Inhibition Of Volume-regulated Anion Channels Research Articles

Smooth Muscle-Specific Knockout of LRRC8A Anion Channels Improves Vasodilation in Renal Arteries from Diabetic Mice

Leucine Rich Repeat Containing 8 (LRRC8) family proteins (LRRC8A, B, C, D and E) comprise volume-regulated anion channels (VRACs). We previously have shown that knockout (KO) or block of LRRC8A anion channel attenuates reactive oxygen species (ROS) production by Nox1, impairs TNFα-induced inflammation, and increases vascular relaxation in mesenteric arteries. These changes are associated with reduced RhoA activation. Diabetic (db/db−/−) mice have previously been shown to develop vascular dysfunction that is characterized by increased ROS production and enhanced RhoA activation. Diabetic nephropathy causes chronic renal disease and can lead to end-stage renal failure. Oxidative stress plays a role in both the initiation and progression of this process. We hypothesized that LRRC8A KO would protect vascular function in db/db−/− mice and promote vascular relaxation in renal arteries. Smooth-muscle specific LRRC8A KO mice were created in a db/db−/− background. Renal arteries were isolated from db/db−/− WT and db/db−/− LRRC8A KO mice vascular contraction and relaxation were measured. Contractile responses to phenylephrine (PE) were unaltered in LRRC8A KO compared to WT vessels. However, both endothelium-dependent (acetylcholine, ACh) and -independent (sodium nitroprusside, SNP) relaxation were increased in LRRC8A KO compared to WT rings that were contracted with PE (Emax ACh: WT 76.8 ± 5.5 vs. KO 92.2 ± 3.3 %, LogEC50 SNP: WT -7.6 ± 0.14 vs. KO -8.4 ± 0.14, n = 4-5, * p<0.05). These data demonstrate preserved vasodilation in smooth muscle-specific LRRC8A KO blood vessels. This may lead to increased renal blood flow in the setting of db/db−/−-related diabetes. Ongoing work will determine if improved vasodilation is associated with preserved renal function in these animals. If so, VRAC inhibition may provide a novel therapeutic approach to the prevention and treatment of diabetes-induced renal dysfunction. This work was supported by DK132948 and HL160975. This is the full abstract presented at the American Physiology Summit 2024 meeting and is only available in HTML format. There are no additional versions or additional content available for this abstract. Physiology was not involved in the peer review process.

Cell volume controlled by LRRC8A-formed volume-regulated anion channels fine-tunes T cell activation and function

Biosynthesis drives the cell volume increase during T cell activation. However, the contribution of cell volume regulation in TCR signaling during T lymphoblast formation and its underlying mechanisms remain unclear. Here we show that cell volume regulation is required for optimal T cell activation. Inhibition of VRACs (volume-regulated anion channels) and deletion of leucine-rich repeat-containing protein 8A (LRRC8A) channel components impair T cell activation and function, particularly under weak TCR stimulation. Additionally, LRRC8A has distinct influences on mRNA transcriptional profiles, indicating the prominent effects of cell volume regulation for T cell functions. Moreover, cell volume regulation via LRRC8A controls T cell-mediated antiviral immunity and shapes the TCR repertoire in the thymus. Mechanistically, LRRC8A governs stringent cell volume increase via regulated volume decrease (RVD) during T cell blast formation to keep the TCR signaling molecules at an adequate density. Together, our results show a further layer of T cell activation regulation that LRRC8A functions as a cell volume controlling “valve” to facilitate T cell activation.

LRRC8A Anion Channel Knockout Stimulates the Nrf2 Antioxidant Response

Leucine Rich Repeat Containing 8A (LRRC8A) is an essential component of volume‐regulated anion channels (VRACs), which regulate cell volume. These anion channels are activated by pro-inflammatory mediators such as angiotensin II and tumor necrosis factor-α (TNFα). We previously demonstrated that VRAC inhibition or LRRC8A knockdown reduced Nox1 activation and impaired the inflammatory response to TNFα in cultured vascular smooth muscle cells. We hypothesized that loss of LRRC8A would also modify the antioxidant status of VSMCs. Vascular smooth muscle-specific (Sm22alpha-Cre) LRRC8A KO mice were created. Primary VSMC cultures were prepared from aortae and mesenteric arteries from male mice. Western blotting of protein from WT and LRRC8A KO mesenteric artery VSMCs revealed significant upregulation of proteins that are known to be under the control of the Nrf-2 antioxidant response; heme oxygenase 1 (2.1 fold), thioredoxin 1 (1.8 fold), and superoxide dismutase 1 (1.3 fold) superoxide dismutase 3 (1.8 fold). There was no change in the abundance of catalase.To further explore the role of LRRC8A in intracellular signaling, LRRC8A was immunoprecipitated from cultured WT and LRRC8A KO VSMCs and proteins were analyzed by Mass Spectrometry (MS). Reads were sorted for proteins identified in wild type but not in KO cells. This approach identified the Trim21 E3 ubiquitin ligase, a known regulator of the antioxidant response, as an LRRC8A-associated protein. Trim21 ubiquitinates and thereby promotes degradation of p62 (Sequestosome-1, SQSTM1). p62 sequesters Kelch-like ECH-associated protein 1 (Keap1), preventing it from promoting ubiquitination and destruction of Nrf-2 by the proteosome. Consistent with this, TRIM21 KO mice have an augmented antioxidant status and are protected from myocardial inflammation in response to doxorubicin or ligation of the left coronary artery. We considered the possibility that association of TRIM21 with LRRC8A (directly or indirectly) regulates its activity. Co-immunoprecipitation and western blotting confirmed association of LRRC8A with TRIM21. Pulldown of ubiquitinated proteins from WT and KO VSMCs failed to provide evidence that LRRC8A is ubiquitinated, however, p62 ubiquitination was markedly reduced in KO VSMCs. Thus, reduced p62 turnover in LRRC8A KO cells may enhance Keap1 sequestration and increase Nrf2 activity. The ability of LRRC8A knockdown to both impair the inflammatory response to TNFα and to enhance the antioxidant status of VSMCs identify this protein as an appealing target for the treatment of vascular inflammation. This work was supported by our both grant from NIDDK (1R01DK132948) and NIH (1 R01 HL160975-01A1). This is the full abstract presented at the American Physiology Summit 2023 meeting and is only available in HTML format. There are no additional versions or additional content available for this abstract. Physiology was not involved in the peer review process.

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.

Transfer of cGAMP into Bystander Cells via LRRC8 Volume-Regulated Anion Channels Augments STING-Mediated Interferon Responses and Anti-viral Immunity

The enzyme cyclic GMP-AMP synthase (cGAS) senses cytosolic DNA in infected and malignant cells and catalyzes the formation of 2'3'cGMP-AMP (cGAMP), which in turn triggers interferon (IFN) production via the STING pathway. Here, we examined the contribution of anion channels to cGAMP transfer and anti-viral defense. A candidate screen revealed that inhibition of volume-regulated anion channels (VRACs) increased propagation of the DNA virus HSV-1 but not the RNA virus VSV. Chemical blockade or genetic ablation of LRRC8A/SWELL1, a VRAC subunit, resulted in defective IFN responses to HSV-1. Biochemical and electrophysiological analyses revealed that LRRC8A/LRRC8E-containing VRACs transport cGAMP and cyclic dinucleotides across the plasma membrane. Enhancing VRAC activity by hypotonic cell swelling, cisplatin, GTPγS, or the cytokines TNF or interleukin-1 increased STING-dependent IFN response to extracellular but not intracellular cGAMP. Lrrc8e-/- mice exhibited impaired IFN responses and compromised immunity to HSV-1. Our findings suggest that cell-to-cell transmission of cGAMP via LRRC8/VRAC channels is central to effective anti-viral immunity.

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.

The LRRC8/VRAC anion channel facilitates myogenic differentiation of murine myoblasts by promoting membrane hyperpolarization

Skeletal muscle myoblast differentiation involves elaborate signaling networks, including the activity of various ion channels and transporters. Several K+ and Ca2+ channels have been shown to affect myogenesis, but little is known about roles of Cl- channels in the associated processes. Here, we report that the leucine-rich repeat containing family 8 (LRRC8)/volume-regulated anion channel (VRAC) promotes mouse myoblast differentiation. All LRRC8 subunits of heteromeric VRAC were expressed during myotube formation of murine C2C12 myoblasts. Pharmacological VRAC inhibitors, siRNA-mediated knockdown of the essential VRAC subunit LRRC8A, or VRAC activity-suppressing overexpression of LRRC8A effectively reduced the expression of the myogenic transcription factor myogenin and suppressed myoblast fusion while not affecting myoblast proliferation. We found that inhibiting VRAC impairs plasma membrane hyperpolarization early during differentiation. At later times (more than 6 h after inducing differentiation), VRAC inhibition no longer suppressed myoblast differentiation, suggesting that VRAC acts upstream of K+ channel activation. Consequently, VRAC inhibition prevented the increase of intracellular steady-state Ca2+ levels that normally occurs during myogenesis. Our results may explain the mechanism for the thinning of skeletal muscle bundles observed in LRRC8A-deficient mice and highlight the importance of the LRRC8/VRAC anion channel in cell differentiation.

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.

The Volume-Regulated Anion Channel LRRC8/VRAC Is Dispensable for Cell Proliferation and Migration.

Cells possess the capability to adjust their volume for various physiological processes, presumably including cell proliferation and migration. The volume-regulated anion channel (VRAC), formed by LRRC8 heteromers, is critically involved in regulatory volume decrease of vertebrate cells. The VRAC has also been proposed to play a role in cell cycle progression and cellular motility. Indeed, recent reports corroborated this notion, with potentially important implications for the VRAC in cancer progression. In the present study, we examined the role of VRAC during cell proliferation and migration in several cell types, including C2C12 myoblasts, human colon cancer HCT116 cells, and U251 and U87 glioblastoma cells. Surprisingly, neither pharmacological inhibition of VRAC with 4-[(2-Butyl-6,7-dichloro-2-cyclopentyl-2,3-dihydro-1-oxo-1H-inden-5-yl)oxy]butanoic acid (DCPIB), carbenoxolone or 5-nitro-2-(3-phenylpropyl-amino)benzoic acid (NPPB), nor siRNA-mediated knockdown or gene knockout of the essential VRAC subunit LRRC8A affected cell growth and motility in any of the investigated cell lines. Additionally, we found no effect of the VRAC inhibition using siRNA treatment or DCPIB on PI3K/Akt signaling in glioblastoma cells. In summary, our work suggests that VRAC is dispensable for cell proliferation or migration.

Natural and synthetic flavonoids, novel blockers of the volume-regulated anion channels, inhibit endothelial cell proliferation

Natural flavonoids are ubiquitous in dietary plants and vegetables and have been proposed to have antiviral, antioxidant, cardiovascular protective, and anticancer effects. Volume-regulated anion channels (VRACs), which are essential for cell volume regulation, have been proposed to play a key role in cell proliferation and migration, apoptosis, transepithelial transport, and cancer development. In this study, we screened a group of 53 structurally related natural flavonoids and three synthetic flavonoids for their inhibitory activities on VRAC currents. A whole-cell patch technique was used to record VRAC currents in the human embryonic kidney (HEK) 293 and human umbilical vein endothelial (HUVEC) cells. The 5'-bromo-2-deoxyuridine (BrdU) assay technique was used to investigate cell proliferation. At 100μM, 34 of 53 compounds significantly inhibited hypotonic extrasolution-induced VRAC currents by > 50% in HEK293 cells. Among these compounds, luteolin, baicalein, eupatorin, galangin, quercetin, fisetin, karanjin, Dh-morin, genistein, irisolidone, and prunetin exhibited the highest efficacy for VRAC blockade (the mean inhibition > 80%) with IC50s of 5-13μM and Emaxs of about 87-99%. We also studied the effects of three synthetic flavonoids on VRAC currents in HEK293 cells. Flavoxate showed high inhibition efficacy toward VRAC currents (IC50 = 2.3 ± 0.3μM; Emax = 91.8% ± 2.7%). Finally, these flavonoids inhibited endogenous VRAC currents and cell proliferation in endothelial cells. This study demonstrates that natural and synthetic flavonoids are potent VRAC current inhibitors, and VRAC inhibition by flavonoids might be responsible for their anti-angiogenic effects.

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.

MPS 18-06 LRRC8A/C VOLTAGE-DEPENDENT ANION CHANNELS ARE REQUIRED FOR TNFALPHA-INDUCED SUPEROXIDE PRODUCTION BY NOX1 IN VASCULAR SMOOTH MUSCLE CELLS

Objective: Tumor necrosis factor-α (TNFα), pro-inflammatory cytokine, is linked to cardiovascular risk, including inflammatory vascular lesions and hypertension. TNFα-induced superoxide (O2−•) production by NADPH oxidase 1 (Nox1) is required for inflammatory signaling in vascular smooth muscle cells (VSMC). TNFα also activates volume-regulated anion channels (VRACs). These multimeric Cl− channels are composed of Leucine Rich Repeat Containing 8A (LRRC8A) protein, combined with various closely-related isoforms (LRRC8B through E). This produces VRACs with differing biophysical properties that support VSMC proliferation and inflammation by an unknown mechanism. We hypothesized that VRAC current is required for Nox1 activation by TNFα. Design and Method: In VSMC, reducing expression (siRNA) of LRRC8A through E or inhibiting VRAC current (DCPIB 30 μM) were used to measure TNFα signaling. Extracellular O2−• production by Nox1 was detected by CAT1H spin probe using electron spin resonance. Biotinylated TNFα was used to visualize TNFα receptor (TNFR1) endocytosis with FITC-conjugated avidin. Immunoprecipitation-western blot analysis was performed with LRRC8A and p22phox subunit of Nox1. Results: siRNA of LRRC8A or C (but not the B, D, or E isoforms) or DCPIB inhibited NF-κB activation (72% decrease), induction of iNOS (52% decrease) and VCAM (36% decrease) expression, and proliferation (siControl 100%, siControl + TNFα 111 ± 1.5%, siLRRC8A + TNFα 101 ± 1.6%). siLRRC8A reduced extracellular O2−• production by Nox1 (siControl + TNFα 1259 ± 80, siLRRC8A + TNFα 792 ± 86 pmol/mg of protein). TNFR1, but not transferrin receptor endocytosis, was impaired by siNox1, siLRRC8A or DCPIB. Extracellular superoxide dismutase (500 U/mL) also potently inhibited TNFR1 endocytosis, while catalase (1000 U/mL) had no effect. Finally, LRRC8A co-immunoprecipitated with the p22phox subunit of Nox1 suggesting a direct functional link to activity of the Nox1 complex. Conclusions: LRRC8A/C VRACs are required for Nox1 activation at the plasma membrane and the extracellular O2−• that it produces is required for TNFR1 endocytosis. These data provide a novel mechanism for the anti-proliferative and anti-inflammatory effects of VRAC inhibition in VSMC.

Leucine Rich Repeat Containing 8A Is Required For NADPH Oxidase 1‐Dependent Superoxide Production and Tumor Necrosis Factor Alpha Receptor Endocytosis

Tumor necrosis factor‐α (TNFα) induces superoxide (O2−•) production by NADPH oxidase 1 (Nox1) and activates volume‐regulated anion channel (VRAC) in vascular smooth muscle cells (VSMC). The Leucine Rich Repeat Containing 8A (LRRC8A) protein is part of VRAC, which supports cell proliferation and inflammation. We hypothesized that LRRC8A supports TNFα signaling. Reduction of LRRC8A expression (siLRRC8A) or inhibition of VRAC (DCPIB 30μM) inhibited responses to TNFα including; VRAC and NF‐kB activation (72% decrease), iNOS (52% decrease) and VCAM (36% decrease) expression, and proliferation (siControl 100%, siControl+TNFα 111±1.5%, siLRRC8A+TNFα 101±1.6%). siLRRC8A also reduced extracellular O2−• production (siControl+TNFα 1259±80, siLRRC8A+TNFα 792±86 pmol/mg of protein). Biotinylated TNFα was used to visualize TNFα receptor (TNFR) endocytosis with FITC‐conjugated avidin. TNFR, but not transferrin receptor endocytosis was impaired by siLRRC8A and DCPIB. Extracellular superoxide dismutase (500 U/mL) also inhibited TNFR endocytosis, while catalase (1000 U/mL) did not. LRRC8A co‐immunoprecipitated with the p22phox subunit of Nox1 suggesting a direct functional link to activity of the Nox1 complex. Thus, LRRC8A is required for Nox1‐mediated O2−• production, and extracellular O2−• is required for TNFR endocytosis. These data provide a novel mechanism for the anti‐proliferative and anti‐inflammatory effects of VRAC inhibition.

Intracellular Na(+) inhibits volume-regulated anion channel in rat cortical astrocytes.

Accumulating evidence indicates that increased intracellular Na(+) concentration ([Na(+) ]i ) in astroglial cells is associated with the development of brain edema under ischemic conditions, but the underlying mechanisms are still elusive. Here, we report that in primary cultured rat cortical astrocytes, elevations of [Na(+) ]i reflecting those achieved during ischemia cause a marked decrease in hypotonicity-evoked current mediated by volume-regulated anion channel (VRAC). Pharmacological manipulations revealed that VRAC inhibition was not due to the reverse mode of the plasma membrane sodium/calcium exchanger. The negative modulation of VRAC was also observed in an astrocytic cell line lacking the predominant astrocyte water channel aquaporin 4, indicating that [Na(+) ]i effect was not mediated by the regulation of aquaporin 4 activity. The inward rectifier Cl(-) current, which is also expressed by cultured astrocytes, was not affected by [Na(+) ]i increase. VRAC depression by high [Na(+) ]i was confirmed in adult astrocytes, suggesting that it was not developmentally regulated. Altogether, these results provide the first evidence that intracellular Na(+) dynamics can modulate astrocytic membrane conductance that controls functional processes linked to cell volume regulation and add further support to the concept that limiting astrocyte intracellular Na(+) accumulation might be a favorable strategy to counteract the development of brain edema.

Effects of octane derivatives on activity of the volume-regulated anion channel in rat pancreatic β-cells

BackgroundSaturated free fatty acids (FFAs) have a dual action on pancreatic β-cells, consisting of an initial enhancement and subsequent suppression of glucose-induced electrical activity and insulin release. These stimulatory and inhibitory effects have been attributed, at least in part, to the activation and inhibition, respectively, of the volume-regulated anion channel (VRAC) by FFAs. Both effects were independent of their metabolism.We have now investigated the effects of related aliphatic compounds in order to further define the determinants of FFA interaction with VRAC. Methodsβ-Cell VRAC and electrical activity were measured by conventional whole-cell and perforated patch recording, respectively. Cell volume was measured using a video-imaging technique. ResultsIn common with octanoic acid, addition of methyl octanoate or n-octanol resulted in a rapid, pronounced and reversible inhibition of VRAC activity. Addition of n-octane had no significant effect on VRAC activity. n-Octanol had a biphasic effect on β-cell membrane potential, namely a small transient depolarization followed by a marked hyperpolarization. n-Octanol was also found to prevent regulatory volume decrease in cells exposed to a hypotonic medium, consistent with VRAC inhibition. ConclusionIt is suggested that methyl octanoate and n-octanol can mimic the effects of FFAs on the pancreatic β-cell via modulation of VRAC activity. The structural requirements for this effect appear to be a medium or long chain aliphatic compound containing at least one oxygen atom.

Distinct expression/function of potassium and chloride channels contributes to the diverse volume regulation in cortical astrocytes of GFAP/EGFP mice.

Recently, we have identified two astrocytic subpopulations in the cortex of GFAP-EGFP mice, in which the astrocytes are visualized by the enhanced green–fluorescent protein (EGFP) under the control of the human glial fibrillary acidic protein (GFAP) promotor. These astrocytic subpopulations, termed high response- (HR-) and low response- (LR-) astrocytes, differed in the extent of their swelling during oxygen-glucose deprivation (OGD). In the present study we focused on identifying the ion channels or transporters that might underlie the different capabilities of these two astrocytic subpopulations to regulate their volume during OGD. Using three-dimensional confocal morphometry, which enables quantification of the total astrocytic volume, the effects of selected inhibitors of K+ and Cl− channels/transporters or glutamate transporters on astrocyte volume changes were determined during 20 minute-OGD in situ. The inhibition of volume regulated anion channels (VRACs) and two-pore domain potassium channels (K2P) highlighted their distinct contributions to volume regulation in HR-/LR-astrocytes. While the inhibition of VRACs or K2P channels revealed their contribution to the swelling of HR-astrocytes, in LR-astrocytes they were both involved in anion/K+ effluxes. Additionally, the inhibition of Na+-K+-Cl− co-transporters in HR-astrocytes led to a reduction of cell swelling, but it had no effect on LR-astrocyte volume. Moreover, employing real-time single-cell quantitative polymerase chain reaction (PCR), we characterized the expression profiles of EGFP-positive astrocytes with a focus on those ion channels and transporters participating in astrocyte swelling and volume regulation. The PCR data revealed the existence of two astrocytic subpopulations markedly differing in their gene expression levels for inwardly rectifying K+ channels (Kir4.1), K2P channels (TREK-1 and TWIK-1) and Cl− channels (ClC2). Thus, we propose that the diverse volume changes displayed by cortical astrocytes during OGD mainly result from their distinct expression patterns of ClC2 and K2P channels.

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.

A dual action of saturated fatty acids on electrical activity in rat pancreatic β‐cells. Role of volume‐regulated anion channel and KATP channel currents

Free fatty acids (FFAs) exert complex actions on pancreatic β-cells. Typically, an initial potentiation of insulin release is followed by a gradual impairment of β-cell function, the latter effect being of possible relevance to hyperlipidaemia in type 2 diabetes mellitus. The molecular actions of FFAs are poorly understood. The present study investigated the acute effects of saturated FFAs on electrophysiological responses of rat pancreatic β-cells. Membrane potential and KATP channel activity were recorded using the perforated patch technique. Volume-regulated anion channel (VRAC) activity was assessed from conventional whole-cell recordings. Cell volume regulation was measured using a video-imaging technique. Addition of octanoate caused a transient potentiation of glucose-induced electrical activity, followed by a gradual hyper-polarisation and a prolonged inhibition of electrical activity. Octanoate caused an initial increase in VRAC activity followed by a secondary inhibition coinciding with increased KATP channel activity. Similar effects were observed with palmitate and 2-bromopalmitate whereas butyrate was virtually ineffective. Octanoate and palmitate also exerted a dual effect on electrical activity evoked by tolbutamide. Octanoate significantly attenuated cell volume regulation in hypotonic solutions, consistent with VRAC inhibition. It is concluded that medium and long chain FFAs have a dual action on glucose-induced electrical activity in rat pancreatic β-cells: an initial stimulatory effect followed by a secondary inhibition. These effects appear to be the result of reciprocal actions on VRAC and KATP channel currents, and could contribute towards the stimulatory and inhibitory actions of FFAs on pancreatic β-cell function.

α1‐Adrenoceptor‐mediated depletion of phosphatidylinositol 4, 5‐bisphosphate inhibits activation of volume‐regulated anion channels in mouse ventricular myocytes

BACKGROUND AND PURPOSE Volume‐regulated anion channels (VRACs) play an important role in cell‐volume regulation. α1‐Adrenoceptor stimulation by phenylephrine (PE) suppressed the hypotonic activation of VRAC current in mouse ventricular cells and regulatory volume decrease (RVD) was also absent in PE‐treated cells. We examined whether the effects of α1‐adrenoceptor stimuli on VRAC current were modulated by phosphatidylinositol signalling.EXPERIMENTAL APPROACH Whole‐cell patch‐clamp method was used to record the hypotonicity‐induced VRAC current in mouse ventricular cells. RVD was analyzed by videomicroscopic measurement of cell images.KEY RESULTS The attenuation of VRAC current by PE was suppressed by α1A‐adrenoceptor antagonists (prazosin and WB‐4101), anti‐Gq protein antibody and a specific phosphoinositide‐specific phospholipase C (PLC) inhibitor (U‐73122), but not by antagonists for α1B‐, α1D‐ or β‐adrenoceptor, or protein kinase C inhibitors. The inhibition of VRAC by PE was antagonized by intracellular excess phosphatidylinositol 4,5‐bisphosphate (PIP2), while intracellular anti‐PIP2 antibody (PIP2 Ab) inhibited the activation of VRAC currents. When cells were loaded with phosphatidylinositol 3,4,5‐trisphosphate (PIP3) with or without PIP2 Ab, PE little affected the VRAC current. Extracellular m‐3M3FBS (an activator of PLC) suppressed VRAC in the absence of PE, and this effect was reversed by intracellular excess PIP2.CONCLUSIONS AND IMPLICATIONS Our results indicate that the stimulation of α1A‐adrenoceptors by PE inhibited the activation of cardiac VRAC current via PIP3 depletion brought about by PLC‐dependent reduction of membrane PIP2 level.

Carbenoxolone inhibits volume-regulated anion conductance in cultured rat cortical astroglia

Accumulating evidence indicate that the gap-junction inhibitor carbenoxolone (CBX) regulates neuronal synchronization, depresses epileptiform activity and has a neuroprotective action. These CBX effects do not depend solely on its ability to inhibit gap junction channels formed by connexins (Cx), but the underlying mechanisms remain to be elucidated. Here we addressed the questions whether CBX modulates volume-regulated anion channels (VRAC) involved in the regulatory volume decrease and regulates the associated release of excitatory amino acids in cultured rat cortical astrocytes. We found that CBX inhibits VRAC conductance with potency comparable to that able to depress the activity of the most abundant astroglial gap junction protein connexin43 (Cx43). However, the knock down of Cx43 with small interfering RNA (siRNA) oligonucleotides and the use of various pharmacological tools revealed that VRAC inhibition was not mediated by interaction of CBX with astroglial Cx proteins. Comparative experiments in HEK293 cells stably expressing another putative target of CBX, the purinergic ionotropic receptor P2X7, indicate that the presence of this receptor was not necessary for CBX-mediated depression of VRAC. Finally, we show that in COS-7 cells, which are not endowed with pannexin-1 protein, another astroglial plasma membrane interactor of CBX, VRAC current retained its sensitivity to CBX. Complementary analyses indicate that the VRAC-mediated release of excitatory amino acid aspartate was decreased by CBX. Collectively, these findings support the notion that CBX could affect astroglial ability to modulate neuronal activity by suppressing excitatory amino acid release through VRAC. They also provide a possible mechanistic clue for the neuroprotective effect of CBX in vivo.