diethyl-maleate and 2-cyclohexen-1-one

The effects of L-buthionine sulfoximine (L-BSO), 2-cyclohexene-1-one and diethylmaleate (DEM) on the concentration of rat brain glutathione (GSH) were investigated. Both DEM and 2-cyclohexene-1-one, administered subcutaneously, produced marked and rapid reduction of brain GSH, but 2-cyclohexene-1-one appeared less toxic than DEM. Six hours after 2-cyclohexene-1-one (100 microliters/kg) the striatal GSH concentration was 35% of control values, whereas the level was 55% of controls at 24 h and 80% of controls at 48 h. Similar results were obtained with DEM (800 microliters/kg). L-BSO (3.2 mg), administered intracerebroventricularly, produced a slower depletion of brain GSH. A 55% reduction of striatal GSH was obtained 24 h after the administration, and the level was approximately 50% of control at 48 h. Thus, the effect of 2-cyclohexene-1-one and DEM is rapid in onset but relatively short lasting, whereas the disappearance of brain GSH after L-BSO is slower but the effect is more long-lasting. By combining L-BSO with either 2-cyclohexene-1-one or DEM both a rapid and long-lasting GSH depletion was obtained that was more profound than after any of the drugs alone. The combination of L-BSO and 2-cyclohexene-1-one was well tolerated, but the combination of L-BSO and DEM led to death in half of the rats the second day after injection. The disappearance rate of GSH after L-BSO alone gives an estimate of the turn-over of GSH. We found the turn-over of GSH to be higher in the substantia nigra pars compacta than in the striatum. The present work suggest that L-BSO and 2-cyclohexene-1-one would be very useful for evaluation of the biological role of GSH in the central nervous system.

Topics: Animals; Buthionine Sulfoximine; Corpus Striatum; Cyclohexanones; Glutathione; Male; Maleates; Methionine Sulfoximine; Rats; Rats, Inbred Strains; Substantia Nigra

To search for a technique to deplete reduced glutathione (GSH) in brain, the influence of various types of compounds on brain GSH levels was investigated in mice. Of the compounds tested, cyclohexene-1-one, cycloheptene-1-one and diethyl maleate were shown to be potent GSH depletors in brain as well as in liver. The depletion of cerebral GSH ranged about 40-60% of control levels at 1 and 3 hr after intraperitoneal injection. Cyclohexene, cycloheptene, phorone, acetaminophen, and benzyl chloride caused mild depletion of cerebral GSH, but buthionine sulfoximine did not alter cerebral GSH levels. Further, intracerebroventricular injection of cyclohexene-1-one and cycloheptene-1-one caused depletion of brain GSH to about 60-80% of control levels at 1 hr after injection, and the effects persisted for at least 6 hr. Under these conditions, hepatic GSH was not altered. These results demonstrated that cyclohexene-1-one and cycloheptene-1-one can cause not only a marked depletion of brain GSH by systemic administration, but also depletion of cerebral GSH by intracerebroventricular injection by virtue of being water-soluble compounds. Thus, methods for depleting brain GSH employing both compounds are available for exploring possible functions of cerebral GSH in in vivo systems.

Topics: Animals; Brain; Cycloheptanes; Cyclohexanones; Dose-Response Relationship, Drug; Glutathione; Injections, Intraventricular; Ketones; Kinetics; Male; Maleates; Mice; Mice, Inbred ICR

The role of glutathione (GSH) in the protection of normal renal function has been investigated using rabbit proximal tubules. Compounds known to deplete GSH in various biological systems by alkylation (via GSH S-transferases, phorone; 2-cyclohexen-1-one, CHX; diethyl maleate, DEM) or by inhibiting GSH synthesis (buthionine sulfoximine, BSO) were added to suspensions of proximal tubules and incubated for 60 min. BSO (1 or 5 mM) did not decrease GSH concentrations, O2 consumption, or cause lactate dehydrogenase release (LDH). Concentrations of CHX (2 mM) and phorone (10 mM) that decreased GSH concentrations also inhibited O2 consumption and caused LDH release. DEM (10 mM) did not significantly decrease GSH concentrations but did inhibit oxygen consumption and cause slight LDH release. Time-course studies using CHX (3 mM) showed that GSH levels and O2 consumption decreased as early as 15 min while LDH release did not occur until 60 min. These results show that: there may be a relationship between O2 consumption and GSH levels; agents that have been used historically to reduce GSH concentrations have other cytotoxic effects; and rabbit renal proximal tubules appear to be resistant to GSH depletion.

Topics: Animals; Buthionine Sulfoximine; Culture Techniques; Cyclohexanones; Female; Glutathione; Ketones; Kidney Concentrating Ability; Kidney Tubules, Proximal; L-Lactate Dehydrogenase; Maleates; Methionine Sulfoximine; Oxygen Consumption; Rabbits

Thiol-oxidizing agents were found to stimulate [14C] aminopyrine accumulation, a reliable index of acid secretory function of isolated canine parietal cells. Glutathione is the predominant intracellular free thiol; thus, its oxidation status largely determines the thiol-disulfide status of the cell by thiol-disulfide interchange reactions. Three agents which alter glutathione oxidation status by different mechanisms were applied to parietal cells in vitro to investigate whether enhanced formation of GSSG alters acid secretory function. The agents studied were diamide (which nonenzymatically oxidizes GSH to GSSG), tert-butyl hydroperoxide (an organic peroxide specifically reduced by glutathione peroxidase, thereby generating GSSG for GSH), and 1,3-bis(2-chloroethyl)-1-nitrosourea (an inhibitor of NADPH:GSSG reductase, which presumably allows the accumulation of GSSG). Each of these agents stimulated aminopyrine accumulation in a dose-dependent fashion. Simple depletion of GSH by diethyl maleate or 2-cyclohexene-1-one did not stimulate aminopyrine accumulation. Likewise, enhanced aminopyrine accumulation occurred at diamide concentrations which did not cause significant depletion of total cellular glutathione. The thiol-reducing agent, dithiothreitol, prevented enhanced aminopyrine accumulation by 1,3-bis(2-chloroethyl)-1-nitrosourea and tert-butyl hydroperoxide. These observations support the hypothesis that thiol-disulfide interchange reactions involving GSSG modulate the acid secretory function of the isolated parietal cell.

Topics: Aminopyrine; Animals; Carmustine; Cyclohexanones; Diamide; Dithiothreitol; Dogs; Dose-Response Relationship, Drug; Gastric Acid; Glutathione; Maleates; Parietal Cells, Gastric; Peroxides; Sulfhydryl Reagents; tert-Butylhydroperoxide

Exogenous thiol compounds have been reported to protect the stomach from ethanol-induced necrotic lesions. The gastric mucosa contains high levels of an endogenous thiol, glutathion (GSH). Because of the known role of glutathione in protecting against hepatic injury, its role in gastric mucosal cytoprotection was of interest. By use of an animal model for acute gastric injury from ethanol, a close parallel relation between depletion of endogenous mucosal GSH and induction of mucosal protection was demonstrated. Surprisingly, mucosal protection varied inversely with the level of mucosal GSH obtained after treatment with specific GSH-depleting agents (diethyl maleate and cyclohexene-1-one). Depletion of gastric mucosal GSH was associated with an increase in the mucosal content of prostaglandins 6-keto F1 alpha and F2 alpha but not E2. The protective effect induced by GSH-depleting agents was partially reversed by indomethacin in some but not all studies. Although GSH depletors increased gastric juice volume, protection with these agents persisted after the volume and mucosal GSH had returned to control levels and also was not reversed by increasing the dose of ethanol threefold to overcome a possible dilutional effect. We conclude that, contrary to apparent predictions, depletion of endogenous gastric GSH protects the stomach from acute ethanol-induced injury. Although the mechanism of this protection is unknown, a mediation by endogenous release of prostaglandins seems to play a minor role since diethyl maleate was protective even in indomethacin-treated animals.

Topics: 6-Ketoprostaglandin F1 alpha; Animals; Cyclohexanones; Dinoprost; Dinoprostone; Ethanol; Gastric Mucosa; Glutathione; Indomethacin; Male; Maleates; Necrosis; Prostaglandins E; Prostaglandins F; Rats; Rats, Inbred Strains

Acetylcholine (ACh) induced dilation of precontracted strips of rabbit aorta by a mechanism dependent on an intact endothelium, probably by releasing an unknown endothelial relaxing factor (ERF). The relaxation was completely inhibited by the lipoxygenase inhibitor nordihydroguaiaretic acid (10(-5) M) but not by the cyclooxygenase inhibitor indomethacin (10(-5) M). The aortic strips were found to release small amounts of a material with a leukotriene-like activity. Its action on the guinea pig ileum was antagonized by FPL 55712 (10(-6) M). However, FPL 55712 (10(-6) - 10(-4) M) did not alter the response of rabbit aortic strips to ACh. Also when decreasing intracellular concentrations of glutathion (GSH) by incubating the strips with diethylmaleat or 2-cyclohexen-1-one (both 10(-3) M) the vasodilator response could still be elicited. Leukotriene (LT) C4 and LTD4 (10(-9) - 10(-6) M) were found to be ineffective on aortic strips under basal or induced tension. The same held true for LTE4 (10(-9) - 10(-7) M). At 10(-6) M, however, LTE4 induced slight relaxations of the vascular tissues. For reasons discussed this is likely to be a pharmacological action independent of the effects of endogenous ERF (e.g. inhibition of the formation of the LTE4 precursor LTD4 by high extracellular GSH concentrations did not reverse the ACh-induced vasodilation). It is concluded from these data, that C-6-sulfidopeptide leukotrienes, although probably produced by vascular tissue, are unlikely to be involved in the ACh-induced relaxation of rabbit aorta.

Topics: Acetylcholine; Animals; Aorta; Cyclohexanones; Endothelium; Female; Guinea Pigs; Ileum; In Vitro Techniques; Indomethacin; Male; Maleates; Muscle Contraction; Muscle Relaxation; Norepinephrine; Peptides; Rabbits; SRS-A; Vasodilation

The role of glutathione (GSH) in the preservation of renal function and the pathogenesis of renal injury has been investigated using the isolated perfused rat kidney as a model. In kidneys perfused for 80 min with 5 mM glucose as the only exogenous substrate, tissue GSH becomes depleted, renal function deteriorates, and a degenerative change appears, restricted to the medullary thick ascending limb. These abnormalities can be ameliorated by providing amino acid supplements or by adding GSH itself to the perfusion. To distinguish between the effects of amino acid supplementation and GSH depletion per se, selective depletion of GSH was accomplished in several different ways. Synthesis of GSH was inhibited by the addition of dl-buthionine-SR-sulfoximine, a specific inhibitor of gamma-glutamyl cysteine synthetase. GSH depletion was also produced by 2-cyclohexene-1-one and diethylmaleate, both known to diminish the concentration of GSH selectively without affecting protein thiols. Perfused kidneys selectively depleted of GSH showed significant impairment of concentrating ability, and less marked decreases in tubular reabsorption of sodium. The degenerative changes in the medullary thick ascending limb, on the other hand, were unaltered.(ABSTRACT TRUNCATED AT 250 WORDS)

Topics: Amino Acids; Animals; Buthionine Sulfoximine; Cyclohexanones; Glomerular Filtration Rate; Glucose; Glutathione; Kidney; Kidney Concentrating Ability; Loop of Henle; Male; Maleates; Methionine Sulfoximine; Natriuresis; Perfusion; Rats; Rats, Inbred Strains

2-cyclohexene-1-one and diethyl maleate specifically decrease reduced glutathione (GSH) levels in human polymorphonuclear leukocytes (PMN) by direct conjugation, and by interaction with the glutathione-s-transferase system. Using these two nontoxic reagents we have examined the effect of decreased GSH levels on five parameters of PMN activation: superoxide generation, release of the lysosomal enzymes lysozyme and beta-glucuronidase, and increases in the influx of Na+ and Ca2+. When PMN pretreated with 2-cyclohexene-1-one or diethyl maleate were incubated with formyl-methionyl-leucyl-phenylalanine (FMLP) or the proteolytic fragment of the fifth component membrane of complement, C5a, agents that interact with surface membrane receptors, increases in all five parameters were inhibited in a dose-dependent manner. For O-2 generation and lysosomal enzyme release the ID50 for 2-CHX-1 was 40--90 micrometers corresponding with a 30--50% decrease in intracellular GHS. In contrast stimulation of treated PMN by the divalent cation ionophore A23187 or 5-hydroxyeicosatetraenoic acid was much less sensitive to depressed GSH; the ID50 for 2-cyclohexene-1-one was 1 mM or greater, corresponding with an 80--90% decrease in GSH. The effect of lowered GSH was not the result of decreased binding of FMLP to surface receptors because [3H]-FMLP binding studies demonstrated a two- to three-fold increase in the number of available binding sites. These data indicate that normal GSH levels are necessary for the transduction of the activation signal from the exterior to the interior of the PMN, but once initiated the activation sequence proceeds normally despite markedly lowered intracellular GSH.

Topics: Calcium; Cells, Cultured; Chemotactic Factors; Chemotaxis, Leukocyte; Complement C5; Cyclohexanes; Cyclohexanones; Glutathione; Humans; Lysosomes; Maleates; N-Formylmethionine; N-Formylmethionine Leucyl-Phenylalanine; Neutrophils; Oligopeptides; Sulfhydryl Compounds; Superoxides

To further elucidate the role of glutathione (GSH) in the biosynthesis of slow reacting substance (SRS), SRS generation was studied in rat basophilic leukemia cells that had been preincubated with 2-cyclohexen-1-one or diethyl maleate to decrease their intracellular GSH concentrations. At low GSH levels SRS formation was markedly inhibited. The formation of other lipoxygenase products was much less affected, although some decrease in 5-hydroxyicosatetraenoic acid formation also occurred, apparently due in part to less rapid reduction of the 5-hydroperoxide.

Topics: Animals; Arachidonic Acids; Autacoids; Cells, Cultured; Cyclohexanones; Glutathione; Leukemia, Experimental; Maleates; Prostaglandins D; Rats