Change in the distribution of acetylcholinesterase molecular forms in frontoparietal cortex of the rat following nucleus basalis lesions with kainic acid.

The unilateral injection of kainic acid into the nucleus basalis magnocellularis (NBM) resulted in an alteration of the distribution of acetylcholinesterase (AChE) molecular forms in frontoparietal cortex ipsilaterally to the lesion. The G4/G1 ratio fell from 5.4 +/- 0.8 in contralateral to 3.0 +/- 0.5 in ipsilateral cortex. The NBM lesion effect thus, mimicks, the loss of tetrameric G4 form reported for various brain cortical areas of Alzheimer's disease (AD) patients. The data support the suggestion that G4 form is enriched in presynaptic nerve terminals.

In vitro and in vivo mutagenicity studies with airborne particulate extracts.

The contribution of nitro compounds to airborne particulate mutagenicity was studied with Salmonella typhimurium strains TA98, TA98NR, TA98/1,8DNP6. The results obtained indicate that nitropyrenes play a minor role in air particulate mutagenicity. Seasonal variations indicate a relatively greater contribution of nitro compounds to the mutagenicity of spring and summer samples. Fractionation of extracts into acidic, neutral and basic components shows that neutral compounds account for about two-thirds of the total mutagenic activity. Attempts to extract mutagens adsorbed onto particulate matter with aqueous media were almost completely negative. No significant mutagenicity was detected in urine and faecal extracts and in plasma samples of Sprague-Dawley rats treated with air particulate extracts at 80 mg/kg either per os or by i.p. injection. Negative results were obtained in the micronucleus test with Swiss mice treated at 200 and 400 mg/kg (twice by i.p. injection). A significant decrease in liver aminopyrine-N-demethylase was observed in Swiss mice injected with air particulate extracts or its basic and neutral fractions. In vitro experiments suggest a direct interaction of test materials with microsomal cytochrome P-450.

Influence of urethane and ketamine on rat hepatic cytochrome P450 in vivo.

The aim of this study was to identify the effects of widely used laboratory anaesthetics on cytochrome (CYP) activity in male Sprague Dawley rats in vivo. The anaesthetics used were urethane and ketamine. 7-Ethoxyresorufin (EROD), 7-pentoxyresorufin (PROD), aniline and ethylmorphine were used as substrates for CYP 1A, CYP 2B, CYP 2E1 and CYP 3A, respectively. Urethane increased EROD (CYP 1A) activity by 40 % (p < 0.01), and hydroxylation of aniline (CYP 2E1) by 14 % in the early phase of anaesthesia and by 60 % (p < 0.01) in the later one. Urethane also reduced the demethylation of ethylmorphine by 37 % (p < 0.01), but did not affect CYP 2B activity significantly. Ketamine did not significantly affect CYP 1A, 2B or 2E1. However, it reduced the demethylation of ethylmorphine (i.e. CYP 3A) by 32 % (p < 0.01). From these data, we concluded that a single dose of urethane inhibits CYP 3A but increases CYP 2E1 and CYP 1A, and that a single dose of ketamine inhibits the activity of CYP 3A.

Synthesis and cholinesterase inhibitory activity of 6-, 7-methoxy-(and hydroxy-) tacrine derivatives.

Some 6- and 7-methoxy-(and hydroxy-) tacrine derivatives were synthesized and evaluated for their in vitro acetylcholinesterase (AChE) and butyrylcholinesterase (BuChE) inhibitory activities. The most potent analogue in our series was the 9-heptylamino-6-methoxytacrine 3af which, in comparison with tacrine (THA), displayed an almost identical inhibitory effect, slightly lower acute toxicity and higher selectivity profile towards AchE when compared with THA.

Metabotropic glutamate receptors and brain ischemia: differential effects on phospoinositide turnover in young and aged rats.

Neuronal damage and phosphoinositide hydrolysis stimulated by neurotransmitter receptor agonists in cerebral cortex of 3- and 24-month Fischer 344 rats, following an episode of brain ischemia, were compared. Transient global ischemia was induced by occlusion of common carotid arteries for 15 minutes in conditions of moderate hypoxia. Seven days after, histological examination of cerebral cortex showed cell loss, neurons with nuclear pyknosis, cytoplasmatic degeneration, and glial proliferation. Ischemic lesions appeared moderate to severe in young rats and intermediate in all the aged animals. In young rats inositol phosphates accumulation stimulated by excitatory amino acids (ACPD, ibotenate and quisqualate), but not by norepinephrine or carbachol, was enhanced significantly with respect to sham-operated animals. No potentiation at all was observed in aged rats. This finding suggests that the events leading to the increased metabotropic response in the post-ischemia period is impaired by the ageing process.

Induction of hepatic microsomal mixed function oxidase system by ethylenethiourea in mice.

Oral administration to mice of ethylenethiourea (ETU) at single doses from 50 to 600 mg/kg caused a dose-dependent increase (up to 200 mg/kg) in hepatic microsomal aniline hydroxylase (AH) without affecting aminopyrine-N-demethylase activity or total microsomal cytochrome P-450 content. Maximal (2.4 fold) enzyme increase was observed 24 h after treatment by an ETU dose of 200 mg/kg and was followed by a return to control levels within 4 days. Pretreatment of mice with actinomycin D completely prevented the increase of AH, while the addition in vitro to the incubation mixture of ETU at concentrations up to 1 mM did not cause any changes of enzymatic activity. These data speak in favour of an induction of AH via de novo protein biosynthesis. A more detailed analysis of the phenomenon indicated that the stimulatory effect of ETU on AH did not add to that produced by beta-naphthoflavone, but added to that due to phenobarbital. Therefore ETU and beta-naphthoflavone appear to affect similar mechanisms inducing aniline hydroxylase activity.

Different effects of harmine on plasma concentrations of L-dopa and on cerebral dopamine metabolism in rabbits and rats.

The effects of short-term intravenous administration of harmine, a monoamine oxidase inhibitor, on the plasma concentrations of L-Dopa and on dopamine levels in the brain striata of rats and rabbits after L-Dopa administration were studied. Harmine affects the L-Dopa plasma concentrations in rabbits but not in rats: in fact in the former species the area under the concentration-time curve observed after administration of L-Dopa alone increased significantly when animals were pretreated with harmine. Dopamine striatal levels increased in concert with plasma L-Dopa concentrations after administration of L-Dopa in rats and rabbits. However pretreatment with harmine resulted in a significant increase of dopamine levels in the brain striata of rabbits only. These results suggest that harmine or one of its metabolites affect the brain dopamine system not merely as a type A monoamine oxidase inhibitor but with a modulatory effect, without a precise indication of site or mode of action of harmine.

Simultaneous high-performance liquid chromatographic analysis of buspirone and its metabolite 1-(2-pyrimidinyl)-piperazine in plasma using electrochemical detection.

A selective and sensitive high-performance liquid chromatographic method with coulometric detection is described for the quantitation of buspirone and its active metabolite, 1-(2-pyrimidinyl)piperazine, in plasma samples of mice treated orally with buspirone (10 mg/kg body weight). The analytes are extracted with a carboxylic acid solid-phase extraction column before chromatography. A dual-electrode electrochemical detector is used. The limit of detection is 50 pg for buspirone and 35 pg for 1-(2-pyrimidinyl)piperazine.

Age-related changes in acetylcholinesterase and its molecular forms in various brain areas of rats.

A previous study conducted in this laboratory revealed a decrease in total cholinesterase (total ChE) in the cerebral cortex, hippocampus and striatum in aged rats (24 months) of various strains, as compared with young animals (3 months). The purpose of the present experiments was to extend the study to other brain areas (hypothalamus, medulla-pons and cerebellum) and to assess whether this decrease was dependent on the reduction of either specific acetylcholinesterase (AChE) or butyrylcholinesterase (BuChE) or both. By using ultracentrifugation on a sucrose gradient, the molecular forms of AChE were evaluated in all the brain areas of young and aged Sprague-Dawley rats. In young rats the regional distribution of total ChE and AChE varied considerably with respect to BuChE. The age-related loss of total ChE was seen in all areas. Although there was a reduction of AChE and, to somewhat lesser extent, of BuChE in the cerebral cortex, hippocampus, striatum, and hypothalamus (but not in the medulla-pons or the cerebellum), the ratio AChE/BuChE was not substantially modified by age. Two molecular forms of AChE, namely G4 (globular tetrameric) and G1 (monomeric), were detected in all the brain areas. Their distribution, expressed as G4/G1 ratio, varied in young rats from about 7.5 for the striatum to about 2.0 for the medulla-pons and cerebellum. The age-related changes consisted in a significant and selective loss of the enzymatic activity of G4 forms in the cerebral cortex, hippocampus, striatum, and hypothalamus, which resulted in a significant decrease of the G4/G1 ratio. No such changes were found in the medulla-pons or the cerebellum.(ABSTRACT TRUNCATED AT 250 WORDS)

Development factors affecting brain acetylcholinesterase inhibition and recovery in DFP-treated rats.

The effect of a single dose of diisopropyl fluorophosphate (DFP; Isoflurophate, 1.1 mg/kg s.c.) administered to rats during pregnancy was evaluated by measuring postpartum maternal and newborn brain-soluble and total acetylcholinesterases (AChE) and their molecular forms at intervals of 1, 2, 3, 4 and 10 days between treatment and sacrifice. Subsequently, the effects of DFP were studied in 18-day-pregnant rats, fetuses and placentae at 90 min and 24 h after treatment. The inhibition of postpartum maternal enzymatic activity did not differ from that previously found in adult males, while inhibition was considerably less pronounced in newborns at all time intervals, with a nearly complete recovery already at 48 h after treatment. An even faster recovery of brain enzyme was observed in 18-day fetuses from DFP-treated mothers (24-hour interval between treatment and sacrifice). In this experiment, a comparable inhibition was observed at 90 min after treatment in the adult and the developing brain, excluding a major influence of disposition factors in the differential recovery phenomena. An experiment on weanling rats yielded intermediate results between those of newborn and those of adult animals. Finally, most data confirmed previous findings that the soluble portion of brain AChE and medium molecular weight enzyme forms may have special significance in the initial phases of recovery.

Interactions between anticholinesterase agents and neuroleptics in terms of cholinesterase inhibition in brain and other tissues of rats.

Treatment of rats with chlorpromazine (CPZ, 15 mg/kg i.p. 60 min before sacrifice) did not modify cholinesterase (ChE) activity, but considerably enhanced the inhibition of total ChE induced by physostigmine (PhS, 0.5 mg/kg i.p. 40 min after CPZ) in brain, skeletal muscle, myocardium, lung, liver, and kidney. Additional experiments also showed a prolongation of PhS inhibition by CPZ in brain. The enhanced inhibition of total ChE due to CPZ depended in most peripheral organs on the effect on pseudoChE (as measured by a spectrophotometric method), except in the case of skeletal muscle in which potentiation of PhS effect was observed on true acetylcholinesterase (AcChE). The results indicate that the potentiation by CPZ of PhS inhibition occurs in all organs tested and is relatively non specific. CPZ was found to potentiate slightly the effects of Mevinphos but did not interact with Carbaryl, Diazinon or Azinphos. Furthermore, haloperidol did not potentiate the effects of physostigmine.

Mechanisms of recovery of brain acetylcholinesterase in rats during chronic intoxication by isoflurophate.

During chronic intoxication by diisopropyl fluorophosphate (Isoflurophate, DFP; s.c. treatment on alternate days - first dose of 1.1 mg/kg, subsequent doses of 0.7 mg/kg each until the 23rd day) a partial recovery of enzymatic activity was found at 24 h after each DFP administration. Relative to maximal AChE depression at 90 min, these rises were more pronounced in the soluble portion of the enzyme than in total enzyme preparation, i.e., that containing mainly membrane-bound AChE. Moreover, from the 2nd DFP administration on, there was a persistent increase of medium-molecular-weight forms both in soluble and in total AChE. The results suggest an important role of the soluble portion of AChE and of medium forms in the process of recovery of enzymatic activity.

Molecular forms of rat brain acetylcholinesterase in DFP intoxication and subsequent recovery.

The effects of acute administration of diisopropyl fluorophosphate, Isofluorophate (DFP) 1.1 mg/kg SC on soluble brain acetylcholinesterase were studied in male Sprague-Dawley rats sacrificed at time intervals ranging from 3 hr to 25 days. Three main molecular forms of AChE were separated by polyacrylamide gel electrophoresis followed by enzymatic reaction with acetylthiocholine, staining and scanning densitometry for their quantitative evaluation. In some experiments the same three forms were separated by chromatography-gel filtration. In the brain of untreated animals the slowly-, medium- and fast-migrating forms accounted respectively for 64, 18 and 18% of the soluble AChE activity. At 3 hr after treatment with DFP, resulting in an 80% reduction of soluble AChE, the relative contribution of slowly-migrating forms to the residual enzymatic activity was decreased, while that of medium-forms was significantly increased. These changes became gradually more pronounced and reached their maximum at 4 days, when AChE had recovered to about 50% of control level. Subsequently, the distribution of the molecular forms showed a progressive return toward the control pattern. The partial recovery in the initial period after maximal enzyme depression was mainly due to an increase of medium-migrating forms. Thus these may be precursors of the biosynthesis of slowly-migrating forms and/or there may be functional specialization of different forms.

Alterations in the distribution of cholinesterase molecular forms in maternal and fetal brain following diisopropyl fluorophosphate treatment of pregnant rats.

Previous work in this laboratory showed that during intoxication of rats with diisopropyl fluorophosphate at day 20 of pregnancy the recovery of ChE activity was faster in fetal than in maternal brain. In the present study the differences between recovery rats in dam and fetus brain were evaluated in terms of molecular forms and spontaneous reactivation. Using ultracentrifugation on sucrose gradient two molecular forms of ChE, namely 10S (tetrameric globular G4 form) and 4S (monomeric G1 form) were detected both in maternal and fetal brain of untreated rats. The ratios 10S/4S were about 5.0 and 0.75 for dams and 20-day fetuses, respectively. DFP administration (1.1 mg/kg sc) inducing at 90 min an about 80% inhibition of ChE in maternal brain caused a shift in its 10S/4S ratio to 1.63, and to 0.53 in fetal brain (in which overall inhibition was about 70%). This means that 10S forms were preferentially inhibited by DFP both in maternal and fetal brain. After 24 and 48 hr there was a negligible recovery of overall ChE in maternal brain with no shift in the ratio. On the other hand, complete recovery of ChE in fetal brain within 48 hr was accompanied by almost total normalization of the 10S/4S ratio. Rapid recovery of fetal ChE appeared not to depend on hydrolysis of DFP-inhibited ChE. In fact, maternal and fetal DFP-inhibited enzyme preparations following the addition of oximes (pralidoxime or obidoxime) in vitro showed similar rates of reactivation. The overall data indicate considerable differences in recovery rate of molecular forms between dams and fetuses, but not in reactivation by dephosphorylation.

Behaviorally augmented tolerance during chronic cholinesterase reduction by paraoxon.

Repeated injection of paraoxon to pretrained rats 2 hr before avoidance sessions, at a dose causing considerable intoxication symptoms and reduction of brain acetylcholinesterase (0.125 mg/kg SC daily), induced marked performance depression followed by progressive development of tolerance. Additional groups treated either after each session (i.e., 23.5 hr before each subsequent session), or treated and not tested, showed a substantial depression when shifted to treatment 2 hr before sessions after achievement of tolerance by the animals tested from the beginning of the experiment at the time of maximal paraoxon effect. This indicates that chronic paraoxon tolerance cannot be ascribed entirely to metabolic and/or physiological changes occurring as a consequence of repeated treatment per se, but must be explained at least in part by postulating a behaviorally augmented (or "learned") component. In an additional experiment chronic paraoxon animals (0.1 mg/kg SC daily) were indistinguishable from control rats with respect to acquisition of light/go, noise-light/no go discrimination, i.e., of an active-passive avoidance task known to be highly sensitive to the disrupting (response-disinhibiting) effect of antimuscarinics. Therefore, the enhanced sensitivity to antimuscarinics in organophosphate tolerant rats, which is usually ascribed to cholinergic receptor changes, does not appear to be associated with a spontaneous "antimuscarinic-like" syndrome.