Physiological regulation of G4 AChe in fast-twitch muscle: effects of exercise and CGRP.

This work addresses the physiological regulation of the tetrameric (G4) form of acetylcholinesterase (AChe) in end-plate regions of anterior gracilis muscles from adult male Sprague-Dawley rats subjected to short-term low-intensity treadmill exercise. Experiments involved analyses of muscle AChe molecular form activities, endogenous calcitonin gene-related peptide (CGRP) levels, and the effect of exogenous CGRP on AChe forms after exercise. Animals were exercised twice per day for 1 or 2 days. Daily training sessions of 135 min (10 min of walking alternating with 5 min of resting) were separated by a 105-min resting period. Results show that exercise causes a slight decline in endogenous CGRP and a selective increase in G4 AChe that is partially reversed by treatment with exogenous CGRP. These findings indicate that CGRP influences the mechanism(s) by which G4 AChe in intact fast-twitch anterior gracilis muscles adapts to enhanced motor activity. They are also consistent with the hypothesis that, in addition to acetylcholine, neurogenic CGRP participates in the regulation of G4 AChe at the neuromuscular junction.

A role for calcitonin gene-related peptide in the regulation of rat skeletal muscle G4 acetylcholinesterase.

Calcitonin gene-related peptide (CGRP) acts as an anterograde trophic agent which regulates skeletal muscle acetylcholine receptor function. We examined whether CGRP also influences other synaptic transmission-related molecules, i.e. acetylcholinesterase (AChE) forms. Results show that: (a) CGRP associated with rat anterior gracilis muscle endplates declines following obturator nerve transection; (b) exogenous CGRP treatment has a selective, innervation-like effect on the globular tetramer (G4) of AChE in gracilis motor endplates; and (c) this effect is reversed by the CGRP receptor antagonist hCGRP8-37. We conclude that exogenous CGRP, and/or a biologically active CGRP fragment(s), influences G4 AChE levels through specific CGRP-CGRP receptor interactions. This conclusion is consistent with the notion that motor nerve-derived CGRP participates in the trophic control of G4 AChE at the neuromuscular junction.

Axoplasmic transport of calcitonin gene-related peptide in rat peripheral nerve as a function of age.

Calcitonin gene-related peptide (CGRP) has been implicated in the trophic regulation of acetyl-choline receptors and G4 acetylcholinesterase at the rat neuromuscular junction. Since these latter molecules exhibit significant changes with advancing age, we examined the possibility that certain aspects of CGRP transport are also influenced by aging. Double nerve ligations and CGRP radio-immunoassay of 3-mm nerve segments permitted the assessment of the peptide's apparent transport rates in sciatic nerves from 3-, 12-, and 24-month-old Fischer 344 rats. Results confirm that CGRP is conveyed by anterograde axoplasmic transport; more importantly, they suggest that CGRP is also transported retrogradely, but in smaller amounts and at slower rates. In addition, our findings indicate that the apparent rates of CGRP transport in both directions significantly decline with advancing age. These data are consistent with the notion that changes in CGRP delivery may contribute to age-related changes in junctional acetylcholine receptors and acetylcholinesterase.

Trophic regulation of acetylcholinesterase isoenzymes in adult mammalian skeletal muscles.

This work addresses the physiological regulation of skeletal muscle acetylcholinesterase (AChE) isoforms by examining endplate-enriched samples from adult rat gracilis muscles 48 h after: low-intensity treadmill exercise; obturator nerve transection; nerve impulse conduction blockade by tetrodotoxin; acetylcholine (ACh) receptor (AChR) inactivation by alpha-bungarotoxin; and, addition of obturator nerve extracts to muscles in organ culture. Results document the important role(s) of functional AChRs and ACh-AChR interactions in the differential control of individual AChE isoenzymes. A theoretical model based on these and other findings considers that: AChR activation by spontaneously released ACh is the only neural factor required for the maintenance of G1 + G2 AChE; the amount of A12 AChE is determined by the combined effects of ACh and another neurogenic substance; although mechanisms intrinsic to myofibers control normal levels of G4 AChE, enhanced production of this isoform is initiated through increasing the frequency of ACh-AChR interactions.

A role for acetylcholine-nicotinic receptor interactions in the selective increase of rat skeletal muscle G4 acetylcholinesterase following short-term denervation.

The present work addresses the effects of short-term denervation on acetylcholinesterase (AChE; EC 3.1.1.7) isoenzymes in anterior gracilis muscles from adult male Sprague-Dawley rats. It examines possible relationships between AChE isoform changes and other denervation phenomena, and evaluates the importance of acetylcholine (ACh)-nicotinic receptor interactions in selectively modulating the activity of G4 AChE. Results confirm that denervation causes a specific, transient increase in G4 AChE and show that: most of the increment can be explained by the hydrophobic species of this isoenzyme; changes in AChE isoforms markedly precede the onset of spontaneous electromechanical activity (fibrillation), as well as acetylcholine receptor (AChR) proliferation; and the G4 AChE response is eliminated when AChRs are blocked by alpha-bungarotoxin treatment performed before but not after (24 h) denervation. These data point to the absence of direct causal relationships between the G4 AChE increment and fibrillation, AChR proliferation, or changes in the release of this isoform from denervated muscle. In turn, they suggest the participation of AChR activation in triggering the G4 AChE response and emphasize the possible role of ACh-AChR interactions in modulating the production of this isoenzyme in not only denervated but also innervated fast-twitch muscles.

Selective increase of tetrameric (G4) acetylcholinesterase activity in rat hindlimb skeletal muscle following short-term denervation.

Acetylcholinesterase (AChE; EC 3.1.1.7) isoenzymes in gracilis muscles from adult Sprague-Dawley rats were studied 24-96 h after obturator nerve transection. Results show a selective denervation-induced increase in the globular G4 isoform, which is predominantly associated with the plasmalemma. This enzymatic increase was (a) transient (occurring between 24 and 60 h) and accompanied by declines in all other identifiable AChE isoforms; (b) observed after concurrent denervation and inactivation of the enzyme with diisopropylfluorophosphate, but not following treatment with cycloheximide; and (c) more prominent in the extracellular compartment of muscle endplate regions. Aside from this transient change, G4 activity did not fall below control levels, indicating that at least the short-term maintenance of G4 AChE (i.e., at both normal and temporarily elevated levels) does not critically depend on the presence of the motor nerve. In addition, this isoform's activity increases in response to perturbations of the neuromuscular system that are known to produce elevated levels of acetylcholine (ACh), such as short-term denervation and exercise-induced enhancement of motor activity. The present study is consistent with the hypothesis that individual AChE isoforms in gracilis muscle are subject to distinct modes of neural regulation and suggests a role for ACh in modulating the activity of G4 AChE at the motor endplate.

Coated vesicles from developing and adult rat skeletal muscles contain multiple molecular forms of acetylcholinesterase.

The main purpose of this work was to determine which of the multiple isoforms of acetylcholinesterase (AChE) are associated with clathrin-coated vesicles (CVs) from developing and adult rat skeletal muscles. CV-enriched preparations were obtained by subcellular fractionation/equilibrium sedimentation and further purified by immunoadsorption to anti-clathrin IgG-coated Staphylococcus aureus cells. Analysis of individual AChE isoforms by velocity sedimentation ultracentrifugation showed that a) while both globular (G-forms) and asymmetric (A-forms) AChE were detected in all subcellular fractions evaluated, the CV-enriched fraction contained a higher proportion of A-forms (mainly the A12 species); b) most of the AChE activity contained in such a CV fraction was recovered following immunoadsorption; c) alkaline extraction conditions (pH 8.5) which depolymerize clathrin were necessary to detect a large proportion of A-forms in both the CV-enriched and immunoprecipitated preparations, while most of the G-forms (especially G1 + G2 AChE) were detected following extraction at pH 6.8; and d) comparison of AChE isoform profiles from neonate and adult muscle CV-enriched fractions showed a greater concentration of A-forms in the former. These data suggest that considerable amounts of A12 and, to a lesser extent, G4 AChE are sequestered within muscle CVs which may be destined for the plasmalemma. Our findings also indicate that the relative proportions of AChE isoenzymes in rat muscle CVs vary according to the extent of muscle development and lend support to the contention that CVs participate in the externalization of functionally important AChE isoenzymes.

Permanent hippocampal mossy fiber hyperdevelopment following prenatal ethanol exposure.

Prenatal exposure to a liquid diet containing 35 percent ethanol derived calories during days 1-21 of gestation resulted in significant alterations in the topographical organization of the zinc-rich mossy fiber system of the rat hippocampus. Using the Timm's sulfide silver histochemical method, permanent alterations were found in the position of the mossy fiber terminal field. Aberrant distal infrapyramidal mossy fiber terminal staining occurred at midtemporal hippocampal levels in ethanol-exposed rats but not in the normal or pair-fed controls; the distribution of distal infrapyramidal mossy fibers in pair-fed rats was even more restricted than in normal rats suggesting that the aberrant mossy fiber topography was the result of the ethanol exposure rather than undernutrition. The abnormal terminal field was found in ethanol-exposed rats up to nine months of age (the oldest animals tested). The results indicate that ethanol exposure in utero, during a period of brain development roughly equivalent to the first and second human trimesters, can produce permanent developmental alterations in brain circuitry.