The cholinergic system in the early development of chickens. 1. Choline acetyltransferase and acetylcholinesterase as the key regulators of acetylcholine metabolism

Acetylcholine esterase (AChE, EC 3.1.1.7) and choline acetyltransferase (CAT, EC 2.3.1.6) activities were studied in the early chick embryos. Gastrulation is accompanied by a sharp increase in the AChE activity which was most pronounced in anterior hypoblast. Three molecular of AChE (4.7, 6.8 and 10.9 S) were identified in the extract of chick embryos using a sucrose density gradient centrifugation. The CAT activity remained unchanged during gastrulation but increased twice at the end of gastrulation.     

The role of extracellular signals in the differentiation of cholinergic neurons from the CNS and PNS in culture

Rat skeletal muscle cells release in culture a macromolecule which stimulates by 25-100 fold the development of choline acetyltransferase (CAT) in cultures of new-born rat sympathetic neurons. This "cholinergic factor" impaired the development of three norepinephrine synthesizing enzymes and of acetylcholinesterase (AChE) in these cultures. The 16S form of AChE failed to develop in cultures grown with the factor, but amounted to 30-40% in 3-week old cultures grown in its absence. Using the development of CAT activity in sympathetic neuron cultures as an assay, the cholinergic factor has been partially purified in 6 steps, and its hydrodynamic parameters determined. The effects of this factor on sympathetic neurotransmitter choice were qualitatively reproduced by 1-10 mM Na butyrate. The cholinergic factor increased CAT activity and decreased AChE in neuron cultures from new-born rat nodose ganglia. The factor also stimulated CAT activity in rat embryo (E14) spinal cord cultures, but stimulated the development of AChE in these cultures.     

Axotomy induced changes in cholinergic enzymes in rat nerve and muscles.

Abstract— The effect of axotomy on acetylcholinesterase (AChE. EC 3.1.1.7). butyrylcholinesterase (BuChE. EC 3.1.1.8) and choline acetyltransferase (CAT, EC 2.3.1.6) activities in sciatic nerve stumps above (proximal) and below (distal) the site of transection. as well as in soleus (SOL) and extensor digitorum longus (EDL) muscles, has been studied in rat. Within 2 weeks. AChE activity decreased on a per mm basis, in proximal nerve by 65%, in distal nerve by 80% and on a per whole muscle basis, in denervated muscle by 85%. BuChE activity increased in proximal nerve and distal nerve to 150% of control and decreased in muscle to 51% of control. CAT activity in the proximal nerve stump was reduced to 70% of control and in the distal stump to less than 1% within 1 week. CAT activity in muscle decreased to 20 and 30% of control for soleus and EDL respectively during a 14 day period. The reduction in AChE and CAT activity observed in the proximal nerve segment may reflect changes in the synthesis, as well as the transport and local degradation of these enzymes. Previous studies on axotomy induced changes have not examined the simultaneous changes in proximal and distal nerve as well as denervated muscle in a single animal species.     

Effects of phenytoin on acetylcholinesterase activity and cell protein in cultured chick embryonic skeletal muscle

Cultured pectoral muscle from 11-day-old chick embryos was treated for 48 h with phenytoin (diphenylhydantoin, DPH) in concentrations ranging from 15 to 270 microgram/ml on days 7-9 in vitro. Acetylcholinesterase (AChE, EC 3.1.1.7), creatine phosphokinase (CPK, EC 2.7.3.2), and lactic dehydrogenase (LDH, EC 1.1.1.27) activities, [3H]leucine incorporation into protein, and total protein of the cultures decreased in a dose-related manner with DPH concentrations of 30 microgram/ml and greater. Total AChE activity and AChE activity released into the medium were specifically decreased with 15 microgram DPH per millilitre. In cultures treated chronically with 15 microgram DPH per millilitre on days 5-13 in vitro, total AChE activity and AChE activity released into the medium were 66.0 +/- 13.2 and 64.7 +/- 11.8% of untreated controls, respectively, but cellular AChE activity, cell protein, and [3H]leucine incorporation into protein were unaffected. The results indicate that DPH specifically decreases the total net synthesis of AChE activity by a direct action on cultured chick embryo muscle.     

16 S acetylcholinesterase in endplate-free regions of developing rat diaphragm

Velocity sedimentation patterns of acetylcholinesterase (AChE, EC 3.7.1.1) in endplate-free regions of the diaphragm were studied in rats during early postnatal development. A significant amount of 16 S AChE, comprising 20% total activity, was found in endplate-free regions of the diaphragm of 8- and 19-day-old rats. By 32 days after birth, 16 S AChE accounted for less than 5% total AChE activity in endplate-free regions. 16 S AChE is, therefore, not strictly an endplate-specific molecular form. Instead, it becomes restricted to the motor endplate region of the rat diaphragm by the end of the first month of life.     

Recovery of acetylcholinesterase activity after irreversible inhibition by organophosphorous compounds in embryonic development

1. Recovery of acetylcholinesterase (AChE) activity was studied using the embryos of sea urchins Strongylocentrotus intermedius and S. nudus, embryos of axolotl Ambystoma mexicanum and in the chick embryo muscle culture treated by "irreversible" organophosphorous inhibitors (OPI). 2. AChE activity was assayed by a modified Ellman's procedure. 3. It follows from the data obtained that, unlike the plutei of sea urchins and the monolayer culture of chick embryo muscle cells, the embryos of axolotl show a compensatory increase in AChE biosynthesis after inhibition by OPI. 4. This mechanism is assumed to be related to the presence of a well developed neuromuscular system in the A. mexicanum embryos. 5. It is possible that acetylcholine accumulated as a result of partial AChE inhibition is responsible for the compensatory increase in AChE biosynthesis.     

Separation in a single step by affinity chromatography of cholinesterases differing in subunit number.

We describe an affinity chromatography method in which dimethylaminoethylbenzoic acid-Sepharose 4B is used, making it possible to separate in one step the molecular forms of globular acetylcholinesterase (AChE, EC 3.1.1.7) or butyrylcholinesterase (ChE, EC 3.1.1.8). A crude extract containing these enzymes was deposited onto the chromatography gel, washed, and eluted by a linear gradient of tetramethylammonium chloride (0-0.3 M). With rat brain AChE, two well-separated peaks were eluted in the presence of 1% Triton X-100; the first peak corresponded to 4 S forms and the second to 11 S forms. This separation was very efficient for salt-soluble activity and less efficient for the detergent-soluble AChE. In this case, the 4 S peak represented only 6.5% of total detergent-soluble activity and was cross-contaminated by the 11 S form. Rat serum ChE was efficiently separated into two peaks of 7 S and 11 S. This method could potentially be adapted to separate other multimeric proteins with varying numbers of affinity sites.     

Monoclonal Antibodies Specific for the Different Subunits of Asymmetric Acetylcholinesterase from Chick Muscle

The asymmetric (20S) acetylcholinesterase (AChE, EC 3.1.1.7) from 1-day-old chick muscle, purified on a column on which was immobilised a monoclonal antibody (mAb) to chick brain AChE, was used to immunise mice. Eight mAbs against the muscle enzyme were hence isolated and characterised. Five antibodies (4A8, 1C1, 10B7, 7G8, and 8H11) recognise a 110-kilodalton (kDa) subunit with AChE catalytic activity, one antibody (7D11) recognises a 72-kDa subunit with pseudocholinesterase or butyrylcholinesterase (BuChE, EC 3.1.1.8) catalytic activity, and two antibodies (6B6 and 7D7) react with the 58-kDa collagenous tail unit. Those three polypeptides can be recognised together in the 20S enzyme used, which is a hybrid AChE/BuChE oligomer. Antibodies 6B6 and 7D7 are specific for asymmetric AChE. Four of the mAbs recognising the 110-kDa subunit were reactive with it in immunoblots. Sucrose density gradient analysis of the antibody-enzyme complexes showed that the anti-110-kDa subunit mAbs cross-link multiple 20S AChE molecules to form large aggregates. In contrast, there is only a 2-3S increase in the sedimentation constant with the mAbs specific for the 72-kDa or for the 58-kDa subunit, suggesting that those subunits are more inaccessible in the structure to intermolecular cross-linking. The 4A8, 10B7, 7D11, and 7D7 mAbs showed cross-reactivity to the corresponding enzyme from quail muscle; however, none of the eight mAbs reacted with either enzyme type from mammalian muscle or from Torpedo electric organ. All eight antibodies showed immunocytochemical localisation of the AChE form at the neuromuscular junctions of chicken twitch muscles.     

Characterization of acetylcholinesterase molecular forms in slow and fast muscle of rat

Multiple molecular forms of acetylcholinesterase (AChE EC 3.1.1.7) from fast and slow muscle of rat were examined by velocity sedimentation. The fast extensor digitorum longus muscle (EDL) hydrolyzed acetylcholine at a rate of 110 mumol/g wet weight/hr and possessed three molecular forms with apparent sedimentation coefficients of 4S, 10S, and 16S which contribute about 50, 35, and 15% of the AChE activity. The slow soleus muscle hydrolyzed acetylcholine at a rate of 55 mumol/g wet weight/hr and has a 4S, 10S, 12S, and 16S form which contribute 22, 18, 34, and 26% of AChE activity, respectively. A single band of AChE activity was observed when a 1M NaCl extract with CsCl (0.38 g/ml) was centrifuged to equilibrium. Peak AChE activity from EDL and SOL extracts were found at 1.29 g/ml. Resedimentation of peak activity from CsCl gradients resulted in all molecular forms previously found in both muscles. Addition of a protease inhibitor phenylmethylsulfonyl chloride did not change the pattern of distribution. The 4S form of both muscles was extracted with low ionic strength buffer while the 10S, 12S, and 16S forms required high ionic strength and detergent for efficient solubilization. All molecular forms of both muscles have an apparent Km of 2 x 10(-4) M, showed substrate inhibition, and were inhibited by BW284C51, a specific inhibitor of AChE. The difference between these muscles in regards to their AChE activity, as well as in the proportional distribution of molecular forms, may be correlated with sites of localization and differences in the contractile activity of these muscles.     

In vitro and in vivo studies of potential biomarkers of lead and uranium contamination: lipid peroxidation, acetylcholinesterase, catalase and glutathione peroxidase activities in three non-mammalian species

The aim of this work was to assess the relationships between lead (Pb) and uranium (U) exposure, lipid peroxidation and some enzyme activities in a mollusc (Cohicula sp.), an earthworm (Eisenia fetida) and a fish (Brachydanio redo). A comparative shrdy was perfotmed both in vibo and in vivo on whole organism postmitochondrial fractions and four potential biomarken were analyted: a marker of neurotoxicity (acetylcholinesterase activity, AChE, EC 3.1.1.7), a marker of oxidative sbss (malondialdehyde (MDA) level), and two markers of hydroperoxide detoxication: catalase (EC 1.11.1.6) and glutathione pemxidase (GPx, EC 1.11.1.9) activities. Our results have shown that the MDA contents were not signilicantty changed by exposures to lead either in vitro or in vivo. During uranium exposure, the MDA content was increased in vitro (particularly in fish samples) whereas this metal failed to sthnulate Spid peroxidation in vivo. With some exceptions, in vitro and in vivo exposures to lead and uranium showed that the AChE, catalase and GPx acbirites were decreased in the three species. These exceptions indicated that different mechanisms occurred in the different species. In conclusion, it was shown that S9 fractions of whole organisms could be useful for environmental contamination biomonitoring. Moreover, it was shown that AChE activities wen modulated by metals in viva and cannot be considered as specific bomarken of organophosphorus or carbaw pedcide exposure. Acetylcholinesterase and catalase activieies cwld be used to survey lead and uranium contamination.     

Recovery of acetylcholinesterase activity in the axolotl embryo following inhibition with the organophosphorus inhibitor Gd-7

The restoration of acetylcholinesterase (AChE) activity in axolotl Ambystoma mexicanum embryo after treatment at 38-42 stages with irreversibly AChE-inhibiting Gd-7 phosphororganic inhibitor in concentrations, significantly decreasing AChE activity level, but not interfering with ontogenesis has been studied. The rate of AChE activity restoration in Gd-7 treated axolotl embryo depends on the level of the enzyme restraint and the stage of the embryo development. The value of maximal restoration of AChE activity differs; it is less in embryos, treated with Gd-7 at later stages of development. The ability of the embryos to swim restores parallel to the increase in AChE activity. The data obtained suggest that axolotl embryo possess compensatory mechanism for increasing AChE biosynthesis after decrease in its activity caused by Gd-7. Acetylcholine, accumulating in the organism at partial inactivation of AChE by phosphororganic inhibitor may participate in this mechanism.     

Acetylcholinesterase activity in the myotome of the early chick embryo

Summary The myotome of early chick embryos was investigated histochemically by means of the acetylcholinesterase (AChE) reaction.Light-microscopically, at the cervical level, the myotome was first recognized and AChE activity demonstrated at stage 13 (2 day-old embryo). Subsequently, the myotome elongated ventro-laterally along the inner surface of the dermomyotome and reached the ventro-lateral end of the dermomyotome at stage 17 to 18 (3 day-old embryo). AChE activity in the myotome showed subsequent increase in intensity during the course of development. The myotome consisted mainly of AChE-positive cells displaying enzymatic activity along the nuclear membrane and within the cytoplasm. In contrast, almost all cells of the dermomyotome and the interstitial cells were AChE-negative.Electron-microscopically, the myotome cells of the 2 day-old embryo and the cells in the dorso-medial portion of the myotome of the 3 day-old embryo were morphologically undifferentiated; AChE activity was detected in the nuclear envelope and in single short profiles of the endoplasmic reticulum (ER). On the other hand, in the 3 day-old embryo the cells in the ventro-lateral portion of the myotome showed AChE activity in the nuclear envelope, numerous profiles of the ER and some Golgi complexes. These AChE-positive cells were regarded as developing myogenic cells based on their morphological characteristics.The present findings indicate (i) that the appearance of AChE activity in the cytoplasm is the first sign of the differentiation of myogenic cells, and (ii) that in these myogenic cells the increase in AChE activity is based on the development of the ER.     

EFFECTS OF SYMPATHECTOMY ON AXOPLASMIC TRANSPORT OF SELECTED ENZYMES INCLUDING MAO AND OTHER MITOCHONDRIAL ENZYMES

Abstract— Axoplasmic transport in guanethidine sympathectomized and control rats was investigated by monitoring the accumulations of various enzyme activities proximal to a ligature placed on the sciatic nerve. Sympathectomy affected the accumulations of three different mitochondrial enzymes quite differently: the accumulation of monoamine oxidase (MAO, EC 1.4.3.4) activity was inhibited 65% or more, that of hexokinase (HK, EC 2.7.1.1) activity was only inhibited 26%, while accumulation of glutamic dehydrogenase (GDH, EC 1.4.1.3) activity was unaffected by Sympathectomy. Accumulation of AChE (EC 3.1.1.7) activity was depressed 40%, but accumulations of the activities of the lysosomal enzyme, acid phosphatase (acid P'tase, EC 3.1.3.2), and of the cytosolic enzyme, choline acetyltransferase (CAT, EC 2.3.1.6) were unchanged.
Despite impressive inhibition of MAO accumulation, the intrinsic activity of this enzyme in sciatic nerve was unaffected by Sympathectomy. The existence in nerve of isozymes of MAO was demonstrated using the inhibitors clorgyline and deprenyl. Transported MAO activity was almost entirely type A; intrinsic activity was 2/3 type A and 1/3 type B.
The differential response of the accumulations of the three mitochondrial enzyme activities measured was interpreted to indicate the existence, within neurons, of mitochondria with different enzyme complements.     

Evaluation of biomarkers in oyster larvae in natural and polluted conditions

Crassostrea gigas D-shaped larvae were subjected to different conditions of temperature and salinity for 24 h and four biomarkers (acetylcholinesterase (AChE) activity, thiobarbituric acid reactive substances (TBARS) levels, glutathione S-transferase (GST) and catalase (CAT) activities) were measured. AChE activity decreased when salinity increased from 25 to 30 and 35 psu at 20 and 25 degrees C. Temperature did not seem to have an influence on AChE activity. TBARS levels increased as a function of salinity when the temperature was maintained at 20 degrees C, whereas at 25 degrees C no effect of salinity could be observed. Variations in GST and CAT activities were not significant with salinity and temperature except that catalase activity was higher at 25 degrees C than at 20 degrees C. Exposure experiments were conducted at 23 degrees C and 30 psu with carbofuran (100 and 1000 microg/l) and malathion (100 and 300 microg/l). There was an inhibition of AChE activity with carbofuran, and a toxic effect shown by an increase in TBARS levels counteracted by increases in GST and CAT activities which protected the larvae. When two pairs of adults producing larvae were taken into consideration, significant differences in biomarker levels were noted between the larval offspring of each pair. Malathion induced a decrease in AChE activity and an increase in CAT activity.     

Neural 16S acetylcholinesterase is solubilized by heparin.

The effect of heparin, a sulphated glycosaminoglycan, on the solubilization of rat sciatic-nerve acetylcholinesterase (acetylcholine acetylhydrolase; AChE; EC 3.1.1.7) was studied. It was found that heparin solubilized esterase activity from ligated nerves. Sedimentation analysis revealed this activity to be mainly the 16S form. Chondroitin sulphate did not solubilize AChE activity, and protamine eliminated the solubilizing effect. Our results suggest the involvement of sulphated glycosaminoglycans in the intra-axonal localization and transport of 16S AChE.     

Differential response of antioxidative enzymes in embryonic axes and cotyledons of germinating lupine seeds

Germination of lupine (Lupinus luteus L.) seeds was accompanied by an increase in concentration of free radicals with g ₁ and g ₂ values of 2.0056 ± 0.0003 and 2.0033 ± 0.0005, respectively. The highest intensity of free radical signal was observed in embryo axes immediately after radicle protruded through the seed coat. Hydrogen peroxide accumulated in embryonic axes and cotyledons during imbibition before the onset of germination in the seed population. The activities of superoxide dismutase (SOD, EC 1.15.1.1) and catalase (CAT, EC 1.11.1.6) rose progressively in embryo axes. In cotyledons SOD activity did not change significantly, while that of CAT increased during germination. The enhancement of Cu, Zn-SODs and Mn-SOD isoforms in embryonic axes was observed. A new isoform of catalase was synthesized, suggesting that it plays a relevant role during germination. SOD and CAT activities were detected in dry seeds. Free radical generation and response of antioxidative enzymes differed between embryo axes and cotyledons during the germination timecourse.     

Modulation of the Acetylcholine System in the Superior Cervical Ganglion of Rat: Effects of GABA and Hypoglossal Nerve Implantation After In Vivo GABA Treatment

gamma-Aminobutyric acid (GABA) was applied to the superior cervical ganglion (SCG) of CFY rats in vitro and in vivo, with or without implantation of a hypoglossal nerve, to evaluate the effects of these experimental interventions on the acetylcholine (ACh) system, which mainly serves the synaptic transmission of the preganglionic input. Long-lasting GABA microinfusion into the SCG in vivo apparently resulted in a "functional denervation." This treatment reduced the acetylcholinesterase (AChE; EC 3.1.1.7) activity by 30% (p less than 0.01) and transiently increased the number of nicotinic acetylcholine receptors, but had no significant effect on the choline acetyltransferase (acetyl-coenzyme A:choline-O-acetyltransferase; EC 2.3.1.6) activity, the ACh level, or the number of muscarinic acetylcholine receptors. The relative amounts of the different molecular forms of AChE did not change under these conditions. In vivo GABA application to the SCG with a hypoglossal nerve implanted in the presence of intact preganglionic afferent synapses exerted a significant modulatory effect on the AChE activity and its molecular forms. The "hyperinnervation" of the ganglia led to increases in the AChE activity (to 142.5%, p less than 0.01) and the 16S molecular form (to 200%, p less than 0.01). It is concluded that in vivo GABA microinfusion and GABA treatment in the presence of additional cholinergic synapses has a modulatory effect on the elements of the ACh system in the SCG of CFY rats.     

Salt and paraquat stress tolerance results from co-expression of the Suaeda salsa glutathione S-transferase and catalase in transgenic rice

GST (Glutathione S-transferase, EC 2.5.1.18) and CAT (Catalase, EC 1.11.1.6) play important roles in oxidative stress resistance. In this study, we transferred both GST and CAT1 of Suaeda salsa into rice (Oryza sativa cv. Zhonghua No.11) by Agrobacterium tumefaciens-mediated transformation under the control of cauliflower mosaic virus (CaMV) 35S promoter, and investigated whether co-expressing the GST and CAT1 in transgenic rice could reduce oxidative damage. Salt and paraquat stresses were applied. The data showed that co-expression of the GST and CAT1 resulted in greater increase of CAT and SOD (Superoxide Dismutase, EC 1.15.1.1) activity in the transgenics compared to non-transgenics following both stress imposition. Whereas the significant increase of GST activity in transgenics only occurred in paraquat stressed plants. While the generation of H₂O₂, Malon dialdehyde and plasma membrane relative electrolyte leakage decreased in the transgenics than in non-transgenics under the same conditions. Moreover, the transgenic rice seedlings showed markedly enhanced tolerance to salt stress compared with non-transgenics upon 200 mM NaCl treatment in greenhouse. The enhancement of the active oxygen-scavenging system that led to increased oxidative stress protection in GST + CAT1-transgenic rice plants could result not only from increased GST and CAT activity but also from the combined increase in SOD activity.     

Inhibitors of procollagen C-terminal proteinase block gastrulation and spicule elongation in the sea urchin embryo

In the sea urchin embryo, inhibition of collagen processing and deposition affects both gastrulation and embryonic skeleton (spicule) formation. It has been found that cell-free extracts of gastrula-stage embryos of Strongylocentrotus purpuratus contain a procollagen C-terminal proteinase (PCP) activity. A rationally designed non-peptidic organic hydroxamate, which is a potent and specific inhibitor of human recombinant PCP (FG-HL1), inhibited both the sea urchin PCP as well as purified chick embryo tendon PCP. In the sea urchin embryo, FG-HL1 inhibited gastrulation and blocked spicule elongation, but not spicule nucleation. A related compound with a terminal carboxylate rather than a hydroxamate (FG-HL2) did not inhibit either chick PCP or sea urchin PCP activity in a procollagen-cleavage assay. However, FG-HL2 did block spicule elongation without affecting spicule nucleation or gastrulation. Neither compound was toxic, because their effects were reversible on removal. It was shown that the inhibition of gastrulation and spicule elongation were independent of tissue specification events, because both the endoderm specific marker Endo1 and the primary mesenchyme cell specific marker SM50 were expressed in embryos treated with FG-HL1 and FG-HL2. These results suggest that disruption of the fibrillar collagen deposition in the blastocoele blocks the cell movements of gastrulation and may disrupt the positional information contained within the extracellular matrix, which is necessary for spicule formation.