Carbonyl cyanide phenylhydrazones as probes of the anionic activator site of the human erythrocyte glutathione adduct transport ATPase.

We have previously shown that the ATPase activity associated with the erythrocyte glutathione adduct transporter is also stimulated by 2,4-dinitrophenol and p-trifluoromethoxy carbonylcyanide phenylhydrazone, both well-known anionic and lipophilic uncouplers of oxidative phosphorylation by mitochondria [C. G. Winter, D. C. DeLuca, and H. Szumilo (1994) Arch. Biochem. Biophys. 314, 17-22]. In this paper, we report the testing of a series of ring-substituted carbonylcyanide phenylhydrazones as activators of the ATPase. All of the compounds tested stimulated the ATPase to similar extents, based on Vmax values. The K0.5 for stimulation of the ATPase depended on the electron-withdrawing characteristics of the ring substituents, resulting in a Hammett linear free energy relationship for the m- and p-substituted derivatives. The slope of this relationship, with lower K0.5 values for electron-withdrawing substituents, suggests that an anionic residue in the active site partially discourages binding of this class of activators. ortho-Substituted carbonylcyanide phenylhydrazones do not follow this relationship, but show lower apparent affinities than expected from their pKa values. This finding suggests that steric effects in that region of the binding site negatively influence the affinity.

Inactivation of an animal and a fungal catalase by hydrogen peroxide.

We have quantitatively compared the rates of peroxide-dependent inactivation of bovine liver catalase and Aspergillus niger catalase as class representatives of catalases that contain tightly bound NADPH and those that do not. Inactivation of these catalases in the presence of ethanol has also been quantitated in an effort to assess the importance of compound II, an inactive form of bovine liver catalase, in the inactivation reaction. The values of k2, the second-order rate constant for inactivation calculated for the bovine enzyme, in the absence and presence of ethanol, respectively, were 8.9 +/- 0.26 and 8.5 +/- 0.27 M-1 min-1. In contrast, the values for the A. niger enzyme were 0.51 +/- 0.069 and 10.5 +/- 0.32 M-1 min-1. The A. niger enzyme is more stable toward hydrogen peroxide-induced inactivation than the liver enzyme. The A. niger enzyme is markedly destabilized by 20 mM ethanol, whereas the inactivation of the liver enzyme is unaffected by ethanol. Reaction of bovine liver catalase with ethyl hydroperoxide produced the characteristic absorption spectrum of compound I and in the absence of ethanol the spectrum associated with compound II. In contrast, the fungal enzyme developed compound I spectrum but spectral changes that might be ascribed to compound II were barely detected in the Soret region. Spectral changes for A. niger catalase in the visible region were modified by the presence of ethanol but could not be clearly correlated with the bovine catalase compound II spectra either in the presence or absence of ethanol. The stability of the fungal and bovine catalases in the presence of hydrogen peroxide is quantitatively documented. The enzymes are also shown to be different in their response to ethanol and in the formation of compound II-like species with ethyl hydroperoxide. It appears unlikely that compound II is an intermediate in the hydrogen peroxide-mediated inactivation reaction of either catalase under catalatic assay conditions.

2,4-Dinitrophenol and carbonylcyanide p-trifluoromethoxyphenylhydrazone activate the glutathione S-conjugate transport ATPase of human erythrocyte membranes.

2,4-Dinitrophenol (DNPOH) and carbonylcyanide p-trifluoromethoxyphenylhydrazone (FCCP), two classical uncouplers of mitochondrial oxidative phosphorylation, were found to stimulate human erythrocyte membrane vesicle ATPase activity. Both compounds competed with S-(2,4-dinitrophenyl)glutathione (DNPSG) for activation of the glutathione S-conjugate transport ATPase. Stimulation of the ATPase by DNPOH or FCCP occurred with Vmax values 4-6 times greater than that with DNPSG. The K0.5 for DNPOH (195 microM) was similar to that of DNPSG (196 microM), while that for FCCP (4.3 microM) was 40 times lower. Vanadate inhibits both the DNPOH- and FCCP-stimulated ATPase activities, as previously reported for the glutathione S-conjugate ATPase. The stimulation of erythrocyte vesicle ATPase activities by these classical uncoupling agents does not result from increased proton conductance across the vesicle membrane: monensin, gramicidin and nystatin, all of which increase proton conductance, but by different mechanisms, do not stimulate erythrocyte vesicle ATPase activity. Verapamil, a known P-glycoprotein ATPase activator also does not stimulate human erythrocyte membrane ATPase activity. These results show that relatively small, monoanionic lipophilic compounds such as DNPOH and FCCP can activate the glutathione S-conjugate transport ATPase. The higher Vmax values for activation by these agents than by DNPSG make possible a more sensitive assay of this transport ATPase activity. The results raise the question of whether these substances and other small anionic, lipophilic compounds are also transported by this system.

Identification of the glutathione conjugate of 4-nitroquinoline 1-oxide formed in the reaction catalyzed by murine glutathione transferases.

The product of the enzyme-catalyzed conjugation of glutathione and 4-nitroquinoline 1-oxide was isolated and its structure determined by MS and NMR. The results indicate that the cysteine sulfur of glutathione replaces the nitro group of 4-nitroquinoline 1-oxide in the reaction with the formation of 4-(glutathion-S-yl)-quinoline 1-oxide. No evidence was found for the binding of glutathione to any other position of 4-nitroquinoline 1-oxide or through any group other than the cysteine sulfur.

Effects of naloxone and neuroleptic drugs on muscle rigidity and heart rate of the nervous Pointer dog.

Muscle rigidity, a persistent physiological characteristic of the nervous Pointer dog, is not only attenuated by the major neuroleptic drugs, haloperidol and pimozide but also by naloxone. In addition, naloxone administration results in a modest but significant increase in heart rate of these subjects. The data obtained in this study add support for the concept that an abnormality in central nervous system dopaminergic function is involved in the genesis of abnormal behavior of this animal model.

Assessment of pointer dog behavior. Drug effects and neurochemical correlates.

Studies of behavioral traits of the "Arkansas Line of nervous pointer dogs" are well documented (Murphree et al. 1969). Demonstrations of drug effects on these traits, as measured on the standardized behavior test and expressed as "Morbidity Score," have been disappointing, despite other evidence of effectiveness of drug treatments such as modification of operant performance (Murphree et al. 1974c). A simple reproducible test with high interrater reliability is presented here. Subjects were nine nervous dogs that received placebo, pimozide, and chlordiazepoxide in a double-blind crossover design. Both drugs significantly attenuated "human avoidance," a nervous trait. Placebo had no effect. The chlordiazepoxide effect was maximal on the first day of administration, returning to baseline three to four days after the last dose. The pimozide maximal effect was on day 4, with return to baseline six to nine days after the last dose. In addition to the aberrant behavior of the nervous pointer, hyperresponsiveness to dopaminergic stimulation and increased levels of L-DOPA in the cisternal spinal fluid were observed.

Probenecid-induced accumulation of cyclic nucleotides, 5-hydroxyindoleacetic acid, and homovanillic acid in cisternal spinal fluid of genetically nervous dogs.

These studies have been conducted on 40 dogs, twenty each of a genetically nervous strain and of a normal strain of short-haired pointers. The nervous strain after about age 3 months displays extreme hypervigilance, timidity, human avoidance, and often shows catatonic-like muscle rigidity when in the presence of humans or novel stimuli. Measurements of probenecid-induced accumulation of acid metabolites in cisternal cerebrospinal fluid (CSF) have been carried out. Among the compounds measured at from 1.5 hr to 6.0 hr after probenecid treatment, homovanillic acid (HVA) was similar for the two strains, 5-hydroxy-indoleacetic acid (5-HIAA) was lower, but cyclic adenosine-3',5'-monophosphate (cAMP) and cyclic guanosine-3',5'-monophosphate (cGMP) were higher for the nervous strain when compared with age- and sex-matched behaviorally normal dogs. Probenecid levels in CSF were similar at all points in time from 1.5 to 6.0 hr after its intravenous administration in a dose of 50 mg/kg body weight. These findings coupled with previously observed differences in the two strains suggest that hyperresponsiveness of the central nervous system (CNS) noradrenergic and cholinergic systems and a hyporesponsiveness of the serotoninergic system are related to the genetically expressed aberrant behavior.

The effects of chlordiazepoxide, amphetamine and cocaine on bar-press behavior in normal and genetically nervous dogs.

Studies on two strains of pointer dogs have demonstrated that administration of a benzodiazepine (chlordiazepoxide) facilitates acquisition of goal-directed behavior in "genetically nervous" subjects. Continued admistration of the drug is required to maintain barpress response in this strain of dogs. The concomitant administration of either cocaine or amphetamine, compounds which inhibit neuronal reuptake of norepinephrine, disrupts the behavioral response of the genetically nervous E-strain subjects to a far greater extent than the stable A-strain subjects. It is also shown that after 14 days of daily administration of chlordiazepoxide, withdrawal of the drug not only re-results in almost complete loss of bar-press response in the E-strain subjects but also results in a temporary decrease in the acquired behavioral response of the stable A-strain subjects.