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Mitochondrial glutathione in liver does not arise by intramitochondrial synthesis, but rather from the cytoplasm, by a process characterized by slow net transport and more rapid exchange transport.Glutathione (GSH) biosynthesis is specifically inhibited by buthionine sulfoximine, a mechanism-based inhibitor ofglutamylcysteine synthetase (1, 2). After administration of this compound, GSH is rapidly depleted from many cells and tissues because it continues to be used by various transport and metabolic processes. Previous experiments showed that the levels ofGSH in the liver and kidney ofmice given a single dose of buthionine sulfoximine decrease within 30-60 min to values that are 15-20% of those of untreated controls. Further decrease of the GSH levels occurs much more slowly even if additional buthionine sulfoximine is administered. In contrast, similar studies with higher homologs of buthionine sulfoximine (hexathionine sulfoximine and heptathionine sulfoximine) showed rapid and virtually complete disappearance of GSH from both liver and kidney (2).The present work elucidates the biphasic disappearance of GSH after administration of buthionine sulfoximine. Previous findings suggested that a separate pool of GSH is sequestered in certain cell types or within cell organelles. The observation that mitochondrial GSH decreases much more slowly than total tissue GSH after buthionine sulfoximine administration (3) suggested that there is a separate mitochondrial pool of GSH. However, since injected buthionine sulfoximine does not readily penetrate into mitochondria (3), it is possible that mitochondrial GSH synthesis occurs but is protected from inhibition by this transport barrier. The present studies exclude this interpretation. We have also examined the rates of incorporation of radiolabel from [35S]cyst(e)ine into cytoplasmic and mitochondrial GSH and have found that these rates are similar. EXPERIMENTAL PROCEDURESMaterials. Reagents used for the isolation of mitochondria and for the assay of enzymes and GSH were obtained from Sigma. Monobromobimane Methods. Isolation of cytoplasmic and mitochondrial fractions for enzyme studies. Mitochondria were isolated from the livers of fed rats by the procedure of Greenawalt (6). The isolation medium (200 mM D-mannitol/70 mM sucrose/2 mM K+ Hepes, pH 7.4) did not contain bovine serum albumin but was supplemented with 5 mM 5-oxo-L-proline for studies of 5-oxoprolinase. The livers of 2 rats (24-27 g of tissue) yielded -2 ml of mitochondrial pellet, which contained 50-60% of the citrate synthase activity (7) of the whole liver homogenate. The mitochondrial wash solutions contained no citrate synthase, indicating that matrix enzymes remained sequestered in the isolated mitochondria. Examination of the mitochondrial pellet by electron microscopy showed intact and generally wellpreserved mitochondria (>90%) as well as some small vesicles and lysosomes. For enzyme studies, isolated mitochondria were suspended in 1 ml of isolation medium and sonicated (Branson model W185D,...

Clinical assessment of the activity of tumor necrosis factor (TNF) against human cancer has been limited by a dose-dependent cardiovascular toxicity, most frequently hypotension. TNF is also thought to mediate the vascular collapse resulting from bacterial endotoxin. The present studies address the mechanism by which TNF causes hypotension and provide evidence for elevated production of nitric oxide, a potent vasodilator initially characterized as endotheliumderived relaxing factor. Nitric oxide is synthesized by several cell types, including endothelial cells and macrophages, from the guanidino nitrogen of L-arginine; the enzymatic pathway is competitively inhibited by -me yl-L-arginine. We found that hypotension induced in pentobarbital-anesthetized dogs by TNF (10 ,ug/kg, i.v., resulting in a fall in mean systemic arterial pressure from 124.7 ± 7 to 62.0 ± 22.9 mmHg; 1 mmHg = 133 Pa) was completely reversed within 2 min following administration ofNG-methyl-L-arginine (4.4 mg/kg, i.v.). In contrast, NG-methyl-L-arginine failed to reverse the hypotensive response to an equivalent depressor dose of nitroglycerin, a compound that acts by forming nitric oxide by a nonenzymatic, arginine-independent mechanism. The effect of NG-methyl-L-arginine on TNF-induced hypotension was antagonized, and the hypotension restored, by administration of excess L-arginine (100 mg/kg, i.v.). Our rmdings suggest that excessive nitric oxide production mediates the hypotensive effect of TNF.Tumor necrosis factor (TNF) is a cytotoxic protein produced by macrophages upon activation by bacterial endotoxin (1, 2). In addition to a spectrum of cytotoxic and immunologic actions, TNF causes marked hypotension in mammals (1, 3). The observations that bacterial endotoxin elicits TNF production (4, 5) and that pretreatment of animals with anti-TNF antibodies abolishes the hypotensive action of endotoxin (6) suggest that TNF is the key mediator of endotoxic shock in vivo. Although TNF is known to promote hemorrhagic necrosis of some animal tumors (7), its clinical promise as an antineoplastic agent is limited by severe dose-dependent side effects, predominantly hypotension (8, 9). Despite the clinical importance of TNF-induced hypotension, its mechanism is unknown.The present study addresses the possibility that increased nitric oxide production accounts for TNF-induced hypotension. Earlier studies established that endothelium-derived nitric oxide is a labile modulator of vascular tone (10, 11). Originally termed endothelium-derived relaxing factor (EDRF, ref. 12), nitric oxide is responsible for the vascular smooth muscle relaxation elicited by acetylcholine, bradykinin, and many other endogenous vasorelaxants. L-Arginine is the biosynthetic precursor of endothelium-derived nitric oxide (13)(14)(15)(16), and NG-methyl-L-arginine (L-MeArg) is a competitive inhibitor of this pathway (14, 15). The finding that administration of L-MeArg causes a moderate increase in blood pressure by an arginine-reversible mechanism in the anesthetized guine...