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1.
H2DIDS, the dihydro analog of DIDS (4,4′-diisothiocyanostilbene-2,2′-disulfonic acid) can interact covalently with membrane sites, resulting in an irreversible inhibition of anion exchange. At low temperatures (0°C) and for relatively short times, however, its interaction is largely reversible, so that a kinetic analysis of the nature of its inhibitory effect on Cl? self exchange can be performed. The effects of variations in the chloride concentration on the inhibitory potency of H2DIDS are consistent with the concept that Cl? and H2DIDS compete for the transport site of the anion exchange system. The value of Ki for H2DIDS is 0.046 μM, indicating that H2DIDS has a higher affinity for the transport system than any other inhibitor so far examined. If, as seems probable, the covalent labelling of H2DIDS occurs at the same site as the reversible binding, H2DIDS can be used as a covalent label for the transport site. The specific localization of H2DIDS in the band-3 protein thus indicates that this protein participates directly in anion exchange.  相似文献   

2.
The effects of phloretin, H2DIDS (4,4′-diisothiocyano-1,2-diphenylethane-2,2′-disulfonate) and SO4?2 on anion transport in Ehrlich ascites tumor cells was studied in an effort to determine whether Cl? and SO4?2 share a common transport mechanism. Sulfate, in the presence of constant extracellular Cl? (100 mM), reduces Cl? self-exchange by 43% (40 mM SO4?2) and Cl??SO4?2 exchange by 36% (25 mM Cl?/O SO4?2) compared to 25 mM Cl?/50 mM SO4?2. Phloretin blocks without delay and to the same extent the self-exchange of both Cl? and SO4?2. For example, at 10?4 M phloretin, anion transport is inhibited 28% which increases to 78% at 5 × 10?4 M. Reversibly bound H2DIDS also inhibits the self-exchange of both Cl? and SO4?2. However, at all H2DIDS concentrations tested (0.5 ? 10 × 10?5 M) SO4?2 transport was far more susceptible to inhibition than that of Cl?. H2DIDS when irreversibly bound to the cell inhibits SO4?2 but not Cl? transport The results of these experiments are consistent with the postulation that both Cl? and SO4?2 are transported by a common mechanism possessing two reactive sites.  相似文献   

3.
Summary Self-exchange of chloride and sulfate in dog and cat red cells has been measured under equilibrium conditions. The rates of efflux for these anions are approximately twofold higher in dog compared to cat red blood cells. Although the rates differ, the anion exchange systems of these two red cell types exhibit many common properties. The dependence of35SO4 efflux on the intracellular SO4 concentration, the pH dependence and the inhibition of35SO4 efflux by Cl and SITS are almost identical in dog and cat red cells. Nystatin treatment was used to study the dependence of36Cl efflux on internal Cl. Chloride efflux exhibits saturation in both cell types with dog red cells possessing a higherV max andK 1/2 than cat red cells. The number of anion transport sites was estimated by extrapolation to the number of molecules of dihydro DIDS (H2DIDS, where DIDS is 4,4-diisothiocyano-2,2 stilbene-disulfonic acid) which were bound at 100% inhibition of transport. The results indicate that either the turnover numbers for anion transport differ in dog, cat, and human red cells or that there is heterogeneity in the function of the membrane components which bind H2DIDS.  相似文献   

4.
Mono-, di-, and trisulfonic acids, including 4,4′-diacetamido stilbene-2,2′-disulfonic acid (DAS) and 2-(4′-amino phenyl)-6-methylbenzene thiazol-3′,7-disulfonic acid (APMB) produce a reversible inhibition of sulfate equilibrium exchange in human red cells. A study of the sidedness of the action of a number of these sulfonic acids in red cell ghosts revealed that some, like DAS, inhibit only at the outer membrane surface while others, like APMB, inhibit at either surface. This finding suggests that at least two different types of membrane sites are involved in the control of anion permeability. The nature of the anion permeability controlling sites in the outer cell surface was investigated by studying the effects of DAS on the inhibition by dinitrofluoro-benzene (DNFB) of anion equilibrium exchange and on the binding of DNFB to the proteins of the red blood cell membrane. After exposure to DNFB in the presence of DAS for a certain period of time, there was a reduction of both the inhibitory effect of DNFB on sulfate exchange and the binding of DNFB to the protein in band 3 of SDS polyacrylamide gel electropherograms (nomenclature of Steck, J. Cell. Biol., 62: 1, 1974). Since binding to other membrane proteins was not affected, this observation supports the assumption that the protein in band 3 plays some role in anion transport. In accordance with the absence of an inhibitory effect at the inner membrane surface, internal DAS does not affect DNFB binding to the protein in band 3. DAS protected the anion exchange system not only against inhibition by DNFB but also by m-isothiocyanato benzene sulfonic acid. In contrast to DAS, the equally inhibitory phlorizin does not reduce the rate of dinitrophenylation of the protein in band 3. This suggests that either not all inhibitors of anion exchange exert their action by a combination with sites on the protein in band 3 or that in spite of the described evidence this protein is not involved in the control of anion movements. The effect of the irreversibly binding inhibitor 4-acetamido-4′-isothiocyanato-stilbene-2,2′-disulfonic acid (SITS) on DNFB binding to the protein in band 3 was studied in an attempt to differentiate DNFB binding related to inhibition of anion permeability from DNFB binding which is not involved. At least three distinguishable populations of DNFB binding sites were found: (1) binding sites common for DNFB and SITS which are probably related to inhibition, (2) other common sites which are not related to inhibition and (3) different sites whose dinitrophenylation is not affected by SITS. The number of sites in population (1) was estimated to be 0.8–1.2 ± 106/cell. A study of the concentration dependence of the inhibition of anion equilibrium exchange with 4,4′-isothiocyanato-2,2′-stilbene disulfonic acid (DIDS) and APMB further suggests that among the sites in population (1) a major fraction is susceptible to modification by APMB and DIDS while the rest is only susceptible to DIDS. It remains undecided whether these differences of susceptibility reflect differences of accessibility or reactivity.  相似文献   

5.
Equilibrium exchange of SO42? was measured prior to and during hemolysis in rabbit erythrocytes exposed to staphylococcal α-hemolysin. The anion-transport protein of the rabbit erythrocyte has also been identified. Equilibrium exchange of SO42? was measured by both efflux and influx of 35SO42?. The rate of influx of SO42? in rabbit erythrocytes exposed to α-hemolysin was twice that of the untreated cells. The rate of SO42? efflux was unchanged by α-hemolysin. Inhibition of anion exchange with 4,4′-diisothiocyano-2,2′-stilbenedisulfonic acid (DIDS) did not inhibit hemolysis, therefore, the increased influx of SO42? may occur through a DIDS-insensitive pathway.  相似文献   

6.
We found that anion channel blockers such as phosphotungstate and 4,4′-diisothiocyanatostilbene-2-2′-disulfonate (DIDS)_enhanced HCO3?-induced activation on porcine epididymal sperm. In the presence of these compounds, HCO3? increased the motility, respiration rate and especially the cAMP content of the sperm to a greater extent than did HCO3? alone. The enhancing effects were not observed in the absence of HCO3?, but were evident when the concentration of HCO3? was low. These compounds did not significantly alter the intracellular pH and did inhibit the adenylate cyclase activity of the sperm plasma membrane. When these compounds were added to sperm homogenate with ATP, the cAMP formed was reduced compared to the control. In addition, these compounds inhibited both the SO42? influx and efflux of the sperm. From these results, we conclude that the anion channel blockers tested principally inhibit the efflux of endogenous HCO3? derived from metabolic CO2, so that HCO3? accumulates intracellularly and stimulates the adenylate cyclase of the sperm.  相似文献   

7.
The interaction of avermectin B1a (AVM) with the γ-aminobutyric acid (GABA) receptor of rat brain was studied using radioactive ligand binding and tracer ion flux assays. Avermectin potentiated the binding of [3H]flunitrazepam and inhibited the binding of both [3H]muscimol and [35S]t-butylbicyclo-phosphorothionate to the GABAA receptor. Inhibition of muscimol binding by AVM suggested competitive displacement. Two kinds of 36chloride (Cl) flux were studied. The 36Cl efflux from preloaded microsacs was potentiated by AVM and was highly inhibited by the Cl-channel blocker 4,4′-diisothiocyano-2,2′-stilbenedisulfonic acid (DIDS). However, it was not potentiated by GABA nor was it sensitive to the convulsants picrotoxin or bicuculline. On the other hand, 36Cl-influx measurement in a different microsac preparation of rat brain was very sensitive to GABA and other GABA-ergic drugs. Avermectin induced 36Cl influx into these microsacs in a dose–dependent manner, but to only 35% of the maximal influx induced by GABA. The AVM-induced 36Cl influx was totally blocked by bicuculline. It is suggested that AVM opens the GABAA-receptor Cl channel by binding to the GABA recognition site and acting as a partial receptor agonist, and also opens a voltage–dependent Cl channel which is totally insensitive to GABA but is very sensitive to DIDS.  相似文献   

8.
A major aim of this investigation was to determine whether, in steady-state ascites cells, Cl? transport can be partitioned into a furosemide-sensitive cotransport with K+ and a separate 4,4′-isothiocyanostilbene-2,2′-disulfonic acid (DIDS) sensitive self-exchange. Both Cl? and K+ fluxes were studied. The furosemide- and Cl? sensitive K+ fluxes were equivalent, both in normal ionic media and when the external K+ concentration, [K+]o, was varied from 4 to 30 mM. The stoichiometry of the furosemide-sensitive Cl? and K+ fluxes was 2 Cl?: 1 K+ at 0.1 and 0.5 mM drug levels but increased to 3 Cl? : 1 K+ at 1.0 mM furosemide. DIDS at 0.1 mM had no effect on the K+ exchange rate but inhibited Cl? exchange by 39% ± 2 (S.E.). The effects of DIDS and 0.5 mM furosemide on Cl? transport were additive but 1.0 mM furosemide and DIDS had overlapping inhibitory actions. Thus furosemide acts on components of K+ and Cl? transport which are linked to each other, but the drug also inhibits an additional DIDS-sensitive Cl? pathway, when present at higher concentrations. The dependence of the furosemide-sensitive K+ and Cl? transport on [K+]o was also studied; both fluxes fell as the [K+]o increased. The latter results recall those in an earlier study by Hempling (Hempling, H.G. (1962) J. Cell. Comp. Physiol. 60, 181–198).  相似文献   

9.
Ionic fluxes in sea urchin sperm plasma membrane regulate cell motility and the acrosome reaction (AR). Although cationic channels mediate some of the ionic movements, little is known about anion channels in these cells. The fusion of sperm plasma membranes into lipid bilayers allowed identification of a 150 pS anion channel. This anion channel was enriched from detergent-solubilized sperm plasma membranes using a wheat germ agglutinin Sepharose column. Vesicles formed from this preparation were fused into black lipid membranes (BLM), yielding single channel anion-selective activity with the properties of those found in the sperm membranes. The following anion selectivity sequence was found: NO3? > CNS? > Br? > CI?. This anion channel has a high open probability at the holding potentials tested, it is partially blocked by 4,4′-diisothiocyano-2,2′ -stilbendisulfonic acid (DIDS), and it often displays substates. The sperm AR was also inhibited by DIDS. © 1993 Wiley-Liss, Inc.  相似文献   

10.
Band 3 of the human erythrocyte is involved in anion transport and binding of the cytoskeleton to the membrane bilayer. Human erythrocytes were treated to incorporate varying concentrations of DIDS (4,4′-diisothiocyanostilbene-2,2′-disulfonic acid) a non-penetrating, irreversible inhibitor of anion transport, and both functions of Band 3 were analyzed. The rate of efflux of 35SO4. was measured and the binding of cytoskeletal components to the membrane was evaluated by extracting the membranes with 0.1 n NaOH and analyzing for the peptides remaining with the membrane. It was found that 0.1 n NaOH extracts all the extrinsic proteins from membranes of untreated cells, while, in the case of the membranes from cells treated with DIDS, a portion of the cytoskeletal components, spectrin (Bands 1 and 2) and Band 2.1 (ankyrin, syndein) remain with the membrane. The amount of these cytoskeletal components remaining with the membrane depends on the concentrations of DIDS incorporated. The effect of DIDS on the extractability of the spectrin-Band 2.1 complex correlates well with DIDS inhibition of anion transport (r = 0.91). At DIDS concentrations which completely inhibit anion transport, about 10% of total spectrin-Band 2.1 complex remains unextracted. Another anion-transport inhibitor, pyridoxal phosphate, has no effect on binding of the cytoskeleton to the membrane. On the other hand, digestion of DIDS-pretreated intact erythrocytes with Pronase, chymotrypsin, or trypsin releases the tight binding of Band 3 to cytoskeleton on the inside of the membrane. Since trypsin does not hydrolyze Band 3 the data suggest that a second membrane protein which is trypsin sensitive may be involved with Band 3 in cytoskeletal binding.  相似文献   

11.
4,4′-dithiocyanatostilbene-2,2′-disulfonic acid (DIDS), an inhibitor of the volume-sensitive anion channel, was used to investigate the role of this channel in the stimulation of rat pancreatic β-cells by glucose and by tolbutamide. Glucose-stimulated electrical activity in β-cells was markedly and reversibly inhibited by DIDS. The increase in cytosolic [Ca2+] and stimulated insulin release evoked by glucose were also inhibited by DIDS. In contrast to its inhibitory effect on glucose-induced β-cell activity, DIDS had no effect on electrical activity, the rise in [Ca2+] i or insulin release induced by tolbutamide. DIDS failed to increase β-cell input conductance, an index of whole-cell K ATP channel activity, or the rate of efflux of 86Rb+ from perifused islets, a measure of net K+ permeability. Furthermore, DIDS had no effect on intracellular pH or on regulatory volume increase following exposure of cells to hypertonic solutions, indicating that the effects of DIDS were not the result of inhibition of Cl transport systems. It is suggested that the DIDS-induced repolarization is caused by inactivation of the volume-sensitive anion channel. The stimulation of β-cell electrical and secretory activity by glucose, but not tolbutamide, may therefore involve activation of the anion channel. Received: 30 November 1999/Revised: 23 June 2000  相似文献   

12.
The permeability of sarcoplasmic reticulum vesicles to sulfate ions was inhibited by diisothiocyano-1,2-diphenylethane-2,2′-disulfonic acid (H2DIDS), which is a potent inhibitor of anion permeability in red blood cell membrane. The amount of H2DIDS bound to the vesicles was determined by using [3H]-H2DIDS. Apparent half inhibition of sulfate permeation was observed on the binding of 2.5 μmol/g protein. SDS-polyacrylamide gel electrophoresis of the vesicles treated with [3H]H2DIDS showed that about 10% of the total bound H2DIDS corresponds to a 100 000-dalton protein, but the remaining 90% to non-protein components. The content of the H2DIDS-binding protein was about 0.5 μmol/g protein. These results suggest that the H2DIDS-binding protein is different from the calcium pump protein and is possibly an anion transport system similar to band 3 in red blood cell membrane.  相似文献   

13.
Summary A chromophoric derivative of phenylglyoxal, 4-hydroxy-3-nitrophenylglyoxal (HNPG), known to be highly selective for modification of arginine residues in aqueous solution is found to be a potent inhibitor of anion transport across the red cell membrane. In contrast to the action of all other arginine-specific reagents used under the experimental conditions in this laboratory, the action of HNPG on sulfate transport is completely reversible. Hence, a kinetic analysis of its inhibitory effect on SO 4 2– self-exchange could be performed. The effect of increasing chloride concentration on the inhibitory potency of HNPG is consistent with the concept that Cl and HNPG compete for the same site on the anion transporter. The IC50 value for the inhibition of SO 4 2– exchange with HNPG is about 0.13mm at pH 8.0 and 0.36mm at pH 7.4, and the Hill coefficient for the interaction between the transporter and the inhibitor is near one at both pH's. HNPG is able to protect the transport system against inhibition with the (under our experimental conditions) irreversibly acting arginine specific reagent, phenylglyoxal. Partial inactivation of the transport system with phenylglyoxal lowers the maximal rates of SO 4 2– and chloride exchange but does not modify the apparentK s for the substrate anions. Reversibly acting anion transport inhibitors known to interact with the DIDS binding site like salicylate, tetrathionate, APMB, DNDS, and flufenamate are able to protect the transport system against phenylglyoxalation. Other inhibitors like phloretin and phlorizin have no effect.  相似文献   

14.
Abstract: 1-Methyl-4-phenylpyridinium (MPP+), the toxic agent in MPTP-induced dopaminergic neurotoxicity, is thought to act by inhibiting mitochondrial electron transport at complex I. This study examined this latter action further with a series of 4′-alkylated analogues of MPP+. These derivatives had IC50 values that ranged from 0.5 to 110 µM and from 1.6 to 3,300 µM in mitochondria and electron transport particles (ETPs), respectively. The IC50 values of corresponding 4′-alkylated phenylpyridine derivatives to inhibit NADH-linked oxidation ranged from 10 to 205 µM in mitochondria and from 1.7 to 142 µM in ETPs. The potencies of both classes of inhibitors directly correlated with their ability to partition between 1-octanol and water. In mitochondria, increased hydrophobicity resulted in greater inhibition of NADH dehydrogenase but a smaller dependence on the transmembrane electrochemical gradient for accumulation of the pyridiniums as evidenced by an ~600-fold, versus only a 36-fold, increase in the IC50 of MPP+ versus 4′-pentyl-MPP+, respectively, in the presence of uncoupler. In ETPs, the analogous increase in potencies of the more hydrophobic analogues was also consistent with an inhibitory mechanism that relied on differential partitioning into the lipid environment surrounding NADH dehydrogenase. However, the pyridinium charge must play a major role in explaining the inhibitory mechanism of the pyridiniums because their potencies are much greater than would be predicted based solely on hydrophobicity. For example, in ETPs, 4′-decyl-MPP+ was nearly 80-fold more potent than phenylpyridine although the latter compound partitions twice as much into 1-octanol. In addition, the lipophilic anion TPB? was a more effective potentiator of inhibition by pyridiniums possessing greater hydrophilicity (0–5 carbons), consistent with facilitation of accumulation of these analogues within the membrane phase of complex I, probably via ion pairing. These studies delineate further the mechanisms by which this class of compounds is able to accumulate in mitochondria, inhibit complex I activity, and thereby, effect neurotoxicity.  相似文献   

15.
Summary Unidirectional fluxes of 204Tl+ through the human red blood cell membrane were measured. The inward rate coefficient measured in a K+-free saline was 15.6±0.6 hr–1. The influx of Tl+ could be partially inhibited with 0.1mm ouabain (by 28%), 0.1mm DIDS (by 50%) or 1mm furosemide (by 51%). The inhibitory effects of ouabain and DIDS or furosemide were additive. Half-maximal responses were seen at 0.72 m and 0.22mm concentrations of DIDS and furosemide, respectively. A similar action of these blockers on Tl+ influx was observed in the erythrocytes incubated in MgCl2-sucrose media. The outward rate coefficient of 204Tl was also inhibited by DIDS and furosemide (by 65 and 52%, respectively). Rate coefficients of 204Tl influx and efflux decreased significantly in the red cells exposed to Cl-free media (NaNO3 or Mg(NO3)2-sucrose). Under these conditions addition of DIDS and furosemide led to only a small inhibition of Tl+ fluxes. There was a linear increase in Tl+ influx with rising of external Cl concentration within 80–155mm or HCO 3 concentration from 20 to 40mm when the sum of anions was kept constant (155mm) with NO 3 . The HCO 3 -stimulated Tl+ influx was completely blocked by 0.05mm DIDS but only 67% by 1mm furosemide. The present study provides direct evidence for the occurrence of Cl (HCO 3 )-dependent, DIDS-sensitive movement of Tl+ across the human erythrocyte membrane in both directions. Under physiological conditions, about half of net Tl+ fluxes occurs due to an anion exchange mechanism. Our data fail to detect a contribution of the Na-K-Cl cotransport system to Tl+ transport in human erythrocytes.  相似文献   

16.
Summary The fluorescence enhancement of 4,4-dibenzamido-2,2-disulfonic stilbene (DBDS) upon binding to membranes was used to examine proximal tubule stilbene binding sites. Equilibrium binding studies of DBDS to renal brush border (BBMV) and basolateral membrane vesicles (BLMV) were performed using a fluorescence enhancement technique developed for red blood cells (A.S. Verkman, J.A. Dix and A.K. Solomon,J. Gen. Physiol. 81:421–449, 1983). In the absence of transportable anions, DBDS bound reversibly to a single class of sites on BLMV isolated from rabbit (K d =3.8 m) and rat (3.2 m); 100 m dihydro-4,4-diisothiocyano-2,2-disulfonic stilbene (H2DIDS) blocked >95% of binding. H2DIDS inhibitable DBDS binding was not detected using rat or rabbit BBMV. In rabbit BLMV, DBDSK d doubled with 10mm SO4, 50mm HCO3 and 100mm Cl, but was not altered by Na or pH (6–8). In stopped-flow experiments the exponential time constant for DBDS binding slowed with SO4, HCO3 and Cl, but was unaffected by Na. These results are consistent with competitive binding of DBDS and anions at an anion transport site. To relate DBDS binding data to anion transport inhibition we used35SO4 uptake to characterize several modes of rabbit BLM anion transport: H/SO4 and Na/SO4 cotransport, and Cl/SO4 countertransport. Each transport process was electroneutral and was inhibited by H2DIDS, furosemide, probenecid, chlorothiazide and DBDS. The apparentK t 's for DBDS (3–20 m) were similar toK d for DBDS binding. These studies define a class of anion transport sites on the proximal tubule basolateral membrane measureable optically by a fluorescent stilbene.  相似文献   

17.
Net nitrate uptake, 36ClO?3/NO?3 influx and 36Cl? influx into Pisum sativum L. cv. Feltham First seedlings have been examined following growth in culture medium containing different combinations of chloride and nitrate. When young (6 days old) seedlings, that had been grown in the absence of N were used, nitrate accumulation stimulated net nitrate uptake and 36ClO?3/NO?3 influx (r2= 0.99) while chloride accumulation inhibited nitrate uptake and 36ClO?3/NO?3 influx (r2= 0.65). When nitrate was provided during growth there was no effect of chloride pretreatment on net nitrate uptake and there was little effect of total [NO?3+ Cl?]i on 36ClO?3/NO?3 influx (r2= 0.26). A direct effect of Cl? on 36ClO?3/NO?3 influx was only found when seedlings had been starved of N for more prolonged periods (14 days). When moderate chloride was supplied during growth, 36Cl? influx was insensitive to nitrate or chloride accumulated, but significantly correlated with loge [NO?3+ Cl?]i (r2= 0.75). When trace amounts of Cl? were supplied during growth 36Cl? influx was inhibited by (a) NO?3 in the external medium and (b) Cl? pretreatment, but was insensitive to NO?3 pretreatment. The sensitivity of 36Cl? influx to external nitrate was not found following Cl? pretreatment in the absence of nitrate. The possibility that there are two populations of chloride carriers which differ in their sensitivity to external nitrate is discussed. Tentative schematic models to account for the regulation of nitrate and chloride uptake are proposed in the context of current hypotheses for regulation of ion transport and control systems theory.  相似文献   

18.
Abstract: The effects of glutamatergic excitotoxins on intracellular Cl? were investigated in the CA1 pyramidal cell layer of the hippocampal slice. Hippocampal slices from rats (14–19 days old) were loaded with 6-methoxy-N-ethylquinolinium chloride (MEQ), a Cl?-sensitive fluorescent probe with a fluorescence intensity that correlates inversely with intracellular [Cl?]. Slices were exposed for at least 10 min at 26–28°C to N-methyl-d -aspartate (NMDA; 100 µM) or α-amino-3-hydroxy-5-methylisoxazole-4-propionic acid (AMPA; 50 µM). A UV laser scanning confocal microscope was used to measure changes in MEQ fluorescence within area CA1 pyramidal cell soma. Both glutamate receptor agonists produced a rapid decrease in MEQ fluorescence that persisted after washout following a 10-min exposure. The effects of NMDA and AMPA were prevented by the competitive antagonists 2-amino-5-phosphonopentanoic acid and 6,7-dinitroquinoxaline-2,3-dione, respectively. Neither tetrodotoxin nor picrotoxin prevented the effect of NMDA or AMPA, indicating the lack of involvement of presynaptic mechanisms. The effects of NMDA and AMPA on MEQ fluorescence were dependent on the levels of extracellular Cl?, but only NMDA responses were dependent on the levels of extracellular Na+. Removal of Ca2+ from the superfusion medium did not alter the effects of NMDA or AMPA on MEQ fluorescence. In addition, neither the Ca2+ ionophore ionomycin nor the L-type voltage-gated Ca2+ channel agonist (Bay K 8644) decreased MEQ fluorescence. The effects of NMDA and AMPA on cell (somal) volume were also assessed with the fluorescent probe calcein acetoxymethyl ester. Both NMDA and AMPA decreased calcein fluorescence (indicating an increased cell volume), but this was preceded by the decrease in MEQ fluorescence (equivalent to an intracellular accumulation of ~20 mM Cl?). Thus, excitotoxins may cause Cl? influx via an anion channel other than the GABAA receptor and/or reduce Cl? efflux mechanisms to produce cell swelling. Such anionic shifts may promote neuronal excitability and cell death following an excitotoxic insult to the hippocampal slice.  相似文献   

19.
The effects of a variety of chemically diverse, reversibly acting inhibitors have been measured on both Cl? and SO42? equilibrium exchange across the human red cell membrane. The measurements were carried out under the same conditions (pH 6.3, 8°C) and in the same medium for both the Cl? and SO24 tracer fluxes. Under these conditions the rate constant for Cl?-Cl? exchange is about 20 000 times larger than that for SO42?-SO42? exchange. Despite this large difference in the rates of transport of the two anions, eight different reversibly acting inhibitors have virtually the same effect on the Cl? and SO42? transport. The proteolytic enzyme papain also has the same inhibitory effect on both the Cl? and SO42? self-exchange. In addition, the slowly penetrating disulfonate 2-(4′-aminophenyl)-6-methylbenzenethiazol-3′,7-disulfonic acid (APMB) is 5-fold more effective from the outer than from the inner membrane surface in inhibiting both Cl? and SO42? self-exchange. We interpret these results as evidence that the rapidly penetrating monovalent anion Cl? and the slowly penetrating divalent anion SO42? are transported by the same system.  相似文献   

20.
The binding of eosin-isothiocyanate (eosin-NCS) and iodoacetamido-eosin (IA-eosin) to band 3 proteins in the membrane of human erythrocytes is characterized by studying the effect of these probes on the anion transport system. Although the unbrominated fluorescein precursors do not affect anion transport, both eosin labels are strong inhibitors of sulphate exchange in intact erythrocytes. 50% inhibition is obtained by binding 4.7 · 105 or 6.0 · 105 molecules/cell for eosin-NCS and IA-eosin, respectively. Both eosin probes are irreversibly bound and occupy common binding sites with 4,4′-diisothiocyano-1,2-diphenyl-ethane-2,2′-disulfonic acid (H2DIDS), although other sites are labeled as well. The inhibition of anion transport is light independent and can therefore not be attributed to a photosensitizing action of the eosin probes. Both eosin derivatives, however, inactivate acetylcholinesterase upon illumination of air-equilibrated samples of hemoglobin-free labeled ghosts. The inactivation of the enzyme is accompanied by the formation of protein aggregates as visualized by polyacrylamide gel electrophoresis. These effects are not observed when intact erythrocytes are illuminated in the presence of eosin probes suggesting a protective effect of hemoglobin during the labeling procedure. Protection of ghosts from photo-oxidation is achieved by displacing air with argon. These results are discussed in relation to the use of these and similar probes to measure protein diffusion in membranes.  相似文献   

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