Anion Channel Blocker Research Articles

Effect of anion channel blockers on l-arginine action in spermatozoa from asthenospermic men

In earlier studies, we have established that l-arginine enhances motility and metabolic rate in spermatozoa of goat, bull and mouse. In the present study this work was extended to human sperm cells obtained from the semen samples of asthenospermic patients, which are characterised by low motility. The metabolic rate was followed by monitoring the glucose consumption (1-(13)C glucose as substrate) and the production of lactate in sperm cells, using (13)C NMR. The stimulatory effect of l-arginine was neutralised on adding an NO-synthase inhibitor like N(omega)-nitro-L-arginine methyl ester. On the other hand, the inactive d-enantiomorph did not affect the stimulatory effect of l-arginine. This strongly suggests that L-arginine acts through the NO signal pathway. We also demonstrated that the stimulatory effect of L-arginine was inhibited in the presence of anion channel inhibitors like 4-acetamido-4'-isothiocyanostilbene-2,2'-disulphonic acid, 2,4-dinitrophenol and carbonyl cyanide m-chlorophenylhydrazone. Furthermore, bicarbonate supplementation was found to be essential for the action of L-arginine. These observations indicate that L-arginine induces NO synthesis and stimulates motility and metabolism only when an active anion transport system is present.

In vitro activation of GAT1 transporters expressed in spinal cord gliosomes stimulates glutamate release that is abnormally elevated in the SOD1/G93A(+) mouse model of amyotrophic lateral sclerosis

The effect of GABA on glutamate release from astrocytes has been studied in healthy mice and in a murine transgenic model of amyotrophic lateral sclerosis (ALS), using mouse spinal cord gliosomes labeled with [(3)H]d-aspartate ([(3)H]d-ASP). GABA concentration-dependently evoked the release of [(3)H]d-ASP. The effect of GABA was not mimicked by GABA(A) or GABA(B) receptor agonists or counteracted by antagonists, excluding receptor involvement. However, it was prevented by the GABA transport inhibitor N-(4,4-phenyl-3-butenyl)-nipecotic acid (SKF 89976A), suggesting participation of GABA transporters type 1 (GAT1) placed on glutamate-releasing astrocyte-derived gliosomes. Accordingly, GAT1 co-expressed with glutamate-aspartate transporter (GLAST) and glutamate transporter type 1 (GLT1) in the majority of glial particles. [(3)H]d-aspartate release was Ca(2+)-independent and not blocked by the glutamate uptake inhibitor dl-threo-b-benzyloxyaspartic acid (dl-TBOA); instead, it was abrogated by the anion channel blockers niflumic acid and 5-nitro-2-(3-phenylpropylamino) benzoic acid (NPPB). The GAT1-mediated release of [(3)H]d-ASP was significantly enhanced in spinal cord gliosomes from the mouse model of ALS. This excessive [(3)H]d-ASP release was very precocious, largely preceding the onset of the disease symptoms. These data indicate that GAT1, GLAST and GLT1 coexist on the same gliosome in mouse spinal cord and that activation of GAT1 transporters elicits glutamate release by anion channel opening. This phenomenon might have pathological relevance, because [(3)H]d-ASP release is enhanced in experimental ALS.

Contribution of Chloride Channel Conductance to the Regulation of Resting Membrane Potential in Chondrocytes

The contribution of Cl(-) conductance relative to that of K(+) in the regulation of membrane potential was examined using OUMS-27 cells, a model cell-line of human chondrocytes. Application of 100 microM niflumic acid (NFA) and other anion-channel blockers induced significant membrane hyperpolarization. The NFA-sensitive membrane current under voltage-clamp was predominantly Cl(-) current. Application of NFA induced small but significant increase in intracellular Ca(2+) concentration ([Ca(2+)](i)) and markedly enhanced the late component of [Ca(2+)](i) rise induced by 1 microM histamine. In conclusion, Cl(-) conductance substantially contributes to the regulation of resting membrane potential and [Ca(2+)](i) in OUMS-27 cells.

Gamma-vinyl GABA increases nonvesicular release of GABA and glutamate in the nucleus accumbens in rats via action on anion channels and GABA transporters.

gamma-Amino butyric acid (GABA) is a well-characterized inhibitory neurotransmitter in the central nervous system, which may also stimulate nonvesicular release of other neurotransmitters under certain conditions. We have recently reported that gamma-vinyl GABA (GVG), an irreversible GABA transaminase inhibitor, elevates extracellular GABA but fails to alter dopamine release in the nucleus accumbens (NAc). Here, we investigated the mechanism(s) by which GVG elevates extracellular GABA levels and whether GVG also alters glutamate release in the NAc. In vivo microdialysis was used to simultaneously measure extracellular NAc GABA and glutamate before and after GVG administration in freely moving rats. Systemic administration of GVG or intra-NAc local perfusion of GVG significantly increased extracellular NAc GABA and glutamate. GVG-enhanced GABA was completely blocked by intra-NAc local perfusion of 5-nitro-2, 3-(phenylpropylamino)-benzoic acid (NPPB), a selective anion channel blocker and partially blocked by SKF89976A, a type 1 GABA transporter inhibitor. GVG-enhanced glutamate was completely blocked by NPPB or SKF89976A. Tetrodotoxin, a voltage-dependent Na(+)-channel blocker, failed to alter GVG-enhanced GABA and glutamate. These data suggest that GVG-enhanced extracellular GABA and glutamate are mediated predominantly by the opening of anion channels and partially by the reversal of GABA transporters. Enhanced extracellular glutamate may functionally attenuate the pharmacological action of GABA and prevent enhanced GABA-induced excess inhibition.

Cryohydrocytosis: increased activity of cation carriers in red cells from a patient with a band 3 mutation

Cryohydrocytosis is an inherited dominant hemolytic anemia characterized by mutations in a transmembrane segment of the anion exchanger (band 3 protein). Transfection experiments performed in Xenopus oocytes suggested that these mutations may convert the anion exchanger into a non-selective cation channel. The present study was performed to characterize so far unexplored ion transport pathways that may render erythrocytes of a single cryohydrocytosis patient cation-leaky. Cold-induced changes in cell volume were monitored using ektacytometry and density gradient centrifugation. Kinetics, temperature and inhibitor-dependence of the cation and water movements in the cryohydrocytosis patient's erythrocytes were studied using radioactive tracers and flame photometry. Response of the membrane potential of the patient's erythrocyte membrane to the presence of ionophores and blockers of anion and cation channels was assessed. In the cold, the cryohydrocytosis patient's erythrocytes swelled in KCl-containing, but not in NaCl-containing or KNO(3)-containing media indicating that volume changes were mediated by an anion-coupled cation transporter. In NaCl-containing medium the net HOE-642-sensitive Na(+)/K(+) exchange prevailed, whereas in KCl-containing medium swelling was mediated by a chloride-dependent K(+) uptake. Unidirectional K(+) influx measurements showed that the patient's cells have abnormally high activities of the cation-proton exchanger and the K(+),Cl(-) co-transporter, which can account for the observed net movements of cations. Finally, neither chloride nor cation conductance in the patient's erythrocytes differed from that of healthy donors. Conclusions These results suggest that cross-talk between the mutated band 3 and other transporters might increase the cation permeability in cryohydrocytosis.

Membrane potential changes during pollen germination and tube growth

We applied quantitative fluorescent microscopy to study membrane potential alterations during pollen germination and in growing pollen tube. Two voltage-sensitive dyes were applied: DiBAC4(3) was used to detect average membrane potential values in pollen grains and isolated protoplasts; Di-4-ANEPPS gave an option of membrane potential mapping on pollen protoplast and pollen tube surfaces. We have found out that tobacco pollen grain activation is accompanied by hyperpolarization of the vegetative cell plasma membrane by about 8 mV. Lily pollen protoplasts were significantly hyperpolarized (-108 mV) with respect to the pollen grains (-23 mV) from which they were isolated. We found polar distribution of the membrane potential along the protoplast surface, and longitudinal potential gradient along the pollen tube. In the presence of plasma membrane H(+)-ATPase inhibitor, sodium orthovanadate (1 mM) or its activator fusicoccin (1 microM), the longitudinal voltage gradient altered but did not disappear. Anion channel blocker, NPPB (40 microM), fully discarded the gradient in pollen tubes. Obtained results give evidence of the plasma membrane hyperpolarization during pollen germination and uneven potential distribution on pollen grain and tube surfaces. Inhibitory analysis showed involvement of the plasma membrane H(+)-ATPase and anion channels in membrane potential regulation.

Thrombin potentiates d‐aspartate efflux from cultured astrocytes under conditions of K+ homeostasis disruption

Thrombin levels increase in brain during ischemia and hemorrhagic episodes, and may contribute to excitotoxic neural damage. This study examined the effect of thrombin on glutamate efflux from rat cortical cultured astrocytes using 3H-D-aspartate as radiotracer. The glutamate efflux was initiated by addition of 100 mM K+ plus 1 mM ouabain (K/O) to replicate extracellular and intracellular ionic changes that occur during cerebral ischemia. Upon exposure to K/O, astrocytes swelled slowly and progressively with no evidence of volume regulation. The K/O-induced swelling was inhibited by 65% with bumetanide and 25% with BaCl2, suggesting contribution of Na+/K+/Cl) co-transporter and Kir channels. K/O-elicited 3H-D-aspartate that consisted of two phases. The first transient component of the release corresponded to 13.5% of total 3H-D-aspartate loaded. It was markedly reduced (61%) by the glutamate transporter blocker DL-threo-b-benzyloxyaspartic acid and weakly inhibited (21%) by the volume-sensitive anion channel blocker 4-[(2-Butyl-6,7-dichloro-2-cyclopentyl-2,3-di-hydro-1oxo-1H-inden-5-yl)oxy] butanoic acid (DCPIB). During the second sustained phase of release, cells lost 45% of loaded of 3H-D-aspartate via a mechanism that was insensitive to DL-threo-b-benzyloxyaspartic acid but nearly completely suppressed by DCPIB. Thrombin (5 U/mL) had only marginal effects on the first phase but strongly potentiated(more than two-fold) 3H-D-aspartate efflux in the second phase. The effect of thrombin effect was proportional to cell swelling and completely suppressed by DCPIB. Overall our data showed that under K/O swelling conditions, thrombin potently enhance glutamate release via volume-sensitive anion channel. Similar mechanisms may contribute to brain damage in neural pathologies which are associated with cell swelling, glutamate efflux and increased thrombin levels.

Bestrophin-1 Encodes for the Ca2+-Activated Anion Channel in Hippocampal Astrocytes

In mammalian brain, neurons and astrocytes are reported to express various chloride and anion channels, but the evidence for functional expression of Ca(2+)-activated anion channel (CAAC) and its molecular identity have been lacking. Here we report electrophysiological evidence for the CAAC expression and its molecular identity by mouse Bestrophin 1 (mBest1) in astrocytes of the mouse brain. Using Ca(2+) imaging and perforated-patch-clamp analysis, we demonstrate that astrocytes displayed an inward current at holding potential of -70 mV that was dependent on an increase in intracellular Ca(2+) after G(alphaq)-coupled receptor activation. This current was mediated mostly by anions and was sensitive to well known anion channel blockers such as niflumic acid, 5-nitro-2(3-phenylpropylamino)-benzoic acid, and flufenamic acid. To find the molecular identity of the anion channel responsible for the CAAC current, we analyzed the expression of candidate genes and found that the mRNA for mouse mBest1 is predominantly expressed in acutely dissociated or cultured astrocytes. Whole-cell patch-clamp analysis using HEK293T cells heterologously expressing full-length mBest1 showed a Ca(2+)-dependent current mediated by mBest1, with a complete impairment of the current by a putative pore mutation, W93C. Furthermore, mBest1-mediated CAAC from cultured astrocytes was significantly reduced by expression of mBest1-specific short hairpin RNA (shRNA), suggesting that the CAAC is mediated by a channel encoded by mBest1. Finally, hippocampal CA1 astrocytes in hippocampal slice also showed mBest1-mediated CAAC because it was inhibited by mBest1-specific shRNA. Collectively, these data provide molecular evidence that the mBest1 channel is responsible for CAAC function in astrocytes.

Electrogenic Cl− secretion does not occur in the ileum of the Australian common brushtail possum, Trichosurus vulpecula, due to low levels of expression of the NaK2Cl cotransporter, NKCC1

The colon of the brushtail possum does not have an electrogenic secretory response. Given the functional significance of electrogenic Cl(-) secretion in the intestine of eutherian mammals, we have investigated the secretory response in the small intestine of this marsupial. In the Ussing chamber cAMP-dependent secretagogues stimulated a sustained increase in ileal short-circuit current (Isc), whereas Ca(2+)-dependent secretagogues induced a transient increase. Both the responses were inhibited by mucosal addition of the anion channel blocker 5-nitro-2-(3-phenylpropylamino) benzoic acid (100 mciromol l(-1)), consistent with an anion secretory response. However, the responses were not inhibited by serosal bumetanide (10 mciromol l(-1)) and were independent of bath Cl(-), indicating that the stimulated ileal Isc does not involve electrogenic Cl(-) secretion driven by the NaK2Cl cotransporter, NKCC1. Consistent with this, there were low levels of NKCC1 expression in the ileal epithelium. In particular, NKCC1 expression in the ileal crypt cells was comparable to that of the villous cells. This differs from eutherian mammals where high levels of NKCC1 expression in the ileal crypt cells are associated with their role in Cl(-) secretion. The cAMP- and Ca(2+)-dependent secretory responses were inhibited by the removal of HCO(3) (-) suggesting that these responses were due to electrogenic HCO(3) (-) secretion. We conclude that the ileum of the possum does not secrete Cl(-) due to low levels of NKCC1 expression. It does however appear to secrete HCO(3) (-). These results are further significant examples of differences in the transport function of the possum intestinal epithelium compared with eutherian mammals.

Activation of a Chloride Channel by a Trophic Ligand Is Required for Development of the Mouse Preimplantation Embryo In Vitro1

Platelet-activating factor (1-o-alkyl-2-acetyl-sn-gylcero-3-phosphocholine [PAF]) is one of several autocrine trophic factors supporting the development of the preimplantation embryo. PAF acts on the embryo to induce receptor-mediated intracellular calcium (Ca(2+))(i) transients, and these coincide with a marked membrane hyperpolarization. Patch-clamp analysis of 2-cell embryos showed that these Ca(2+)(i) transients resulted in an outward membrane current. The present study characterizes this current and assesses its role in embryo development. The outward current was dependent upon the presence of anions in the extracellular medium and occurred as a consequence of the PAF-induced Ca(2+)(i) transients. The anion current induced by PAF was inhibited by niflumic acid (NFA), a selective blocker of Ca(2+)-activated Cl(-) channels, but this drug did not block the PAF-induced Ca(2+)(i) transients. Voltage ramp analysis showed that the Cl(-) conductance was outwardly rectifying and inactivated at holding potentials more positive than +30 mV. Culture in NFA or 4,4'-diisothiocyanatostilbene-2,2'-disulfonic acid (a broad-specificity anion channel blocker) from the zygote stage significantly reduced development to blastocysts, with most arresting at the 4-cell and 8-cell stages. Niflumic acid exposure only from the zygote to the late 2-cell stage also reduced the subsequent development to blastocysts. By contrast, treatment from the late 2-cell stage or the 8-cell stage had no effect on development to the blastocyst stage. This study demonstrates the activation of a Ca(2+)-sensitive Cl(-) channel in the 2-cell embryo by PAF and shows that this current activity during the zygote to 2-cell stage is required for normal embryo development in vitro.

Bradykinin‐induced astrocyte–neuron signalling: glutamate release is mediated by ROS‐activated volume‐sensitive outwardly rectifying anion channels

Glial cells release gliotransmitters which signal to adjacent neurons and glial cells. Previous studies showed that in response to stimulation with bradykinin, glutamate is released from rat astrocytes and causes NMDA receptor-mediated elevation of intracellular Ca(2+) in adjacent neurons. Here, we investigate how bradykinin-induced glutamate release from mouse astrocytes signals to neighbouring neurons in co-cultures. Astrocyte-to-neuron signalling and bradykinin-induced glutamate release from mouse astrocytes were both inhibited by the anion channel blocker 4,4'-diisothiocyanatostilbene-2,2'-disulfonic acid (DIDS) and phloretin. Glutamate release was also sensitive to 4-(2-Butyl-6,7-dichlor-2-cyclopentylindan-1-on-5-yl) oxybutyric acid (DCPIB), a specific blocker of the volume-sensitive outwardly rectifying anion channel (VSOR). Astrocytes, but not neurons, responded to bradykinin with activation of whole-cell Cl- currents. Although astrocytes stimulated with bradykinin did not undergo cell swelling, the bradykinin-activated current exhibited properties typical of VSOR: outward rectification, inhibition by osmotic shrinkage, sensitivity to DIDS, phloretin and DCPIB, dependence on intracellular ATP, and permeability to glutamate. Bradykinin increased intracellular reactive oxygen species (ROS) in mouse astrocytes. Pretreatment of mouse astrocytes with either a ROS scavenger or an NAD(P)H oxidase inhibitor blocked bradykinin-induced activation of VSOR, glutamate release and astrocyte-to-neuron signalling. By contrast, pretreatment with BAPTA-AM or tetanus neurotoxin A failed to suppress bradykinin-induced glutamate release. Thus, VSOR activated by ROS in mouse astrocytes in response to stimulation with bradykinin, serves as the pathway for glutamate release to mediate astrocyte-to-neuron signalling. Since bradykinin is an initial mediator of inflammation, VSOR might play a role in glia-neuron communication in the brain during inflammation.

Transfer of ascorbic acid across the vascular endothelium: mechanism and self-regulation

To determine how ascorbic acid moves from the bloodstream into tissues, we assessed transfer of the vitamin across the barrier generated by EA.hy926 endothelial cells when these were cultured on semipermeable filter supports. Ascorbate transfer from the luminal to the abluminal compartment was time dependent, inhibited by anion channel blockers and by activation of protein kinase A, but was increased by thrombin. Ascorbate transfer occurred by a paracellular route, since it did not correlate with intracellular ascorbate contents and was not rectified or saturable. Nonetheless, intracellular ascorbate inhibited the transfer of both ascorbate and radiolabeled inulin across the endothelial barrier. The increase in barrier function due to ascorbate was dependent on its intracellular concentration, significant by 15 min of incubation, prevented by the cytoskeletal inhibitor colchicine, associated with F-actin stress fiber formation, and not due to collagen deposition. These results show that ascorbate traverses the endothelial barrier by a paracellular route that is regulated by cell metabolism, ion channels, and ascorbate itself. Since the latter effect occurred over the physiological range of ascorbate plasma concentrations, it could reflect a role for the vitamin in control of endothelial barrier function in vivo.

Net and Unidirectional Liquid Fluxes Across Porcine Bronchial Airways in Health and During Bacterial Pneumonia

The epithelium of the tracheobronchial airways is capable of both active absorption and secretion of ions and liquid to maintain adequate volumes of airway surface liquid to support mucociliary transport and prevent airway obstruction. Using cannulated, liquid‐filled bronchi from pigs, rates of liquid flux across the airway wall were measured in the presence and absence of NPPB (an anion channel blocker that inhibits liquid secretion). Net and unidirectional liquid fluxes were determined. Responses from healthy animals were compared to animals diagnosed with bacterial pneumonia (Pasteurella multocida and Streptococcus suis). Unidirectional liquid secretion in infected airways (2.35± 0.55 μl/cm2/h) was significantly (p<.05) greater than in healthy airways (0.40±0.66 μl/cm2/h). Unidirectional liquid absorption in infected airways (3.29±0.47 μl/cm2/h) was reduced relative to healthy airways (4.65±0.55 μl/cm2/h), but this difference was not significant. In healthy airways, the net liquid flux was absorptive (4.25±0.62 μl/cm2/h). In the infected airways, the net liquid flux was significantly reduced (0.93±0.43 μl/cm2/h). We conclude that the bronchial airways of animals infected with these pathogens exhibit a net reduction in absorption of airway fluid that results from both reduced unidirectional absorption and augmented unidirectional secretion. (Support: NIH 063302, CFF BALLAR07XX0)

The anti-inflammatory agent flufenamic acid depresses store-operated channels by altering mitochondrial calcium homeostasis

Fenamates like flufenamic acid (FFA) are anti-inflammatory drugs known to alter ion fluxes through the plasma membrane. They are for instance potent blockers of cation and anion channels, and FFA is now commonly used to block currents through TRP channels and receptor-operated channels. However, FFA exerts complex and multifaceted actions on ion transport systems and, in most instances, a molecular understanding of these FFA-dependent modulations is lacking. In addition, FFA is also to known to perturb the homeostasis of Ca 2+. In the present report, we investigated whether the FFA-induced alterations of the Ca 2+ homeostasis could play a role in the FFA-dependent modulation of transmembrane ion fluxes. Experiments performed with the Ca 2+ indicator Fluo-4 on cultured cortical neurons and HEK-293 cells showed that FFA increased the cytosolic concentration of Ca 2+ even in cells kept in a Ca 2+-free medium or when the endoplasmic reticulum was depleted with thapsigargin. The FFA-dependent Ca 2+ responses were, however, strongly reduced by bongkrekic acid, a specific ligand of the mitochondrial ADP/ATP carrier which, in addition, inhibits the permeability transition pore. Like FCCP, FFA released Ca 2+ from isolated brain mitochondria and indirectly modulates store-operated Ca 2+ channels. We suggest that some of the effects of FFA on plasma membrane ion channels could be explained, at least partially, by its ability to modulate the mitochondrial Ca 2+ homeostasis.

R-type anion channel activation is an essential step for ROS-dependent innate immune response in Arabidopsis suspension cells.

Plants are constantly exposed to environmental biotic and abiotic stresses. Plants cells perceive these factors and trigger early responses followed by delayed and complex adaptation processes. Using cell suspensions of Arabidopsis thaliana (L.) as a cellular model, we investigated the role of plasma membrane anion channels in Reactive Oxygen Species (ROS) production and in cell death which occurs during non-host pathogen infection. Protoplasts derived from Arabidopsis suspension cells display two anion currents with characteristics very similar to those of the slow nitrate-permeable (S-type) and rapid sulfate-permeable (R-type) channels previously characterised in hypocotyl cells and other cell types. Using seven inhibitors, we showed that the R-type channel and ROS formation in cell cultures present similar pharmacological profiles. The efficiency of anion channel blockers to inhibit ROS production was independent of the nature of the triggering signal (osmotic stress or general elicitors of plant defence), indicating that the R-type channel represents a crossroad in the signalling pathways leading to ROS production. In a second step, we show that treatment with R-type channel blockers accelerates cell death triggered by the non-specific plant pathogen Xanthomonas campestris. Finally, we discuss the hypothesis that the R-type channel is involved in innate immune response allowing cell defence via antibacterial ROS production.

ATP release through connexin hemichannels and gap junction transfer of second messengers propagate Ca2+ signals across the inner ear.

Extracellular ATP controls various signaling systems including propagation of intercellular Ca(2+) signals (ICS). Connexin hemichannels, P2x7 receptors (P2x7Rs), pannexin channels, anion channels, vesicles, and transporters are putative conduits for ATP release, but their involvement in ICS remains controversial. We investigated ICS in cochlear organotypic cultures, in which ATP acts as an IP(3)-generating agonist and evokes Ca(2+) responses that have been linked to noise-induced hearing loss and development of hair cell-afferent synapses. Focal delivery of ATP or photostimulation with caged IP(3) elicited Ca(2+) responses that spread radially to several orders of unstimulated cells. Furthermore, we recorded robust Ca(2+) signals from an ATP biosensor apposed to supporting cells outside the photostimulated area in WT cultures. ICS propagated normally in cultures lacking either P2x7R or pannexin-1 (Px1), as well as in WT cultures exposed to blockers of anion channels. By contrast, Ca(2+) responses failed to propagate in cultures with defective expression of connexin 26 (Cx26) or Cx30. A companion paper demonstrates that, if expression of either Cx26 or Cx30 is blocked, expression of the other is markedly down-regulated in the outer sulcus. Lanthanum, a connexin hemichannel blocker that does not affect gap junction (GJ) channels when applied extracellularly, limited the propagation of Ca(2+) responses to cells adjacent to the photostimulated area. Our results demonstrate that these connexins play a dual crucial role in inner ear Ca(2+) signaling: as hemichannels, they promote ATP release, sustaining long-range ICS propagation; as GJ channels, they allow diffusion of Ca(2+)-mobilizing second messengers across coupled cells.

Two Distinct Modes of Hypoosmotic Medium-Induced Release of Excitatory Amino Acids and Taurine in the Rat Brain In Vivo

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

Pharmacological “cross‐inhibition” of connexin hemichannels and swelling activated anion channels

The study of ion channels has relied heavily on the use of pharmacological blocking agents. However, many of these agents have multiple effects, which may compromise interpretation of results when the affected mechanisms/pathways mediate similar functions. Volume regulated anion channels (VRAC) and connexin hemichannels can both mediate the release of glutamate and taurine, although these channels have distinct activation stimuli and hemichannels, but not VRAC, are permeable to Lucifer Yellow (LY). It has been reported that some anion channel blockers may inhibit connexin hemichannels. We further examined the effects of classic gap junction/hemichannel blockers and anion channel blockers on these channels. The typical VRAC blockers, NPPB, IAA-94, and tamoxifen blocked low divalent cation-induced glutamate and taurine release and LY loading, presumed due to hemichannel opening. The blocking action of these compounds on hemichannels was concentration dependent and fell within the same range where the drugs classically block VRACs. Conversely, carbenoxolone (CBX), the most widely used gap junction/hemichannel blocker, was an effective blocker of VRAC-mediated glutamate and taurine release, and blocked these channels at similar concentrations at which it blocked hemichannels. The CBX effect on VRACs was verified using astrocytes from connexin 43 knock out (Cx43 KO) animals. In these cells, the hypotonic induced amino acid flux was retained whereas the low divalent cation solution-induced flux was lost. These results extend our knowledge about "cross-inhibition" of VRACs and gap junctions/hemichannels by certain pharmacological agents. Given the overlap in function of these two types of channels, great care must be exerted in using pharmacological blockers to identify one channel from the other.

The Plasmodium falciparum-induced anion channel of human erythrocytes is an ATP-release pathway

Infection with the malaria parasite Plasmodium falciparum induces osmolyte and anion channels in the host erythrocyte membrane involving ATP release and autocrine purinergic signaling. P. falciparum-parasitized but not unstimulated uninfected erythrocytes released ATP in a 5-nitro-2-(3-phenylpropylamino)-benzoic acid (NPPB; 7 microM)-sensitive and serum album (SA; 0.5% w/v)-stimulated manner. Since Plasmodium infection of human erythrocytes induces SA-dependent outwardly (OR) and SA-independent inwardly rectifying (IR) anion conductances, we tested whether the infection-induced OR channels directly generate an ATP release pathway. P. falciparum-parasitized erythrocytes were recorded in whole-cell mode with either Cl(-) or ATP as the only anion in the bath or pipette. In parasitized cells with predominant OR activity, replacement of bath NaCl by Na-ATP (NMDG-Cl pipette solution) shifted the current reversal potential (V (rev)) from -2 +/- 1 to +51 +/- 3 mV (n = 15). In cells with predominant IR activity, in contrast, the same maneuver induced a shift of V (rev) to significantly larger (p < or = 0.05, two-tailed t test) values (from -3 +/- 1 to +66 +/- 8 mV; n = 5) and an almost complete inhibition of outward current. The anion channel blocker NPPB reversibly decreased the ATP-generated OR currents from 1.1 +/- 0.1 nS to 0.2 +/- 0.05 nS and further shifted V (rev) to +87 +/- 7 mV (n = 12). The NPPB-sensitive fraction of the OR reversed at +48 +/- 4 mV suggesting a relative permeability of P (ATP)/P (Cl) approximately 0.01. Together, these data raise the possibility that the OR might be the electrophysiological correlate of an erythrocyte ATP release pathway.

Evidence for a Superoxide Permeability Pathway in Endosomal Membranes

The compartmentalized production of superoxide (*O(2)(-)) by endosomal NADPH oxidase is important in the redox-dependent activation of NF-kappaB following interleukin 1beta (IL-1beta) stimulation. It remains unclear how *O(2)(-) produced within endosomes facilitates redox-dependent signaling events in the cytoplasm. We evaluated *O(2)(-) movement out of IL-1beta-stimulated endosomes and whether SOD1 at the endosomal surface mediates redox-signaling events required for NF-kappaB activation. The relative outward permeability of NADPH-dependent *O(2)(-) from fractionated endosomes was assessed using membrane-permeable (luminol and lucigenin) and -impermeable (isoluminol) luminescent probes for *O(2)(-). In these studies, approximately 60% of *O(2)(-) efflux out of endosomes was inhibited by treatment with either of two anion channel blockers, 4'-diisothiocyano-2,2'-disulfonic acid stilbene (DIDS) or niflumic acid (NFA). Furthermore, radioisotopic electrodiffusion flux assays on endomembrane proteoliposomes suggested that *O(2)(-) and Cl(-) are transported through the same DIDS-sensitive channel(s). Rab5-based immunoaffinity isolation of IL-1beta-stimulated early endosomes demonstrated SOD1 recruitment to endosomes harboring the IL-1 receptor. Finally, SOD1-deficient cells were found to be defective in their ability to activate NF-kappaB following IL-1beta stimulation. Together, these results suggest that *O(2)(-) exits endosomes through a DIDS-sensitive chloride channel(s) and that SOD1-mediated dismutation of *O(2)(-) at the endosomal surface may produce the localized H(2)O(2) required for redox-activation of NF-kappaB.