Antidepressants inhibit human acetylcholinesterase and butyrylcholinesterase activity

Magnesium deficiency in experimental animals leads to inflammation, exacerbated immune stress response and a decrease of specific immune response. It also results in a significant increase in free radical species and subsequent tissue injury. An accelerated thymus involution was observed in Mg-deficient rats in relation to enhanced apoptosis and enhanced susceptibility to oxidative stress. To examine the stress-inducing effects of low Mg status on thymocytes, cDNA arrays were used to evaluate changes in gene expression in weaning rats submitted to Mg deficiency of short duration (2 days). Several genes exhibited changes in their expression caused by Mg deficiency before any perceptible modification in cell integrity and functions. The up-regulated genes included cytochrome c oxidase, glutathione transferase, CuZn superoxide dismutase, genes associated with the stress response (HSP70 and HSP84) and a gene involved in DNA synthesis and repair (GADD45). The down-regulated genes included Na/P cotransporter 1. These findings are consistent with altered cell growth, modifications of ion fluxes and oxidative stress described during Mg deficiency. The observation of induction of genes involved in protection and repair in cells from Mg-deficient animals provides additional evidence of the role of oxidative stress in the pathobiology of this deficiency.     

Human butyrylcholinesterase components differ in aryl acylamidase activity

Apart from its esterase activity, butyrylcholinesterase (BuChE) displays aryl acylamidase (AAA) activity able to hydrolyze o-nitroacetanilide (ONA) and its trifluoro-derivative (F-ONA). We report here that, despite amidase and esterase sites residing in the same protein, in human samples depleted of acetylcholinesterase the ratio of amidase to esterase activity varied depending on the source of BuChE. The much faster degradation of ONA and F-ONA by BuChE monomers (G1) of colon and kidney than by the tetramers (G4) suggests aggregation-driven differences in the AAA site between single and polymerized subunits. The similar ratio of F-ONAto butyrylthiocholine hydrolysis by serum G1 and G4 forms support structural differences in the amidase site according to the source of BuChE. The changing ratios of amidase to esterase activities in the human sources probably arise from post-translational modifications in BuChE subunits, the specific proportion of monomers and oligomers and the variable capacity of the tetramers for degrading ONA and F-ONA. The elevated amidase activity of BuChE monomers and the scant activity of the tetramers justify the occurrence of single BuChE subunits in cells as a means to sustain the AAA activity of BuChE which otherwise could be lost by tetramerization.     

Increased serum butyrylcholinesterase activity in type IIb hyperlipidaemic patients

The inheritance of the apolipoprotein E4 (APOE4) allele has been shown to increase the plasma cholesterol level, but little information is as concerns the association of the APOE genotype and hyperlipidaemia and the activities of two serum enzymes, acetylcholinesterase (AChE) and butyrylcholinesterase (BChE). Blood samples from 55 type IIb hyperlipidaemic, non-demented patients and 55 age- and sex-matched controls were therefore examined in this pilot study. A significantly increased BChE activity was found in the serum of type IIb hyperlipidaemic patients, but the AChE activity did not differ significantly as compared with that in the control group. The APOE4 allele was significantly overrepresented among the hyperlipidaemic probands, but neither serum cholinesterase activity was affected by the dosage of the APOE4 gene. Our results point to a possible association between an abnormal lipid metabolism and the BChE activity and might have implications as regards the pathomechanism of both Alzheimer's and vascular dementias and the cholinesterase inhibitor therapy of dementing disorders.     

Acetylcholinesterase/butyrylcholinesterase inhibition activity of some new carbacylamidophosphate derivatives

Eight newly synthesized carbacylamidophosphates with the general formula RC(O)NHP(O)Cl2 with R = pCl-C6H4 1a, pBr-C6H4 2a, C6H5 3a, and pMe-C6H4 4a and RC(O)NHP(O)(NC4H8O)2 R = pCl-C6H4 1b, pBr-C6H4 2b, C6H5 3b, pMe-C6H4 4b, were selected to compare the inhibition kinetic parameters, IC50, Ki, kp and KD, on human erythrocyte acetylcholinesterase (hAChE) and bovine serum butyrylcholinesterase (BuChE), Also, the in vivo inhibition potency of compound 2a, 2b and 3a, were studied. The data demonstrates that compound 2a and compound 2b are the potent sensitive as AChE and BuChE inhibitors respectively, and the inhibition of hAChE is about 10-fold greater than that of BuChE.     

Cerebrospinal fluid gradients of acetylcholinesterase and butyrylcholinesterase activity in healthy aging

Acetylcholinesterase (AChE) and butyrylcholinesterase (BChE) activities were measured in 13 sequential 2 ml aliquots of cerebrospinal fluid (CSF) obtained by lumbar puncture from 7 young and 7 elderly healthy normal subjects. The slopes of the rostrocaudal gradients of AChE and BChE were calculated and compared to those of total protein concentration and the major dopaminergic metabolite homovanillic acid (HVA), for which a pronounced rostrocaudal gradient (with highest concentrations of HVA in more rostral CSF) is consistent with HVA originating primarily from the brain. AChE activity was higher in more caudal fractions of young, but not elderly subjects and there was a significant difference between the mean AChE gradient slopes in the young and old groups. These results suggest that the spinal cord makes an important contribution to AChE activity in lumbar CSF. Furthermore, the absence of a negative AChE gradient in elderly subjects may be the result of a greater rate of entry of cerebral AChE into CSF, possibly as a consequence of an increased ventricular surface area and shorter diffusion distances in atrophic elderly brains. In contrast to AChE, BChE activity and total protein concentrations were higher in more caudal CSF fractions of not only young but also old subjects. In addition, there was a significant correlation between the gradient slopes of BChE activity and total protein concentrations, suggesting that the majority of BChE activity in lumbar CSF derives from the same source as the majority of total protein, namely plasma. The diffuse (i.e. brain and spinal cord) origin of AChE in lumbar CSF would explain the relatively modest changes in lumbar CSF AChE activity in diseases involving certain central cholinergic systems, most notably Alzheimer's disease.     

Synthesis and acetylcholinesterase/butyrylcholinesterase inhibition activity of new tacrine-like analogues

The synthesis and preliminary results for acetylcholinesterase and butyrylcholinesterase inhibition activity of a series of pyrano[2,3-b]quinolines (2, 3) and benzonaphthyridines (5, 6) derivatives are described. These molecules are tacrine-like analogues which have been prepared from readily available polyfunctionalized ethyl [6-amino-5-cyano-4H-pyrans and 6-amino-5-cyanopyridines]-3-carboxylates via Friedlander condensation with selected ketones. These compounds showed moderate acetylcholinesterase inhibition activity, the more potent (2e, 5b) being 6 times less active than tacrine. The butyrylcholinesterase activity of some of these molecules is also discussed.     

NO-donating tacrine derivatives as potential butyrylcholinesterase inhibitors with vasorelaxation activity

To search for potent anti-Alzheimer’s disease (AD) agents with multifunctional effects, 12 NO-donating tacrine–flurbiprofen hybrid compounds (2a–l) were synthesized and biologically evaluated. It was found that all the new target compounds showed selective butyrylcholinesterase (BuChE) inhibitory activity in vitro comparable or higher than tacrine and the tacrine–flurbiprofen hybrid compounds 1a–c, and released moderate amount of NO in vitro. The kinetic study suggests that one of the most active and highest BuChE selective compounds 2d may not only compete with the substrate for the same catalytic active site (CAS) but also interact with a second binding site. Furthermore, 2d and 2l exhibited significant vascular relaxation effect, which is beneficial for the treatment of AD. All the results suggest that 2d and 2l might be promising lead compounds for further research.     

Nippostrongylus brasiliensis: Infection decreases plasma butyrylcholinesterase activity in rats

Plasma butyrylcholinesterase (BChE) hydrolyzes ester-containing compounds such as succinylcholine, as well as acting as a scavenger against neurotoxic organophosphates (OPs). We previously found that Nippostrongylus brasiliensis infection makes rats more susceptible to OP toxicity by decreasing serum paraoxonase-1 (PON1) activity. In the present study, we examined the effects of N.brasiliensis infection on acetylcholinesterase (AChE) activity in plasma, red blood cells (RBCs), brain and diaphragm, as well as serum PON1 activity, in rats at day 7 after infection. N.brasiliensis infection significantly decreased plasma BChE and PON1 activities without significantly altering AChE activity in RBCs, brain and diaphragm. These results provide further insight into the unusual deleterious effects of intestinal nematode infections on body homeostasis.     

Excessive hippocampal acetylcholine levels in acetylcholinesterase-deficient mice are moderated by butyrylcholinesterase activity

Central cholinergic systems are involved in a plethora of brain functions and are severely and selectively damaged in neurodegenerative diseases such as Alzheimer's disease and dementia with Lewy bodies. Cholinergic dysfunction is treated with inhibitors of acetylcholinesterase (AChE) while the role of butyrylcholinesterase (BChE) for brain cholinergic function is unclear. We have used in vivo microdialysis to investigate the regulation of hippocampal acetylcholine (ACh) levels in mice that are devoid of AChE (AChE-/- mice). Extracellular ACh levels in the hippocampus were 60-fold elevated in AChE-/- mice compared with wild-type (AChE+/+) animals. In AChE-/- mice, calcium-free conditions reduced hippocampal ACh levels by 50%, and infusion of tetrodotoxin by more than 90%, indicating continuous ACh release. Infusion of a selective AChE inhibitor (BW284c51) caused a dose-dependent, up to 16-fold increase of extracellular ACh levels in AChE+/+ mice but did not change ACh levels in AChE-/- mice. In contrast, infusion of a selective inhibitor of BChE (bambuterol) caused up to fivefold elevation of ACh levels in AChE-/- mice, but was without effect in AChE+/+ animals. These results were corroborated with two other specific inhibitors of AChE and BChE, tolserine and bis-norcymserine, respectively. We conclude that lack of AChE causes dramatically increased levels of extracellular ACh in the brain. Importantly, in the absence of AChE, the levels of extracellular ACh in the brain are controlled by the activity of BChE. These results point to a potential usefulness of BChE inhibitors in the treatment of central cholinergic dysfunction in which brain AChE activity is typically reduced.     

Association of the CHE2 locus with body mass index and butyrylcholinesterase activity

The butyrylcholinesterase (BChE; EC 3.1.1.8) activities of two electrophoretic bands of the CHE2 C5+ phenotype--C5 and C(OF) (other forms)--were quantified by densitometry in 100 individuals. The activity data suggested that, in addition to determining C5, the CHE2*C5+ allele also increases the level of other BChE forms. Since the relative activity of C5 showed the highest correlation coefficient with weight when compared with the other BChE activity variables (total, absolute C5, and absolute C(OF)), its median activity level was used for the classification of CHE2 C5+ phenotypes (faint and intense). Mean body mass index (BMI) was compared among the CHE2 locus phenotypes-controlled by sex, age, and ethnic group. It was shown that the intense CHE2 C5+ phenotype presents a significantly lower (p < 0.001) mean BMI (23.2) than the other phenotypes (faint CHE2 C5+ = 25.2; CHE2 C5- = 25.4). It seems that the relative COF activity is positively associated with fat storage, since CHE2 C5- and faint CHE2 C5+ phenotypes showed higher mean BMI than the intense CHE2 C5+ phenotype. Our hypothesis is that the presence of C5 in a relatively high proportion leads to less fat storage.     

Acetylcholinesterase and butyrylcholinesterase inhibitory activity of Pinus species essential oils and their constituents

This study aimed to investigate the acetylcholinesterase (AChE) and butyrylcholinesterase (BChE) inhibitory activity of the essential oils from Pinus nigra subsp. nigra, P. nigra var. calabrica, and P. heldreichii subsp. leucodermis. This activity is relevant to the treatment of Alzheimer’s disease (AD), since cholinesterase drugs are currently the only drugs available to treat AD. P. heldreichii subsp. leucodermis exhibited the most promising activity, with IC₅₀ values of 51.1 and 80.6?μg/mL against AChE and BChE, respectively. An interesting activity against AChE was also observed with P. nigra subsp. nigra essential oil, with an IC₅₀ value of 94.4?μg/mL. Essential oils were analyzed by GC and GC-MS with the purpose of investigating their relationships with the observed activities. Among the identified constituents, terpinolene, β-phellandrene, linalyl acetate, trans-caryophyllene, and terpinen-4-ol were tested. trans-Caryophyllene and terpinen-4-ol inhibited BChE with IC₅₀ values of 78.6 and 107.6?μg/mL, respectively. β-Phellandrene was selective against AChE (IC₅₀ value of 120.2?μg/mL).     

Acetylcholinesterase and butyrylcholinesterase activity in the atria of the heart of adult albino rats

In experiments on adult albino rats the authors used the substances BW 284 C51 (1.5-bis(allyldimethylammoniumphenyl)-pentane-3-one-dibromide) as a specific inhibitor of acetylcholinesterase (AChE) and ethopropazine (10-(2-diethylaminopropyl) phenothiazine hydrochloride) as a specific inhibitor of butyrylcholinesterase (BuChE) to determine the two enzyme activities in atrial homogenates and to investigate changes after AChE or BuChE inhibition of the negative chronotropic effect of acetylcholine (ACh) on atria incubated in vitro. AChE accounted for only 12% and BuChE for 88% of the total ability of atrial homogenates to hydrolyse acetylcholine. The concentration of exogenous ACh needed to reduce the spontaneous frequency of contractions of the isolated right atrium by 30, 60, or 90/min fell by 78%, 79% and 84% respectively after BW 284 C51 inhibition of AChE and by 95%, 94% and 94% after simultaneous inhibition of AChE and BuChE. The significance of AChE in control of the negative chronotropic effect of ACh is thus evidently significantly greater than would correspond to the percentual proportion of AChE in cholinesterase activities in the atria of the rat heart. In can be assumed that AChE is functionally associated with parasympathetic innervation of the heart and that it is probably present in a high concentration in the primary pacemaker region.     

Microbial transformation of (-)-isolongifolol and butyrylcholinesterase inhibitory activity of transformed products

The microbial transformation of (-)-isolongifolol (1) by using the standard two-stage fermentation technique with Fusarium lini afforded polar oxygenated metabolites: 10-oxoisolongifolol (2), 10alpha-hydroxyisolongifolol (3), and 9alpha-hydroxyisolongifolol (4). Metabolites 3 and 4 were also formed with the incubation of 1 with Aspergillus niger. All three metabolites were found to be new. Compounds 3 and 4 inhibited butyrylcholinesterase enzyme in a concentration-dependent manner with IC50 values 13.6 and 299.5 microM, respectively. Compound 3 showed un-competitive mode of inhibition against butyrylcholinesterase with Ki value 15.0 microM. The structures of metabolites 2-4 were deduced on the basis of spectroscopic techniques and single-crystal X-ray diffraction techniques.     

Chronic methylphenidate administration alters antioxidant defenses and butyrylcholinesterase activity in blood of juvenile rats

Methylphenidate (MPH), a psychostimulant that affects both dopaminergic and noradrenergic systems, is one of the most frequently prescribed treatments for attention-deficit hyperactivity disorder. The present study investigated the effects of chronic administration of MPH on some parameters of oxidative stress, as well as on butyrylcholinesterase (BuChE) activity in blood of young rats. Rats received intraperitoneal injections of MPH (2.0 mg/kg) once a day, from the 15th to the 45th day of age or an equivalent volume of 0.9% saline solution (controls). Two hours after the last injection, animals were euthanized, and blood was collected. Results demonstrated that MPH did not alter the dichlorofluorescein formed, decreased both thiobarbituric acid reactive substances and total non-enzymatic radical-trapping antioxidant, and increased superoxide dismutase and catalase activities, suggesting that this psychostimulant may alter antioxidant defenses. BuChE activity was increased in blood of juvenile rats subjected to chronic MPH administration. These findings suggest that MPH may promote peripheral oxidative adaptations and cholinergic changes.     

Ultrafiltration-based assay for heparanase activity

Heparanase, a mammalian endoglycosidase that specifically cleaves heparan sulfate (HS), has been found in many tissues. Platelet, liver, and placenta have been abundant sources for the study of the enzyme. Notably, certain malignant cells also have been found to produce large amounts of the enzyme, the levels of which often correlate with their invasive and metastatic properties. To study roles of heparanase in various biological situations, a reliable method measuring the enzyme activity is indispensable. In the past, measurement of heparanase enzyme activity was done either by the detection of the degradation of fluorescent or radiolabeled HS chains by gel filtration procedures or by the use of radiolabeled substrate conjugated to solid matrices for the easy separation of degraded HS chains. A newly developed procedure, presented in this article, measures degradation of radiolabeled HS chains in the aqueous buffer by detecting their degradation products using an ultrafiltration device, the Centricon 30. This procedure has several advantages over previous assay procedures that involved tedious processing such as gel filtration chromatography of each sample or the preparation of substrate HS proteoglycans conjugated to a solid matrix. The simplicity of the new procedure allows a short setup time and a rapid processing of a large number of samples. Furthermore, the enzymatic reaction during the aqueous phase allows kinetic analyses in standard conditions.     

Cell-based assay for beta-secretase activity

The cerebral deposition of amyloid beta-peptide (Abeta) is a major factor in the etiology of Alzheimer's disease. beta-Secretase (BACE) initiates the generation of Abeta by cleaving the amyloid precursor protein at the beta-site and is therefore a prime target for therapeutic intervention. Here we report a cell-based method suitable for monitoring BACE activity and the efficacy of protease inhibitors. A fusion protein containing the amino-terminal transmembrane domain of Golgi alpha-mannosidase II, a Drosophila Golgi integral membrane protein, linked to human alkaline phosphatase (AP) by a short beta-site sequence, was expressed in Drosophila S2 cells. While the uncleaved fusion protein was retained in the Golgi apparatus, cleavage of the beta-site by BACE resulted in the release of AP to the culture medium, where it was easily detected and quantified. Three peptidomimetic inhibitors (LB83190, LB83192, LB83202) were tested for their efficacy with this cell-based assay. While LB83190 and LB83192 effectively blocked BACE activity, LB83202, a carboxylated derivative of LB83192, did not. This is consistent with the inability of LB83202 to permeate the cell membrane. The present cell-based assay could provide a convenient tool for high-throughput screening of substances that can interfere with BACE in living cells.