Assay Neuromuscular junction

A variety of methods have been developed to study exocytosis. Neurotransmitter and hormone release can be measured by the electrical effects of released neurotransmitter or hormone on postsynaptic membrane receptors, such as the neuromuscular junction (NMJ see below), and directly by biochemical assay. Another direct measure of exocytosis is the increase in membrane area due to the incorporation of the secretory granule or vesicle membrane into the plasma membrane. This can be measured by increases in membrane capacitance (Cm). Cm is directly proportional to membrane area and is defined as Cm = QAJV, where Cm is the membrane capacitance in farads (F), Q is the charge across the membrane in coulombs (C), V is voltage (V) and Am is the area of the plasma membrane (cm2). The specific capacitance, Q/V, is the amount of charge that must be deposited across 1 cm2 of membrane to change the potential by IV. The specific capacitance, mainly determined by the thickness and dielectric constant of the phospholipid bilayer membrane, is approximately 1 pF/cm2 for intracellular organelles and the plasma membrane. Therefore, the increase in plasma membrane area due to exocytosis is proportional to the increase in Cm. 

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The best known clinical application of cholinesterase assay concerns the abnormally prolonged effect of the muscle relaxant succinylcholine that is found in a small proportion of patients. This compound, which was introduced into clinical medicine in the early 1950s (B29, B41, T47), owes its relaxant action to competition with acetylcholine for the receptors at the neuromuscular junction both cause depolarization of the muscle fibers, which contract. Acetylcholine is rapidly destroyed by acetylcholinesterase, so that repeated stimuli are applied to the muscle, causing a controlled contraction which persists as long as the nerve is stimulated. When, however, succinylcholine is administered, it is not destroyed by acetylcholinesterase, and its action persists until a large proportion of the dose has been hydrolyzed in the plasma. After initial contraction, the muscle fibers passively elongate to give the relaxation required by the anesthetist. 

The physiological basis of action of organophosphates and carbamates is inhibition of acetylcholinesterase at the insect neuromuscular junction. Accordingly, for almost 30 years attempts have been made to analyse these pesticides using cholinesterase inhibition assays(14 >. Many of these assays have... 

In a study of the stereochemical preferences for curarimimetic neuromuscular junction blockade, the / ,i -isomer of A -methylpavine methiodide showed a modest statistical superiority in potency in two of the four assay systems employed. ... 

Figure 15.2. Drosophila larval neuromuscular junction system. A wandering third-instar larva is dissected open to reveal the ventral neuromusculature (see Figure 15.1). The peripheral nerve is severed and stimulated with a glass suction electrode. The muscle is recorded from in two-electrode voltage-clamp (TEVC) configuration. The postsynaptic excitatory junctional current (EJC) is recorded to assay synaptic transmission bottom inset). Top inset) Basis of synaptic transmission event being evoked by nerve stimulation and recorded via ion flux through muscle glutamate receptors.

Of all the synapses which have cholinergic transmission, that at the motor end-plate (at the volimtary neuromuscular junction) has been most studied. Its structure, visible even in the light microscope and sketched in Fig. 7.1, has revealed yet further complexities to the electron microscope, to electro-physiological measurements, to assays of acetylcholine vesicles and of acetylcholinesterase, and to autoradiography. The chemical nature of the receptors in the end-plate is imperfectly known, but a disulphide (S-S) group is essential for its functioning. This follows from the inhibition of the receptor by dithiothreitol (a disulphide reducer) and restoration of sensitivity by 5,5 -dithio-fe 5-2-nitrobenzoic acid (which restores a dithiol to the disulphide state) (Karlin and Bartels, 1966). 

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