Unique Role of Vimentin in the Intermediate Filament Proteins Family.

Intermediate filaments (IFs), being traditionally the least studied component of the cytoskeleton, have begun to receive more attention in recent years. IFs are found in different cell types and are specific to them. Accumulated data have shifted the paradigm about the role of IFs as structures that merely provide mechanical strength to the cell. In addition to this role, IFs have been shown to participate in maintaining cell shape and strengthening cell adhesion. The data have also been obtained that point out to the role of IFs in a number of other biological processes, including organization of microtubules and microfilaments, regulation of nuclear structure and activity, cell cycle control, and regulation of signal transduction pathways. They are also actively involved in the regulation of several aspects of intracellular transport. Among the intermediate filament proteins, vimentin is of particular interest for researchers. Vimentin has been shown to be associated with a range of diseases, including cancer, cataracts, Crohn's disease, rheumatoid arthritis, and HIV. In this review, we focus almost exclusively on vimentin and the currently known functions of vimentin intermediate filaments (VIFs). This is due to the structural features of vimentin, biological functions of its domains, and its involvement in the regulation of a wide range of basic cellular functions, and its role in the development of human diseases. Particular attention in the review will be paid to comparing the role of VIFs with the role of intermediate filaments consisting of other proteins in cell physiology.

Huntingtin and Other Neurodegeneration-Associated Proteins in the Development of Intracellular Pathologies: Potential Target Search for Therapeutic Intervention.

Neurodegenerative diseases are currently incurable. Numerous experimental data accumulated over the past fifty years have brought us closer to understanding the molecular and cell mechanisms responsible for their development. However, these data are not enough for a complete understanding of the genesis of these diseases, nor to suggest treatment methods. It turns out that many cellular pathologies developing during neurodegeneration coincide from disease to disease. These observations give hope to finding a common intracellular target(s) and to offering a universal method of treatment. In this review, we attempt to analyze data on similar cellular disorders among neurodegenerative diseases in general, and polyglutamine neurodegenerative diseases in particular, focusing on the interaction of various proteins involved in the development of neurodegenerative diseases with various cellular organelles. The main purposes of this review are: (1) to outline the spectrum of common intracellular pathologies and to answer the question of whether it is possible to find potential universal target(s) for therapeutic intervention; (2) to identify specific intracellular pathologies and to speculate about a possible general approach for their treatment.

The Cytoplasmic Actins in the Regulation of Endothelial Cell Function.

The primary function of the endothelial cells (EC) lining the inner surface of all vessels is to regulate permeability of vascular walls and to control exchange between circulating blood and tissue fluids of organs. The EC actin cytoskeleton plays a crucial role in maintaining endothelial barrier function. Actin cytoskeleton reorganization result in EC contraction and provides a structural basis for the increase in vascular permeability, which is typical for many diseases. Actin cytoskeleton in non-muscle cells presented two actin isoforms: non-muscle ß-cytoplasmic and γ-cytoplasmic actins (ß-actins and γ-actins), which are encoded by ACTB and ACTG1 genes, respectively. They are ubiquitously expressed in the different cells in vivo and in vitro and the ß/γ-actin ratio depends on the cell type. Both cytoplasmic actins are essential for cell survival, but they perform various functions in the interphase and cell division and play different roles in neoplastic transformation. In this review, we briefly summarize the research results of recent years and consider the features of the cytoplasmic actins: The spatial organization in close connection with their functional activity in different cell types by focusing on endothelial cells.

Synthesis and biological evaluation of novel mono- and bivalent ASGP-R-targeted drug-conjugates.

Asialoglycoprotein receptor (ASGP-R) is a promising biological target for drug delivery into hepatoma cells. Nevertheless, there are only few examples of small-molecule conjugates of ASGP-R selective ligand equipped by a therapeutic agent for the treatment of hepatocellular carcinoma (HCC). In the present work, we describe a convenient and versatile synthetic approach to novel mono- and multivalent drug-conjugates containing N-acetyl-2-deoxy-2-aminogalactopyranose and anticancer drug - paclitaxel (PTX). Several molecules have demonstrated high affinity towards ASGP-R and good stability under physiological conditions, significant in vitro anticancer activity comparable to PTX, as well as good internalization via ASGP-R-mediated endocytosis. Therefore, the conjugates with the highest potency can be regarded as a promising therapeutic option against HCC.

Magnetocontrollability of Fe7C3@C superparamagnetic nanoparticles in living cells.

BACKGROUND: A new type of superparamagnetic nanoparticles with chemical formula Fe7C3@C (MNPs) showed higher value of magnetization compared to traditionally used iron oxide-based nanoparticles as was shown in our previous studies. The in vitro biocompatibility tests demonstrated that the MNPs display high efficiency of cellular uptake and do not affect cyto-physiological parameters of cultured cells. These MNPs display effective magnetocontrollability in homogeneous liquids but their behavior in cytoplasm of living cells under the effect of magnetic field was not carefully analyzed yet. RESULTS: In this work we investigated the magnetocontrollability of MNPs interacting with living cells in permanent magnetic field. It has been shown that cells were capable of capturing MNPs by upper part of the cell membrane, and from the surface of the cultivation substrate during motion process. Immunofluorescence studies using intracellular endosomal membrane marker showed that MNP agglomerates can be either located in endosomes or lying free in the cytoplasm. When attached cells were exposed to a magnetic field up to 0.15 T, the MNPs acquired magnetic moment and the displacement of incorporated MNP agglomerates in the direction of the magnet was observed. Weakly attached or non-attached cells, such as cells in mitosis or after cytoskeleton damaging treatments moved towards the magnet. During long time cultivation of cells with MNPs in a magnetic field gradual clearing of cells from MNPs was observed. It was the result of removing MNPs from the surface of the cell agglomerates discarded in the process of exocytosis. CONCLUSIONS: Our data allow us to conclude for the first time that the magnetic properties of the MNPs are sufficient for successful manipulation with MNP agglomerates both at the intracellular level, and within the whole cell. The structure of the outer shells of the MNPs allows firmly associate different types of biological molecules with them. This creates prospects for the use of such complexes for targeted delivery and selective removal of selected biological molecules from living cells.

Centrosome nucleates numerous ephemeral microtubules and only few of them participate in the radial array.

It is generally accepted that long microtubules (MTs) grow from the centrosome with their minus ends anchored there and plus ends directed towards cell membrane. However, recent findings show this scheme to be an oversimplification. To further analyze the relationship between the centrosome and the MT array we undertook a detailed study on the MTs growing from the centrosome after microinjection of Cy3 labeled tubulin and transfection of cells with EB1-GFP. To evaluate MTs around the centrosome two approaches were used: path photobleaching across the centrosome area (Komarova et al., ) and sequential image subtraction analysis (Vorobjev et al., ). We show that about 50% of MTs had been nucleated at the centrosome are short-living: their mean length was 1.8 ± 0.8 µm and their life span - 7 ± 2 s. MTs initiated from the centrosome also rarely reach cell margin, since their elongation was limited and growth after shortening (rescue) was rare. After initial growth all MTs associated with the centrosome converted to pause or shortening. After pause MTs associated with the centrosome mainly depolymerized via the plus end shortening. Stability of the minus ends of cytoplasmic MTs was the same as for centrosomal ones. We conclude that in fibroblasts (1) the default behavior of free MTs in the cell interior is biased dynamic instability (i.e., random walk of the plus ends with significant positive drift); (2) MTs born at the centrosome show "dynamic instability" type behavior with no boundary; and (3) that the extended radial array is formed predominantly by MTs not associated with the centrosome.

The leading role of microtubules in endothelial barrier dysfunction: disassembly of peripheral microtubules leaves behind the cytoskeletal reorganization.

Disturbance of the endothelial barrier is characterized by dramatic cytoskeleton reorganization, activation of actomyosin contraction and, finally, leads to intercellular gap formation. Here we demonstrate that the edemagenic agent, thrombin, causes a rapid increase in the human pulmonary artery endothelial cell (EC) barrier permeability accompanied by fast decreasing in the peripheral microtubules quantity and reorganization of the microtubule system in the internal cytoplasm of the EC within 5 min of the treatment. The actin stress-fibers formation occurs gradually and the maximal effect is observed relatively later, 30 min of the thrombin treatment. Thus, microtubules reaction develops faster than the reorganization of the actin filaments system responsible for the subsequent changes of the cell shape during barrier dysfunction development. Direct microtubules depolymerization by nocodazole initiates the cascade of barrier dysfunction reactions. Nocodazole-induced barrier disruption is connected directly with the degree of peripheral microtubules depolymerization. Short-term loss of endothelial barrier function occurs at the minimal destruction of peripheral microtubules, when actin filament system is still intact. Specifically, we demonstrate that the EC microtubule dynamics examined by time-lapse imaging of EB3-GFP comets movement has changed under these conditions: microtubule plus ends growth rate significantly decreased near the cell periphery. The microtubules, apparently, are the first target in the circuit of reactions leading to the pulmonary EC barrier compromise. Our results show that dynamic microtubules play an essential role in the barrier function in vitro; peripheral microtubules depolymerization is necessary and sufficient condition for initiation of endothelial barrier dysfunction.

Protein kinase C-α and arginase I mediate pneumolysin-induced pulmonary endothelial hyperpermeability.

Antibiotics-induced release of the pore-forming virulence factor pneumolysin (PLY) in patients with pneumococcal pneumonia results in its presence days after lungs are sterile and is a major factor responsible for the induction of permeability edema. Here we sought to identify major mechanisms mediating PLY-induced endothelial dysfunction. We evaluated PLY-induced endothelial hyperpermeability in human lung microvascular endothelial cells (HL-MVECs) and human lung pulmonary artery endothelial cells in vitro and in mice instilled intratracheally with PLY. PLY increases permeability in endothelial monolayers by reducing stable and dynamic microtubule content and modulating VE-cadherin expression. These events, dependent upon an increased calcium influx, are preceded by protein kinase C (PKC)-α activation, perturbation of the RhoA/Rac1 balance, and an increase in myosin light chain phosphorylation. At later time points, PLY treatment increases the expression and activity of arginase in HL-MVECs. Arginase inhibition abrogates and suppresses PLY-induced endothelial barrier dysfunction by restoring NO generation. Consequently, a specific PKC-α inhibitor and the TNF-derived tonoplast intrinsic protein peptide, which blunts PLY-induced PKC-α activation, are able to prevent activation of arginase in HL-MVECs and to reduce PLY-induced endothelial hyperpermeability in mice. Arginase I (AI)(+/-)/arginase II (AII)(-/-) C57BL/6 mice, displaying a significantly reduced arginase I expression in the lungs, are significantly less sensitive to PLY-induced capillary leak than their wild-type or AI(+/+)/AII(-/-) counterparts, indicating an important role for arginase I in PLY-induced endothelial hyperpermeability. These results identify PKC-α and arginase I as potential upstream and downstream therapeutic targets in PLY-induced pulmonary endothelial dysfunction.

The "Third Violin" in the Cytoskeleton Orchestra-The Role of Intermediate Filaments in the Endothelial Cell's Life.

The endothelium plays an important role in the transcytosis of lipoproteins. According to one of the theories, endothelial injury is a triggering factor for the development of atherosclerosis, and intracellular structures, including components of the endotheliocyte cytoskeleton (microtubules, actin, and intermediate filaments), are involved in its development. In contrast to the proteins of tubulin-based microtubules and actin microfilaments, intermediate filaments are comprised of various tissue-specific protein members. Vimentin, the main protein of endothelial intermediate filaments, is one of the most well-studied of these and belongs to type-III intermediate filaments, commonly found in cells of mesenchymal origin. Vimentin filaments are linked mechanically or by signaling molecules to microfilaments and microtubules by which coordinated cell polarisation and migration are carried out, as well as control over several endotheliocyte functions. Moreover, the soluble vimentin acts as an indicator of the state of the cardiovascular system, and the involvement of vimentin in the development and course of atherosclerosis has been demonstrated. Here we discuss current concepts of the participation of vimentin filaments in the vital activity and functioning of endothelial cells, as well as the role of vimentin in the development of inflammatory processes and atherosclerosis.

Colocalization Analysis of Cytoplasmic Actin Isoforms Distribution in Endothelial Cells.

Actin cytoskeleton is an essential component of living cells and plays a decisive role in many cellular processes. In mammals, ß- and γ-actin are cytoplasmic actin isoforms in non-muscle cells. Despite minor differences in the amino acid sequence, ß- and γ-actin localize in different cell structures and perform different functions. While cytoplasmic ß-actin is involved in many intracellular processes including cell contraction, γ-actin is responsible for cell mobility and promotes tumor transformation. Numerous studies demonstrate that ß- and γ-actin are spatially separated in the cytoplasm of fibroblasts and epithelial cells; this separation is functionally determined. The spatial location of ß/γ-actin in endothelial cells is still a subject for discussion. Using super-resolution microscopy, we investigated the ß/γ-actin colocalization in endotheliocytes and showed that the ß/γ-actin colocalization degree varies widely between different parts of the marginal regions and near the cell nucleus. In the basal cytoplasm, ß-actin predominates, while the ratio of isoforms evens out as it moves to the apical cytoplasm. Thus, our colocalization analysis suggests that ß- and γ-actin are segregated in the endotheliocyte cytoplasm. The segregation is greatly enhanced during cell lamella activation in the nocodazole-induced endothelial barrier dysfunction, reflecting a different functional role of cytoplasmic actin isoforms in endothelial cells.

Microtubules growth rate alteration in human endothelial cells.

To understand how microtubules contribute to the dynamic reorganization of the endothelial cell (EC) cytoskeleton, we established an EC model expressing EB3-GFP, a protein that marks microtubule plus-ends. Using this model, we were able to measure microtubule growth rate at the centrosome region and near the cell periphery of a single human EC and in the EC monolayer. We demonstrate that the majority of microtubules in EC are dynamic, the growth rate of their plus-ends is highest in the internal cytoplasm, in the region of the centrosome. Growth rate of microtubule plus-ends decreases from the cell center toward the periphery. Our data suggest the existing mechanism(s) of local regulation of microtubule plus-ends growth in EC. Microtubule growth rate in the internal cytoplasm of EC in the monolayer is lower than that of single EC suggesting the regulatory effect of cell-cell contacts. Centrosomal microtubule growth rate distribution in single EC indicated the presence of two subpopulations of microtubules with "normal" (similar to those in monolayer EC) and "fast" (three times as much) growth rates. Our results indicate functional interactions between cell-cell contacts and microtubules.

Huntington's Disease-An Outlook on the Interplay of the HTT Protein, Microtubules and Actin Cytoskeletal Components.

Huntington's disease is a severe and currently incurable neurodegenerative disease. An autosomal dominant mutation in the Huntingtin gene (HTT) causes an increase in the polyglutamine fragment length at the protein N-terminus. The consequence of the mutation is the death of neurons, mostly striatal neurons, leading to the occurrence of a complex of motor, cognitive and emotional-volitional personality sphere disorders in carriers. Despite intensive studies, the functions of both mutant and wild-type huntingtin remain poorly understood. Surprisingly, there is the selective effect of the mutant form of HTT even on nervous tissue, whereas the protein is expressed ubiquitously. Huntingtin plays a role in cell physiology and affects cell transport, endocytosis, protein degradation and other cellular and molecular processes. Our experimental data mining let us conclude that a significant part of the Huntingtin-involved cellular processes is mediated by microtubules and other cytoskeletal cell structures. The review attempts to look at unresolved issues in the study of the huntingtin and its mutant form, including their functions affecting microtubules and other components of the cell cytoskeleton.

Drug Targeting the Actin Cytoskeleton Potentiates the Cytotoxicity of Low Dose Vincristine by Abrogating Actin-Mediated Repair of Spindle Defects.

Antimicrotubule vinca alkaloids are widely used in the clinic but their toxicity is often dose limiting. Strategies that enhance their effectiveness at lower doses are needed. We show that combining vinca alkaloids with compounds that target a specific population of actin filaments containing the cancer-associated tropomyosin Tpm3.1 result in synergy against a broad range of tumor cell types. We discovered that low concentrations of vincristine alone induce supernumerary microtubule asters that form transient multi-polar spindles in early mitosis. Over time these asters can be reconstructed into functional bipolar spindles resulting in cell division and survival. These microtubule asters are organized by the nuclear mitotic apparatus protein (NuMA)-dynein-dynactin complex without involvement of centrosomes. However, anti-Tpm3.1 compounds at nontoxic concentrations inhibit this rescue mechanism resulting in delayed onset of anaphase, formation of multi-polar spindles, and apoptosis during mitosis. These findings indicate that drug targeting actin filaments containing Tpm3.1 potentiates the anticancer activity of low-dose vincristine treatment. IMPLICATIONS: Simultaneously inhibiting Tpm3.1-containing actin filaments and microtubules is a promising strategy to potentiate the anticancer activity of low-dose vincristine.

The Centriolar Adjunct⁻Appearance and Disassembly in Spermiogenesis and the Potential Impact on Fertility.

During spermiogenesis, the proximal centriole forms a special microtubular structure: the centriolar adjunct. This structure appears at the spermatid stage, which is characterized by a condensed chromatin nucleus. We showed that the centriolar adjunct disappears completely in mature porcine spermatozoa. In humans, the centriolar adjunct remnants are present in a fraction of mature spermatids. For the first time, the structure of the centriolar adjunct in the cell, and its consequent impact on fertility, were examined. Ultrastructural analysis using transmission electron microscopy was performed on near 2000 spermatozoa per person, in two patients with idiopathic male sterility (IMS) and five healthy fertile donors. We measured the average length of the "proximal centriole + centriolar adjunct" complex in sections, where it had parallel orientation in the section plane, and found that it was significantly longer in the spermatozoa of IMS patients than in the spermatozoa of healthy donors. This difference was independent of chromatin condensation deficiency, which was also observed in the spermatozoa of IMS patients. We suggest that zygote arrest may be related to an incompletely disassembled centriolar adjunct in a mature spermatozoon. Therefore, centriolar adjunct length can be potentially used as a complementary criterion for the immaturity of spermatozoa in the diagnostics of IMS patients.

Identification of Cancer-Targeted Tropomyosin Inhibitors and Their Synergy with Microtubule Drugs.

Actin filaments, with their associated tropomyosin polymers, and microtubules are dynamic cytoskeletal systems regulating numerous cell functions. While antimicrotubule drugs are well-established, antiactin drugs have been more elusive. We previously targeted actin in cancer cells by inhibiting the function of a tropomyosin isoform enriched in cancer cells, Tpm3.1, using a first-in-class compound, TR100. Here, we screened over 200 other antitropomyosin analogues for anticancer and on-target activity using a series of in vitro cell-based and biochemical assays. ATM-3507 was selected as the new lead based on its ability to disable Tpm3.1-containing filaments, its cytotoxicity potency, and more favorable drug-like characteristics. We tested ATM-3507 and TR100 alone and in combination with antimicrotubule agents against neuroblastoma models in vitro and in vivo Both ATM-3507 and TR100 showed a high degree of synergy in vitro with vinca alkaloid and taxane antimicrotubule agents. In vivo, combination-treated animals bearing human neuroblastoma xenografts treated with antitropomyosin combined with vincristine showed minimal weight loss, a significant and profound regression of tumor growth and improved survival compared with control and either drug alone. Antitropomyosin combined with vincristine resulted in G2-M phase arrest, disruption of mitotic spindle formation, and cellular apoptosis. Our data suggest that small molecules targeting the actin cytoskeleton via tropomyosin sensitize cancer cells to antimicrotubule agents and are tolerated together in vivo This combination warrants further study. Mol Cancer Ther; 16(8); 1555-65. ©2017 AACR.

Where are the limits of the centrosome?

The centrosome is a key component of the cell is involved in the processes of cell division, cell motility, intracellular transport, organization of the microtubules (MT) network and the production of cilia and flagella. The peculiarity of this organelle is that its boundaries are not clearly defined, the centrioles at the center of the centrosome are surrounded by electron dense pericentriolar material, the size and protein composition of this centrosome component experiences significant transformation during the cell cycle. It has been shown in this study that within the centrosome different proteins occupy different areas corresponding to: MT nucleation region (defined as gamma-tubulin-stained area), regulatory region (defined as kinase pEg2-stained area) and motor proteins region (kinesin-like motor XlEg5-stained area). The boundary of pEg2 is near 1.3 times greater while XlEg5 is 3.0 times greater than that of gamma-tubulin. Thus, the size of the centrosome, determined according to the structural electron microscopy (EM) analysis (about 1 µm) corresponds to the regulatory proteins area, but the actual functional centrosome size defined at the motor proteins region, is more than twice the size.

Protective effect of Growth Hormone-Releasing Hormone agonist in bacterial toxin-induced pulmonary barrier dysfunction.

RATIONALE: Antibiotic treatment of patients infected with G(-) or G(+) bacteria promotes release of the toxins lipopolysaccharide (LPS) and pneumolysin (PLY) in their lungs. Growth Hormone-releasing Hormone (GHRH) agonist JI-34 protects human lung microvascular endothelial cells (HL-MVEC), expressing splice variant 1 (SV-1) of the receptor, from PLY-induced barrier dysfunction. We investigated whether JI-34 also blunts LPS-induced hyperpermeability. Since GHRH receptor (GHRH-R) signaling can potentially stimulate both cAMP-dependent barrier-protective pathways as well as barrier-disruptive protein kinase C pathways, we studied their interaction in GHRH agonist-treated HL-MVEC, in the presence of PLY, by means of siRNA-mediated protein kinase A (PKA) depletion. METHODS: Barrier function measurements were done in HL-MVEC monolayers using Electrical Cell substrate Impedance Sensing (ECIS) and VE-cadherin expression by Western blotting. Capillary leak was assessed by Evans Blue dye (EBD) incorporation. Cytokine generation in broncho-alveolar lavage fluid (BALF) was measured by multiplex analysis. PKA and PKC-α activity were assessed by Western blotting. RESULTS: GHRH agonist JI-34 significantly blunts LPS-induced barrier dysfunction, at least in part by preserving VE-cadherin expression, while not affecting inflammation. In addition to activating PKA, GHRH agonist also increases PKC-α activity in PLY-treated HL-MVEC. Treatment with PLY significantly decreases resistance in control siRNA-treated HL-MVEC, but does so even more in PKA-depleted monolayers. Pretreatment with GHRH agonist blunts PLY-induced permeability in control siRNA-treated HL-MVEC, but fails to improve barrier function in PKA-depleted PLY-treated monolayers. CONCLUSIONS: GHRH signaling in HL-MVEC protects from both LPS and PLY-mediated endothelial barrier dysfunction and concurrently induces a barrier-protective PKA-mediated and a barrier-disruptive PKC-α-induced pathway in the presence of PLY, the former of which dominates the latter.

Stimulation of kainate toxicity by zinc in cultured cerebellar granule neurons and the role of mitochondria in this process.

Zinc chloride (0.01 mM kept for 3h) is not toxic to cultured cerebellar granule neurons (CGNs) while kainate (0.1mM kept for 3h) demonstrates some but very low toxicity towards these cells. Measurements of the relative intraneuronal zinc ion concentration showed that increase in [Zn(2+)](i) under the simultaneous action of ZnCl(2) and kainate was significantly stronger compared to their separate action. Simultaneous treatment of CGNs with kainate and zinc chloride caused the swelling of neuronal mitochondria and consequent intensive neuronal death, which was totally prevented by NBQX (an AMPA/kainate-receptors blocker) or ruthenium red (a mitochondrial Ca(2+) uniporter blocker). These data imply that Zn(2+) synergistically to kainate increase their separate toxic effects on mitochondria leading to rapid neuronal death.

Molecular mechanisms involved in adenosine-induced endothelial cell barrier enhancement.

Extracellular adenosine is a physiologically relevant agonist released by various sources, including endothelial cells (EC) and activated platelets, with complex effects mediated via activation of P1 purinergic receptors. Adenosine-induced EC production of glutathione peroxidase1 and nitric oxide is recognized, and an anti-inflammatory mechanism has been described. Effects of extracellular adenosine on the pulmonary EC barrier function and vascular permeability, however, remain poorly characterized. In this study, we demonstrated the adenosine-induced rapid dose-dependent barrier enhancement in human pulmonary artery EC (HPAEC) as measured by an increase in transendothelial electrical resistance (TER). We have shown that HPAEC express only A2A and A2B adenosine receptors. Pharmacological and siRNA depletion studies indicate that A2A, but not A2B receptor activation is required for the adenosine-induced TER increase. Depletion of Galphas with a specific siRNA significantly attenuated the adenosine-induced TER response in HPAEC. In contrast, depletion of either Galphaq or Galphai2 did not affect the adenosine-induced TER increase. This suggests that the adenosine-induced TER increase is cAMP-dependent. The adenosine-induced barrier enhancement effects were associated with a rearrangement of the EC F-actin component of the cytoskeleton, enhanced cell-surface presentation of cell-cell junctional protein VE-cadherin and an involvement of Myosin-light-chain phosphatase (MLCP). Our results suggest, for the first time, that adenosine regulates the EC barrier function via A2A receptors followed by Galphas engagement and is associated with cytoskeletal activation.

Effect of transitory glucose deprivation on mitochondrial structure and functions in cultured cerebellar granule neurons.

We found that 60-min glucose deprivation leads to progressive decrease in the mitochondrial membrane potential and increase in [Ca(2+)](i) in cultured cerebellar granule neurons. The latter effect was fully reversible, returning to the basal level 60 min after restoration of normal glucose level in the incubation medium, whereas mitochondrial membrane potential remained at 10.0+/-1.8% below the initial value. Electron microscopy indicated that glucose deprivation induced appearance of mitochondria with local lightening of the matrix and destruction of cristae. This mitochondrial conformation was preserved during the restoration phase after glucose level in the cultivation medium returned to the normal level. Neuronal death within a 24-h period after 60-min glucose deprivation was relatively small, being 14.0+/-4.4%.