Chloride Channel Blocker Research Articles

Anoctamin-1 in the juvenile rat urinary bladder.

PurposeTo investigate presence, location and functional role of calcium-activated chloride channel (CaCC) Anoctamin-1 (Ano1) in rat urinary bladder.Materials and MethodsBladders from 3 week old Wistar rats were studied. End-point PCR on total mRNA was used to assess the expression of Ano1. Immunofluorescent labelling of whole mount bladder tissue imaged with confocal microscope allowed localization of Ano1 and vimentin immunopositive cells. The effects of CaCC blockers: niflumic acid (NFA) (3,10,30 µM) and 5-Nitro-2-(3-phenylpropylamino)benzoic acid (NPPB) (10, 30 µM) on spontaneous phasic contractile activity of intact (with mucosa) and denuded (without mucosa) detrusor strips were measured under isometric tension in organ baths (n = 141, N = 60).ResultsAno1 expression was found at mRNA level in mucosa and detrusor layers. Confocal microscopy revealed presence of Ano1 immunopositive cells in mucosa and in detrusor layers; a subpopulation of vimentin positive cells expressed Ano1. Both chloride channel blockers reduced the amplitude and frequency of phasic contractions in denuded and intact strips.ConclusionsAno1 is expressed in rat urinary bladder and is present in cells sharing markers with interstitial cells. CaCC blockers reduced phasic activity of the bladder tissue. Ano1 is expressed in the bladder and plays a role in its spontaneous phasic contractile activity.

Functional expression of chloride channels and their roles in the cell cycle and cell proliferation in highly differentiated nasopharyngeal carcinoma cells.

We previously demonstrated that the growth of the poorly differentiated nasopharyngeal carcinoma cells (CNE‐2Z) was more dependent on the activities of volume‐activated chloride channels than that of the normal nasopharyngeal epithelial cells (NP69‐SV40T). However, the activities and roles of such volume‐activated chloride channels in highly differentiated nasopharyngeal carcinoma cells (CNE‐1) are not clarified. In this study, it was found that a volume‐activated chloride current and a regulatory volume decrease (RVD) were induced by 47% hypotonic challenges. The current density and the capacity of RVD in the highly differentiated CNE‐1 cells were lower than those in the poorly differentiated CNE‐2Z cells, and higher than those in the normal cells (NP69‐SV40T). The chloride channel blockers, 5‐nitro‐2‐(3‐phenylpropylamino) benzoic acid (NPPB) and tamoxifen inhibited the current and RVD. Depletion of intracellular Cl− abolished the RVD. The chloride channel blockers reversibly inhibited cell proliferation in a concentration‐ and time‐dependent manner, and arrested cells at the G0/G1 phases, but did not change cell viability. The sensitivity of the three cell lines to the chloride channel blockers was different, with the highest in poorly differentiated cells (CNE‐2Z) and the lowest in the normal cells (NP69‐SV40T). ClC‐3 proteins were expressed in the three cells and distributed inside the cells as well as on the cell membrane. In conclusion, the highly differentiated nasopharyngeal carcinoma CNE‐1 cells functionally expressed the volume‐activated chloride channels, which may play important roles in controlling cell proliferation through modulating the cell cycle, and may be associated with cell differentiation. Chloride channels may be a potential target of anticancer therapy.

Probing the location of methanol in methanol/AOT/n-heptane system: true microemulsion or bi-continuous medium?

Whether methanol can be effectively encapsulated within a reverse micellar core to form a true microemulsion is still a matter of debate. Thus, in this contribution, we have reported the modified photophysics of a potential chloride channel blocker 9-methyl anthroate (9-MA) in methanol/AOT/n-heptane reverse micelles and methanol/n-heptane binary mixture in order to assess the location of methanol by means of steady-state and time-resolved fluorescence spectroscopic techniques. By comparing the striking similarity between the photophysics of the probe in the methanol/AOT/n-heptane system and in the methanol/n-heptane binary mixture, we conclude that methanol does not form a true microemulsion but rather remains dissolved in the bulk nonpolar phase forming a bi-continuous medium owing to an appreciable mutual solubility between n-heptane and methanol.

APOL1 risk variants enhance podocyte necrosis through compromising lysosomal membrane permeability.

Development of higher rates of nondiabetic glomerulosclerosis (GS) in African Americans has been attributed to two coding sequence variants (G1 and G2) in the APOL1 gene. To date, the cellular function and the role of APOL1 variants (Vs) in GS are still unknown. In this study, we examined the effects of overexpressing wild-type (G0) and kidney disease risk variants (G1 and G2) of APOL1 in human podocytes using a lentivirus expression system. Interestingly, G0 inflicted podocyte injury only at a higher concentration; however, G1 and G2 promoted moderate podocyte injury at lower and higher concentrations. APOL1Vs expressing podocytes displayed diffuse distribution of both Lucifer yellow dye and cathepsin L as manifestations of enhanced lysosomal membrane permeability (LMP). Chloroquine attenuated the APOL1Vs-induced increase in podocyte injury, consistent with targeting lysosomes. The chloride channel blocker DIDS prevented APOL1Vs- induced injury, indicating a role for chloride influx in osmotic swelling of lysosomes. Direct exposure of noninfected podocytes with conditioned media from G1- and G2-expressing podocytes also induced injury, suggesting a contributory role of the secreted component of G1 and G2 as well. Adverse host factors (AHFs) such as hydrogen peroxide, hypoxia, TNF-α, and puromycin aminonucleoside augmented APOL1- and APOL1Vs-induced podocyte injury, while the effect of human immunodeficiency virus (HIV) on podocyte injury was overwhelming under conditions of APOLVs expression. We conclude that G0 and G1 and G2 APOL1 variants have the potential to induce podocyte injury in a manner which is further augmented by AHFs, with HIV infection being especially prominent.

Effects of San-Huang-Xie-Xin-tang, a traditional Chinese prescription for clearing away heat and toxin, on the pacemaker activities of interstitial cells of Cajal from the murine small intestine

Ethnopharmacological relevanceSan-Huang-Xie-Xin-Tang (SHXXT) is a traditional Chinese medicinal formula composed of Coptidis rhizoma (Coptis chinesis Franch), Scutellariae radix (Scutellaria baicalensis Georgi), and Rhei rhizoma (Rheum officinale Baill) and is widely used in Eastern Asia, especially to ameliorate the symptoms of gastrointestinal (GI) disorders related to gastritis, gastric bleeding, peptic ulcers, and abnormal GI motility Aim of the studyInterstitial cells of Cajal (ICCs) are pacemaker cells in the GI tract that generate rhythmic oscillations in membrane potentials known as slow waves. Because GI disorders, especially abnormal GI motility, are major lifelong problems, the authors investigated the effects of SHXXT on mouse small intestine ICCs, and sought to identify the receptors and the action mechanisms involved. Materials and methodsEnzymatic digestions were used to dissociate ICCs from small intestines, and the whole-cell patch-clamp configuration was used to record potentials generated by cultured ICCs. ResultsSHXXT produced membrane depolarization in current-clamp mode, and Y25130 (a 5-HT3 receptor antagonist) and RS39604 (a 5-HT4 receptor antagonist) blocked SHXXT-induced membrane depolarizations, whereas SB269970 (a 5-HT7 receptor antagonist) did not. However, during external Ca2+ free conditions or in the presence of thapsigargin, SHXXT did not exhibit membrane depolarization. Furthermore, the application of flufenamic acid (a nonselective cation channel (NSCC) blocker) or DIDS (a chloride channel blocker) abolished pacemaker potential generation and blocked SHXXT-induced membrane depolarizations. In addition, SHXXT-induced membrane depolarizations, which are dependent on G-protein, in ICCs were blocked by PD 98059 (a p42/44 mitogen-activated protein kinase (MAPK) inhibitor), SB203580 (a p38 MAPK inhibitor), and by a c-jun NH2-terminal kinase (JNK) II inhibitor. Regarding the components of SHXXT, Coptidis rhizome and Rhei rhizoma modulated ICC pacemaking activity, whereas Scutellariae radix did not. ConclusionSHXXT modulates pacemaker potentials via 5-HT3 and 5-HT4 receptor-mediated pathways, external Ca2+ influx, and Ca2+ release from internal stores. Furthermore, NSCCs and Cl- channels play important roles in the regulation of pacemaking activity in a MAPK dependent manner in ICCs. The regulation of pacemaking activity by SHXXT may be due to the activity of Coptidis rhizome and Rhei rhizome. The study shows SHXXT can modulate the pacemaking activity of ICCs in the GI tract, and thus, suggests SHXXT has potential pharmacological relevance for the treatment of GI motility disorders.

Adrenomedullin increases the short-circuit current in the mouse seminal vesicle: actions on chloride secretion.

Adrenomedullin (ADM) may regulate seminal vesicle fluid secretion, and this may affect sperm quality. In this study, we investigated the effect of ADM on chloride secretion in the mouse seminal vesicle. The presence of ADM in mouse seminal vesicle was confirmed using immunostaining, and the molecular species was determined using gel filtration chromatography coupled with enzyme-linked assay for ADM. The effects of ADM on chloride secretion were studied by short-circuit current technique in a whole-mount preparation of mouse seminal vesicle in an Ussing chamber. The effects of specific ADM and calcitonin gene-related peptide (CGRP) receptor antagonists were investigated. Whether the ADM effect depended on the cAMP- and/or calcium-activated chloride channel was also studied using specific chloride channel blockers. The results showed that ADM was present in seminal vesicle epithelial cells. The major molecular species was precursor in the mouse seminal vesicle. ADM increased short-circuit current through the calcium-activated chloride channel in mouse seminal vesicle, and CGRP receptor was involved. We conclude that ADM may regulate chloride and fluid secretion from the seminal vesicle, which may affect the composition of the seminal plasma bathing the sperm and, hence, fertility.

Chloride channel blockade relaxes airway smooth muscle and potentiates relaxation by β-agonists.

Severe bronchospasm refractory to β-agonists continues to cause significant morbidity and mortality in asthmatic patients. We questioned whether chloride channels/transporters are novel targets for the relaxation of airway smooth muscle (ASM). We have screened a library of compounds, derivatives of anthranilic and indanyloxyacetic acid, that were originally developed to antagonize chloride channels in the kidney. We hypothesized that members of this library would be novel calcium-activated chloride channel blockers for the airway. The initial screen of this compound library identified 4 of 20 compounds that relaxed a tetraethylammonium chloride-induced contraction in guinea pig tracheal rings. The two most effective compounds, compounds 1 and 13, were further studied for their potential to either prevent the initiation of or relax the maintenance phase of an acetylcholine (ACh)-induced contraction or to potentiate β-agonist-mediated relaxation. Both relaxed an established ACh-induced contraction in human and guinea pig ex vivo ASM. In contrast, the prevention of an ACh-induced contraction required copretreatment with the sodium-potassium-chloride cotransporter blocker bumetanide. The combination of compound 13 and bumetanide also potentiated relaxation by the β-agonist isoproterenol in guinea pig tracheal rings. Compounds 1 and 13 hyperpolarized the plasma cell membrane of human ASM cells and blocked spontaneous transient inward currents, a measure of chloride currents in these cells. These functional and electrophysiological data suggest that modulating ASM chloride flux is a novel therapeutic target in asthma and other bronchoconstrictive diseases.

Anoctamin 1 induces calcium-activated chloride secretion and proliferation of renal cyst–forming epithelial cells

Polycystic kidney diseases are characterized by multiple bilateral renal cysts that gradually enlarge and lead to a decline in renal function. Cyst enlargement is driven by transepithelial chloride secretion, stimulated by enhanced levels of cyclic adenosine monophosphate, which activates apical cystic fibrosis transmembrane conductance regulator chloride channels. However, chloride secretion by calcium-dependent chloride channels, activated through stimulation of purinergic receptors, also has a major impact. To identify the molecular basis of calcium-dependent chloride secretion in cyst expansion, we determined the role of anoctamin 1 and 6, two recently discovered calcium-activated chloride channels both of which are expressed in epithelial cells. We found that anoctamin 1, which plays a role in epithelial fluid secretion and proliferation, is strongly expressed in principal-like MDCK cells (PLCs) forming cysts within a collagen matrix, in an embryonic kidney cyst model, and in human autosomal dominant polycystic kidney disease tissue. Knockdown of anoctamin 1 but not anoctamin 6 strongly diminished the calcium-dependent chloride secretion of PLCs. Moreover, two inhibitors of anoctamin ion channels, tannic acid and a more selective inhibitor of anoctamin 1, significantly inhibited PLC cyst growth and cyst enlargement in an embryonic kidney cyst model. Knockdown of ANO1 by morpholino analogs also attenuated embryonic cyst growth. Thus, calcium-activated chloride secretion by anoctamin 1 appears to be a crucial component of renal cyst growth.

Preclinical evaluation of marketed sodium channel blockers in a rat model of myotonia discloses promising antimyotonic drugs

Although the sodium channel blocker mexiletine is considered the first-line drug in myotonia, some patients experiment adverse effects, while others do not gain any benefit. Other antimyotonic drugs are thus needed to offer mexiletine alternatives. In the present study, we used a previously-validated rat model of myotonia congenita to compare six marketed sodium channel blockers to mexiletine. Myotonia was induced in the rat by injection of anthracen-9-carboxylic acid, a muscle chloride channel blocker. The drugs were given orally and myotonia was evaluated by measuring the time of righting reflex. The drugs were also tested on sodium currents recorded in a cell line transfected with the human skeletal muscle sodium channel hNav1.4 using patch-clamp technique. In vivo, carbamazepine and propafenone showed antimyotonic activity at doses similar to mexiletine (ED50 close to 5mg/kg); flecainide and orphenadrine showed greater potency (ED50 near 1mg/kg); lubeluzole and riluzole were the more potent (ED50 near 0.1mg/kg). The antimyotonic activity of drugs in vivo was linearly correlated with their potency in blocking hNav1.4 channels in vitro. Deviation was observed for propafenone and carbamazepine, likely due to pharmacokinetics and multiple targets. The comparison of the antimyotonic dose calculated in rats with the current clinical dose in humans strongly suggests that all the tested drugs may be used safely for the treatment of human myotonia. Considering the limits of mexiletine tolerability and the occurrence of non-responders, this study proposes an arsenal of alternative drugs, which may prove useful to increase the quality of life of individuals suffering from non-dystrophic myotonia. Further clinical trials are warranted to confirm these results.

Possibility of inhibition of calcium-activated chloride channel rescuing erectile failures in diabetes

Although calcium-activated chloride channel (CaCC) blockers, niflumic acid (NFA) and anthracene-9-carboxylic acid (A9C), have been shown as potential erectogenic agents in healthy corpus cavernosum (CC) tissues, the pharmacological characteristics of CaCC blockers in diabetic state are relatively unknown. This study compares the direct muscle relaxant property of NFA and A9C with their influence on contraction and nitrergic relaxation as elicited by electrical field stimulation in normal and 16-week-old diabetic rabbit CC (n=8). Mean blood glucose level in alloxan-treated rabbits was elevated threefold (21.9±0.5 mmol l(-1) vs 7.1±0.2 mmol l(-1) in untreated rabbits; P<0.05). There was no significant alteration in the efficacies of NFA and A9C in eliciting a concentration-dependent relaxation of noradrenaline-induced cavernosum tone and in inhibiting neurogenic contraction of CC from diabetic rabbits. The capability of NFA (100 μM) and A9C (1 mM) in augmenting nitrergic transmission was also not adversely affected by diabetes. However, in CC from diabetic rabbits, A9C markedly increased nitrergic relaxation response to 1-10 Hz by 10.6-36.6% (vs -5.1-0.8% in nondiabetic control). CaCC sensitivity to A9C appears to be enhanced in diabetic CC tissue. Inhibiting the CaCC activity in diabetes-related ED may tip the balance between proerectile/relaxant and antierectile/contractile mechanisms in favor of cavernosum relaxation.

A Chloride Channel Blocker Prevents Inorganic Phosphate Accumulation and Its Effects in the Sarcoplasmic Reticulum of Frog Permeabilized Skeletal Muscle Fibers

In skeletal muscle intense activity is accompanied by an increase in intracellular inorganic phosphate (Pi) due to breakdown of ATP and phoshocreatine. It is widely accepted that Pi enters the sarcoplasmic reticulum (SR) where it precipitates as Ca2+ salt, thus contributing to the impairment of Ca2+ release in muscle fatigue (Fryer et al, J Physiol 1995; Launikonis et al., Biophys J 2005). We studied the effects of increasing Pi concentrations on elementary Ca2+ release events (sparks) in permeabilized fibers. Spark frequency F/F0 increased with [Pi] up to 7-10 mM/l and decreased monotonically reaching 0.1 at [Pi] = 55 mM. These changes were highly correlated with the level of [Ca2+]SR, monitored with Fluo5N or Mag-Fluo4. SR anion channels, probably chloride channels (Laver et al.,J Physiol2001), provide the principal entry path of Pi to the SR. The chloride channel blocker 9-anthracenecarboxylic acid (9AC, at 400 uM) increased spark frequency by about 15% and shifted the Pi effects to slightly higher concentrations. Because 9AC blocks the channel from the SR-luminal side another approach was taken to enhance its action. Fibers were incubated in the presence of 5 mM Mg2+ and 2 mM [9AC] for 30 minutes. There after 9AC was reduced to 400 uM and Mg2+ to 0.4 mM/l. After this protocol, Pi concentrations up to 55 mM/l had no effect on frequency or spark morphology. Correspondingly, [Ca2+]SR-monitoring signals were not affected. These results indicate that chloride channels constitute the main entrance of Pi to the SR, confirm that Pi impairs Ca2+ release by accumulating and precipitating with Ca2+ inside the SR and provide approaches to study these mechanisms quantitatively.Funded by CSIC and NIH.

Interaction of a potential chloride channel blocker with a model transport protein: a spectroscopic and molecular docking investigation

The present work demonstrates a detailed characterization of the interaction of a potential chloride channel blocker, 9-methyl anthroate (9-MA), with a model transport protein, Bovine Serum Albumin (BSA). The modulated photophysical properties of the emissive drug molecule within the microheterogeneous bio-environment of the protein have been exploited spectroscopically to monitor the probe-protein binding interaction. Apart from evaluating the binding constant, the probable location of the neutral molecule within the protein cavity (subdomain IB) is explored by an AutoDock-based blind docking simulation. The absence of the Red-Edge Effect has been corroborated by the enhanced lifetime of the probe, being substantially greater than the solvent reorientation time. A dip-and-rise characteristic of the rotational relaxation profile of the drug within the protein has been argued to originate from a significant difference in the lifetime as well as amplitude of the free and protein-bound drug molecule. Unfolding of the protein in the presence of the drug molecule has been probed by the decrease of the α-helical content, obtained via circular dichroism (CD) spectroscopy, which is also supported by the gradual loss of the esterase activity of the protein in the presence of the drug molecule.

Cystic fibrosis transmembrane conductance regulator chloride channel blockers: Pharmacological, biophysical and physiological relevance

Dysfunction of the cystic fibrosis transmembrane conductance regulator (CFTR) chloride channel causes cystic fibrosis, while inappropriate activity of this channel occurs in secretory diarrhea and polycystic kidney disease. Drugs that interact directly with CFTR are therefore of interest in the treatment of a number of disease states. This review focuses on one class of small molecules that interacts directly with CFTR, namely inhibitors that act by directly blocking chloride movement through the open channel pore. In theory such compounds could be of use in the treatment of diarrhea and polycystic kidney disease, however in practice all known substances acting by this mechanism to inhibit CFTR function lack either the potency or specificity for in vivo use. Nevertheless, this theoretical pharmacological usefulness set the scene for the development of more potent, specific CFTR inhibitors. Biophysically, open channel blockers have proven most useful as experimental probes of the structure and function of the CFTR chloride channel pore. Most importantly, the use of these blockers has been fundamental in developing a functional model of the pore that includes a wide inner vestibule that uses positively charged amino acid side chains to attract both permeant and blocking anions from the cell cytoplasm. CFTR channels are also subject to this kind of blocking action by endogenous anions present in the cell cytoplasm, and recently this blocking effect has been suggested to play a role in the physiological control of CFTR channel function, in particular as a novel mechanism linking CFTR function dynamically to the composition of epithelial cell secretions. It has also been suggested that future drugs could target this same pathway as a way of pharmacologically increasing CFTR activity in cystic fibrosis. Studying open channel blockers and their mechanisms of action has resulted in significant advances in our understanding of CFTR as a pharmacological target in disease states, of CFTR channel structure and function, and of how CFTR activity is controlled by its local environment.

Sign‐preserving and sign‐inverting synaptic interactions between rod and cone photoreceptors in the dark‐adapted retina

We show that various types of rods and cones in the dark-adapted salamander retina are electrically coupled with linear and symmetrical junctional conductances G(j) (40-223 pS) and a rank order: Rod(C)-large single cone, rod-large single cone, rod-small single cone, rod-accessory double cone and rod-principal double cone. By systematically comparing the transjunctional current-voltage (I(j)-V(j)) relations and average G(j) values of the five types of rod-cone pairs recorded at day and night times, our results suggest that the differences in G(j) values among various types of rod-cone pairs are not caused by circadian differences, and the circadian-dependent changes in rod-cone coupling observed in the fish and rodent retinas are not present in the tiger salamander. In addition to rod-cone coupling, there is a sign-inverting, unidirectional rod→cone current I(RC), and the I(RC)-V(Cone) relations are linear, with a reversal potential near the chloride reversal potential E(Cl). I(RC) can be observed in rods and cones separated by at least 260 μm, and its waveform resembles that of the rod-elicited horizontal cell (HC) response I(HC). A glutamate transporter-associated chloride channel blocker TBOA suppresses I(RC) but not I(HC). These results suggest that I(RC) is largely mediated by HCs via a sign-inverting feedback chemical synapse associated with a chloride channel. I(RC) significantly reduced rod→cone coupling in the frequency range below 15 Hz, allowing better separation of rod and cone signals in the dark-adapted retina.

The ClC-3 chloride channel associated with microtubules is a target of paclitaxel in its induced-apoptosis

Recent evidences show that cationic fluxes play a pivotal role in cell apoptosis. In this study, the roles of Cl− channels in paclitaxel-induced apoptosis were investigated in nasopharyngeal carcinoma CNE-2Z cells. Chloride current and apoptosis were induced by paclitaxel and inhibited by chloride channel blockers. Paclitaxel-activated current possessed similar properties to volume-activated chloride current. After ClC-3 was knocked-down by ClC-3-siRNA, hypotonicity-activated and paclitaxel-induced chloride currents were obviously decreased, indicating that the chloride channel involved in paclitaxel-induced apoptosis may be ClC-3. In early apoptotic cells, ClC-3 was up-regulated significantly; over-expressed ClC-3 was accumulated in cell membrane to form intercrossed filaments, which were co-localized with α-tubulins; changes of ultrastructures and decrease of flexibility in cell membrane were detected by atomic force microscopy. These suggest that ClC-3 is a critical target of paclitaxel and the involvement of ClC-3 in apoptosis may be associated with its accumulation with membrane microtubules and its over activation.

Depolarization-induced depression of inhibitory transmission in cerebellar Purkinje cells

Several forms of depolarization-induced plasticity in inhibitory transmission have been reported to occur in cerebellar Purkinje cells (PCs), namely depolarization-induced suppression of inhibition (DSI), depolarization-induced potentiation of inhibition (DPI), and rebound potentiation (RP). Here, we describe another form of synaptic plasticity for gamma-amino butyric acid (GABA)ergic transmission in PCs. Immediately following depolarization trains in a PC, evoked inhibitory postsynaptic currents (eIPSCs) changed their direction from outward to inward currents under a recording condition in which eIPSCs were elicited as an outward current. Subsequently, the eIPSC amplitude remained depressed (depolarization-induced depression of inhibition [DDI]) for more than 20 min under the blockade of cannabinoid and N-methyl-D-aspartic acid (NMDA) receptor-mediated DSI and DPI, respectively. This DDI was completely abolished by intracellular infusion of the fast Ca2+-chelating agent BAPTA and by inhibition of Ca2+/calmodulin-dependent protein kinase II (CaMKII). Furthermore, DDI was strongly suppressed by calcium-activated chloride channel (CaCC) blockers, while an inhibitor of cation-chloride cotransporters (CCCs) partially blocked DDI during the early phase. Exogenous GABA-induced inhibition of spontaneous spike activity was attenuated in ∼50% of the PCs by climbing fiber stimulation-induced depolarization. These results suggest that activation of both CaCCs and CCCs was necessary for alteration of [Cl-]i after activation of CaMKII following elevation of [Ca2+]i in PCs. DDI may provide another mechanism for regulation of inhibitory inputs to PCs within the neuronal networks of the cerebellar cortex.

Involvement of Volume-Activated Chloride Channels in H2O2 Preconditioning Against Oxidant-Induced Injury Through Modulating Cell Volume Regulation Mechanisms and Membrane Permeability in PC12 Cells

The functions of chloride channels in preconditioning-induced cell protection remain unclear. In this report, we show that the volume-activated chloride channels play a key role in hydrogen peroxide (H2O2) preconditioning-induced cell protection in pheochromocytoma PC12 cells. The preconditioning with 100 μM H2O2 for 90 min protected the cells from injury induced by long period exposure to 300 μM H2O2. The protective effect was attenuated by pretreatment with the chloride channel blockers, 5-nitro-2-3-phenylpropylamino benzoic acid (NPPB) and tamoxifen. H2O2 preconditioning directly activated a chloride current, which was moderately outward-rectified and sensitive to the chloride channel blockers and hypertonicity-induced cell shrinkage. H2O2 preconditioning functionally up-regulated the activities of volume-activated chloride channels and enhanced the regulatory volume decrease when exposure to extracellular hypotonic challenges. In addition, acute application of H2O2 showed distinctive actions on cell volume and membrane permeability in H2O2 preconditioned cells. In H2O2 preconditioned cells, acute application of 300 μM H2O2 first promptly induced a decrease of cell volume and enhancement of cell membrane permeability, and then, cell volume was maintained at a relatively stable level and the facilitation of membrane permeability was reduced. Conversely, in control cells, 300 μM H2O2 induced a slow but persistent apoptotic volume decrease (AVD) and facilitation of membrane permeability. H2O2 preconditioning also significantly up-regulated the expression of ClC-3 protein, the molecular candidate of the volume-activated chloride channel. These results suggest that H2O2 preconditioning can enhance the expression and functional activities of volume-activated chloride channels, thereby modulate cell volume and cell membrane permeability, which may contribute to neuroprotection against oxidant-induced injury.

Electrophysiological evidence for 4-isobutyl-3-isopropylbicyclophosphorothionate as a selective blocker of insect GABA-gated chloride channels

Invertebrate γ-aminobutyric acid (GABA)-gated chloride channels (GABACls) and glutamate-gated chloride channels (GluCls), which function as inhibitory neurotransmitter receptors, are important targets of insecticides and antiparasitic agents. The antagonism of GABACls and GluCls by 4-isobutyl-3-isopropylbicyclophosphorothionate (PS-14) was examined in cultured cockroach and rat neurons using a whole-cell patch-clamp method. The results indicated that PS-14 selectively blocks cockroach GABACls relative to cockroach GluCls and rat GABACls. PS-14 represents a useful probe for the study of insect GABA receptors.

Excitatory actions of GABA in developing chick vestibular afferents: Effects on resting electrical activity

The aim of this study was to characterize the effect of γ-aminobutyric acid (GABA) in the resting multiunit activity of the vestibular afferents during development using the isolated inner ear of embryonic and postnatal chickens (E15-E21 and P5). GABA (10(-3) to 10(-5) M; n = 133) and muscimol (10(-3) M) elicited an increase in the frequency of the basal discharge of the vestibular afferents. We found that GABA action was dose-dependent and inversely related to animal age. Thus, the largest effect was observed in embryonic ages such as E15 and E17 and decreases in E21 and P5. The GABAA receptor antagonists, bicuculline (10(-5) M; n = 10) and picrotoxin (10(-4) M; n = 10), significantly decreased the excitatory action of GABA and muscimol (10(-3) M). Additionally, CNQX 10(-6) M, MCPG 10(-5) M and 7ClKyn 10(-5) M (n = 5) were co-applied by bath substitution (n = 5). Both the basal discharge and the GABA action significantly decreased in these experimental conditions. The chloride channel blocker 9-AC 0.5 mM produced an important reduction in the effect of GABA 10(-3) (n = 5) and 10(-4) M (n = 5). Thus, our results suggest an excitatory role of GABA in the resting activity of the vestibular afferents that can be explained by changes in the gradient of concentration of Cl(-) during development. We show for the first time that the magnitude of this GABA effect decreases at later stages of embryonic and early postnatal development. Taking into account the results with glutamatergic antagonists, we conclude that GABA has a presynaptic action but is not the neurotransmitter in the vestibular afferent synapses, although it could act as a facilitator of the spontaneous activity and may regulate glutamate release.

Opening of Chloride Channels by 1α,25-Dihydroxyvitamin D3 Contributes to Photoprotection against UVR-Induced Thymine Dimers in Keratinocytes

UVR produces vitamin D in skin, which is hydroxylated locally to 1α,25-dihydroxyvitamin D(3) (1,25(OH)(2)D(3)). 1,25(OH)(2)D(3) protects skin cells against UVR-induced DNA damage, including thymine dimers, but the mechanism is unknown. As DNA repair is inhibited by nitric oxide (NO) products but facilitated by p53, we examined whether 1,25(OH)(2)D(3) altered the expression of nitrotyrosine, a product of NO, or p53 after UVR in human keratinocytes. 1,25(OH)(2)D(3) and the nongenomic agonist 1α,25-dihydroxylumisterol(3) reduced nitrotyrosine 16 hours after UVR, detected by a sensitive whole-cell ELISA. p53 was enhanced after UVR, and this was further augmented in the presence of 1,25(OH)(2)D(3). DIDS (4,4'-diisothiocyanatostilbene-2,2'-disulfonic acid), a chloride channel blocker previously shown to prevent 1,25(OH)(2)D(3)-induced chloride currents in osteoblasts, had no effect on thymine dimers on its own but prevented the 1,25(OH)(2)D(3)-induced protection against thymine dimers. Independent treatment with DIDS, at concentrations that had no effect on thymine dimers, blocked UVR-induced upregulation of p53. In contrast, reduction of nitrotyrosine remained in keratinocytes treated with 1,25(OH)(2)D(3) and DIDS at concentrations shown to block decreases in post-UVR thymine dimers. These results suggest that 1,25(OH)(2)D(3)-induced chloride currents help protect from UVR-induced thymine dimers, but further increases in p53 or reductions of nitrotyrosine by 1,25(OH)(2)D(3) are unlikely to contribute substantially to this protection.