Glutathione S-transferase in human organs

Glutathione S-transferase (GSH-T) distribution has been investigated in human tissues. The relative contribution of each species to total enzyme activity of the various tissues has been compared. "Cationic" (pI greater than 7.5) "neutral" (pI 6-6.5) and "anionic" (pI less than 5.4) species of GSH-T were separated by isoelectric focusing. "Cationic" GSH-Ts (ligandin) quantitated by radioimmunoassay were present in all tissues studied. Highest concentrations were in liver, kidney, duodenum, testis and adrenal. "Neutral" and "anionic" GSH-Ts were not present in every tissue or in every specimen of some tissues studied. Marked inter-organ and inter-individual variation in the relative concentration of the 3 GSH-T species may explain individual and organ susceptibility to drugs and toxins and underlines the need for future studies to examine individual enzymes rather than total activity.     

alpha-Tocopherol inhibits human glutathione S-transferase pi

alpha-Tocopherol is the most important fat-soluble, chain-breaking antioxidant. It is known that interplay between different protective mechanisms occurs. GSTs can catalyze glutathione conjugation with various electrophiles, many of which are toxic. We studied the influence of alpha-tocopherol on the activity of the cytosolic pi isoform of GST. alpha-Tocopherol inhibits glutathione S-transferase pi in a concentration-dependent manner, with an IC(50)-value of 0.5 microM. At alpha-tocopherol additions above 3 microM there was no GST pi activity left. alpha-Tocopherol lowered the V(max) values, but did not affect the K(m) for either CDNB or GSH. This indicates that the GST pi enzyme is noncompetitively inhibited by alpha-tocopherol. An inhibition of GST pi by alpha-tocopherol may have far-reaching implications for the application of vitamin E.     

Glutathione S-transferase is a novel target for mood stabilizing drugs in primary cultured neurons

Oligonucleotide microarray technology was used to analyze gene expression profiles after chronic treatment with the mood stabilizing drug valproate at a therapeutically relevant concentration in primary cultured rat cerebral cortical cells. We discovered that valproate regulates expression of 28 genes, including three isoenzymes (M1, A3 and A4) of glutathione S-transferase (GST), an important protective factor against oxidative stress. Because previous studies in our laboratory found that chronic valproate treatment protected cultured neurons against oxidative stress, further experiments on the regulation of GST were performed. Regulation of GST M1, GST A3 and GST A4 was verified using northern blotting hybridization. Chronic valproate treatment increased mRNA levels of M1 and A4, but decreased the A3 mRNA level dose-dependently, indicating further complexities in the regulation of GST by valproate. The level of GST M1 protein and GST activity were also increased by chronic valproate treatment. In addition, chronic treatment with lithium, another commonly prescribed mood stabilizer, also increased levels of GST M1 mRNA and protein. The present findings suggest that regulation of GST M1, and possibly GST A4, may mediate the anti-oxidative effects of valproate treatment, and regulation of GST may be involved in the mood stabilizing effect of valproate and lithium.     

Cortical alpha-adrenoceptor downregulation by tricyclic antidepressants in the rat brain

The aim of the present study was to examine the effect of chronic tricyclic antidepressants (TCAs) treatment on the density of -adrenoceptors in the rat brain. Density of ₁- and ₂-adrenoceptors was measured in cortex and hippocampus of rats treated with imipramine (IMI, 5 mg/kg body weight), desipramine (DMI, 10 mg/kg body weight), clomipramine (CMI, 10 mg/kg body weight) and amitriptyline (AMI, 10 mg/kg body weight), for 40 days, using [³H]prazosin and [³H]clonidine, respectively. The density of cortical ₁-adrenoceptors was significantly decreased with IMI (46%), DMI (21%), CMI (50%) and AMI (67%) treatment, without altering the affinity of the receptor. The density of cortical ₂-adrenoceptors was also significantly decreased with DMI (69%), CMI (81%) and AMI (80%) treatment, without affecting the affinity for [³H]clonidine. The density of hippocampal ₁-adrenoceptors was significantly decreased only with AMI treatment (47%), without affecting the affinity for [³H]prazosin. However, no change in hippocampal ₂-adrenoceptor density was observed with any of these TCAs. The results suggest that chronic antidepressant (AD) treatment downregulates the cortical, but not hippocampal, ₁- and ₂-adrenoceptors in rat brain. The region-specific downregulation of ₁- and ₂-adrenoceptors density, which occur after prolonged AD treatment, may underline the therapeutic mechanism of action.     

Glutathione S-transferase distribution and concentration in human organs

The concentration of basic, near-neutral and acid GSH S-transferase was measured in 18 organs from each of 9 male human subjects using radial immunodiffusion. Basic transferases were detectable in all tissues studied. Highest concentrations were found in liver, testis, kidney, adrenal and jejunum while low levels were found in bladder, muscle and thyroid. The concentration in liver was 230 times higher than that in thyroid. Near-neutral GSH S-transferase were absent in all tissues in 5 of the 9 individuals studied. When present they were widely distributed, highest concentrations being found in liver, testis, muscle, adrenal and brain and lowest levels in thyroid, lung, duodenum, stomach, heart and kidney. Acid GSH S-transferases were present in every individual studied although they were undetectable in the liver of a single subject. Highest concentrations were present in colon, jejunum, ileum, bladder, spleen and lung while low concentrations were found in liver. Our study provides conclusive evidence of marked inter-individual and inter-organ variation of the three groups of human GSH S-transferase.     

Effects of tricyclic antidepressants upon human platelet monoamine oxidase

Tricyclic antidepressant drugs were found to inhibit human platelet MAO. The I50 for the inhibition by amitriptyline was 4 × 10^?6 M, 1.6 × 10^?5 M, and 2 × 10^?4 M when phenylethylamine, tryptamine, and benzylamine were used as substrates. Amitriptyline exhibited noncompetitive inhibition with the substrates phenylethylamine and tryptamine but competitive inhibition with benzylamine. Solubilization and partial purification of platelet MAO did not alter the inhibitory effects of tricyclics. Treatment of the partially purified enzyme with the chaotropic agent sodium perchlorate produced only a slight increase in the inhibition constant for amitriptyline. Our findings suggest that selective inhibition of phenylethylamine oxidation may mediate the antidepressant actions of tricyclics. In addition, our studies provide some evidence for the existence of multiple catalytic sites of MAO activity in the human platelet.     

Effects of tricyclic antidepressants on muscarinic cholinergic receptor binding in mouse brain

Tricyclic antidepressants (TADs) were administered (10 mg/kg/day, i.p.) to mice for 2 or 4 weeks. Tolerance to the antimuscarinic effects of these agents was demonstrated by comparing their ability to supress oxotremorine-induced tremors in treated and in control animals. ED50's increased nearly three-fold after four weeks of treatment. CNS muscarinic acetylcholine receptor binding was also examined after 2 to 7 weeks of treatment by measurement of 3H-quinuclidinyl benzilate (QNB) binding. No change was found in either density or affinity of these receptors. The development of tolerance to the antimuscarinic effects of TADs is not due to alteration of either the number or the conformation of central muscarinic receptors. Evidence is presented that this phenomenon may instead be the result of an unidentified mechanism by which the post-synaptic effect of a single receptor-agonist interaction is magnified.     

Glutathione S-transferase variants as risk factor for essential hypertension in Italian patients

Involvement of genetic polymorphisms in arterial hypertension has already been reported, including GST genes, with contrasting results. The present research evaluates the possible association between GST gene polymorphisms and essential hypertension (EH) in an Italian population sample. 193 hypertensive subjects and 210 healthy controls were recruited. Buccal cells were collected from each subject using an oral swab and DNA was extracted using the phenol:chloroform:isoamilic alcohol method. GST SNPs were determined using the PCR-RFLP method, while GST null polymorphisms were determined using a Multiplex PCR. Among GST polymorphisms, only the frequency of the GSTT1 null phenotype was significantly higher in hypertensive patients than in normotensive participants. GSTT1 null individuals were significantly associated with increased risk of hypertension [P < 0.001; adjusted OR 2.24 (1.43-3.50)]. In sex-based analysis, the risk was significantly higher in female hypertensives [P < 0.001; adjusted OR 3.25 (1.78-5.95)] but not in male subjects. This study analyzed all GST gene that, in other research, have been studied in relation to arterial hypertension and the GSTO polymorphisms, showing an association only with GSTT1. The results for the GSTO genes represent the first analysis of this GST class in relation to blood pressure regulation. The association between the GSTT1 null phenotype and EH was confirmed in the overall population and in women, but not in men. These data suggest that GSTT1 could be a sex-specific candidate gene for EH.     

Selective interaction of tricyclic antidepressants with a subclass of rat brain cholinergic muscarinic receptors

Experiments were undertaken to determine whether the anticholinergic actions of tricyclic antidepressants are mediated by a selective interaction with a subclass of muscarinic receptors. To this end, the potencies of these antidepressants to inhibit [3H]-QNB binding to rat brain cerebral cortical membranes was compared to their potencies as antagonists of carbachol-stimulated inositol phosphate accumulation in cerebral cortical slices and carbachol-induced inhibition of GTP-stimulated adenylate cyclase in striatal membranes. Whereas amitriptyline was more potent than pirenzepine, a selective muscarinic M1 receptor antagonist, in competing for [3H]-QNB binding sites and as an antagonist of carbachol-induced inhibition of adenylate cyclase, pirenzepine was substantially more active (ten-fold) than amitriptyline in blocking carbachol-stimulated phosphatidyl inositol turnover. Atropine was more potent than all other agents in these assays, failing to display any significant degree of selectivity. The results suggest that the tricyclic antidepressants, in particular amitriptyline, appear to be selective antagonists for muscarinic receptors associated with adenylate cyclase in striatal membranes. Given the current classification of cholinergic receptors, these findings indicate that the tricyclic antidepressants may be useful for defining the properties of M2 receptors in brain.     

Glutathione S-transferase of the malarial parasite Plasmodium falciparum: characterization of a potential drug target

Glutathione S-transferases (GSTs), which occur abundantly in most organisms, are essentially involved in the intracellular detoxification of numerous substances including chemotherapeutic agents, and thus play a major role in the development of drug resistance. A gene encoding a protein with sequence identity of up to 37% with known GSTs was identified on chromosome 14 of the malarial parasite Plasmodium falciparum. It was amplified using gametocyte cDNA and expressed in Escherichia coli as a hexahistidyl-tagged protein of 26 kDa subunit size. The homodimeric enzyme (PfGST) was found to catalyse the glutathione (GSH)-dependent modification of 1-chloro-2,4-dinitrobenzene and other typical GST substrates such as o-nitrophenyl acetate, ethacrynic acid, and cumene hydroperoxide. The Km value for GSH was 164+/-20 microM. PfGST was inhibited by cibacron blue (Ki=0.5 microM), S-hexylglutathione (Ki=35 microM), and protoporphyrin IX (Ki=10 microM). Hemin, a most toxic compound for parasitised erythrocytes, was found to be an uncompetitive ligand of PfGST with a Ki of 6.5 microM. Based on the activity of PfGST in extracts of P. falciparum, the enzyme represents 1 to 10% of cellular protein and might therefore serve as an efficient in vivo buffer for parasitotoxic hemin. Destabilising ligands of GST are thus expected to be synergistic with the antimalarial drug chloroquine, which itself was found to be a very weak inhibitor of PfGST (IC50>200 microM). X-ray quality crystals of PfGST (250x200x50 microm) will serve as starting point for structure-based drug design.     

Glutathione S-transferase activities in rat and mouse sperm and human semen

Glutathione $\text{S}$-transferase activity was found in sperm of the rat and $\text{DBA}\text{2J}$ and C57 $\text{BL}\text{6J}$ mice. In rat sperm activities with benzo(a)pyrene 4,5-oxide, styrene 7,8-oxide, and 1-chloro-2,4-dinitrobenzene were 0.88, 1.07, and 26.1 nmoles/min/mg protein, respectively. Δ⁵-3-Ketosteroid isomerase activity of rat sperm was 4.9 nmoles/min/mg protein. These specific glutathione $\text{S}$-transferase and Δ⁵-3-ketosteroid isomerase activities in sperm represent 0.4–4.1% of rat liver cytosol values. Human semen also contained significant glutathione $\text{S}$-transferase activity. It is postulated that these enzymes could function in the metabolism and detoxification of certain electrophilic xenobiotics, if present in sperm.     

Uptake and/or binding of tricyclic antidepressants in human red cells

The factors affecting the red blood cells (RBC) uptake and/or binding of tricyclic antidepressants imipramine (IMI) and desmethylimipramine (DMI) were investigated under in vitro conditions. The time course of drug distribution indicated that equilibrium between RBC and medium is reached rapidly. The uptake and/or binding of IMI and DMI to RBC was not saturable and RBC/medium ratio was unaffected by pH, nature of monovalent cations or by the presence of therapeutic concentrations (5-50 ng/ml) of antipsychotic drugs. In the protein-free medium, RBC/medium ratio of DMI was X +/- SE = 2.58 +/- 0.08 (n = 5) and that of IMI was 7.38 +/- 0.12 (n = 6). However, RBC/plasma ratio of DMI was 0.78 +/- 0.04 (n = 7) and IMI ratio was 0.64 +/- 0.03 (n = 9). The RBC/medium ratio was linearly related to free fraction of IMI in a buffer containing 60 mg/ml of human serum albumin with various concentrations of alpha 1-acid glycoprotein (r = 0.971, n = 7). These results suggest that in the absence of proteins the lipid solubility of antidepressants is the main determinant factor in RBC uptake and/or binding, whereas, in the presence of proteins, free fraction of the drug plays the major role. Hence, the RBC/plasma ratio of antidepressants may correlate with treatment outcome in clinical studies better than total drug concentration in plasma.     

Interactions of tricyclic antidepressants with a synaptic ion channel

The interactions of four tricyclic antidepressants, including two dibenzazepines, imipramine and desipramine, and two dibenzocyclo-heptenes, amitriptyline and nortryptiline, were studied with the ion channel associated with the nicotinic acetylcholine receptor from the electric organs of $\text{Torpedo ocellata}$. These drugs inhibited the binding of tritiated perhydrohistrionicotoxin and phencyclidine to sites on the ion channel. All four compounds interacted with the ion channel with approximately equal affinities, inhibition constants for the two sites ranging from 1.1 to 3.4 μM in the absence and from 0.16 to 0.75 μM in the presence of 1 μM acetylcholine. The affinities of the secondary amines were increased by acetylcholine to a greater degree than were those of the tertiary amine compounds.     

Inhibition of sparteine oxidation in human liver by tricyclic antidepressants and other drugs

Testing for competitive inhibition of sparteine oxidation in the 9000 × g supernatant fraction from human liver provides an in vitro means to identify drugs which can bind to the same form of cytochrome P450 which oxidizes sparteine. There has so far been only two outcomes of this test: either the drug examined competed with sparteine for a common binding site, or it did not inhibit the reaction. The results of such in vitro testing implicated the involvement of guanoxan, nortriptyline, desipramine, imipramine, amitriptyline and chlorpromazine with this enzyme. Amobarbital, tolbutamide and guanethidine in therapeutic concentrations did not interfere with sparteine oxidation by this preparation.     

Glutathione S-transferase in human lymphoid cell lines and fractionated peripheral leucocytes.

Glutathione S-transferase activity was identified in cytosol from human lymphoid-cell lines and peripheral leucocytes (polymorphonuclear-leucocyte/monocyte and small-lymphocyte fractions) and compared with human liver enzyme. The findings of closely similar elution volume in gel filtration, substrate (1-chloro-2,4-dinitrobenzene) and inhibitory (probenecid) kinetics indicate that the liver, leucocyte and lymphoid-cell transferases are closely related. The interaction of reduced glutathione and 1-chloro-2,4-dinitrobenzene was shown to occur in intact-lymphoid-cell culture, to be linear with time and quantity of cells and to have kinetics similar to those of the enzyme reaction catalysed by cytosol.