Rapid chemical antagonism of neuromuscular blockade by L-cysteine adduction to and inactivation of the olefinic (double-bonded) isoquinolinium diester compounds gantacurium (AV430A), CW 002, and CW 011.

BACKGROUND: The ultra-short-acting neuromuscular blocker gantacurium is chemically degraded in vitro by rapid adduction of L-cysteine to its central olefinic double bond. Preliminary data have suggested that exogenous (intravenous) L-cysteine abolishes gantacurium blockade. Two new analogues of gantacurium (CW 002 and CW 011) have been synthesized to undergo slower L-cysteine adduction, yielding intermediate duration. L-cysteine adduction to and antagonism of these novel agents is further defined herein. METHODS: Comparative reaction half-time for L-cysteine adduction in vitro of the three compounds was determined by high-performance liquid chromatography. ED95 for twitch inhibition in monkeys under isoflurane was calculated, and duration at approximately 4-5x ED95 was correlated with reaction half-time for adduction. Speed of L-cysteine antagonism was contrasted with anticholinesterase reversal. Potencies of CW 002 and its adduction product were compared to provide a basis for L-cysteine antagonism. RESULTS: Rate of L-cysteine adduction in vitro (reaction half-time) was 11.4 and 13.7 min for CW 002 and CW 011 versus 0.2 min for gantacurium, and was inversely related to duration of block (P < 0.0001). CW 002 and CW 011 were 3x longer acting than gantacurium (28.1 and 33.3 min vs. 10.4 min), but only half the duration of cisatracurium. The adduct of CW 002 was approximately 70x less potent than CW 002. L-cysteine (10-50 mg/kg intravenously) given 1 min after approximately 4-5x ED95 doses of all the three compounds abolished block within 2-3 min. CONCLUSIONS: L-cysteine adduction occurs at different rates by design in olefinic isoquinolinium diester neuromuscular blockers, yielding corresponding durations of action. Antagonism by exogenous L-cysteine is superior to anticholinesterases, inducing inactivation of the active molecules to restore function rapidly at any time.

Clofibrate-induced in vitro hepatoprotection against acetaminophen is not due to altered glutathione homeostasis.

Prior induction of peroxisome proliferation protects mice against the in vivo hepatotoxicity of acetaminophen and various other bioactivation-dependent toxicants. The mechanisms underlying such chemoresistance are poorly understood, although they have been suggested to involve alterations in glutathione homeostasis. To clarify the role of glutathione in this phenomenon, we isolated hepatocytes from mice in which hepatic peroxisome proliferation had been induced with clofibrate. The cells were incubated with a range of acetaminophen concentrations and the extent of cell killing after up to 8 h was assessed by measuring lactate dehydrogenase leakage from the cells. Hepatocytes from clofibrate-pretreated mice were much less susceptible to acetaminophen than cells from vehicle-treated controls. However, the extent of glutathione depletion during exposure to acetaminophen was similar in both cell types, as were rates of excretion of the product of glutathione-mediated detoxication of acetaminophen's quinoneimine metabolite, 3-glutathionyl-acetaminophen. The glutathione-replenishing ability of clofibrate-pretreated cells after a brief exposure to diethyl maleate also resembled that of control cells. More importantly, prior depletion of glutathione by diethyl maleate did not abolish the resistance of clofibrate-pretreated cells to acetaminophen. Taken together, these findings indicate that although glutathione-dependent pathways may contribute to hepatoprotection during peroxisome proliferation, the resistance phenomenon is not due exclusively to this mechanism.

Retinoblastoma protein dephosphorylation is an early event of cellular response to prooxidant conditions.

The modification of intracellular redox conditions with diethylmaleate (DEM), a glutathione-depleting agent, induces a p53-independent growth arrest mediated by the accumulation of p21(waf1) mRNA and protein. The same treatment also induces the retinoblastoma protein (pRb) dephosphorylation. This dephosphorylation (i) is very fast, being observed already 5 min after the exposure of the cells to DEM, (ii) is dependent on the prooxidant effects of DEM, being prevented by the treatment with N-acetylcysteine and (iii) is completely reversible, since the rephosphorylation of pRb is promptly obtained upon the removal of the glutathione-depleting agent from the culture medium. The dephosphorylation of pRb is independent of the accumulation of p21(waf1) induced by DEM; in fact, p21(waf1) levels start to increase much later after DEM treatment and accordingly cyclin-dependent kinase activities are not yet induced when pRb is already dephosphorylated following DEM treatment. Finally, pRb dephosphorylation is catalyzed by phosphatases activated by DEM treatment.

Glutathione synthesis in the exocrine pancreas.

Glutathione is essential for cellular cytoprotection, and in the exocrine pancreas, it is required for digestive enzyme synthesis. The purpose of these studies was to measure the capacity of the exocrine pancreas to synthesize glutathione, determine whether the pancreatic transsulfuration pathway has a role in providing cysteine needed for glutathione synthesis, and determine whether the glutathione synthetic capacity of the pancreas responds to pathologically relevant stresses. The activity of gamma-glutamylcysteine synthetase, the key regulatory enzyme for glutathione synthesis, was 3.56 +/- 0.29 mU/mg protein in the pancreas of fed rats, compared to 31 +/- 4 in the liver and 116 +/- 5 in the kidney. Studies using dispersed rat pancreatic acinar cells showed that the exocrine pancreas synthesizes glutathione from precursor amino acids and that the transsulfuration pathway is functionally intact in the pancreas and may serve as an important source of pancreatic cysteine. In mice, pancreatic gamma-glutamylcysteine synthetase activity was induced 37% by corn oil, 77% by ethanol, and 88% by both treatments. Thus, the glutathione synthetic capacity of the pancreas is quantitatively less than that of the kidney or liver, but its key regulatory enzyme responds dynamically to pathologically relevant metabolic stresses, suggesting that glutathione is a key pancreatic cytoprotectant.

Maleylated human serum albumin inhibits HIV-1 infection in vitro.

Maleylated-human serum albumin (Mal-HSA) inhibited human immunodeficiency virus type-1 (HIV-1) infection of MT-4 cells in vitro. It was also found to inhibit the fusion between uninfected CD4+ cells (Molt-4 clone 8 cells) and HIV-1 infected cells (Molt-4/HIV-1) to form syncytia. To investigate the mechanism of the inhibition, a study was designed to determine whether Mal-HSA could bind to CD4+ cells. Mal-HSA could bind to both MT-4 cells and Molt-4 clone 8 cells with high affinity, Kd = 2.0 nM and Kd = 5.8 nM, respectively. However, Mal-HSA could neither inhibit anti CD4 antibody Leu 3a binding to Molt-4 clone 8 cells nor modulate the expression of CD4 molecules on the surface of the cells. Mal-HSA binding to Molt-4 clone 8 cells was completely inhibited by sulfated polysaccharides bearing anti-HIV activity, such as dextran sulfate, fucoidan and carrageenan. Other HIV-1 susceptible human T-cell lines, such as Molt-4, CEM-5, H-9 and HuT-78 cells, also have Mal-HSA binding sites showing a high affinity, Kd = 0.9 +/- 0.4 nM. Mal-HSA binding proteins of Molt-4 clone 8 cells were identified by ligand blotting as 155 and 220 kDa proteins. Unlike dextran sulfate, Mal-HSA could not inhibit reverse transcriptase activity of HIV-1. These results indicate that Mal-HSA inhibits HIV-1 infection and syncytia formation, and suggest that 155 and/or 220 kDa proteins of target cells are involved in HIV-1 adsorption and/or the membrane fusion between HIV-1 and target cells.

Inhibition by colchicine of biliary secretion of diethylmaleate in the rat: evidence for microtubule-dependent vesicular transport.

It has been proposed that a microtubule-dependent transport of vesicles derived from the Golgi apparatus may play a role in biliary secretion of bile salts and other cholephilic anions. To test this hypothesis, we examined the influence of colchicine and vinblastine, two microtubule inhibitors, on diethylmaleate-induced bile flow and on the biliary secretion of diethylmaleate, an organic anion whose glutathione conjugates may be secreted into bile through the Golgi apparatus and Golgi-derived vesicles. Rats were pretreated with colchicine or vinblastine, and diethylmaleate was injected intraperitoneally at doses of 28 to 400 mumol/100 gm body wt. Basal bile flow was unaffected by colchicine or vinblastine. In contrast, diethylmaleate-induced bile flow and the secretion into bile of diethylmaleate conjugates (estimated by the cation-anion gap in bile) were significantly lower in colchicine-treated and vinblastine-treated animals than in controls. Diethylmaleate-induced bile flow was reduced in proportion to diethylmaleate conjugate secretion. A linear relationship was seen between bile flow and biliary output of diethylmaleate conjugates: this relationship was similar in colchicine-treated or vinblastine-treated animals and in controls. At electron microscopy, diethylmaleate had induced distension of the Golgi saccules of the hepatocytes. In conclusion, colchicine and vinblastine inhibited the secretion into bile of diethylmaleate conjugates and diethylmaleate-induced bile flow. These results support the view that microtubule-dependent transport of Golgi-derived vesicles is involved in the biliary secretion of diethylmaleate and, perhaps, other cholephilic organic anions.

Inhibition by aldehydes as a possible further mechanism for glucose-6-phosphatase inactivation during CCl4-poisoning.

Diffusable aldehydes are known to be produced during lipoperoxidative deterioration of unsaturated fatty acids. Malealdehyde (MLA) and 4-hydroxy-2,3-trans-penten-1-al (4-HPE) inhibit rat liver glucose-6-phosphatase activity in vitro. With MLA inhibition is significant at 0.25 mM concentration. With 4-HPE inhibition takes place at 0.5 mM. 1 mM MLA inhibited by about 89%, 6 mM -HPE by about 67%. Maximal inhibition is present as early as 5 min after addition of both aldehydes. Preincubation of aldehydes with 2 mM cystein or glycine in the absence of microsomes almost completely prevents the inhibitory influence. Previous incubation of microsomes with 2 mM glutathione or 2 mM dithiothreitol or 2 mM cysteine affords a good protection towards the inhibitory action of the aldehydes; on the contrary, no protection is seen when microsomes are preincubated in the presence of either 2 mM glycine or asparagine. The total content of microsomes -SH groups is strongly decreased after incubation with 2mM malealdehyde. These results support the idea that the two aldehydes inhibit glucose-6-phosphatase mostly through interaction with protein -SH groups. The possibility that aldehydes derivated from the peroxidative decomposition of lipids may play a cooperative role in the inhibition of glucose-6-phosphatase occurring early after CCl4-poisoning is discussed.