Acetylcholine as a Neurotransmitter
Synthesis of acetylcholine
Synthesis of acetylcholine is facilitated by the enzyme, choline acetyltransferase (CAT). This enzyme combines choline with acetate derived from acetyl coenzyme A (CoA). Choline is taken up into cholinergic nerves by a high affinity transport process (sodium-choline cotransport) that is indirectly coupled to the energy stored by the Na/K pump ATPase. This transporter process is inhibited by hemicholinium-3 (HC-3). HC-3 has no immediate effect on neurotransmission, but can cause cholinergic nerve fibers eventually to run out of transmitter. In the presence of HC-3, the more rapidly cholinergic fibers are stimulated, the more rapidly they run out of ACh.
Storage of acetylcholine
ACh in cholinergic nerve fibers is taken up into synaptic vesicles by an uptake process that is inhibited by the drug vesamicol. In the presence of vesamicol, cholinergic fibers soon have no ACh stored in vesicles for release. Transmission fails although other functions of the fiber are still intact.
Release of acetylcholine
Release of acetylcholine, like synaptic release at other junctions, is based on quantal release of vesicles containing preformed neurotransmitter molecules. Vesicular release depends on depolarization of the nerve terminal and the influx of calcium ion. In ways not yet understood in detail, the influx of calcium promotes simultaneous exocytosis of many vesicles. At the motor end-plate in the neuromuscular junction this results in a relatively massive release of ACh (hundreds of vesicles and thousands of ACh molecules per vesicle) and an end-plate potential that normally results in depolarization of the muscle cell and contraction. The effect of background quantal release of ACh-containing vesicles can be observed as miniature end plate potentials (mepps). The release of ACh at various cholinergic junctions can be blocked by certain toxins, most notably those produced by Clostridium species. Botulinum toxin A, from Clostridium botulinum binds to cholinergic nerve terminals and is internalized. Once internalized it acts on the vesicle release process and prevents exocytosis. All junctional release of ACh is inhibited by such toxins. In patients poisoned by Clostridium botulinum the immediate clinical problem is flaccid paralysis and respiratory failure.
Actions of acetylcholine
Acetylcholine (ACh) has diverse actions on a number of cell types mediated by two major classes of receptors. Nicotinic receptors are ligand-gated ion channels. Muscarinic receptors are part of the transmembrane, G protein coupled receptor family.
There are two major subtypes of nicotinic receptors; those found in the neuromuscular junction of skeletal muscle (nicotinic muscle, Nm) and those found in autonomic ganglia and other parts of the nervous system (nicotinic neuronal, Nn). When ACh or other agonists occupy the receptor site on the external surface of the cell membrane there is a conformational change in the ion channel and an increase in conductance to the ion(s) for which that channel is selective. Thus, when Nm receptors are activated, there is an influx of cations through the ion channel and depolarization of the motor end plate. In short, nicotinic receptors rather directly transduce the ACh external messenger into an action on the cell.
Transduction of the ACh message is more complex in the muscarinic family of receptors. And the family of muscarinic receptors is more complex than the nicotinic family. There are at least 5 muscarinic receptor subtypes expressed in humans. For most purposes it is sufficient to concentrate on M1, M2 and M3 receptors. M1 receptors are located in autonomic ganglia and the central nervous system. M2 receptors are located mainly in the supraventricular parts of the heart. M3 receptors are located in smooth muscles and glands, and on endothelial cells in the vasculature.
M1 and M3 receptors are coupled to the enzyme phospholipase C (PLC) through a G protein. When the receptor is activated the enzyme increases splitting of phosphatidylinositol polyphosphates of the cell membrane into (mainly) inositol-1,4,5-trisphosphate (IP3) and diacylglycerol (DAG). IP3 contains many charged phosphate groups and is water soluble. It is thus released into the interior of the cell and acts on IP3 receptors on the surface of the endoplasmic (or sarcoplasmic) reticulum. IP3 receptors increase the release of Ca from the ER and increased cytosolic Ca is thus part of the intracellular message from ACh at the surface membrane. In the example illustrated here, an M3 receptor on a smooth muscle cell promotes smooth muscle contration by promoting increased cytosolic free Ca ion. Another part of the transduced message from ACh at the cell surface is DAG. Because DAG is lipid soluble it remains in the cell membrane. Its presence in the membrane, along with increased intracellular Ca activates a protein kinase, protein kinase C (PKC). PKC is involved in turn in regulating a number of other enzyme activities.
The bottom line is that M1 and M3 receptors generally mediate excitatory responses in effector cells. Thus, M1 receptors promote depolarization of postganglionic autonomic nerves, and M3 receptors mediate contraction of all smooth muscles (an apparent exception to be noted below) and increased secretion in glands. It is useful to remember that excess ACh levels in the body (for example caused by inhibition of AChE) are associated with GI cramping, salivation, lacrimation, urination, etc.
An apparent, but important, exception to the general rule that ACh stimulates all smooth muscle is the effect of ACh on blood vessels. When injected intravenously (see Virtual Lab pathway for examples), ACh causes vasodilation and decreased blood pressure. This is mediated by an effect of ACh on the endothelial cells of the vasculature. In response to activation of M3 receptors on the surface of the endothelial cells there is increased intracellular Ca and activation of the enzyme nitric oxide synthase (NOS). This results in increased synthesis of the highly diffusable free radical, nitric oxide (NO). NO diffuses from endothelial cells into the adjacent smooth muscle cells of the vasculature. In those cells, NO activates the cytoplasmic enzyme, guanylate cyclase. This increases intracellular cyclic-3',5'-guanosine monophosphate (cyclic GMP or cGMP), which promotes relaxation of the vascular smooth muscle cells. Note that relaxation of vascular smooth muscle by ACh is an indirect effect that is utterly dependent on the presence of intact endothelial cells. If the endothelium is removed, ACh exerts a stimulatory effect on vascular smooth muscle cells, as it does on other smooth muscle cells. The interaction between endothelial and vascular smooth muscle cells was demonstrated by Robert Furchgott who was awarded the Nobel Prize for his work in this area.
M2 muscarinic receptors, in contrast to M1 and M3 receptors, tend to mediate inhibition of cellular activity. They do so through G proteins that inhibit adenylyl cyclase (opposite of the activation of adenylyl by beta adrenergic receptors) and by activation of K channels in the plasma membrane. Clinically important examples of K channel activation by ACh are especially prominent in the supraventricular parts of the heart. Thus, it is important to be familiar with the effects of ACh on the cellular electrophysiology of the heart. This diagram shows the respective SA node, atrial and AV nodal action potentials as they might occur in a control situation. The SA node action potentials appear first in time, because this is the site of the normal pacemaker. The diastolic depolariztion that leads smoothly into a relatively slow upstroke action potential. This is characteristic of a pacemaker cell and a cell with a relatively low membrane voltage. The slow upstroke action potential happens because Na channels are inactivated to a significant extent, leaving the Ca channels to carry the 'spikes' in these nodal cells.
The second tracing shows that the wave of excitation has passed a cell in the atrium. The upstroke is fast showing that Na channels were 'ready to go' in the atrial cell. The action potential repolarizes after some delay, showing that K channels activate somewhat slowly in a voltage and time dependent manner.
The bottom tracing shows that the wave of excitation has been conducted to a cell in the AV node. The electrophysiological properties of the AV node are similar to those of the SA node. Thus AV nodal cells also show spontaneous diastolic depolarization and slow upstroke action potentials. It should not be surprising that AV nodal rhythms are among the most common arrhythmias.
These tractings demonstrate the effects of ACh on supraventricular cells of the heart, most easily accomplished by stimulating the vagus nerve. Activation of K channels by muscarinic receptors brings about important changes in the electrophysiology of supraventricular cells. This diagram shows decreased heart rate, characteristic of vagal or ACh effects. The top tracing shows that K channel activation inhibits spontaneous activity of the SA node cells (slower spontaneous diastolic depolarization, a greater maximal diastolic potential [more negative and further from threshold]). These effects combine to decrease heart rate.
In the second tracing, atrial cells display decreased action potential duration because there is no 'delay' for increasing K permeability of the membrane when the membrane becomes depolarized (K permeability is already increased by ACh action). The effect on action potential duration shortens the atrial refractory period. This is critical in patients who may have atrial flutter or fibrillation.
As shown in the bottom tracing, the AV node cells respond to ACh much as SA node cells. They show a decreased rate of spontaneous diastolic depolarization and a greater maximal diastolic potential (closer to the K equilibrium potential). Although it is not apparent in this diagram, the AV nodal cells also conduct more slowly (would be seen as an increased P-R interval on EKG) and, when appropriately tested, display increased refractory period of the AV node. This latter phenomenon is extremely important for limiting ventricular rate in patients with supraventricular tachyarrhythmias.
Note that all of these seemingly diverse effects of ACh are mediated by an increase in membrane permeability to K.
Inactivation of acetylcholine
Acetylcholine (ACh) is terminated by hydrolysis, which is greatly accelerated by one or more of the cholinesterase enzymes. Acetylcholinesterase (AChE) is present in high concentration in cholinergic synapses. Butyrylcholinesterase, also known as pseudocholinesterase is important for hydrolyzing ACh in the circulation. It is important to recognize that the neurotransmitter actions of acetylcholine are terminated by a chemical reaction that forms two products (choline and acetate) which are essentially inactive. Diffusion of ACh from the synaptic region plays a minor role because AChE is so active. By contrast, the neurotransmitter actions of catecholamines are terminated mainly by diffusion away from the postsynaptic receptors; a process greatly facilitated by the active re-uptake of the catecholamine into the presynaptic nerve terminal. AChE inhibitors, also designated AChEIs, include echothiophate, edrophonium, neostigmine, physostigmine. Other AChEIs include various so-called nerve gas agents such as sarin and soman.
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