Volume-regulated Anion Channels Inhibitors Research Articles

Smooth Muscle LRRC8A Anion Channel Knockout Promotes Vasodilation and Protects Against TNFα‐induced Vascular Dysfunction

Tumor necrosis factor‐α (TNFα), a pro‐inflammatory cytokine, is associated with cardiovascular risk. Volume‐regulated anion channels (VRACs) are activated by TNFα and are composed of Leucine Rich Repeat Containing 8 (LRRC8) family proteins (LRRC8A, B, C, D and E). LRRC8A is required for all VRACs. We previously determined that VRAC inhibitors or LRRC8A knockdown suppress the inflammatory response to TNFα in cultured vascular smooth muscle cells, however, the role of these channels in vasomotor function has not been studied. We hypothesized that loss of LRRC8A would prevent the impairment of vascular function by TNFα. We created vascular smooth muscle‐specific (Sm22alpha‐Cre) LRRC8A knockout (KO) mice and isolated mesenteric arteries from male mice for wire myograph recording. Vessel segments were incubated with TNFα (10 ng/mL) or vehicle for 2 days in tissue culture. Contractile responses to norepinephrine (NE) were unaltered in LRRC8A KO mice but enhanced by TNFα in wild type (WT) than in KO rings (WT+TNFα 284 ± 18.9 %, KO+TNFα 222 ± 7.1 % of KCl contraction, *p<0.05, n = 5‐6). In the vehicle‐treated group both endothelium‐dependent (acetylcholine, ACh) and ‐independent (sodium nitroprusside, SNP) relaxation were increased in LRRC8A KO compared to WT rings contracted with phenylephrine (PE). Exposure to TNFα impaired relaxation to both ACh and SNP in WT vessels but had no effect on relaxation of LRRC8A KO vessels (black *p<0.05 compared to WT control, red *p<0.05 compared to TNFα‐treated, n = 6‐8). The enhanced relaxation responses of KO vessels were recapitulated in WT rings by 20 min of exposure to a VRAC inhibitor, carbenoxolone (CBX; 100 μM). Thus, in addition to its established role in TNFα signaling, LRRC8A anion channel activity modulates vascular reactivity with loss of function enhancing vasodilator function. VRAC inhibition may provide a novel therapeutic approach to the prevention and treatment of inflammation‐induced vascular dysfunction.

Leucine rich repeat containing 8A anion channels modulate vascular reactivity in the pudendal artery

ABSTRACT Introduction Erectile dysfunction (ED) is characterized by an inability to develop or maintain penile erection during sexual activity. The internal pudendal artery (IPA) is the primary supplier of genital blood flow. Reactive oxygen species (ROS) generation is necessary for intracellular signaling, but excessive ROS production can promote ED. NADPH oxidase (Nox) produces extracellular superoxide (O2 •-) in the vasculature and we have demonstrated that Leucine Rich Repeat Containing 8 (LRRC8) Volume Regulated Anion Channels (VRACs) physically associate with and regulate Nox1. LRRC8A downregulation or channel inhibition decreases extracellular O2 •- production by Nox1 and dramatically reduces the inflammatory response to tumor necrosis factor alpha. Objective We hypothesize that smooth muscle-specific LRRC8A knockout (KO) increases vascular relaxation in IPA. Methods The IPAs from male wildtype (WT) and LRRC8A KO (smooth muscle-specific) mice were used in wire myograph studies. Results Following phenylephrine (PE, 2x10-6 M)-induced contraction, cumulative relaxation-response curves to acetylcholine (ACh, 10-9 to 3x10-6 M), sodium nitroprusside (SNP, 10-9 to 10-5 M), and a soluble guanylyl cyclase activator (BAY60-2770, 10-9 to 10-6 M) were measured. Endothelium-dependent relaxation to ACh was increased in LRRC8A KO compared to WT (WT LogEC50 -7.3 ± 0.08; LRRC8A KO LogEC50 -7.7 ± 0.12; *p<0.05, n=5). Relaxation to SNP or BAY60-2770 showed no difference between WT and KO. The LRRC8A-targeted VRAC inhibitor, montelukast (10 μM) reduced PE (10-6 M)-induced contractile responses in the IPA (56 % decrease). Conclusions These data demonstrate that LRRC8A plays an important role in controlling vascular reactivity that may be associated with ED. We speculate that LRRC8 channels are novel pharmacologic targets. Inhibition of the channels may reduce oxidative stress and improve sexual function. Disclosure Work supported by industry: no.

Novel DCPIB analogs as dual inhibitors of VRAC/TREK1 channels reduced cGAS-STING mediated interferon responses

The enzyme cyclic GMP-AMP synthase (cGAS) senses cytosolic DNA and catalyzes the formation of 2′3′-cyclic-GMP-AMP (cGAMP), which in turn triggers interferon (IFN) production. Inappropriate activation of cGAS and production of cGAMP have been linked to a diversity of autoimmune diseases. The volume-regulated anion channels (VRACs) have been recently demonstrated to permeate cGAMP, thus making the channel essential for the activation of the cGAS-cGAMP-STING axis. DCPIB, a prominent inhibitor of VRAC channel, has been recently reported to also significantly activate TREK1 channel. Herein, in this study, we have designed and synthesized a series of novel DCPIB derivatives and investigated their potential regulatory effects on VRAC/TREK1 channels. Our results manifested that compound 6u was a dual inhibitor of VRAC/TREK1 channels with IC50s of 7.11 ± 0.94 μM and 4.43 ± 0.90 μM, respectively. On top of that, our data demonstrated that 6u impaired interferon production in a concentration-dependently manner by dampening cGAS-cGAMP-STING pathway without any cytotoxicity when it comes to herpes simplex virus type 1 (HSV1) infection. To sum up, our study not only discovered a novel DCPIB analog with dual inhibitory effects on VRAC/TREK1 channels but also provided a new strategy for the design and development of newly potent VRAC inhibitors, which benefits the treatment of cGAS-STING related autoimmune and inflammatory diseases.

CysLT1 Receptor Antagonists Pranlukast and Zafirlukast Inhibit LRRC8-Mediated Volume Regulated Anion Channels Independently of the Receptor

Volume Regulated Anion Channels (VRACs) encoded by the LRRC8 gene family play critical roles in myriad cellular processes and might represent druggable targets. The dearth of pharmacological compounds available for studying VRAC physiology led us to perform a high-throughput screen (HTS) of 1,184 of FDA-approved drugs for novel VRAC modulators. We discovered the cysteinyl leukotriene receptor 1 (CysLT1R) antagonist, Pranlukast, as a novel inhibitor of endogenous VRAC expressed in HEK293 cells. Pranlukast inhibits VRAC voltage-independently, reversibly, and dose-dependently with a maximal efficacy of only ∼50%. The CysLT1R pathway has been implicated in activation of VRAC in other cell types, prompting us to test whether Pranlukast requires the CysLT1R for inhibition of VRAC. Quantitative PCR analysis demonstrated that CYSLTR1 mRNA is virtually undetectable in HEK293 cells. Furthermore, the CysLT1R agonist LTD4 had no effect on VRAC activity and failed to stimulate Gq-coupled receptor signaling. Heterologous expression of the CysLT1R reconstituted LTD4-CysLT1R-Gq-calcium signaling in HEK293 cells but had no effect on VRAC inhibition by Pranlukast. Finally, we show the CysLT1R antagonist Zafirlukast inhibits VRAC with an IC50 of ∼17 µM and does so with full efficacy. To assess the binding the site of Zafirlukast on VRAC we used homomeric LRRC8A, and various homomeric LRRC8 chimeras, showing Zafirlukast inhibits each construct equally, suggesting Zafirlukast may be binding in a conserved region among LRRC8A, LRRC8C, LRRC8D, and LRRC8E subunits. In summary, our data suggest that both Pranlukast and Zafirlukast are likely direct channel inhibitors that work independently of the CysLT1R. This study provides clarifying insights into the putative role of leukotriene signaling in modulation of VRAC and identifies two new chemical scaffolds that can be used for development of more potent and specific VRAC inhibitors.

CysLT1 receptor antagonists pranlukast and zafirlukast inhibit LRRC8-mediated volume regulated anion channels independently of the receptor.

Volume-regulated anion channels (VRACs) encoded by the leucine-rich repeat containing 8 (LRRC8) gene family play critical roles in myriad cellular processes and might represent druggable targets. The dearth of pharmacological compounds available for studying VRAC physiology led us to perform a high-throughput screen of 1,184 of US Food and Drug Administration-approved drugs for novel VRAC modulators. We discovered the cysteinyl leukotriene receptor 1 (CysLT1R) antagonist, pranlukast, as a novel inhibitor of endogenous VRAC expressed in human embryonic kidney 293 (HEK293) cells. Pranlukast inhibits VRAC voltage-independently, reversibly, and dose-dependently with a maximal efficacy of only ~50%. The CysLT1R pathway has been implicated in activation of VRAC in other cell types, prompting us to test whether pranlukast requires the CysLT1R for inhibition of VRAC. Quantitative PCR analysis demonstrated that CYSLTR1 mRNA is virtually undetectable in HEK293 cells. Furthermore, the CysLT1R agonist leukotriene D4 had no effect on VRAC activity and failed to stimulate Gq-coupled receptor signaling. Heterologous expression of the CysLT1R reconstituted LTD4-CysLT1R- Gq-calcium signaling in HEK293 cells but had no effect on VRAC inhibition by pranlukast. Finally, we show the CysLT1R antagonist zafirlukast inhibits VRAC with an IC50 of ~17 µM and does so with full efficacy. Our data suggest that both pranlukast and zafirlukast are likely direct channel inhibitors that work independently of the CysLT1R. This study provides clarifying insights into the putative role of leukotriene signaling in modulation of VRAC and identifies two new chemical scaffolds that can be used for development of more potent and specific VRAC inhibitors.

Discovery of Pranlukast in a High‐Throughput Screen for Novel Inhibitors of LRRC8 Volume Regulated Anion Channels

Cells critically regulate their volume in response to hypotonic cell swelling by transporting chloride and small organic osmolytes out of the cell through volume regulated anion channels (VRACs) encoded by members of the recently defined LRRC8 gene family. VRACs are ubiquitously expressed in mammalian cells and have been implicated in diverse cellular functions in addition to cell volume regulation, including endothelial cell calcium signaling and pancreatic beta cell insulin secretion. The lack of potent and specific pharmacological tool compounds represents a critical barrier to evaluating the integrative physiology and therapeutic potential of VRACs. We therefore carried out a high‐throughput screen (HTS) of 1,184 of FDA approved drugs using a fluorescence‐based assay for novel inhibitors of native VRACs expressed in HEK‐293 cells. The assay measures the quenching of intracellular YFP by iodide influx through VRACs activated by hypotonic‐induced cell swelling. In this screen, the cysteinyl leukotriene receptor 1 (CysLT1R) antagonist, Pranlukast, was identified as a novel inhibitor of VRAC. In YFP quenching assays and patch clamp electrophysiology experiments, Pranlukast inhibits HEK‐293 cell VRAC reversibly and dose‐dependently with an IC50 of ~6 μM and ~239 nM, respectively. Interestingly, the CysLT1R pathway has been implicated in activation of VRAC in other cell types, raising the possibility that Pranlukast inhibits VRAC indirectly via the CysLT1R pathway. Using a series of fluorescence assays and qPCR we show that CysLT1R is not functionally expressed in HEK‐293 cells. Additionally, using a structurally distinct CysLT1R inhibitor, Zafirlukast, did not inhibit VRAC. These studies provide clarifying insights into the putative role of CysLT1 receptor signaling in regulation of VRACs and identify Pranlukast as a structurally novel small‐molecule inhibitor of LRRC8 channels.Support or Funding InformationRuth L. Kirschstein National Research Service Award F31 (1F31DK120225‐01)This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

DCPIB, an Inhibitor of Volume-Regulated Anion Channels, Distinctly Modulates K2P Channels.

K2P potassium channels stabilize the resting membrane potential in nearly all cells and have been implicated in several neuronal, cardiovascular, and immune diseases. DCPIB, a known specific and potent inhibitor of volume-regulated anion channels (VRAC), has been reported to activate TREK1 and TREK2 in astrocytes and in vitro recently. In the present study, we demonstrated DCPIB also voltage dependently activated TRAAK besides TREK1/TREK2, showing DCPIB activated all TREK subfamily members. In contrast, the compound potently inhibited several other K2P channels with no voltage dependence, including TRESK, TASK1, and TASK3. DCPIB displayed superior selectivity toward TRESK with an IC50 of 0.14 μM, demonstrating at least 100-fold higher affinity over TREK1/TRAAK channels. Furthermore, the impaired ion selectivity filter region greatly impaired the activating effect of DCPIB on TREK1 but not the inhibitory effect of DCPIB on TRESK. This indicates distinct molecular determinants underlying the effect of DCPIB on TREK1 or TRESK channels. Our results showed that DCPIB played diverse effects on K2P channels and could be a useful tool for further investigating structure-function studies of K2P channels.

Development of novel inhibitors of swelling‐activated LRRC8 anion channels

Cells critically regulate their volume in response to hypoosmotic swelling by transporting chloride and small organic osmolytes out of the cell through so‐called volume‐regulated anion channels (VRACs). The 5 genes encoding VRACs were recently identified in two independent genome‐wide siRNA screens and are named LRRC8 (for Leucine Rich Repeat Containing) A‐E. LRRC8‐containing VRACs have recently been implicated in drug uptake, adipocyte size, and insulin signaling. Unfortunately, the pharmacological tools available to study the physiology and therapeutic potential of LRRC8 channels is limited to low‐affinity small‐molecule inhibitors that exhibit promiscuous activity toward other anion channels, cation channels, and myriad other off‐targets. To circumvent this barrier, we are employing high‐throughput screening (HTS) of small‐molecule libraries and medicinal chemistry to develop more potent and specific inhibitors of LRRC8 channels. HTS will be performed using a fluorescence assay based on halide quenching of a YFP mutant (i.e. F46L, H148Q, I152L) termed Ozzy. Briefly, HEK‐293 cells stably transfected with Ozzy will be plated in black‐walled, clear‐bottomed 384‐well plates, treated with hypotonic solution containing compounds from the Vanderbilt Institute of Chemical Biology library, and then treated with sodium iodide (NaI). Cell swelling opens endogenously expressed VRAC channels, through which iodide enters the cell and quenches Ozzy fluorescence. Small‐molecule inhibitors should block the iodide‐induced quenching of Ozzy. Checkerboard analysis using the best‐in‐class VRAC inhibitor, DCPIB, yielded a Z′ value of 0.74, indicating the assay is reproducible and robust enough to ensure high‐confidence hit‐picking in a large‐scale screen. The assay also reports dose‐dependent inhibition of iodide influx by DCPIB with an IC50 of 11 micromolar, which is close to values derived from electrophysiological experiments. We are currently using this assay to interrogate an analog library based on the DCPIB scaffold, with the goal of identifying more potent and specific inhibitors of VRAC.Support or Funding InformationDepartment of Anesthesiology Faculty Development FundsThis abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

LRRC8A channels support TNFα-induced superoxide production by Nox1 which is required for receptor endocytosis

Leucine Rich Repeat Containing 8A (LRRC8A) is a required component of volume-regulated anion channels (VRACs). In vascular smooth muscle cells, tumor necrosis factor-α (TNFα) activates VRAC via type 1 TNFα receptors (TNFR1), and this requires superoxide (O2•-) production by NADPH oxidase 1 (Nox1). VRAC inhibitors suppress the inflammatory response to TNFα by an unknown mechanism. We hypothesized that LRRC8A directly supports Nox1 activity, providing a link between VRAC current and inflammatory signaling. VRAC inhibition by 4-(2-butyl-6,7-dichlor-2-cyclopentylindan-1-on-5-yl) oxobutyric acid (DCPIB) impaired NF-κB activation by TNFα. LRRC8A siRNA reduced the magnitude of VRAC and inhibited TNFα-induced NF-κB activation, iNOS and VCAM expression, and proliferation of VSMCs. Signaling steps disrupted by both siLRRC8A and DCPIB included; extracellular O2•- production by Nox1, c-Jun N-terminal kinase (JNK) phosphorylation and endocytosis of TNFR1. Extracellular superoxide dismutase, but not catalase, selectively inhibited TNFR1 endocytosis and JNK phosphorylation. Thus, O2•- is the critical extracellular oxidant for TNFR signal transduction. Reducing JNK expression (siJNK) increased extracellular O2•- suggesting that JNK provides important negative feedback regulation to Nox1 at the plasma membrane. LRRC8A co-localized by immunostaining, and co-immunoprecipitated with, both Nox1 and its p22phox subunit. LRRC8A is a component of the Nox1 signaling complex. It is required for extracellular O2•- production, which is in turn essential for TNFR1 endocytosis. These data are the first to provide a molecular mechanism for the potent anti-proliferative and anti-inflammatory effects of VRAC inhibition.

Ascorbic acid efflux from human brain microvascular pericytes: role of re-uptake.

Microvascular pericytes take up ascorbic acid on the ascorbate transporter SVCT2. Intracellular ascorbate then protects the cells against apoptosis induced by culture at diabetic glucose concentrations. To investigate whether pericytes might also provide ascorbate to the underlying endothelial cells, we studied ascorbate efflux from human pericytes. When loaded with ascorbate to intracellular concentrations of 0.8-1.0 mM, almost two-thirds of intracellular ascorbate effluxed from the cells over 2 H. This efflux was opposed by ascorbate re-uptake from the medium, since preventing re-uptake by destroying extracellular ascorbate with ascorbate oxidase increased ascorbate loss even further. Ascorbate re-uptake occurred on the SVCT2, since its blockade by replacing medium sodium with choline, by the SVCT2 inhibitor sulfinpyrazone, or by extracellular ascorbate accelerated ascorbate loss from the cells. This was supported by finding that net efflux of radiolabeled ascorbate was increased by unlabeled extracellular ascorbate with a half-maximal effect in the range of the high affinity Km of the SVCT2. Intracellular ascorbate did not inhibit its efflux. To assess the mechanism of ascorbate efflux, known inhibitors of volume-regulated anion channels (VRACs) were tested. These potently inhibited ascorbate transport into cells on the SVCT2, but not its efflux. An exception was the anion transport inhibitor DIDS, which, despite inhibition of ascorbate uptake, also inhibited net efflux at 25-50 µM. These results suggest that ascorbate efflux from vascular pericytes occurs on a DIDS-inhibitable transporter or channel different from VRACs. Further, ascorbate efflux is opposed by re-uptake of ascorbate on the SVCT2, providing a potential regulatory mechanism.

Regulated phosphorylation of the K-Cl cotransporter KCC3 is a molecular switch of intracellular potassium content and cell volume homeostasis.

The defense of cell volume against excessive shrinkage or swelling is a requirement for cell function and organismal survival. Cell swelling triggers a coordinated homeostatic response termed regulatory volume decrease (RVD), resulting in K+ and Cl− efflux via activation of K+ channels, volume-regulated anion channels (VRACs), and the K+-Cl− cotransporters, including KCC3. Here, we show genetic alanine (Ala) substitution at threonines (Thr) 991 and 1048 in the KCC3a isoform carboxyl-terminus, preventing inhibitory phosphorylation at these sites, not only significantly up-regulates KCC3a activity up to 25-fold in normally inhibitory isotonic conditions, but is also accompanied by reversal of activity of the related bumetanide-sensitive Na+-K+-2Cl− cotransporter isoform 1 (NKCC1). This results in a rapid (<10 min) and significant (>90%) reduction in intracellular K+ content (Ki) via both Cl-dependent (KCC3a + NKCC1) and Cl-independent [DCPIB (VRAC inhibitor)-sensitive] pathways, which collectively renders cells less prone to acute swelling in hypotonic osmotic stress. Together, these data demonstrate the phosphorylation state of Thr991/Thr1048 in KCC3a encodes a potent switch of transporter activity, Ki homeostasis, and cell volume regulation, and reveal novel observations into the functional interaction among ion transport molecules involved in RVD.

The Phosphorylation State of KCC3 Encodes a Potent Molecular Switch of Transporter Activity, Cell Volume, and K + Content

Defense of cell volume against excessive shrinkage or swelling is a requirement for multiple essential cellular functions and organismal survival. Cell swelling triggers a homeostatic counter-response. regulatory volume decrease (RVD), resulting in K+ and Cl– efflux (and osmotically-obliged water) via the activation of K+ channels, volume-regulated anion channels (VRACs)), and K+-Cl– cotransporters (KCCs) such as KCC3 (SLC12A6). Here, we characterize a molecular switch of KCC3 activity, cell volume, and intracellular K+ ion content (Ki) encoded in the relative phosphorylation state of two threonines (T991 and T1048) located in the cytoplasmic KCC3 C-terminus. Constitutive dephosphorylation of T991/T1048 stimulates KCC3 activity >25-fold, is accompanied by a complete inhibition (and reversal) of the related bumetanide-sensitive Na+-K+-2Cl- cotransporter (NKCC1), and results in a rapid (<10 min) and significant (>90%) reduction in Ki via both Cl–-dependent (KCC3 + NKCC1) and Cl–-independent (DCPIB [VRAC inhibitor]-sensitive) pathways. Together, these effects render cells less prone to swelling in even extreme hypotonic stress. We speculate that stimulating KCC3 by antagonizing T991/T1048 phosphorylation might be a novel strategy of countering pathological cell swelling, as seen in cerebral edema, by “re-setting” the cellular osmostat.

The inhibitor of volume‐regulated anion channels DCPIB activates TREK potassium channels in cultured astrocytes

The ethacrynic acid derivative, 4-(2-butyl-6,7-dichlor-2-cyclopentylindan-1-on-5-yl) oxobutyric acid (DCPIB) is considered to be a specific and potent inhibitor of volume-regulated anion channels (VRACs). In the CNS, DCPIB was shown to be neuroprotective through mechanisms principally associated to its action on VRACs. We hypothesized that DCPIB could also regulate the activity of other astroglial channels involved in cell volume homeostasis. Experiments were performed in rat cortical astrocytes in primary culture and in hippocampal astrocytes in situ. The effect of DCPIB was evaluated by patch-clamp electrophysiology and immunocytochemical techniques. Results were verified by comparative analysis with recombinant channels expressed in COS-7 cells. In cultured astrocytes, DCPIB promoted the activation of a K(+) conductance mediated by two-pore-domain K(+) (K(2P) ) channels. The DCPIB effect occluded that of arachidonic acid, which activates K(2P) channels K(2P) 2.1 (TREK-1) and K(2P) 10.1 (TREK-2) in cultured astrocytes. Immunocytochemical analysis suggests that cultured astrocytes express K(2P) 2.1 and K(2P) 10.1 proteins. Moreover, DCPIB opened recombinant K(2P) 2.1 and K(2P) 10.1 expressed in heterologous system. In brain slices, DCPIB did not augment the large background K(+) conductance in hippocampal astrocytes, but caused an increment in basal K(+) current of neurons. Our results indicate that the neuroprotective effect of DCPIB could be due, at least in part, to activation of TREK channels. DCPIB could be used as template to build new pharmacological tools able to increase background K(+) conductance in astroglia and neuronal cells.

Does NAD(P)H oxidase-derived H2O2 participate in hypotonicity-induced insulin release by activating VRAC in β-cells?

NAD(P)H oxidase (NOX)-derived H(2)O(2) was recently proposed to act, in several cells, as the signal mediating the activation of volume-regulated anion channels (VRAC) under a variety of physiological conditions. The present study aims at investigating whether a similar situation prevails in insulin-secreting BRIN-BD11 and rat β-cells. Exogenous H(2)O(2) (100 to 200μM) at basal glucose concentration (1.1 to 2.8mM) stimulated insulin secretion. The inhibitor of VRAC, 5-nitro-2-(3-phenylpropylamino)-benzoate (NPPB) inhibited the secretory response to exogenous H(2)O(2). In patch clamp experiments, exogenous H(2)O(2) was observed to stimulate NPPB-sensitive anion channel activity, which induced cell membrane depolarization. Exposure of the BRIN-BD11 cells to a hypotonic medium caused a detectable increase in intracellular level of reactive oxygen species (ROS) that was abolished by diphenyleneiodonium chloride (DPI), a universal NOX inhibitor. NOX inhibitors such as DPI and plumbagin nearly totally inhibited insulin release provoked by exposure of the BRIN-BD11 cells to a hypotonic medium. Preincubation with two other drugs also abolished hypotonicity-induced insulin release and reduced basal insulin output: 1) N-acetyl-L-cysteine (NAC), a glutathione precursor that serves as general antioxidant and 2) betulinic acid a compound that almost totally abolished NOX4 expression. As NPPB, each of these inhibitors (DPI, plumbagin, preincubation with NAC or betulinic acid) strongly reduced the volume regulatory decrease observed following a hypotonic shock, providing an independent proof that VRAC activation is mediated by H(2)O(2). Taken together, these data suggest that NOX-derived H(2)O(2) plays a key role in the insulin secretory response of BRIN-BD11 and native β-cells to extracellular hypotonicity.

DCPIB, A Specific Inhibitor of Volume-Regulated Anion Channels (VRACs), Inhibits Astrocyte Proliferation and Cell Cycle Progression Via G1/S Arrest

Volume-regulated anion channels (VRACs) are involved in regulating the proliferation and cell cycle progression of a number of cell types. However, little is known about its role on astrocyte proliferation, although numerous VRACs were reported to play important roles in astrocytic neurotransmitters release during swelling. In the present study, we demonstrated that VRAC inhibition by 4-(2-butyl-6,7-dichloro-2-cyclopentyl-indan-1-on-5-yl) oxobutyric acid (DCPIB) can attenuate astrocyte proliferation and DNA synthesis in vitro. Simultaneously, Western blots showed that VRAC inhibition led to a decrease in CDK 4 and cyclin D1 expression accompanied with an increase in p27 expression, which caused the proliferating astrocytes arrested at the G1/S checkpoint. Furthermore, we showed that DCPIB-induced astrocyte proliferation inhibition was modulated through the ERK signaling cascade. These observations demonstrated that VRACs play essential roles in the proliferation of astrocyte.

Volume Regulated Anion Channel Currents of Rat Hippocampal Neurons and Their Contribution to Oxygen-and-Glucose Deprivation Induced Neuronal Death

Volume-regulated anion channels (VRAC) are widely expressed chloride channels that are critical for the cell volume regulation. In the mammalian central nervous system, the physiological expression of neuronal VRAC and its role in cerebral ischemia are issues largely unknown. We show that hypoosmotic medium induce an outwardly rectifying chloride conductance in CA1 pyramidal neurons in rat hippocampal slices. The induced chloride conductance was sensitive to some of the VRAC inhibitors, namely, IAA-94 (300 µM) and NPPB (100 µM), but not to tamoxifen (10 µM). Using oxygen-and-glucose deprivation (OGD) to simulate ischemic conditions in slices, VRAC activation appeared after OGD induced anoxic depolarization (AD) that showed a progressive increase in current amplitude over the period of post-OGD reperfusion. The OGD induced VRAC currents were significantly inhibited by inhibitors for glutamate AMPA (30 µM NBQX) and NMDA (40 µM AP-5) receptors in the OGD solution, supporting the view that induction of AD requires an excessive Na+-loading via these receptors that in turn to activate neuronal VRAC. In the presence of NPPB and DCPIB in the post-OGD reperfusion solution, the OGD induced CA1 pyramidal neuron death, as measured by TO-PRO-3-I staining, was significantly reduced, although DCPIB did not appear to be an effective neuronal VRAC blocker. Altogether, we show that rat hippocampal pyramidal neurons express functional VRAC, and ischemic conditions can initial neuronal VRAC activation that may contribute to ischemic neuronal damage.

Purinergic activation of anion conductance and osmolyte efflux in cultured rat hippocampal neurons

The majority of mammalian cells demonstrate regulatory volume decrease (RVD) following swelling caused by hyposmotic exposure. A critical signal initiating RVD is activation of nucleotide receptors by ATP. Elevated extracellular ATP in response to cytotoxic cell swelling during pathological conditions also may initiate loss of taurine and other intracellular osmolytes via anion channels. This study characterizes neuronal ATP-activated anion current and explores its role in net loss of amino acid osmolytes. To isolate anion currents, we used CsCl as the major electrolyte in patch electrode and bath solutions and blocked residual cation currents with NiCl(2) and tetraethylammonium. Anion currents were activated by extracellular ATP with a K(m) of 70 microM and increased over fourfold during several minutes of ATP exposure, reaching a maximum after 9.0 min (SD 4.2). The currents were blocked by inhibitors of nucleotide receptors and volume-regulated anion channels (VRAC). Currents showed outward rectification and inactivation at highly depolarizing membrane potentials, characteristics of swelling-activated anion currents. P2X agonists failed to activate the anion current, and an inhibitor of P2X receptors did not block the effect of ATP. Furthermore, current activation was observed with extracellular ADP and 2-(methylthio)adenosine 5'-diphosphate, a P2Y(1) receptor-specific agonist. Much less current activation was observed with extracellular UTP, suggesting the response is mediated predominantly by P2Y(1) receptors. ATP caused a dose-dependent loss of taurine and alanine that could be blocked by inhibitors of VRAC. ATP did not inhibit the taurine uptake transporter. Thus extracellular ATP triggers a loss of intracellular organic osmolytes via activation of anion channels. This mechanism may facilitate neuronal volume homeostasis during cytotoxic edema.

DCPIB, a specific inhibitor of volume regulated anion channels (VRACs), reduces infarct size in MCAo and the release of glutamate in the ischemic cortical penumbra

Previous studies have indicated that volume regulated anion channels (VRACs) may be involved in the pathology of the ischemic brain cortical penumbra due to activation of VRAC-mediated excitatory amino-acid (EAA) release. To assess this we had studied neuroprotection and EAA release inhibition by a potent VRAC inhibitor, tamoxifen. However, tamoxifen inhibits several other neurodamaging processes. In the present study we use an ethacrynic acid derivative, 4-(2-butyl-6,7-dichloro-2-cyclopentyl-indan-1-on-5-yl) oxobutyric acid (DCPIB), that has recently been shown to be a specific antagonist of volume regulated anion channels (VRAC), to measure the extent of neuroprotection provided and thus to better assess the role of VRAC-mediated release of excitatory amino acids in an intraluminal suture, reversible middle cerebral artery occlusion (rMCAO) model in adult rats. Rats given DCPIB intracisternally had significantly better neurobehavioral scores after 24 h and showed significantly reduced infarct volumes. Mean infarct volumes were 208.0 (SD = 38.3) mm 3 for the vehicle groups, compared with 68.5 (SD = 22.7) mm 3 for intracisternally DCPIB-treated groups ( p = 0.02, Mann–Whitney test), a reduction of around 75%. However, a 500-fold higher dose of DCPIB given intravenously did not reduce infarct volume or improve behavior. The microdialysis study demonstrated statistically significant reduced brain extracellular fluid glutamate when DCPIB was present in the probe. Thus DCPIB, a specific inhibitor of VRACs, given i.c., provides strong neuroprotection in brain ischemia, but it appears to not cross the blood brain barrier as it is not effective when given i.v. These experiments support the hypothesis that EAA released via VRACs contributes to later ischemic-induced damage.

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.

Characterization of the Pharmacology of YM-198313 on Volume-Regulated Anion Channels

Activation of the volume-regulated anion channels (VRAC) is considered to be involved in arrhythmia, but it has not yet been fully elucidated because of the lack of its high affinitive and selective compounds. A newly synthesized compound, YM-198313 (sodium 4-({[2-(methylthio)benzyl]amino}-5-[(1-phenylethyl)thio]isothiazol-3-olate), strongly inhibited VRAC in HeLa cells with an IC50 of 3.03+/-0.05 microM. However, YM-198313 weakly affected both the Ca2+-activated Cl- channels in HTC cells and the cAMP-activated Cl- channels in T84 cells, demonstrating that this compound is selective for VRAC among Cl- channels. At 10 microM, YM-198313 almost completely (100+/-7.8%) inhibited the VRAC current in guinea pig atrial myocytes. However, at the same concentration, YM-198313 showed little inhibitory effect on the cardiac cation currents in ventricular myocytes. We believe that YM-198313 is a potent and selective VRAC inhibitor, therefore, it should be use to clarify the role VRAC plays in arrhythmia.