36Cl- flux measurements on GABAA receptor-activated chloride exchange. Multiple mechanisms of the chloride channel inactivation

The technique of radiotracer 36Cl- influx in primary culture of rat cerebellar granule cells was applied to study the mechanism of inactivation of the GABAA receptor-activated chloride channel. During sustained application of GABA, muscimol and THIP the specific bicuculline-sensitive 36Cl- influx tends to decline with time. The sequence in decay half-time is GABA less than muscimol less than THIP. Diazepam accelerates the rate of decay of the peak response to GABA. (-)-Baclofen enhances the rate of decline of the response to muscimol in a dose-dependent manner. Treatment of the cells with pertussis toxin antagonized the effect of (-)-baclofen. It is concluded that rat neonatal cerebellar neurons maintained in tissue culture exhibit complex inactivation of the GABAA channel, indicating some interaction with the GABAB receptor system.     

GABAA receptor-mediated increase in membrane chloride conductance in rat paratracheal neurones.

1. The actions of gamma-aminobutyric acid (GABA) on the intramural neurones of 14-18 day old rats were studied in situ by use of intracellular current- and voltage-clamp techniques. The ionic conductance changes and the effects of various GABA-receptor agonists and antagonists on these neurones were also investigated. 2. Prolonged application of GABA either by ionophoresis (10 pC-10 nC) or superfusion (10-100 microM), evoked a biphasic membrane depolarization in over 90% of all paratracheal neurones studied. Typically, the response consisted of an initial rapid depolarization (18-45 ms) that subsequently faded over a period of 15-25 s to reveal a second smaller depolarization which was maintained for the duration of GABA application. Both components of the evoked response resulted in an increase in membrane conductance and an inward flow of current. 3. The amplitude of the transient inward current, recorded during the initial phase of the response, was linearly related to the membrane potential at which it was elicited and reversed symmetrically at a membrane potential of -32.7 mV. The underlying increase in conductance was largely independent of membrane potential. The equilibrium potential for the sustained inward current was -38.7 mV. Replacement of extracellular chloride with gluconate ions initially enhanced the GABA-evoked inward current. With successive applications of GABA in low chloride, the evoked current and conductance changes declined markedly. 4. Muscimol superfusion (1-10 microM) or ionophoresis (10 pC-10 nC) mimicked both the initial and late phases of the GABA-induced conductance change and inward current.(ABSTRACT TRUNCATED AT 250 WORDS)     

Effects of insecticides on GABA-induced chloride influx into rat brain microsacs

The actions of different insecticides known to affect binding of ligands to gamma-aminobutyric acid (GABA) receptors were studied on the function of GABAA receptors in rat brain as assayed by GABA-induced 36Cl- influx into membrane microsacs. This flux was inhibited by the competitive antagonist bicuculline and the noncompetitive antagonist t-butylbicyclophosphorothionate, and the GABA effect was potentiated by the tranquilizer flunitrazepam and the depressant pentobarbital, as expected for effects on a GABAA receptor. The GABA-induced 36Cl- flux was inhibited by several cyclodienes and gamma-hexachlorocyclohexane (gamma-BHC) with the following order of decreasing potency: endosulfan I greater than endrin greater than endosulfan II greater than dieldrin greater than heptachlor epoxide greater than gamma-BHC greater than heptachlor. The noninsecticidal beta-BHC had no effect, while the IC 50 values for gamma-BHC and endrin were 1 and 0.2 microM, respectively. The four pyrethroids tested also inhibited the GABA-induced 36Cl- flux with the following decreasing potencies: 1R,cis,alpha S-cypermethrin greater than 1R,trans, alpha S-cypermethrin greater than fluvalinate greater than allethrin. Avermectin B1a was the only insecticide tested that, in the absence of GABA, stimulated 36Cl- flux in a dose-dependent manner, and this flux was inhibited by bicuculline. The stereospecific inhibition of the GABA-induced 36Cl- influx by the cyclodienes and gamma-BHC supports previously published data on their binding to mammalian brain GABAA receptor and suggests that these insecticides inhibit this receptor's function. It is also suggested that type II pyrethroids are potent inhibitors of the same receptor. However, avermectin B1a appears to act as a partial agonist of GABAA receptors.     

Pyrethroid insecticides indirectly inhibit GABA-dependent Cl influx in synaptoneurosomes from the trout brain

Rainbow trout (Oncorhynchus mykiss) are extremely sensitive to the neurotoxic activity of pyrethroid insecticides. One possible target for pyrethroids is the GABA_A receptor of brain of the trout, the function of which can be tested by measurement of influx of ³⁶Cl ⁻ into synaptoneurosomes, in response to the application of agonists. γ-Aminobutyric acid produced a time- and concentration-dependent increase in influx of ³⁶Cl⁻ in synaptoneurosomes from the brain of the trout, which exhibited the pharmacology characteristic of a response mediated by activation of a GABA_A receptor. Deltamethrin, (1RS)-cia-cypermethrin and permethrin produced a dose-dependent increase in the basal uptake and a corresponding decrease in GABA-dependent influx, with a maximum inhibition of 70–82%. This effect of pyrethroid was stereospecific, of high potency and inhibited by tetrodotoxin (TTX) and t-butylbicy-clophosphorothionate (TBPS). The sensitivity of the effect of the pyrethroid to TTX suggested an activation by pyrethroid of the voltage-dependent sodium channel. Veratridine, a sodium channel activator, elicited similar changes in the basal uptake of chloride, which were TTX-sensitive. Neither deltamethrin nor veratridine had a measurable effect on the efflux of ³⁶Cl⁻ from synaptoneurosomes. Thus, pyrethroid insecticides may interfere with the function of GABA_A receptors indirectly through an interaction with the voltage-dependent sodium channel in the brain of the trout and consequently perturb chloride influx, possibly through a voltage-dependent chloride channel.     

Ion transport blockers inhibit human rhinovirus 2 release

Picornavirus replication causes leakage of cytoplasmic K+ and an influx of Na+ and Ca2+. In this study, we have explored the possibility that a blockade of Ca2+ and Na+ influx would reduce rhinovirus production and/or release. The Ca2+-channel blockers, verapamil and diltiazem, as well as the blocker of Na+/H+ exchange and the epithelial Na+ channel, EIPA, inhibited both virus production and release. The effect on virus release was more pronounced than the effect on production, thus raising the possibility that rhinovirus release may serve as a target for antiviral agents. Unexpectedly, our results also showed that the antiviral activity of the Ca2+-channel blockers was not due to the block of Ca2+ influx. Similarly, the antiviral activity of EIPA appeared to be unrelated to the blockade of cellular Na+/H+ exchanger or the epithelial Na+ channel. Potential alternative mechanisms of the antiviral activity of these compounds are discussed.     

二甲双胍介导胆固醇逆转运抑制动脉粥样硬化

目的探讨二甲双胍通过对胆固醇逆转运抑制ApoE^-/-小鼠动脉粥样硬化的分子机制。方法18只ApoE^-/-小鼠随机分为三组,对照组、模型组和二甲双胍组,每周监测体质量变化。取血检测血清血脂水平;小动物超声检测腹主动脉管壁厚度;HE和油红O染色评价肝脏脂肪病变程度;Western blot检测肝脏胆固醇逆转运相关蛋白LXRɑ、ABCA1的表达。结果与对照组比,模型组体质量、血清TC、TG、LDL升高,HDL降低(P<0.05),腹主动脉管壁增厚(P<0.05),肝脏脂肪沉积加重及LXRɑ、ABCA1的表达降低。二甲双胍组体质量、血清TC、TG、LDL降低,HDL升高(P<0.05),肝脏脂肪沉积及腹主动脉管壁厚度显著减轻(P<0.05),LXRɑ、ABCA1表达显著增高(P<0.05)。结论二甲双胍通过上调肝脏胆固醇逆向转运相关蛋白LXRɑ和ABCA1的表达,增强肝脏胆固醇逆转运能力,调节血脂代谢、减轻肝脏脂质沉积,从而延缓动脉粥样硬化的进展。     

Pyrethroid insecticides and radioligand displacement from the GABA receptor chloride ionophore complex

Radioligand binding displacement studies were conducted to determine the effects of Type I and II pyrethroids on [3H]flunitrazepam (FLU), [3H]muscimol (MUS), and [35S]t-butylbicyclophosphorothionate (TBPS) binding. Competition experiments with [3H]FLU and [3H]MUS indicate a lack of competition for binding by the pyrethroids. Type I pyrethroids failed to compete for the binding of [35S]TBPS at concentrations as high as 50 microM. Type II pyrethroids inhibited [35S]TBPS binding to rat brain synaptosomes with Ki values ranging from 5-10 microM. The data presented here suggest that the interaction of Type II pyrethroids with the gamma-aminobutyric acid (GABA) receptor-ionophore complex is restricted to a site near the TBPS/picrotoxinin binding site.     

Stimulated chloride transport by isolated parietal cells

A preparation of rabbit parietal cells has been shown to respond to effectors of gastric acid secretion. The cells were isolated, aligned at the interface of two aqueous phases, and the transport of Cl^? from one phase to the other effected by the cells under various pharmacological stimuli, was measured. Ca²⁺, in contrast to Mg²⁺, was shown to be necessary to elicit a response with histamine. This response was inhibited by prostaglandin E₁. Dibutyryl cyclic AMP was without effect. Carbachol and catecholamines also stimulated secretion, but this was not inhibited by prostaglandin E₁. Because basal Cl^? transport in the presence of Ca²⁺ is in a direction opposite to that of the stimulated transport (and to the low basal transport in the presence of Mg²⁺), at least two secrection mechanisms appear operative.     

Chlorotoxin does not inhibit volume-regulated, calcium-activated and cyclic AMP-activated chloride channels

It was the aim of this study to look for a high-affinity and selective polypeptide toxin, which could serve as a probe for the volume-regulated anion channel (VRAC) or the calcium-activated chloride channel (CaCC). We have partially purified chlorotoxin, including new and homologous short chain insectotoxins, from the crude venom of Leiurus quinquestriatus quinquestriatus (Lqq) by means of gel filtration chromatography. Material eluting between 280 and 420 min, corresponding to fractions 15-21, was lyophilized and tested on VRAC and CaCC, using the whole-cell patch-clamp technique. We have also tested the commercially available chlorotoxin on VRAC, CaCC, the cystic fibrosis transmembrane conductance regulator (CFTR) and on the glioma specific chloride channel (GCC). VRAC and the correspondent current, I(Cl,swell), was activated in Cultured Pulmonary Artery Endothelial (CPAE) cells by a 25% hypotonic solution. Neither of the fractions 16-21 significantly inhibited I(Cl,swell) (n=4-5). Ca(2+)-activated Cl(-) currents, I(Cl,Ca), activated by loading T84 cells via the patch pipette with 1 microM free Ca(2+), were not inhibited by any of the tested fractions (15-21), (n=2-5). Chlorotoxin (625 nM) did neither effect I(Cl,swell) nor I(Cl,Ca) (n=4-5). The CFTR channel, transiently transfected in COS cells and activated by a cocktail containing IBMX and forskolin, was not affected by 1.2 microM chlorotoxin (n=5). In addition, it did not affect currents through GCC. We conclude that submicromolar concentrations of chlorotoxin do not block volume-regulated, Ca(2+)-activated and CFTR chloride channels and that it can not be classified as a general chloride channel toxin.     

Actions of anaesthetics and avermectin on GABAA chloride channels in mammalian dorsal root ganglion neurones.

1. The gamma-aminobutyric acid (GABA)-mimetic actions of some anaesthetics and the antehelminthic avermectin B1a were examined on freshly isolated mammalian dorsal root ganglion (DRG) neurones by use of suction electrodes and a single electrode voltage clamp. 2. Pentobarbitone (60 microM-3 mM), chloralose (600 microM-1 mM), etomidate (10-100 microM), alphaxalone (10-60 microM) and avermectin (10-60 microM) directly activated chloride channels in GABA-sensitive DRG neurones. The agonist action was sensitive to block by bicuculline and picrotoxinin. 3. Steady-state current-voltage (I-V) curves for the anaesthetics were either linear, or rectified in the opposite direction to steady-state I-V curves obtained with GABA. Current relaxations in response to voltage jumps were also of the opposite direction. An extra surge of current ('bounce') was commonly observed on washout of some of these agonists. 4. Pentobarbitone was ineffective as an agonist at alkali pH (10.4 and 9.4), but was approximately twice as effective at acid (5.4) than at normal (7.4) pH values. 5. These results suggest that some anaesthetics and avermectin are capable of 'blocking' GABA channels in addition to activating them.     

Methylxanthines inhibit glucose transport in rat adipocytes by two independent mechanisms

In summary, this study characterized the biphasic inhibition of fat cell glucose transport by the lipolytic agents caffeine and theophylline. Like the lipolytic drug forskolin, both methylxanthines produced an immediate inhibition of glucose transport that was not seen with 8-phenyltheophylline, a pure adenosine receptor antagonist. The immediate inhibition was therefore not mediated by the adenosine receptor antagonism but seems to be due to a direct interaction with the hexose transporter. This conclusion is supported by the immediate onset of the inhibition and additionally by the interference of theophylline and caffeine with the binding of cytochalasin B, a ligand of the glucose transporter that binds to an intracellular site of the transporter molecule. In addition, a second, delayed inhibitory effect of theophylline and caffeine on glucose transport was observed. This portion shared many aspects of the inhibitory effect of lipolytic hormones. It developed over a period of about 5 min and was antagonized by the simultaneous addition of the antilipolytic hormone PGE2. This component of transport inhibition could be attributed to the antagonistic effect of methylxanthines at the fat cell A1-adenosine receptor since it was also seen with 8-phenyltheophylline. This conclusion is further supported by data showing that the removal of endogenous adenosine with adenosine deaminase resulted in a comparable 25-30% inhibition of insulin-stimulated glucose transport. In addition, the time course of glucose transport inhibition by the subsequent addition of adenosine deaminase is similar to that of the delayed portion of the inhibition seen with theophylline and caffeine. Both treatments produced their maximal inhibition after 5 min. In conclusion, the methylxanthines theophylline and caffeine inhibit glucose transport by a combination of two different modes of action. The immediate major component is mediated via a direct interaction with the hexose transporter whereas the delayed component involves adenosine receptor antagonism and thereby the interaction with G-proteins.     

Carbapenem antibiotics inhibit valproic acid transport in Caco-2 cell monolayers

The concomitant use of carbapenem antibiotics with valproic acid has been prohibited because carbapenems induced a decrease in plasma concentration of valproic acid in epileptic patients during valproic acid therapy. Our previous in vivo study in rats proposed that inhibition by carbapenem of the intestinal absorption of valproic acid might be a possible mechanism for the drug-drug interaction. To demonstrate the hypothesis, we examined the effects of imipenem and panipenem on intestinal transepithelial transport of valproic acid using Caco-2 cell monolayers. Imipenem and panipenem inhibited the transport of [14C]-valproic acid across the Caco-2 cell monolayers from apical-to-basolateral side in a concentration-dependent manner, although they had no effect on the uptake of [14C]-valproic acid by Caco-2 cells. The inhibition by the carbapenems of the valproic acid transport was found even when they were added to only the basolateral side. From these results, the carbapenems may inhibit the absorption of valproic acid at the basolateral membrane of intestinal epithelial cells, which contributes to the decrease in plasma concentration of valproic acid after oral administration.     

Inhibitors of renal chloride transport do not block toxicant-induced chloride influx in the proximal tubule

We have previously demonstrated that chloride influx occurs during the late stages of mitochondrial inhibitor-induced renal proximal tubule (RPT) cell injury. The purpose of this study was to determine if chloride influx is a common pathway in toxicant-induced cell injury and if inhibitors of renal chloride transport block the chloride influx. Chloride influx occurred in the late stages of RPT cell injury induced by the diverse toxicants mercuric chloride, t-butyl hydroperoxide, bromohydroquinone, and tetrafluoroethyl-l-cysteine. Specific inhibitors of known renal chloride transport did not prevent antimycin A-induced chloride influx. Toxicant-induced chloride influx occurred prior to cell swelling and decreasing the extracellular chloride concentration diminished toxicant-induced cell death. Thus, chloride influx is a common pathway in the late stages of toxic cell injury and does not occur through known mechanisms of renal chloride transport. Further, we propose that toxicant-induced chloride influx is mediated by a novel receptor related to the neuronal strychnine-sensitive glycine receptor and that chloride influx is a key step in cell swelling and lysis.     

Cobaltous chloride and hypoxia inhibit aryl hydrocarbon receptor-mediated responses in breast cancer cells

The aryl hydrocarbon receptor (AhR) is expressed in estrogen receptor (ER)-positive ZR-75 breast cancer cells. Treatment with 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) induces CYP1A1 protein and mRNA levels and also activates inhibitory AhR-ERalpha crosstalk associated with hormone-induced reporter gene expression. In ZR-75 cells grown under hypoxia, induction of these AhR-mediated responses by TCDD was significantly inhibited. This was not accompanied by decreased nuclear AhR levels or decreased interaction of the AhR complex with the CYP1A1 gene promoter as determined in a chromatin immunoprecipitation assay. Hypoxia-induced loss of Ah-responsiveness was not associated with induction of hypoxia-inducible factor-1alpha or other factors that sequester the AhR nuclear translocation (Arnt) protein, and overexpression of Arnt under hypoxia did not restore Ah-responsiveness. The p65 subunit of NFkappaB which inhibits AhR-mediated transactivation was not induced by hypoxia and was primarily cytosolic in ZR-75 cells grown under hypoxic and normoxic conditions. In ZR-75 cells maintained under hypoxic conditions for 24 h, BRCA1 (an enhancer of AhR-mediated transactivation in breast cancer cells) was significantly decreased and this contributed to loss of Ah-responsiveness. In cells grown under hypoxia for 6 h, BRCA1 was not decreased, but induction of CYP1A1 by TCDD was significantly decreased. Cotreatment of ZR-75 cells with TCDD plus the protein synthesis inhibitor cycloheximide for 6 h enhanced CYP1A1 expression in cells grown under hypoxia and normoxia. These results suggest that hypoxia rapidly induces protein(s) that inhibit Ah-responsiveness and these may be similar to constitutively expressed inhibitors of Ah-responsiveness (under normoxia) that are also inhibited by cycloheximide.     

Many P-glycoprotein substrates do not inhibit the transport process across cell membranes

1. The critical role of P-glycoprotein (P-gp) in the clinical exposure of many pharmaceuticals and toxins has become widely appreciated. The P-gp-mediated influence can often be more significant than that of other well-known xenobiotic defence enzymes in both breadth and impact. The inhibition of P-gp, therefore, has often been examined by testing a compound for its influence on the P-gp-mediated transport of some marker substrate, often the compound is also evaluated for its active efflux mediated by P-gp. 2. Although a substrate for a xenobiotic defence enzyme is logically presumed to be an inhibitor of that enzyme toward an alternate substrate, that is not necessarily the case with a transmembrane active efflux transporter. A substrate that is ejected from the cytosolic side of the membrane bilayer that does not rapidly cross the membrane by passive diffusion back into the cell interior will not occlude the substrate binding site. Hence, some substrates may not significantly affect the overall P-gp function of causing a concentration gradient by efficient net transport. A wide variety of compounds that are documented as substrates of P-gp are characterized here as having no effect on the ability of P-gp to transport several conventional P-gp marker substrates. 3. Transbilayer passive diffusion apparently dictates the ability of a P-gp substrate to be an inhibitor, as described herein based on relative rates of transport (active efflux versus passive re-entry) and the interaction of amphipathic compounds with the cell membrane. 4. The portion of P-gp substrates whose disposition is dependent on P-gp function and which are not also inhibitors is striking. It is therefore important to characterize both the efflux rate parameters and those of inhibition. 5. This report affords a valuable list of known P-gp substrates that are non-inhibitors.     

Evidence for furosemide-sensitive active chloride transport in vascular smooth muscle

An inhibitor of Cl^? transport, furosemide, interfered potently with the ³⁶Cl steady state exchange in rabbit aortic tissue. On long-term incubation with furosemide, [Cl^?]_i decreased and E_Cl increased, thereby approaching the resting membrane potential of vascular smooth muscle. Electrophysiological microelectrode studies on rabbit pulmonary arteries revealed a hyperpolarization of 5.5 mV in the presence of furosemide. These findings provide evidence for an inwardly directed chloride pump operating in vascular smooth muscle.     

Mechanism of action of the insecticides, lindane and fipronil, on glycine receptor chloride channels

BACKGROUND AND PURPOSE

Docking studies predict that the insecticides, lindane and fipronil, block GABA_A receptors by binding to 6′ pore-lining residues. However, this has never been tested at any Cys-loop receptor. The neurotoxic effects of these insecticides are also thought to be mediated by GABA_A receptors, although a recent morphological study suggested glycine receptors mediated fipronil toxicity in zebrafish. Here we investigated whether human α1, α1β, α2 and α3 glycine receptors were sufficiently sensitive to block by either compound as to represent possible neurotoxicity targets. We also investigated the mechanisms by which lindane and fipronil inhibit α1 glycine receptors.

EXPERIMENTAL APPROACH

Glycine receptors were recombinantly expressed in HEK293 cells and insecticide effects were studied using patch-clamp electrophysiology.

KEY RESULTS

Both compounds completely inhibited all tested glycine receptor subtypes with IC₅₀ values ranging from 0.2–2 µM, similar to their potencies at vertebrate GABA_A receptors. Consistent with molecular docking predictions, both lindane and fipronil interacted with 6′ threonine residues via hydrophobic interactions and hydrogen bonds. In contrast with predictions, we found no evidence for lindane interacting at the 2′ level. We present evidence for fipronil binding in a non-blocking mode in the anaesthetic binding pocket, and for lindane as an excellent pharmacological tool for identifying the presence of β subunits in αβ heteromeric glycine receptors.

CONCLUSIONS AND IMPLICATIONS

This study implicates glycine receptors as novel vertebrate toxicity targets for fipronil and lindane. Furthermore, lindane interacted with pore-lining 6′ threonine residues, whereas fipronil may have both pore and non-pore binding sites.     

盐酸戊乙奎醚治疗急性有机磷农药中毒的临床研究

目的：观察盐酸戊乙奎醚（长托宁）在抢救急性有机磷农药中毒患者中的临床疗效。方法：将本院进行抢救的47例急性有机磷农药中毒患者作为观察组,给予盐酸戊乙奎醚联合氯解磷定治疗;既往本科应用传统的阿托品加氯解磷定进行抢救的47例急性有机磷中毒患者作为对照组,比较两组患者的临床疗效。结果：观察组的临床疗效、平均住院时间、CHE恢复时间、中毒症状消失时间及意识清醒时间与对照组比较,差异均有统计学意义（P〈0.05）;并且不良反应的发生率仅为10.6%,明显比对照组少（P〈0.05）。结论：盐酸戊乙奎醚（长托宁）治疗急性有机磷农药中毒患者疗效确切,不良反应少,值得广泛应用。