SUBCELLULAR ANALYSIS OF THE MOLECULAR FORMS OF ACETYLCHOLINESTERASE IN RAT SKELETAL MUSCLE

Acetylcholinesterase (AChE, EC 3.1.1.7) activity of rat gastrocnemius muscle homogenized in 1 M-NaCl and 0.5% Triton X-100 was separated by velocity sedimentation in sucrose gradients into three molecular forms with sedimentation coefficients of about 4S, 10S and 16S. The distribution of homogenate AChE activity among the three peaks was 53, 34 and 13% respectively. The different molecular forms were found to be heterogeneously distributed in subcellular fractions prepared from sucrose homogenates of muscle, as follows: Subfractions of the crude sarcolemmal fraction were prepared by discontinuous sucrose gradient centrifugation. AChE was recovered in the greatest yield and with the highest specific activity in a light density subfraction (0.6/0.8 M-sucrose interface). The AChE activity in this light density subfraction was mainly (81-88%) the 10S form of the enzyme. The velocity sedimentation profiles of the AChE activity in the more dense subfractions were markedly different in that 16S AChE was a major component.     

LOCALIZATION AND MULTIPLE FORMS OF ACETYLCHOLINESTERASE IN SEA URCHIN EMBRYOS

Acetylcholinesterase during the development of the sea urchin Pseudocentrotus depressus was examined by enzyme assay (the colorimetric method of E llman et al. ), histochemistry (a Cu-thiocholine method), polyacrylamide gel electrophoresis and DEAE-Sephadex ion exchange chromatography.
The enzyme activity is detected in the unfertilized egg, remains low during cleavage, elevates slightly through gastrulation, and then increases rapidly thereafter. The intense activity is localized in the mesenchyme cells associated with the larval skeleton of young pluteus larvae, and their cell membranes and nuclear envelops. Soluble enzyme accounts for 60% of the total activity. The additional 34% is extracted by 1% Triton X-100 from particulates. The soluble enzyme consists of two forms. Both are strongly acidic proteins which are similar in electric charge, but dissimilar in size, being 180,000 and 280,000 in molecular weights. The enzyme released from the membrane by the detergent possesses a component which is not present in the soluble complement of the enzyme. It is not a secondary product of the soluble enzyme interacting with the detergent.
Acetylcholinesterase serves as a marker of late differentiation and regional differentiation in the sea urchin embryo.     

DEVELOPMENT OF ACETYLCHOLINESTERASE MULTIPLE MOLECULAR FORMS IN CHICKEN MUSCLES

Abstract— AChE activity and protein content in chicken ALD and PLD muscles was studied during pre- and postnatal development. Protein content in both muscles increased whereas AChE activity increased in ALD and decreased in PLD during development. All studied values reached the steady-state 3 weeks after hatching.
Electrophoretic separation of the samples showed three molecular forms of AChE present in both adult ALD and PLD muscles. Two molecular forms in ALD muscle increased slowly, one form quickly. On the other hand, the activity of AChE forms in PLD muscle decreased with different rates. It appears from these results that the multiple molecular forms of AChE in muscles are not of the same physiological importance.     

CATECHOLAMINES IN SYMPATHETIC GANGLIA OF RAT: EFFECTS OF DEXAMETHASONE AND RESERPINE1

The superior cervical sympathetic ganglion of rats contains a finite amount of epinephrine all of which is of ganglionic origin. Treatment of new-born rats with dexamethasone for 8 days results in a 112-fold increase in the epinephrine concentration, whereas the norepinephrine and dopamine are increased by only 1·4- and 1·9-fold respectively. This epinephrine increase in newborn rats is reversible if treatment is discontinued, and it fails to occur in adult animals. The epinephrine store of normal and dexamethasone treated animals is resistant to the depletion by reserpine. There is no increase in the epinephrine content in organs innervated by axons emanating from the ganglion. The data presented support the localization of epinephrine in small intensely fluorescent cells in the ganglion and we propose that epinephrine may be released from these cells and function as a modulator of ganglionic transmission.     

IDENTIFICATION OF MULTIPLE FORMS OF AMINOBUTYRATE TRANSAMINASE IN MOUSE AND RAT BRAIN : SUBCELLULAR LOCALIZATION

—(1) At least four distinct molecular forms of 4-aminobutyrate: 2-oxoglutarate aminotransferase from mouse and rat brain, have been separated by electrophoresis on paper, cellogel, agargel, silicagel and by immunoelectrophoresis. (2) The existence of specific typical electrophoretic profiles in mitochondrial and extramitochondrial compartments was shown. (3) A differential effect of pH on the anionic and cationic 4-aminobutyrate:2-oxoglutarate aminotransferase transaminase activities has been shown. (4) The possible consequences of the 4-aminobutyrate: 2-oxoglutarate aminotransferase isozyme compartimentation on the local availability of γ-aminobutyric acid pools has been discussed.     

ACETYLCHOLINESTERASE IN THE SUBSTANTIA NIGRA AND CAUDATE-PUTAMEN OF THE RAT: PROPERTIES AND LOCALIZATION IN DOPAMINERGIC NEURONS

Abstract— In order to examine the hypothesis that acetylcholinesterase (AChE) is contained within dopaminergic neurons of the nigro-striatal projection, the effects of selective destruction of these neurons by 6-hydroxydopamine (6-OHDA) on cholinesterase, tyrosine hydroxylase, and choline acetyltransferase in substantia nigra (SN) and caudate-putamen (CP) were studied in the rat. Four to five weeks after intraventricular or intracerebral 6-OHDA injections tyrosine hydroxylase in these structures was reduced by 90% or more. Choline acetyltransferase was not affected in the SN or CP, but cholinesterase was reduced by about 40% in the SN and by 12% in the CP. To determine that the observed decreases in cholinesterase activity reflected true AChE and not butyrylcholinesterase (BChE), further experiments were conducted on tissues from animals with intracerebral 6-OHDA lesions. (1) Substrate specificity. Acetylcholine (ACh) was replaced by either acetyl-β-methyl-choline (AcβMeCh) or butyrylcholine (BCh) in the cholinesterase assay. SN and CP from 6-OHDA lesioned rats showed 54% and 92% of control tissue cholinesterase activity respectively with AcβMeCh as substrate, in good agreement with values found using ACh. No decrease in activity toward BCh was observed. (2) Kinetics. The decrease in cholinesterase activities at different concentrations of ACh was determined. Analysis of the data revealed that cholinesterase in dopaminergic neurons was inhibited by high ACh concentrations, a characteristic property of AChE but not BChE. (3) Selective inhibitors. In the SN, cholinesterase in dopaminergic neurons was inhibited by the selective AChE inhibitors BW284C51 and ambenonium with a dose-response curve similar to erythrocyte AChE but different from serum BChE. The selective BChE inhibitor, tetraisopropylpyrophosphoramide, inhibited the enzyme in dopaminergic neurons only at concentrations which inhibited erythrocyte AChE, concentrations somewhat higher than those which inhibited serum BChE. These results support recent histochemical observations indicating that AChE is contained in dopaminergic neurons of the SN. Moreover, these experiments represent the first characterization of AChE from a homogeneous population of non-cholinergic neurons in mammalian CNS.     

NOREPINEPHRINE-STIMULATED BREAKDOWN OF TRIPHOSPHOINOSITIDE OF RABBIT IRIS SMOOTH MUSCLE: EFFECTS OF SURGICAL SYMPATHETIC DENERVATION AND IN VIVO ELECTRICAL STIMULATION OF THE SYMPATHETIC NERVE OF THE EYE

Abstract— Paired iris smooth muscles from rabbits were prelabelled either in vitro by incubation for 30 min at 37°C in an iso-osmotic salt medium containing glucose, inositol, cytidine and ³²Pi, or in vivo by administration of the isotope intracamerally into each eye 1 h before death. One of the pair was then incubated at 37°C for 10 min in an unlabelled medium containing 10 mm of 2-deoxyglucose and the other was incubated in the presence of norepinephrine (NE) or other adrenergic agents. Triphosphoinositide (TPI) was found to contain more ³²P than any other phospholipid (almost 39% of total lipid radioactivity) in both the in vitro and in vivo experiments. NE (50 μm ) increased the loss of ³²P from TPI (the TPI effect') by 28–30% in the ³²P-labelled muscle. The TPI effect was accompanied by a significant increase in ³²P labelling of phosphatidic acid (PA) and phosphatidylinositol (PI) but not phosphatidylcoholine. In this tissue the TPI effect was found to be mediated through α-adrenergic receptors. At 14 days after surgical sympathetic denervation, incorporation of ³²P into phospholipids of the denervated muscle increased by an average of 6% over that of the normal muscle. The increase in TPI, PI and PA was 7%, 4% and 9% of that of the control respectively. There was little change in phospholipid content of the denervated muscle. The increase in sensitivity to NE (12.5 μm ) caused by denervation produced about 18% increase in the TPI effect and a 25% increase in the ³²P labelling of PA, but not PI. In view of our previous findings on the requirement of the TPI effect for Ca²⁺, this observation could suggest that an increase in Ca²⁺ influx, following the interaction between the neurotransmitter and its receptor could stimulate TPI-phosphodiesterase, thus leading to increased PA via increased diglyceride. This denervation-induced supersensitivity to NE appears to be postsynaptic in nature. ³²Pi was injected intracamerally into each eye 1 h before electrical stimulation of one of the sympathetic trunks. After stimulation for 30 min there was a significant loss of ³²P from TPI and a significant increase in the labelling of PI and PA of the stimulated muscle. It is concluded that TPI and its enzymes could play an important role in neurotransmission at the neuromuscular junction of smooth muscle.     

交感传出在大鼠糖尿病性痛过敏中的作用

交感传出和前列腺素（ＰＧｓ）在周围神经不全损伤和炎症所引起的痛过敏中起重要作用,它们对糖尿病性痛过敏影响尚不清楚。本工作先给大鼠腹腔注射６－羟多巴胺（６－ＯＨＤＡ）损毁交感节后神经元（ＳＰＧＮｓ）末梢后,再给予链脲佐菌素（ＳＴＺ）以建立６－ＯＨＤＡ糖尿病大鼠模型,在连续４周的观察中这组大鼠伤害性爪回缩阈值（ＮＰＷＴ）和甩尾反向潜伏期（ＴＥＬ）没有明显变化,而糖尿 病组大鼠的痛阈显著降低,并伴有痛过     

MEMBRANE AFFINITIES AND SUBCELLULAR DISTRIBUTION OF THE DIFFERENT MOLECULAR FORMS OF CHOLINE ACETYLTRANSFERASE FROM RAT

Choline acetyltransferase from rat brain is present in three different molecular forms with isoelectric points at pH 7·4-7.6, 7·7-7·9 and 8·3. The three forms were identified in a highly purified enzyme preparation, in a preparation of synaptosomes and in a cyto-plasmic preparation from disrupted axons and perikarya (fraction S₃). The three molecular forms differed in their affinities for synaptosome membranes and for a cation exchange resin (CM-Sephadex C-50). The positive surface charges of the different molecular forms and their affinities for membranes correlated well with their isoelectric points. The molecular form with jsoelectric point 8·3 had the largest positive surface charge and the highest membrane affinity. On isoelectric focusing of an extract from rat brain synaptosomes, the molecular form with isoelectric point 8·3 formed a complex with a negatively charged compound, presumably a protein. A method was developed to remove this compound by treatment with DEAE-Sephadex or by precipitation with vinblastine. These procedures are similar to methods known to remove the neurotubular protein. The complex formation did not occur in fraction S₃.     

LOCALIZATION OF TRITIATED NOREPINEPHRINE IN VASCULAR SYMPATHETIC AXONS OF THE RAT INTESTINE AND MESENTERY BY ELECTRON MICROSCOPE RADIOAUTOGRAPHY

The distribution of infused tritiated norepinephrine (NE-³H) in small mesenteric arteries and intestinal arterioles in rats was investigated with electron microscopic radioautography. Silver grains, indicating the presence of the tritium label on the sections, were found lying mainly over axon bundles, but some were present over collagen and smooth muscle cells. Axons with the highest concentrations of silver grains had been sectioned at points where they were naked of Schwann cell sheath, were dilated into varicosities, and contained small granular vesicles. This finding was taken as confirmatory circumstantial evidence that the small granular vesicles were the sites of uptake and storage of NE. The short interval between the start of infusion and the fixation of the tissue appeared to rule out any process other than a direct uptake of NE by the peripheral axons. If axonal sites of uptake of NE-³H correspond to sites of release of NE, then the evidence suggests that such sites of release are widespread over the terminal part of the axon and are not confined to those parts of the axon which are in close contact with smooth muscle cells. Since the fixation and embedding procedures will remove NE which is not strongly bound to tissues, the localization of NE-³H in the radioautographs does not necessarily correspond to the distribution of all the NE present in vivo.     

THE EFFECTS OF SOLUBILIZATION PROCEDURES ON THE RELEASE AND MOLECULAR STATE OF ACETYLCHOLINESTERASE FROM ELECTRIC ORGAN TISSUE

Abstract— A study was made of the effect of various solubilization procedures on the release of AChE from electric organ tissue of the electric eel and on the molecular state of the enzyme. The procedures employed included homogenization in different ionic media or in the presence of detergents, etuymic treatment and chemical modification. Studies were performed on intact electroplax, tissue homogenates and membrane fractions. The apparent AChE activity of intact cells, homogenates and membrane fractions was shown to be governed by diffusion-controlled substrate and hydrogen ion gradients, generated by AChE-catalyscd hydrolysis, leading to a lower substrate concentration and a lower pH in the vicinity of the particulate enzyme.
Treatment of homogenates with NaCl solutions or with NaCl solutions containing the nonionic detergent Triton X-100 causes release of the native'molecular forms of the enzyme (primarily the 18 S species) which aggregate at low ionic strength. For optimal extraction both high ionic strength (e.g. 1 M-NaCl) and the detergent are needed AChE is also solubilized by treatment of tissue homogenates with trypsin, bacterial protease or collagenase. The first two enzymes caused its release as an 11 S non-aggregating form, while collagenase also produces a minor non-aggregating - 16 S component. Treatment of tissue homogenates with maleic anhydride causes release of AChE as a non-aggregating 18 S species. On the basis of the solubilization experiments it is concluded that the interaction of AChE with the excitable membrane is primarily electrostatic. The possible orientation of the enzyme within the synaptic gap is discussed.     

尖吻蝮蛇毒对蟾蜍交感神经节细胞电活动的影响

用细胞内电位记录技术,以离体蟾蜍交感链为标本,观察了尖吻蝮蛇毒(AAV)的神经毒性作用。结果表明,该蛇毒(10-200μg/ml)对交感神经元的静息电位、膜电阻和膜电容没有显著的作用,对动作电位形状也无可测出的影响,但能使阈电位轻皮升高,即使神经元兴奋性稍降低。AAV(>25μg/ml)对胆碱能性的快兴奋性突触后电位有剂量依从性的,部分可逆性的抑制作用。其作用机制至少应部分归之于AAV对突触后膜上的N型胆碱能受体的阻断作用。本研究发现的AAV神经毒性作用可能有一定实践意义。     

THE RELEASE AND MOLECULAR STATE OF MAMMALIAN BRAIN ACETYLCHOLINESTERASE

Abstract— By incubating the particulate fraction of caudate nucleus from calf brain in ion-free media, about 90 per cent of the AChE activity was brought into solution. The effects of different salts, EDTA and tetracaine on the release were studied. The mol. wt. of the enzyme was determined by gel filtration. About 90 per cent of the activity in a fresh preparation appeared in a form with mol. wt. 80,000. During storage this form was gradually transformed into forms with higher mol. wts. The effects of changes in the ionic environment on the aggregation were investigated. Purification attempts always resulted in the transformation of the enzyme into high mol. wt. forms. If the release was performed in the presence of DEAE-Sephadex-A25, the enzyme no longer aggregated. The cytosol fraction always contained some AChE activity; the significance of the presence of AChE in this fraction is discussed.     

EFFECTS OF SURGICAL DECENTRALIZATION AND NERVE GROWTH FACTOR ON THE MATURATION OF ADRENERGIC NEURONS IN A MOUSE SYMPATHETIC GANGLION

Abstract— The increase in tyrosine hydroxylase activity in mouse superior cervical ganglion during postnatal development was prevented by administration of the protein synthesis inhibitor cycloheximide. Surgical section of the preganglionic nerves in 4-day-old mice prevented the normal increases in tyrosine hydroxylase and monoamine oxidase activity in the ganglion during development. Surgical decentralization also prevented the developmental increases in ganglion size and cell numbers. The preganglionic fibres thus appear to exert a general regulatory effect on the growth and biochemical maturation of postganglionic adrenergic neurons in sympathetic ganglia. Administration of nerve growth factor to young mice failed to eliminate the differences in ganglion size, cell numbers and tyrosine hydroxylase activity between normally innervated and decentralized ganglia. Nerve growth factor, however, caused an increase in all these parameters in both control and decentralized ganglia–the magnitude of these increases being greatest in the control ganglia. Administration of carbachol and physostigmine to neonatal mice did not influence the normal development of tyrosine hydroxylase activity in the superior cervical ganglion.     

刺激交感神经对大鼠多觉型伤害性感受器诱发和自发放电的不同作用

多觉型伤害性感受器是皮肤内专一性较强的痛觉感受器。本实验用剥制神经细束的技术,引导大鼠尾神经C类纤维的传入放电反映多觉型伤害性感受器的活动,以判定刺激交感神经对外周痛觉感受过程的调制作用。测试了57个该类感受器的单位放电,发现下述两个主要事实:(1)刺激腰骶部交感干外周端,可以显著抑制伤害性刺激(包括机械压力,直流电-钾离子,热烫等)诱发的多觉型伤害性感受器的单位放电,其作用出现较快,可使放电数减少1/3左右,后作用延续十多分钟。局部动脉注射去甲肾上腺素也产生类似的抑制效应。从而证实交感神经具有抑制痛觉感受器的作用。(2)交感神经对部分多觉型伤害性感受器活动的调制具有双重作用的特点,即对同一单位因外加刺激引起的诱发放电有抑制作用,对其自发放电则有易化作用。讨论了交感神经这一双重作用的临床意义以及针刺通过交感神经调制外周痛觉感受过程的设想。     

棉铃虫AChE不同分子型对抑制剂的敏感度及其与抗药性的关系(英文)

在分离到的棉铃虫AChE五种不同的分子型中 ,2 .1s和 8.7sAChE抗性品系对毒扁豆碱的敏感度明显低于敏感品系 ,成虫头部I50 值分别相差 1 86.3和 84.8倍 ,幼虫I50 值分别相差 1 0 1 0 倍和 1 0 5 倍。幼虫 5.3sAChE对毒扁豆碱的敏感度抗性品系和敏感品系相差达 1 2 3倍 ,而成虫则没有差异。研究结果表明 2 .1s、5.3s和 8.7sAChE敏感度降低可能是造成棉铃虫对有机磷和氨基甲酸酯类杀虫药剂产生抗性的主要原因     

EFFECT OF DENERVATION AND OF AXOPLASMIC TRANSPORT BLOCKAGE ON THE IN VITRO RELEASE OF MUSCLE ENDPLATE ACETYLCHOLINESTERASE

Abstract— Cat geniohyoid muscle samples containing endplate regions, when incubated in vitro at 37°C in phosphate buffer (pH 73, release acetylcholinesterase (AChE; EC 3.1.1.7) to the bathing medium. By treating the muscle samples with collagenase (EC 3.4.4.19), it was confirmed that most of the AChE released came from the endplates. Enzyme liberation was studied 10 days after either local injection of 10mM-cokhicine into the hypoglossal nerve or following nerve transection. Results showed that the rate of release is increased by denervation, but is not affected by axoplasmic transport blockage. It is postulated that the cellular contact between nerve and muscle—altered by denervation but not by interruption of axoplasmic transport—is an essential factor in maintaining the localization of end-plate AChE within the synaptic cleft substance. This does not invalidate the possible participation of ACh and muscle activity in such enzyme localization.     

PROPERTIES OF ACETYLCHOLINESTERASE FROM RAT BRAIN

—Acetylcholinesterase (EC 3.1.1.7) from cerebral cortex of mature rats was purified by means of affinity chromatography, to a specific activity of 4.5 mmol acetylthiocholine hydrolysed × min^?1× mg^?1 protein. The enzyme is a glycoprotein and contains a single subunit with a mol. wt of about 80,000. Electrofocusing either a pure or a crude preparation of the enzyme produces six enzymatically active bands with a range of isoelectric points from 5.04 to 5.54. Gel filtration yields oligomers with molecular weights of about 150,000, 320,000, 500,000 and 650,000, with 60 per cent of the activity in the 150,000 fraction. The gel fractions with molecular weights 150,000 and 320,000 produce the same isoelectric patterns. Different subcellular fractions of the cortex show different characteristic isoenzyme patterns.     

MOLECULAR PROPERTIES AND DIFFERENTIATION OF ACETYLCHOLINESTERASE IN SEA URCHIN EMBRYOS*

The two molecular forms of acethylcholinesterase (EC 3.1.1.7) in sea urchin embryos were characterized by several physical methods. The sedimentation coefficients determined by sucrose gradient centrifugation are 7.6S and 10.6S. The Stokes radii determined by gel filtration are 65 Å and 91 Å. From these parameters, molecular weights were estimated as 190,000 and 380,000; the one is twice as large as the other. Both forms have similar electric property and buoyant density in a CsCl gradient. When the enzyme solution was concentrated, the 10.6S form became predominant. These results suggest that the two forms are monomer and dimer. The sea urchin enzymes resemble globular forms of acetylcholinesterase of the electric organ of fishes. The activity of the enzyme abruptly increases in post-gastrulation embryos. Inhibition of concomitant protein synthesis by a specific inhibitor, emetine, does not affect the increase in enzyme activity. The result suggests that post-translational processes may be involved in the differentiation of this enzyme in sea urchin development. The following sea urchins were used in the study: Strongylocentrotus purpuratus, Strongylocentrotus franciscanus, and Dendraster excentricus.     

THE EFFECTS OF PENTOBARBITONE-SODIUM ANAESTHESIA, SHOCK AVOIDANCE CONDITIONING AND ELECTRICAL SHOCK ON ACETYLCHOLINESTERASE ACTIVITY AND PROTEIN CONTENT IN REGIONS OF THE RAT BRAIN

Abstract— Pentobarbitone sodium anaesthesia was found to produce an increase in protein content in some regions of the rat brain, i.e. posterior cortex, caudate nucleus, and a decrease in protein content in the ventral cortex.
Acetylcholinesterase expressed in terms of wet weight was found to increase in the cerebellum, medulla, and to decrease in the medial cortex, hippocampus, thalamus and caudate nucleus. The changes in activity were not explicable in terms of a direct effect of the anaesthetic on the enzyme. A decrease in protein content of rat brain was observed in the frontal cortex, ventral cortex, hippocampus and caudate nucleus after electrical shocks. Following shock avoidance conditioning procedure (shuttle-box), decreases in protein content were observed in the medial cortex, posterior cortex, cerebellum and ventral cortex; in the thalamus an increase in protein content was observed.
Changes in AChE activity were observed following footshock in the frontal cortex and medulla where there was an increase in activity and in the caudate nucleus, hypothalamus, thalamus, and olfactory tubercle where there was a decrease in activity.
Following shock avoidance conditioning the activity of the AChE increased in posterior cortex, hippocampus, thalamus and hypothalamus and the activity of the enzyme decreased in the ventral cortex.