Co-opting functions of cholinesterases in neural, limb and stem cell development

Acetylcholinesterase (AChE) is a most remarkable protein, not only because it is one of the fastest enzymes in nature, but also since it appears in many molecular forms and is regulated by elaborate genetic networks. As revealed by sensitive histochemical procedures, AChE is expressed specifically in many tissues during development and in many mature organisms, as well as in healthy and diseased states. Therefore it is not surprising that there has been a long-standing search for additional, "non-classical" functions of cholinesterases (ChEs). In principle, AChE could either act nonenzymatically, e.g. exerting cell adhesive roles, or, alternatively, it could work within the frame of classic cholinergic systems, but in non-neural tissues. AChE might be considered a highly co-opting protein, since possibly it combines such various functions within one molecule. By presenting four different developmental cases, we here review i) the expression of ChEs in the neural tube and their close relation to cell proliferation and differentiation, ii) that AChE expression reflects a polycentric brain development, iii) the retina as a model for AChE functioning in neural network formation, and iv) nonneural ChEs in limb development and mature bones. Also, possible roles of AChE in neuritic growth and of cholinergic regulations in stem cells are briefly outlined.     

Cholinesterases in development and disease

Cholinesterases (ChEs) including acetylcholinesterase (AChE) and butyrylcholinesterase (BuChE) are abundant in the nervous system and other tissues. Here we describe two different aspects of ChEs and the cholinergic system. The first aspect concerns the role of cholinergic transmission in central pattern generation in the neonatal rat spinal cord and the second one describes the involvement of ChEs in the pathologies of dystrophin-deficient mutant (mdx) mice, the animal model of Duchenne muscular dystrophy. Thus, this study is divided into two distinct parts. In the first part we show that AChE is abundant in ventral horn neurons, central canal-adjacent and partition neurons in all the observed segments (L2, L5, S1, and S2). AChE was also found in the intermediolateral and sacral parasympathetic nuclei of L2 and S1, respectively. Blocking the AChE by edrophonium produced non-stationary bursting in spinal cord preparations of developing rats. Cross-wavelet/coherence analyses of the data revealed epochs of locomotor-like activity (left-right and flexor-extensor alternation) followed by other rhythmic or non-rhythmic bursting patterns. Addition of exogenous ACh stabilized the rhythm and increased the incidence of locomotor-like pattern in the preparations. Thus, the cholinergic system in the spinal cord is capable of producing and modulating functional rhythmic bursts. Moreover, bath-applied edrophonium and exogenous ACh were found as potent means of modulation of the locomotor rhythm produced by stimulation of sacrocaudal afferents (SCAs). We show that a subclass of sacral neurons with contralateral funicular projections to the thoracolumbar cord is associated with the cholinergic system. This group of neurons may play a major role in the observed enhancement of the SCA-induced motor rhythm. In the second part we show that adult mdx-muscles are malformed with distorted neuromuscular junctions (nmjs) and impaired regulation of acetylcholine receptors. Examination of circulating ChE levels revealed that in mdx-sera, while AChE activity was elevated, BuChE activity was markedly lower than in wild-type (wt) sera. Thus, BuChE to AChE ratio in mouse sera decreased from 6:1 in wt control to 3:1 in mdx. Because serum ChE levels may be modulated by gonadal steroids, it is possible that lack of dystrophin in mdx-mice may affect this regulation. Further studies are in progress to determine the potential endocrine regulation of ChEs in circulation and at the nmjs of mdx- and wt-mice. These studies will help clarify whether the hormonal regulation is impaired in the mdx mutant, and whether changes in circulating ChE reflect or influence the functional deficits observed in excitable tissues of diseased states.     

Ultrastructural distribution of AChE in Catenula leptocephala (Nuttycombe, 1956)

Summary Acetylcholinesterase activity (AChE, E.C. 3.1.1.7) was examined in different tissues of Catenula leptocephala (Nuttycombe, 1956). Eserine and iso-OMPA were used to distinguish AChE from non-specific cholinesterases (ChE, E.C. 3.1.1.8). The enzyme was located mainly in the brain neuropil, the peripheral nervous system, neuromuscular junctions, on the membrane of muscle cells and of cells with rhabdites. The distribution of the enzyme suggests that cholinergic transmission occurs in Catenula leptocephala, while simultaneously the presence of AChE on the membranes of muscle cells points to the receipt of cholinergic stimulation. The role of AChE in differentiation and maturation of cells with rhabdites is also discussed in this paper.     

Non-classical actions of cholinesterases: Role in cellular differentiation, tumorigenesis and Alzheimer''s disease

The cholinesterases are members of the serine hydrolase family, which utilizes a serine residue at the active site. Acetylcholinesterase (AChE) is distinguished from butyrylcholinesterase (BChE) by its greater specificity for hydrolysing acetylcholine. The function of AChE at cholinergic synapses is to terminate cholinergic neurotransmission. However, AChE is expressed in tissues that are not directly innervated by cholinergic nerves. AChE and BChE are found in several types of haematopoietic cells. Transient expression of AChE in the brain during embryogenesis suggests that AChE may function in the regulation of neurite outgrowth. Overexpression of cholinesterases has also been correlated with tumorigenesis and abnormal megakaryocytopoiesis. Acetylcholine has been shown to influence cell proliferation and neurite outgrowth through nicotinic and muscarinic receptor-mediated mechanisms and thus, that the expression of AChE and BChE at non-synaptic sites may be associated with a cholinergic function. However, structural homologies between cholinesterases and adhesion proteins indicate that cholinesterases could also function as cell-cell or cell-substrate adhesion molecules. Abnormal expression of AChE and BChE has been detected around the amyloid plaques and neurofibrillary tangles in the brains of patients with Alzheimer's disease. The function of the cholinesterases in these regions of the Alzheimer brain is unknown, but this function is probably unrelated to cholinergic neurotransmission. The presence of abnormal cholinesterase expression in the Alzheimer brain has implications for the pathogenesis of Alzheimer's disease and for therapeutic strategies using cholinesterase inhibitors.     

Ultrastructural distribution of AChE in Catenula leptocephala (Nuttycombe, 1956).

Acetylcholinesterase activity (AChE, E.C. 3.1.1.7) was examined in different tissues of Catenula leptocephala (Nuttycombe, 1956). Eserine and iso-OMPA were used to distinguish AChE from non-specific cholinesterases (ChE, E.C. 3.1.1.8). The enzyme was located mainly in the brain neuropil, the peripheral nervous system, neuromuscular junctions, on the membrane of muscle cells and of cells with rhabdites. The distribution of the enzyme suggests that cholinergic transmission occurs in Catenula leptocephala, while simultaneously the presence of AChE on the membranes of muscle cells points to the receipt of cholinergic stimulation. The role of AChE in differentiation and maturation of cells with rhabdites is also discussed in this paper.     

Indophenol Chromogenic Substrates of Cholinesterases of Different Origin

An analysis of influence of indophenol substrate structure on rate of their enzymatic hydrolysis under action of cholinesterases (ChE) of different animals is carried out for the first time. Study of indophenylacetate (IPhA) and a group of isomeric dichloroderivatives as substrates of erythrocyte acetylcholinesterase, serum butyrylcholinesterase, and ChE from optical ganglia of the Pacific squid Todarodes pacificus allowed us to reveal a role of steric and inductive effects of the substrates molecule in enzymatic catalysis, as well as differences in substrate specificity of the studied ChE. This comparative enzymologic aspect of the work was evident to a greater degree at studying hydrolysis of choline (acetylcholine, acetylthiocholine) and indophenol (IPhA, 2,6-dichloroindophenylacetate, 2,6-dichloro-3´-methyl indophenylacetate) esters under action of mammalian blood ChEs, ChE from hemolymph of the gastropod mollusc Neptunea, and also of ChE from the nervous tissue of different species of Pacific squids and of the cabbage root fly. Differences in values of the kinetic parameters characterizing sorption and catalytic stages of the hydrolysis process are revealed. Comparison of substrate properties of choline and indophenol esters enabled us to compare enzymes in terms of hydrophobic-hydrophilic interactions.     

Cross-Homologies and Structural Differences Between Human Cholinesterases Revealed by Antibodies Against cDNA-Produced Human Butyrylcholinesterase Peptides

To study the polymorphism of human cholinesterases (ChEs) at the levels of primary sequence and three-dimensional structure, a fragment of human butyrylcholinesterase (BuChE) cDNA was subcloned into the pEX bacterial expression vector and its polypeptide product analyzed. Immunoblot analysis revealed that the clone-produced BuChE peptides interact specifically with antibodies against human and Torpedo acetylcholinesterase (AChE). Rabbit polyclonal antibodies prepared against the purified clone-produced BuChE polypeptides interacted in immunoblots with denatured serum BuChE as well as with purified and denatured erythrocyte AChE. In contrast, native BuChE tetramers from human serum, but not AChE dimers from erythrocytes, interacted with these antibodies in solution to produce antibody-enzyme complexes that could be precipitated by second antibodies and that sedimented faster than the native enzyme in sucrose gradient centrifugation. Furthermore, both AChE and BuChE dimers from muscle extracts, but not BuChE tetramers from muscle, interacted with these antibodies. To reveal further whether the anti-cloned BuChE antibodies would interact in situ with ChEs in the neuromuscular junction, bundles of muscle fibers were microscopically dissected from the region in fetal human diaphragm that is innervated by the phrenic nerve. Muscle fibers incubated with the antibodies and with 125I-Protein A were subjected to emulsion autoradiography, followed by cytochemical ChE staining. The anti-cloned BuChE antibodies, as well as anti-Torpedo AChE antibodies, created patches of silver grains in the muscle endplate region stained for ChE, under conditions where control sera did not. These findings demonstrate that the various forms of human AChE and BuChE in blood and in neuromuscular junctions share sequence homologies, but also display structural differences between distinct molecular forms within particular tissues, as well as between similarly sedimenting molecular forms from different tissues.     

High aryl acylamidase activity associated with cobra venom acetylcholinesterase: Biological significance

Investigation of the non-classical functions of cholinesterases (ChEs) has been the subject of interest in the past three decades. One of which is aryl acylamidase (AAA) activity associated with ChEs, but characterized in in vitro, as an enzyme, splitting the artificial substrate o-nitroacetanilide with unknown physiological function. In the present study, we have compared levels of AAA activity of AChE from different sources like goat brain, electric eel organ and from venoms of different snakes. Remarkably cobra venom showed the highest AAA activity and also high AAA/AChE ratio. Both serotonergenic and cholinergic inhibitors inhibited the cobra venom AAA activity in a concentration dependent manner, which also underlines the association of AAA with AChE of cobra venom. The study becomes interesting because of i) the cobra venom AChE exists in monomeric globular forms; ii) in Alzheimer's disease too the most abundant forms of cholinesterases are monomeric globular forms, thought to be involved in the pathogenesis of Alzheimer's disease; iii) the effect of Alzheimer's disease drugs on the AAA activity of cobra venom, indicated that AAA activity of cobra venom was more sensitive than AChE and iv) Huperzine and Tacrine showed more pronounced effect on AAA. Thus, this study elucidates the high AAA associated with cobra venom AChE may serve as one of the prominent activity to test the pharmacological effect of AD drugs, as other sources were found to have lower activity.     

Characterization and use of the total head soluble cholinesterases from mosquitofish (Gambusia holbrooki) for screening of anticholinesterase activity

Inhibition of cholinesterases (ChE) has been widely used as an environmental biomarker of exposure to organophosphates (OP) and carbamate (CB) pesticides. Different ChE isoforms may be present in the same tissue and may present distinct sensitivities towards environmental contaminants. The present work characterises the soluble ChE present in mosquitofish (Gambusia holbrooki) total head homogenates, through the use of different substrates and selective inhibitors of cholinesterasic activity. Furthermore, the effects of sodium dodecylsulphate (SDS) on the enzymatic activity were investigated, both in vivo and in vitro. These results showed that acetylcholinesterase (AChE) seemed to be the predominant form present in head homogenates of G. holbrooki, despite the inhibition by tetraisopropylpyrophosphoramide (iso-OMPA) found at high concentrations. SDS was responsible for in vitro, but not in vivo, inhibitory effects. The in vitro AChE inhibitory effects of SDS was partially prevented by the use of increasing amounts of ethanol, suggesting that the inhibition was induced by an emulsion effect, which may explain the lack of effect in vivo.     

The effect in vitro of volatile anesthetics on the activity of cholinesterases.

Because the mechanism of anesthesia is unknown, the relationship between anesthetics and enzymes essential to brain function may be an important one. Therefore, the effect of 8 volatile anesthetics on the enzymatic activity of solubilized, purified dog brain and human erythrocyte acetylcholinesterase (AChE) and human serum cholinesterase (ChE) was studied in vitro. Serum ChE was found to be insensitive to saturated solutions of all the anesthetics studied. However, brain and erythrocyte AChE were reversibly inhibited in a dose-dependent manner by all 8 anesthetics in concentrations exceeding those used in clinical practice. Kinetic analysis revealed a mixed (competitive, non-competitive) type of inhibition with the exception of the ether-crythrocyte AChE interaction which was characterized by competitive inhibition. Ether and methoxyflurane were found to depress the AChE activity the most and isoflurane and enflurane the least. The concentrations of anesthetic in the gas phase necessary for 50% inhibition of erythrocyte AChE activity (I₅₀) were calculated for 5 anesthetics and found to correlate with their water-gas partition coefficients. These data suggest that the effect in vitro of volatile anesthetics on the catalytic activity of cholinesterases is a variable one and may be unrelated to anesthetic potency in vivo. The implications of these data concerning anesthetic-active site interactions are discussed.     

Molecular characterization of Hydra acetylcholinesterase and its catalytic activity

A full-length cDNA encoding an acetylcholinesterase (AChE) from Hydra magnipapillata was isolated. All of the important aromatic residues that line a catalytic gorge in cholinesterases of other species were conserved, but the sequences of peripheral anionic and choline binding sites were not. Hydra AChE, expressed in Xenopus oocytes, showed AChE activity. The gene was expressed in both ectodermal and endodermal epithelial cells except for the tentacles and basal disk. AChE gene expression was not detected in the regenerating tips in either the head or the foot, indicating that regeneration is controlled by the non-neuronal cholinergic system in Hydra.     

The expression of cholinesterases in human renal tumours varies according to their histological types

The change in the expression of acetylcholinesterase (AChE) and butyrylcholinesterase (BuChE) activities in neoplastic colon and lung prompted us to study the possible effect of cancer on the expression of cholinesterases (ChEs) in kidney. Samples of papillary renal cell carcinoma (pRCC), conventional RCC (cRCC), chromophobe RCC (chRCC) and renal oncocytoma (RON), beside adjacent non-cancerous tissues, were analyzed. In pRCC both AChE and BuChE activities were statistically increased; in cRCC and chRCC only AChE activity increased and in RON neither AChE nor BuChE activities were affected. Abundant amphiphilic AChE dimers (G(2)(A)) and fewer monomers (G(1)(A)) were identified in healthy kidney as well as in all tumour classes. Incubation with PIPLC revealed glycosylphosphatidylinositol in AChE forms. BuChE is distributed between principal G(4)(H), fewer G(1)(H), and much fewer G(4)(A) and G(1)(A) species. RT-PCR showed similar amounts of AChE-H, AChE-T and BuChE mRNAs in healthy kidney. Their levels increased in pRCC but not in the other tumour types. The data support the idea that, as in lung tumours, in renal carcinomas expression of ChE mRNAs, biosynthesis of molecular components and level of enzyme activity change according to the specific kind of cell from which tumours arise.     

Expression of cholinesterase gene(s) in human brain tissues: translational evidence for multiple mRNA species

To resolve the origin(s) of the molecular heterogeneity of human nervous system cholinesterases (ChEs), we used Xenopus oocytes, which produce biologically active ChE when microinjected with unfractionated brain mRNA. The RNA was prepared from primary gliomas, meningiomas and embryonic brain, each of which expresses ChE activity with distinct substrate specificities and molecular forms. Sucrose gradient fractionation of DMSO-denatured mRNA from these sources revealed three size classes of ChE-inducing mRNAs, sedimenting at approximately 32S, 20S and 9S. The amounts of these different classes of ChE-inducing mRNAs varied between the three tissue sources examined. To distinguish between ChEs produced in oocytes and having different substrate specificities, their activity was determined in the presence of selective inhibitors. Both 'true' (acetylcholine hydrolase, EC 3.1.1.7) and 'pseudo' (acylcholine acylhydrolase, EC 3.1.1.8) multimeric cholinesterase activities were found in the mRNA-injected oocytes. Moreover, human brain mRNAs inducing 'true' and 'pseudo' ChE activities had different size distribution, indicating that different mRNAs might be translated into various types of ChEs. These findings imply that the heterogeneity of ChEs in the human nervous system is not limited to the post-translational level, but extends to the level of mRNA.     

Cholinesterases: New Roles in Brain Function and in Alzheimer's Disease

The most important therapeutic effect of cholinesterase inhibitors (ChEI) on approximately 50% of Alzheimer's disease (AD) patients is to stabilize cognitive function at a steady level during a 1-year period of treatment as compared to placebo. Recent studies show that in a certain percentage (approximately 20%) of patients this cognitive stabilizing effect can be prolonged up to 24 months. This long-lasting effect suggests a mechanism of action other than symptomatic and cholinergic. In vitro and in vivo studies have consistently demonstrated a link between cholinergic activation and APP metabolism. Lesions of cholinergic nuclei cause a rapid increase in cortical APP and CSF. The effect of such lesions can be reversed by ChEI treatment. Reduction in cholinergic neurotransmission–experimental or pathological, such as in AD–leads to amyloidogenic metabolism and contributes to the neuropathology and cognitive dysfunction. To explain the long-term effect of ChEI, mechanisms based on -amyloid metabolism are postulated. Recent data show that this mechanism may not necessarily be related to cholinesterase inhibition. A second important aspect of brain cholinesterase function is related to enzymatic differences. The brain of mammals contains two major forms of cholinesterases: acetylcholinesterase (AChE) and butyrylcholinesterase (BuChE). The two forms differ genetically, structurally, and for their kinetics. Butyrylcholine is not a physiological substrate in mammalian brain, which makes the function of BuChE of difficult interpretation. In human brain, BuChE is found in neurons and glial cells, as well as in neuritic plaques and tangles in AD patients. Whereas, AChE activity decreases progressively in the brain of AD patients, BuChE activity shows some increase. To study the function of BuChE, we perfused intracortically the rat brain with a selective BuChE inhibitor and found that extracellular acetylcholine increased 15-fold from 5 nM to 75 nM concentrations with little cholinergic side effect in the animal. Based on these data and on clinical data showing a relation between cerebrospinal fluid (CSF) BuChE inhibition and cognitive function in AD patients, we postulated that two pools of cholinesterases may be present in brain, the first mainly neuronal and AChE dependent and the second mainly glial and BuChE dependent. The two pools show different kinetic properties with regard to regulation of ACh concentration in brain and can be separated with selective inhibitors. Within particular conditions, such as in mice nullizygote for AChE or in AD patients at advanced stages of the disease, BuChE may replace AChE in hydrolizing brain acetylcholine.     

Comparison of methods used for the determination of cholinesterase activity in whole blood

Cholinesterases (ChEs) are classified as either acetylcholinesterase (AChE) or butyrylcholinesterase (BChE) based on their substrate and inhibitor specificity. Organophosphate and carbamate compounds commonly represented by herbicides, pesticides, and nerve gases irreversibly inhibit ChEs. Therefore, exposure to organophosphates and carbamates is normally assessed by measuring ChE activity in blood. There are two approaches for measuring AChE and BChE activity present in whole blood: (1) separating blood into erythrocytes, which contain only AChE, and plasma which contains only BChE, to measure their activity individually, or (2) use a BChE-specific inhibitor to measure the activity of AChE in whole blood. A number of studies have reported the use of different inhibitors for the simultaneous measurement of AChE and BChE activities. However, the inhibitors used for completely inhibiting BChE activity also inhibited AChE activity leading to errors in reported values. The goal of this study was to find the most accurate and simple method for the simultaneous determination of AChE and BChE activity in animal whole blood. Solutions containing human AChE and BChE in various proportions were prepared and AChE and BChE activities were measured using three reported methods. Results demonstrate that ethopropazine and (-) huperzine A appear to be the most specific ChE inhibitors. Preliminary results with human and animal whole blood suggest that 20muM ethopropazine and 500nM (-) huperzine A can be used for measuring AChE and BChE activities across species.     

Characterization and use of the total head soluble cholinesterases from mosquitofish (Gambusia holbrooki) for screening of anticholinesterase activity

Inhibition of cholinesterases (ChE) has been widely used as an environmental biomarker of exposure to organophosphates (OP) and carbamate (CB) pesticides. Different ChE isoforms may be present in the same tissue and may present distinct sensitivities towards environmental contaminants. The present work characterises the soluble ChE present in mosquitofish (Gambusia holbrooki) total head homogenates, through the use of different substrates and selective inhibitors of cholinesterasic activity. Furthermore, the effects of sodium dodecylsulphate (SDS) on the enzymatic activity were investigated, both in vivo and in vitro. These results showed that acetylcholinesterase (AChE) seemed to be the predominant form present in head homogenates of G. holbrooki, despite the inhibition by tetraisopropylpyrophosphoramide (iso-OMPA) found at high concentrations. SDS was responsible for in vitro, but not in vivo, inhibitory effects. The in vitro AChE inhibitory effects of SDS was partially prevented by the use of increasing amounts of ethanol, suggesting that the inhibition was induced by an emulsion effect, which may explain the lack of effect in vivo.     

How various substrates activate the process of enzymatic hydrolysis by different cholinesterases

Kinetic analysis of the activating effect of substrate on the cholinesterase catalysis is performed. There are determined values of coefficient of activation A in the pH zone 5 for the process of hydrolysis of acetylcholine, indophenylacetate (IPhA), and 2,6-dichlorophenolindoph enylacetate (DIPhA) by cholinesterase (ChE) of horse blood serum, as well as of IPhA and DIPhA by ChE of optical ganglia of the Pacific squid Todarodes pacificus. The phenomenon of activation has not been revealed at hydrolysis of phenylacetate by the horse blood serum ChE. The conclusion is made that the cause of the activating effect of substrate on the process of enzymatic hydrolysis by ChEs of different origin is the presence of the onium grouping in the structure of substrates.     

Anthelmintic efficacy of flemingia vestita (fabaceae): Genistein-induced alterations in the esterase activity in the cestode,raillietina echinobothrida

To ascertain the anthelmintic efficacy ofFlemingia vestita (an indigenous leguminous plant of Meghalaya, having putative anthelmintic usage), its crude root-tuber peel extract and active chemical principle, genistein, were testedin vitro with reference to esterase activity in the fowl tapeworm,Raillietina echinobothrida. With the localization of non-specific esterases (NSE) and cholinesterase (ChE), the organization of the cholinergic components of the nervous system in toto could be visualized in the cestodeo The specific ChE in the parasite is acetylcholinesterase (AChE). Both NSE and ChE were found in close association with the central and peripheral nervous components, besides being present in the tegument and muscular parts of the terminal male genitalia. The whole tissue homogenate of the parasite also showed a high AChE activity. After exposure to the crude peel extract (50 mg/ml of the incubation medium) and to genistein (0.5 mg/ml), a pronounced decline in the visible stain intensity in the cholinergic components of the nervous system and in the tegument was noticeable, indicating extremely reduced activity of NSE and ChE in these sites. The total AChE activity was also reduced to 4907% and 56–77%, following treatment with the peel extract and genistein, respectively. The reference drug, praziquantel (0.01 mg/ml) also caused reduction in the enzyme activity, somewhat at par with the genistein treatment. Genistein appears to have a transtegumental mode of action. Alteration in the AChE activity points towards acetylcholine, an inhibitory neurotransmitter in cestodes, as the potential target of action.