Dantrolene inhibits human erythrocyte glutathione reductase

Dantrolene, a drug used to treat malignant hyperthermia, inhibits abnormal Ca2+ release from the sarcoplasmic reticulum. Glutathione reductase (Glutathione: NADP+ oxidoreductase, EC 1.8.1.7), a member of the pyridine-nucleotide disulfide oxidoreductase family of flavoenzymes, catalyzes the reduction of glutathione disulfide (GSSG) to reduced form (GSH) in the presence of nicotinamide adenine dinucleotide phosphate (NADPH). In the present study, the in vitro effects of dantrolene on human erythrocyte glutathione reductase were investigated. For this purpose, initially, human erythrocyte glutathione reductase was purified 2555.56 fold in a yield of 29.74% using both 2',5'-ADP Sepharose-4B affinity gel chromatography and Sephadex G-200 gel filtration chromatography. The purity of the enzyme was controlled by sodium dodecyle sulfate (SDS)-polyacrylamide gel electrophoresis (SDS-PAGE) which showed a single band. A constant temperature (+4 degrees C) was maintained during the purification process. Enzyme activity was determined with the Beutler method at 340 nm by means of a spectrophotometer. Dantrolene showed remarkable in vitro inhibitory effects on the enzyme. Ki constant and 50% inhibitory concentration (IC50) value for dantrolene were determined by Lineweaver-Burk graphs and plotting activity % vs. [I], respectively. Ki constant for dantrolene was found to be 0.1116+/-0.04 mM; IC50 value was 0.0523 mM. Dantrolene displayed non-competitive inhibition.     

Inhibition of human erythrocyte glutathione S-transferase by some flavonoid derivatives

Glutathione transferase is one of the important enzymes for xenobiotic metabolism. Glutathione transferases catalyze the conjugation of various electrophilic endogenous and exogenous xenobiotics with γ-glutamyl-cysteinyl-glycine (GSH). GST activity can be changed by natural plant products. The present study’s goal is to examine the interaction of human erythrocyte GST with the natural plant compounds baicalin, baicalein, phloridzin, and phloretin. First, GST was purified from human erythrocyte with 1654-fold purification and 19.27% recovery by glutathione agarose affinity chromatography. The purity of the enzyme was checked by SDS-PAGE, showing single band, because it had a homodimer structure. Baicalin, baicalein, phloridzin, and phloretin flavonoids were shown to inhibit human erythrocyte GST with the IC₅₀ values of 28.75, 57.50, 769.10, and 99.02?μM, respectively. The K_i constants for baicalin, baicalein, phloridzin, and phloretin flavonoids were 14.50?±?0.71, 24.33?±?2.08, 762.50?±?85.97, and 86.49?±?1.11?μM, respectively. According to these results, baicalin is the best inhibitor for human erythrocyte GST.     

Stimulation of a human erythrocyte membrane ATPase by glutathione conjugates

An ATP-dependent transport process for S-(2,4-dinitrophenyl) glutathione (Dnp-SG) mediated by a novel ATPase designated as Dnp-SG ATPase has been demonstrated in human erythrocytes (LaBelle et al., FEBS Lett. 228, 53-51, 1988). In order to investigate whether the Dnp-SG ATPase system represents a generalized mechanism for the transport of xenobiotic conjugates of glutathione (GSH), stimulation of this ATPase by different GSH conjugates was studied in membrane vesicles prepared from human erythrocytes. Kinetic parameters for several GSH conjugates including S-(methyl)glutathione, S-(n-propyl)glutathione, S-(n-pentyl)glutathione, S-(n-decyl)glutathione, S-(p-chlorophenacyl)glutathione, S-(p-nitrobenzyl)glutathione, and the GSH conjugate of 9,10-epoxystearic acid were determined in order to evaluate their affinity for Dnp-SG ATPase. These studies reveal that all these conjugates stimulated Dnp-SG ATPase of human erythrocyte membrane. The apparent Km values of Dnp-SG ATPase for different conjugates were found to be in the range of 0.26-0.66 mM with Vmax values ranging from 0.55 to 4.44 nmol/min/mg protein. The results of these studies indicate that erythrocyte membrane Dnp-SG ATPase represents a generalized mechanism for the transport of GSH conjugates formed with xenobiotics as well as with the endogenously generated electrophilic compounds such as epoxystearic acid. It is suggested that Dnp-SG ATPase in conjunction with GSH and GSH S-transferase may play an important role in the protection of erythrocytes from exogenous as well as endogenous electrophilic toxicants.     

Irreversible inhibition of human glutathione S-transferase isoenzymes by tetrachloro-1,4-benzoquinone and its glutathione conjugate

The quinones tetrachloro-1,4-benzoquinone (1,4-TCBQ) and its glutathione conjugate (GS-1,4-TCBQ) are potent irreversible inhibitors of most human glutathione S-transferase (GST) isoenzymes. Human pi, psi, and mu are almost completely inhibited at a molar ratio 1,4-TCBQ/GST = 2/1. The isoenzyme B1B1 was inhibited up to 75%, and higher concentrations (1,4-TCBQ/GST = 6/1) were needed to reach this maximum effect. For these isoenzymes 75-85% of the maximal amount of inhibition was already reached on incubation of equimolar ratios of 1,4-TCBQ and subunit GST, while approximately 1 nmol (0.82-0.95) 1,4-[U-14C]TCBQ per nmol subunit GST could be covalently bound. These results suggest that these GST isoenzymes possess only one cysteine in or near the active site of GST, which is completely responsible for the inhibition. In agreement, human isoenzyme B2B2 which possesses no cysteine, was not inhibited and no 1,4-TCBQ was bound to it. The rate of inhibition was studied at 0 degrees: 1,4-TCBQ, trichloro-1,4-benzoquinone and GS-1,4-TCBQ all inhibit GST very fast. Especially for B1B1, the inhibition by the glutathione conjugate is significantly faster than inhibition by 1,4-TCBQ: the glutathione moiety seems to target the quinone to the enzyme. For the other isoenzymes only minor differences are observed between 1,4-TCBQ and its glutathione conjugate under the conditions used.     

Inhibition of rat and human glutathione S-transferase isoenzymes by ethacrynic acid and its glutathione conjugate

Ethacrynic acid, a potent inhibitor of glutathione S-transferases (GST), has been shown to enhance the cytotoxicity of chlorambucil in drug resistant cell lines, but a definite mechanism has not been established. Both covalent binding to GST and reversible inhibition of GST have been reported. In the present study no irreversible inhibition was observed: for all rat GST tested, inactivation was complete within 15 sec at 0 degree, and dialysis of GST after incubation with ethacrynic acid gave complete recovery of enzyme activity for all isoenzymes tested. Moreover, the inhibition was competitive towards 1-chloro-2,4-dinitrobenzene and non-competitive towards glutathione for rat isoenzyme 1-1. Strong inhibition of both human and rat GST of the alpha-, mu- and pi-classes was obtained with ethacrynic acid, while conjugation of ethacrynic acid with glutathione did not abolish its inhibiting properties. For the alpha-, mu- and pi-class I50 values (microM) were 4.6-6.0, 0.3-1.9 and 3.3-4.8, respectively for ethacrynic acid, and 0.8-2.8, less than 0.1-1.2 and 11.0, respectively for its glutathione conjugate. Of all isoenzymes tested the human isoenzyme mu is most sensitive to the action of both ethacrynic acid and its glutathione conjugate.     

Serine-O-sulphate transport by the human glutamate transporter,EAAT2

1. Expression of the recombinant human excitatory amino aid transporters, EAAT1 and EAAT2, in Xenopus laevis oocytes allows electrogenic transport to be studied under voltage clamp conditions.
2. We have investigated the transport of the pharmacological substrate, L-serine-O-sulphate transport by EAAT1 and EAAT2. The EC₅₀ values for L-serine-O-sulphate transport by EAAT2 showed a steep voltage-dependence, increasing from 152±11 μM at −100 mV to 1930±160 μM at 0 mV. In contrast to EAAT2, EC₅₀ values for L-serine-O-sulphate transport by EAAT1 were relatively constant over the membrane potential range of −100 mV to 0 mV. The EC₅₀ values for L-glutamate and D-aspartate transport, by EAAT2, were also relatively constant over this membrane potential range.
3. Chloride ions modulated the voltage-dependent changes in EC₅₀ values for transport by EAAT2. This effect was most apparent for L-serine-O-sulphate transport, and to a lesser extent for L-glutamate and not at all for D-aspartate transport by EAAT2.
4. Extracellular sodium and proton concentrations also modulated the voltage-dependence of L-serine-O-sulphate EC₅₀ values for EAAT2.
5. We speculate that these different properties of L-serine-O-sulphate transport by EAAT2 compared to other substrates may be due to the much stronger acidity of the sulphate group of L-serine-O-sulphate compared to carboxyl groups of L-glutamate or D-aspartate.
6. These results highlight some of the differences in the way different glutamate transporter subtypes transport substrates. This may be used to understand further the transport process and develop subtype selective inhibitors of glutamate transport.

     

Comparison of inhibitory effects between acetaminophen–glutathione conjugate and reduced glutathione in human glutathione reductase

Acetaminophen overdose is the most frequent cause of acute liver injury. The main mechanism of acetaminophen toxicity has been attributed to oxidation of acetaminophen. The oxidation product is very reactive and reacts with glutathione generating acetaminophen–glutathione conjugate (APAP‐SG). Although this conjugate has been recognized to be generally nontoxic, we have found recently that APAP‐SG could produce a toxic effect. Therefore, the aim of our study was to estimate the toxicity of purified APAP‐SG by characterizing the inhibitory effect in human glutathione reductase (GR) and comparing that to the inhibitory effect of the natural inhibitor reduced glutathione. We used two types of human GR: recombinant and freshly purified from red blood cells. Our results show that GR was significantly inhibited in the presence of both APAP‐SG and reduced glutathione. For example, the enzyme activity of recombinant and purified GR was reduced in the presence of 4 mm APAP‐SG (with 0.5 mm glutathione disulfide) by 28% and 22%, respectively. The type of enzyme inhibition was observed to be competitive in the cases of both APAP‐SG and glutathione. As glutathione inhibits GR activity in cells under physiological conditions, the rate of enzyme inhibition ought to be weaker in the case of glutathione depletion that is typical of acetaminophen overdose. Notably, however, enzyme activity likely remains inhibited due to the presence of APAP‐SG, which might enhance the pro‐oxidative status in the cell. We conclude that our finding could reflect some other pathological mechanism that may contribute to the toxicity of acetaminophen. Copyright © 2013 John Wiley & Sons, Ltd.     

Lead and calcium transport in human erythrocyte.

In this paper we report the lead (Pb) and calcium (Ca) uptake by erythrocyte ghosts. In both cases the transport was carried out by a passive transport system with two kinetic components (Michaelis-Menten and Hill). Pb and Ca were capable of inhibiting the transport of the other metal in a non-competitive way. Under hyperpolarization, the uptakes of Ca and Pb were enhanced and the Michaelis-Menten component prevailed. Both Ca and Pb uptakes were inhibited by N-ethyl-maleimide to the same extent. These results indicate that Pb and Ca share the same permeability pathway in human erythrocytes and that this transport system is electrogenic.     

Inhibition of erythrocyte calcium transport by cetiedil

The elevated calcium content found in red cells from patients with sickle cell anemia may be of pathophysiologic importance in the hemolysis and vasoocclusion which characterize this disorder. Cetiedil, an antisickling agent, has been reported to inhibit the activity of enzymes that are stimulated by the calcium regulatory protein calmodulin. To investigate the mechanism by which cetiedil modifies calcium-mediated erythrocyte function, the effect of the drug on the active transport of calcium into inside-out erythrocyte vesicles was examined and its influence on the activities of phosphodiesterase and Ca-ATPase studied. Cetiedil, in the presence of calmodulin, significantly inhibited calcium transport into inside-out vesicles that were prepared with erythrocytes from normal controls and from patients with sickle cell anemia. However, in the absence of calmodulin, no inhibition was observed. Likewise, cetiedil inhibited calmodulin-stimulated, but not basal, activities of phosphodiesterase and Ca-ATPase. These data, along with previous reports, suggest that cetiedil does not act by lowering the intracellular calcium content. It is, therefore, likely that the beneficial effect of cetiedil is due to its ability to protect the red cell from the deleterious consequences of an elevated concentration of intracellular calcium.     

Effect of lead on the calcium transport in human erythrocyte

In this paper we study the calcium uptake in the erythrocyte, a non-excitable cell. This uptake is performed through a passive transport system with two kinetic components (Michaelis-Menten and Hill). The uptake of calcium seems to be driven by voltage through its electrophoretical effect. Lead is capable of inhibiting calcium uptake in a non-competitive manner. As it has been described in other systems, lead is also capable of inhibiting calcium efflux by inhibiting Ca(Mg)-ATPase. Under physiological conditions, the function of ATPase reduces the effect of lead on calcium influx. However, in chronic intoxication a small increment of intracellular calcium is observed, indicating that lead is affecting calcium efflux mainly. We discuss the effects of lead on calcium equilibrium in erythrocytes.     

Nephrotoxicity of 4-aminophenol glutathione conjugate.

4-Aminophenol (p-aminophenol, PAP) causes selective necrosis to the pars recta of the proximal tubule in Fischer 344 rats. The basis for this selective toxicity is not known, but PAP can undergo oxidation in a variety of systems to form the 4-aminophenoxy free radical. Oxidation or disproportionation of this radical will form 1,4-benzoquinoneimine which can covalently bind to tissue macromolecules. Recent studies have shown that certain benzoquinol-glutathione conjugates can cause renal necrosis in rats. We have synthesized a putative glutathione conjugate of PAP. The effect on the kidney of this conjugate and the sulphate and N-acetyl conjugates, known metabolites of PAP, have been examined in Fischer 344 rats. 4-Amino-3-S-glutathionylphenol produced a dose-dependent (92-920 mumol kg-1) necrosis of the proximal tubular epithelium and altered renal excretory function. The lesion at the low dose was restricted to the pars recta of the proximal tubule in the medullary rays, while at the higher doses it affected the pars recta region of all nephrons. In contrast, PAP-O-sulphate and N-acetyl-4-aminophenol (paracetamol) caused no histological or functional alteration to the kidney at 920 mumol kg-1. The renal necrosis produced by 4-amino-3-S-glutathionylphenol was very similar to that produced by PAP (367-920 mumol kg-1), both functionally and histologically, except that smaller doses of the glutathione conjugate were required. These studies indicate that glutathione conjugation of PAP generates a metabolite that is more toxic to the kidney than the parent compound. A possible mechanism of toxicity (analogous to that reported for glutathione conjugates of certain quinones) involving oxidation to form a 1,4-benzoquinoneimine thioether that could redox cycle is discussed.     

Formation of a glutathione conjugate from butylated hydroxytoluene by rat liver microsomes

Butylated hydroxytoluene (BHT) was converted to S-(3,5-di-tert-butyl-4-hydroxybenzyl)-glutathione (BHT-glutathione) by rat liver microsomes in the presence of NADPH, molecular oxygen, and glutathione. NADH was far less effective than NADPH and exhibited little synergistic effect when used together with NADPH. Cytochrome P-450 inhibitors, such as SKF 525-A, alpha-naphthoflavone, metyrapone, and carbon monoxide, significantly inhibited BHT-glutathione formation. Liver microsomes from phenobarbital-treated rats catalyzed the formation of BHT-glutathione at a rate that was nine times the rate of adduct formation by control microsomes. No stimulation of BHT-glutathione formation was observed with the addition of liver cytosol fraction to the microsomal incubation mixtures even at low glutathione concentrations. These results support the view that BHT is converted by the cytochrome P-450 monooxygenases to a chemically reactive metabolite, possibly BHT-quinone methide, which forms BHT-glutathione by nonenzymatic conjugation with glutathione.     

Metformin transport by a newly cloned proton-stimulated organic cation transporter (plasma membrane monoamine transporter) expressed in human intestine.

Metformin is a widely used oral antihyperglycemic drug for the treatment of type II diabetes mellitus. The intestinal absorption of metformin is dose-dependent and involves an active, saturable uptake process. Metformin has been shown to be transported by the human organic cation transporters 1 and 2 (hOCT1-2). We recently cloned and characterized a novel proton-activated organic cation transporter, plasma membrane monoamine transporter (PMAT). We previously showed that PMAT transports many classic organic cations (e.g., monoamine neurotransmitters, 1-methyl-4-phenylpyridinium) in a pH-dependent manner and its mRNA is expressed in multiple human tissues. The goal of this study is to investigate whether metformin is a substrate of PMAT and whether PMAT plays a role in the intestinal uptake of metformin. Using Madin-Darby canine kidney cells stably expressing human PMAT, we showed that metformin is avidly transported by PMAT, with an apparent affinity (K(m) = 1.32 mM) comparable to those reported for hOCT1-2. Interestingly, the concentration-velocity profile of PMAT-mediated metformin uptake is sigmoidal, with a Hill coefficient of 2.64. PMAT-mediated metformin transport is greatly stimulated by acidic pH, with the uptake rate being approximately 4-fold higher at pH 6.6 than at pH 7.4. Using a polyclonal antibody against PMAT, we showed that the PMAT protein (58 kDa) was expressed in human small intestine and concentrated on the tips of the mucosal epithelial layer. Taken together, our results suggest that PMAT transports metformin, is expressed in human intestine, and may play a role in the intestinal absorption of metformin and possibly other cationic drugs.     

A human serotonin transporter mutation causes constitutive activation of transport activity

A rarely occurring variant of human serotonin transporter (hSERT) was tested for its functional consequences in HeLa and COS-7 cells. The variant, in which Ile-425 is converted to Val, was significantly different from wild type with respect to its catalytic properties. In both cell types, rates of serotonin (5-HT) transport were higher for the I425V variant. Both an increase in Vmax and a decrease in KM caused this increase in rate. The increase in Vmax was not accounted for by increases in transporter expression or in the distribution of transporter between the cell surface and intracellular pools. The decrease in KM was accompanied by a decrease in the KD for binding of the cocaine analog 2beta-carbomethoxy-3beta-(4-[125I]iodophenyl)tropane. In both HeLa and COS-7 cells, the nitric oxide donor S-nitroso-N-acetylpenicillamine increased the activity of wild-type hSERT to that of the variant but did not change the activity of the I425V variant. This stimulation was prevented by the presence of oxyhemoglobin, which quenches nitric oxide, and by an inhibitor of guanylyl cyclase.     

Mutagenicity of a glutathione conjugate of butadiene diepoxide

The mutagenicity and carcinogenicity of the important commodity chemical 1,3-butadiene are attributed to the epoxide products. We confirmed our previous work showing that expression of rat glutathione (GSH) transferase 5-5 enhances the mutagenicity of butadiene diepoxide in Salmonella typhimurium TA1535. A GSH-butadiene diepoxide conjugate was isolated and fully characterized by mass spectrometry and nuclear magnetic resonance as S-(2-hydroxy-3,4-epoxybutyl)GSH. The conjugate had a t(1/2) of 2.6 h (pH 7.4, 37 °C) and was considerably more mutagenic than butadiene diepoxide or monoepoxide in S. typhimurium. We propose that the GSH conjugate may be a major species involved in butadiene genotoxicity, not a detoxication product.     

The formation of glutathione conjugate derived from propranolol

Mixed function oxidases in liver microsomes from 3-methylcholanthrene-pretreated guinea pigs converted S-propranolol to 4-hydroxypropranolol, 5-hydroxypropranolol, and desisopropyl-propranolol. The addition of glutathione and cytosol from rat liver to the system resulted in the formation of a water-soluble metabolite. Evidence that the metabolite was a glutathione adduct was obtained by showing that treatment with gamma-glutamyltranspeptidase changed its HPLC retention time. The formation of the glutathione adduct required glutathione S-transferases in cytosol and was accompanied by a decrease in the formation of 5-hydroxypropranolol, but not of 4-hydroxypropranolol or desisopropylpropranolol. The formation of the propranolol-glutathione adduct was confirmed by two independent approaches. When [14C]glutathione and [3H]S-propranolol were used, the relationship between 14C and 3H in the metabolite indicated that it contained equal amounts of glutathione and propranolol. When a pseudoracemic mixture of S-propranolol and [2,4-2H2]R-propranolol was used, thermospray LC/MS analysis of the glutathione adduct revealed two peaks having different retention times. The first peak, representing a [2,4-2H2]R-propranolol-GSH adduct, gave fragment ions at m/z 294, 278, 262, while the second, which represented the S-propranolol-GSH adduct, gave fragment ions having 2 mass units less than those obtained with [2,4-2H2]R-propranolol. There was no evidence of any loss of deuterium during the formation of these fragment ions. The material in both peaks gave ions of m/z 308, 147, and 130, which is consistent with the presence of a glutathione group.     

Identification of a novel glutathione conjugate of flutamide in incubations with human liver microsomes.

Flutamide, a nonsteroidal antiandrogen drug widely used in the treatment of prostate cancer, has been associated with rare incidences of hepatotoxicity in patients. It is believed that bioactivation of flutamide and subsequent covalent binding to cellular proteins is responsible for its toxicity. Current in vitro studies were undertaken to probe the cytochrome P450 (P450)-mediated bioactivation of flutamide and identify the possible reactive species using reduced glutathione (GSH) as a trapping agent. NADPH- and GSH-supplemented human liver microsomal incubations of flutamide gave rise to a novel GSH conjugate where GSH moiety was conjugated to the flutamide molecule via the amide nitrogen, resulting in a sulfenamide. The structure of the conjugate was characterized by liquid chromatography-tandem mass spectrometry and NMR experiments. The conjugate formation was primarily catalyzed by heterologously expressed CYP2C19, CYP1A2, and, to a lesser extent, CYP3A4 and CYP3A5. The mechanism for the formation of this conjugate is unknown; however, a tentative bioactivation mechanism involving a P450-catalyzed abstraction of hydrogen atom from the amide nitrogen of flutamide and the subsequent trapping of the nitrogen-centered radical by GSH or oxidized glutathione (GSSG) was proposed. Interestingly, the same adduct was formed when flutamide was incubated with human liver microsomes in the presence of GSSG and NADPH. This finding suggests that P450-mediated oxidation of flutamide via a nitrogen-centered free radical could be one of the several bioactivation pathways of flutamide. Even though the relationship of the GSH conjugate to flutamide-induced toxicity is unknown, the results have revealed the formation of a novel, hitherto unknown, GSH adduct of flutamide.