The effects of persistent articholinesterase action at the neuromuscular junction

The effects of organophosphorus compounds which form a rapidly-ageing complex with acetylcholinesterase (AChE) (e.g. pinacolyl S-(2- trimethylaminoethyl)methylphosphonothioate (BOS)) and hence exert a persistent anticholinesterase (anti-ChE) action have been compared with other compounds with a shorter time course of inhibition (e.g. ecothiopate iodide (ECO)). Although the inhibition of AChE produced by BOS lasted longer than that seen with ECO, the time course of the myopathy appeared very similar. BOS also possessed a number of properties which have been seen with other anti-ChEs. BOS and ECO produced significant increases in neuromuscular "jitter" 5 days after injection, not only in the diaphragm but also in the soleus and extensor digitorum longus muscles. Increases in "jitter" produced by ECO could be prevented by pyridostigmine prophylaxis or rapid treatment with pyridine-2- aldoxime methiodide. Some protection from the BOS-induced increases in "jitter" could be gained by repeated treatment with pyridine-2-aldoxime methiodide, an effect which could not be accounted for simply by enzyme reactivation. From experiments performed in Rej 129 mice it was determined that increases in "jitter", although demonstrated in some dystrophic muscles, could not be used as an early diagnostic tool. Because sequalae of inhibition were present some time after intoxication, by which time AChE appeared biochemically normal, experiments were performed to investigate inactivation of physiologically important AChE. The time course of extracellular MEPPs was utilised as an indicator of physiologically important AChE and compared with the AChE activity measured by the technique of Ellman et al. (1961). It was concluded that the degree of persistence of anti-ChE action was unimportant for the induction of myopathy with a time course of 3-24 hours, but had some importance in events of longer duration.

The neurotoxic effects of enzyme inhibitors at the neuromuscular junction

Current knowledge of the long-term, low dose effects of carbamate (CB) anti-cholinesterases on skeletal muscle or on the metabolism and regulation of the molecular forms of acetylcholinesterase (AChE) is limited. This is largely due to the reversible nature of these inhibitors and the subtle effects they induce which has generally made their study difficult and preliminary investigations were conducted to determine suitable study methods. A sequential extraction technique was used to rapidly analyse AChE molecular form activity at the mouse neuromuscular junction and also in peripheral parts of muscle fibres. AChE in the synaptic cleft involved in the termination of cholinergic transmission was successfully assessed by the assay method and by an alternative method using a correlation equation which represented the relationship between synaptic AChE and the prolongation of extra-cellular miniature endplate potentials. It was found that inhibition after in vivo Carbamate (CB) dosing could not be maintained during tissue analysis because CB-inhibited enzyme complexes decarbamoylated vary rapidly and could not be prevented even when maintained on ice. The methods employed did not therefore give a measure of inhibition but presented a profile of metabolic responses to continual, low dose CB treatment. Repetitive and continual infusion with low doses of the CBs: pyridostigmine and physostigmine induced a variety of effects on mouse skeletal muscle. Both compounds induced a mild myopathy in the mouse diaphragm during continual infusion which was characterised by endplate deformation without necrosis; such deformation persisted on termination of treatment but had recovered slightly 14 days later. Endplate and non-endplate AChE molecular forms displayed selective responses to CB treatment. During treatment endplate AChE was reduced whereas non-endplate AChE was largely unaffected, and after treatment, endplate AChE recovered, whereas non-endplate AChE was up-regulated. The mechanisms by which these responses become manifest are unclear but may be due to CB-induced effects on nerve-mediated muscle activity, neurotrophic factors or morphological and physiological changes which arise at the neuromuscular junction. It was concluded that, as well as inhibiting AChE, CBs also influence the metabolism and regulation of the enzyme and induce persistent endplate deformation; possible detrimental effects of long-term, low-dose determination requires further investigation.

The toxic effects of anticholinesterases on muscle

It has been shown that acute administration of ecothiopate iodine in vivo caused an approximate 80% depression of acetylcholinesterase activity in the diaphragms of mice. Inhibition of acetylcholinesterase was accompanied by an influx of calcium at the junctional region of the diaphragm, which continued during subsequent progressive development of a severe myopathy located in the same region. Myopathy was accompanied by loss of creatine kinase from the muscle and was represented, at the light microscope level, by hypercontraction, Procion Yellow staining and loss of cross striations within the muscle fibres. It appeared to reach a point of maximum severity approximately 3-6 hours after ecothiopate administration and then, by means of some repair/regeneration process, regained an apparently normal morphology within 72 hours of the intoxication. At the ultrastructural level, ecothiopate-induced myopathy was recognised by loss of Z-lines, swelling and vacuolation of mitochondria and sarcoplasmic reticulum, dissarray of myofilaments, crystal formation, and sometimes, by the complete obliteration of sarcomeric structure. The development of myopathy in vitro was shown to be nerve-mediated and to require a functional acetylcholine receptor for its development It was successfully treated therapeutically in vivo by pyridine-2-aldoxime methiodide and prophylactically by pyridostigmine bromide. However, the use of a range of membrane-on channel blockers, and of leupeptin, an inhibitor of calcium-activated-neutral-protease, have been unsuccessful in the prevention of ecothiopate-induced myopathy.

Mechanisms of anticholinesterase-induced myopathy and its prevention

In the introduction a brief outline of the possible mechanisms involved in the process of cellular necrosis with particular emphasis on skeletal muscle necrosis after antiChE is discussed. Ecothiopate (ECO), an antiChE, was shown to produce dose-dependent inhibition of both AChE and BuChE in diaphragm and blood of mice. Inhibition of AChE resulted in dose-dependent influx of calcium at the junctional region with the consequent development of morphological and biochemical alterations. Non-necrotising doses of ECO caused hypercontractions of varying severity, distorted end plate and slight elevation of serum creatine kinase (CK). Necrotising doses of ECO further caused contraction clumps, loss of striations and procion staining with high serum CK. The extent of ECO-induced myopathy depended on entry of extracellular calcium rather than the degree of AChE inhibition. The essential Ca2+ mediated process(es) in ECO-induced myopathy was thought to be the generation of superoxide and superoxide-derived free radicals and/or lipid peroxidation. Mitochondria and xanthine oxidase may be the major contributors to the generation of superoxide. No evidence was found for the depletion of high energy phosphates. ECO-induced myopathy could be successfully prevented by prior administration of pyridostigmine or various antioxidants, the most effective being Vit E or Vit E + N-acetylcysteine. Allopurinol or N-acetylcysteine alone were also effective. However, the use of a wide range of membrane end plate channel blockers or non-quantal release blockers were unsuccessful in the prevention of ECO-induced myopathy.

Study of the cholinergic factors affecting the flash and pattern reversal visual evoked potentials

The effects of cholinergic agents undergoing clinical trials for the treatment of Alzheimer's disease and the anticholinergic agent scopolamine, were investigated on the components of the flash and pattern reversal visual evoked potentials (VEPs) in young healthy volunteers. The effect of recording the flash and pattern reversal VEPs for 13 hours in 5 healthy male volunteers, revealed no statistically significant change in the latency or amplitude measures. Administration of the muscarinic agonist SDZ 210-086 to 16 healthy male volunteers resulted in the reduction of the flash N2-P2 and pattern reversal N75-P100 peak-to-peak amplitudes. These effects on the flash VEP occurred at both doses (0.5 and 1.0 mg/day), but only at the higher dose on the pattern reversal VEP. Administration of the antimuscarinic agent scopolamine to 11 healthy young male volunteers, resulted in a delay of the flash P2 latency but no effect on the pattern reversal P100 latency. The pattern reversal N75-P100 peak-to-peak amplitude was also increased post dosing. The combination of scopolamine with the acetylcholinesterase inhibitor SDZ ENA 713 resulted in no significant effect on the flash and pattern reversal VEPs, suggesting that the effects of scopolamine may have been partially reversed. Topical application of scopolamine in 6 young healthy volunteers also resulted in no statistically significant effects on the flash and pattern reversal VEPs. The selective effect of scopolamine on the flash P2 latency but not on the pattern reversal P100 latency, provided a model whereby new cholinergic agents developed for the treatment of Alzheimer's disease can be investigated on a physiological basis. In addition, the results of this study led to the hypothesis that the selective flash P2 delay in Alzheimer's disease was probably due to a cholinergic deficit in both the tectal pathway from the retina to the visual cortex and the magnocellular path of the geniculostriate pathway, whereas the lack of an effect on the pattern reversal P100 component was probably due to a sparing of the parvocellular geniculostriate pathway.