The study on structure-activity relationship between chromone derivatives and inhibition of superoxide anion generating from human neutrophils.

Over activation of neutrophils has been linked to many inflammatory diseases; one of critical pathologic mechanisms is that generation and exocellular release of superoxide anion from neutrophils results in peripheral tissues damage. Besides, in this study, 2-(3,5-dimethoxyphenoxy)-5,7-dimethoxy-chromen-4-one (4), a 2-phexnoychromone from our compound bank, was demonstrated to have the moderate inhibitory effect on superoxide anion generating. Therefore, serial chromones substituted with phenols or 3-flourothiophenol were designed, synthesized, and examined for suppression of superoxide anion generation. In accordance with the results, the methoxy group at 7 position (R3) of the chromone, as well as a hydrogen bond donor at a meta site of the phenyl ring greatly impacted on the activity. 2-(3-fluorophenyl)sulfanyl-7-methoxy-chromen-4-one (16), a successful example of bioisosteres from a phenol to a thiophenol, exhibited prominent anti-inflammatory effects with the IC50 value against superoxide anion generation of 5.0 ± 1.4 µM.

The gasotransmitter hydrogen sulfide inhibits transepithelial anion secretion of pregnant mouse endometrial epithelium.

Endometrial epithelium exhibits a robust ion transport activity required for dynamical regulation of uterine fluid environment and thus embryo implantation. However, there still lacks a thorough understanding of the ion transport processes and regulatory mechanism in peri-implantation endometrial epithelium. As a gaseous signaling molecule or gasotransmitter, hydrogen sulfide (H2S) regulates a myriad of cellular and physiological processes in various tissues, including the modulation of ion transport proteins in epithelium. This study aimed to investigate the effects of H2S on ion transport across mouse endometrial epithelium and its possible role in embryo implantation. The existence of endogenous H2S in pregnant mouse uterus was tested by the detection of two key H2S-generating enzymes and measurement of H2S production rate in tissue homogenates. Transepithelial ion transport processes were electrophysiologically assessed in Ussing chambers on early pregnant mouse endometrial epithelial layers, demonstrating that H2S suppressed the anion secretion by blocking cystic fibrosis transmembrane conductance regulator (CFTR). H2S increased intracellular Cl- concentration ([Cl-]i) in mouse endometrial epithelial cells, which was abolished by pretreatment with the CFTR selective inhibitor CFTRinh-172. The cAMP level in mouse endometrial epithelial cells was not affected by H2S, indicating that H2S blocked CFTR in a cAMP-independent way. In vivo study showed that interference with H2S synthesis impaired embryo implantation. In conclusion, our study demonstrated that H2S inhibits the transepithelial anion secretion of early pregnant mouse endometrial epithelium via blockade of CFTR, contributing to the preparation for embryo implantation.

The soluble curcumin derivative NDS27 inhibits superoxide anion production by neutrophils and acts as substrate and reversible inhibitor of myeloperoxidase.

A water-soluble curcumin lysinate incorporated into hydroxypropyl-ß-cyclodextrin (NDS27) has been developed and shown anti-inflammatory properties but no comparative study has been made in parallel with its parent molecule, curcumin on polymorphonuclear neutrophils (PMNs) and myeloperoxidase (MPO) involved in inflammation. The effect of NDS27, its excipients (hydroxypropyl-ß-cyclodextrin and lysine), curcumin lysinate and curcumin were compared on the release of superoxide anion by PMNs using a chemiluminescence assay and on the enzymatic activity of MPO. It was shown that curcumin and NDS27 exhibit similar inhibition activities on superoxide anion release by stimulated PMNs but also on MPO peroxidase and halogenation activities. The action mechanism of curcumin and NDS27 on the MPO activity was refined by stopped-flow and docking analyses. We demonstrate that both curcumin and NDS27 are reversible inhibitors of MPO by acting as excellent electron donors for redox intermediate Compound I (∼107 M-1 s-1) but not for Compound II (∼103 M-1 s-1) in the peroxidase cycle of the enzyme, thereby trapping the enzyme in the Compound II state. Docking calculations show that curcumin is able to enter the enzymatic pocket of MPO and bind to the heme cavity by π-stacking and formation of hydrogen bonds involving substituents from both aromatic rings. Hydroxypropyl-ß-cyclodextrin is too bulky to enter MPO channel leading to the binding site suggesting a full release of curcumin from the cyclodextrin thereby allowing its full access to the active site of MPO. In conclusion, the hydroxypropyl-ß-cyclodextrin of NDS27 enhances curcumin solubilization without affecting its antioxidant capacity and inhibitory activity on MPO.

Comparative Reduction of Egg Yolk Cholesterol Using Anionic Chelating Agents.

Egg yolk is used as an emulsifying agent. Nevertheless, its high concentration of cholesterol is linked to chronic degenerative diseases that cause cardiovascular disease. In this study, three methods for reducing the level of cholesterol in egg yolks were studied. The first method consisted of physical separation of the granules contained in the yolk (NaG). The second method applied was the use of anionic chelating biopolymers, such as arabic gum solution (AG) and mesquite gum solution (MG), and the third method was extraction with a solvent (SA). For this purpose, the cholesterol present in egg yolks, the microstructure, particle size, zeta potential, and its emulsifying capacity were determined. The amount of cholesterol removed was 97.24% using 1% mesquite gum (MG1%), and 93.26% using 1% Arabic gum (AG1%). The zeta potential was determined, and the isoelectric point (ζ = 0) of egg yolk was identified as pH 4.6. While, at this pH, the zeta potential of mesquite gum was -14.8 mV, the zeta potential for the arabic gum was -16 mV. The emulsifying capacity of MG1% was 62.95%, while the emulsifying capacity of AG1% was 63.57%. The complex obtained can be used in the development of functional foods reduced in cholesterol.

Toxicity of lupane derivatives on anionic membrane models, isolated rat mitochondria and selected human cell lines: Role of terminal alkyl chains.

Triterpenoids have multiple biological properties, although little information is available regarding their toxicity. The present study evaluates the toxicity of two new synthetic lupane derivatives using distinct biological models including synthetic lipids membranes, isolated liver and heart mitochondria fractions, and cell lines in culture. The two novel triterpenoids caused perturbations in the organization of synthetic lipid bilayers, leading to changes in membrane fluidity. Inhibition of cell proliferation and mitochondrial and nuclear morphological alterations were also identified. Inhibition of mitochondrial oxygen consumption, transmembrane electric potential depolarization and induction of the mitochondrial permeability transition pore was observed, although effects on isolated mitochondrial fractions were tissue-dependent (e.g. liver vs. heart). The size and length of hydrocarbon chains in the two molecules appear to be determinant for the degree of interaction with mitochondria, especially in the whole cell environment, where more barriers for diffusion exist. The results suggest that the positively charged triterpenoids target mitochondria and disrupt bioenergetics.

7-Hydroxycoumarin modulates the oxidative metabolism, degranulation and microbial killing of human neutrophils.

In the present study, we assessed whether 7-hydroxycoumarin (umbelliferone), 7-hydroxy-4-methylcoumarin, and their acetylated analogs modulate some of the effector functions of human neutrophils and display antioxidant activity. These compounds decreased the ability of neutrophils to generate superoxide anion, release primary granule enzymes, and kill Candida albicans. Cytotoxicity did not mediate their inhibitory effect, at least under the assessed conditions. These coumarins scavenged hypochlorous acid and protected ascorbic acid from electrochemical oxidation in cell-free systems. On the other hand, the four coumarins increased the luminol-enhanced chemiluminescence of human neutrophils stimulated with phorbol-12-myristate-13-acetate and serum-opsonized zymosan. Oxidation of the hydroxylated coumarins by the neutrophil myeloperoxidase produced highly reactive coumarin radical intermediates, which mediated the prooxidant effect observed in the luminol-enhanced chemiluminescence assay. These species also oxidized ascorbic acid and the spin traps α-(4-pyridyl-1-oxide)-N-tert-butylnitrone and 5-dimethyl-1-pyrroline-N-oxide. Therefore, 7-hydroxycoumarin and the derivatives investigated here were able to modulate the effector functions of human neutrophils and scavenge reactive oxidizing species; they also generated reactive coumarin derivatives in the presence of myeloperoxidase. Acetylation of the free hydroxyl group, but not addition of the 4-methyl group, suppressed the biological effects of 7-hydroxycoumarin. These findings help clarify how 7-hydroxycoumarin acts on neutrophils to produce relevant anti-inflammatory effects.

Synthesis and biological evaluation of new active For-Met-Leu-Phe-OMe analogues containing para-substituted Phe residues.

In the present study, we report synthesis and biological evaluation of the N-Boc-protected tripeptides 4a-l and N-For protected tripeptides 5a-l as new For-Met-Leu-Phe-OMe (fMLF-OMe) analogues. All the new ligands are characterized by the C-terminal Phe residue variously substituted at position 4 of the aromatic ring. The agonism of 5a-l and the antagonism of 4a-l (chemotaxis, superoxide anion production, lysozyme release as well as receptor binding affinity) have been examined on human neutrophils. No synthesized compounds has higher activity than the standard fMLF-OMe tripeptide to stimulate chemotaxis, although compounds 5a and 5c with -CH(3) and -C(CH(3))(3), respectively, in position 4 on the aromatic ring, are better than the standard tripeptide to stimulate the production of superoxide anion, in higher concentration. Compounds 4f and 4i, containing -F and -I in position 4, respectively, on the aromatic ring of phenylalanine, exhibit significant chemotactic antagonism. The influence of the different substitution at the position 4 on the aromatic ring of phenylalanine is discussed.

Anion inhibition studies of an α-carbonic anhydrase from the living fossil Astrosclera willeyana.

An α-carbonic anhydrase (CA, EC 4.2.1.1) isolated from the living fossil sponge Astrosclera willeyana, Astrosclerin, was investigated for its inhibition profile with simple inorganic anions, complex anions and other small molecules known to interact with these zinc enzymes. Astrosclerin is a catalytically highly efficient enzyme, and is inhibited in the low micromolar range by sulfamide, sulfamic acid, phenylboronic acid and phenylarsonic acid, and in the submillimolar range by a variety of anions including fluoride, chloride, cyanate, thiocyanate, cyanide, hydrogen sulfide, bisulfate, stannate, perosmate, divanadate, perrhenate, perruthenate, selenocyanide, trithiocarbonate, diethyldithiocarbamate and iminodisulfonate. Less efficient Astrosclerin inhibitors were sulfate, bromide, iodide, azide, bicarbonate, carbonate, tetraborate and perchlorate (K(I)s of 5.11-30.6mM) whereas tetrafluoroborate was not at all inhibitory. Because Astrosclerin is involved in calcification processes in vivo, its anion inhibition profile may be important for future studies designed to shed light on the physiologic functions of α-CAs in marine organisms.

Anion-exchange blocker enhances cytoplasmic vacuole formation and cell death in serum-deprived mouse kidney epithelial cells in mice.

An anion exchange blocker, DIDS, exhibits anti-apoptotic activity in response to several apoptotic stimuli, but has an opposite effect when apoptosis is induced by serum deprivation. After adding DIDS, serum-deprived MCT cells exhibited vacuole formation in their cytoplasm and underwent cell death. Caspase activity increased with the addition of DIDS to serum-deprived MCT cells, but Z-Asp-CH2-DCB, a caspase inhibitor, did not inhibit cell death in DIDS-treated, serum-deprived MCT cells. Cathepsin is considered to be important for vacuole formation and cell lysis, and pepstatin A, a cathepsin D inhibitor, partially inhibited vacuole formation in DIDS-treated, serum-deprived MCT cells, although caspase activation was not inhibited. 3-Methyladenin inhibited vacuole formation and cell death in DIDS-treated, serum-deprived MCT cells. These results suggest that DIDS-treated serum-deprived MCT cells undergo autophagy, not apoptosis.

Ca2+-activated Cl- current reduces transmural electrical heterogeneity within the rabbit left ventricle.

OBJECTIVE: Various cationic membrane channels contribute to the heterogeneity of action potential configuration between the transmural layers of the left ventricle. The role of anionic membrane channels is less intensively studied. We investigated the role of the Ca2+-activated Cl- current, ICl(Ca), in transmural electrical heterogeneity. METHODS AND RESULTS: We determined the density of ICl(Ca) and its physiological role in subepicardial and subendocardial ventricular myocytes of rabbit using the patch-clamp technique. ICl(Ca) was measured as the 4,4'diisothiocyanatostilbene-2,2'-disulphonic acid (DIDS) sensitive current. The current-voltage relationships and the densities of ICl(Ca) were similar in subepicardial and subendocardial myocytes. However, the functional role of ICl(Ca) exhibited striking differences. In subendocardial myocytes, blockade of ICl(Ca) by DIDS increased action potential duration (APD) significantly at all measured stimulus frequencies (3.33-0.2 Hz). In subepicardial myocytes, ICl(Ca) blockade increased APD only at 3.33 Hz, but not at the lower stimulus frequencies. At 1 Hz, ICl(Ca) blockade in subepicardial myocytes only caused an APD increase when the transient outward K+ current, Ito1, was blocked. CONCLUSIONS: The densities and gating properties of ICl(Ca) are similar in subepicardial and subendocardial myocytes. ICl(Ca) contributes to APD shortening in subendocardial, but not in subepicardial myocytes except at 3.33 Hz. These differences in functional expression of ICl(Ca) reduce the electrical heterogeneity in rabbit left ventricle.

Pharmacological characterization of a 7-benzylidenenaltrexone-preferring opioid receptor in porcine ileal submucosa.

In the intestine, opioids produce antidiarrhoeal and constipating actions that are mediated by enteric neurones. Through interactions with opioid receptors (ORs) on submucosal neurones, opioids suppress active ion transport evoked by transmural electrical stimulation (TES) in mucosa-submucosa sheets from the porcine ileum. In this study, we examined the pharmacological characteristics of the previously described OR, which is sensitive to the delta1-OR antagonist 7-benzylidenenaltrexone and modulates neurogenic transepithelial ion transport in this tissue preparation. Increases in short-circuit current (Isc, a measure of active anion transport) evoked by TES in ileal mucosa-submucosa sheets were inhibited by opioid agonists possessing high selectivity for either delta- or micro-ORs including [d-Pen2,5]enkephalin (DPDPE), [d-Ala2, Glu4]deltorphin II, and [d-Ala2, N-Me-Phe4, Gly5-ol]enkephalin (DAMGO). As determined by the Schild analysis, the actions of these agonists were competitively inhibited by 7-benzylidenenaltrexone. The nonequilibrium micro-OR antagonist beta-funaltrexamine inhibited the actions of DAMGO only at a high concentration (1 microm) but did not alter DPDPE or deltorphin II action. At concentrations up to 10 microm, the nonequilibrium delta-OR antagonist naltrindole 5'-isothiocyanate did not alter the actions of delta- or micro-OR agonists. Radioligand binding analyses of neuronal homogenates from the ileal submucosa revealed that the nonselective OR ligand [3H]diprenorphine bound to two populations of specific binding sites. One of these sites possessed binding characteristics similar to the delta-OR. In summary, neurogenic ion transport in the porcine intestine is modulated by an OR which shares pharmacological characteristics of both micro- and delta-ORs and may represent a novel receptor entity.

Bicarbonate-dependent chloride secretion in Calu-3 epithelia in response to 7,8-benzoquinoline.

Stimulation of Calu-3 epithelia with 7,8-benzoquinoline, under short circuit current conditions, produced a current increase that was completely accounted for by the net flux of chloride, measured simultaneously with 36Cl-. Nevertheless the current stimulated by 7,8-benzoquinoline was sensitive to acetazolamide, which caused up to 50 % inhibition of the stimulated current, the remainder being sensitive to the Na+-K+-2Cl- cotransport inhibitor bumetanide. The effects of acetazolamide could be mimicked by either amiloride or by the di-sodium salt of 4,4'-dinitrostilbene-2,2'-disulphonic acid (DNDS) added to the basolateral side of the epithelium, but their actions were not additive. Amiloride was needed in sufficient concentration to inhibit the sodium-proton exchanger NHE1. DNDS blocks both the chloride-bicarbonate exchanger AE2 and the sodium-bicarbonate transporter NBC1. However, since 7,8-benzoquinoline activates basolateral K+ channels, causing hyperpolarisation, it is unlikely NBC1 is active after addition of 7,8-benzoquinoline. The effect of DNDS is, therefore, mainly on AE2. It is concluded that chloride enters the basolateral aspect of the cells using the Na+-K+-2Cl- cotransporter and a parallel arrangement of NHE1 with AE2, these latter two being sensitive to acetazolamide because of their association with the cytoplasmic form of carbonic anhydrase CAII. The effects of acetazolamide could be mimicked by removal of HCO3-/CO2 from the bathing medium, and furthermore showed that the NHE1-AE2 mechanism is particularly important when the transport rate is high. Thus part of the current stimulated by 7,8-benzoquinoline and inhibited by acetazolamide or HCO3-/CO2 removal can be said to represent bicarbonate-dependent chloride secretion.

Modifying the bitterness of selected oral pharmaceuticals with cation and anion series of salts.

PURPOSE: NaCl has proven to be an effective bitterness inhibitor, but the reason remains unclear. The purpose of this study was to examine the influence of a variety of cations and anions on the bitterness of selected oral pharmaceuticals and bitter taste stimuli: pseudoephedrine, ranitidine, acetaminophen, quinine, and urea. METHOD: Human psychophysical taste evaluation using a whole mouth exposure procedure was used. RESULTS: The cations (all associated with the acetate anion) inhibited bitterness when mixed with pharmaceutical solutions to varying degrees. The sodium cation significantly (P < 0.003) inhibited bitterness of the pharmaceuticals more than the other cations. The anions (all associated with the sodium cation) also inhibited bitterness to varying degrees. With the exception of salicylate, the glutamate and adenosine monophosphate anions significantly (P < 0.001) inhibited bitterness of the pharmaceuticals more than the other anions. Also, there were several specific inhibitory interactions between ammonium, sodium and salicylate and certain pharmaceuticals. CONCLUSIONS: We conclude that sodium was the most successful cation and glutamate and AMP were the most successful anions at inhibiting bitterness. Structure forming and breaking properties of ions, as predicted by the Hofmeister series. and other physical-chemical ion properties failed to significantly predict bitterness inhibition.

State-dependent cross-inhibition between anionic GABA(A) and glycine ionotropic receptors in rat hippocampal CA1 neurons.

Using whole-cell patch-clamp recording of acutely isolated rat hippocampal CA1 neurons, we studied the state-dependent cross-inhibition between anionic GABA(A) and glycine (Gly) ionotropic receptors. Co-application of relatively high concentrations of GABA and Gly produced a total current (IGABA + Gly) much smaller than the linear summation of the two individual responses. The mutual inhibition between IGABA and IGly was voltage-independent. However, no current inhibition was observed with co-application of low concentrations of these two agonists. The hippocampal neurons lacking Gly response (IGly) showed no current inhibition with GABA current (IGABA), whereas the neurons losing GABA response (IGABA) (after complete rundown of IGABA) showed no current inhibition with IGly. The current inhibition was also absent in the presence of Gly or GABA(A) receptor antagonists, strychnine or bicuculline, respectively. The results indicate that cross-inhibition between GABA(A) and Gly receptors is state-dependent and a direct receptor-receptor interaction might enable the cross-inhibition to occur.

Inhibitory effect of ambroxol on superoxide anion production and generation by murine lung alveolar macrophages.

We examined the effect of ambroxol on superoxide anion production before and generation after phorbol-myristate acetate (PMA) stimulation of lung alveolar macrophages. Lung free cells including lung alveolar macrophages were obtained from Fischer 344 rats and guinea pigs using bronchoalveolar lavage. The superoxide anion produced by lung alveolar macrophages with or without stimulation of PMA was measured by lucigenin-dependent chemiluminescence method using a photon counter. Ambroxol inhibited the superoxide anion production and generation by lung alveolar macrophages harvested from both F344 rats and guinea pigs in a dose-dependent fashion. Approximately 16 mumol/L of ambroxol inhibited 50% of superoxide production of lung alveolar macrophages in rats and guinea pigs, whereas a slightly greater dose of ambroxol, i.e., 18-26 mumol/L, was necessary to inhibit 50% of PMA-enhanced superoxide generation by lung alveolar macrophages. These results suggest that ambroxol acts as an antioxidant in murine lungs and may be a potential therapeutic option for reactive oxygen species-associated lung disorders including bronchial asthma.

Effect of anion secretion inhibitors on mucin content of airway submucosal gland ducts.

In porcine bronchi, inhibition of both Cl- and HCO3- transport is required to block the anion secretion response to ACh and to cause mucus accumulation within ACh-treated submucosal gland ducts [S. K. Inglis, M. R. Corboz, A. E. Taylor, and S. T. Ballard. Am. J. Physiol. 272 (Lung Cell. Mol. Physiol. 16): L372-L377, 1997]. In this previous study, a combination of three potential HCO3- transport inhibitors [1 mM acetazolamide, 1 mM DIDS, and 0.1 mM dimethylamiloride (DMA)] was used to block carbonic anhydrase, Cl-/HCO3- exchange, and Na+/H+ exchange, respectively. The aim of the present study was to obtain a better understanding of the mechanism of ACh-induced HCO3- secretion in airway glands by determining which of the three inhibitors, in combination with bumetanide, is required to block anion secretion and so cause ductal mucin accumulation. Gland duct mucin content was measured in distal bronchi isolated from domestic pigs. Addition of either bumetanide alone, bumetanide plus acetazolamide, or bumetanide plus DIDS had no significant effect on ACh-induced mean gland duct mucin content. In contrast, glands treated with bumetanide plus DMA as well as glands treated with all four anion transport blockers were almost completely occluded with mucin after the addition of ACh. These data suggest that mucin is cleared from the ducts of bronchial submucosal glands by liquid generated from Cl(-)- and DMA-sensitive HCO3- transport.

Ethanol inhibition of phagocytosis and superoxide anion production by microglia.

Ethanol consumption has been associated with aberrant immune responses resulting in increased susceptibility to infection including opportunistic infections of the central nervous system. We have investigated the effects of chronic ethanol treatment on phagocytosis and production of superoxide anion by microglia. Phagocytosis of radio-labeled opsonized E. coli was markedly suppressed by treating microglia with ethanol. The unstimulated synthesis of superoxide anion was not altered by ethanol treatment of microglia, but ethanol treatment effectively suppressed phorbol-12 myristate-13 acetate-stimulated microglia superoxide anion production. The results indicate that ethanol inhibition of microglia function may play a role in increased susceptibility for central nervous system infections, particularly in immunocompromised subjects.

Effect of anion transport inhibition on mucus secretion by airway submucosal glands.

To model the airway glandular defect in cystic fibrosis (CF), the effect of anion secretion blockers on submucosal gland mucus secretion was investigated. Porcine distal bronchi were isolated, pretreated with a Cl- secretion blocker (bumetanide) and/or a combination of blockers to inhibit HCO3- secretion (dimethylamiloride, acetazolamide, and 4,4'-diisothiocyanostilbene-2,2'-disulfonic acid), and then treated with acetylcholine (ACh), a glandular liquid and mucus secretagogue. Bronchi were then fixed, sectioned, and stained for mucins. Each gland duct was ranked for mucin content from zero (no mucin) to five (duct completely occluded with mucin). Untreated bronchi, bronchi treated only with ACh, and ACh-treated bronchi that received either bumetanide or the HCO3- secretion blockers all exhibited low gland duct mucin content (1.18 +/- 0.34, 0.59 +/- 0.07, 0.65 +/- 0.03, and 0.83 +/- 0.11, respectively). However, pretreatment with both Cl- and HCO3- secretion blockers before ACh addition resulted in substantial and significant ductal mucus accumulation (3.57 +/- 0.22). In situ videomicroscopy studies of intact airways confirmed these results. Thus inhibition of the anion (and presumably liquid) secretion response to ACh leads to mucus obstruction of submucosal gland ducts that resembles the early pathological changes observed in CF.

Chloride and taurine effluxes occur by different pathways in skate erythrocytes.

The aim of this study was to determine whether volume-activated taurine and Cl- effluxes occur via the same system in skate (Raja erinacea) red blood cells (RBC). The effluxes were measured in isotonic and hypotonic elasmobranch Ringer solutions, in which NaCl was replaced by mannitol and the remaining exchangeable anions with gluconate. Methazolamide (0.1 mM) was added to minimize HCO3- formation. RBC Cl- content fell approximately 50%/h in both isotonic and hypotonic media, with no detectable K- loss in either medium. The observed Cl- loss was accompanied by an increase in pH. Both the Cl- loss and pH rise were inhibited by 4,4'- diisothiocyanostilbene-2,2'-disulfonic acid (0.1 mM), suggesting that Cl- efflux was due to H(+)-Cl- cotransport. 36Cl- effluxes in isotonic and hypotonic media were (means +/- SE, n = 11) 2.8 +/- 0.6 and 3.5 +/- 0.9 mumol.g dry wt RBC-1.min-1, respectively, whereas [3H]taurine effluxes in the same media were 0.045 +/- 0.02 and 2.1 +/- 0.05 mumol.g dry wt RBC-1.min-1, respectively (n = 6). These results indicate that taurine and Cl- effluxes occur via different pathways in skate RBC. In addition, the swelling-activated Cl- channel reported in epithelial cells does not appear to be present in skate RBC. This conclusion was confirmed by Western blots with an antibody to swelling-activated Cl- channels. Taurine and Cl- fluxes are apparently under different pathway influences in these RBC: taurine diffuses via a channel, whereas Cl- is transported by cotransporters.

Tenidap and other anion transport inhibitors disrupt cytolytic T lymphocyte-mediated IL-1 beta post-translational processing.

LPS-activated murine peritoneal macrophages produce IL-1 beta but externalize little mature cytokine in the absence of a secondary stimulus, and CTLs previously were reported to serve this capacity. The release of 17-kDa IL-1 beta from LPS-activated BALB/c macrophages occurred rapidly after the addition of C57/B1-derived allogeneic CTLs; within 30 min of coculture, mature IL-1 beta was observed in the medium, and maximum release was achieved within 4 h. CTL-induced post-translational processing was efficient, and >80% of newly synthesized pro-IL-1 beta was released into the medium as the 17-kDa species. Externalization of IL-1 beta required active recognition of the macrophage target by the CTL preparation; C57/B1 CTLs promoted the release of mature IL-1 beta from allogeneic BALB/c macrophages, but not from syngeneic C57/B1 macrophages. In contrast, extracellular ATP promoted mature IL-1 beta release from both macrophage populations. CTL-induced cytokine post-translational processing was blocked by anion transport inhibitors, including 4,4'-diisothiocyanatostilbene-2,2'-disulfonic acid, UK5099, and the anti-inflammatory agent tenidap. An analogue of tenidap, CP-100,829, was more effective as an inhibitor of both IL-1 beta post-translational processing and anion transport. In contrast, the close structural analogue CP-236,492 inhibited neither process. Tenidap's activity was reversible and was not mimicked by cyclooxygenase inhibitors or by cycloheximide. Therefore, tenidap disrupted CTL-induced IL-1 beta post-translational processing by a mechanism dependent on anion transport inhibition. Multiple stimuli are likely to operate in vivo to promote IL-1 beta post-translational processing, and anion transport inhibitors such as tenidap that suppress cytokine processing independently of the initiating stimulus thus represent attractive candidates as therapeutic regulators of IL-1 production.