Acetylcholine vesicles

Lambert-Eaton syndrome is an antibody-mediated neuromuscular junction disorder. This disorder, which is most frequently encountered in patients with small-cell lung carcinoma, is characterized clinically by weakness and hyporeflexia. The impaired release of acetylcholine vesicles from presynaptic terminals at neuromuscular junctions that causes the weakness is a consequence of autoantibodies against small cell carcinoma epitopes that cross-react with and downregulate the expression of motor nerve terminal Ca2+ channels [39]. (See in Ch. 43.)... 

The acetylcholine vesicle release process is blocked by botulinum toxin through the enzymatic removal of two amino acids from one or more of the fusion proteins. 

At least two additional types of acetylcholine receptors are found within the neuromuscular apparatus. One type is located on the presynaptic motor axon terminal, and activation of these receptors mobilizes additional transmitter for subsequent release by moving more acetylcholine vesicles toward the synaptic membrane. The second type of receptor is found on perijunctional cells and is not normally involved in neuromuscular transmission. However, under certain conditions (eg, prolonged immobilization, thermal burns), these receptors may proliferate sufficiently to affect subsequent neuromuscular transmission. 

The terminals of cholinergic neurons contain large numbers of small membrane-bound vesicles concentrated near the synaptic portion of the cell membrane (Figure 6-3) as well as a smaller number of large dense-cored vesicles located farther from the synaptic membrane. The large vesicles contain a high concentration of peptide cotransmitters (Table 6-1), while the smaller clear vesicles contain most of the acetylcholine. Vesicles are initially synthesized in the neuron soma and transported to the terminal. They may also be recycled several times within the terminal. 

Acetylcholine vesicles fuse with the presynaptic membrane at a low rate to release their packets of transmitter even in the absence of nerve action potentials. This spontaneous release is random. It is insufficient to trigger an action potential in the muscle but can cause a small depolarisation of the membrane, termed a miniature endplate potential The smallest depolarisation is caused by release of the contents of a single vesicle, or one quantum of acetylcholine. After release the synaptic vesicle membrane is rapidly taken up into the nerve terminal and reutilised. The acetylcholine is broken down in the cleft to acetate and choline in a reaction catalysed by the enzyme acetylcholine esterase which resides in the neuromuscular cleft and the choline is taken back up into the nerve terminal where it participates in the synthesis of new transmitter. 

Inside the cytosol, the L chain of the toxin acts as a proteolytic enzyme whose activity is dependent on the Zn ions present in the molecules. It hydrolyses protein components of the exocytosis apparatus to block the release of the transmitter. It is known that botulinum toxins B, D and G can cleave vesicle associated membrane protein (VAMP), a protein present in the membranes of acetylcholine vesicles. Botulinum toxins A, C and E, on the other hand, act on proteins of the pres maptic plasma membrane. A and E cleave synaptosomal associated protein 25 (SNAP 2s) and C cleaves... 

Toxins produced by Clostridium botulinum interacts with synaptobrevin to block release of acetylcholine vesicles. Tox neuromuscular paralysis. 

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). 

The exocytotic release of neurotransmitters from synaptic vesicles underlies most information processing by the brain. Since classical neurotransmitters including monoamines, acetylcholine, GABA, and glutamate are synthesized in the cytoplasm, a mechanism is required for their accumulation in synaptic vesicles. Vesicular transporters are multitransmembrane domain proteins that mediate this process by coupling the movement of neurotransmitters to the proton electrochemical gradient across the vesicle membrane. 

Synaptic vesicles isolated from brain exhibit four distinct vesicular neurotransmitter transport activities one for monoamines, a second for acetylcholine, a third for the inhibitory neurotransmitters GABA and glycine, and a fourth for glutamate [1], Unlike Na+-dependent plasma membrane transporters, the vesicular activities couple to a proton electrochemical gradient (A. lh+) across the vesicle membrane generated by the vacuolar H+-ATPase ( vacuolar type proton translocating ATPase). Although all of the vesicular transport systems rely on ApH+, the relative dependence on the chemical and electrical components varies (Fig. 1). The... 

Chromaffin granules, platelet dense core vesicles, and synaptic vesicles accumulate ATP. ATP uptake has been demonstrated using chromaffin granules and synaptic vesicles and the process appears to depend on A(.lh+. It has generally been assumed that ATP is costored only with monoamines and acetylcholine, as an anion to balance to cationic charge of those transmitters. However, the extent of ATP storage and release by different neuronal populations remains unknown, and the proteins responsible for ATP uptake by secretory vesicles have not been identified. 

To achieve their different effects NTs are not only released from different neurons to act on different receptors but their biochemistry is different. While the mechanism of their release may be similar (Chapter 4) their turnover varies. Most NTs are synthesised from precursors in the axon terminals, stored in vesicles and released by arriving action potentials. Some are subsequently broken down extracellularly, e.g. acetylcholine by cholinesterase, but many, like the amino acids, are taken back into the nerve where they are incorporated into biochemical pathways that may modify their structure initially but ultimately ensure a maintained NT level. Such processes are ideally suited to the fast transmission effected by the amino acids and acetylcholine in some cases (nicotinic), and complements the anatomical features of their neurons and the recepter mechanisms they activate. Further, to ensure the maintenance of function in vital pathways, glutamate and GABA are stored in very high concentrations (10 pmol/mg) just as ACh is at the neuromuscular junction. 

Ceccarelli, B and Hurlbut, WP (1980) Vesicle hypothesis of the release of quanta of acetylcholine. Physiol. Rev. 60 396-441. 

The release of some peptides may differ from that of other transmitters, depending on the firing rate of the neurons. The large vesicles needed to store a peptide may need a greater rate of depolarisation for membrane fusion and release of the contents. In the salivary gland the release of vasoactive intestinal polypeptide requires high-frequency stimulation whereas acetylcholine is released by all stimuli. Due to the complexities and problems of access to CNS synapses it is not known if the same occurs here but there is no reason why this should not. In sensory C-fibres a prolonged stimulus appears to be a prerequisite for the release of substance P. 

Acetylcholine, which is set free from vesicles present in the neighbourhood of the presynaptic membrane, is transferred into the recipient cell through this channel (Fig. 6.25). Once transferred it stimulates generation of a spike at the membrane of the recipient cell. The action of acetylcholine is inhibited by the enzyme, acetylcholinesterase, which splits acetylcholine to choline and acetic acid. 

Salin-Pascual, R. J. Jimenez-Anguiano, A (1995). Vesamicol, an acetylcholine uptake blocker in presynaptic vesicles, suppresses rapid eye movement (REM) sleep in the rat. Psychopharmacology, fieri. 121, 485-7. 

A second transport system concentrates acetylcholine in the synaptic vesicle 193... 

Choline is supplied to the neuron either from plasma or by metabolism of choline-containing compounds 193 A slow release of acetylcholine from neurons at rest probably occurs at all cholinergic synapses 194 The relationship between acetylcholine content in a vesicle and the quanta of acetylcholine released can only be estimated 194 Depolarization of the nerve terminal by an action potential increases the number of quanta released per unit time 194 All the acetylcholine contained within the cholinergic neuron does not behave as if in a single compartment 194... 

FIGURE18-4 Neuropeptides and conventional neurotransmitters are released from different parts of the nerve terminal. A neuromuscular junction containing both large dense-core vesicles (containing the neuropeptide SCP) and also small synaptic vesicles (containing acetylcholine) was stimulated for 30 min at 12 Hz (3.5 s every 7 s). Depletion of the small clear vesicles at the muscle face and of the peptide granules at the nonmuscle face of the nerve terminal was observed. After stimulation, there was an increase in the number of large dense-core vesicles within one vesicle diameter of the membrane. (Adapted from reference [37].)... 

Measuring muscle-evoked responses to repetitive motor nerve electrical stimulation permits detection of presyn-aptic neuromuscular junction dysfunction. In botulism and the Lambert-Eaton syndrome, repetitive stimulation elicits a smaller than normal skeletal muscle response at the beginning of the stimulus train, due to impaired initial release of acetylcholine-containing vesicles from presyn-aptic terminals of motor neurons followed by a normal or accentuated incremental muscle response during repeated stimulation. This incremental response to repetitive stimulation in presynaptic neuromuscular disorders can be distinguished from the decremental response that characterizes autoimmune myasthenia gravis, which affects the postsynaptic component of neuromuscular junctions. 

Lipid-protein interactions are of major importance in the structural and dynamic properties of biological membranes. Fluorescent probes can provide much information on these interactions. For example, van Paridon et al.a) used a synthetic derivative of phosphatidylinositol (PI) with a ris-parinaric acid (see formula in Figure 8.4) covalently linked on the sn-2 position for probing phospholipid vesicles and biological membranes. The emission anisotropy decays of this 2-parinaroyl-phosphatidylinositol (PPI) probe incorporated into vesicles consisting of phosphatidylcholine (PC) (with a fraction of 5 mol % of PI) and into acetylcholine receptor rich membranes from Torpedo marmorata are shown in Figure B8.3.1. 

---