LONG-TERM INFLUENCE OF NERVE ON MUSCLE

The long-term objective of the research proposed in this application is to determine, at a molecular level, how motor neurons exert their trophic influence on rat skeletal muscle acetylcholinesterase (AChE). There is good evidence that the influence of nerve on muscle AChE is mediated in part by the activity (action potentials and/or contraction) set up in muscle by nerve and in part by a factor (AIF) that is delivered to muscle by nerve. We plan to improve our bioassay for AIF so that we can purify and characterize this factor efficiently. Muscle cells produce three major types of AChE; soluble globular forms that are secreted, integral sarcolemmal globular forms that are externalized with their active sites exposed, and asymmetric forms that appear to associate with the basal lamina that ensheathes each muscle fiber. Our working hypothesis is that each of these forms is assembled intracellularly from the monomeric form of the enzyme, externalized, and then lost by secretion or local degradative processes. We propose that the activity set up in muscle by nerve increases the rate at which all forms of the enzyme are synthesized (we have good evidence for this) and that innervation selectively increases the rate at which asymmetric forms are produced at the end-plate and decreases the rate at which asymmetric forms are degraded at the endplate. AIF selectively maintains endplate enzyme and probably acts by altering the metabolism of endplate asymmetric forms in one of the ways postulated. Using two different methods to evaluate AChE metabolism, we plan to test these hypotheses in various ways by examining three preparations - cultured embryonic rat myotubes, organ-cultured rat diaphragm, and model innervated muscles prepared by explanting phrenic nerve - hemidiaphragm preparations into organ culture and stimulating the nerve to maintain AChE at innervated levels. In an effort to extend our analysis to the mRNA level, we will evaluate the Xenopus oocyte as a system for determining if the increased rate of AChE synthesis that we have observed in active (fibrillating) embryonic rat myotubes is correlated with an increased amount of AChE mRNA.