Unbiased expression mapping identifies a link between the complement and cholinergic systems in the rat central nervous system.

The complement system is activated in a wide spectrum of CNS diseases and is suggested to play a role in degenerative phenomena such as elimination of synaptic terminals. Still, little is known of mechanisms regulating complement activation in the CNS. Loss of synaptic terminals in the spinal cord after an experimental nerve injury is increased in the inbred DA strain compared with the PVG strain and is associated with expression of the upstream complement components C1q and C3, in the absence of membrane attack complex activation and neutrophil infiltration. To further dissect pathways regulating complement expression, we performed genome-wide expression profiling and linkage analysis in a large F2(DA × PVG) intercross, which identified quantitative trait loci regulating expression of C1qa, C1qb, C3, and C9. Unlike C1qa, C1qb, and C9, which all displayed distinct coregulation with different cis-regulated C-type lectins, C3 was regulated in a coexpression network immediately downstream of butyrylcholinesterase. Butyrylcholinesterase hydrolyses acetylcholine, which exerts immunoregulatory effects partly through TNF-α pathways. Accordingly, increased C3, but not C1q, expression was demonstrated in rat and mouse glia following TNF-α stimulation, which was abrogated in a dose-dependent manner by acetylcholine. These findings demonstrate new pathways regulating CNS complement expression using unbiased mapping in an experimental in vivo system. A direct link between cholinergic activity and complement activation is supported by in vitro experiments. The identification of distinct pathways subjected to regulation by naturally occurring genetic variability is of relevance for the understanding of disease mechanisms in neurologic conditions characterized by neuronal injury and complement activation.

Use of neuromuscular monitoring to detect prolonged effect of succinylcholine or mivacurium: three case reports.

Mutations in the butyrylcholinesterase gene can lead to a prolonged effect of the neuromuscular blocking agents, succinylcholine and mivacurium. If the anaesthesiologist is not aware of this condition, it may result in insufficient respiration after tracheal extubation. However, this can be avoided with the use of objective neuromuscular monitoring if used adequately. Three case reports of prolonged effect of succinylcholine or mivacurium were presented to illustrate the importance of neuromuscular monitoring during anaesthesia. In the first case, continuous intraoperative neuromuscular monitoring allowed a prolonged neuromuscular blockade to be discovered prior to tracheal extubation of the patient. The patient was extubated after successful reversal of the neuromuscular blockade. On the contrary, neuromuscular monitoring was not used during anaesthesia in the second patient; hence, the prolonged effect of the neuromuscular blocking agent was not discovered until after extubation. In the third patient, the lack of response to nerve stimulation was interpreted as a technical failure and the prolonged effect of succinylcholine was discovered when general anaesthesia was terminated. Both patients had insufficient respiration. They were therefore re-sedated, transferred to the intensive care unit and the tracheas were extubated after full recovery from neuromuscular blockade. We recommend the use of monitoring every time these agents are used, even with short-acting drugs like succinylcholine and mivacurium.

Prolonged toxic effects after cocaine challenge in butyrylcholinesterase/plasma carboxylesterase double knockout mice: a model for butyrylcholinesterase-deficient humans.

Death and toxicity after cocaine use do not correlate with cocaine blood levels. One explanation for this observation is that cocaine abusers may posses one or more of the 58 possible known mutations in the butyrylcholinesterase gene (BCHE). Butyrylcholinesterase (BChE) serves as the primary cocaine hydrolase producing a nontoxic product ecgonine methyl ester. A reduction in endogenous levels of BChE may result in increased metabolism by hepatic carboxylesterase to produce norcocaine, a toxic product. Humans have carboxylesterase in tissues but not in plasma, whereas wild-type mice have significant amounts of carboxylesterase in tissues and plasma. Knockout mice with no plasma carboxylesterase were created to eliminate the contribution of plasma carboxylesterase in cocaine hydrolysis, thereby simulating human enzyme levels. This study tested the hypothesis that reductions in BChE such as those in humans with BChE mutations contribute to increased toxicity after cocaine use. Carboxylesterase and BChE double knockout mice, models for humans with BChE deficiency, were challenged with a nonlethal dose of 100 mg/kg (-)-cocaine. Carboxylesterase/BChE double knockout mice demonstrated toxic signs significantly longer than did wild-type and carboxylesterase knockout mice. The carboxylesterase/BChE-deficient mice took approximately 2.5 times as long to recover from cocaine toxicities, including the following: hypothermia, hyperactivity, stereotypical behavior, ocular effects, and dorsiflexion of the tail. The carboxylesterase/BChE double knockout mouse model demonstrates the importance of endogenous BChE for protection against cocaine toxicity and provides an in vivo system for studying drug sensitivity of humans who carry a BChE mutation.

Scutellarin protects against Aß-induced learning and memory deficits in rats: involvement of nicotinic acetylcholine receptors and cholinesterase.

AIM: To examine the protective effects of scutellarin (Scu) on rats with learning and memory deficit induced by ß-amyloid peptide (Aß). METHODS: Fifty male Wistar rats were randomly divided into 5 groups: control, sham operation, Aß, Aß+Scu, and Aß+piracetam groups. Aß(25-35) was injected into the lateral ventricle (10 µg each side). Scu (10 mg/2 mL) or piracetam (10 mg/2 mL was intragastrically administered per day for 20 consecutive days following Aß treatment. Learning and memory was assessed with Morris water maze test. The protein and mRNA levels of nicotinic acetylcholine receptor (nAChR) α4, α7, and ß2 subunits in the brain were examined using Western blotting and real-time PCR, respectively. The activities of acetylcholinesterase (AChE) and butyrylcholinesterase (BuChE) in the brain and plasma were measured using Ellman's colorimetric method. RESULTS: In Aß group, the escape latency period and first platform cross was significantly increased, and the total number of platform crossings was significantly decreased, as compared with the control and the sham operation groups. Both Scu and piracetam treatment significantly reduced the escape latency period and time to cross platform, and increased the number of platform crosses, but there were no significant differences between Aß+Scu and Aß+piracetam groups. In Aß group, the protein levels of nAChR α4 and α7 subunits in the cerebral cortex were significantly decreased by 42%-47% and 58%-61%, respectively, as compared to the control and the sham operation groups. Scu treatment caused upregulation of α4 and α7 subunit proteins by around 24% and 30%, respectively, as compared to Aß group, but there were no significant differences between Aß+Scu and Aß+piracetam groups. The protein level of nAChR ß2 subunit had no significant difference among different groups. The mRNA levels of nAChR α4, α7, and ß2 subunits were not significantly changed. In Aß group, the activities of AChE and BuChE in the brain were significantly increased, but were significantly decreased in the plasma, as compared to the control and the sham operation groups. Scu or piracetam treatment restored the activities in brain and plasma nearly to the levels in the control group. CONCLUSION: The results suggest that Scu may rescue some of the deleterious effects of Aß, possibly by stimulating nAChR protein translation and regulating cholinesterase activity.

Mechanism of hydrolysis of dicholine esters with long polymethylene chain by human butyrylcholinesterase.

Human butyrylcholinesterase hydrolyzes long chain dicholine esters more rapidly than short chain dicholine esters. The active site of butyrylcholinesterase is deeply buried within the enzyme molecule and there is limited space for binding of large compounds. Our goal was to understand how butyrylcholinesterase accommodates long chain dicholine esters to make them better substrates than short chain dicholine esters. For this purpose we studied the rate of hydrolysis of adipyldicholine (n=4) and sebacyldicholine (n=8) with mass spectrometry, a method that allowed monitoring the dicholine substrates, the monocholine intermediates, the dicarboxylic acid and choline products. It was shown that hydrolysis of adipyldicholine involves two consecutive steps, dicholine ester hydrolysis followed by relatively slow monocholine ester hydrolysis. However, sebacyldicholine was hydrolyzed at both choline ester sites, though hydrolysis of dicholine was faster than hydrolysis of monocholine. Sebacyldicholine was completely converted to sebacic acid and choline within 90 min, whereas only 15% of the adipyldicholine was converted to adipic acid in this time. Molecular modeling indicated that these dicholine esters can bind to butyrylcholinesterase in two energetically equivalent alternative conformations that may theoretically lead to hydrolysis. The long chain dicholine ester makes closer contact than the short chain ester between one of its carbonyl carbons and the catalytic Ser198, thus explaining why long-chain dicholine esters are hydrolyzed more rapidly by butyrylcholinesterase.

Genetic analysis of collagen Q: roles in acetylcholinesterase and butyrylcholinesterase assembly and in synaptic structure and function.

Acetylcholinesterase (AChE) occurs in both asymmetric forms, covalently associated with a collagenous subunit called Q (ColQ), and globular forms that may be either soluble or membrane associated. At the skeletal neuromuscular junction, asymmetric AChE is anchored to the basal lamina of the synaptic cleft, where it hydrolyzes acetylcholine to terminate synaptic transmission. AChE has also been hypothesized to play developmental roles in the nervous system, and ColQ is also expressed in some AChE-poor tissues. To seek roles of ColQ and AChE at synapses and elsewhere, we generated ColQ-deficient mutant mice. ColQ-/- mice completely lacked asymmetric AChE in skeletal and cardiac muscles and brain; they also lacked asymmetric forms of the AChE homologue, butyrylcholinesterase. Thus, products of the ColQ gene are required for assembly of all detectable asymmetric AChE and butyrylcholinesterase. Surprisingly, globular AChE tetramers were also absent from neonatal ColQ-/- muscles, suggesting a role for the ColQ gene in assembly or stabilization of AChE forms that do not themselves contain a collagenous subunit. Histochemical, immunohistochemical, toxicological, and electrophysiological assays all indicated absence of AChE at ColQ-/- neuromuscular junctions. Nonetheless, neuromuscular function was initially robust, demonstrating that AChE and ColQ do not play obligatory roles in early phases of synaptogenesis. Moreover, because acute inhibition of synaptic AChE is fatal to normal animals, there must be compensatory mechanisms in the mutant that allow the synapse to function in the chronic absence of AChE. One structural mechanism appears to be a partial ensheathment of nerve terminals by Schwann cells. Compensation was incomplete, however, as animals lacking ColQ and synaptic AChE failed to thrive and most died before they reached maturity.

The butyrylcholinesterase knockout mouse is obese on a high-fat diet.

Butyrylcholinesterase (BChE) inactivates the appetite stimulating hormone octanoyl-ghrelin. The hypothesis was tested that BChE-/- mice would have abnormally high body weight and high levels of octanoyl-ghrelin. It was found that BChE-/- mice fed a standard 5% fat diet had normal body weight. However, BChE-/- mice fed a diet containing 11% fat became obese. Their obesity was not explained by increased levels of octanoyl-ghrelin, or by increased caloric intake, or by decreased exercise. Instead, a role for BChE in fat utilization was suggested.

Human recombinant butyrylcholinesterase purified from the milk of transgenic goats interacts with beta-amyloid fibrils and suppresses their formation in vitro.

BACKGROUND: In Alzheimer's disease (AD), brain butyrylcholinesterase (BChE) co-localizes with beta-amyloid (Abeta) fibrils. AIMS: In vitro testing of the significance of this phenomenon to AD progress. METHODS: A thioflavine T (ThT) fluorogenic assay, photo-induced cross-linking and quantifiable electron microscopy served to compare the effect on Abeta fibril formation induced by highly purified recombinant human BChE (rBChE) produced in the milk of transgenic goats with that of serum-derived human BChE. RESULTS: Both proteins at 1:50 and 1:25 ratios to Abeta dose-dependently prolonged the ThT lag time and reduced the apparent rate of Abeta fibril formation compared to Abeta alone. Photo-induced cross-linking tests showed that rBChE prolonged the persistence of amyloid dimers, trimers and tetramers in solution, whereas Abeta alone facilitated precipitation of such multimers from solution. Transmission electron microscopy showed that rBChE at 1:100 to Abeta prevented the formation of larger, over 150-nm-long, Abeta fibrils and reduced fibril branching compared to Abeta alone as quantified by macro programming of Image Pro Plus software. CONCLUSION: Our findings demonstrate that rBChE interacts with Abeta fibrils and can attenuate their formation, extension and branching, suggesting further tests of rBChE, with unlimited supply and no associated health risks, as a therapeutic agent for delaying the formation of amyloid toxic oligomers in AD patients.

Comparison of cognitive functions between people with silent and wild-type butyrylcholinesterase.

In the human brain, butyrylcholinesterase (BuChE) is expressed in neurons and glia. For example, many nuclei in the human thalamus, with projections to the cerebral cortex, contain a large number of neurons with intense BuChE activity. Thalamocortical projections subserve a variety of cognitive functions. Due to genetic mutations, there are individuals who do not have detectable BuChE activity (silent BuChE). While the prevalence of silent BuChE is only 1:100,000 in European and American populations, it is 1:24 in the Vysya community in Coimbatore, India. To examine whether there are differences in cognitive functions between individuals with silent BuChE and those expressing normal BuChE (wild-type), twelve healthy individuals with silent BuChE and thirteen healthy individuals with wild-type BuChE, all from the Vysya community in Coimbatore, were tested for cognitive function using the Automated Neuropsychological Assessment Metrics test battery. The silent BuChE group was slightly faster on simple reaction tasks, but slower on a visual perceptual matching task. Furthermore, discriminant function analyses correctly classified 11/12 silent and 8/13 wild-type BuChE subjects (76% correct classification overall) based on BuChE status. Different profiles of cognitive test performance between individuals with silent and wild-type BuChE were observed. These observations suggest a function for BuChE in cognition.

A medical health report on individuals with silent butyrylcholinesterase in the Vysya community of India.

BACKGROUND: Butyrylcholinesterase (BChE; gi:116353) deficiency has adverse effects on the response to succinylcholine and mivacurium. A physiological function of BChE is to inactivate octanoyl ghrelin. We determined the health effect of complete absence of BChE in humans. METHODS: Clinical tests of cardiac, lung, liver, and kidney function, body weight, sperm counts and motility were performed on 5 men, age 20-32 y, in the Vysya community of Coimbatore, India who had silent BChE. Postmortem tissues from 2 cadavers with wild-type BChE were assayed. RESULTS: Test results were normal, except for lung function, which indicated mild obstruction in silent as well as in wild-type BChE subjects. Creatine kinase-MB levels were high in 2 subjects, but there were no other indications of damage to the heart. Body weight was normal. Family histories revealed no trend in disease susceptibility. The human body contains 10 times more BChE than acetylcholinesterase molecules. CONCLUSION: Individuals completely deficient in BChE have only minor abnormalities in clinical test results. However, they respond abnormally to standard doses of succinylcholine and mivacurium. It is expected, but not proven, that they are unusually susceptible to the toxicity of cocaine and organophosphorus pesticides, and resistant to bambuterol and irinotecan. Their normal body weight suggests alternative routes for deactivation of octanoyl ghrelin.

Sensitivity of butyrylcholinesterase knockout mice to (--)-huperzine A and donepezil suggests humans with butyrylcholinesterase deficiency may not tolerate these Alzheimer's disease drugs and indicates butyrylcholinesterase function in neurotransmission.

Butyrylcholinesterase (EC 3.1.1.8 BChE) is present in all human and mouse tissues, and is more abundant than acetylcholinesterase (EC 3.1.1.7 AChE) in all tissues except brain. People who have no BChE activity due to a genetic variation are healthy. This has led to the hypothesis that BChE has no physiological function. We tested this hypothesis by challenging BChE and AChE knockout mice, as well as wild-type mice, with the AChE specific inhibitors, (--)-huperzine A and donepezil, and with serine hydrolase inhibitors, echothiophate and chlorpyrifos oxon. (--)-Huperzine A and donepezil caused mortality and significant toxicity in the BChE-/- animals. The BChE heterozygote (BCHE+/-) mice with approximately one-half the BChE activity of the BChE wild type (BChE+/+) exhibited intermediate toxic symptoms, and survived a longer period. The BChE+/+ animals displayed comparatively minor toxic symptoms and recovered by 24h post-dosing. Plasma AChE activity was inhibited to the same extent in BChE-/-, +/-, and +/+ mice, whereas BChE activity was not inhibited. This indicated that the protective effect of BChE was not due to scavenging (--)-huperzine A. AChE-/- mice were unaffected by (--)-huperzine A and donepezil, demonstrating the specificity of these inhibitors for AChE. AChE-/- mice treated with chlorpyrifos oxon lost all BChE activity, had severe cholinergic symptoms and died of convulsions. This showed that BChE activity was essential for survival of AChE-/- mice. In conclusion, we propose that the protective effect of BChE is explained by hydrolysis of excess acetylcholine in physiologically relevant regions such as diaphragm, cardiac muscle, and brain. Thus, BChE has a function in neurotransmission. People with BChE deficiency are expected to be intolerant of standard doses of the anti-Alzheimer's drugs, (--)-huperzine A and donepezil.

Endochondral Ossification Is Accelerated in Cholinesterase-Deficient Mice and in Avian Mesenchymal Micromass Cultures.

Most components of the cholinergic system are detected in skeletogenic cell types in vitro, yet the function of this system in skeletogenesis remains unclear. Here, we analyzed endochondral ossification in mutant murine fetuses, in which genes of the rate-limiting cholinergic enzymes acetyl- (AChE), or butyrylcholinesterase (BChE), or both were deleted (called here A-B+, A+B-, A-B-, respectively). In all mutant embryos bone growth and cartilage remodeling into mineralizing bone were accelerated, as revealed by Alcian blue (A-blu) and Alizarin red (A-red) staining. In A+B- and A-B- onset of mineralization was observed before E13.5, about 2 days earlier than in wild type and A-B+ mice. In all mutants between E18.5 to birth A-blu staining disappeared from epiphyses prematurely. Instead, A-blu+ cells were dislocated into diaphyses, most pronounced so in A-B- mutants, indicating additive effects of both missing ChEs in A-B- mutant mice. The remodeling effects were supported by in situ hybridization (ISH) experiments performed on cryosections from A-B- mice, in which Ihh, Runx2, MMP-13, ALP, Col-II and Col-X were considerably decreased, or had disappeared between E18.5 and P0. With a second approach, we applied an improved in vitro micromass model from chicken limb buds that allowed histological distinction between areas of cartilage, apoptosis and mineralization. When treated with the AChE inhibitor BW284c51, or with nicotine, there was decrease in cartilage and accelerated mineralization, suggesting that these effects were mediated through nicotinic receptors (α7-nAChR). We conclude that due to absence of either one or both cholinesterases in KO mice, or inhibition of AChE in chicken micromass cultures, there is increase in cholinergic signalling, which leads to increased chondroblast production and premature mineralization, at the expense of incomplete chondrogenic differentiation. This emphasizes the importance of cholinergic signalling in cartilage and bone formation.

The key role of butyrylcholinesterase during neurogenesis and neural disorders: an antisense-5'butyrylcholinesterase-DNA study.

The wide tissue distribution of butyrylcholinesterase (BChE) in organisms makes specific roles possible, although no clear physiologic function has yet been assigned to this enzyme. In vertebrates, it appears e.g. in serum, hemopoietic cells, liver, lung, heart, at cholinergic synapses, in the central nervous system. in tumors and not at least (besides acetylcholinesterase, AChE) in developing embryonic tissues. Here, a functional role of BChE can be found in regulation of cell proliferation and the onset of differentiation during early neuronal development--independent of its enzymatic activity. For studies concerning this point, we have established a strategy for a specific and efficient inhibition of BChE to investigate how the expected decrease of enzyme and, therefore, the manipulation of cellular cholinesterase-equilibrium influences embryonic neurogenesis--among others to gain information about the significance of noncholinergic, activity-independent and cell growth functions of BChE. The antisense-5'BChE-DNA strategy is based on inhibition of BChE mRNA transcription and protein synthesis. For this, the BChE gene is cloned into a suitable vector system; this is done in antisense-orientation, so that a transfected cell will produce their own antisense mRNA to inhibit gene expression. For such investigations in neurogenesis, the developing retina is a good model and we are able to create organotypic, three-dimensional retinal aggregates in vitro (retinospheroids) using isolated retinal cells of 6-day-old chicken embryos. Using this in vitro retina and "knock out" of BChE gene expression, we could show a key role of BChE during neurogenesis. The results are of great interest because in tumorigenesis and some neuronal disorders, the BChE gene is amplified or abnormally expressed. It has to be discussed how the antisense-5'BChE strategy can play a role in the development of new and efficient therapy forms.

Learning performances and vulnerability to amyloid toxicity in the butyrylcholinesterase knockout mouse.

Butyrylcholinesterase (BChE) is an important enzyme for detoxication and metabolism of ester compounds. It also hydrolyzes the neurotransmitter acetylcholine (ACh) in pathological conditions and may play a role in Alzheimer's disease (AD). We here compared the learning ability and vulnerability to Aß toxicity in male and female BChE knockout (KO) mice and their 129Sv wild-type (Wt) controls. Animals tested for place learning in the water-maze showed increased acquisition slopes and presence in the training quadrant during the probe test. An increased passive avoidance response was also observed for males. BChE KO mice therefore showed enhanced learning ability in spatial and non-spatial memory tests. Intracerebroventricular (ICV) injection of increasing doses of amyloid-ß[25-35] (Aß25-35) peptide oligomers resulted, in Wt mice, in learning and memory deficits, oxidative stress and decrease in ACh hippocampal content. In BChE KO mice, the Aß25-35-induced deficit in place learning was attenuated in males and blocked in females. No change in lipid peroxidation or ACh levels was observed after Aß25-35 treatment in male or female BChE KO mice. These data showed that the genetic invalidation of BChE in mice augmented learning capacities and lowered the vulnerability to Aß toxicity.

Modeling effects of oxyanion hole on the ester hydrolysis catalyzed by human cholinesterases.

Molecular dynamics (MD) simulations and hydrogen bonding energy (HBE) calculations have been performed on the prereactive enzyme-substrate complexes (ES), transition states (TS1), and intermediates (INT1) for acetylcholinesterase (AChE)-catalyzed hydrolysis of acetylcholine (ACh), butyrylcholinesterase (BChE)-catalyzed hydrolysis of ACh, and BChE-catalyzed hydrolysis of (+)/(-)-cocaine to examine the protein environmental effects on the catalytic reactions. The hydrogen bonding of cocaine with the oxyanion hole of BChE is found to be remarkably different from that of ACh with AChE/BChE. Whereas G121/G116, G122/G117, and A204/A199 of AChE/BChE all can form hydrogen bonds with ACh to stabilize the transition state during the ACh hydrolysis, BChE only uses G117 and A199 to form hydrogen bonds with cocaine. The change of the estimated total HBE from ES to TS1 is ca. -5.4/-4.4 kcal/mol for AChE/BChE-catalyzed hydrolysis of ACh and ca. -1.7/-0.8 kcal/mol for BChE-catalyzed hydrolysis of (+)/(-)-cocaine. The remarkable difference of approximately 3 to 5 kcal/mol reveals that the oxyanion hole of AChE/BChE can lower the energy barrier of the ACh hydrolysis significantly more than that of BChE for the cocaine hydrolysis. These results help to understand why the catalytic activity of AChE against ACh is considerably higher than that of BChE against cocaine and provides valuable clues on how to improve the catalytic activity of BChE against cocaine.

Butyrylcholinesterase regulates laminar retinogenesis of the chick embryo in vitro.

During chicken neurogenesis, the sequential expression of butyrylcholinesterase (BChE) and acetylcholinesterase (AChE) between final cell proliferation and differentiation is functionally not understood. Recently, cholinesterases have been shown to regulate neurite growth in vitro. Here, we investigated the effects of inhibition of BChE on laminar histogenesis in retinospheroids that arise from dissociated embryonic chicken retinal cells in rotation culture. In the presence of the BChE inhibitor iso-OMPA (tetraisopropyl pyrophosphoramide), the number of spheroids/dish is increased, and their diameter is decreased by about 20%, corresponding to about 50% volume size. As a corollary, the course of histotypical differentiation is dramatically accelerated. Thus as a consequence of BChE inhibition both, organization of nuclear cell layers and of plexiform-like (neuropile) areas, as detected by an antibody to the fiber fasciculation protein F11, is temporally advanced by at least two days. Moreover, AChE is almost fully diminished in these areas. The results further demonstrate novel roles of cholinesterases during laminar histogenesis of coherent neural networks in vitro.

Role of butyrylcholinesterase in canine tracheal smooth muscle function.

The role of butyrylcholinesterase (BuChE) and acetylcholinesterase (AChE) in in regulating acetylcholine (ACh) lifetime was investigated by use of selective cholinesterase (ChE) inhibitors. Addition of 1 microM tetraisopropylpyrophosphoramide (iso-OMPA) led to a 98% inhibition of BuChE activity with little or no effect on AChE activity. This inhibition was accompanied by a 26% increase in the amplitude and a 43% prolongation in the half-relaxation time of contractions elicited by electric field stimulation (EFS). Coapplication of BW 284C51 (a selective AChE inhibitor) and 1 microM iso-OMPA resulted in increases of 2-fold in the amplitude and 10-fold in the half-relaxation time of EFS-induced contractions. These alterations were accompanied by small but sustained baseline contractures that were antagonized completely by incubation with exogenous BuChE (2.5 U/ml). The results suggest that BuChE serves to coregulate the lifetime of ACh in canine tracheal smooth muscle.

Slow axonal transport of the molecular forms of butyrylcholinesterase in a peripheral nerve.

Butyrylcholinesterase was found in chick sciatic nerve in four main molecular forms--G1, G2, G4 and A12--distinguishable by thier sedimentation coefficients in sucrose gradients (4.2S, 6.4S, 11.3S and 19S, respectively). Axonal transport of butyrylcholinesterase was studied by measuring the accumulation of its molecular forms on each side of a transected sciatic nerve. Twenty-four hours after transection, butyrylcholinesterase activity had risen by about 32% at the extremity of the proximal stump, and by 20% at the extremity of the distal stump. Proximal accumulation was due to a two-fold rise in G4 activity and to a six-fold rise in A12 activity, whereas distal accumulation was exclusively due to a 50% increase in G4 activity, accompanied by the complete loss of A12. The activities of G1 and G2 remained stable in both directions. Under our experimental conditions, the accumulation of butyrylcholinesterase activity cannot be attributable to local protein synthesis, cross-contamination with accumulated acetylcholinesterase or the presence of plasma butyrylcholinesterase. Hence we conclude that all A12 butyrylcholinesterase molecules were carried in the anterograde direction, moving at 11.6 +/- 4.2 mm/day, and that probably some of the G4 molecules were slowly transported in both directions. These findings suggest that some of the butyrylcholinesterase is located in the axonal mitochondria and/or axolemma.

Cholinesterases in neural development: new findings and toxicologic implications.

Developing animals are more sensitive than adults to acute cholinergic toxicity from anticholinesterases, including organophosphorus pesticides, when administered in a laboratory setting. It is also possible that these agents adversely affect the process of neural development itself, leading to permanent deficits in the architecture of the central and peripheral nervous systems. Recent observations indicate that organophosphorus exposure can affect DNA synthesis and cell survival in neonatal rat brain. New evidence that acetylcholinesterase may have a direct role in neuronal differentiation provides additional grounds for interest in the developmental toxicity of anticholinesterases. For example, correlative anatomic studies show that transient bursts of acetylcholinesterase expression often coincide with periods of axonal outgrowth in maturing avian, rodent, and primate brain. Some selective cholinesterase inhibitors effectively suppress neurite outgrowth in model systems like differentiating neuroblastoma cells and explanted sensory ganglia. When enzyme expression is altered by genetic engineering, acetylcholinesterase levels on the outer surface of transfected neurons correlate with ability to extend neurites. Certain of these "morphogenic" effects may depend on protein-protein interactions rather than catalytic acetylcholinesterase activity. Nonetheless, it remains possible that some pesticides interfere with important developmental functions of the cholinesterase enzyme family.

The neurochemistry of Parkinson's disease--comments on present and future developments.

The neurochemistry of Parkinson's disease is currently undergoing considerable changes. From the analysis of diseased tissue and of lesion models, attention has turned to basic questions of neuronal development and neuronal death including the role played by trophic factors in the protection and regeneration of nervous tissue. Here some aspects are highlighted which emphasize molecular targets of possible causes and of future therapies, e.g., transmitter transporters and novel trophic effectors.