Alexander A. Mongin

STIM1 and Orai1 mediate CRAC channel activity and are essential for human glioblastoma invasion

The Ca2+ sensor stromal interacting molecule 1 (STIM1) and the Ca2+ channel Orai1 mediate the ubiquitous store-operated Ca2+ entry (SOCE) pathway activated by depletion of internal Ca2+ stores and mediated through the highly Ca2+-selective, Ca2+ release-activated Ca2+ (CRAC) current. Furthermore, STIM1 and Orai1, along with Orai3, encode store-independent Ca2+ currents regulated by either arachidonate or its metabolite, leukotriene C4. Orai channels are emerging as important contributors to numerous cell functions, including proliferation, migration, differentiation, and apoptosis. Recent studies suggest critical involvement of STIM/Orai proteins in controlling the development of several cancers, including malignancies of the breast, prostate, and cervix. Here, we quantitatively compared the magnitude of SOCE and the expression levels of STIM1 and Orai1 in non-malignant human primary astrocytes (HPA) and in primary human cell lines established from surgical samples of the brain tumor glioblastoma multiforme (GBM). Using Ca2+ imaging, patch-clamp electrophysiology, pharmacological reagents, and gene silencing, we established that in GBM cells, SOCE and CRAC are mediated by STIM1 and Orai1. We further found that GBM cells show upregulation of SOCE and increased Orai1 levels compared to HPA. The functional significance of SOCE was evaluated by studying the effects of STIM1 and Orai1 knockdown on cell proliferation and invasion. Utilizing Matrigel assays, we demonstrated that in GBM, but not in HPA, downregulation of STIM1 and Orai1 caused a dramatic decrease in cell invasion. In contrast, the effects of STIM1 and Orai1 knockdown on GBM cell proliferation were marginal. Overall, these results demonstrate that STIM1 and Orai1 encode SOCE and CRAC currents and control invasion of GBM cells. Our work further supports the potential use of channels contributed by Orai isoforms as therapeutic targets in cancer.

Pharmacological comparison of swelling‐activated excitatory amino acid release and Cl− currents in cultured rat astrocytes

Ubiquitously expressed volume‐regulated anion channels (VRACs) are chloride channels which are permeable to a variety of small organic anions, including the excitatory amino acids (EAAs) glutamate and aspartate. Broad spectrum anion channel blockers strongly reduce EAA release in cerebral ischaemia and other pathological states associated with prominent astrocytic swelling. However, it is uncertain whether VRAC serves as a major pathway for EAA release from swollen cells. In the present study, we measured swelling‐activated release of EAAs as d‐[3H]aspartate efflux, and VRAC‐mediated Cl− currents by whole‐cell patch clamp in cultured rat astrocytes. We compared the pharmacological profiles of the swelling‐activated EAA release pathway and Cl− currents. The expression of candidate Cl− channels was confirmed by RT‐PCR. The maxi Cl− channel (p‐VDAC) blocker Gd3+, the ClC‐2 inhibitor Cd2+, and the MDR‐1 blocker verapamil did not affect EAA release or VRAC currents. An antagonist of calcium‐sensitive Cl− channels (CaCC), niflumic acid, had little effect on EAA release and only partially inhibited swelling‐activated Cl− currents. The phorbol ester PDBu, which blocks ClC‐3‐mediated Cl− currents, had no effect on VRAC currents and up‐regulated EAA release. In contrast, DCPIB, which selectively inhibits VRACs, potently suppressed both EAA release and VRAC currents. Two other relatively selective VRAC inhibitors, tamoxifen and phloretin, also blocked the VRAC currents and strongly reduced EAA release. Taken together, our data suggest that (i) astrocytic volume‐dependent EAA release is largely mediated by the VRAC, and (ii) the ClC‐2, ClC‐3, ClC‐4, ClC‐5, VDAC, CaCC, MDR‐1 and CFTR gene products do not contribute to EAA permeability.

Mechanisms of cell volume regulation and possible nature of the cell volume sensor

In animal organisms, cell volume undergoes dynamic changes in many physiological and pathological processes. To protect themselves against lysis and apoptosis and to maintain an optimal concentration of intracellular enzymes and metabolites, most animal cells actively regulate their volume. In the present review, we shortly summarize the data on ion transport mechanisms involved in regulatory volume decrease (RVD) and regulatory volume increase (RVI) with an emphasis on unresolved aspects of this problem such as: (i) how cells sense their volume changes; (ii) what signals are generated upon cell volume alterations; and (iii) how these signals are transferred to the ion transport systems executing cell volume regulation.

Activation of microglia with zymosan promotes excitatory amino acid release via volume-regulated anion channels: the role of NADPH oxidases

Microglia are the resident immune cells of the CNS, which are important for preserving neural tissue functions, but may also contribute to neurodegeneration. Activation of these cells in infection, inflammation, or trauma leads to the release of various toxic molecules, including reactive oxygen species (ROS) and the excitatory amino acid glutamate. In this study, we used an electrophysiologic approach and a d‐[3H]aspartate (glutamate) release assay to explore the ROS‐dependent regulation of glutamate‐permeable volume‐regulated anion channels (VRACs). Exposure of rat microglia to hypo‐osmotic media stimulated Cl− currents and d‐[3H]aspartate release, both of which were inhibited by the selective VRAC blocker, DCPIB. Exogenously applied H2O2 potently increased swelling‐activated glutamate release. Stimulation of microglia with zymosan triggered production of endogenous ROS and strongly enhanced glutamate release via VRAC in swollen cells. The effects of zymosan were attenuated by the ROS scavenger, MnTMPyP, and by two inhibitors of NADPH oxidase (NOX), diphenyliodonium and thioridazine. However, zymosan‐stimulated glutamate release was insensitive to other NOX blockers, apocynin and HEBSF. This pharmacologic profile pointed to the potential involvement of apocynin‐insensitive NOX4. Using RT‐PCR we confirmed that NOX4 is expressed in rat microglial cells along with NOX1 and NOX2. To check for potential involvement of phagocytic NOX2, we stimulated this isoform using protein kinase C (PKC) activator, phorbol 12‐myristate 13‐acetate or inhibited it with the broad spectrum PKC blocker, Gö6983. Both agents potently modulated endogenous ROS production by NOX2 but not VRAC activity. Taken together, these data suggest that the anion channel VRAC may contribute to microglial glutamate release and that its activity is regulated by endogenous ROS originating from NOX4.

Tamoxifen inhibits nitrotyrosine formation after reversible middle cerebral artery occlusion in the rat

Tamoxifen (TAM), a widely used non‐steroidal anti‐estrogen, has recently been shown to be neuroprotective in a rat model of reversible middle cerebral artery occlusion (rMCAo). Tamoxifen has several potential mechanisms of action including inhibition of the release of excitatory amino acids (EAA) and nitric oxide synthase (NOS) activity. The question addressed in this study was whether TAM reduces ischemia‐induced production of nitrotyrosine, considered as a footprint of the product of nitric oxide and superoxide, peroxynitrite. In rat brain, 2 h rMCAo produced a time‐dependent increase in nitrotyrosine content in the cerebral cortex, as measured by Western blot analysis. Compared with vehicle, TAM significantly reduced nitrotyrosine levels in the ischemic cortex at 24 h. The neuronal (n)NOS inhibitor, 7‐nitroindazole also tended to reduce nitrotyrosine, but this reduction was not statistically significant. Immunostaining for nitrotyrosine was seen in cortical neurons in the MCA territory and this immunostaining was reduced by TAM. In vitro, TAM and the calmodulin inhibitor trifluoperazine inhibited, with similar EC50 values, the activity of recombinant nNOS as well as NOS activity in brain homogenates, measured by conversion of [3H]arginine to [3H]citrulline. There was marginal inhibition of recombinant inducible (i)NOS activity up to 100 µm TAM. These data suggest that TAM is an effective inhibitor of Ca2+/calmodulin‐dependent NOS and the derived peroxynitrite production in transient focal cerebral ischemia and this may be one mechanism for its neuroprotective effect following rMCAo.

[3H]taurine andd-[3H]aspartate release from astrocyte cultures are differently regulated by tyrosine kinases

Volume-dependent anion channels permeable for Cl- and amino acids are thought to play an important role in the homeostasis of cell volume. Astrocytes are the main cell type in the mammalian brain showing volume perturbations under physiological and pathophysiological conditions. We investigated the involvement of tyrosine phosphorylation in hyposmotic medium-induced [3H]taurine andd-[3H]aspartate release from primary astrocyte cultures. The tyrosine kinase inhibitors tyrphostin 23 and tyrphostin A51 partially suppressed the volume-dependent release of [3H]taurine in a dose-dependent manner with half-maximal effects at ∼40 and 1 μM, respectively. In contrast, the release ofd-[3H]aspartate was not significantly affected by these agents in the same concentration range. The inactive analog tyrphostin 1 had no significant effect on the release of both amino acids. The data obtained suggest the existence of at least two volume-dependent anion channels permeable to amino acids in astrocyte cultures. One of these channels is permeable to taurine and is under the control of tyrosine kinase(s). The other is permeable to both taurine and aspartate, but its volume-dependent regulation does not require tyrosine phosphorylation.

Calcium-Activated Potassium Channels BK and IK1 Are Functionally Expressed in Human Gliomas but Do Not Regulate Cell Proliferation

Gliomas are morbid brain tumors that are extremely resistant to available chemotherapy and radiology treatments. Some studies have suggested that calcium-activated potassium channels contribute to the high proliferative potential of tumor cells, including gliomas. However, other publications demonstrated no role for these channels or even assigned them antitumorogenic properties. In this work we characterized the expression and functional contribution to proliferation of Ca2+-activated K+ channels in human glioblastoma cells. Quantitative RT-PCR detected transcripts for the big conductance (BK), intermediate conductance (IK1), and small conductance (SK2) K+ channels in two glioblastoma-derived cell lines and a surgical sample of glioblastoma multiforme. Functional expression of BK and IK1 in U251 and U87 glioma cell lines and primary glioma cultures was verified using whole-cell electrophysiological recordings. Inhibitors of BK (paxilline and penitrem A) and IK1 channels (clotrimazole and TRAM-34) reduced U251 and U87 proliferation in an additive fashion, while the selective blocker of SK channels UCL1848 had no effect. However, the antiproliferative properties of BK and IK1 inhibitors were seen at concentrations that were higher than those necessary to inhibit channel activity. To verify specificity of pharmacological agents, we downregulated BK and IK1 channels in U251 cells using gene-specific siRNAs. Although siRNA knockdowns caused strong reductions in the BK and IK1 current densities, neither single nor double gene silencing significantly affected rates of proliferation. Taken together, these results suggest that Ca2+-activated K+ channels do not play a critical role in proliferation of glioma cells and that the effects of pharmacological inhibitors occur through their off-target actions.

Two distinct modes of hypoosmotic medium-induced release of excitatory amino acids and taurine in the rat brain in vivo.

A variety of physiological and pathological factors induce cellular swelling in the brain. Changes in cell volume activate several types of ion channels, which mediate the release of inorganic and organic osmolytes and allow for compensatory cell volume decrease. Volume-regulated anion channels (VRAC) are thought to be responsible for the release of some of organic osmolytes, including the excitatory neurotransmitters glutamate and aspartate. In the present study, we compared the in vivo properties of the swelling-activated release of glutamate, aspartate, and another major brain osmolyte taurine. Cell swelling was induced by perfusion of hypoosmotic (low [NaCl]) medium via a microdialysis probe placed in the rat cortex. The hypoosmotic medium produced several-fold increases in the extracellular levels of glutamate, aspartate and taurine. However, the release of the excitatory amino acids differed from the release of taurine in several respects including: (i) kinetic properties, (ii) sensitivity to isoosmotic changes in [NaCl], and (iii) sensitivity to hydrogen peroxide, which is known to modulate VRAC. Consistent with the involvement of VRAC, hypoosmotic medium-induced release of the excitatory amino acids was inhibited by the anion channel blocker DNDS, but not by the glutamate transporter inhibitor TBOA or Cd2+, which inhibits exocytosis. In order to elucidate the mechanisms contributing to taurine release, we studied its release properties in cultured astrocytes and cortical synaptosomes. Similarities between the results obtained in vivo and in synaptosomes suggest that the swelling-activated release of taurine in vivo may be of neuronal origin. Taken together, our findings indicate that different transport mechanisms and/or distinct cellular sources mediate hypoosmotic medium-induced release of the excitatory amino acids and taurine in vivo.

LRRC8A protein is indispensable for swelling-activated and ATP-induced release of excitatory amino acids in rat astrocytes

Swelling‐activated release of amino acids in the CNS is thought to be mediated by an unidentified volume‐regulated anion channel. Two recent studies discovered that LRRC8 family members form a volume‐regulated anion channel in non‐neural cells. In this work we established a critical contribution of the LRRC8A gene product to swelling‐activated glutamate and taurine release from primary rat astrocytes. We also found that LRRC8A is indispensable for glutamate and taurine release from non‐swollen astrocytes when they are stimulated with ATP. These findings suggest that LRRC8A may play a role in physiological release of gliotransmitters, and mediate pathological glutamate release in the CNS disorders associated with cellular swelling.

Enhanced GLT-1 mediated glutamate uptake and migration of primary astrocytes directed by fibronectin-coated electrospun poly-L-lactic acid fibers.

Bioengineered fiber substrates are increasingly studied as a means to promote regeneration and remodeling in the injured central nervous system (CNS). Previous reports largely focused on the ability of oriented scaffolds to bridge injured regions and direct outgrowth of axonal projections. In the present work, we explored the effects of electrospun microfibers on the migration and physiological properties of brain astroglial cells. Primary rat astrocytes were cultured on either fibronectin-coated poly-L-lactic acid (PLLA) films, fibronectin-coated randomly oriented PLLA electrospun fibers, or fibronectin-coated aligned PLLA electrospun fibers. Aligned PLLA fibers strongly altered astrocytic morphology, orienting cell processes, actin microfilaments, and microtubules along the length of the fibers. On aligned fibers, astrocytes also significantly increased their migration rates in the direction of fiber orientation. We further investigated if fiber topography modifies astrocytic neuroprotective properties, namely glutamate and glutamine transport and metabolism. This was done by quantifying changes in mRNA expression (qRT-PCR) and protein levels (Western blotting) for a battery of relevant biomolecules. Interestingly, we found that cells grown on random and/or aligned fibers increased the expression levels of two glutamate transporters, GLAST and GLT-1, and an important metabolic enzyme, glutamine synthetase, as compared to the fibronectin-coated films. Functional assays revealed increases in glutamate transport rates due to GLT-1 mediated uptake, which was largely determined by the dihydrokainate-sensitive GLT-1. Overall, this study suggests that aligned PLLA fibers can promote directed astrocytic migration, and, of most importance, our in vitro results indicate for the first time that electrospun PLLA fibers can positively modify neuroprotective properties of glial cells by increasing rates of glutamate uptake.