Alena Rudkouskaya

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.

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.

Two conventional protein kinase C isoforms, α and βI, are involved in the ATP-induced activation of volume-regulated anion channel and glutamate release in cultured astrocytes

Volume‐regulated anion channels (VRACs) are activated by cell swelling and are permeable to inorganic and small organic anions, including the excitatory amino acids glutamate and aspartate. In astrocytes, ATP potently enhances VRAC activity and glutamate release via a P2Y receptor‐dependent mechanism. Our previous pharmacological study identified protein kinase C (PKC) as a major signaling enzyme in VRAC regulation by ATP. However, conflicting results obtained with potent PKC blockers prompted us to re‐evaluate the involvement of PKC in regulation of astrocytic VRACs by using small interfering RNA (siRNA) and pharmacological inhibitors that selectively target individual PKC isoforms. In primary rat astrocyte cultures, application of hypoosmotic medium (30% reduction in osmolarity) and 20 μM ATP synergistically increased the release of excitatory amino acids, measured with a non‐metabolized analog of l‐glutamate, d‐[3H]aspartate. Both Go6976, the selective inhibitor of Ca2+‐sensitive PKCα, βI/II, and γ, and MP‐20‐28, a cell permeable pseudosubstrate inhibitory peptide of PKCα and βI/II, reduced the effects of ATP on d‐[3H]aspartate release by ∼45–55%. Similar results were obtained with a mixture of siRNAs targeting rat PKCα and βI. Surprisingly, down‐regulation of individual α and βI PKC isozymes by siRNA was completely ineffective. These data suggest that ATP regulates VRAC activity and volume‐sensitive excitatory amino acid release via cooperative activation of PKCα and βI.

Two conventional PKC isoforms, α and βI, are involved in the ATP-induced regulation of VRAC and glutamate release in cultured astrocytes

Volume‐regulated anion channels (VRACs) are activated by cell swelling and are permeable to inorganic and small organic anions, including the excitatory amino acids glutamate and aspartate. In astrocytes, ATP potently enhances VRAC activity and glutamate release via a P2Y receptor‐dependent mechanism. Our previous pharmacological study identified protein kinase C (PKC) as a major signaling enzyme in VRAC regulation by ATP. However, conflicting results obtained with potent PKC blockers prompted us to re‐evaluate the involvement of PKC in regulation of astrocytic VRACs by using small interfering RNA (siRNA) and pharmacological inhibitors that selectively target individual PKC isoforms. In primary rat astrocyte cultures, application of hypoosmotic medium (30% reduction in osmolarity) and 20 μM ATP synergistically increased the release of excitatory amino acids, measured with a non‐metabolized analog of l‐glutamate, d‐[3H]aspartate. Both Go6976, the selective inhibitor of Ca2+‐sensitive PKCα, βI/II, and γ, and MP‐20‐28, a cell permeable pseudosubstrate inhibitory peptide of PKCα and βI/II, reduced the effects of ATP on d‐[3H]aspartate release by ∼45–55%. Similar results were obtained with a mixture of siRNAs targeting rat PKCα and βI. Surprisingly, down‐regulation of individual α and βI PKC isozymes by siRNA was completely ineffective. These data suggest that ATP regulates VRAC activity and volume‐sensitive excitatory amino acid release via cooperative activation of PKCα and βI.

Long-lasting inhibition of presynaptic metabolism and neurotransmitter release by protein S-nitrosylation

Nitric oxide (NO) and related reactive nitrogen species (RNS) play a major role in the pathophysiology of stroke and other neurodegenerative diseases. One of the poorly understood consequences of stroke is a long-lasting inhibition of synaptic transmission. In this study, we tested the hypothesis that RNS can produce long-term inhibition of neurotransmitter release via S-nitrosylation of proteins in presynaptic nerve endings. We examined the effects of exogenous sources of RNS on the vesicular and nonvesicular L-[(3)H]glutamate release from rat brain synaptosomes. NO/RNS donors, such as spermine NONOate, MAHMA NONOate, S-nitroso-L-cysteine, and SIN-1, inhibited only the vesicular component of glutamate release with an order of potency that closely matched levels of protein S-nitrosylation. Inhibition of glutamate release persisted for >1h after RNS donor decomposition and washout and strongly correlated with decreases in the intrasynaptosomal ATP levels. Post-NO treatment of synaptosomes with thiol-reducing reagents decreased the total content of S-nitrosylated proteins but had little effect on glutamate release and ATP levels. In contrast, post-NO application of the end-product of glycolysis, pyruvate, partially rescued neurotransmitter release and ATP production. These data suggest that RNS suppress presynaptic metabolism and neurotransmitter release via poorly reversible modifications of glycolytic and mitochondrial enzymes, one of which was identified as glyceraldehyde-3-phosphate dehydrogenase. A similar mechanism may contribute to the long-term suppression of neuronal communication during nitrosative stress in vivo.

Targeted inactivation of integrin-linked kinase in hair follicle stem cells reveals an important modulatory role in skin repair after injury

Inactivation of integrin-linked kinase in the stem cells of the hair follicle bulge results in impaired skin regeneration after injury but does not affect hair follicle entry into anagen.

Staphylococcus aureus keratinocyte invasion is mediated by integrin-linked kinase and Rac1

Staphylococcus aureus is a major component of the skin microbiota and causes a large number of serious infections. S. aureus first interacts with epidermal keratinocytes to breach the epidermal barrier through mechanisms not fully understood. By use of primary keratinocytes from mice with epidermis‐restricted Ilk gene inactivation and control integrin‐linked kinase (ILK)‐expressing litter‐mates, we investigated the role of ILK in epidermal S. aureus invasion. Heat‐killed, but not live, bacteria were internalized to Rab5‐ and Rab7‐positive phagosomes, and incubation with keratinocyte growth factor increased their uptake 2.5‐fold. ILK‐deficient mouse keratinocytes internalized bacteria 2‐ to 4‐fold less efficiently than normal cells. The reduced invasion by live S. aureus of ILK‐deficient cells was restored in the presence of exogenous, constitutively active Rac1. Thus, Rac1 functions downstream from ILK during invasion. Further, invasion by S. aureus of Rac1‐deficient cells was 2.5‐fold lower than in normal cells. Paradoxically, staphylococcal cutaneous penetration of mouse skin expiants with ILK‐deficient epidermis was 35‐fold higher than that of normal skin, indicating defects in epidermal barrier function in the absence of ILK. Thus, we identified an ILK‐Rac1 pathway essential for bacterial invasion of keratinocytes, and established ILK as a key contributor to prevent invasive staphylococcal cutaneous infection.—Sayedyahossein, S., Xu, S. X., Rudkouskaya, A., McGavin, M. J., McCormick, J. K., Dagnino, L. Staphylococcus aureus keratinocyte invasion is mediated by integrin‐linked kinase and Rac1. FASEB J. 29, 711‐723 (2015). www.fasebj.org

ILK modulates epithelial polarity and matrix formation in hair follicles

Integrin-linked kinase–deficient hair follicles fail to develop apical–basal polarity and show impaired specification of the hair matrix cell lineage. Exogenous laminin-511 restores matrix cell formation.