Ethanol enhancement of the motor-stimulating effect of nicotine in the rat.

Although ethanol stimulates locomotion in mice, it has been difficult to demonstrate such an action in rats. In contrast, nicotine has been shown to enhance locomotion, including ipsiversive rotation in nigral-lesioned rats. We found no significant effect of ethanol alone on rat rotation at doses of 0.125, 0.50, 1.0, and 2.0 g/kg, IP, during a 30-min observation period. However, there was a dose-dependent effect of ethanol enhancing the rotation induced by nicotine (0.4 mg/kg, SC) given 30 min after the ethanol. The interaction of ethanol and nicotine on locomotion most likely involves the release of dopamine and may be related to the motor abnormalities sometimes seen clinically.

Action of nicotine on accumbens dopamine and attenuation with repeated administration.

The behavioral and physiological effects of repeated nicotine administration are complex; sedation and hypothermia are present early but become attenuated while locomotor activity increases. Maximal blood levels and behavioral changes occur within 10 min of s.c. injection. We examined the effects of 10 nicotine injections (0.8 mg/kg) in 14 days on the levels of brain amines following challenge with either saline or nicotine on the 15th day. Dopamine, DOPAC, HVA, 3-methoxytyramine, norepinephrine, 5-hydroxytyramine, and 5-HIAA were measured in the frontal cortex, olfactory tubercle, nucleus accumbens, caudate-putamen, substantia nigra and ventral tegmental area. Ten minutes after nicotine was given to rats that had previously received only saline the levels of dopamine and its metabolite DOPAC indicated an increase in dopamine turnover in the nucleus accumbens. Of the areas examined the accumbens was the most sensitive to nicotine, with few significant amine changes in other regions. Twenty-four hours after the last nicotine injection the levels of dopamine and its metabolites indicated a sustained decrease in dopamine turnover in the accumbens induced by repeated administration. Following repeated nicotine a nicotine challenge still induced an acute increase in dopamine turnover in the accumbens, but the response was less than in animals not previously given nicotine. The results confirm earlier studies indicating that the accumbens is a major site of nicotine action.

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.

Chronic nicotine administration increases binding of [3H]domperidone in rat nucleus accumbens.

An apparent inverse relationship between smoking and Parkinson's disease prompted an investigation of the effect of chronic nicotine administration on dopaminergic and serotonergic receptors in rat brain. Nicotine, 0.8 mg/kg, was injected once daily, five times per week, for 6 weeks. In nucleus accumbens the Kd for [3H]domperidone was increased 2-4-fold, and the Bmax was increased 1.5-2-fold. No changes were observed in the binding of [3H]domperidone in caudate-putamen or in that of [3H]ketanserin in frontal cortex. It is concluded that chronic nicotine administration may have a suppressant effect on central nervous system release of dopamine that in pre-parkinsonian persons causes an aversion to the effects of smoking.

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 influence of systemic factors on acrylamide-induced changes in brain, nerve, and other tissues.

In order to define the locus of acrylamide neurotoxicity, the effects of chronic intoxication (total dose 500 mg/kg) on cholinergic synthesis and transport, the Schwann cell-myelin complex, lysosomal activity, and several metabolic pathways were determined in rat sciatic nerve, spinal cord, and brain. No changes were found in hematological measures or in the levels of clinically important blood enzymes, indicating no major damage to other organs. The activities of choline acetyltransferase (ChAT), 2',3'-cyclic nucleotide phosphohydrolase, beta-glucuronidase, and lactate dehydrogenase were unaffected in acrylamide paralyzed animals, but creatine kinase (CK) decreased in sciatic nerve, muscle, and brain, particularly in animals dying of the intoxication. CK blood and the CK isoenzyme patterns in blood were unchanged. The synthesis of protein in brain and spinal cord (measured in vivo) were decreased in rats exposed to high-dose acrylamide. However, in brain and cord, CK decreased only after animals became systemically ill and suffered weight loss, with the lowest activities in those animals sick enough to die. The degree of stress to which the animals had been subjected was indicated by enlargement of the adrenal glands and decreased sulfolipid synthesis in the adrenals. Rats exposed to 25 mg/kg/day acrylamide to a total dose of 250 mg/kg developed leg weakness but not paralysis or weight loss and had a 25% decrease in CK only in the distal sciatic nerve. Because of the apparently stress-related or agonal loss of CK, no specific effect of acrylamide on the enzyme could be definitely demonstrated. Neither could the changes in protein synthesis be attributed solely to a direct effect of the toxin. These results illustrate the difficulties encountered in interpreting intoxication studies that produce systemic illness and support the suggestion that CK activity may be a useful marker of the severity and duration of the agonal state in studies of postmortem human brain.

The effects of BCNU (1,3-bis(2-chloroethyl)-1-nitrosourea) and CCNU (1-(2-chloroethyl)-3-cyclohexyl-1-nitrosourea) on glutathione reductase and other enzymes in mouse tissue.

BCNU has been reported to cause a rapid, irreversible inhibition of human erythrocyte glutathione reductase (GR) at chemotherapeutic dosage, without affecting metabolic enzymes. This inhibition may mediate some of the therapeutic and toxic effects of BCNU. Thiol containing agents such as reduced glutathione can protect cells against BCNU and a change in glutathione concentrations could modify BCNU effectiveness. At doses of 50 mg/kg (LD-50) and 100 mg/kg, i.p., BCNU decreased the activity of GR in mouse kidney, liver, brain, and heart with a greater loss in those animals which died from drug administration. GR activity tended to recover but still remained below control at 96 hours. Erythrocyte GR activity was reduced only at the higher BCNU dose. CCNU (100 mg/kg, i.p.) did not affect GR activity. BCNU also decreased creatine kinase, malate dehydrogenase, and lactate dehydrogenase activities. The inhibition of GR in vitro occurred only after biochemical reduction of the enzyme with NADPH. The oxidation state of GR may determine its sensitivity to BCNU in the human erythrocyte but we were unable to demonstrate an unusually high sensitivity or a specific effect of BCNU on GR in mouse tissues.

Enzyme changes in axon, myelin, and Schwann cells in injured sciatic nerve.

Four enzymes related to specific cell functions were assayed in rat sciatic nerve injury by crush (cr) or crush and ligation (cr-lig) after 2, 7, and 15 days in situ. Enzyme activities in segments of sciatic nerve proximal and distal to the injury were compared to those in corresponding segments of the contralateral nerve. Choline acetyltransferase (CAT) activity in the distal portion decreased by 65% for cr and almost to zero for cr-lig by day 7, while in the proximal portions CAT decreased to 70% of control values by 7 days and to 50% at 15 days after cr-lig. The activity of the Schwann cell-myelin-associated enzyme 2',3'-cyclic nucleotide phosphohydrolase (CNP) decreased slowly distal to the injury. Distal to both types of injury the lysosomal enzyme beta-glucuronidase (GLR) increased six- to eightfold by 15 days. Proximal to injury GLR also increased (P cr X 2.5, P cr-lig X 5) but the peak proximally was attained by day 7. Despite interruption of axonally transported enzymes, the activities of the metabolic enzyme creatine kinase (CK) increased distal to injury apparently reflecting changes in the functions of the Schwann cells. The loss of metabolic enzymes from the axonal compartment may be completely obscured by reciprocal changes in the non-neuronal compartments if the activity is present in both compartments.

Norepinephrine stimulation of pentose cycle in rat brain prisms.

Using the C1/C6 14CO2 ratio as a relative measure of pentose shunt metabolism, rat brain prisms were incubated with (1-14C) or (6-14C) glucose and respired 14CO2 collected. Shunt metabolism was stimulated with 10-4M but not 10-5M norepinephrine. MAO inhibitors and reserpine blocked norepinephrine stimulation but uptake inhibition did not. These data demonstrate that, under the tested conditions, MAO mediated norepinephrine metabolism does stimulate shunt activity but not at physiological concentrations.

Postmortem stability of enzymes detoxifying peroxide in brain.

Glutathione peroxidase, glutathione reductase, and catalase activities were measured to 48 h after death in mouse brains held at temperatures replicating the cooling occurring in human cadaver brain. Glutathione peroxidase was stable for 48 h; catalase was stable for 24 h and then declined 20% in activity. Glutathione reductase was stable for 4 h and then decreased to 55% of its initial activity by 48 h. Perfusion of mouse brain with 0.9% (wt/vol) NaCl did not decrease enzyme activities, indicating that erythrocyte contamination has little effect on measured brain activities. The results suggest that glutathione peroxidase would not be affected by moderate time delays in obtaining human postmortem brains but catalase activity may be affected if brains are not promptly removed. Glutathione reductase is not stable and measurements would require controls carefully matched for postmortem conditions.

2', 3'-cyclic nucleotide 3'-phosphodiesterase in cerebrospinal fluid.

The activity of the myelin-associated enzyme 2',3'-cyclic nucleotide 3'-phosphodiesterase (CNP) was assayed in the cerebrospinal fluid (CSF) of 107 neurologic patients by a new and sensitive fluorometric method. The activity of CNP was about 20 nmol per hour per milligram protein or 12 nmol per hour per milliliter CSF. At these extremely low levels, the presence of even a small amount of blood (which has slightly greater activity) significantly elevated CNP values. Patients with radicular syndromes had slightly higher than average CNP activities, but there was no difference in enzyme activities of 47 patients with multiple sclerosis and the general neurologic population. CNP activity was not related to stage of demyelinating illness or intrathecal injection of steroid. CNP-like myelin basic protein may be released into the CSF after destruction of myelin, but our results suggest that the enzyme activity is lost in the process.

Regional activities of metabolic enzymes and glutamate decarboxylase in human brain.

Interpretation of biochemical measurements in the human brain after death is complicated by a variety of premortem, perimortem, and postmortem factors. The activity of glutamic acid decarboxylase (GAD) in particular has been found to vary considerably among human brains. In contrast to neurotransmitter-associated enzymes, metabolic enzymes are present in all brain cells and should not be specifically lost by patterned neuronal cell loss such as that which occurs in Parkinson disease. We compared the activity of GAD to that of the metabolic enzymes creatine kinase (CK), adenylate kinase, hexokinase, beta-glucuronidase, and malate, lactate, glucose-6-phosphate, and isocitrate dehydrogenases in 24 regions of six human brains. Of the metabolic enzymes, only CK showed a 5-fold variation approaching that of GAD. Like GAD, CK activity was stable postmortem, but its activity was apparently inversely related to the severity and duration of the preterminal illness. CK may be a useful marker of agonal deterioration.

The activity of 2',3'-cyclic nucleotide 3'-phosphodiesterase in rat tissues.

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 mumol/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 mumol/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 subfractions, 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.

Coupling of dopamine oxidation (monoamine oxidase activity) to glutathione oxidation via the generation of hydrogen peroxide in rat brain homogenates.

Homogenates of perfused rat brain generated oxidized glutathione from reduced glutathione during incubation with dopamine or serotonin. This activity was blocked by pargyline, a monoamine oxidase inhibitor, or by catalase, a scavenger of hydrogen peroxide. These results demonstrate formation of hydrogen peroxide by monoamine oxidase and the coupling of the peroxide to glutathione peroxidase activity. Oxidized glutathione was measured fluorometrically via the oxidation of NADPH by glutathione reductase. In the absence of added dopamine or serotonin, a much smaller amount of reduced glutathione was oxidized; this activity was blocked by catalase, but not by pargyline. Therefore, endogenous production of hydrogen peroxide, not linked to monoamine oxidase activity, was present. These results indicate that glutathione peroxidase (linked to hexose monophosphate shunt activity) can function to eliminate hydrogen peroxide generated by monoamine oxidase and other endogenous sources in aminergic neurons.

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.

Developmental study of rat brain glutathione peroxidase and glutathione reductase.

Glutathione peroxidase and glutathione reductase activities were measured in whole rat brains at selected ages from birth to adulthood. On a wet weight basis glutathione peroxidase activity increased 70% during development and glutathione reductase activity increased 160%. On a protein basis glutathione peroxidase declined slightly in activity during the first two weeks of life and then maintained the 14-day activity into adulthood while glutathione reductase showed a 30% increase in activity. While less than the developmental changes in many enzyme involved in aerobic glycolysis or catecholamine metabolism, these increases do suggest a role in CNS metabolism.

Regional distribution of glutathione reductase in the adult rat brain.

Glutathione reductase was measured in 10 selected areas of the adult rat brain. Activitives varied over a 2.5-fold range. The caudate-putamen and cortical areas contained the highest activities. Several gray matter structures contained intermediate activities. The substantia nigra and the corpus callosum, a white matter structure, contained the lowest activities. The caudate-putamen, a catecholamine rich area, may produce H2O2 via MAO and would require glutathione reductase to recycle glutathione oxidized by glutathione peroxidase. However, the distribution of glutathione reductase activity also suggests a role in reducing oxidants formed during tissue respiration.

Regional distribution of glutathione peroxidase in the adult rat brain.

Glutathione peroxidase activity was measured in 10 areas of perfused adult rat brain with the use of a fluorometric assay coupled to NADPH oxidation. The caudate-putamen and the substantia nigra had the highest activities. Cortical areas and several nuclear areas had somewhat lower activity. Activity was lowest in a white matter structure (corpus callosum). High activity of glutathione peroxidase may be related to the need to reduce hydrogen peroxide arising in the course of monoamine metabolism.