O Cholinergic Neurochemistry

Cholinergic neurons synthesize, store, and release ACh (Fig. 17.5). The neurons also form choline acetyltransferase (ChAT) and AChE. These enzymes are synthesized in the soma of the neuron and distributed throughout the neuron by axoplasmic flow. AChE is also located outside the neuron and is associated with the neuroglial cells in the synaptic cleft. ACh is prepared in the nerve ending by the transfer of an acetyl group from acetyl-coenzyme A (CoA) to choline. The reaction is catalyzed by ChAT. Cell fractionation studies show that much of the ACh is contained in synaptic vesicles in the nerve ending but that some is also free in the cytosol. Choline is the limiting substrate for the synthesis of ACh. Most choline for ACh synthesis comes from the hydrolysis of ACh in the synapse. Choline is recaptured by the presynaptic terminal as part of a high-affinity uptake system under the influence of sodium ions25 to synthesize ACh.

Several quaternary ammonium bases act as competitive inhibitors of choline uptake. Hemicholinium (HC-3), a bisquaternary cyclic hemiacetal, and the triethyl analog of choline, 2-hydroxyethyltriethylammonium, act at the presyn-aptic membrane to inhibit the high-affinity uptake of choline into the neuron. These compounds cause a delayed paralysis at repetitively activated cholinergic synapses and can produce respiratory paralysis in test animals. The delayed block is caused by the depletion of stored ACh, which may be

Neurochemistry Acetylcholine

Figure 17.5 • Hypothetical model of synthesis, storage, and release of ACh. 1. ACh is released from storage granules under the influence of the nerve action potential and Ca2+. 2. ACh acts on postsynaptic cholinergic receptors. 3. Hydrolysis of ACh by AChE occurs in the synaptic cleft. 4. A high-affinity uptake system returns choline to the cytosol. 5. ChAT synthesizes ACh in the cytosol, and the ACh is stored in granules. 6. Glucose is converted to pyruvate, which is converted to acetyl-CoA in the mitochondria. Acetyl-CoA is released from the mitochondria by an acetyl carrier. 7. Choline is also taken up into the neuron by a low-affinity uptake system and converted partly to phosphorylcholine.

Figure 17.5 • Hypothetical model of synthesis, storage, and release of ACh. 1. ACh is released from storage granules under the influence of the nerve action potential and Ca2+. 2. ACh acts on postsynaptic cholinergic receptors. 3. Hydrolysis of ACh by AChE occurs in the synaptic cleft. 4. A high-affinity uptake system returns choline to the cytosol. 5. ChAT synthesizes ACh in the cytosol, and the ACh is stored in granules. 6. Glucose is converted to pyruvate, which is converted to acetyl-CoA in the mitochondria. Acetyl-CoA is released from the mitochondria by an acetyl carrier. 7. Choline is also taken up into the neuron by a low-affinity uptake system and converted partly to phosphorylcholine.

reversed by choline. The acetyl group used for the synthesis of ACh is obtained by conversion of glucose to pyruvate in the cytosol of the neuron and eventual formation of acetyl-CoA. Because of the impermeability of the mitochondrial membrane to acetyl-CoA, this substrate is brought into the cytosol by the aid of an acetyl "carrier."

The synthesis of ACh from choline and acetyl-CoA is catalyzed by ChAT. Transfer of the acetyl group from acetyl-CoA to choline may be by a random or an ordered reaction of the Theorell-Chance type. In the ordered sequence, acetyl-CoA first binds to the enzyme, forming a complex (EA) that then binds to choline. The acetyl group is transferred, and the ACh formed dissociates from the enzyme active site. The CoA is then released from the enzyme complex, EQ, to regenerate the free enzyme. The scheme is diagrammed in Figure 17.6. ChAT is inhibited in vitro by trans-N-methyl-4-(1-naphthylvinyl)pyridinium iodide26; however, its inhibitory activity in whole animals is unreliable.27

Newly formed ACh is released from the presynaptic membrane when a nerve action potential invades a presynaptic nerve terminal.28 The release of ACh results from depolarization of the nerve terminal by the action potential, which alters membrane permeability to Ca2+. Calcium enters the nerve terminal and causes release of the contents of several synaptic vesicles containing ACh into the synaptic cleft. This burst, or quantal release, of ACh causes depolarization of the postsynaptic membrane. The number of quanta of ACh released may be as high as several hundred at a neuromuscular junction, with each quantum containing between 12,000 and 60,000 molecules. ACh is also released spontaneously in small amounts from presynaptic membranes. This small amount of neurotransmitter maintains muscle tone by acting on the cholinergic receptors on the postsynaptic membrane.

Figure 17.6 • Ordered synthesis of acetylcholine (ACh) by choline acetyltransferase (ChAT).

Figure 17.6 • Ordered synthesis of acetylcholine (ACh) by choline acetyltransferase (ChAT).

After ACh has been released into the synaptic cleft, its concentration decreases rapidly. It is generally accepted that there is enough AChE at nerve endings to hydrolyze into choline and acetate any ACh that has been liberated. For example, there is sufficient AChE in the nerve junction of rat intercostal muscle to hydrolyze about 2.7 X 108 ACh molecules in 1 ms; this far exceeds the 3 X 106 molecules released by one nerve impulse.29

O CHOLINERGIC AGONISTS Cholinergic Stereochemistry

Three techniques have been used to study the conformational properties of ACh and other cholinergic chemicals: x-ray crystallography, nuclear magnetic resonance (NMR), and molecular modeling by computation. Each of these methods may report the spatial distribution of atoms in a molecule in terms of torsion angles. A torsion angle is defined as the angle formed between two planes, for example, by the O1—C5—C4—N atoms in ACh. The angle between the oxygen and nitrogen atoms is best depicted by means of Newman projections (Fig. 17.7). A torsion angle has a positive sign when the bond of the front atom is rotated to the right to eclipse the bond of the rear atom. The spatial orientation of ACh is described by four torsion angles (Fig. 17.8).

The conformation of the choline moiety of ACh has drawn the most attention in studies relating structure and

Newman projection Figure 17.7 • Spatial orientation of 01—C5—C4—N atoms in ACh.

pharmacological activity. The torsion angle (r2) determines the spatial orientation of the cationic head of ACh to the ester group. X-ray diffraction studies have shown that the torsion angle (r2) on ACh has a value of +77°. Many compounds that are muscarinic receptor agonists containing a choline component (e.g., O—C—C—N+[CH3]3) have a preferred synclinal (gauche) conformation, with r2 values ranging from 68° to 89° (Table 17.2). Intermolecular packing forces in the crystal as well as electrostatic interactions between the charged nitrogen group and the ether oxygen of the ester group are probably the two dominant factors that lead to a preference for the synclinal conformation in the crystal state. Some choline esters display an antiperiplanar (trans) conformation between the onium and ester groups. For example, carbamoyl choline chloride (r2, +178°) is stabilized in this trans conformation by several hydrogen bonds. Acetylthiocholine iodide (r2, +171°) is in this conformation because of the presence of the bulkier and less electronegative sulfur atom, and (+) trans-(1S,2S)-acetoxy-cyclopropyltrimethylammonium iodide (r2, +137°) is fixed in this conformation by the rigidity of the cyclopropyl ring.

NMR spectroscopy of cholinergic molecules in solution is more limited than crystallography in delineating the conformation of compounds and is restricted to determining the torsion angle O1—C5—C4—N. Most NMR data are in agreement with the results of x-ray diffraction studies. NMR studies indicate that ACh and methacholine apparently are not in their most stable trans conformation but exist in one of two gauche conformers30 (Fig. 17.9). This may result from strong intramolecular interactions that stabilize the conformation of these molecules in solution.31

Molecular orbital calculations based on the principles of quantum mechanics may be used to determine energy minima of rotating bonds and to predict preferred conformations for the molecule. By means of molecular mechanics, theoretical conformational analysis has found that ACh has an energy minimum for the r2 torsion angle at about 84° and that the preferred conformation of ACh corresponds closely in aqueous solution to that found in the crystal state.

The study of interactions between bimolecules and small molecules is of great interest and importance toward the understanding of drug action. These studies are challenging t3

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