Alanine reverses the inhibitory effect of phenylalanine on acetylcholinesterase activity

The aim of this work was to evaluate, in vitro, the effect of L-alanine (Ala) on suckling rat brain acetylcholinesterase (AChE) and on eel Electrophorus electricus pure AChE inhibited by L-phenylalanine (Phe) as well as to investigate whether Phe or Ala is a competitive inhibitor or an effector of the enzyme. AChE activity was determined in brain homogenates and in the pure enzyme after 1 h preincubation with 1.2 mM of Phe or Ala as well as with Phe plus Ala. The activity of the pure AChE was also determined using as a substrate different amounts of acetylthiocholine. Ala reversed completely the inhibited AChE by Phe (18-20% in 500-600 microM substrate, p<0.01). Lineweaver-Burk plots showed that Vmax remained unchanged. However, Km was found increased with Phe (150%, p<0.001), decreased with Ala alone (50%, p<0.001) and unaltered with Phe plus Ala. It is suggested that: a) Phe presents a competitive inhibitory action with the substrate whereas Ala a competitive activation; b) Ala competition with Phe might unbind the latter from AChE molecule inducing the enzyme stimulation; c) Ala might reverse the inhibitory effect of Phe on brain AChE in phenylketonuric patients, if these results are extended into the in vivo reality.     

A comparison of tabun-inhibited rat brain acetylcholinesterase reactivation by three oximes (HI-6, obidoxime,and K048) in vivo detected by biochemical and histochemical techniques

Tabun belongs to the most toxic nerve agents. Its mechanism of action is based on acetylcholinesterase (AChE) inhibition at the peripheral and central nervous systems. Therapeutic countermeasures comprise administration of atropine with cholinesterase reactivators able to reactivate the inhibited enzyme. Reactivation of AChE is determined mostly biochemically without specification of different brain structures. Histochemical determination allows a fine search for different structures but is performed mostly without quantitative evaluation. In rats intoxicated with tabun and treated with a combination of atropine and HI-6, obidoxime, or new oxime K048, AChE activities in different brain structures were determined using biochemical and quantitative histochemical methods. Inhibition of AChE following untreated tabun intoxication was different in the various brain structures, having the highest degree in the frontal cortex and reticular formation and lowest in the basal ganglia and substantia nigra. Treatment resulted in an increase of AChE activity detected by both methods. The highest increase was observed in the frontal cortex. This reactivation was increased in the order HI-6 < K048 < obidoxime; however, this order was not uniform for all brain parts studied. A correlation between AChE activity detected by histochemical and biochemical methods was demonstrated. The results suggest that for the mechanism of action of the nerve agent tabun, reactivation in various parts of the brain is not of the same physiological importance. AChE activity in the pontomedullar area and frontal cortex seems to be the most important for the therapeutic effect of the reactivators. HI-6 was not a good reactivator for the treatment of tabun intoxication.     

A comparison of tabun-inhibited rat brain acetylcholinesterase reactivation by three oximes (HI-6, obidoxime, and K048) in vivo detected by biochemical and histochemical techniques

Tabun belongs to the most toxic nerve agents. Its mechanism of action is based on acetylcholinesterase (AChE) inhibition at the peripheral and central nervous systems. Therapeutic countermeasures comprise administration of atropine with cholinesterase reactivators able to reactivate the inhibited enzyme. Reactivation of AChE is determined mostly biochemically without specification of different brain structures. Histochemical determination allows a fine search for different structures but is performed mostly without quantitative evaluation. In rats intoxicated with tabun and treated with a combination of atropine and HI-6, obidoxime, or new oxime K048, AChE activities in different brain structures were determined using biochemical and quantitative histochemical methods. Inhibition of AChE following untreated tabun intoxication was different in the various brain structures, having the highest degree in the frontal cortex and reticular formation and lowest in the basal ganglia and substantia nigra. Treatment resulted in an increase of AChE activity detected by both methods. The highest increase was observed in the frontal cortex. This reactivation was increased in the order HI-6 < K048 < obidoxime; however, this order was not uniform for all brain parts studied. A correlation between AChE activity detected by histochemical and biochemical methods was demonstrated. The results suggest that for the mechanism of action of the nerve agent tabun, reactivation in various parts of the brain is not of the same physiological importance. AChE activity in the pontomedullar area and frontal cortex seems to be the most important for the therapeutic effect of the reactivators. HI-6 was not a good reactivator for the treatment of tabun intoxication.     

The effect of galactose metabolic disorders on rat brain acetylcholinesterase activity

To evaluate whether in classical galactosemia galactose (Gal), galactose-1-phosphate (Gal-1-P) and galactitol (Galtol) affect brain acetylcholinesterase (AChE) activity, various concentrations (1-16 mM) of these compounds were preincubated with brain homogenates of suckling rats as well as with pure eel Electroforus electricus AChE at 37 degrees C for 1 h. Initially, Galtol (up to 2.0 mM) increased (25%) AChE activity which decreased. thereafter, reaching the control value in high Galtol concentrations. Gal-1-P decreased gradually the enzyme activity reaching a plateau (38%), when incubated with 8-16 mM. However, when the usually found 2 mM of Galtol and 2 mM of Gal-1-P, concentrations in galactosemia were added in the incubation mixture simultaneously, brain AChE was stimulated (16%). Galtol or Gal-1-P modulated brain AChE as well as enzyme activity of E.electricus in the same way. Gal, Glucose (Glu) and glucose-1-phosphate (Glu-1-P) had no effect on AChE activity. It is suggested that Galtol as well as Gal-1-P can affect acetylcholine degradation acting directly on AChE molecule. Consequently the direct action of these substances on the enzyme might explain the brain cholinergic dysfunction in untreated galactosemia patients.     

Induction of acetylcholinesterase by 17- estradiol in the brain of rats of various ages

The induction of acetylcholinesterase (AChE) was studied in the cerebral hemisphere and cerebellum of immature (9-), adult (29-) and old (65-week) female rats. The specific activity of AChE of the cerebral hemisphere of normal rats is highest at 9 weeks and decreases thereafter. There is no such change in the cerebellum. Ovariectomy decreases its activity in the cerebral hemisphere of adult, and cerebellum of immature and adult rats, but not of old rats. Administration of estradiol to ovariectomized rats increases the activity in the cerebral hemisphere and cerebellum of immature and adult rats but not of old rats. The magnitude of stimulation, which is actinomycin D-sensitive, is highest in immature rats.     

An attempt to assess functionally minimal acetylcholinesterase activity necessary for survival of rats intoxicated with nerve agents

Acetylcholinesterase (AChE, EC 3.1.1.7) is an important enzyme for cholinergic nerve transmission. The action of toxic organophosphates such as nerve agents is based on AChE inhibition. The death following acute nerve agent poisoning is due to central or peripheral respiratory/cardiac failure. Therefore, the changes in AChE activity following nerve agents acting predominantly on the central (sarin, soman) or peripheral (VX) level were studied. It is known that AChE activity in different structures exists in relative excess. Female Wistar rats intoxicated with sarin, soman, and VX in different doses (0.5-2.0xLD(50)) were divided into groups of survived and died animals. AChE activities in diaphragm, brain parts (pontomedullar area, frontal cortex, basal ganglia, in some cases other parts of the brain) were determined and the rest of activity (in %) was correlated with survival/death of animals. More precise elucidation of action of nerve agents and the assessment of minimal AChE activity in different organs compatible with the survival of organism poisoned with nerve agents were the aims of this study.     

Effect of neuroleptics on acetylcholine metabolism in the basal ganglia of the brain

Neuroleptics (haloperidol) closapine, pimozid, chlorpromazine) diminished the level of free (functionally active) form of acetylcholine (ACh), and, to some extent, the bound form of ACh; they changed the content of the labile-bound (vesicular) form of ACh and weakly influenced the choline-acetyltranspherase activity in the basal ganglia of the rat brain 5 to 30 min after the injection. In contrast to the inhibitory action on the acetyl-cholinesterase (AChE) activity in vitro, most of the neuroleptics, except closapine, increased the AChE activity in vivo. These results indicate that the neuroleptics activate ACh-metabolism and probably stimulate the cholinergic structure in the basal ganglia of the brain; the AChE activity may serve as a criterion of such stimulating action of neuroleptics.     

The depolarizing action of GABA in cultured hippocampal neurons is not due to the absence of ketone bodies

Two recent reports propose that the depolarizing action of GABA in the immature brain is an artifact of in vitro preparations in which glucose is the only energy source. The authors argue that this does not mimic the physiological environment because the suckling rats use ketone bodies and pyruvate as major sources of metabolic energy. Here, we show that availability of physiologically relevant levels of ketone bodies has no impact on the excitatory action of GABA in immature cultured hippocampal neurons. Addition of β-hydroxybutyrate (BHB), the primary ketone body in the neonate rat, affected neither intracellular calcium elevation nor membrane depolarizations induced by the GABA-A receptor agonist muscimol, when assessed with calcium imaging or perforated patch-clamp recording, respectively. These results confirm that the addition of ketone bodies to the extracellular environment to mimic conditions in the neonatal brain does not reverse the chloride gradient and therefore render GABA hyperpolarizing. Our data are consistent with the existence of a genuine "developmental switch" mechanism in which GABA goes from having a predominantly excitatory role in immature cells to a predominantly inhibitory one in adults.     

Ecdysterone and an analogue of juvenile hormone on the autophagy in the cells of fat body of mamestra brassicae.

The effect of ecdysterone and a juvenile hormone analogue (JHa) on autophagy and heterophagy was investigated in the fat body cells of the last larval instar of Mamestra brassicae. In the course of normal development autophagic vacuoles and protein granules of heterophagic origin begin to accumulate in these cells, on the 4th and 5th day of the last larval stage respectively. When ecdysterone (10 mug/g body weight) was administered to the larvae for 24 h either on the 1st or on the 2nd day of the last larval stage, autophagic and heterophagic vacuoles appeared in the cells as early as on the 2nd or 3rd days. Autophagy was also observed in the cells of one-or two-day-old last larval fat body after a 5 h incubation in a medium containing 10 mug/ml ecdysterone, in vitro. Ligation of the last thoracic segment resulted in inhibition of metamorphic changes in the fat body lobules of the isolated abdomen. Injection of 10 mug ecdysterone into the isolated abdomen resulted in an appearence of autophagic vacuoles in these cells, too. JHa treatment, when started on the 2nd or 3rd day of the last larval stage, inhibited both auto- and heterophagy and the fat bodies maintained their larval character. Treatment started on the 4th or 5th day proved either ineffective or lethal. It is concluded that the auto- and heterophagy taking place in the larval fat body cells are stimulated by ecdysterone and inhibited by JHa. Experiments performed in vitro or on ligated animals in vivo provided evidence for a direct action of ecdysterone at the cellular level.     

Glutamyl transferase and acetylcholinesterase activity in various areas of the rat brain in alcoholic intoxication

Albino mongrel rats were used for the determination of the gamma-glutamyl transferase (gamma-GTF) and acetylcholine esterase (AChE) activities in various brain areas (cerebral hemispheres, cerebellum, hippocampus, brain stem) during acute (1.5; 4 and 6 g/kg i. p.) and chronic (15 months) alcoholic intoxication and alcohol withdrawal (24-48 h, 4 and 8 days). An increase or a decrease in the activity of these two enzymes in the various rat brain areas depends on the dose of ethanol and the time of its action. The activity of gamma-GTF grew in all brain areas during chronic ethanol intoxication; the activity of AChE was also enhanced in three brain areas but it was diminished in cerebral hemispheres. Alcohol withdrawal caused diverse changes in the activities of these two enzymes in various areas of the brain. A tendency to normalization of the gamma-GTF and AChE activities is manifested 4-8 days after alcohol withdrawal.     

Protection from quinidine or physostigmine against in vitro inhibition by sarin of acetylcholinesterase activity

We have studied the relative effectiveness of quinidine and physostigmine in protecting against the inhibition of acetylcholinesterase (AChE) by sarin, an organophosphate (OP) compound. The protective effects of these compounds were studied in vitro in both synaptosomal and soluble samples obtained from various regions of sarin-administered or control isolated, perfused canine brain. Although AChE activities in the sarin-administered brain were substantially lower than in the control brain, we observed regional differences in the AChE activity in both. The AChE in the control brain and the AChE remaining in sarin-administered brain had different susceptibilities to inhibition from OP compounds in vitro and, therefore, have different properties. Quinidine partially protected AChE from the inhibitory effects of sarin in vitro possibly by altering the sarin binding sites. Addition of sarin to physostigmine-treated control brain samples allowed partial recovery of the AChE activity. The protective effects of quinidine or physostigmine were lost when samples from sarin-administered brain were treated in vitro with these compounds and then again exposed to sarin. Therefore, both quinidine and physostigmine provided partial protection against the inhibitory effects of sarin in vitro if they were added prior to sarin.     

Effect of intratesticular administration of TRH or anti-TRH antiserum on function of rat testis

The effect of intratesticular administration of thyrotropin-releasing hormone (TRH) and anti-TRH antiserum on steroidogenesis was studied in immature and adult rats. In 9-day-old animals local administration of the neuropeptide resulted in an increase in basal testosterone secretion in vitro. Similar treatment of 15-day-old rats suppressed hCG-stimulated testosterone secretion with no change in basal testosterone production. In both immature groups the treatment did not affect serum testosterone concentration. By contrast, in adults TRH decreased serum testosterone level, but did not influence basal and hCG-stimulated testosterone secretion. Both in immature and adult rats, the changes in steroidogenesis were evident 1 hour posttreatment. Five days after the administration of anti-TRH antiserum into the remaining testis of immature rats subjected to hemicastration just prior to the antiserum treatment, the alterations in steroidogenesis were opposite to those detected after treatment with TRH. In 9-day-old rats the antiserum suppressed steroidogenesis, while in 15-day-old animals it stimulated testosterone secretion. The results suggest that testicular TRH might exert a local action on testicular steroidogenesis, and the effect is age-dependent.     

Atypical effect of some spin trapping agents: reversible inhibition of acetylcholinesterase

N-tert-butyl-alpha-phenylnitrone (PBN), a widely used nitrone-based free radical trap was recently shown to prevent acetylcholinesterase (AChE) inhibitors induced muscle fasciculations and brain seizures while being ineffective against glutamergic or cholinergic receptor agonist induced seizures. In the present study we compared the effects on AChE activity of four free radical spin traps PBN, alpha-(4-pyridil-1)-N-tert-butyl nitrone (POBN), N-tert-butyl-alpha-(2-sulfophenyl)-nitrone (S-PBN) and 5-diethoxyphosphoryl-5-methyl-1-pyrroline-N-oxide (DEPMPO). The kinetics of AChE inhibition were studied in vitro using a spectrophotometric kinetic assay with AChE from rat brain, diaphragm, electric eel and mouse brain. Spin trapping compounds S-PBN and DEPMPO, in concentrations up to 3 mM did not inhibit hydrolysis of ACh, while PBN and POBN inhibited hydrolysis of ACh in a reversible and concentration-dependent manner. Double reciprocal plots of the reaction velocity against varying ACh concentrations at each inhibitor concentration were linear and generally indicated mixed type inhibition. PBN was the most potent inhibitor of mouse AChE with Ki and Ki' of 0.58 and 2.99 mM, respectively, and the weakest inhibitor of electric eel AChE. In contrast, POBN showed the highest affinity for electric eel enzyme, with Ki and Ki' values of 1.065 and 3.15 mM, respectively. These findings suggest that the effect of PBN and POBN on AChE activity does not depend on trapping of damaging reactive oxygen and that in addition to their antioxidant action other pharmacological effects of these compounds should be considered when neuroprotective actions of PBN or POBN are investigated.     

Alpha7-nicotinic acetylcholine receptors mediate an Abeta(1-42)-induced increase in the level of acetylcholinesterase in primary cortical neurones

The beta-amyloid protein (Abeta) is the major protein component of amyloid plaques found in the Alzheimer brain. Although there is a loss of acetylcholinesterase (AChE) from both cholinergic and non-cholinergic neurones in the brain of Alzheimer patients, the level of AChE is increased around amyloid plaques. Previous studies using P19 cells in culture and transgenic mice which overexpress human Abeta have suggested that this increase may be due to a direct action of Abeta on AChE expression in cells adjacent to amyloid plaques. The aim of the present study was to examine the mechanism by which Abeta increases levels of AChE in primary cortical neurones. Abeta1-42 was more potent than Abeta1-40 in its ability to increase AChE in primary cortical neurones. The increase in AChE was unrelated to the toxic effects of the Abeta peptides. The effect of Abeta1-42 on AChE was blocked by inhibitors of alpha7 nicotinic acetylcholine receptors (alpha7 nAChRs) as well as by inhibitors of L- or N-type voltage-dependent calcium channels (VDCCs), whereas agonists of alpha7 nAChRs (choline, nicotine) increased the level of AChE. The results demonstrate that the effect of Abeta1-42 on AChE is due to an agonist effect of Abeta1-42 on the alpha7 nAChR.     

Partial agonist of benzodiazepine receptors Ro 19-2088 elicits withdrawal symptoms after short-term administration in immature rats

Repeated administration of partial agonist of benzodiazepine receptors Ro 19-8022 (a derivative of quinolizine class) does not elicit withdrawal in adult rats. Our older data demonstrated that single injection of Ro 19-2088 to immature rats induces increased sensitivity to convulsant action of pentylenetetrazol as a withdrawal phenomenon. To know if repeated administration of the partial agonist has the same effect we injected rats at postnatal days 7 to 11 with an anticonvulsant dose of Ro 19-2088 (0.5 mg/kg i.p.) and tested them 24 h, 48 h and 4 days after the last injection. Repeated administration of Ro 19-8022 resulted also in an increased sensitivity to convulsant action of pentylenetetrazol in immature rats (higher incidence and severity of seizures). This effect was significant 24 h after the last injection but only outlined 48 h after administration. No signs of hypersensitivity were seen at 4-day interval. There is a difference between immature and adult brain in an appearance of withdrawal symptom after administration of the partial agonist of benzodiazepine receptors Ro 19-2088.     

Acute effects of acephate and methamidophos on acetylcholinesterase activity, endocrine system and amino acid concentrations in rats

Acute effects of acephate (Ace) and methamidophos (Met) on acetylcholinesterase activity, endocrine system and amino acid concentrations were studied in rats. The rats were injected intraperitoneally with Ace (500 mg/kg) or Met (5 mg/kg) and then sacrificed at 15 or 60 min after the injection (A15 and A60 for Ace and M15 and M60 for Met). The primary aim of this study was to determine whether the mammalian toxicity of Ace is solely due to its conversion to Met or the protection of Ace against Met-inhibited AChE is also an important factor. The second aim of this study was to study the effects of Ace and Met on the endocrine system and amino acid concentrations and whether or not these effects correlate with AChE inhibition and Met accumulation. The Ace or Met injected animals did not exhibit the signs of organophosphate (OP) poisoning within 15 min after the injection, but exhibited tremors at 45 min after the injection. Blood and brain AChE activity in A15 and M15 rats exhibited 55 to 75% inhibition while the enzyme activity in A60 and M60 rats exhibited 80 to 95% inhibition. Ace was metabolized to Met in rats both in vivo and in vitro. A 5 rats had significantly higher Met concentration in their liver, brain and adrenal glands compared to M 5 rats, and A60 rats had significantly higher Met concentrations in their blood, liver, brain and adrenal glands compared to M60 rats. Thus, tissue Met concentrations in Ace-treated rats were significantly higher than in Met-treated rats and the inhibition of AChE activity was not consistent with the amount of metabolically formed Met, supporting the hypothesis that the Ace protection plays a role in the overall toxicity. Ace and Met both impaired circulating blood hormone and amino acid concentrations in rats. The endocrine effects of Ace and Met differed from their cholinergic effects, and were not proportional to the amount of Met present in different tissues obtained from the treatment groups. Plasma ACTH concentration was elevated in M60 rats but not in A60 rats. Thus, Ace may indirectly protect the pituitary against the toxic effects of Met. Unlike plasma ACTH levels, serum corticosterone and aldosterone levels were elevated in both A60 and M60 rats. Therefore, the effect of Met on the adrenal cortex may be mediated by the pituitary gland, while the effect of Ace may be due to direct Ace-gland interaction. The decrease in the levels of some of the serum amino acids showed an increase in the energy demands in the treatment groups.     

Changes in glucocorticoid receptors in different regions of brain of immature and mature male rats

Specific cytosolic binding for synthetic glucocorticoid dexamethasone was studied in several brain regions (hypothalamus, hippocampus, caudate nucleus, cerebellum, cerebral cortex) of immature (3-week) and mature (26-week) male rats, intact and adrenalectomized. A significant regional difference was observed in the concentration of in vitro [3H] dexamethasone binding in the brain of adrenalectomized rats at both ages, with the highest levels in the hippocampus. A marked decrease in specific binding was observed in all brain regions of adrenalectomized mature rats as compared to immature. The dexamethasone binding was significantly lower in all brain regions of normal intact animals as compared to adrenalectomized rats in both ages.     

Evaluation of Acetylcholinesterase in an Animal Model of Maple Syrup Urine Disease

Maple syrup urine disease is an inherited metabolic disease predominantly characterized by neurological dysfunction. However, the mechanisms underlying the neuropathology of this disease are still not defined. Therefore, the aim of this study was to investigate the effect of acute and chronic administration of a branched-chain amino acids (BCAA) pool (leucine, isoleucine, and valine) on acetylcholinesterase (AChE) activity and gene expression in the brain and serum of rats and to assess if antioxidant treatment prevented the alterations induced by BCAA administration. Our results show that the acute administration of a BCAA pool in 10- and 30-day-old rats increases AChE activity in the cerebral cortex, striatum, hippocampus, and serum. Moreover, chronic administration of the BCAA pool also increases AChE activity in the structures studied, and antioxidant treatment prevents this increase. In addition, we show a significant decrease in the mRNA expression of AChE in the hippocampus following acute administration in 10- and 30-day-old rats. On the other hand, AChE expression increased significantly after chronic administration of the BCAA pool. Interestingly, the antioxidant treatment was able to prevent the increased AChE activity without altering AChE expression. In conclusion, the results from the present study demonstrate a marked increase in AChE activity in all brain structures following the administration of a BCAA pool. Moreover, the increased AChE activity is prevented by the coadministration of N-acetylcysteine and deferoxamine as antioxidants.     

Alterations in rat lipid metabolism following ecdysterone treatment

The influence of ecdysterone on the lipid metabolism in liver and adipose tissue from rat was investigated using 14C-acetate and 32P-orthophosphate as precursors. Ecdysterone produced an increase in 14C-acetate incorporation into triglycerides. A concomitant decrease in free fatty acids and diglycerides was observed. The effect of ecdysterone on triglyceride lipase activity was investigated and a significant decrease was found. Ecdysterone produced a significant increase in the specific activity of phosphatidylethanolamine and phosphatidylserine in liver. On the contrary, the specific activity of phosphatidylcholine was reduced. In adipose tissue, the most evident effect observed was the increase of specific activity of phosphatidylcholine. These results contribute to knowledge of the heterophylic action of ecdysterone.     

Profile of acetylcholinesterase in brain areas of male and female rats of adult and old age

Inhibition of acetylcholinesterase (AChE)-metabolizing enzyme of acetylcholine, is presently the most important therapeutic target for development of cognitive enhancers. However, AChE activity in brain has not been properly evaluated on the basis of age and sex. In the present study, AChE activity was investigated in different brain areas in male and female Sprague-Dawley rats of adult (3 months) and old (18-22 months) age. AChE was assayed spectrophotometrically by modified Ellman's method. Specific activity (micromoles/min/mg of protein) of AChE was assayed in salt soluble (SS) and detergent soluble (DS) fractions of various brain areas, which consists of predominantly G1 and G4 molecular isoforms of AChE respectively. The old male rats showed a decrease (40-55%) in AChE activity in frontal cortex, striatum, hypothalamus and pons in DS fraction and there was no change in SS fraction in comparison to adult rats. In the old female rats the activity was decreased (25-40%) in frontal cortex, cerebral cortex, striatum, thalamus, cerebellum and medulla in DS fraction whereas in SS fraction the activity was decreased only in hypothalamus as compared to adult. On comparing with old male rats, old female rats showed increase in AChE activity in cerebral cortex, hippocampus and hypothalamus of DS fraction and decrease in hypothalamus of SS fraction. There was a significant increase in AChE activity in DS fraction of cerebral cortex, hippocampus, hypothalamus, thalamus and cerebellum in female as compared to male adult rats. However, no significant change in AChE activity was found in the SS fraction, except hypothalamus between these groups. Thus it appears that age alters AChE activity in different brain regions predominantly in DS fraction (G4 isoform) that may vary in male and female. These observations have significant relevance to age related cognitive deficits and its pharmacotherapy.