Physiology, Acetylcholine

Issues of Concern

Acetylcholine intervenes in numerous physiological functions, such as regulating cardiac contractions and blood pressure, intestinal peristalsis, glandular secretion, etc. Typically, acetylcholine is an excitatory mediator. The system of cholinergic nerve fibers that release acetylcholine at their endings is widespread in both the central and peripheral nervous systems.In the periphery, all the preganglionic fibers are cholinergic, sympathetic, parasympathetic, the parasympathetic postganglionic, and the motor fibers that innervate the voluntary skeletal muscle. In the central nervous system, the cholinergic system has extensive branches in the spinal cord, thalamus, limbic system, and cortex. Acetylcholine ensures rapid but generally fleeting neurotransmission due to the prompt inactivation of the mediator by acetylcholinesterase. Acetylcholine receptors subdivide into two types: nicotinic - ion channels for sodium and calcium, and muscarinic -coupled with G proteins.

Acetylcholine is also involved in the immune system because it is secreted by T lymphocytes.

Cellular Level

Acetylcholine derives from two constituents, choline, and an acetyl group, the latter derived from the coenzyme acetyl-CoA.[2] Choline is naturally present in foods such as egg yolks, liver, seeds of various vegetables, and legumes. Choline is also produced by the liver natively. Once choline is circulating in the plasma, it can readily cross the blood-brain barrier and be taken up by cholinergic nerve terminals via sodium-dependent uptake channels.[3][4] The rate-limiting step in acetylcholine production is the availability of acetate derived from mitochondrial acetyl-CoA and choline derived from the plasma directly and from reuptake from the synaptic cleft. 

The synthesis of acetylcholine occurs in the terminal ends of axons. Choline acetyltransferase (CAT) is the enzyme that catalyzes the reaction of choline with acetyl-CoA to create a new molecule of acetylcholine. CAT is produced in the neuronal soma (body) and subsequently transported to the axon terminus via axoplasmic transport in which vesicles full of various proteins are “hitched” to actin filaments that span the length of the neuron for transport. Although localized mainly to the axon terminus, CAT is present throughout the neuron itself.[5][6]

In the axon terminal, newly formed acetylcholine will be placed in vesicles with a minuscule number of free molecules still free in the cytosol. The vesicles are acidified via an energy-dependent pump (H-ATPase), which is utilized to create a gradient for acetylcholine to enter via vesicular acetylcholine transporter (VAChT), which exchanges one vesicular proton for one molecule of acetylcholine.[7]

The release of acetylcholine occurs when an action potential is relayed and reaches the axon terminus in which depolarization causes voltage-gated calcium channels to open and conduct an influx of calcium, which will allow the vesicles containing acetylcholine for release into the synaptic cleft. This release is highly dependent upon the SNARE protein system.[8][9] Synaptobrevin is often referred to as a “v” SNARE since it is in the vesicular membrane, and SNAP-25 along with syntaxin-1, often called “t” SNAREs since they are part of the presynaptic membrane, are different types of SNARE proteins that work together with calcium to perform vesicle membrane fusion and release. Importantly, synaptogamin is another vesicle-bound SNARE protein that will act as the calcium sensor for this system. Once the vesicle docks close enough to the presynaptic membrane, the cytosolic protein Munc18 will serve as an activating “clasp” that will attach synaptobrevin to SNAP-25 and syntaxin-1, bringing the vesicle and presynaptic membrane into close apposition as their free helical ends begin to twist around each other. The cytosolic protein complexin will then insert itself into this newly formed SNARE complex and prevent spontaneous fusion of the vesicle with the presynaptic membrane to prevent spontaneous fusion. When calcium is finally introduced into the cell after neuronal depolarization, it will bind synaptogamin and allow this molecule to bind to acidic phospholipids in the presynaptic membrane and displace the complexin molecules, which will then promote the fusion-block to be lifted. Only with the introduction of calcium into the cell can vesicular fusion to the presynaptic membrane be accomplished.[10] After completion of the fusion process, Ca-ATPase (PMCA) will pump calcium out of the neuron, and neuronal mitochondria will uptake calcium, both processes aiming to decrease intracellular calcium concentration. With the decrease in calcium, we see that synaptogamin will disassociate from the SNARE complex and that other SNARE proteins will be recruited to break down and recycle the constructed complex to get ready for the next round of vesicle fusion.[11] Once in the synaptic cleft, acetylcholine can bind either acetylcholine or muscarinic cholinergic receptors.

There are two subtypes of nicotinic receptors, the muscular type (N1) and the neuronal type (N2). The muscular type is found specifically on the surface of muscle cells at the neuromuscular junction. The neuronal subtype is in the peripheral and central nervous systems. Specifically, N2 receptors are present in the adrenal medulla, on the postsynaptic cell bodies of neurons within the sympathetic and parasympathetic nervous systems, as well as in various locations in the brain such as the ventral tegmental area, hippocampus, prefrontal cortex, amygdala, and the nucleus accumbens.[12]

 There are five different types of muscarinic receptors, M1, M2, M3, M4, and M5. All these subtypes are metabotropic receptors, contrasting them from the nicotinic type of acetylcholine receptors. Furthermore, each subtype is either stimulatory or inhibitory. Subtypes M1, M3, and M5 function through the phospholipase C second messenger pathway, while M2 and M4 function through a second messenger pathway that inhibits adenylate cyclase and prevents the formation of cAMP from ATP.[13] Generally, subtypes M1, M3, and M5 are stimulatory, and their G-alpha subunit of their GPCR will go on to activate downstream proteins while subtypes M2 and M4 are inhibitory with their G-alpha subunit going on to cause adenylate cyclase inhibition. All five subtypes of muscarinic receptors are present in the CNS, but M1-M4 can be found in a multitude of other organ systems as well. The M1 muscarinic receptor is in the cerebral cortex, salivary, and gastric glands. M2 receptors are present in smooth muscle as well as cardiac tissue. M3 receptors are in smooth muscle cells, particularly of the bronchioles, iris, bladder, and small intestines. M4 and M5 receptors have a less clear distribution but have been found in the hippocampus, substantia nigra, and other locations within the brain.[14][15]

Termination of acetylcholine action in the synaptic junction occurs when acetylcholine rapidly binds, then unbinds from its receptor in the target cell’s surface and gets subsequently cleaved by acetylcholinesterase into choline and acetate. Acetylcholinesterase is present in the synaptic cleft as a free molecule or GPI-linked protein on the surface of the postsynaptic cell surface.[16]

Development

Acetylcholine is found in the first moments of developing the ectodermal system (neural plate), as its action is fundamental for the differentiation of neural cells. The neurotransmitter acts as a morphogen.

Organ Systems Involved

Acetylcholine performs its actions by binding the cholinergic receptors (muscarinic and nicotinic). Acetylcholine performs various functions through cholinergic muscarinic receptors.

In the cardiovascular system, it determines generalized vasodilation; decrease in heart rate (negative chronotropic effect); reduction of cardiac contraction force (negative inotropic effect), at the ventricular level, this effect is less pronounced; decrease in the speed of conduction in the specialized tissue of the sinoatrial and atrioventricular nodes (negative dromotropic effect).

• In the gastrointestinal system, through stimulation of the vagus nerve, the tone, the amplitude of contractions, and the secretory activity of the stomach and intestine increase, sphincters are released.
• In the respiratory system, it determines bronchoconstriction and stimulation of the chemoreceptors of the aortic and carotid glomus, with consequent reflex hyperpnea.
• In the urinary system, parasympathetic sacral stimulation causes contraction of the detrusor muscle of the bladder, increasing the emptying pressure and ureteral peristalsis, the release of sphincters.
• In the exocrine glands, it stimulates the secretion of all the exocrine glands that receive a parasympathetic innervation, including the lacrimal, tracheobronchial, salivary, digestive glands, and the exocrine sweat glands.
• In the eye, it determines miosis and accommodation of the lens in close vision, inducing the contraction of the sphincter muscle of the pupil and the ciliary muscle.
• In the male reproductive system, it causes erection.

However, through the nicotinic cholinergic receptors, acetylcholine allows for skeletal muscle contraction; in the adrenal glands, the release of adrenaline and norepinephrine; and in the peripheral sympathetic ganglia, activation of the sympathetic system with the release of norepinephrine.

Mechanism

The nicotinic acetylcholine receptors are ligand-gated ion channels. They are composed of five polypeptide subunits that always have two or more α subunits and may have β, δ, and γ subunits. The muscular type (N1) is composed of 2α, β, δ, and one γ subunit for a total of five subunits while the neuronal type (N2) is composed of 2α and 3β subunits. Upon binding 2 acetylcholine molecules to the nicotinic acetylcholine receptor, the pentameric structure will change its internal conformation, creating a transmembrane pore for the passage of sodium, potassium, and calcium ions. The passage of these ions will then give rise to the depolarization of the cell, depending on the strength of the initial stimulus.[17]

Muscarinic acetylcholine receptors are G-protein coupled receptors (GPCRs) composed of a single polypeptide with seven discrete regions arranged in an α-helix. In these α-helices are hydrophobic residues that allow the polypeptide to span the neuronal cell membrane seven times. The carboxy-tail and fifth cytoplasmic facing loop of this polypeptide are the regions that interact with the second messenger system G proteins. G proteins are composed of an α, β, and γ subunit. When the muscarinic receptor is bound by acetylcholine, it will change its conformation, causing the α subunit to release the natively bound guanosine diphosphate molecule (GDP) and trade it for guanosine triphosphate (GTP). Once GTP binds to the α subunit, the α subunit will dissociate from the β and γ subunits and interact with other downstream effector molecules. The α subunit has intrinsic GTPase activity and will eventually catalyze GTP back into GDP, thus turning the second messenger system “off” intrinsically with time. The use of second messenger pathways to exert their effects allows muscarinic receptors to have diverse functions in human physiology that are tissue-dependent and can be either inhibitory or stimulatory. 

Muscarinic receptor subtypes M2 and M4 exert their effects through inhibition of adenylate cyclase. Once the M2/M4 receptors are bound by acetylcholine, the now activated α subunit of the G protein will cause inhibition of adenylate cyclase and, thus, a reduction in intracellular cAMP. Reduction in cAMP has widespread effects as this molecule is central to many downstream effector’s activation or inhibition. 

Muscarinic receptors M1, M3, and M5 all exert their functions through the stimulation of phospholipase C. When the α subunit of the G protein complex becomes activated, it will then be able to interact with phospholipase C (PLC) and activate it. Activated PLC will then hydrolyze phosphatidylinositol bisphosphate into two separate second messengers, inositol triphosphate (IP3) and diacylglycerol (DAG). IP3 binds the IP3 receptors in the smooth endoplasmic reticulum to release calcium into the cell's cytosol, thus increasing the intracellular calcium concentration. DAG serves to activate protein kinase C, which can phosphorylate a multitude of downstream effector molecules that exert tissue-specific functions.[18]  

Pathophysiology

Acetylcholine in the Peripheral Nervous System

At the presynaptic terminal, acetylcholine storage occurs within the presynaptic vesicle. With the stimulation of the presynaptic terminal, acetylcholine is released from the vesicles and into the synaptic cleft, where the neurotransmitter is free to bind with receptors.  Binding with receptors can have different effects depending on the area of the nervous system that acetylcholine is affecting. Acetylcholine can cause an action potential, or it could activate a secondary messenger system. Free acetylcholine within the synaptic cleft is degraded by an enzyme called acetylcholinesterase. This enzyme assures that no excess of acetylcholine remains in the synaptic cleft to cause the continuous activation of receptors. 

Acetylcholine has different roles and functions at different synapses throughout the body. In the somatic nervous system, acetylcholine is used at the neuromuscular junctions, triggering the firing of motor neurons and affecting voluntary movements. Within both the sympathetic and parasympathetic systems, acetylcholine is utilized by presynaptic neurons of the intermediate horn of the spinal cord to communicate with post-synaptic neurons. Within the parasympathetic nervous system alone, the postganglionic neuron releases acetylcholine as its primary neurotransmitter. Within the sympathetic nervous system, the only postganglionic neurons that release acetylcholine as their primary neurotransmitter are those found innervating the sudoriferous (sweat) glands and some blood vessels of non-apical skin.  

Acetylcholine in the Central Nervous System

Within the brain, acetylcholine has involvement in memory, motivation, arousal, and attention.  Acetylcholine originates from two major places in the brain: 1) basal forebrain and 2) the mesopontine tegmentum area. Acetylcholine originates in the basal forebrain from both the basal nucleus of Meynert and the medial septal nucleus. The basal nucleus of Meynert works on the M1 receptors within the neocortex. The medial septal nucleus functions in the hippocampus and parts of the cerebral cortex at the M1 receptors.   

The mesopontine tegmentum is in the brain stem, and acetylcholine comes from its pedunculopontine nucleus and laterodorsal tegmental nucleus. The mesopontine tegmentum mainly activates the M1 receptors in the brainstem.  The M1 receptors in the brainstem are present in the raphe nucleus, lateral reticular nucleus, deep cerebellar nuclei, pontine nuclei, locus coeruleus, and the inferior olive. However, the mesopontine tegmentum also projects to the basal ganglia, thalamus, basal forebrain, and tectum.

Acetylcholine is known to have effects on a person's memory. For example, drugs such as scopolamine, an anticholinergic that works primarily at M1 receptors, prevent the learning of new information. Also, studies have shown that acetylcholine is essential in the neocortex to learn simple tasks of discrimination.  In the hippocampus, the absence of acetylcholine causes forgetfulness. 

Altering the Acetylcholine Pathway

Alteration or interference with acetylcholine in the nervous system can result in several different pathologies. Pharmacology frequently targets the acetylcholine receptors, pathway, or acetylcholinesterase to correct human physiology during various pathologies. Acetylcholine itself has few uses pharmacologically because it is non-selective for all of the various nicotinic and muscarinic receptors. Additionally, it undergoes rapid inactivation by acetylcholinesterase. One way that acetylcholine is useful pharmacologically is as eye-drops to constrict the pupil during cataract surgery; this occurs because the constrictor pupillae and ciliary muscles of the eyes are both controlled by cranial nerve III, which has a parasympathetic function and can thus activate the M3 receptors of these muscles by acetylcholine secretion.[19]

Cholinesterase inhibitors cause an increase in activity at acetylcholine receptors by blocking the breakdown of acetylcholine. Because the blocking of acetylcholinesterase causes a build-up of acetylcholine in the synaptic cleft, there is continuous activation of the cholinergic receptors. Pharmacologically, cholinesterase inhibitors can help to treat Alzheimer disease and myasthenia gravis since, in both conditions, there is a severe reduction in the amount of native acetylcholine receptor stimulation. Specifically, in Alzheimer disease, there is a decrease in acetylcholine in the neocortex. In myasthenia gravis, there is a severe reduction in the amount of N1 receptors at the neuromuscular junction due to the aberrant production of autoantibodies. Many toxins are cholinesterase inhibitors as well, and these toxins can cause death if given in high enough dosages.

Botulinum toxin works by preventing acetylcholine release from the presynaptic terminals.  Hence, local injections can be useful in treating muscle spasticity, cosmetic wrinkles, and migraines. Black widow spider venom has the opposite effect of botulinum toxin. It causes the cells to release all of their acetylcholine, causing excessive muscle contraction. If all acetylcholine supplies are exhausted due to the venom, then paralysis occurs.

Clinical Significance

Acetylcholine (ACh) is clinically significant in many disease processes, the most commonly seen of which include Alzheimer disease (AD), Lambert-Eaton myasthenic syndrome (LEMS), and myasthenia gravis (MG).

Patients with AD have reduced cerebral content of choline acetyltransferase, which leads to a decrease in acetylcholine synthesis and impaired cortical cholinergic function. Cholinesterase inhibitors (donepezil, rivastigmine, and galantamine) increase cholinergic transmission by inhibiting cholinesterase at the synaptic cleft and provide modest symptomatic benefit in some patients with dementia.[20] 

MG is an autoimmune disorder recognized by the rapid weakening of the skeletal muscles after repeated use. The weakness is due to an antibody-mediated process in which antibodies are produced that have a tropism for acetylcholine receptors or their associated proteins located in the postsynaptic membrane of the neuromuscular junction.[21]

LEMS is a disorder of reduced Ach release from the presynaptic nerve terminals, despite normal ACh vesicle number, normal ACh presynaptic concentration, and normal postsynaptic acetylcholine receptors. This condition occurs when there is autoimmunity (production of autoantibodies) to the voltage-gated calcium channels found on presynaptic neurons' axon terminus.[22]

MG is an autoimmune disorder recognized by the rapid weakening of the skeletal muscles after repeated use. The weakness is due to an antibody-mediated process in which some antibodies have a tropism for acetylcholine receptors or their proteins located in the postsynaptic membrane of neuromuscular junctions.