancy and 8 women with PE. Results Expressions of 15-LO-1 and 15-LO-2 in placentas and HUA rings in PE increased more than that in normal groups ( P<0.01). 15-HETE increased HUA rings tension in a dose-dependence manner, which were significantly greater in PE than in normal pregnant controls ( P<0.01). However, the constriction of HUA rings was completely eliminated by 2-aminoethoxydiphenyl borate (2-APB) in both normal pregnancy and PE ( P<0.01) and attenuated partly by nifedipine in dose-dependence in normal pregnancy (10 −8 mol/L P>0.05; 10 −7, 10 −6 mol/L P<0.05) and in PE ( P<0.01). Conclusion PE upregulates 15-LO pathway via 15-HETE, which increased intercellular calcium level to cause constriction of HUA rings.

Silencing MaxiK Activity in Corporal Smooth Muscle Cells Initiates Compensatory Mechanisms to Maintain Calcium Homeostasis

The MaxiK potassium channel is regulated by voltage and intracellular calcium, and plays a critical role in regulating intracellular calcium concentration ([Ca(2+) ](i)), which is the ultimate determinant of smooth muscle tone. Tight control of corpus cavernosum smooth muscle (CCSM) tone is critically important and misregulation can result in erectile dysfunction. Because of the tight functional linkage of MaxiK and calcium channel activity, the aim of this study was to determine the effects of silencing and pharmacological inhibition of MaxiK on calcium homeostasis and intercellular calcium signaling in CCSM cells. We compared changes in the basal intracellular [Ca(2+) ](i) and parameters defining intercellular calcium wave (ICW) spread in 48 hours MaxiK silenced CCSM cells vs. acute blockade of the channel with iberiotoxin. To analyze changes occurring in gene expression we performed micro-array analysis following MaxiK silencing for 48 hours. Changes in Fura-2 fluorescence intensities were measured to evaluate basal [Ca(2+) ](i) levels and ICW parameters. Microarray analysis of global gene expression was performed. Forty-eight hours after MaxiK silencing the basal [Ca(2+) ](i) , the ICW amplitude and spread among CCSM cells were not markedly different in silenced compared to mock transfected controls, whereas short-term blockade significantly increased basal [Ca(2+) ](i) level and amplified Ca(2+) signaling among CCSM cells. Micro-array analysis showed that several genes within Ca(2+) homeostasis and smooth muscle tone regulation pathways had significantly altered expression. Our results indicate that while short-term blockade of the MaxiK channel is associated with an increase in basal [Ca(2+) ](i), Ca(2+) homeostasis is restored during the 48 hours period following silencing. We hypothesize that the different pathways regulating [Ca(2+) ](i) and CCSM tone are linked through molecular crosstalk and that their coordinated regulation is part of a compensatory mechanism aimed to maintain Ca(2+) homeostasis and CCSM tone.

Intercellular calcium waves in glial cells with bistable dynamics

A two-dimensional model is proposed for intercellular calcium (Ca2 +) waves with Ca2 +-induced IP3 regeneration and the diffusion of IP3 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 Ca2 + oscillation state coexisting with a fixed point, can cause a prolonged plateau of Ca2 + signals in the cells nearby the stimulated cell when the cell network responds to the local mechanical stimulation.