Ethylcholine mustard aziridinium blocks the axoplasmic transport of acetylcholinesterase in cholinergic nerve fibres of the rat

A cholinotoxin, ethylcholine mustard aziridinium ion, (AF64A) specifically and irreversibly blocks the intraaxonal transport of acetylcholinesterase in the rat. Impairment of the transport of this enzyme in the septo-hippocampal cholinergic fibres and in the sciatic nerve has been studied, using different doses of AF64A. It is demonstrated that the effect on the axonal transport is dose-dependent, but is not related to the mode of drug application. AF64A thus may exert its neurotoxic effects on cholinergic neurons at several target sites of action. In addition to the localized presynaptic mechanisms, it may also be compromising cholinergic function by inhibiting axonal transport in vivo.     

Ethylcholine mustard aziridinium blocks the axoplasmic transport of acetylcholinesterase in cholinergic nerve fibres of the rat

Summary A cholinotoxin, ethylcholine mustard aziridinium ion, (AF64A) specifically and ireversibly blocks the intraaxonal transport of acetylcholinesterase in the rat. Impairment of the transport of this enzyme in the septo-hippocampal cholinergic fibres and in the sciatic nerve has been studied, using different doses of AF64A. It is demonstrated that the effect on the axonal transport is dose-dependent, but is not related to the mode of drug application. AF64A thus may exert its neurotoxic effects on cholinergic neurons at several target sites of action. In addition to the localized presynaptic mechanisms, it may also be compromising cholinergic function by inhibiting axonal transport in vivo.     

Ethylcholine mustard aziridinium ion lesions of the rat cortex result in retrograde degeneration of basal forebrain cholinergic neurons: Implications for animal models of neurodegenerative disease

Multiple injections of 2 nmols of cyclised ethylcholine mustard aziridinium ion (ECMA), a putative cholinergic neurotoxin, were made (unilaterally) into the cortical terminal field of cholinergic neurons projecting from the nucleus basalis of Meynert (NBM) in the rat basal forebrain. After 30 days, choline acetyltransferase enzymatic activity, a marker for cholinergic function, was significantly lowered in both ipsilateral cortex and NBM, and cholinergic cell bodies in the latter reduced in cross-sectional area, a spectrum of effects characteristic of retrograde degeneration of this pathway. These results are discussed in the context of neurodegenerative diseases affecting cholinergic function.     

Kinetics of irreversible inhibition of choline transport in synaptosomes by ethylcholine mustard aziridinium

Summary Ethylcholine mustard aziridinium (ECMA) inhibits choline transport in synaptosomes at a half-maximal concentration of about 20 m. The rate of inhibition falls off rapidly after 10 min and the concentration dependency reaches a plateau at about 100 m. The inhibition is not removed by washing the synaptosomes, and choline and hemicholinium-3 protect the carrier against attack by the mustard. Choline efflux, particularly that stimulated by choline in the medium (transactivation) is also inhibited by the aziridinium compound. Similarly choline influx activated by preloaded internal choline is inhibited by ECMA. The mustard can enter the synaptosomes in an active form but most of the carrier is alkylated when facing the outside. Prior depolarization of the synaptosomes causes an increase in the rate of inhibition by ECMA which is proportionally about the same as the increase in choline influx also caused by depolarization. At low ECMA concentrations the rate of inhibition is that of a first-order reaction with the carrier but at high ECMA concentrations the translocation of the carrier to the outward-facing conformation controls the rate of inhibition. Using a model of choline transport with some simplifying assumptions it is possible to estimate the amount of carrier; cholinergic synaptosomes carry about six times the concentration of carrier found in noncholinergic ones. In noncholinergic synaptosomes the carrier faces predominately out, the reverse in cholinergic ones. The rate constant of carrier translocation is increased by combination with choline some six- to sevenfold to about 3.5 min^–1. The rate constant of ECMA attack on the carrier is about 440m ^–1 sec^–1.     

Bidirectional axonal transport of 16S acetylcholinesterase in rat sciatic nerve

Axonal transport of the 16S Molecular form of acetylcholinesterase (16S-AChE) in doubly ligated rat sciatic nerves was studied by means of velocity sedimentation analysis on sucrose gradients. This form of AChE was selectively confined to motor, and not to sensory, fibers in the sciatic nerve, where it represented 3--4% of total AChE. Its activity increased linearly with time (4--20 hr) in nerve segments (7 mm) proximal to the central ligature (4.5 mU/24hr) and distal to the peripheral ligature (2.0 mU/24 hr). From the linear rates of accumulation of 16S-AChE, we conclude that the enzyme is conveyed by anterograde and retrograde axonal transport at velocities close to those previously defined for the movement of total AChE (410 mm/day, anterograde; 220 mm/day, retrograde). The transport of AChE molecular forms, other than the 16S form, could not be resolved presumably due to their presence in blood as well as at extraaxonal sites. The present findings are consistent with the view that in rat sciatic nerve most, if not all, of the small portion of total AChE (approximately 3%) which is transported may be accounted for by 16S-AChE.     

Inhibition of choline transport into human erythrocytes by choline mustard aziridinium ion.

Acetylcholine mustard aziridinium ion inhibited the transport of [3H]choline into human erythrocytes. Treatment of the erythrocytes with 1 X 10(-4) M tetraethylpyrophosphate prevented the inhibition of [3H]choline transport by acetylcholine mustard aziridinium ion. Hydrolyzed acetylcholine mustard aziridinium ion inhibited choline transport both in the presence and absence of 1 X 10(-4) M tetraethylpyrophosphate. The product of hydrolysis was equipotent with acetylcholine mustard in its ability to inhibit choline transport; incubation of this product with sodium thiosulfate prevented inhibition of choline transport thereby indicating the presence of an aziridinium ion. The hydrolysis product is likely to be choline mustard aziridinium ion. Results on the efflux of [3H]choline from erythrocytes in the presence of the proposed choline mustard aziridinium ion showed that the mustard moiety was transported into the red cells on the choline carrier. The rate of efflux of [3H]choline produced by choline mustard aziridinium ion was 55% of that produced by the same concentration of choline. It is concluded that acetylcholinesterase (EC 3.1.1.7) of red cells rapidly hydrolyzes acetylcholine mustard aziridinium ion to acetate and choline mustard aziridinium and the latter compound can act as a potent inhibitor of choline transport. This finding would indicate that the hemicholinium-like toxicity of acetylcholine mustard in the mouse is due to the formation of choline mustard aziridinium ion.     

Monoaminergic and cholinergic nerve fibres in the human dental pulp

Summary Distribution of cholinesterase-containing nerve fibres was studied in 15 human dental pulps by the thiocholine method. Falk's fluorescent method was used to demonstrate catecholamines (8 dental pulps).Cholinesterases were localized partly in the subodontoblastic plexus sending out fine branches towards odontoblasts, and partly in the nerve fibres attached to the blood vessel walls. These fibres in contrast to those of the subodontoblastic plexus were finer and showed fine varicosities.Monoaminergic terminals were localized mainly along blood vessel walls, however, some fibres having no relation to the blood vessels were also found.Cholinesterase-containing nerve fibres in the periphery of the pulp are considered to be sensitive nerve fibres originating from n.V. Distribution of cholinesterase-containing nerve fibres and monoaminergic terminals along the blood vessel walls indicates that the blood vessels in the human dental pulp might be under both parasympathetic and sympathetic control.     

Choline mustard: an irreversible ligand for use in studies of choline transport mechanisms at the cholinergic nerve terminal

It has been shown in our laboratory that choline mustard aziridinium ion is a potent and irreversible inhibitor of choline transport into rat brain synaptosomes; this compound showed selectivity for the sodium-dependent, high affinity carrier in that it was 30 times more potent as an inhibitor when compared with the effect on sodium-independent, low affinity choline uptake. In the present study, this mustard analogue did not inhibit synaptosomal uptake of 5-hydroxytryptamine, noradrenaline, or gamma-aminobutyric acid, thereby confirming further the specificity of this compound for the choline carrier. Studies of the effect of depolarization of the nerve terminals on the inactivation of choline carriers by choline mustard were performed. It was determined that alkylation of the carrier was significantly increased in nerve endings previously depolarized. The enhancing effect of depolarization on choline transport velocity and on the alkylation of choline carriers by choline mustard was dependent upon the presence of sodium in the external medium. Possible mechanisms for the enhanced inactivation of choline carriers by choline mustard aziridinium ion are proposed, and kinetic interactions of choline mustard with the high affinity choline carrier and with choline acetyltransferase are reviewed and discussed.     

Distribution of acetylcholinesterase in extraocular muscle fibres of the rat

Summary The distribution of acetyleholinesterase in the fine structure of the extraocular muscle of the rat was studied using acetylthiocholine iodide as a substrate together with selective inhibitors.Acetylcholinesterase was observed in the membranes of the transverse elements of the sarcoplasmic reticulum and in the region of the A—I junction.The author thanks to Professor Antti Telkkä, M. D., Head of the Electron Microscope Laboratory, University of Helsinki, for permission to use the facilities of the laboratory.     

Capsaicin-induced inhibition of axoplasmic transport is prevented by nerve growth factor

Summary Capsaicin injected into the scrotal skin of rats was observed to induce a decrease in the amount of horseradish peroxidase (HRP) transported in the pudendal nerve to the sixth lumbar dorsal root ganglion on the pretreated side. This was seen as a decrease in the number of HRP-labelled neurones compared to the control side. A morphometric study confirmed that the effect of capsaicin was exerted predominantly on the small neurones. Injection of nerve growth factor (NGF) into the pudendal nerve prevented the deleterious effects of capsaicin, thereby suggesting a possible site of action and mechanism for the effect of capsaicin on peripheral nerves.     

Dose- and time-dependent hippocampal cholinergic lesions induced by ethylcholine mustard aziridinium ion: Effects of nerve growth factor,GM1 ganglioside,and vitamin E

Ethylcholine mustard aziridinium ion (ECMA) was infused intracerebroventricularly (icv) to rats followed by measurement of two markers of presynaptic cholinergic neurons, choline acetyltransferase (ChAT) activity and high affinity choline transport (HAChT), in the hippocampus and cortex. Bilateral icv administration of 1, 2, or 3 nmol of ECMA per side produced dose-dependent reductions in each marker in the hippocampus, but not in the cortex, one week after treatment. Reductions of 52% and 46% for ChAT activity and HAChT, respectively, were produced in the hippocampus by 3 nmol ECMA. Measurement of these two markers at different times after icv infusion of 2 nmol ECMA/ventricle revealed that the activity of ChAT was reduced to a greater extent than was HAChT in the hippocampus 1 day and 1, 2, 4, and 6 weeks after treatment. The maximal reductions of ChAT activity and HAChT (61% and 53%, respectively) were reached between 1 and 2 weeks after ECMA administration. There was no evidence of regeneration of either marker at 4 or 6 weeks posttreatment. HAChT and ChAT activity in the cortex were not altered at any of the posttreatment times examined.ECMA-induced deficits in hippocampal ChAT activity and HAChT were not counteracted by the following treatments: (i) daily administration of GM₁ ganglioside (10 mg/kg, intraperitoneally (ip)) from the day prior to infusion of ECMA until 2 weeks later; (ii) daily administration of GM₁ ganglioside between 2 and 6 weeks after infusion of ECMA; and (iii) icv administration of nerve growth factor (NGF) twice per week for 2 weeks after ECMA treatment. Since similar treatments with NGF and GM₁ ganglioside ameliorate lesions induced by other methods, these results indicate that the mechanism of lesion formation and the surviving cellular components influence the functional effects of neurotrophic factors. In contrast to the above results, treatment with vitamin E significantly attenuated ECMA-induced deficits of ChAT activity and HAChT. Further studies of the effects of vitamin E on the development of ECMA-induced deficits may help to elucidate the mechanism action of ECMA.     

神经再生过程中细胞骨架蛋白的轴浆转运与再生神经构筑的变化

神经再生是长期未能解决的问题,原因之一是对再生的规律和调控缺乏足够的认识。近代神经生物学的进展表明,神经再生的特点之一是轴突缺乏合成蛋白的能力,其再生所需的     

Localization of catecholamines and acetylcholinesterase in the terminal nerve fibres of the rabbit myometrium

Summary The autonomic nerves of the myometrium of the rabbit were studied in order to demonstrate simultaneously the adrenergic nature of an axon and the localization of acetylcholinesterase (AChE) in the same axons. The synaptic vesicles of the adrenergic axons and nerve terminals remained partially filled with the electron dense material typical for them after formaldehyde fixation and short incubation time for AChE. AChE stain was localized regularly on the axons which contained agranular synaptic vesicles and also on axons which contained dense cored synaptic vesicles beeing probably adrenergic. The role of AChE on the adrenergic axons is discussed.     

Anoxic block and recovery of axoplasmic transport and electrical excitability of nerve

Axoplasmic transport of cat sciatic nerves was studied in vitro in a chamber in which maximal α action potentials could also be elicited. After initiation of N₂ anoxia, electrical responses fell to zero at an average time of 22 min. A shorter time to zero of 11 min was seen during a second period of anoxia. A good recovery of both action potential responses and axoplasmic transport occurs after a period of anoxia lasting 1–1.5 hr. An apparent failure of recovery of axoplasmic transport was seen after 2 hr of anoxia with a good recovery of electrical responses. Axoplasmic transport tended to return toward normal when more time was allowed for recovery after anoxia. An adequate supply of ～P was shown to be present by measurement of ATP and creatine phosphate levels. The delay in recovery of transport thus signifies a failure of utilization of ～P by the transport mechanism. Longer periods of anoxia and recovery were limited in vitro and for this reason, ischemic anoxia was produced in vivo. Blood pressure cuffs were placed on the upper thigh of cats and maintained for times of 1–8 hr at pressures of 300–310 mm Hg. Then, recovery times up to 7 days were allowed. It was shown that axoplasmic transport could gradually recovery after an anoxia lasting up to 6–7 hr if sufficient recovery times were allowed. A possible explanation for the delay in the recovery of axoplasmic transport and the disassociation in the earlier recovery of electrical responses as against the recovery of transport was discussed.