Volume-regulated Chloride Channels Research Articles

Electrophysiological and Molecular Mechanisms of Sinoatrial Node Mechanosensitivity.

The understanding of the electrophysiological mechanisms that underlie mechanosensitivity of the sinoatrial node (SAN), the primary pacemaker of the heart, has been evolving over the past century. The heart is constantly exposed to a dynamic mechanical environment; as such, the SAN has numerous canonical and emerging mechanosensitive ion channels and signaling pathways that govern its ability to respond to both fast (within second or on beat-to-beat manner) and slow (minutes) timescales. This review summarizes the effects of mechanical loading on the SAN activity and reviews putative candidates, including fast mechanoactivated channels (Piezo, TREK, and BK) and slow mechanoresponsive ion channels [including volume-regulated chloride channels and transient receptor potential (TRP)], as well as the components of mechanochemical signal transduction, which may contribute to SAN mechanosensitivity. Furthermore, we examine the structural foundation for both mechano-electrical and mechanochemical signal transduction and discuss the role of specialized membrane nanodomains, namely, caveolae, in mechanical regulation of both membrane and calcium clock components of the so-called coupled-clock pacemaker system responsible for SAN automaticity. Finally, we emphasize how these mechanically activated changes contribute to the pathophysiology of SAN dysfunction and discuss controversial areas necessitating future investigations. Though the exact mechanisms of SAN mechanosensitivity are currently unknown, identification of such components, their impact into SAN pacemaking, and pathological remodeling may provide new therapeutic targets for the treatment of SAN dysfunction and associated rhythm abnormalities.

ClC-3: A Novel Promising Therapeutic Target for Atherosclerosis.

Chloride channel 3 (ClC-3), a Cl-/H+ antiporter, has been well established as a member of volume-regulated chloride channels (VRCCs). ClC-3 may be a crucial mediator for activating inflammation-associated signaling pathways by regulating protein phosphorylation. A growing number of studies have indicated that ClC-3 overexpression plays a crucial role in mediating increased plasma low-density lipoprotein levels, vascular endothelium dysfunction, pro-inflammatory activation of macrophages, hyper-proliferation and hyper-migration of vascular smooth muscle cells (VSMCs), as well as oxidative stress and foam cell formation, which are the main factors responsible for atherosclerotic plaque formation in the arterial wall. In the present review, we summarize the molecular structures and classical functions of ClC-3. We further discuss its emerging role in the atherosclerotic process. In conclusion, we explore the potential role of ClC-3 as a therapeutic target for atherosclerosis.

Volume-regulated chloride channel regulates cell proliferation and is involved in the possible interaction between TMEM16A and LRRC8A in human metastatic oral squamous cell carcinoma cells

ObjectivesVolume-regulated anion channels (VRACs), expressed in various cells, play an important role in cell volume regulation. Despite being physiologically defined almost half a century ago, only the molecular candidates of VRAC, TMEM16A, LRRC8A, and bestrophin-1 (BEST1), are known. Here, we aimed to explore the functional significance of VRAC in, HST-1, an oral squamous cell carcinoma (OSCC) cell line. MethodsCell proliferation assays, RT-PCR, Western blot, and flow cytometry were used to estimate changes in gene expression and cell proliferation. Ion channel activity was recorded using the patch-clamp technique. Specific genes were knocked-down by siRNA assays. ResultsVRAC, identified as a hypotonicity-induced current, was highly functional and associated with the proliferation of HST-1 cells but not of HaCaT (a normal keratinocyte) cells. The pharmacological profile of VRAC in HST-1 was similar to that reported previously. DCPIB, a specific VRAC inhibitor, completely inhibited VRAC and proliferation of HST-1 cells, eventually leading to apoptosis. VRAC in HST-1 was attenuated by the knockdown of TMEM16A and LRRC8A, while knockdown of BEST1 affected cell proliferation. In situ proximity ligation assay showed that TMEM16A and LRRC8A co-localized under isotonic conditions (300 mOsM) but were separated under hypotonic conditions (250 mOsM) on the plasma membrane. ConclusionsWe have found that VRAC acts to regulate the proliferation of human metastatic OSCC cells and the composition of VRAC may involve in the interactions between TMEM16A and LRRC8A in HST-1 cells.

Anti-angiogenic activity of chloride and potassium channel modulators: repurposing ion channel modulators

BackgroundExcessive angiogenesis can be the root cause of many pathological conditions. Various types of ion channels are found on the endothelial cells. These ion channels play a vital role in the multi-stepped process of angiogenesis. The study aims to investigate the anti-angiogenic effects of specific ion channel modulators mefloquine (volume-regulated chloride channel blocker), lubiprostone (ClC-2 channel agonist), and 4-aminopyridine (voltage-gated potassium channel blocker).ResultsThe anti-angiogenic activity of ion channel modulators was screened by measuring its effects on the area of neovascularization and histopathological studies by in vivo (corneal neovascularization) method and by in vitro assays, endothelial cell proliferation assay, cell migration assay, and matrigel cord-like morphogenesis assay. The test and standard drug (bevacizumab) groups were compared with the control group using one-way ANOVA, followed by post hoc test, and Dunnett’s test to compare the mean of all the groups with the control mean. The results revealed that mefloquine at the dose of 0.6% w/v and 1.0% w/v, lubiprostone at the dose of 0.5% w/v and 1.0% w/v, and 4-aminopyridine at the dose of 2% w/v and 4% w/v showed significant anti-angiogenic property. In the studies on human umbilical vein endothelial cells, the test drugs (100 nM) showed significant inhibition of proliferation, migration, and decrease in network length of cord-like tubes.ConclusionThe scientific findings indicate that the test drugs have potent anti-angiogenic activity by inhibiting the cell proliferation, inhibiting the cell volume increase, arresting the cell cycle progression and by causing membrane hyperpolarization. The potent anti-angiogenic drugs obtained by repurposing these ion channel modulators, in the further studies, will be able to treat the diseases due to excess angiogenesis from the root cause.Graphical abstract

The Y227N mutation affects bestrophin-1 protein stability and impairs sperm function in a mouse model of Best vitelliform macular dystrophy.

ABSTRACTHuman bestrophin-1 (BEST1) is an integral membrane protein known to function as a Ca2+-activated and volume-regulated chloride channel. The majority of disease-associated mutations in BEST1 constitute missense mutations and were shown in vitro to lead to a reduction in mutant protein half-life causing Best disease (BD), a rare autosomal dominant macular dystrophy. To further delineate BEST1-associated pathology in vivo and to provide an animal model useful to explore experimental treatment efficacies, we have generated a knock-in mouse line (Best1Y227N). Heterozygous and homozygous mutants revealed no significant ocular abnormalities up to 2 years of age. In contrast, knock-in animals demonstrated a severe phenotype in the male reproductive tract. In heterozygous Best1Y227N males, Best1 protein was significantly reduced in testis and almost absent in homozygous mutant mice, although mRNA transcription of wild-type and knock-in allele is present and similar in quantity. Degradation of mutant Best1 protein in testis was associated with adverse effects on sperm motility and the capability to fertilize eggs. Based on these results, we conclude that mice carrying the Best1 Y227N mutation reveal a reproducible pathologic phenotype and thus provide a valuable in vivo tool to evaluate efficacy of drug therapies aimed at restoring Best1 protein stability and function.

The AQP-3 water channel is a pivotal modulator of glycerol-induced chloride channel activation in nasopharyngeal carcinoma cells

Aquaporin (AQP) and chloride channels are ubiquitous in virtually all living cells, playing pivotal roles in cell proliferation, migration and apoptosis. We previously reported that AQP-3 aquaglyceroporin and ClC-3 chloride channels could form complexes to regulate cell volume in nasopharyngeal carcinoma cells. In this study, the roles of AQP-3 in their hetero-complexes were further investigated. Glycerol entered the cells via AQP-3 and induced two different Cl− currents through cell swelling-dependent or -independent pathways. The swelling-dependent Cl− current was significantly inhibited by pretreatment with CuCl2 and AQP-3-siRNA. After siRNA-induced AQP-3 knock-down, the 140mM glycerol isoosmotic solution swelled cells by 22% (45% in AQP-3-intact cells) and induced a smaller Cl− current; this current was smaller than that activated by 8% cell volume swelling, which induced by the 140mM glycerol hyperosmotic solution in AQP-3-intact cells. This suggests that the interaction between AQP-3 and ClC-3 plays an important role in cell volume regulation and that AQP-3 may be a modulator that opens volume-regulated chloride channels. The swelling-independent Cl− current, which was activated by extracellular glycerol, was reduced by CuCl2 and AQP-3-siRNA pretreatment. Dialyzing glycerol into cells via the pipette directly induced the swelling-independent Cl− current; however this current was blocked by AQP-3 down-regulation, suggesting AQP-3 is essential for the opening of chloride channels. In conclusion, AQP-3 is the pathway for water, glycerol and other small solutes to enter cells, and it may be an essential modulator for the gating of chloride channels.

Reduction of Intracellular Chloride Concentration Promotes Foam Cell Formation.

Previous work has demonstrated that the volume-regulated chloride channel is activated during foam cell formation, and inhibition of chloride movement prevents intracellular lipid accumulation. However, the mechanism explaining how chloride movement promotes foam cell formation is not clear. Foam cell formation was determined by Oil Red O staining. Western blotting and co-immunoprecipitation were used to examine protein expression and protein-protein interaction. [Cl(-)]iwas measured using 6-methoxy-N-ethylquinolinium iodide dye. The results showed that [Cl(-)]iwas decreased in monocytes/macrophages from patients with hypercholesterolemia and from apoE(-/-)mice fed with a high-fat diet. Lowering [Cl(-)]iupregulated scavenger receptor A (SR-A) expression, increased the binding and uptake of oxLDL, enhanced pro-inflammatory cytokine production and subsequently accelerated foam cell formation in macrophages from humans and mice. In addition, low Cl(-)solution stimulated the activation of JNK and p38 mitogen-activated protein kinases. Inhibition of JNK and p38 blocked Cl(-)reduced medium-induced SR-A expression and lipid accumulation. In contrast, reduction of [Cl(-)]ipromoted the interaction of SR-A with caveolin-1, thus facilitating caveolin-1-dependent SR-A endocytosis. Moreover, disruption of caveolae attenuated SR-A internalization, JNK and p38 activation, and ultimately prevented SR-A expression and foam cell formation stimulated by low Cl(-)medium. This data provide strong evidence that reduction of [Cl(-)]iis a critical contributor to intracellular lipid accumulation, suggesting that modulation of [Cl(-)]iis a novel avenue to prevent foam cell formation and atherosclerosis.

Integrin β3 mediates cerebrovascular remodelling through Src/ClC-3 volume-regulated Cl−channel signalling pathway

Cerebrovascular remodelling is one of the important risk factors of stroke. The underlying mechanisms are unclear. Integrin β3 and volume-regulated ClC-3 Cl(-) channels have recently been implicated as important contributors to vascular cell proliferation. Therefore, we investigated the role of integrin β3 in cerebrovascular remodelling and related Cl(-) signalling pathway. Cl(-) currents were recorded using a patch clamp technique. The expression of integrin β3 in hypertensive animals was examined by Western blot and immunohistochemisty. Immunoprecipitation, cDNA and siRNA transfection were employed to investigate the integrin β3/Src/ClC-3 signalling. Integrin β3 expression was up-regulated in stroke-prone spontaneously hypertensive rats, 2-kidney 2-clip hypertensive rats and angiotensin II-infused hypertensive mice. Integrin β3 expression was positively correlated with medial cross-sectional area and ClC-3 expression in the basilar artery of 2-kidney 2-clip hypertensive rats. Knockdown of integrin β3 inhibited the proliferation of rat basilar vascular smooth muscle cells induced by angiotensin II. Co-immunoprecipitation and immunofluorescence experiments revealed a physical interaction between integrin β3, Src and ClC-3 protein. The integrin β3/Src/ClC-3 signalling pathway was shown to be involved in the activation of volume-regulated chloride channels induced by both hypo-osmotic stress and angiotensin II. Tyrosine 284 within a concensus Src phosphorylation site was the key point for ClC-3 channel activation. ClC-3 knockout significantly attenuated angiotensin II-induced cerebrovascular remodelling. Integrin β3 mediates cerebrovascular remodelling during hypertension via Src/ClC-3 signalling pathway.

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Voltage-sensitive chloride ion channels in Anopheles gambiae Sua-1B cells

In this study, we performed electrophysiological analysis of Anopheles gambiae Sua-1B cells having "neuron-like" morphologies using the patch clamp method. The recorded cells (n = 79) had processes resembling axons/dendrites, with 63 % unipolar, 22 % bipolar, and 15 % multipolar. While no inward currents were observed following step depolarizations (holding potential = -80 mV), a slowly activating outward current was observed in 96 % of the cells, especially at depolarized potentials. The amplitude of the current was attenuated nearly 70 % by reducing extracellular Cl⁻ ion concentration, or by incubating with 100 μM DIDS, a known voltage-sensitive chloride channel blocker, suggesting that the current was mediated by chloride ions. No qualitative difference was found between recordings made with Cs⁺ ions in the intracellular pipette solution (inhibits K⁺ currents) and those made with normal physiological solution, indicating a deficiency of potassium channels. Additionally, recordings made with Ca²⁺-free extracellular bath solution eliminated the slowly activating outward current. A subset of cells (n = 3) lacked this current, but had outward currents with voltage-dependent properties similar to those of volume-regulated chloride channels. Taken together, our results suggest that the voltage-sensitive currents observed in the majority of Sua-1B cells are mediated primarily by chloride channels of the calcium-dependent subtype.

Differences between femoral artery and vein smooth muscle cells in volume-regulated chloride channels

The purpose of the present study was to compare the differences between the role of volume-regulated Cl⁻ channels (VRCCs) in veins and arteries. We used the whole cell patch clamp and fluorescence imaging techniques to evaluate swelling-induced Cl⁻ current (I(Cl,vol)) and changes in the intracellular concentrations of Cl⁻ ([Cl⁻](i)) induced by hypotonic solutions in rat femoral artery cells (FASMCs) and vein smooth muscle cells (FVSMCs). I(Cl,vol) and [Cl⁻](i) decline induced by hypotonic solution were more prominent in FASMCs than in FVSMCs. I(Cl,vol) and the alterations in [Cl⁻](i) were gradually increased as the number of cell passages increased. However, the regulatory function of tyrosine protein phosphorylation in volume-regulated chloride movement is prominent in veins. The expression of ClC-3 was higher in FASMCs than in FVSMCs. VRCC activity is more pronounced in rat femoral arteries than in veins. VRCC activity and tyrosine protein phosphorylation regulative function increase gradually as vascular cells switch from contractile to proliferative phenotypes.

Ion channel electrophysiology via integrated planar patch‐clamp chip with on‐demand drug exchange

Planar patch clamp has revolutionized characterization of ion channel behavior in drug discovery primarily via advancement in high throughput. Lab use of planar technology, however, addresses different requirements and suffers from inflexibility to enable wide range of interrogation via a single cell. This work presents integration of planar patch clamp with microfluidics, achieving multiple solution exchanges for tailor-specific measurement and allowing rapid replacement of the cell-contacting aperture. Studies via endogenously expressed ion channels in HEK 293T cells were commenced to characterize the device. Results reveal the microfluidic concentration generator produces distinct solution/drug combination/concentrations on-demand. Volume-regulated chloride channel and voltage-gated potassium channels in HEK 293T cells immersed in generated solutions under various osmolarities or drug concentrations show unique channel signature under specific condition. Excitation and blockage of ion channels in a single cell was demonstrated via serial solution exchange. Robustness of the reversible bonding and ease of glass substrate replacement were proven via repeated usage of the integrated device. The present approach reveals the capability and flexibility of integrated microfluidic planar patch-clamp system for ion channel assays.

Alteration of volume-regulated chloride channel during macrophage-derived foam cell formation in atherosclerosis

ObjectiveVolume-regulated Cl− channel (VRCC) plays a critical role in regulation of a variety of physiological functions. However, little is known whether VRCC is involved in atherosclerosis. In this study, we investigated the functions of VRCC during foam cell formation in macrophages. Methods and resultsTreatment of RAW264.7 cells with ox-LDL increased intracellular cholesterol content as well as cell volume. After ox-LDL treatment, the resting [Cl−]i in isotonic solution was decreased. Hypotonic solution reduced [Cl−]i and evoked volume-regulated Cl− current in all the cells, however, the swelling-induced reduction of [Cl−]i and increase of Cl− current were more prominent in ox-LDL treated cells than that in control. The increases of volume-regulated Cl− movement positively correlated with the intracellular cholesterol content. Moreover, in peritoneal macrophages isolated from high-fat diet ApoE−/− mice, the swelling-induced Cl− movement and current were enhanced compared with those in control group, and their increments positively correlated with atherosclerotic plaque area. Finally, activation of VRCC by hypotonic medium significantly accelerated, whereas, inhibition of VRCC with Cl− channel blockers remarkably attenuated, ox-LDL-induced macrophage-derived foam cell formation. ConclusionThe activity of VRCC is augmented during macrophage-derived foam cell formation. Activation of VRCC accelerated, whereas, inhibition of VRCC attenuated, ox-LDL-induced lipid accumulation in macrophages, suggesting VRCC is involved in the regulation of foam cell formation.

Effects of Tween 80 on volume-regulated chloride channel and cell proliferation in rat basilar artery smooth muscle cell

We have previously found that volume-regulated chloride current (VRCC) is involved in cell cycle progression and cell proliferation. This study was to examine the effect of Tween 80, a nonionic surfactant, on VRCC and cell proliferation in rat basilar artery smooth muscle cells (BASMCs). VRCC was recorded using a whole-cell patch clamp. Cell proliferation and cell cycle were determined by CCK-8, cell count and flow cytometry. The results showed that endothelin-1 promotes cell cycle transition from the G0/G1 phase to the S phase and significantly increases VRCC in BASMCs. The effect of Tween 80 on VRCC is reversible and concentration dependent. However, this chemical has no effect on the calcium-activated chloride channel. Tween 80 also concentration-dependently inhibits BASMCs proliferation and arrests cells in the G1/S checkpoint. The antiproliferative effect is paralleled with the inhibitory effect on VRCC. Our study demonstrates that the inhibitory effect of Tween 80 on VRCC contributes importantly to arrest of the cell cycle and prevention of cell proliferation.

Activation of Volume Regulated Chloride Channels Protects Myocardium from Ischemia/reperfusion Damage in Second-window Ischemic Preconditioning

Activation of volume regulated chloride channels (VRCCs) has been shown to be cardioprotective in ischemic preconditioning (IPC) of isolated hearts but the underlying molecular mechanisms remain unclear. Recent independent studies support that ClC-3, a ClC voltage-gated chloride channel, may function as a key component of the VRCCs. Thus, ClC-3 knockout (Clcn3^-/-) mice and their age-matched heterozygous (Clcn3^+/-) and wild-type (Clcn3^+/+) littermates were used to test whether activation of VRCCs contributes to cardioprotection in early and/or second-window IPC. Targeted disruption of ClC-3 gene caused a decrease in the body weight but no changes in heart/body weight ratio. Telemetry ECG and echocardiography revealed no differences in ECG and cardiac function under resting conditions among all groups. Under treadmill stress (10 m/min for 10 min), the Clcn3^-/- mice had significant slower heart rate (648±12 bpm) than Clcn3^+/+ littermates (737±19 bpm, n=6, P<0.05). Ex vivo IPC in the isolated working-heart preparations protected cardiac function during reperfusion and significantly decreased apoptosis and infarct size in all groups. In vivo early IPC significantly reduced infarct size in all groups including Clcn3^-/- mice (22.7±3.7% vs control 40.1±4.3%, n=22, P=0.004). Second-window IPC significantly reduced apoptosis and infarction in Clcn3^+/+ (22.9±3.2% vs 45.7±5.4%, n=22, P<0.001) and Clcn3^+/- mice (27.5±4.1% vs 42.2±5.7%, n=15, P<0.05) but not in Clcn3^-/- littermates (39.8±4.9% vs 41.5±8.2%, n=13, P>0.05). Impaired cell volume regulation of the Clcn3^-/- myocytes may contribute to the failure of cardioprotection by second-window IPC. These results strongly support that activation of VRCCs may play an important cardioprotective role in second-window IPC.

The research progress of volume-sensitive Cl- channel in tumor

Cell swelling activates or upregulates a number of anion channels. Of the volume-activated or-regulated anion channels, the volume-sensitive outwardly rectifying anion channel (VSOR) is most prominently activated and ubiquitously expressed. Volume regulated chloride channels play significant roles in a variety of physiological processes including volume regulation, proliferation, differentiation, migration,apoptosis and resistance of tumor cells. Molecular identification and activation mechanisms of VSOR are poorly understood. Many signaling molecules have been shown to regulate VSOR activity in research, playing permissive or modulatory roles. Therefore, VSOR may be a new anticancer target by identifying its signaling molecules and activation mechanisms. Key words: Chloride channels; Neoplasms; Research

Simvastatin Ameliorates Rat Cerebrovascular Remodeling During Hypertension via Inhibition of Volume-Regulated Chloride Channel

Statins have pleiotropic actions against the development of vascular remodeling and the incidence of ischemic stroke. Although previous studies have suggested that posttranslational modification of several proteins, such as Rho by mevalonate-derived isoprene groups, geranylgeranyl pyrophosphate or farnesyl pyrophosphate, underlie the pleiotropic effects of statins, the detailed mechanisms remain elusive. Recent growing evidence demonstrated that ClC-3 volume-regulated chloride channel plays an important role in cell proliferation, and the activity of this channel is increased in basilar smooth muscle cells from a hypertensive rat. We hypothesized that inhibition of volume-regulated chloride channel may contribute to the beneficial effects of statins on cerebrovascular remodeling during hypertension. Our study here demonstrated that simvastatin ameliorated hypertension-caused cerebrovascular remodeling. In rat basilar smooth muscle cells, simvastatin inhibited cell proliferation and activation of volume-regulated chloride channel, and these effects of simvastatin were abolished by pretreatment with mevalonate or geranylgeranyl pyrophosphate. In addition, Rho A inhibitor C3 exoenzyme and Rho kinase inhibitor Y-27632 both reduced cell proliferation and activation of volume-regulated chloride channel. Moreover, ClC-3 overexpression decreased the suppressive effect of simvastatin on cell proliferation and increased estimated IC(50) of simvastatin on endothelin 1- and hypo-osmolarity-induced cell proliferation from 3.40+/-0.08 and 3.50+/-0.10 micromol/L to 5.30+/-0.70 and 5.60+/-0.70 micromol/L, respectively (P<0.01; n=6). Furthermore, the expression of ClC-3 was increased in basilar artery during hypertension, and simvastatin normalized the upregulation of ClC-3. Our data suggested that simvastatin ameliorates cerebrovascular remodeling in the hypertensive rat through inhibition of vascular smooth muscle cell proliferation by suppression of volume-regulated chloride channel.

Functional and molecular characterizations of chloride channels in rat pleural mesothelial cells

The purposes of the present study were to clarify whether some Cl− channels exist in rat pleural mesothelial cells, and to investigate functional and molecular characteristics of these channels. Electrophysiological recordings were performed at room temperature using the whole-cell configuration of the patch-clamp technique. We could observe outwardly rectifying Cl− currents in rat pleural mesothelial cells under isotonic conditions. These currents exhibited time-dependent inactivation at potential over +60 mV and were inhibited by NPPB. It suggests the presence of voltage-dependent Cl− channels. Moreover, we observed the currents activated under hypotonic conditions. Their biophysical and pharmacological properties exhibited as follows; moderate outward rectification of whole-cell currents; time-dependent inactivation at large positive potential; anion selectivity with a type-I Eisenman's permeability sequence (I−>Br−>Cl−>F−>glutamate−); inhibited by NPPB. These properties are consistent with volume-regulated chloride channels (VRCCs), even though molecular identity of VRCCs could not have been determined, the molecular expressions of mRNA of the Cl− channels ClC-2, ClC-3, pICln, MDR1 were confirmed. The properties of VRCCs in the pleural mesothelial cells were consistent with those of ClC-3 channels, and different from those of ClC-2. Therefore, these results suggest that ClC-3 might contribute to the modulation of VRCCs in rat pleural mesothelial cells.

Alteration of Volume-Regulated Chloride Movement in Rat Cerebrovascular Smooth Muscle Cells During Hypertension

The cerebrovascular remodeling is a prominent feature of hypertension and considered a major risk factor for stroke. Cerebrovascular smooth muscle cells meet volume challenge during this pathophysiological process. Our previous studies suggest that volume regulated chloride channels may be critical to the cell cycle of vascular smooth muscle cells. However, it is unknown whether the volume-regulated chloride movement is altered in hypertension. Therefore, we directly measured the concentration of intracellular chloride ([Cl(-)](i)) in rat basilar arterial smooth muscle cells isolated from control rats and rats that were made hypertensive for 1 to 12 weeks after partial renal artery constriction (2-kidney, 2-clip method) using a 6-methoxy-N-ethylquinolinium iodide fluorescence probe. The [Cl(-)](i) in isotonic solution showed no difference in all of the groups. After hypotonic perfusion, the reduction in [Cl(-)](i) was more prominent in hypertensive cerebrovascular smooth muscle cells than in sham control cells. Genistein, a protein tyrosine kinase inhibitor, inhibited hypotonic-induced reduction in [Cl(-)](i), whereas sodium orthovanadate, a protein-tyrosine phosphatase inhibitor, enhanced hypotonic-induced reduction in [Cl(-)](i) in both groups. The percentage inhibition of reduction in [Cl(-)](i) by genistein on volume-regulated chloride movement has a positive correlation with blood pressure levels in the 2-kidney, 2-clip hypertensive group, as is the case for the percentage increase of reduction in [Cl(-)](i) by sodium orthovanadate. Antihypertensive therapy with the angiotensin-converting enzyme inhibitor captopril completely reversed abnormal volume-regulated chloride movement in hypertensive rats. We conclude that volume-regulated chloride movement is augmented in rat cerebrovascular smooth muscle cells in proportion to the severity of hypertension.

Cancer Cell Cycle Modulated by a Functional Coupling between Sigma-1 Receptors and Cl– Channels

The sigma-1 receptor is an intracellular protein characterized as a tumor biomarker whose function remains mysterious. We demonstrate herein for the first time that highly selective sigma ligands inhibit volume-regulated chloride channels (VRCC) in small cell lung cancer and T-leukemia cells. Sigma ligands and VRCC blockers provoked a cell cycle arrest underlined by p27 accumulation. In stably sigma-1 receptor-transfected HEK cells, the proliferation rate was significantly lowered by sigma ligands when compared with control cells. Sigma ligands produced a strong inhibition of VRCC in HEK-transfected cells but not in control HEK. Surprisingly, the activation rate of VRCC was dramatically delayed in HEK-transfected cells in the absence of ligands, indicating that sigma-1 receptors per se modulate cell regulating volume processes in physiological conditions. Volume measurements in hypotonic conditions revealed indeed that the regulatory volume decrease was delayed in HEK-transfected cells and virtually abolished in the presence of igmesine in both HEK-transfected and T-leukemic cells. Moreover, HEK-transfected cells showed a significant resistance to staurosporine-induced apoptosis volume decrease, indicating that sigma-1 receptors protect cancer cells from apoptosis. Altogether, our results show for the first time that sigma-1 receptors modulate "cell destiny" through VRCC and cell volume regulation.