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 he 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, 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.

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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. However, the absence of innervation during development is strongly reflected in the pattern of acetylcholinesterase molecular forms. The activity of the 16 S molecular form of AChE remains high in the developing superior cervical ganglion, decentralized at birth, in contrast to the substantial absolute and relative decrease of specific activity of this form during development of a normally innervated ganglion. A high proportion of 16 S AChE probably reflects a shift of decentralized immature ganglion nerve cells toward a cholinergic character. In accordance with this assumption, choline acetyltransferase activity in early decentralized ganglia is significantly higher than that in the ganglia decentralized in adult animals.

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. The 4 S molecular form, predominant in this extract (corresponding to the fastest electrophoretic band), is very sensitive to papain proteolysis but can be protected by a detergent. This molecular form therefore carries an important hydrophobic domain and is probably membrane bound in situ. The 10 S form of ganglionic AChE, extracted in Ringer's solution, is probably a soluble enzyme since, like soluble plasma enzymes, it is not amphiphile dependent and is rather resistant to proteolysis. Ganglion nsChE is more water soluble, less amphiphile dependent and more protease resistant than AChE.

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. Comparing in innervated and decentralized ganglia the pattern of recovery rate and ultrastructural reappearance of ChEs after local inhibition, with that reported after systemic ChEs inhibition, it was possible to distinguish between the indirect effects of innervation on the recovery rate and pattern of ChEs of ganglion origin and the direct contribution to the total ganglion enzyme activity of ChEs originating in the preganglionic elements.The absence of nerve contracts affects mostly extracellular activity, particularly AChE, whereas the intracellular activity of AChE was only slightly decreased and the activity of nsChE was somewhat increased. This increase coincides with the enhanced cytochemical reaction of nsChE in some nonneuronal cells in the ganglion.Actinomycyn D decreased the rate of initial rapid phase of recovery of intracellular ChEs when injected in the ganglion daily for three days, whereas the recovery of extracellular ChEs was already decreased the first day of Actinomycyn D application. This indicates that the externalization of the enzyme is more affected than its synthesis by inhibition the translation step.The results further suggest that functional innervation of the ganglion down-regulate the rate of reappearance of the new enzyme at the extracellular and intracellular locations and changes the ratio between AChE and nsChE. ChEs synthesized in preganglionic neurons begins to contribute directly to the total ganglion ChEs activity in the late stages of enzyme recovery in spite of the presence of the preganglionic ChEs @ 1987 Alan R. Liss, Inc.which was not inhibited after intraganglionic injection of soman. This finding suggests that the externalization and extracellular attachment of ChEs and not their preganglionic resynthesis may be the cause for delayed reappearance of ChEs affiliated with nerve terminals in the ganglion.

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