Narrow Extracellular Spaces Research Articles

Lipid Monolayer on Cell Surface Protein Templates Functional Extracellular Lipid Assembly.

When the ancestors of men moved from aquatic habitats to the drylands, their evolutionary strategy to restrict water loss is to seal the skin surface with lipids. It is unknown how these rigid ceramide-dominated lipids with densely packed chains squeeze through narrow extracellular spaces and how they assemble into their complex multilamellar architecture. Here it is shown that the human corneocyte lipid envelope, a monolayer of ultralong covalently bound lipids on the cell surface protein, templates the functional barrier assembly by partly fluidizing and rearranging the free extracellular lipids in its vicinity during the sculpting of a functional skin lipid barrier. The lipid envelope also maintains the fluidity of the extracellular lipids during mechanical stress. This local lipid fluidization does not compromise the permeability barrier. The results provide new testable hypotheses about epidermal homeostasis and the pathophysiology underlying diseases with impaired lipid binding to corneocytes, such as congenital ichthyosis. In a broader sense, this lipoprotein-mediated fluidization of rigid (sphingo)lipid patches may also be relevant to lipid rafts and cellular signaling events and inspire new functional materials.

Role of aquaporins in corneal healing post chemical injury

Aquaporins (AQPs) are transmembrane water channel proteins that regulate the movement of water through the plasma membrane in various tissues including cornea. The cornea is avascular and has specialized microcirculatory mechanisms for homeostasis. AQPs regulate corneal hydration and transparency for normal vision. Currently, there are 13 known isoforms of AQPs that can be subclassified as orthodox AQPs, aquaglyceroporins (AQGPs), or supraquaporins (SAQPs)/unorthodox AQPs. AQPs are implicated in keratocyte function, inflammation, edema, angiogenesis, microvessel proliferation, and the wound-healing process in the cornea. AQPs play an important role in wound healing by facilitating the movement of corneal stromal keratocytes by squeezing through tight stromal matrix and narrow extracellular spaces to the wound site. Deficiency of AQPs can cause reduced concentration of hepatocyte growth factor (HGF) leading to reduced epithelial proliferation, reduced/impaired keratocyte migration, reduced number of keratocytes in the injury site, delayed and abnormal wound healing process. Dysregulated AQPs cause dysfunction in osmolar homeostasis as well as wound healing mechanisms. The cornea is a transparent avascular tissue that constitutes the anterior aspect of the outer covering of the eye and aids in two-thirds of visual light refraction. Being the outermost layer of the eye, the cornea is prone to injury. Of the 13 AQP isoforms, AQP1 is expressed in the stromal keratocytes and endothelial cells, and AQP3 and AQP5 are expressed in epithelial cells in the human cornea. AQPs can facilitate wound healing through aid in cellular migration, proliferation, migration, extracellular matrix (ECM) remodeling and autophagy mechanism. Corneal wound healing post-chemical injury requires an integrative and coordinated activity of the epithelium, stromal keratocytes, endothelium, ECM, and a battery of cytokines and growth factors to restore corneal transparency. If the chemical injury is mild, the cornea will heal with normal clarity, but severe injuries can lead to partial and/or permanent loss of corneal functions. Currently, the role of AQPs in corneal wound healing is poorly understood in the context of chemical injury. This review discusses the current literature and the role of AQPs in corneal homeostasis, wound repair, and potential therapeutic target for acute and chronic corneal injuries.

Afterhyperpolarization potential modulated by local [K+]o in K+ diffusion-restricted extracellular space in the central clock of suprachiasmatic nucleus

BackgroundIntercellular coupling is essential for the suprachiasmatic nucleus (SCN) to serve as a coherent central clock. Synaptic release of neurotransmitters and neuropeptides is critical for synchronizing SCN neurons. However, intercellular coupling via non-synaptic mechanisms has also been demonstrated. In particular, the abundant perikaryal appositions with morphological specializations in the narrow extracellular space (ECS) may hinder molecular diffusion to allow for ion accumulation or depletion. MethodsThe SCN neurons were recorded in the whole-cell current-clamp mode, with pipette filled with high (26 mM)-Na+ or low (6 mM)-Na+ solution. ResultsCells recorded with high-Na+ pipette solution could fire spontaneous action potentials (AP) with peak AHP more negative than the calculated value of K+ equilibrium potential (EK) and with peak AP more positive than calculated ENa. Cells recorded with low-Na+ pipette solution could also have peak AHP more negative than calculated EK. In contrast, the resting membrane potential (RMP) was always less negative to calculated EK. The distribution and the averaged amplitude of peak AHP, peak AP, or RMP was similar between cells recorded with high-Na+ and low-Na+ solution pipette. In a number of cells, the peak AHP could increase from more positive to become more negative than calculated EK spontaneously or after treatments to hyperpolarize the RMP. TTX blocked the Na+ -dependent APs and tetraethylammonium (TEA), but not Ba2+ or Cd2+, markedly reduced the peak AHP. Perforated-patch cells could also but rarely fire APs with peak AHP more negative than calculated EK. ConclusionThe result of peak AHP negative to calculated EK indicates that local [K+]o sensed by the TEA-sensitive AHP K+ channels must be lower than bulk [K+]o, most likely due to K+ clearance from K+ diffusion-restricted ECS by the Na+/K+-ATPase. The K+ diffusion-restricted ECS may allow for K+-mediated ionic interactions among neurons to regulate SCN excitability.

Neutrophils and aquatic pathogens.

IntroductionNeutrophilic granulocytes are short‐lived cells continuously circulating in the vascular system of vertebrates. They play a basic and decisive role in the innate immune defence of the hosts against all types of pathogenic microorganisms.MethodsBased on a literature study, the functions of neutrophils and cells with similar functions are described. The study places special emphasis on organisms in the aquatic environment and the pathogens occurring in that particular environment.ResultsThe evolutionary origin of this specific cell type is not clear, but its most basic traits (recognition of foreign elements, extracellular trap release, phagocytosis and elimination of ingested material) are found in phagocytes in members of evolutionary ancient invertebrate groups spanning from amoebae, sponges, sea‐anemones, mollusks (snails and mussels), arthropods (crustaceans and insects) to echinoderms (sea stars and sea urchins). Their functions as innate immune sentinels and effector cells in these groups are well described. Neutrophilic granulocytes with elongated and lobed nuclei (possibly allowing cell movements through narrow extracellular spaces and leaving space for phagosomes) occur in vertebrates including fish, amphibians, reptiles, birds and mammals although the morphology of the nucleus, stainability of cytoplasmic granula, and the antimicrobial armament vary among groups. Following the pathogen invasion of a fish host, the neutrophils migrates from the vascular system into the infection focus. They apply their PRRs (including TLRs) to recognize the invader as non‐self, produce netosis by casting extracellular chromatin containing traps in the microenvironment. These nets assist the immobilization of invading microbes and prevents their further spread. The cells attach to and engulf the microbes by phagocytosis, whereafter they eliminate the pathogen in phagolysosomes equipped with a range of killing mechanisms and attract, by release of chemokines, additional immune cells (monocytes, macrophages and lymphocytes) to the site of invasion. Their role in innate immunity of fish hosts towards aquatic pathogens has been elucidated by in vivo and in vitro studies. Neutrophils interact with virus (e.g. IPNV and VHSV), bacteria (e.g. Aeromonas, Vibrio, Edwardsiella, Mycobacterium and Renibacterium) and parasites, including monogeneans (Gyrodactylus), cestodes (Diphyllobothrium), trematodes (Diplostomum) and ciliates (Ichthyophthirius and Philasterides). Despite the decisive function of neutrophils in innate immunity and early protection, the excessive production of ROS, RNS and NETs may lead to pathological disturbances in the host, which are exacerbated if the pathogens evolve immune evasion mechanisms.ConclusionNeutrophils in aquatic organisms play a central role in innate immunity but may serve as a toll and a support in acquired protection. The strong impact of the cellular reactions not only on pathogen but also on host tissues emphasizes that an optimal immune reaction is balanced, involves targeted and specific effector mechanisms, which leaves a minimum of collateral damage in host organs.

Sodium channels and the intercalated disk - it is all about location, location, location.

Sodium channels and the intercalated disk - it is all about location, location, location.

Signaling Determinants of Glioma Cell Invasion.

Tumor cell invasiveness is a critical challenge in the clinical management of glioma patients. In addition, there is accumulating evidence that current therapeutic modalities, including anti-angiogenic therapy and radiotherapy, can enhance glioma invasiveness. Glioma cell invasion is stimulated by both autocrine and paracrine factors that act on a large array of cell surface-bound receptors. Key signaling elements that mediate receptor-initiated signaling in the regulation of glioblastoma invasion are Rho family GTPases, including Rac, RhoA and Cdc42. These GTPases regulate cell morphology and actin dynamics and stimulate cell squeezing through the narrow extracellular spaces that are typical of the brain parenchyma. Transient attachment of cells to the extracellular matrix is also necessary for glioblastoma cell invasion. Interactions with extracellular matrix components are mediated by integrins that initiate diverse intracellular signalling pathways. Key signaling elements stimulated by integrins include PI3K, Akt, mTOR and MAP kinases. In order to detach from the tumor mass, glioma cells secrete proteolytic enzymes that cleave cell surface adhesion molecules, including CD44 and L1. Key proteases produced by glioma cells include uPA, ADAMs and MMPs. Increased understanding of the molecular mechanisms that control glioma cell invasion has led to the identification of molecular targets for therapeutic intervention in this devastating disease.

A Bidomain Model for Lens Microcirculation

There exists a large body of research on the lens of the mammalian eye over the past several decades. The objective of this work is to provide a link between the most recent computational models and some of the pioneering work in the 1970s and 80s. We introduce a general nonelectroneutral model to study the microcirculation in the lens of the eye. It describes the steady-state relationships among ion fluxes, between water flow and electric field inside cells, and in the narrow extracellular spaces between cells in the lens. Using asymptotic analysis, we derive a simplified model based on physiological data and compare our results with those in the literature. We show that our simplified model can be reduced further to the first-generation models, whereas our full model is consistent with the most recent computational models. In addition, our simplified model captures in its equations the main features of the full computational models. Our results serve as a useful link intermediate between the computational models and the first-generation analytical models. Simplified models of this sort may be particularly helpful as the roles of similar osmotic pumps of microcirculation are examined in other tissues with narrow extracellular spaces, such as cardiac and skeletal muscle, liver, kidney, epithelia in general, and the narrow extracellular spaces of the central nervous system, the “brain.” Simplified models may reveal the general functional plan of these systems before full computational models become feasible and specific.

Hyaluronan metabolism enhanced during epidermal differentiation is suppressed by vitamin C.

Hyaluronan is a large, linear glycosaminoglycan present throughout the narrow extracellular space of the vital epidermis. Increased hyaluronan metabolism takes place in epidermal hypertrophy, wound healing and cancer. Hyaluronan is produced by hyaluronan synthases and catabolized by hyaluronidases, reactive oxygen species and KIAA1199. To investigate the changes in hyaluronan metabolism during epidermal stratification and maturation, and the impact of vitamin C on these events. Hyaluronan synthesis and expression of the hyaluronan-related genes were analysed during epidermal maturation from a simple epithelium to a fully differentiated epidermis in organotypic cultures of rat epidermal keratinocytes using quantitative reverse transcriptase polymerase chain reaction, immunostaining and Western blotting, in the presence and absence of vitamin C. With epidermal stratification, both the production and the degradation of hyaluronan were enhanced, resulting in an increase of hyaluronan fragments of various sizes. While the mRNA levels of Has3 and KIAA1199 remained stable during the maturation, Has1, Has2 and Hyal2 showed a transient upregulation during stratification, Hyal1 transcription remained permanently increased and transcription of the hyaluronan receptor, Cd44, decreased. At maturation, vitamin C downregulated Has2, Hyal2 and Cd44, whereas it increased high-molecular-mass hyaluronan in the epidermis, and reduced small fragments in the medium, suggesting stabilization of epidermal hyaluronan. Epidermal stratification and maturation is associated with enhanced hyaluronan turnover, and release of large amounts of hyaluronan fragments. The high turnover is suppressed by vitamin C, which is suggested to enhance normal epidermal differentiation in part through its effect on hyaluronan.

Shear-Induced Amyloid Formation in the Brain: I. Potential Vascular and Parenchymal Processes.

Shear distortion of amyloid-beta (Aβ) solutions accelerates amyloid cascade reactions that may yield different toxic oligomers than those formed in quiescent solutions. Recent experiments indicate that cerebrospinal fluid (CSF) and interstitial fluid (ISF) containing Aβ flow through narrow brain perivascular pathways and brain parenchyma. This paper suggests that such flow causes shear distortion of Aβ molecules involving conformation changes that may be one of the initiating events in the etiology of Alzheimer’s disease. Aβ shearing can occur in or around brain arteries and arterioles and is suggested as the origin of cerebral amyloid angiopathy deposits in cerebrovascular walls. Comparatively low flow rates of ISF within the narrow extracellular spaces (ECS) of the brain parenchyma are suggested as a possible initiating factor in both the formation of neurotoxic Aβ42 oligomers and amyloid fibrils. Aβ42 in slow-flowing ISF can gain significant shear energy at or near the walls of tortuous brain ECS flow paths, promoting the formation of a shear-distorted, excited state hydrophobic Aβ42* conformation. This Aβ42* molecule could possibly be involved in one of two paths, one involving rapid adsorption to a brain membrane surface, ultimately forming neurotoxic oligomers on membranes, and the other ultimately forming plaque within the ECS flow pathways. Rising Aβ concentrations combined with shear at or near critical brain membranes are proposed as contributing factors to Alzheimer’s disease neurotoxicity. These hypotheses may be applicable in other neurodegenerative diseases, including tauopathies and alpha-synucleinopathies, in which shear-distorted proteins also may form in the brain ECS.

High Resolution Imaging Reveals Hematopoietic Stem Cells in the Perivascular Niche Are Anchored to Mesenchymal Stromal Cells That Orient Their Divisions

Hematopoietic stem cells (HSC) reside in a highly structured microenvironment called the niche. There is two-way communication between a stem cell and its niche that determines important cell fate decisions. HSC must remain quiescent to persist throughout life but also divide and contribute progenitors that will replenish the blood supply. Although there have been a number of elegant studies that have imaged the mammalian bone marrow, we still lack a high-resolution real-time view of endogenous HSC behaviors and interactions within the niche. To overcome these challenges, we developed a transgenic zebrafish line that expresses GFP or mCherry in HSC. We generated this line using the previously described mouse Runx1 +23 kb intronic enhancer. We confirmed the purity of these stem cells by adult-to-adult limiting dilution transplantation with as few as one cell. Based on long-term multi-lineage engraftment, we estimated a stem cell purity of approximately 1/35, which is similar to the KSL (Kit+Sca1+Lin-) population in mouse. Using a novel embryo-to-embryo transplantation assay that is unique to zebrafish, we estimated an even higher stem cell purity of 1/2. These experiments have defined the most pure HSC population in the zebrafish. Using this novel transgenic reporter we have tracked HSC as they migrate in the live zebrafish embryo. This allowed us to image HSC as they interact with other cell types in their microenvironment, including endothelial cells and mesenchymal stromal cells. We have shown that a small group of endothelial cells remodel around a single HSC soon after it lodges in the niche. Recently, we have also found that a single stromal cell can anchor an HSC as it divides. In most cases, we observed that an HSC divides perpendicular to the stromal cell, with one daughter cell remaining attached to the stromal cell and the other migrating away. To gain a much higher resolution view of these cellular events than is possible with confocal microscopy we looked for an alternative approach. A combined method is called “Correlative Light and Electron Microscopy” (CLEM), and involves identification of cells by confocal microscopy, followed by processing of the same sample for EM scanning. We have applied this method by: 1) tracking endogenous HSC in the live embryo; 2) fixing the same embryo for serial block-face scanning EM; 3) reconstructing 3D models from high resolution serial EM sections. We used easily visible blood vessels as anatomical markers that allowed us to pinpoint a single cell in a relatively large block of scanned tissue. As expected, the identified HSC was round, had a distinctive large nucleus, scant cytoplasm, and ruffled membrane. The HSC was surrounded by a small group of 5-6 endothelial cells, as predicted from our confocal live imaging. However at this very high resolution (10 nm/pixel), we could see that only part of the HSC surface was contacted and wrapped by an endothelial cell. Other regions of the HSC surface were contacted by small endothelial cell protrusions. Much of the HSC surface was surrounded by a narrow extracellular space with endothelial and stromal cells lying opposite. Strikingly, we were able to identify the firm anchored attachment between a single stromal cell and HSC that we showed previously oriented the plane of division. By combining confocal live imaging of a novel zebrafish HSC reporter, and serial block-face scanning EM, we have created the first high-resolution 3D model of an endogenous stem cell in its niche. DisclosuresTamplin:Boston Children’s Hospital: Patents & Royalties. Zon:FATE Therapeutics, Inc: Equity Ownership, Membership on an entity’s Board of Directors or advisory committees, Other; Scholar Rock: Equity Ownership, Membership on an entity’s Board of Directors or advisory committees, Other; Stemgent: Equity Ownership, Membership on an entity’s Board of Directors or advisory committees.

Immunolocalization of a beta-defensin (Tu-BD-1) in the skin and subdermal granulocytes of turtles indicate the presence of an antimicrobial skin barrier

The resistance of turtle skin to infections is likely due to the production of a thick corneous layer but also of soluble antimicrobial molecules released in the stratum corneum. The present study has determined the localization of beta-defensin peptides previously identified by biomolecular analysis in turtle skin. After the production of a specific antibody for one peptide indicated as Ts-BD-1, light and ultrastructural localization indicates that the peptide is mainly present in some variably dense primary granules of granulocytes. These cells are sparse in the connective tissues and within blood vessels present in the dermis and subdermal inter-muscular connective tissue. A weak labeling is also present in the epidermis, especially localized in sparse secretory granules discharged among corneocytes of the stratum corneum. This localization suggests that antimicrobial peptides are common constituents of the narrow extracellular spaces located among superficial corneocytes where bacteria or their remnants are frequently detected. The study suggests that turtle beta-defensins are mainly contained in granulocytes involved in the immunological surveillance of the skin and that they become active after skin wounding.

Adenosine Enhances Sweet Taste through A2B Receptors in the Taste Bud

Mammalian taste buds use ATP as a neurotransmitter. Taste Receptor (type II) cells secrete ATP via gap junction hemichannels into the narrow extracellular spaces within a taste bud. This ATP excites primary sensory afferent fibers and also stimulates neighboring taste bud cells. Here we show that extracellular ATP is enzymatically degraded to adenosine within mouse vallate taste buds and that this nucleoside acts as an autocrine neuromodulator to selectively enhance sweet taste. In Receptor cells in a lingual slice preparation, Ca(2+) mobilization evoked by focally applied artificial sweeteners was significantly enhanced by adenosine (50 μM). Adenosine had no effect on bitter or umami taste responses, and the nucleoside did not affect Presynaptic (type III) taste cells. We also used biosensor cells to measure transmitter release from isolated taste buds. Adenosine (5 μM) enhanced ATP release evoked by sweet but not bitter taste stimuli. Using single-cell reverse transcriptase (RT)-PCR on isolated vallate taste cells, we show that many Receptor cells express the adenosine receptor, Adora2b, while Presynaptic (type III) and Glial-like (type I) cells seldom do. Furthermore, Adora2b receptors are significantly associated with expression of the sweet taste receptor subunit, Tas1r2. Adenosine is generated during taste stimulation mainly by the action of the ecto-5'-nucleotidase, NT5E, and to a lesser extent, prostatic acid phosphatase. Both these ecto-nucleotidases are expressed by Presynaptic cells, as shown by single-cell RT-PCR, enzyme histochemistry, and immunofluorescence. Our findings suggest that ATP released during taste reception is degraded to adenosine to exert positive modulation particularly on sweet taste.

Ion channels and tranporters in cancer. 2. Ion channels and the control of cancer cell migration

A hallmark of high-grade cancers is the ability of malignant cells to invade unaffected tissue and spread disease. This is particularly apparent in gliomas, the most common and lethal type of primary brain cancer affecting adults. Migrating cells encounter restricted spaces and appear able to adjust their shape to accommodate to narrow extracellular spaces. A growing body of work suggests that cell migration/invasion is facilitated by ion channels and transporters. The emerging concept is that K+ and Cl− function as osmotically active ions, which cross the plasma membrane in concert with obligated water thereby adjusting a cell's shape and volume. In glioma cells Na+-K+-Cl− cotransporters (NKCC1) actively accumulate K+ and Cl−, establishing a gradient for KCl efflux. Ca2+-activated K+ channels and voltage-gated Cl− channels are largely responsible for effluxing KCl promoting hydrodynamic volume changes. In other cancers, different K+ or even Na+ channels may function in concert with a variety of Cl− channels to support similar volume changes. Channels involved in migration are frequently regulated by Ca2+ signaling, most likely coupling extracellular stimuli to cell migration. Importantly, the inhibition of ion channels and transporters appears to be clinically relevant for the treatment of cancer. Recent preclinical data indicates that inhibition of NKCC1 with an FDA-approved drug decreases neoplastic migration. Additionally, ongoing clinical trials demonstrate that an inhibitor of chloride channels may be a therapy for the treatment of gliomas. Data reviewed here strongly indicate that ion channels are a promising target for the development of novel therapeutics to combat cancer.

Self-Gating Effect at the Rat Neuromuscular Junction

Event Abstract Back to Event Self-Gating Effect at the Rat Neuromuscular Junction Mohammed Mostafizur Rahman1*, Mufti Mahmud1 and Stefano Vassanelli1 1 University of Padova, NeuroChip Lab, Italy The neuromuscular Junction (NMJ) is the place where the axon terminal of a motor neuron and the motor end plate of a muscle fiber's functionality meet creating a synapse to transmit messages from the brain which cause the muscle to contract. The pulse carried by the presynaptic neuron when reaches the synapses causes release of Acetylcholine into the synaptic cleft. The flow of current along the narrow extracellular space between nerve cell and muscular fiber (cleft) could affect the local extracellular electrical potential. As a consequence, voltage-dependent processes may be affected in the adjacent membranes. According to this hypothesis the voltage drop in the cleft increase the opening rate of voltage-gated ion channels. This phenomenon of current passing through weakly opened sodium channels and decreasing the potential of the extracellular space by a positive feedback was previously described in a general cell-to-substrate adhesion contact and termed “self-gating” [1]. To model the rat NMJ a barrel shaped muscle is considered (50 µm diameter × 500 µm long). To form a synapse, an axon terminal (10 µm radius) is connected to the highly-excitable muscle fiber with a synaptic cleft (distance: 50 nm). The potential at the cleft (Vj) is calculated using total sodium conductance of the endplate motoneuron (Glob) and the resistance of the cleft. The endplate sodium current (EPINa) is calculated using the potential of the junction and the Glob. The Acetylcholine Receptor current (AChR) together with the EPINa act as stimulus current for the Hodgkin-Huxley based membrane potential (Vm) calculation at the muscle fiber. The conductance associated to voltage-gated sodium channels depends on the voltage drop of Vm–Vj across the membrane [1]. The Vj was calculated for different membrane potentials which showed that the voltage-dependent sodium conductance at the NMJ undergoes self-gating due to the drop of Vj in the synaptic cleft and in the following depolarization of the potential (Vm–Vj) across the membrane. During the self-gating condition a switching behavior was noticed in the sodium channels conductance: from low to high during depolarization and from high to low during repolarization of membrane potential. At this time bistability and hysteresis of sodium channels occurred which explains the transmission efficiency of the NMJ under self-gating condition. During self-gating, the channels remain open for a longer period letting inward currents to flow during repolarization. This sustained current flow favors cell depolarization and action potential firing in the muscle. The higher number of open sodium channels during the self-gating increases sodium current intensity during the first phase of action potential which directly affects the depolarization of the cell, thus, making it fire at a lower firing threshold. Also, the muscle unit’s firing rate increases under self-gating condition due to the fact that the longer inactivation time is compensated by the switching behavior of the sodium current. This allows fast and larger entrance of sodium current in every successive spike. [1] P. Fromherz, Self-Gating of Ion Channels in Cell Adhesion. Phys. Rev. Lett., 78(21): 4131-4134, 1997. Figure 1 Keywords: computational neuroscience, General neuroinformatics Conference: 4th INCF Congress of Neuroinformatics, Boston, United States, 4 Sep - 6 Sep, 2011. Presentation Type: Poster Presentation Topic: General neuroinformatics Citation: Rahman M, Mahmud M and Vassanelli S (2011). Self-Gating Effect at the Rat Neuromuscular Junction. Front. Neuroinform. Conference Abstract: 4th INCF Congress of Neuroinformatics. doi: 10.3389/conf.fninf.2011.08.00018 Copyright: The abstracts in this collection have not been subject to any Frontiers peer review or checks, and are not endorsed by Frontiers. They are made available through the Frontiers publishing platform as a service to conference organizers and presenters. The copyright in the individual abstracts is owned by the author of each abstract or his/her employer unless otherwise stated. Each abstract, as well as the collection of abstracts, are published under a Creative Commons CC-BY 4.0 (attribution) licence (https://creativecommons.org/licenses/by/4.0/) and may thus be reproduced, translated, adapted and be the subject of derivative works provided the authors and Frontiers are attributed. For Frontiers’ terms and conditions please see https://www.frontiersin.org/legal/terms-and-conditions. Received: 17 Oct 2011; Published Online: 19 Oct 2011. * Correspondence: Dr. Mohammed Mostafizur Rahman, University of Padova, NeuroChip Lab, Padova, Italy, mostafiz.math@gmail.com Login Required This action requires you to be registered with Frontiers and logged in. To register or login click here. Abstract Info Abstract The Authors in Frontiers Mohammed Mostafizur Rahman Mufti Mahmud Stefano Vassanelli Google Mohammed Mostafizur Rahman Mufti Mahmud Stefano Vassanelli Google Scholar Mohammed Mostafizur Rahman Mufti Mahmud Stefano Vassanelli PubMed Mohammed Mostafizur Rahman Mufti Mahmud Stefano Vassanelli Related Article in Frontiers Google Scholar PubMed Abstract Close Back to top Javascript is disabled. Please enable Javascript in your browser settings in order to see all the content on this page.

Role of cation‐chloride cotransporter activity in glial tumor proliferation and migration

Primary central nervous system tumors account for 2.2% of all cancer‐related deaths. Glioblastoma multiformes, the most aggressive form of brain tumor have relatively short median survival times (9–12 months in most cases). Like most aggressive cancers, malignant gliomas exhibit a strong tendency to migrate through narrow extracellular spaces in brain tissue by the functional shape‐volume changes mediated by net salt fluxes across cell membranes. The electroneutral‐coupled movement of ions by the cation‐chloride cotransporter family may, therefore, have an important role in this process. In this study, we used the ‘scratch wound’ assay and time‐lapse microscopy to examine the role of cotransporter activity on the proliferation and migration of rat C6 glioma cells, an immortalized astrocytoma tumor cell line. Untreated C6 cells completely filled the ‘wound area’ within 6 hours, significantly faster than their doubling time. Application of 2 mM furosemide (loop diuretic) completely inhibited the ‘closure’ of the wound area. Four hours after removal of the loop diuretic, the C6 cells were able to migrate into the ‘wound area’, similar to untreated cells. These studies suggest a significant role for cation‐chloride cotransporter activity in the physiology of primary CNS tumors and may help identify novel targets for the development of drugs and therapies to combat these aggressive and fatal neoplasms.

La pompe à sodium pourrait représenter une nouvelle cible pour le traitement des glioblastomes

Malignant gliomas of which glioblastomas represent the ultimate grade of malignancy are characterized by dismal prognoses because malignant glioma cells present both important proliferation and neoangiogenesis processes and can actively migrate through the narrow extra-cellular spaces in the brain, often travelling relatively long distances, making them elusive targets for effective surgical management. Invasive malignant glioma cells show a decrease in their proliferation rates and a relative resistance to apoptosis (type I programmed cell death) as compared to the highly cellular centre of the tumour, and this may contribute to their resistance to conventional proapoptotic chemotherapy and radiotherapy. The multidisciplinary up to date treatment for glioblastoma patients combined maximal surgical removal of the tumor with postoperative radiotherapy and concomitant chemotherapy with temozolomide. Temozolomide is a proautophagic (type II programmed cell death) drug and can thus circumvent part of the glioblastoma resistance to apoptosis. Another way to potentially overcome apoptosis resistance is to decrease the migration of malignant glioma cells in the brain, which then should restore a level of sensitivity to proapoptotic drugs. The Na(+)/K(+)-ATPase or sodium pump is an ion transporter which in addition to exchanging cations, is also the ligand for cardenolides and is directly involved in the migration of cancer cells in general and of glioma cells in particular. We have shown that the alpha1 subunit of the sodium pump is highly expressed in glioma cells compared to normal brain tissues and we are the first to propose the alpha1 subunit of the sodium pump as a new target in the context of malignant glioma treatment. Using a novel cardenolide with unique structural features, which markedly inhibits sodium pump activity and binds to the alpha1 subunit, we have shown marked anti-proliferative and anti-migratory effects on human glioblastoma cells (and other cancer cell types). We have characterized at least partially the anti-cancer mechanism of action of the novel cardenolide. It is a ligand of the alpha1 subunit of the pump which impairs the proliferation and migration of glioblastoma cells by disorganizing the actin cytoskeleton and inducing severe autophagic process in glioblastoma cells. Collectively, these data suggests that the novel cardenolide is an attractive candidate for preclinical and clinical development, at least in the area of glioblastoma. This compound should reach phase I clinical trials in the summer of 2008.

Identification and functional significance of a brain aquaporin‐4/Na+, K+‐ATPase/mGluR5 macromolecular complex

The water channel aquaporin‐4 (AQP4) is expressed in astrocytes, which play a major role in clearing the narrow extracellular space (ES) between neurons and astrocyte endfeet from K+ and glutamate released during neuronal activity. Compelling evidence suggests that AQP4 may play a role in the K+ clearance. Here we show that AQP4 co‐immunoprecipitates with Na+, K+‐ATPase and inwardly rectifying K+ channel Kir4.1; transporters of key importance for the K+ clearance. Using GST pull down assay, we identified the MVAFKGVWTQ of the AQP4 N‐terminus as the site for interaction with Na+,K+‐ATPase. The metabotropic glutamate receptor mGluR5 was also pulled down by AQP4. Using rubidium uptake and astrocyte water permeability measurements, we found that the mGluR agonist DHPG increased both Na+,K+‐ATPase‐mediated K+ transport and AQP4‐mediated water transport. Finally, we created a mathematical model, of K+ transport across the plasma membrane in the ES. This model predicts that AQP4‐driven water transport will facilitate K+ clearance and stabilize the membrane potential. Thus we have demonstrated a multiprotein K+‐transporting microdomain in the astrocyte that includes AQP4. This microdomain is functionally regulated by glutamate and serves to optimize the clearance of K+ from the ES.

The vicissitudes of the pacemaker current I Kdd of cardiac purkinje fibers

The mechanisms underlying the pacemaker current in cardiac tissues is not agreed upon. The pacemaker potential in Purkinje fibers has been attributed to the decay of the potassium current I (Kdd). An alternative proposal is that the hyperpolarization-activated current I (f) underlies the pacemaker potential in all cardiac pacemakers. The aim of this review is to retrace the experimental development related to the pacemaker mechanism in Purkinje fibers with reference to findings about the pacemaker mechanism in the SAN as warranted. Experimental data and their interpretation are critically reviewed. Major findings were attributed to K(+) depletion in narrow extracellular spaces which would result in a time dependent decay of the inward rectifier current I (K1). In turn, this decay would be responsible for a "fake" reversal of the pacemaker current. In order to avoid such a postulated depletion, Ba(2+) was used to block the decay of I (K1). In the presence of Ba(2+) the time-dependent current no longer reversed and instead increased with time and more so at potentials as negative as -120 mV. In this regard, the distinct possibility needs to be considered that Ba(2+) had blocked I (Kdd) (and not only I (K1)). That indeed this was the case was demonstrated by studying single Purkinje cells in the absence and in the presence of Ba(2+). In the absence of Ba(2+), I (Kdd) was present in the pacemaker potential range and reversed at E (K). In the presence of Ba(2+), I (Kdd) was blocked and I (f) appeared at potentials negative to the pacemaker range. The pacemaker potential behaves in a manner consistent with the underlying I (Kdd) but not with I (f). The fact that I (f) is activated on hyperpolarization at potential negative to the pacemaker range makes it suitable as a safety factor to prevent the inhibitory action of more negative potentials on pacemaker discharge. It is concluded that the large body of evidence reviewed proves the pacemaker role of I (Kdd) (but not of I (f)) in Purkinje fibers.

A Cell-Semiconductor Synapse: Transistor Recording of Vesicle Release in Chromaffin Cells

The release of dense-core vesicles in bovine chromaffin cells is a model for the presynaptic process in neurons. It is usually studied by microamperometry of catecholamines with carbon fibers. Here we introduce transistor recording as a tool to study vesicle release. When we stimulate a chromaffin cell placed on a field-effect transistor, the gate voltage exhibits peaks that correlate with a simultaneously performed amperometric recording. We attribute the transistor signal to a release of protons from the extruded matrix of vesicles that lowers the extracellular pH and changes the electrical surface potential of the gate oxide. The rise time of the transistor signals is similar to that of amperometric responses, whereas their duration is distinctly longer. In a model computation, the rise time is identified with the extrusion of vesicle matrix into the narrow extracellular space between cell and gate oxide, and the decay time is attributed to pH equilibration through slow diffusion in the extruded matrix. Because the transistor recording relies on protons, it can be applied to acidic vesicles with electrochemically inactive hormones or transmitters.

The Extracellular Electrical Resistivity in Cell Adhesion

The interaction of cells in a tissue depends on the nature of the extracellular matrix. The electrical properties of the narrow extracellular space are unknown. Here we consider cell adhesion mediated by extracellular matrix protein on a solid substrate as a model system. We culture human embryonic kidney (HEK293) cells on silica coated with fibronectin and determine the electrical resistivity in the cell-solid junction ρ J = r J d J by combining measurements of the sheet resistance r J and of the distance d J between membrane and substrate. The sheet resistance is obtained from phase fluorometry of the voltage-sensitive dye ANNINE-5 by alternating-current stimulation from the substrate. The distance is measured by fluorescence interference contrast microscopy. We change the resistivity of the bath in a range from 66 ⁡ Ω ⁡ cm to 750 ⁡ Ω ⁡ cm and find that the sheet resistance r J is proportionally enhanced, but that the distance is invariant around d J = 75 ⁡ nm . In all cases, the resulting resistivity ρ J is indistinguishable from the resistivity of the bath. A similar result is obtained for rat neurons cultured on polylysine. On that basis, we propose a “bulk resistivity in cell adhesion” model for cell-solid junctions. The observations suggest that the electrical interaction between cells in a tissue is determined by an extracellular space with the electrical properties of bulk electrolyte.