Comparison between the effects of botulinum toxin-induced paralysis and denervation on molecular forms of acetylcholinesterase in muscles.

Velocity sedimentation analysis of acetylcholinesterase (AChE) molecular forms in the fast extensor digitorum longus muscle and in the slow soleus muscle of the rat was carried out on days 4, 8, and 14 after induction of muscle paralysis by botulinum toxin type A (BoTx). The results were compared with those observed after muscle denervation. In addition, the ability of BoTx-paralyzed muscles to resynthesize AChE was studied after irreversible inhibition of the preexistent enzyme by diisopropyl phosphorofluoridate.

The effects of pretreatment with soman simulator in the skeletal muscle: direct interactions with acetylcholinesterase.

Soman simulator PDP is a compound that has a chemical structure identical to soman, except that the fluorine atom is replaced by a methyl group which makes PDP unable to bind covalently to the AChE active center. In rats, late mortality observed after treatment with high doses of soman could be prevented by PDP pretreatment. Such pretreatment has been much less efficient in primates.

Influence of denervation on the molecular forms of junctional and extrajunctional acetylcholinesterase in fast and slow muscles of the rat.

Acetylcholinesterase (AChE) molecular forms in denervated rat muscles, as revealed by velocity sedimentation in sucrose gradients, were examined from three aspects: possible differences between fast and slow muscles, response of junctional vs extrajunctional AChE, and early vs late effects of denervation. In the junctional region, the response of the asymmetric AChE forms to denervation is similar in fast extensor digitorum longus (EDL) and slow soleus (SOL) muscle: (a) specific activity of the A12 form decreases rapidly but some persists throughout and even increases after a few weeks; (b) an early and transient increase of the A4 AChE form lasting for a few weeks may be due to a block in the synthetic process of the A12 form. In the extrajunctional regions, major differences with regard to AChE regulation exist already between the normal EDL and SOL muscle.

Biochemical, morphological, and functional changes during peripheral nerve regeneration.

The success of axon regeneration after nerve injury should be judged by the extent to which the target organs regain their function. Recovery of muscle contraction involves axon regeneration, reestablishment of nerve-muscle connections, recovery of transmission, and muscle force. All these processes were investigated under the same experimental conditions and correlated in order to better understand their time-course and interdependence.

Influence of innervation on molecular forms of acetylcholinesterase in regenerating fast and slow skeletal muscles.

Nerve-intact muscle regenerates were prepared by ischemic-toxic injury of slow soleus (SOL) and fast extensor digitorum longus (EDL) muscles of the rat. Rapid innervation of regenerating myotubes modified intrinsic patterns of AChE molecular forms, revealed by velocity sedimentation in linear sucrose gradients. Regarding their onset, the effects of innervation can be classified as early and late.

Interactions between intrinsic regulation and neural modulation of acetylcholinesterase in fast and slow skeletal muscles.

1. Initiation of subsynaptic sarcolemmal specialization and expression of different molecular forms of AChE were studied in fast extensor digitorum longus (EDL) and slow soleus (SOL) muscle of the rat under different experimental conditions in order to understand better the interplay of neural influences with intrinsic regulatory mechanisms of muscle cells. 2.

Prevention of diisopropylphosphorofluoridate-induced myopathy by botulinum toxin type A blockage of quantal release of acetylcholine.

Botulinum toxin type A (BTx), which blocks quantal and partially reduces spontaneous nonquantal acetylcholine (ACh) release at neuromuscular junctions, was tested for its possible attenuating effect on diisopropylphosphorofluoridate (DFP)-induced muscle lesions. The extent of muscle lesion in extensor digitorum longus and soleus muscle of DFP injected rats with and without BTx pretreatment was evaluated using light and electron microscopic procedures. In parallel experiments, acetylcholinesterase (AChE) activity was measured and the functional state of muscles in experimental groups was determined by electrophysiological methods.

[The "Young Researchers" project--the effect on scientific research and educational potential in Slovenia].

The aims and the development of the "Young Researchers" project, financed and headed by the "Research Association of Slovenia", have been described in this article. The goal of this project is to obtain new personnel resources for scientific research and developmental functions with a substantial expansion of personnel potential in research activity; a proportion of the 2000 young researchers is supposed to take part in the project by the end of 1990. They will renew the advisory personnel (researchers for renovation), and the other part, together with a defined number of already qualified researchers from research and university organizations, should be involved directly in immediate development and new production (researchers for development).

Attenuation of soman-induced lesions of skeletal muscle by acetylcholinesterase reactivating and non-reactivating antidotes.

It has been reported recently that some oximes reactivating acetylcholinesterase (AChE) exhibit concomitant ganglion-blocking effects which presumably could contribute independently to their powerful antidotal action in organophosphate inhibitor (OPI) poisoning, thus mimicking some unrelated substances which are effective antidotes without reactivating AChE. This raises the question whether OPI-induced muscle lesions, like some other symptoms could also be attenuated by oximes and other antidotes in the absence of AChE reactivation. To test this possibility, the oxime HI-6 was applied at increasing time intervals after the injection of soman until and beyond the point when soman-AChE complex becomes completely "aged" and not capable of reactivation.

Two types of focal accumulations of acetylcholinesterase appear in noninnervated regenerating skeletal muscles of the rat.

Muscle fibers in the soleus muscle of the rat, injured by bupivacaine and free autografting, were allowed to regenerate within their old basal laminae. Histochemical and cytochemical analysis of newly synthesized acetylcholinesterase (AChE) revealed that two kinds of focal accumulations of AChE appeared in regenerating myotubes. First, AChE gets concentrated at the sites of the former motor endplates.

Iso-OMPA-induced potentiation of soman toxicity in rat correlates with the inhibition of plasma carboxylesterases.

Recently, the question was raised as to why iso-OMPA, generally known as a selective irreversible inhibitor of butyrylcholinesterase (BuChE), potentiates soman toxicity in rats but not in mice. Mice are known to have higher carboxylesterase (CarbE) and lower BuChE activity in plasma than rat. It could be hypothesized that it is the iso-OMPA inhibition of plasma CarbE, and not of BuChE, which is responsible for potentiation of soman toxicity in iso-OMPA-pretreated rats.

Molecular forms and localization of acetylcholinesterase and nonspecific cholinesterase in regenerating skeletal muscles.

Molecular forms and histochemical localization of acetylcholinesterase and nonspecific cholinesterase were analysed in muscle regenerates obtained from rat EDL and soleus muscles after ischaemic-toxic degeneration and irreversible inhibition of preexistent enzymes. Regenerating myotubes and myofibres produce the 16S AChE form in the absence of innervation. The 10S AChE form prevails over 4S form with maturation into striated fibres.

Recovery of cholinesterases in soman-injected superior cervical ganglion of the rat in the presence and absence of innervation.

We have previously described the procedure for quantitative separation of extracellular and intracellular ChEs using mild treatment of rat superior cervical ganglion with papain. Here, this procedure was used in order to investigate the recovery of ChEs in the two pools after irreversible inhibition by soman which was directly injected into the ganglion. After such treatment only ganglion ChEs were totally inhibited, whereas the activity of ChEs in preganglionic neurons and their axons remained unaffected.

Effect of HI-6, applied into the cerebral ventricles, on the inhibition of brain acetylcholinesterase by soman in rats.

When applied to rats (intraperitoneally) immediately after subcutaneous injection of soman (120 micrograms/kg) HI-6 (100 mg/kg) protected about 40% of the activity of acetylcholinesterase (AChE) in the motor end plate region of the diaphragm but did not protect AChE in the brain. However, a partial protection of AChE in brain against inhibition by soman was obtained in anaesthetized, atropinized rats by the oxime injected into the cerebral ventricle 5 min before parenteral exposure to soman. The AChE activity in brain of rats pretreated with HI-6, analyzed 60 min after the injection of soman was between 10 and 19%, while that in non-protected animals did not exceed 1% of the control.

Properties of acetylcholinesterase and non-specific cholinesterase in rat superior cervical ganglion and plasma.

Amphiphile dependency, solubility in aqueous solutions, and sensitivity to proteolysis of acetylcholinesterase (AChE) and nonspecific cholinesterase (nsChE) in the rat superior cervical ganglion were studied and compared to properties of soluble plasma cholinesterases. Ganglion AChE shows strong amphiphile dependency: an amphyphilic substance must be present in the homogenizing medium in order to obtain maximal apparent enzyme activity. Apparent activity of AChE solubilized in Ringer's solution was also increased after subsequent addition of a detergent.

Asymmetric molecular forms of acetylcholinesterase in mammalian skeletal muscles.

Velocity sedimentation analysis of acetylcholinesterase (AChE) molecular forms was performed separately in endplate-rich and endplate-free regions of the diaphragm muscle of the rat, guinea pig, rabbit, dog, and pig, and in mm. erectores trunci and m. vastus lateralis in man.

Plasma acetylcholinesterase in Duchenne muscular dystrophy.

Muscle acetylcholinesterase (AChE) in unregulated in animal and human muscular dystrophies and its activity is elevated in plasma of dystrophic chickens, probably due to a leakage from affected muscles. It is possible to measure AChE activity in human plasma in spite of high butyrylcholinesterase activity if acetyl-beta-methylcholine is used as the substrate and butyrylcholinesterase is inhibited by iso-OMPA. It has been found that, unlike in chickens, the plasma AChE activity in human newborns is not higher than that in adults.

Presynaptic modulation of activity and molecular forms of acetylcholinesterase in the rat superior cervical ganglion during early postnatal development.

Preganglionic nerve trunk of the rat superior cervical ganglion was transected shortly after birth in order to evaluate the influence of preganglionic nerves on the development of acetylcholinesterase and choline acetyltransferase in ganglionic neurons. In spite of an early decentralization, specific activity of acetylcholinesterase in the ganglion is increasing during the first 3 wk of life until it is about equal to the activity which remains in the superior cervical ganglion decentralized in an adult animal. Thus, the preganglionic nerves, which per se contribute the presynaptic fraction of total ganglionic AChE activity in normal innervated ganglia, apparently exert no significant regulatory effect on the specific activity of the fraction of acetylcholinesterase affiliated with the developing ganglionic cells.

Activity, molecular forms, and cytochemistry of cholinesterases in developing rat diaphragm.

Acetylcholinesterase (AChE) and butyrylcholinesterase (BuChE) were studied in the diaphragm of early postnatal rats, using radiometric determination of enzyme activities, velocity sedimentation for separation of molecular forms, and electron microscopic cytochemistry to localize enzymes. AChE activity did not change significantly during the first 2 months after birth. The level of 16 S AChE was high at early stages, but decreased to adult levels between days 19 and 32 after birth.

Recovery of acetylcholinesterase in the diaphragm, brain, and plasma of the rat after irreversible inhibition by soman: a study of cytochemical localization and molecular forms of the enzyme in the motor end plate.

Recovery of AChE activity in the motor end plate region and end plate free region of the rat diaphragm was studied after irreversible inhibition by soman. Recovery was slow during the first 2 days and only 4 S and 10 S molecular forms of AChE were present in the end plate region. However, cytochemical evidence indicates that synaptic AChE has already started to accumulate and that the synthesis of AChE in muscle and Schwann cell might even be enhanced.

16 S acetylcholinesterase in endplate-free regions of developing rat diaghragm.

Velocity sedimentation patterns of acetylcholinesterase (AChE, EC 3.7.1.

Cholinesterases of neuromuscular junction.

The ultrastructural localization of AChE and BuChE was studied in motor endplates of the rat and mouse diaphragms, using the CNS(?) modification of the one-step Cu-thiocholine procedure. AChE activity was observed on pre- and postsynaptic membranes, in the basal lamina and in the Schwann cell-nerve terminal interspace. The localization of BuChE was similar but much less pronounced in the adult muscle, while for the developing muscle, the reverse was true.

Cholinesterases and choline acetyltransferase in the longitudinal muscle of the guinea pig ileum.

In order to gain insight into the possible role of the ACh-system in the smooth muscle cell, the presence of choline acetyltransferase, acetylcholinesterase and butyrylcholinesterase was studied in the longitudinal muscle of the guinea-pig ileum after the mechanical removal of Auerbach's plexus. Such treatment completely removes all nerve elements as confirmed by histochemistry and electron-microscopic examination. It was found that in the longitudinal muscle devoid of all nervous elements a substantial percentage of the activity of all three enzymes still remained.

Attachment of acetylcholinesterase to structures of the motor endplate.

The kinetics of AChE solubilization from intact motor endplates of mouse diaphragm, by collagenase, papain and hyaluronidase, was studied in parallel with the ultrastructural localization of AChE in treated neuromuscular junctions. Hyaluronidase did not solubilize more AChE from isolated motor endplate regions than Ringer's solution itself. Residual AChE activity could be demonstrated histochemically in motor endplates even after the plateau of solubilization by collagenase or papain was reached.