Howard S. Maker

Regional Distribution of Catalase in the Adult Rat Brain

Catalase activity was measured in 11 areas of perfused adult rat brain. The hypothalamus and substantia nigra contained the highest activities. The corpus callosum. a white‐matter structure, contained intermediate activity. The caudate‐putamen and frontal cortex contained the lowest activities. Regional catalase bears some relationship to the reported distribution of microperoxisomes, but considerable activity is present in areas with few microperoxisomes. Catalase may function as one of the systems detoxifying H2O2 formed in CNS amine metabolism.

Dopamine-like action of nicotine: lack of tolerance and reverse tolerance

Rats with unilateral 6-hydroxydopamine lesions of the substantia nigra became briefly sedated and hypothermic after the acute injection of nicotine s.c. (0.4 or 0.8 mg/kg free base). When nicotine was repeated 5 days per week there was rapid tolerance for the sedation and slower tolerance for the hypothermia and the lesioned animals began to rotate ipsiversively after each injection. Stereotypic behavior was also noted. Rats injected with nicotine 5 days per week and nigrally lesioned on the 24th day rotated promptly on their first postoperative injection of nicotine. The nicotinic antagonist, mecamylamine (1.0 mg/kg i.p.), completely blocked the induced rotation. The appearance of rotation did not seem to depend on tolerance to sedation. The direction of rotation indicated enhancement of activity in the intact nigrostriatal system. However, 10 min after the acute injection of 0.8 mg/kg nicotine no change was found in the ratios of dopamine to its metabolites DOPAC and homovanillic acid in the substantia nigra, caudate-putamen, nucleus accumbens, olfactory tubercle, frontal cortex, or ventral tegmental area. Rats given 0.4 or 0.8 mg/kg nicotine 5 days per week and either lesioned prior to nicotine or lesioned during the third week rotated during the sixth week without any sign of tolerance. One day after the 30th injection in intact or lesioned rats the ratios of dopamine to its metabolites did not differ from those in saline controls on either the right or left side of any of the regions examined. There was no evidence of a change in dopamine metabolism after an acute challenge with nicotine or of a sustained change after repeated injection. The possibility remains that repeated nicotine modifies the dopaminergic response to nicotine without causing a sustained change in metabolism.

The Activity of 2′,3′-Cyclic Nucleotide 3′-Phosphodiesterase in Rat Tissues

Abstract: The activity of the myelin‐associated enzyme 2′,3′‐cyclic nucleotide 3′‐phosphodiesterase (CNP) was measured in 14 rat tissues and in subcellular fractions of rat liver by a sensitive fluorometric method, using cyclic NADP as substrate. CNP activity in brain (339 μmol/h/mg protein) was fourfold that of the sciatic nerve. The activities in tissues outside the nervous system ranged from a low of 0.42 μmol/h/mg protein in the unwashed red blood cell to a high of 9.96 in the spleen. The activity was highest in tissues containing cells with membranes capable of undergoing transformation and elaboration (spleen and thymus) and low in those in which the cell membranes are morphologically stable (muscle and red cell). The enzyme was found in all major liver subtractions, with the highest activities in the microsomal and nuclear fractions. Despite the large difference in the maximal velocities of CNP in brain and liver, the affinity of the liver enzyme for the substrate (km) was similar to that of brain enzyme. Brain CNP was stable over a 48‐h postmortem period.

Sensitive fluorometric assays for glutathione peroxidase and reductase

Abstract A microfluorometric adaptation of the method of D. E. Paglia and W. N. Valentine has been made which can assay glutathione peroxidase activity in less than 100 μg of tissue. As in the original method, the oxidized glutathione produced in the reaction is coupled to the oxidation of NADPH by glutathione reductase. No inhibition by NADPH was found. A similar method can be used to measure glutathione reductase. These methods have been used to assay glutathione peroxidase and reductase in rat brain and in a neuronal and a glial cell line using samples containing 15 μg of protein. The assays are sensitive enough to allow multiple determinations of the enzymes in brain regions, organotypic tissue cultures, and microwell cell cultures.

Amine-mediated toxicity: The effects of dopamine, norepinephrine, 5-hydroxytryptamine, 6-hydroxydopamine, ascorbate, glutathione and peroxide on the in vitro activities of creatine and adenylate kinases in the brain of the rat

The effects of several concentrations of amines and reducing agents on the activity of creatine (CK) and adenylate (AK) kinases were determined in homogenates of the brain of the rat at 0 and 37 degrees C. The order of decreasing irreversible inhibition of the enzymes was peroxide, 6-hydroxydopamine, dopamine, norepinephrine, 5-hydroxytryptamine. At 37 degrees C, approx. 50% of the activity of creatine kinase was lost in 30 min in the presence of 20 microM dopamine. 5-Hydroxytryptamine was several orders of magnitude less toxic. The action of dopamine was not prevented by inhibition of monoamine oxidase, chelation of metals or the addition of a catalase, indicating that formation of peroxide by monoamine oxidase was not the primary cause of the loss of enzyme. Although auto-oxidation of dopamine to a toxic quinone was considered, the degree of inhibition of creatine kinase was not affected when auto-oxidation was prevented under anaerobic conditions. Glutathione (GSH), present during the incubation, protected the enzymes but could not restore activity after exposure to amine. Concentrations of glutathione above 5 mM and of oxidized glutathione as low as 10 microM inhibited creatine kinase. Ascorbate protected the enzymes even when present at a concentration much less than that of the amine, but ascorbate was itself toxic. The findings indicate that dopamine, at concentrations attained after drug-induced release or ischemia, can be toxic to a metabolic enzyme present in the synaptosomal membrane.

The sensitivities of creatine and adenylate kinases to the neurotoxins acrylamide and methyl n-butyl ketone

Abstract It has recently been proposed that the similar multifocal axonopathies caused by several industrial toxins are due to the inhibition of metabolic enzymes with sensitive sulfhydryl groups. We find that the neurotoxins acrylamide and methyl n -butyl ketone in millimolar concentrations irreversibly inhibit rat brain and rabbit muscle creatine kinase and brain adenylate kinase. Brain creatine kinase was more sensitive to the toxins than was muscle creatine kinase or brain adenylate kinase. The chemically related but “nonneurotoxic” agents, N,N ′-methylene bisacrylamide and methyl isobutyl ketone, were somewhat less effective than the known toxins. Dithiothreitol protected the enzymes against the toxins. However, differences in the effects of temperature and the protection by dithiothreitol indicated complex enzyme -toxin -dithiothreitol interaction. Protection by dithiothreitol does not necessarily implicate enzyme sulfhydryl groups or the site of toxin action. The lack of of cificity of toxin action and the concentrations of toxin required make it unlikely that metabolic enzyme inhibition alone accounts for hydrocarbon axonopathy.