Miro Brzin

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

Abstract 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. Tubular structures, observed underneath the motor end plate, may serve to transport the enzyme from its sites of synthesis in the sarcoplasmic reticulum. Asymmetric molecular forms of AChE in the end plate region appeared later during recovery and, one week after poisoning, their activity was only about 50% of normal value. The limited ability of newly synthesized AChE to attach to the subcellular structures and, therefore, to be retained in the muscle, may explain the phase of slow recovery. In accordance with this view, AChE activity in brain recovered in a similar way as in muscle, whereas soluble plasma cholinesterases recovered faster, apparently without a slow initial phase.

A comparison of the effect of cholinesterase inhibitors on end-plate current and on cholinesterase activity in frog muscle

Abstract The effect of various cholinesterase inhibitors on the end-plate current was studied in glycerol-treated frog skeletal muscle. In the same muscle, the activity of cholinesterases was estimated histochemically and measured quantitatively. By using the irreversibile cholinesterase inhibitor methanesulphonyl fluoride, cholinesterases were completely inhibited. This resulted in an increase of the amplitude, of the rise time and half-time of the end-plate current. By using the reversible cholinesterase inhibitors eserine and prostigmine, the changes of the end-plate current seemed complex and not well understood. At concentrations of these drugs which did not inhibit the activity of cholinesterases completely, the amplitude, the rise time and the half-time of the end-plate current were increased by a variable extent. At a higher concentration, which almost completely inhibited the cholinesterases, the amplitude and the half-time of the end-plate current were depressed. This was particularly well pronounced with the cholinesterase inhibitor BW284C51. The changes in the end-plate current, observed at a relatively high concentration of reversible inhibitors, are thought to be related either to a presynaptic, or to a postsynaptic “curare-like” action of these drugs.

Electron Microscopic-Cytochemical and Biochemical Studies of Acetylcholinesterase Activity in Denervated Muscle of Rabbits

Acetylcholinesterase (AChE) activity has been studied in normal, control and denervated muscle of rabbits by electron microscopic-cytochemistry and radiometric assay. A small amount of butyrylcholinesterase (BuChE) activity is also found in biochemical assay of unfixed muscle, but it is not demonstrable cytochemically in fixed specimens by the method used in this study. Both a soluble and particulate AChE activity are present in all specimens examined. The particulate activity is probably due to enzyme localized in the sarcotubular system and at the motor end-plate. Soluble AChE activity may represent those sites exhibiting random cytochemical end product, such as some areas of normal and denervated muscle and muscle nuclei, Schwann cells, and AChE-containing mononuclear cells in the connective tissue. There is a greater proportion of particulate than soluble AChE activity in normal and control muscle, a finding which is compatible with the well localized cytochemical sites. Four to six weeks post-denervation, there is a marked increase in extrajunctional AChE activity to peak values 15 to 30 fold above control values. The increase is accompanied by a reversal in the proportion of particulate to soluble enzyme, so that there is almost twice as much soluble as particulate AChE. There are also numerous “spots” of random cytochemical end product throughout extrajunctional muscle. The increase in levels of AChE activity, the change to predominantly soluble form, and the large numbers of new cytochemically active sites indicate that synthesis of new enzyme has taken place. Changes in AChE activity in denervated rabbit have been compared to those occurring in dystrophic mouse muscle. It has been suggested that there might be a relationship between the formation of new extrajunctional sarcoplasmic sites of AChE activity and the spread of α-bungarotoxin binding sites and chemosensitivity in developing and denervated muscle.

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

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.

Iodide, thiocyanate and cyanide ions as capturing reagents in one-step copper-thiocholine method for cytochemical localization of cholinesterase activity

SummaryThe necessity of the presence of iodide in Cu-ThCh reaction was investigated by following the precipitate formation “in vitro” and by evaluating the ultrastructural localization of the precipitate in sympathetic ganglion cells of the frog and in the end-plate regions of the rat diaphragm.It was found that thiocyanate or cyanide is the only anion that can be substituted for iodide as the capturing agent in precipitation. The optimal concentration in the preincubation and incubation media of any one of the three anions is from 2 to 5 mM. At a concentration below 1 mM precipitation “in vitro” is considerably delayed as a result of which in electron microscopy diffusion artefacts appear in tissue sections.The unconverted primary precipitate obtained in the presence of iodide had been used for ultrastructural localization of ChE activity and now this use has been extended to precipitates obtained in the presence of CN− or CNS−. Better-quality localization in the presence of either one of the latter anions suggests that they, and particularly CN−, should be substituted for I− in the one-step Cu-ThCh method for the cytochemistry of cholinesterases.

Attachment of acetylcholinesterase to structures of the motor endplate

SummaryThe 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 Ringers solution itself. Residual AChE activity could be demonstrated histochemically in motor endplates even after the plateau of solubilization by collagenase or papain was reached. Less than 35% of junctional AChE is left after collagenase, and less than 20% after papain treatment, as estimated by the percentage of AChE activity left in the isolated endplate region of the diaphragm after protease treatment. Cytochemically, both proteases had a similar effect on postsynaptic AChE. Residual AChE activity was distributed randomly, adhering to the sarcolemma of junctional clefts. Presynaptic AChE localized in the gap between axon terminal and Schwann cell appears to be resistant to collagenase but not to papain treatment. The mode of AChE attachment or the composition of the intercellular material in this gap may differ from that of the primary and secondary clefts.

Interactions Between Intrinsic Regulation and Neural Modulation of Acetylcholinesterase in Fast and Slow Skeletal Muscles

Summary1.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.Former junctional sarcolemma still accumulated AChE and continued to differentiate morphologically for at least 3 weeks after early postnatal denervation of EDL and SOL muscles. In noninnervated regenerating muscles, postsynapticlike sarcolemmal specializations with AChE appeared (a) in the former junctional region, possibly induced by a substance in the former junctional basal lamina, and (b) in circumscribed areas along the whole length of myotubes. Therefore, the muscle cells seem to be able to produce a postsynaptic organization guiding substance, located in the basal lamina. The nerve may enhance the production or accumulation of this substance at the site of the future motor end plate.3.Significant differences in the patterns of AChE molecular forms in EDL and SOL muscles arise between day 4 and day 10 after birth. The developmental process of downregulation of the asymmetric AChE forms, eliminating them extrajunctionally in the EDL, is less efficient in the SOL. The presence of these AChE forms in the extrajunctional regions of the SOL correlates with the ability to accumulate AChE in myotendinous junctions. The typical distribution of the asymmetric AChE forms in the EDL and SOL is maintained for at least 3 weeks after muscle denervation.4.Different patterns of AChE molecular forms were observed in noninnervated EDL and SOL muscles regeneratingin situ. In innervated regenerates, patterns of AChE molecular forms typical for mature muscles were instituted during the first week after reinnervation.5.These results are consistent with the hypothesis that intrinsic differences between slow and fast muscle fibers, concerning the response of their AChE regulating mechanism to neural influences, may contribute to different AChE expression in fast and slow muscles, in addition to the influence of different stimulation patterns.

Electron microscopic cytochemistry and microgasometric analysis of cholinesterase in the nervous system.

Publisher Summary Techniques from different disciplines have been combined in the present investigation to provide more meaningful information. Electron microscopic-Cytochemistry and microgasometric analysis have been used to study acetyl cholinesterase in the sympathetic and dorsal root ganglia of the frog, using tissue blocks or isolated cells. In addition, some observations on the development of cholinesterase in the nervous system of the embryonic rabbit and human are presented. Cholinesterase activity of isolated sympathetic and dorsal root neurons has been studied by use of the Cartesian diver, which permits a more sensitive method for quantitative determination of the activity of the enzyme. It is shown that the neural plasma lemma is the ultimate permeability barrier to the substrates acetylcholine and acetylthiocholine. The review also mentions about the materials and methods used in these investigation studies. Cervical sympathetic ganglia and lumbar dorsal root ganglia of the frog were used for cytochemical studies. For developmental studies embryonic tissue of rabbits from 9 to 18 days gestation and of human fetuses from 2 to 3 months gestation were employed. From the microgasometric analysis and cytochemistry of isolated neurons studies, the chapter puts forward a proved theory that the intact neural plasma lemma, and possibly, the satellite sheath formed a permeability barrier to the substrate. The enzyme, Acetyl cholinesterase was demonstrated in two principle areas depending on whether the tissue was fixed or unfixed prior to incubation.

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. The extrajunctional asymmetric AChE forms are absent in the EDL because they became completely repressed during the first month after birth, but they persist in the SOL. Differences remain also after denervation and are, therefore, not directly due to different neural stimulation patterns in both muscles: (a) an early but transient increase of the G4 AChE occurs in the denervated EDL but not in the SOL; (b) no significant extrajunctional activity of the asymmetric AChE forms reappears in the EDL up till 7 wk after denervation. In the SOL, activity of the asymmetric AChE forms is decreased early after denervation but increases thereafter.(ABSTRACT TRUNCATED AT 250 WORDS)

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. In order to test this hypothesis two doses of iso-OMPA were administered to rats prior to soman. The two doses were selected in such a way that both were high enough to inhibit more than 90% of plasma BuChE activity; plasma CarbE activity, however, was only slightly inhibited by the lower and substantially by the higher dose of iso-OMPA. Our results demonstrate that iso-OMPA-induced potentiation of soman toxicity correlates with the inhibition of CarbE and not with the inhibition of BuChE activity in rat plasma. Relative resistance of mice to iso-OMPA-induced potentiation of soman toxicity could therefore be explained by a higher proportion of CarbE activity remaining uninhibited after iso-OMPA pretreatment. By having their active centers unoccupied, CarbE molecules can bind soman and reduce its concentration in neuronal tissue and motor end-plates.