Acetylcholine (ACh) Synthesis and Storage PDF

ACh SYNTHESIS and STORAGE Acetylcholine (ACh) is the oldest neurotransmitter  Detail of ACh synthesis:  Two precursors are required to synthesize ACh: choline and acetyl coenzyme A (acetyl CoA)  A single synthetic step by a synthesizing enzyme, i.e., choline acetyltransferase (...

ACh SYNTHESIS and STORAGE Acetylcholine (ACh) is the oldest neurotransmitter  Detail of ACh synthesis:  Two precursors are required to synthesize ACh: choline and acetyl coenzyme A (acetyl CoA)  A single synthetic step by a synthesizing enzyme, i.e., choline acetyltransferase (ChAT), which transfers an acetyl group (-COCH3) from acetyl CoA to choline to form ACh.  Cholinergic neurons: Synthesizing enzyme ChAT is located ONLY in the cholinergic neurons. This specificity allows us to identify cholinergic neurons by staining for ChAT.  Control of the ACh synthesis rate: a. The availability of its precursors inside the cholinergic neurons make more ACh when more choline and/or acetyl CoA are available. b. Cell firing rate: when neurons are stimulated to fire at a higher rate, they make more ACh. How is ACh stored?  ACh is packaged into synaptic vesicles by the vesicular acetylcholine transporter (VAChT).  Vesamicol is a selective blocker of VAChT.  If synthesized ACh molecules remain outside the vesicles due to the presence of vasamicol to block the VAChT, the amount of ACh for the release will be reduced leading to blockade of ACh-mediated synaptic transmission. 1  ACh RELEASE and BREAKDOWN  Neural (Ca2+)-dependent release: Upon stimulating neurons, ACh-containing vesicles fuse with the plasma membrane in response to elevated intracellular Ca2+.  Non-neural dependent release - neurotoxins affect the ACh release: The release of ACh is dramatically affected by various animal and bacterial toxins. 1. A toxin found in the venom of the black widow spider, Latrodectus mactans, leads to a massive release of ACh at synapses in the PNS.  Over-activity of the PNS cholinergic system causes numerous symptoms, including muscle pain in the abdomen or chest, tremors, nausea and vomiting, salivation, and copious sweating.  Physiologically the neuromuscular junction, where the axon of a motor nerve terminal synapses with cell membrane of the muscle fiber, transmits messages to cause the muscle contraction. In vertebrates, the signal passes through the neuromuscular junction by the release of ACh. 2. Botulinum toxin blocks ACh release at the neuromuscular junction by preventing fusion of synaptic vesicles with the terminal membrane.  Botulinum toxins target at neuromuscular junction by hydrolyzing several functional proteins that span between vesicle membrane and presynaptic membrane, which are normally involved in priming vesicles for fusing with the presynaptic plasma membrane. Hydrolyzation of these proteins is involved in the inhibition of ACh release. Such inhibition of the release deadly leads to muscular paralysis. 2  ACh RELEASE and BREAKDOWN (continued) Enzymatic break-down of ACh is catalyzed by acetylcholinesterase (AChE)  AChE breaks down ACh into choline and acetic acid.  AChE is located in several locations within the cell: 1). inside the presynaptic axons, where it can metabolize excess ACh that has been synthesized. 2). present on the membrane of the postsynaptic neuron to break down molecules of ACh after their release into the synaptic cleft (after use). 3). at neuromuscular junctions, AChE is secreted by muscle cells into the synaptic cleft. Immediately after a squirt of ACh causes a particular muscle to contract, ACh is metabolized extremely rapidly so that the muscle can relax until the next command arrives to squirt out some more ACh and contract that muscle once again.  Take-up of choline: Once ACh has been broken down within the synaptic cleft, the liberated choline is then taken back up into the cholinergic nerve terminal by a choline transporter in the membrane of the terminal.  Hemicholinium-3 (HC-3): The choline transporter is subject to the blockade by the drug hemicholinium-3 (HC-3). As a result, the rate of ACh production declines due to lack of precursor, choline. 3  AChE inhibitors and their clinical significance AChE inhibitors are used clinically for the treatment of mild to moderate Alzheimer's disease  The rationale for the use of AChE inhibitors in patients with Alzheimer is a significant loss of forebrain cholinergic neurons in this disorder. Such loss of cholinergic neurons contributes to the profound cognitive deficits suffered by these patients. Thus, increasing the availability of the remaining ACh offers some cognitive benefit, although the effects are modest. However, AChE inhibitors do not prevent progression of the disease. Some AChE inhibitors used for dealing with myasthenia gravis in clinics  Physostigmine, neostigmine and pyridostigmine selectively inhibit activity of AChE, which blocks enzymatic breakdown of ACh → maintaining sufficient concentration of ACh.  Physostigmine is able to cross the blood-brain barrier and exerts on the CNS. Such poisoning leads to an over-activity of central cholinergic synapses due to accumulation of ACh in the brain. The symptoms include slurred speech, mental confusion, hallucinations, loss of reflexes, convulsions, and even coma and death.  Neostigmine and pyridostigmine have therapeutic significance: Since they do NOT cross the BBB, they are used for treatment of an autoimmune neuromuscular disorder, myasthenia gravis that happens in neuromuscular junctions of the PNS  Patients with myasthenia gravis develop antibodies by one’s own immune system against their own muscle cholinergic receptors. 4  AChE inhibitors and their clinical significance (continued)  In this case, the antibodies block cholinergic receptors in the muscle, and the loss of receptor function causes the patient’s muscles to be less sensitive to ACh, which in turn leads to severe weakness and fatigue.  By inhibiting AChE, neostigmine or pyridostigmine prolongs the action of ACh at the neuromuscular junction, which causes increased stimulation of the remaining undamaged cholinergic receptors.  Multiple administration is needed because these AChE inhibitors are reversible. The effect is reduced when drug concentration drops due to being metabolized. Irreversible AChE inhibitors:  Organophosphates are irreversible AChE inhibitors (therefore it is permanent), which have been developed as “nerve gases” used as “chemical weapon”, such as Sarin and Soman.  These highly lipid soluble agents readily and quickly enter the body through skin contact or by inhalation and readily cross the BBB to the brain.  Symptoms of nerve gas poisoning due to a rapid ACh accumulation and overstimulation of cholinergic synapses throughout both the CNS and PNS include slurred speech, mental confusion, hallucinations, loss of reflexes, convulsions, and even coma and death.  Treatments: A reversible AChE inhibitor, pyridostigmine, is used as an antidote against Sarin or Soman. The strategy is that temporary interaction of pyridostigmine with the enzyme AChE protects AChE from permanent inactivation by the irreversible AChE inhibitors. 5  Organization and Function of the Cholinergic System The peripheral nervous system (PNS, review CH-2):  Cholinergic synapses are in neuromuscular junctions responsible for initiating/controlling muscle contraction.  Cholinergic synapses are in the parasympathetic and sympathetic branches of the autonomic NS. i. Preganglionic neurons of both sympathetic and parasympathetic branches are cholinergic and located within the CNS and send their axons to the autonomic post-ganglia in the PNS that in turn innervate target organs. ii. The postganglionic neurons of parasympathetic branches are cholinergic, too. iii. The postganglionic neurons of sympathetic branches are adrenergic. 6  Organization and Function of the Cholinergic System (continued)  Several cholinergic systems with cholinergic pathways in the brain: 1. Basal forebrain cholinergic system (BFCS) contains two groups of cholinergic neurons: (1) the medial septal group that project cholinergic axons to the hippocampus; (2) the nucleus basalis group that project cholinergic axons to the prefrontal cortex, cingulate cortex and amygdala. o The BFCS plays an important role in cognitive functioning. Damage to the BFCS cholinergic system due to ACh cell loss contributes to the dementia observed in Alzheimer. 2. Basal ganglia: The cholinergic interneurons in the striatum (basal ganglia) play a role of balance with the dopaminergic neurons. A neurotransmitter imbalance happened in Parkinson’s disease (PD) due to neurodegeneration of dopaminergic neurons (anti-cholinergic drugs are used in the early stages of PD). 3. The lateraldorsal tegmental (LDTg) and pedunculopontine tegmental (PPTg) nuclei in brainstem: o Axons from the LDTg exert a powerful excitatory influence on DA neuron activity of the midbrain VTA by activating nicotinic cholinergic receptors expressed on DA neurons involved in the reinforcing effects of nicotine, and by muscarinic cholinergic receptors involved in rewarding-producing effects of morphine and cocaine – These are done via facilitate the mesolimbic dopaminergic pathway. o Other ACh pathways from the pons project to reticular formation in the brainstem and thalamic areas, playing important roles in behavioral arousal, sensory processing, and initiation of rapid-eye-movement sleep. BFCS 7  Subtypes of cholinergic receptors: Nicotinic Two cholinergic receptor subtypes: nicotinic and muscarinic Nicotinic receptors are ionotropic with ion channels allowing Na+ and Ca2+ influx. In addition to ACh, this subtype of receptors respond selectively to the agonist nicotine, an alkaloid found in the leaves of the tobacco plant, so named.  Highly concentrated on 1) muscle cells at neuromuscular junctions, 2) post-ganglionic neural cell bodies of both sympathetic and parasympathetic systems, and 3) many neurons in the brain.  Comprise 5 subunits (2 α, 1 β, 1 γ & 1 δ) that come together in the cell membrane forming the ion channel in the center. Two α-subunits form an ACh binding site on the receptor. Both binding sites must be occupied by ACh to open the receptor channel.  Different combinations of subunits: The exact subunits that make up muscle and neuronal receptors are different. This structural difference leads to significant pharmacological differences between neuronal and muscle receptors.  In fact, muscle nicotinic receptors are not as sensitive to nicotine as the nicotinic receptors located in the brain and autonomic nervous system. This is why smokers only obtain the psychological effects of nicotine without experiencing muscle contractions or spasms.  Activation of nicotinic receptors opens channels very rapidly and Na+ and Ca2+ enter the neuron or muscle cell. This will depolarize the cell membrane, thereby increasing the cell's excitability. 8  Subtypes of cholinergic receptors: Nicotinic (continued)  Nicotinic receptors mediate fast excitatory cellular responses.  In the CNS, the nicotinic receptors can be located presynaptically on the terminals (axoaxonic synapse). ACh binding to nicotinic receptors (serve as heteroreceptors, see CH-3) enhances the release of other type neurotransmitter from this nerve terminal.  Postsynapticlly on the dendrites or cell bodies of receiving neurons (axondendritic or axonsomatic synapse): activation of receptors by ACh can stimulate cell firing (excitation). Nicotinic receptor desensitization, resensitization and blockade  Desensitization means an inactivated state of the receptor in which the channel remains closed regardless of whether an agonist (ACh or nicotine) are bound to the receptor.  This occurs when receptors are subject to continuous or persistent agonist exposure, they may become desensitized due to persistent depolarization. When the agonist concentration drops, this status will spontaneously switch to re-sensitization to be able to respond again to a nicotinic agonist.  Depolarization block: Continuous nicotinic stimulation may cause a persistent depolarization of the cell membrane, in which the resting potential is lost, and the cell cannot be excited again n tio until the agonist is removed and the membrane re-polarized. tiz a nsi  A chemical relative of Ach, succinylcholine, is AChE resistant, so - se re it continuously stimulates the nicotinic receptors and induces a depolarization block of the muscle cells, good used for surgical procedures to provide sufficient relaxation.  Blockade: Muscle relaxation or paralysis can also be induced by blocking muscle nicotinic receptors, like curare-induced poisoning. 9  Subtypes of cholinergic receptors: Muscarinic Muscarinic receptors are metabotropic with 5 subtypes designated as M1, M2, M3, M4 and M5 1. Muscarinic receptors operate through several different second-messenger systems. a. PKC activation: Some activate the phosphoinositide second-messenger system (PKC). b. PKA inhibition: Others inhibit the formation of cyclic adenosine monophosphate (cAMP)-PKA. 2. Muscarinic receptors open K+ channel via G-protein linked K+ channels - hyperpolarization.  Muscarinic receptors are highly expressed in the neocortex, hippocampus thalamus, striatum, and basal forebrain. (B) 1). The receptors in the forebrain and hippocampus, activated by the BFCS, play an important role in the cognitive effects of ACh. 2). Those in the striatum (basal ganglia) are involved in modulation of smooth locomotor activity initiated by the motor cortex. 3). Midbrain LDTg activates DA neurons in the VTA via M5 receptors expressed on DA neurons involved in the rewarding and dependence-producing effects of abused drugs, like morphine, that are studied using animal models (A).  M5 receptor knockout mice show deficits in both morphine and cocaine reward responses in a study using place conditioning (Refer to Figure 4.10 on page 129 for detailed apparatus setup and rationale).  The place conditioning was conducted in (B). Morphine doses were paired with one chamber and produced a robust place preference in normal animals but had no effect on the M5 knockout mice. o Loss of M5 receptors reduced withdrawal symptoms in mice that were made dependent on morphine, but it had no effect on morphine-induced analgesia Mesolimbic pathway - A way to develop non-addictive analgesic drugs? 10  Muscarinic receptors in the PNS:  Most visceral organs are expressed with high density of muscarinic receptors: like cardiac muscle of the heart and the smooth muscle of many organs (bronchioles, stomach, intestines, bladder, etc., but not blood vessels. Refer to Figure 2.18 on page 69). These peripheral muscarinic receptors are activated by ACh released from postganglionic axonal fibers of the parasympathetic NS.  M2 expressed in cardiac muscles: Activation of the muscarinic receptors M2 subtype in the cardiac muscles slows heart rate (bradycardia) and decreases the strength of contraction.  M3 expressed in smooth muscles of the gut: Activation of the muscarinic receptors M3 subtype activates smooth muscle of the gut, increasing gut movements.  Activation of M3 muscarinic receptors in the secretory glands produces salivation, sweating, and lacrimation. Drugs for treatments of psychiatric disorders are muscarinic antagonists that cause a side effect, “dry-mouth’.  Pancreatic β-cells receive their parasympathetic innervation through the vagus nerve, which increases activity at the beginning of a meal. The resulting release of ACh acts on M3 receptors in β-cells to stimulate insulin secretion to regulate blood glucose levels during and following food consumption.  The incidence of type 2 diabetes is due to that the pancreas secretes insufficient insulin, which results in chronically elevated concentrations of glucose in the bloodstream. Scientists become interested in exploring the possibility that muscarinic regulation of β-cell insulin release might be a future target of medications being developed to treat the insulin-insufficiency form of type 2 diabetes. 11  Muscarinic receptor agonists-like substances in nature and their clinical significance  Muscarinic receptor agonist-like substances are from many plant’s leaves or seeds. Because their ingestion mimics many effects of parasympathetic activation by activating muscarinic receptors, they are named as parasympathomimetic agents.  Poisoning due to accidental ingestion of these agonist like-substances leads to exaggerated parasympathetic responses including lacrimation, salivation, sweating, pinpoint pupils (related to constriction of the iris), severe abdominal pain and painful diarrhea due to strong contractions of the smooth muscles of the viscera. High doses can even cause cardiovascular collapse, convulsions, coma and death. Muscarinic receptor antagonist-like substances in nature and their clinical significance  Since antagonists inhibit the actions of the parasympathetic system by blocking muscarinic receptors, they are named as parasympatholytic agents.  Peripherally, muscarinic antagonists have several medical applications: pupil dilation used for eye exam; secretion reduction in the patient's airways for anesthesia procedure during surgery; counter effect on poisoning with a cholinergic agonist, etc.  Centrally, toxic effects of muscarinic antagonists are complex: when high doses, the CNS effects include restlessness, irritability, disorientation, hallucinations and delirium. The worst case would be the CNS depression, coma and eventually death by respiratory failure. 12 A model of cholinergic synapse to demonstrate the pharmacology of ACh (good for guiding your review) 13  Study Questions 1. What are the chemical reactions involved in ACh synthesis and breakdown? Include the names of the enzymes catalyzing each reaction. Which of these enzymes is used as a bio-chemical marker for cholinergic neurons? 2. Briefly discuss the factors that regulate the rate of ACh synthesis. Are there any current therapeutic interventions targeting the process of ACh synthesis? 3. By what mechanism is ACh taken up and stored in synaptic vesicles? Name a drug that interferes with vesicular ACh uptake and describe the effects of this drug on cholinergic transmission. 4. List and discuss the effects of toxins that either stimulate or inhibit ACh release. Include in your answer the therapeutic applications of toxins that inhibit the release of this neurotransmitter. 5. Unlike the monoamine transmitters discussed in previous chapters, there is no reuptake system for ACh itself. Describe the alternative mechanism that has evolved to help cholinergic neurons recycle/reutilize the transmitter. How do we know that this recycling process plays an important role in normal functioning of the nervous system? 6. List and discuss the various drugs that function as AChE inhibitors. In general, what is the effect of these drugs on cholinergic transmission? Include in your answer a consideration of the different consequences of peripheral versus central AChE inhibition and also the different consequences of reversible versus irreversible inhibition of the enzyme. 7. Describe the localization and functions of ACh in the peripheral nervous system. 8. Name and indicate the location of the principle cholinergic cell groups in the brain. Discuss the role of the BFCS in cognitive function, including relevant experimental findings. 9. Describe the molecular structure and signaling mechanism of nicotinic cholinergic receptors. 10. A nicotinic receptor complex can be in one of three different states: open, closed, and desensitized. Describe the properties of each state, including whether agonist is bound or not as well as the state of the receptor channel. 14  Study Questions 11. Discuss the molecular structure, signaling mechanisms, and localization of muscarinic cholinergic receptors in the brain. 12. Discuss the role of M5 muscarinic receptors in the control of dopaminergic cell firing and the rewarding and reinforcing effects of abused drugs. Include relevant experimental findings in your answer. 13. Describe the locations and functional roles of muscarinic receptors expressed by peripheral organs and glands. Include in your answer a discussion of the so-called dry mouth effect. 14. Discuss the role of pancreatic M3 muscarinic receptors in the control of insulin secretion and the involvement of this receptor subtype in the development of insulin resistance that accompanies the administration of certain anti-psychotic drugs. 15. What is meant by the terms parasympathomimetic and parasympatholytic agents? List examples of drugs belonging to these categories along with their physiological effects and medical or other uses. 15