Reversed isoniazids: Design, synthesis and evaluation against Mycobacterium tuberculosis.

Novel reversed isoniazid (RINH) agents were synthesized by covalently linking isoniazid with various efflux pump inhibitor (EPI) cores and their structural motifs. These RINH agents were then evaluated for anti-mycobacterial activity against sensitive, isoniazid mono-resistant and MDR clinical isolates of M. tuberculosis and a selected number of compounds were also tested ex vivo for intracellular activity as well as in the ethidium bromide (EB) assay for efflux pump inhibition efficacy. The potency of some compounds against various strains of M. tuberculosis (4a-c, 7 and 8; H37Rv-MIC99 ≤1.25 µM, R5401-MIC99 ≤2.5 µM, X_61-MIC99 ≤5 µM) demonstrated the potential of the reversed anti-TB agent strategy towards the development of novel anti-mycobacterial agents to address the rapidly growing issue of resistance. Further, macrophage activity with >90% inhibition by 1a-c and 3b (MIC90 ≤13.42 µM) and inhibition of EB efflux demonstrated by these compounds are encouraging.

Efflux inhibitor suppresses Streptococcus mutans virulence properties.

It is well established that efflux pumps play important roles in bacterial pathogenicity and efflux inhibitors (EIs) have been proved to be effective in suppressing bacterial virulence properties. However, little is known regarding the EI of Streptococcus mutans, a well-known caries-inducing bacterium. In this study, we identified the EI of S. mutans through ethidium bromide efflux assay and investigated how EI affected S. mutans virulence regarding the cariogenicity and stress response. Results indicated that reserpine, the identified EI, suppressed acid tolerance, mutacin production and transformation efficiency of S. mutans, and modified biofilm architecture and extracellular polysaccharide distribution. Suppressed glycosyltransferase activity was also noted after reserpine exposure. The data from quantitative real-time-PCR demonstrated that reserpine significantly altered the expression profile of quorum-sensing and virulence-associated genes. These findings suggest that reserpine represents a promising adjunct anticariogenic agent in that it suppresses virulence properties of S. mutans.

Anion Channel Inhibitor NPPB-Inhibited Fluoride Accumulation in Tea Plant (Camellia sinensis) Is Related to the Regulation of Ca²âº, CaM and Depolarization of Plasma Membrane Potential.

Tea plant is known to be a hyper-accumulator of fluoride (F). Over-intake of F has been shown to have adverse effects on human health, e.g., dental fluorosis. Thus, understanding the mechanisms fluoride accumulation and developing potential approaches to decrease F uptake in tea plants might be beneficial for human health. In the present study, we found that pretreatment with the anion channel inhibitor NPPB reduced F accumulation in tea plants. Simultaneously, we observed that NPPB triggered Ca(2+) efflux from mature zone of tea root and significantly increased relative CaM in tea roots. Besides, pretreatment with the Ca(2+) chelator (EGTA) and CaM antagonists (CPZ and TFP) suppressed NPPB-elevated cytosolic Ca(2+) fluorescence intensity and CaM concentration in tea roots, respectively. Interestingly, NPPB-inhibited F accumulation was found to be significantly alleviated in tea plants pretreated with either Ca(2+) chelator (EGTA) or CaM antagonists (CPZ and TFP). In addition, NPPB significantly depolarized membrane potential transiently and we argue that the net Ca(2+) and H⁺ efflux across the plasma membrane contributed to the restoration of membrane potential. Overall, our results suggest that regulation of Ca(2+)-CaM and plasma membrane potential depolarization are involved in NPPB-inhibited F accumulation in tea plants.

[The importance of efflux systems in antibiotic resistance and efflux pump inhibitors in the management of resistance]. / Antibiyotik direncinde disa atim sistemlerinin ve dirençle mücadelede disa atim pompa inhibitörlerinin önemi.

The process of active efflux, the last described resistance mechanism in bacteria, is one of the important factors of acquired antibiotic resistance. Efflux systems which consist of membrane-located pump proteins that exist in all eukaryotic and prokaryotic cells, are responsible for extrusion of the various exogenous and endogenous substances. Bacterial pump proteins, namely ATP Binding Cassette (ABC), Major Facilitator (MFS), Small Multidrug Resistance (SMR), Multidrug and Toxic Compound Extrusion (MATE) and Resistance - Nodulation - Division (RND) are grouped in five super families. Efflux pumps play a role in intrinsic resistance against antibiotics among some pathogens. Besides, pumps give rise to acquired resistance by over-expression and contribute to other resistance mechanisms. Furthermore, pumps can directly or indirectly intensify the virulence properties of bacteria. Thus, for inhibition of efflux-mediated resistance or conversion of resistant bacteria to the susceptible phenotype, potential pump inhibitors combined with traditional antibiotics is one of the research areas of interest in the fight against resistance. There are some compounds known to inhibit bacterial pump systems, such as phenyl-arginine beta naphthylamide (PAßN), INF271, INF55, carbonyl cyanide m-chlorophenyl hydrazone (CCCP), reserpine, 1-(1-naphthylmethyl)-piperazine (NMP), biricodar, timcodar, verapamil, milbemycin, chlorpromazine, paroxetine and omeprazole. However, particularly due to the toxicity problems, there has been no clinical use of a pump inhibitor yet. Nevertheless, in the upcoming period, derivatives of efflux pump inhibitor compounds with acceptable level of toxicity and efficacy may be included in antibacterial formulations. Additionally, a number of natural and synthetic compounds with pump inhibition activity are reported by many researchers in industrial and academic areas. In this context, various antibiotic derivatives (tetracycline, fluoroquinolone and aminoglycoside analogs) or non-antibiotic compounds (indole, urea, aromatic acid, piperidine-carboxylic acid, quinoline derivatives and peptidomimetics), some of which already have patent applications, are often studied. Pump inhibitors are shown to reduce the minimum inhibitory concentration (MIC) values of various antibiotics in some studies. In this review article, the structure and functions of bacterial efflux pumps have been summarized and the efflux pump inhibitors with natural and synthetic origins have been evaluated.

Possible involvement of cationic-drug sensitive transport systems in the blood-to-brain influx and brain-to-blood efflux of amantadine across the blood-brain barrier.

The purpose of this study was to characterize the brain-to-blood efflux transport of amantadine across the blood-brain barrier (BBB). The apparent in vivo efflux rate constant for [(3) H]amantadine from the rat brain (keff ) was found to be 1.53 × 10(-2) min(-1) after intracerebral microinjection using the brain efflux index method. The efflux of [(3) H]amantadine was inhibited by 1-methyl-4-phenylpyridinium (MPP(+) ), a cationic neurotoxin, suggesting that amantadine transport from the brain to the blood across the BBB potentially involves the rat plasma membrane monoamine transporter (rPMAT). On the other hand, other selected substrates for organic cation transporters (OCTs) and organic anion transporters (OATs), as well as inhibitors of P-glycoprotein (P-gp), did not affect the efflux transport of [(3) H]amantadine. In addition, in vitro studies using an immortalized rat brain endothelial cell line (GPNT) showed that the uptake and retention of [(3) H]amantadine by the cells was not changed by the addition of cyclosporin, which is an inhibitor of P-gp. However, cyclosporin affected the uptake and retention of rhodamine123. Finally, the initial brain uptake of [(3) H]amantadine was determined using an in situ mouse brain perfusion technique. Notably, the brain uptake clearance for [(3) H]amantadine was significantly decreased with the co-perfusion of quinidine or verapamil, which are cationic P-gp inhibitors, while MPP(+) did not have a significant effect. It is thus concluded that while P-gp is not involved, it is possible that rPMAT and the cationic drug-sensitive transport system participate in the brain-to-blood efflux and the blood-to-brain influx of amantadine across the BBB, respectively.

Ion channels and transporters as therapeutic targets in the pulmonary circulation.

Pulmonary circulation is a low pressure, low resistance, high flow system. The low resting vascular tone is maintained by the concerted action of ion channels, exchangers and pumps. Under physiological as well as pathophysiological conditions, they are targets of locally secreted or circulating vasodilators and/or vasoconstrictors, leading to changes in expression or to posttranslational modifications. Both structural changes in the pulmonary arteries and a sustained increase in pulmonary vascular tone result in pulmonary vascular remodeling contributing to morbidity and mortality in pediatric and adult patients. There is increasing evidence demonstrating the pivotal role of ion channels such as K(+) and Cl(-) or transient receptor potential channels in different cell types which are thought to play a key role in vasoconstrictive remodeling. This review focuses on ion channels, exchangers and pumps in the pulmonary circulation and summarizes their putative pathophysiological as well as therapeutic role in pulmonary vascular remodeling. A better understanding of the mechanisms of their actions may allow for the development of new options for attenuating acute and chronic pulmonary vasoconstriction and remodeling treating the devastating disease pulmonary hypertension.

Repeated administration of inhibitors for ion pumps reduce markedly tumor growth in vivo.

INTRODUCTION: Measurements of extracellular pH show that the micro environment of malignant tumors is more acidic than that of normal cells, whereas pH does not differ appreciable in normal and malignant cells. The acid micro environment of tumors is created by the secretion of tumor factors and ATP hydrolysis in hypoxic tumor tissue. In order to survive in a low pH-environment tumor cells develop regulatory mechanisms which keep their intracellular pH stable. Two of the most important systems are the Na+/H+ ion pump and the Na-dependent HCO3-/Cl- pump of stilbenian derivatives. MATERIAL AND METHODS: Experiments were carried out on DBA mice of both sexes at the age of 4 month. Laboratory animals were grown in our institute and supplied with food and aqua ad libitum. RESULTS: After termination of the experiments the mean tumor diameter in the control group was 12.4 +/- 0.8mm, in group A it was 6.9 +/- 0.6mm, and in group B we measured 6.6 +/- 3.1mm. At the final day the tumor size in treated animals was twice as small as in the control group. In addition we observed the rate of survival. In the control group only 18% of the animals were still alive at day 18. Considering the rate of survival a statistically significant difference between treated and untreated animals was observed. The survival of tumor cells is dependent on the function of these ion pumps which keep their intracellular pH values constant in the setting of an acid extracellular environment. CONCLUSION: The activity of the ion pump is especially important at the beginning of cell division and in cell proliferation. Our in vivo experiments demonstrate that prolonged administration of intratumoral ion pump inhibitors suppresses tumor growth as well as enhances survival of tumor-bearing animals. Research of inhibitors of ion pumps and their action in tumor growth opens new perspectives into pathophysiology of malignant tumors and may create new therapeutic options.

Extracellular alkaline pH leads to increased metastatic potential of estrogen receptor silenced endocrine resistant breast cancer cells.

INTRODUCTION: Endocrine resistance in breast cancer is associated with enhanced metastatic potential and poor clinical outcome, presenting a significant therapeutic challenge. We have established several endocrine insensitive breast cancer lines by shRNA induced depletion of estrogen receptor (ER) by transfection of MCF-7 cells which all exhibit enhanced expression profile of mesenchymal markers with reduction of epithelial markers, indicating an epithelial to mesenchymal transition. In this study we describe their behaviour in response to change in extracellular pH, an important factor controlling cell motility and metastasis. METHODS: Morphological changes associated with cell exposure to extracellular alkaline pH were assessed by live cell microscopy and the effect of various ion pumps on this behavior was investigated by pretreatment with chemical inhibitors. The activity and expression profile of key signaling molecules was assessed by western blotting. Cell motility and invasion were examined by scratch and under-agarose assays respectively. Total matrix metalloproteinase (MMP) activity and specifically of MMP2/9 was assessed in conditioned medium in response to brief alkaline pH exposure. RESULTS: Exposure of ER -ve but not ER +ve breast cancer cells to extracellular alkaline pH resulted in cell shrinkage and spherical appearance (termed contractolation); this was reversed by returning the pH back to 7.4. Contractolation was blocked by targeting the Na(+)/K(+) and Na(+)/H(+) pumps with specific chemical inhibitors. The activity and expression profile of key signaling molecules critical for cell adhesion were modulated by the exposure to alkaline pH. Brief exposure to alkaline pH enhanced MMP2/9 activity and the invasive potential of ER -ve cells in response to serum components and epithelial growth factor stimulation without affecting unhindered motility. CONCLUSIONS: Endocrine resistant breast cancer cells behave very differently to estrogen responsive cells in alkaline pH, with enhanced invasive potential; these studies emphasise the crucial influence of extracellular pH and caution against indiscriminate application of alkalinising drug therapy.

Ion pumps as biological targets for decavanadate.

The putative applications of poly-, oligo- and mono-oxometalates in biochemistry, biology, pharmacology and medicine are rapidly attracting interest. In particular, these compounds may act as potent ion pump inhibitors and have the potential to play a role in the treatment of e.g. ulcers, cancer and ischemic heart disease. However, the mechanism of action is not completely understood in most cases, and even remains largely unknown in other cases. In the present review we discuss the most recent insights into the interaction between mono- and polyoxometalate ions with ion pumps, with particular focus on the interaction of decavanadate with Ca(2+)-ATPase. We also compare the proposed mode of action with those of established ion pump inhibitors which are currently in therapeutic use. Of the 18 classes of compounds which are known to act as ion pump inhibitors, the complete mechanism of inhibition is only known for a handful. It has, however, been established that most ion pump inhibitors bind mainly to the E2 ion pump conformation within the membrane domain from the extracellular side and block the cation release. Polyoxometalates such as decavanadate, in contrast, interact with Ca(2+)-ATPase near the nucleotide binding site domain or at a pocket involving several cytoplasmic domains, and therefore need to cross through the membrane bilayer. In contrast to monomeric vanadate, which only binds to the E2 conformation, decavanadate binds to all protein conformations, i.e. E1, E1P, E2 and E2P. Moreover, the specific interaction of decavanadate with sarcoplasmic reticulum Ca(2+)-ATPase has been shown to be non-competitive with respect to ATP and induces protein cysteine oxidation with concomitant vanadium reduction which might explain the high inhibitory capacity of V10 (IC50 = 15 µM) which is quite similar to the majority of the established therapeutic drugs.

Regulation of colonic ion transport by gasotransmitters.

Gaseous molecules such as nitric oxide (NO), hydrogen sulfide (H2S), or carbon monoxide (CO) are involved in the regulation of colonic water and salt transport, which can be switched between absorption and secretion. Nitric oxide is produced from the amino acid L-arginine by different isoforms of the enzyme NO synthase, which are expressed both by enteric neurones and by the colonic epithelium. NO donors evoke a transepithelial Cl⁻ secretion in vitro. Most actions of NO are mediated by a stimulation of guanosine 5' cyclic monophosphate (cGMP) synthesis via activation of the soluble guanylate cyclase. In rat colon, NO possesses several main action sites: a stimulation of apical Cl⁻ channels most probably not related to cGMP-dependent phosphorylation, and an increase in the cytosolic Ca²âº concentration, which stimulates a Ca²âº-dependent K⁺ conductance in the basolateral membrane. Hydrogen sulfide, produced during the metabolism of the amino acid L-cysteine, also evokes a Cl⁻ secretion, either by stimulation of secretomotor submucosal neurones as in guinea-pig colon or by activating Ca²âº-dependent and ATP-sensitive K⁺ channels as in rat colon. The third gasotransmitter, CO, produced during the degradation of heme, evokes anion secretion carried by Cl⁻ and HCO3⁻. This response is mainly caused by the activation of apical anion channels and a stimulation of Ca²âº-dependent K⁺ channels via an increase of the cytosolic Ca²âº concentration. Consequently, gaseous molecules produced by enteric neurones, epithelial cells, as well-in the case of H2S-the microbial flora affect key transport enzymes involved in colonic ion transport.

Organic anion and cation transporters are possibly involved in renal excretion of entecavir in rats.

AIMS: The purpose of the present study was to investigate the roles of transporters in the renal excretion of entecavir. MAIN METHODS: We analyzed the effect of probenecid, cimetidine, sulfobromophthalein sodium (BSP), verapamil, inhibitors of organic anion transporter (OAT), organic cation transporter (OCT), multidrug resistance-associated protein 2 (MRP2) and P-glycoprotein respectively, on the excretion of entecavir. The area under plasma concentration-time curve (AUC), body clearance, and renal clearance of entecavir was examined in each group. KEY FINDINGS: After intravenous coadministration with entecavir in conscious rats, cimetidine, probenecid, BSP and verapamil significantly increased the AUC of entecavir by 40.07%, 48.78%, 37.49%, and 54.58%, and reduced the body clearance by 27.14%, 31.69%, 29.79%, and 42.17%, respectively. Then the effects of these inhibitors on the renal clearance of entecavir in unconscious rats were studied. Coadministration of cimetidine and probenecid increased the steady plasma concentration of entecavir by 127.61% and 169.46%, reduced the renal clearance by 50.47% and 67.76%, and decreased the excretion ratio by 44.81% and 64.16% compared to initial values. However, the effects of BSP and verapamil were slight. Cimetidine and probenecid also increased the concentration of entecavir in kidney from 34.00±0.80ng/mL to 55.19±4.92ng/mL and 49.92±1.53ng/mL, while the concentration of entecavir in kidney from BSP and verapamil groups was 30.96±0.81ng/mL and 35.72±7.30ng/mL, respectively. SIGNIFICANCE: These results suggest that cimetidine and probenecid inhibit the renal excretion of entecavir in rats, which indicates the most likely involvement of organic anion and cation transporters in the renal excretion of entecavir.

Mechanisms of carbacholine and GABA action on resting membrane potential and Na+/K+-ATPase of Lumbricus terrestris body wall muscles.

This work was aimed to identify the action of several ion channel and pump inhibitors as well as nicotinic, GABAergic, purinergic and serotoninergic drugs on the resting membrane potential (RMP) and assess the role of cholinergic and GABAergic sensitivity in earthworm muscle electrogenesis. The nicotinic agonists acetylcholine (ACh), carbacholine (CCh) and nicotine depolarize the RMP at concentrations of 5 µM and higher. The nicotinic antagonists (+)tubocurarine, α-bungarotoxin, muscarinic antagonists atropine and hexamethonium do not remove or prevent the CCh-induced depolarization. Verapamil, tetrodotoxin, removal of Cl(-) and Ca(2+) from the solution also cannot prevent the depolarization by CCh. In a Na(+)-free medium, however, CCh lost this depolarization ability and this indicates that the drug opens the sodium permeable pathway. Serotonin, glutamate, glycine, adenosine triphosphate (ATP) and cis-4-aminocrotonic acid (GABA(C) receptor antagonist) had no effect on the RMP. On the other hand, isoguvacin, γ-aminobutyric acid (GABA) and baclofen (GABA(B) receptor agonist) hyperpolarized the RMP. Ouabain, bicucullin (GABA(A) antagonist) and phaclofen (GABA(B) antagonist), as well as the removal of Cl(-), suppressed the effect of GABA and baclofen. CCh did not enhance the depolarization generated by ouabain but, on the other hand, hindered the hyperpolarizing activity of baclofen both in the absence and presence of atropine and (+)tubocurarine. The long-term application of CCh depolarizes the RMP primarily by inhibiting the Na(+)/K(+)-ATPase. The muscle membrane also contains A and B type GABA binding sites, the activation of which increases the RMP at the expense of increasing the action of ouabain- and Cl(-) -sensitive electrogenic pumps.

Carrier-mediated uptake of Levofloxacin by BeWo cells, a human trophoblast cell line.

OBJECTIVE: Placental transfer of Levofloxacin (LF), a broad spectrum fluoroquinolone antibiotic, and its inhibition was investigated in BeWo cells, a human trophoblast cell line. METHODS: The experiments of LF uptake by BeWo cells were performed after preincubation and in the presence of the P-glycoprotein inhibitors (Cyclosporin A, Verapamil and Quercetin), the organic anion/cation transporter inhibitor (Cimetidine) and the MCT substrates (lactic acid and salicylic acid). RESULTS: P-glycoprotein inhibitors increased the uptake of LF by BeWo cells. The increase in LF accumulation by Cyclosporin A, Verapamil and Quercetin was by 30, 90 and 80%, respectively. Cimetidine, the organic cation inhibitor, increased the transport of LF by 48%. Lactic acid and salicylic acid, the MCT substrates, initially decreased the accumulation of LF by 30% and subsequently increased the uptake of LF by 500 and 53%, respectively. CONCLUSIONS: The uptake of LF by human trophoblast cells is mediated by multiple transporters as well as passive diffusion.

The NADPH oxidase inhibitor diphenyleneiodonium is also a potent inhibitor of cholinesterases and the internal Ca(2+) pump.

BACKGROUND AND PURPOSE: Diphenyleneiodonium (DPI) is often used as an NADPH oxidase inhibitor, but is increasingly being found to have unrelated side effects. We investigated its effects on smooth muscle contractions and the related mechanisms. EXPERIMENTAL APPROACH: We studied isometric contractions in smooth muscle strips from bovine trachea. Cholinesterase activity was measured using a spectrophotometric assay; internal Ca(2+) pump activity was assessed by Ca(2+) uptake into smooth muscle microsomes. KEY RESULTS: Contractions to acetylcholine were markedly enhanced by DPI (10(-4) M), whereas those to carbachol (CCh) were not, suggesting a possible inhibition of cholinesterase. DPI markedly suppressed contractions evoked by CCh, KCl and 5-HT, and also unmasked phasic activity in otherwise sustained responses. Direct biochemical assays confirmed that DPI was a potent inhibitor of acetylcholinesterase and butyrylcholinesterase (IC(50) approximately 8 x 10(-6) M and 6 x 10(-7) M, respectively), following a readily reversible, mixed non-competitive type of inhibition. The inhibitory effects of DPI on CCh contractions were not mimicked by another NADPH oxidase inhibitor (apocynin), nor the Src inhibitors PP1 or PP2, ruling out an action through the NADPH oxidase signalling pathway. Several features of the DPI-mediated suppression of agonist-evoked responses (i.e. suppression of peak magnitudes and unmasking of phasic activity) are similar to those of cyclopiazonic acid, an inhibitor of the internal Ca(2+) pump. Direct measurement of microsomal Ca(2+) uptake revealed that DPI modestly inhibits the internal Ca(2+) pump. CONCLUSIONS AND IMPLICATIONS: DPI inhibits cholinesterase activity and the internal Ca(2+) pump in tracheal smooth muscle.

[Advances in the study of the microbial efflux pumps and its inhibitors development].

Drug resistant bacteria is an increasingly urgent challenge to public health. Bacteria adaptation and extensive abuse of antibiotics contribute to this dilemma. Active efflux of antibiotics is employed by the bacteria to survive the antibiotic pressure. Efflux pump is one of the hot spots of current drug related studies and ideal targets for the improvement of treatment. The efflux pumps and related mechanisms of action, regulation of expression and methodologies were summarized. Comparative genomics analyses were employed to elucidate the underlying mechanisms of action and evolution of efflux pump as exemplified by the Mycobacterium in our lab, which is a crucial re-emerging threat to global public health. The pathway and state-of-art drug development of efflux pump related drugs are included too.

Effect of hibernation on sodium and chloride ion transport in isolated frog skin.

The aim of the study was to evaluate the effect of hibernation on electrophysiological parameters of isolated frog skin under control incubation (Ringer solution) and after inhibition of Na+ and CI- transepithelial transport by application of amiloride and bumetanide. The transepithelial electrical potential difference (PD in mV) was measured before and after mechanical stimulation of isolated frog skin. The tissues were mounted in a modified Ussing chamber. The results revealed a reduced PD of frog skin during hibernation. In February, as compared with November, PD of frog skin incubated in Ringer solution decreased by about 50%. Hibernation also affected hyperpolarization (dPD) of frog skin after mechanical stimulation. In November and December, dPD was about 50% and 30% lower, respectively, compared with the subsequent two months of the experiment. The incubation of frog skin with amiloride, a sodium ion channel blocker, resulted in reduced values of all measured electrophysiological parameters irrespective of the phase of hibernation. After application of chloride ion transport inhibitor (bumetanide), the PD in November and December decreased compared with the control incubation by about 80% and 75%, while in January and February by about 40% and 25%, respectively. In January and February dPD increased by four times and three times as compared with November and December. Hibernation reduces net ion flow in isolated frog skin. During the initial period of hibernation the sensitivity of the skin to mechanical stimulation also decreases. Towards the end of hibernation, on the other hand, excitation of mechanosensitive ion channels takes place.

Effect of an inactivator of glyceraldehyde-3-phosphate dehydrogenase, a fortuitous arsenate reductase, on disposition of arsenate in rats.

The environmentally prevalent arsenate (AsV) is reduced in the body to the much more toxic arsenite (AsIII). Recently, we have demonstrated that the glycolytic enzyme glyceraldehyde-3-phosphate dehydrogenase (GAPDH) catalyzes the reduction of AsV in the presence of glutathione, yet the role of GAPDH in AsV reduction in vivo is unknown. Therefore, we examined the effect of (S)-alpha-cholorhydrin (ACH), which forms a GAPDH-inhibitory metabolite, on the reduction of AsV in rats. These studies confirmed the in vitro role of GAPDH as an AsV reductase, inasmuch as 3 h after administration of ACH (100 or 200 mg/kg, ip) to rats both the cytosolic GAPDH activity and the AsV-reducing activity dramatically fell in the liver, moderately decreased in the kidneys, and remained unchanged in the muscle. Moreover, the AsV-reducing activity closely correlated with the GAPDH activity in the hepatic cytosols of control and ACH-treated rats. Two confounding effects of ACH (i.e., a slight fall in hepatic glutathione levels and a rise in urinary AsV excretion) prompted us to examine its influence on the disposition of injected AsV (50 micromol/kg, iv) in rats with ligated bile duct as well as in rats with ligated bile duct and renal pedicles. These experiments demonstrated that the hepatic retention of AsV significantly increased, and the combined levels of AsV metabolites (i.e., AsIII plus methylated arsenicals) in the liver decreased in response to ACH; however, ACH failed to delay the disappearance of AsV from the blood of rats with blocked excretory routes. Thus, the GAPDH inactivator ACH inhibits AsV reduction by the liver, but not by the whole body, probably because the impaired hepatic reduction is compensated for by hepatic and extrahepatic AsV-reducing mechanisms spared by ACH. It is most likely that ACH inhibits hepatic AsV reduction predominantly by inactivating GAPDH in the liver; however, a slight ACH-induced glutathione depletion may also contribute. While this study seems to support the conclusion that GAPDH in the liver is involved in AsV reduction in rats, confirmation of the in vivo role of GAPDH as an AsV reductase is desirable.

Inhibitors of efflux pumps in Gram-negative bacteria.

In Gram-negative bacteria, efflux complexes, consisting of an inner-membrane pump, a periplasmic adaptor protein and outer-membrane channel, provide an efficient means for the export of structurally unrelated drugs, causing the multidrug-resistance phenotype. Resistance due to this antibiotic efflux is an increasing problem worldwide. A new molecular challenge is to combat this transport by searching for new molecules to block efflux and thus restore drug susceptibility to resistant clinical strains. Recent data shed new light on the structure and activity of the archetypal efflux pumps AcrAB-TolC and MexAB-OprM. Here, we describe recent insights into the molecular mechanisms of bacterial efflux pumps and their inhibitors. Current progress for the clinical use of efflux-pump inhibitors and new strategies to combat the drug-efflux mechanisms will be discussed.

Glutathione-dependent reduction of arsenate in human erythrocytes--a process independent of purine nucleoside phosphorylase.

Reduction of arsenate (AsV) to the more toxic arsenite (AsIII) is toxicologically important, yet its mechanism is unknown. To clarify this, AsV reduction was investigated in human red blood cells (RBC), as they possess a simple metabolism. RBC were incubated with AsV in gluconate buffer, and the formed AsIII was quantified by high performance liquid chromatography-hydride generation-atomic fluorescence spectrometry (HPLC-HG-AFS). The observations are compatible with the following conclusions. (1) Human RBC reduce AsV intracellularly, because 4,4'-diisothiocyanatostilbene-2,2'-disulfonic acid (DIDS, inhibitor of the chloride-bicarbonate exchanger, which also mediates phosphate and AsV uptake), as well as chloride and phosphate, countered AsIII formation. (2) Purine nucleoside phosphorylase (PNP), whose AsV reductase activity has been directly demonstrated, cannot be a physiologically relevant AsV reductase, because its inhibitor (BCX-1777) failed to decrease the basal erythrocytic AsV reduction, although it prevented the increase in AsIII formation caused by artificial activation of PNP with inosine and dithiothreitol. (3) The basal (PNP-independent) AsV reduction requires glutathione (GSH), because the GSH depletor diethylmaleate strongly diminished AsIII formation. (4) The erythrocytic AsV reduction apparently depends on NAD(P) supply, because oxidants of NAD(P)H (i.e., pyruvate, ferricyanide, methylene blue, nitrite, tert-butylhydroperoxide, dehydroascorbate, 4-dimethylaminophenol) enhanced AsIII formation from AsV. The oxidant-stimulated AsV reduction is PNP-independent, because BCX-1777 failed to affect it, but is GSH-dependent, because diethylmaleate impaired it. (5) Pyruvate-induced glucose depletion, which causes NAD enrichment in the erythrocytes at the expense of NADH, enhanced AsV reduction. This suggests that the erythrocytic AsV reduction requires both NAD supply and operation of the lower part of the glycolytic pathway starting from glyceraldehyde-3-phosphate dehydrogenase (GAPDH) that, unlike the upper part, remains fed with substrates originating from the degradation of 2,3-bisphosphoglycerate in RBC depleted of glucose by pyruvate. (6) Fluoride, which arrests glycolysis at enolase and thus prevents NAD formation, inhibited AsV reduction in glucose-sufficient RBC, but increased it in glucose-deficient (NAD-enriched) cells, suggesting that the section of glycolysis coupled to AsV reduction lies between GAPDH and enolase. In conclusion, besides the artificial PNP-dependent AsV reduction, human RBC contain a PNP-independent AsV-reducing mechanism. This appears to require the supply of GSH, NAD, and substrate to one or more of the glycolytic enzymes localized between GAPDH and enolase.