Hugo L. Fernandez

Cellular distribution of 16S acetylcholinesterase.

Multiple molecular forms of acetylcholinesterase (AChE; EC 3.1.1.7), in crude extracts of various tissues from the rat, were distinguished by velocity sedimentation analysis on linear sucrose gradients. Skeletal muscle samples containing end‐plate regions showed three different forms of AChE with apparent sedimentation coefficients of 16, 10 and 4s. The 16s form was not detected in non‐innervated regions of skeletal muscle, large intestine smooth muscle, whole brain tissue, red blood cells or plasma. Spinal cord, a predominantly motor cranial nerve and mixed (sensory and motor) peripheral nerves contained 16, 10, 6.5 and 4S AChE. Ventral motor roots, supplying the sciatic nerve, contained these four forms of the enzyme, while corresponding dorsal sensory roots were devoid of the 16S form. The 16s‐AChE confined to ventral roots can be attributed totally to motor neurons and not to Schwann cells composing these roots. Whether the 16s‐AChE presently found in motor nerves has chemical identity with that found at motor end‐plates is the basis of future experiments.

Muscle fibrillation induced by blockage of axoplasmic transport in motor nerves.

Abstract Colchicine injection under the epineurium of the cats hypoglossal nerve induced fibrillation and ACh hypersensitivity in the geniohyoid muscle. Experiments performed 1–47 days after the injection, showed that both effects were reversible and followed a similar time course, i.e. , they were detected between 6 and 30 days. These muscle alterations were brought about by colchicine acting directly on the nerve; the time of onset was related to the length of nerve distal to the injection site. Similar results were obtained after denervation but changes induced were irreversible and started earlier than those evoked by the drug. In addition, colchicine treatment did not affect nerve conduction or effective neuromuscular transmission. The present results can be ascribed to a differential and reversible blockage of axoplasmic transport by colchicine.

Subcellular localization of acetycholinesterase molecular forms in endplate regions of adult mammalian skeletal muscle

The characterization of individual acetylcholinesterase (AChE) molecular form subcellular pools in adult mammalian skeletal muscle is a critical point when considering such questions as the origin, assembly, and neurotrophic regulation of these molecules. By correlating the results of differential extraction, in vitro collagenase digestion, and in situ pharmacologic probes of AChE molecular forms in endplate regions of adult rat anterior gracilis muscle, we have shown that: 1) 4.0S (G1) and 6.0S (G2) AChE are predominantly membrane-bound and intracellular; if an extracellular and/or soluble fraction of these forms exists, it cannot be adequately resolved by our methods; 2) 9–11S (globular) AChE activity is distributed between internal and external pools, as well as membrane-associated and soluble fractions; 3) 16.0S (A12) AChE is not an integral membrane protein and exists both intracellularly (25–30%) and extracellularly (70–75%).

EFFECT OF DENERVATION AND OF AXOPLASMIC TRANSPORT BLOCKAGE ON THE IN VITRO RELEASE OF MUSCLE ENDPLATE ACETYLCHOLINESTERASE

Abstract— Cat geniohyoid muscle samples containing endplate regions, when incubated in vitro at 37°C in phosphate buffer (pH 73, release acetylcholinesterase (AChE; EC 3.1.1.7) to the bathing medium. By treating the muscle samples with collagenase (EC 3.4.4.19), it was confirmed that most of the AChE released came from the endplates. Enzyme liberation was studied 10 days after either local injection of 10mM‐cokhicine into the hypoglossal nerve or following nerve transection. Results showed that the rate of release is increased by denervation, but is not affected by axoplasmic transport blockage. It is postulated that the cellular contact between nerve and muscle—altered by denervation but not by interruption of axoplasmic transport—is an essential factor in maintaining the localization of end‐plate AChE within the synaptic cleft substance. This does not invalidate the possible participation of ACh and muscle activity in such enzyme localization.

Protease Inhibitors Reduce Effects of Denervation on Muscle End‐Plate Acetylcholinesterase

Abstract: The effects of certain protease inhibitors on end‐plate acetyl‐cholinesterase (AChE) activity, as well as on wet weight and total protein, were studied in vivo in intact and denervated anterior gracilis muscles from the rat. A combination of leupeptin, pepstatin, and aprotinin, administered intraarterially, partly prevented the early (24 h) denervation‐induced decrease in muscle weight and protein content. In turn, leupeptin and aprotinin, either alone or in combination, markedly reduced the decay of AChE activity in the denervated muscles, whereas pepstatin alone was ineffective. Such effects were additive in that the inhibitors in combination were more effective than when they were used separately. Additional experiments indicated that none of the inhibitors, at the concentrations used, affected AChE activity directly, nor did they have a significant effect during processing of the muscle samples. These findings indicate that the initial decay of AChE activity with denervation was effectively reduced by the inhibitors, probably through inactivation of proteolytic enzymes which, otherwise, would be increased in denervated muscle.

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 acetylcholine 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 radioimmunoassay of 3-mm nerve segments permitted the assessment of the peptides 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: lowintensity treadmill exercise; obturator nerve transection; nerve impulse conduction blockade by tetrodotoxin; acetylcholine (ACh) receptor (AChR) inactivation by α-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.

Selective Increase of Tetrameric (G4) Acetylcholinesterase Activity in Rat Hindlimb Skeletal Muscle Term Denervation

Abstract: 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 G4AChE (i.e., at both normal and temporarily elevated levels) does not critically depend on the presence of the motor nerve. In addition, this isoforms 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.

Retrograde axonal transport mediates the onset of regenerative changes in the hypoglossal nucleus

The hypoglossal nucleus was assayed for [14C]2-deoxyglucose uptake 24 h after axotomy with and without colchicine injections into the nerve proximal to the nerve transection. Colchicine effectively blocked the usual increase in [14C]2-deoxyglucose uptake seen after axotomy. The drug also blocked the transport of horseradish peroxidase from the tongue muscles to the hypoglossal nucleus which otherwise occurred within this time period. Distribution of [3H]colchicine showed that the drug remained localized within the nerve close to the injection point. These results suggest that retrograde axoplasmic flow is involved in the mechanism which initiates hypoglossal neuron regeneration.

Intra- versus extracellular recovery of 16S acetylcholinesterase following organophosphate inactivation in the rat

Acetylcholinesterase (AChE) inhibitor treatments were used to study the temporal course of intra- versus extracellular 16S AChE recovery in endplate regions of adult rat anterior gracilis muscles previously exposed to a brief, in situ application of diisopropylfluorophosphate (DFP). Following such enzymatic inactivation (95-100%), extracellular 16S AChE recovery began significantly later than that of intracellular (onset at approximately 36 and 12 h, respectively) but, once begun, progressed at approximately the same rate (1.32%/h). The recovery of AChE molecular form activities subsequent to identical DFP-inactivation was blocked to a large extent (65-85%) by in vivo treatment with cycloheximide, a protein synthesis inhibitor. These results support the hypothesis that extracellular 16S AChE at mammalian skeletal muscle motor endplates is primarily derived from complete, previously assembled 16S molecules originating in myofibers.