6/10/ CHOLINERGIC TRANSMISSION

6/10/ Learning objectives At the end of the session students must be able to have:  A clear understanding of the cholinergic transmission in the periphery and the ways in which drugs affect it.

6/10/ CHOLINERGIC TRANSMISSION Acetylcholine receptors are subdivided into : Nicotinic (nAChR) Muscarinic (mAChR) subtypes

6/10/ Nicotinic receptors Directly coupled to cation channels Mediate fast excitatory synaptic transmission at the NMJ (neuromuscular junction), autonomic ganglia and at various sites in the CNS. Nicotinic receptors are subdivided into: - Muscle – type NM ( muscle nAChR) - Neuronal type NN (neuronal nAChR)

6/10/ Nicotinic Receptors (cont)… Muscle nAChR and Neuronal nAChR differ in their molecular structure and pharmacology

6/10/ The nicotinic actions correspond to those of Ach acting on autonomic ganglia of the symphathetic and parasympathetic systems, the motor end place of voluntary muscle and the secretory cells of the adrenal medulla.

6/10/ Muscarinic Receptors Muscarinic receptors (mAChRs) are G- protein coupled receptors, causing: - Activation of phospholipase C (hence formation inositol triphosphate and diacylglycerol as second messengers) - activation of K + channels or inhibition of Ca + channels. - Inhibition of adenylate cyclase

6/10/ Muscarinic Receptors… Mediate Ach effects at postganglionic para sympathetic synapses (mainly heart, smooth muscle, glands) and contribute to ganglionic excitation.

6/10/ Exceptions: 1) Ach causes generalized vasodilatation, even though most blood vessels have no parasympathetic innervation. 2) Ach evokes secretion from sweat glands which are innervated by cholinergic fibres of the sympathetic nervous system.

6/10/ Muscarinic receptor-Subtypes Gene cloning has revealed five distinct types of muscarinic receptor : M1, M2, M3, M4, M5 but only four have been distinguished functionally and pharmacologically. Three of this (M1, M2, M3) are well characterised

6/10/ M1-receptors (‘neural’) found mainly on CNS, peripheral neurones and gastric parietal cells They mediate excitatory effects, for example the slow muscarinic excitation mediated by acetylcholine in sympathetic ganglia and central neurones. this excitation is produced by a decrease in K+ conductance, which causes membrane depolarisation.

6/10/ M1-receptors (‘neural’)… Defficiency of this kind of acetylcholine- mediated effect in the brain is possibly associated with dementia. Also involved in the increase of gastric acid secretion following vagal stimulation are selectively blocked by pirenzepine.

6/10/ M2-receptors (‘cardiac’) Occur in the heart, and also on the presynaptic terminals of periphery and central neurons. Exert inhibitory effects, mainly by increasing K+ conductance and inhibiting calcium channels

6/10/ M2-receptors (‘cardiac’) cont… causing decrease in cardiac rate and force of contraction (mainly of atria) and also mediate presynaptic inhibition They are selectively blocked by gallamine.

6/10/ M3-receptors (‘glandular/smooth muscle’) Produce mainly excitatory effects i.e. stimulation of glandular secretions (salivary, bronchial, sweat etc.) and contraction of visceral smooth muscle Mediate relaxation (mainly vascular) of smooth muscle, which results from the release of nitric oxide from neighboring endothelial cells

6/10/ M1-; M2- and M3- receptors occur also in specific locations in the CNS

6/10/ M4 and M5 Receptors Are largely confined to the CNS Their functional role is not well characterized.

6/10/  All mAChR are activated by Ach and blocked by atropine The pharmacological classification of these receptor types relies on the limited selectivity of certain agonists and antagonists that can be distinguished between them. (Refer: Pharmacology by Rang & Dale-Table 10.2 of chapter 10- ”mAchR Subtypes)

6/10/ Synthesis and release of Acetylcholine. Ach is synthesized within the nerve terminal from choline, which enters the neuron via carrier-mediated transport.

6/10/ Ach Synthesis (cont..) Requires acetylation of choline, utilizing acetyl CoA ( coenzyme A) as source of acetyl groups, which requires choline acetyl transferase (CAT), a cytosolic enzyme found only in cholinergic neurons.

6/10/ Most of the ACh synthesized is packaged into synaptic vesicles at high concentration ( about 100mmol/l) by carrier-mediated transport. Ach release occurs by Ca ++ - mediated exocytosis. At the neuromuscular junction (NMJ), one presynaptic nerve impulse releases vesicles.

6/10/ Ach Release: Requires calcium Triggers an interaction between several proteins associated with the vesicle [VAMPs = vesicle – associated membrane proteins, synaptobrevin synaptogmin] and nerve ending membrane (SNAPS, synaptosome- associated proteins, SNAPS25, syntazin and others).

6/10/ Ach Release (cont) This interaction results in the fusion of the membranes of the vesicle with the nerve ending membranes, the opening of a pore to the extracellular space and the release of the stored transmitter.

6/10/ At the NMJ, Ach acts on nicotinic receptor to open cation channels, producing a rapid depolarisation (end plate potential), which normally initiates an action potential in the muscle fibre. Transmission at other “fast” cholinergic synapses (e.g. ganglionic) is similar.

6/10/ At “fast” cholinergic synapses, ACh is hydrolysed within about 1ms by acetylcholinesterase, so a presynaptic action potential produces only one post synaptic action potential.

6/10/ Transmission mediated by muscarinic receptors is much slower in its time course and synaptic structures are less clearly defined. In most cases Ach function as a modulator rather than as a direct transmitter.

6/10/ Main mechanisms of pharmacological block: inhibition of choline uptake, inhibition of Ach release, block of postsynaptic receptors or ion channels, persistent postsynaptic depolarisation

6/10/ EFFECTS OF DRUGS ON CHOLINERGIC TRANSMISSION

6/10/ Effect of Drugs on Cholinergic Transmission Drugs can influence cholinergic transmission either by acting on postsynaptic acetylcholine receptors as agonists or antagonists, or by affecting the release or destruction of endogenous acetylcholine.

6/10/ Groups of Drugs according to their Physiological Site of Action Drugs affecting muscarinic receptors -muscarinic agonists -muscarinic antagonists Drugs affecting ganglia -ganglion stimulating drugs -ganglion blocking drugs

6/10/ Groups of Drugs according to their Physiological Site of Action… Drugs blocking neuromuscular transmission -non-depolarising drugs -depolarising drugs -inhibitors of acetylcholine synthesis or release

6/10/ Groups of Drugs according to their Physiological Site of Action… Drugs that enhance cholinergic transmission -inhibitors of cholinestrase -stimulants of acetylcholine release

6/10/ Drugs that can act on acetylcholine receptors may:  Mimic the action of Ach e.g. muscarine, nicotine.  Block the action of Ach e.g. atropine, tubocurarine.

6/10/ Drugs Affecting Release or Destruction of ACh Examples:  4-aminopyridine enhance release  Neostigmine-inhibit cholinesterase  Inhibit Ach release, by inhibiting synthesis (e.g. hemicholinium, which blocks choline uptake) or vesicular storage (e.g. vesamicol).

6/10/ Drugs Affecting Release or Destruction of Ach (cont.)  Or by inhibiting the release mechanism itself (e.g. botulinum toxin, magnesium ion, aminoglycoside antibiotics).

6/10/ DRUGS AFFECTING MUSCARINIC RECEPTORS Muscarinic agonists: -as a group, are often referred to as parasympathomimetic because the main effects that they produce resemble those of parasympathetic stimulation.

6/10/ Muscarinic Agonists… Include ACh, carbachol, methacholine muscarine and pilocarpine. They vary in muscarinic/nicotinic selectivity, and in susceptibility to cholinesterase.

6/10/ Muscarinic Agonists- Main Effects Cardiovascular effects: bradycardia and vasodilatation (endothelium-dependent), leading to fall in BP. Smooth muscle: Contraction of visceral smooth muscle (gut, bladder, bronchi etc) exocrine secretions: sweating, lacrimation, salivation, bronchial secretion Effects on the eye: pupillary constriction and ciliary muscle contraction leading to decrease of intraocular pressure.

6/10/ Muscarinic agonists… Clinical uses is in the treatment of glaucoma, by local instillation in the form of eye drops. ( e.g. pilocarpine).

6/10/ Muscarinic Antagonists Are referred to as parasympatholytic because they selectively block the effects of parasympathetic nerve activity All of them are competitive antagonists, and their chemical structures usually contain ester and basic groups in the same relationship as Ach, but they have a bulky aromatic group in place of the acetylgroup. Include: Atropine, hyoscine, ipratropium & pirenzepine.

6/10/ EFFECTS OF MUSCARINIC ANTAGONISTS All of the muscarinic antagonists produce basically similar peripheral effects, though some show a degree of selectivity (e.g. for the heart or the gastrointestinal tract), reflecting heterogeneity among muscarinic receptors

6/10/ EFFECTS OF MUSCARINIC ANTAGONISTS … Main effects of atropine include:  Inhibition of secretions (salivary, lacrimal, bronchial and sweat glands) are inhibited by very low doses producing dry mouth and skin  Effects on heart rate: tachycardia through block of cardiac muscarinic receptors.

6/10/ Effects of Muscarinic Antagonists… Main Effects of Atropine (cont)  Effects on the eye: pupillary dilatation (mydriasis) and becomes unresponsive to light. Also relaxation of the ciliary muscle causes paralysis of accomodation (cycloplegia); as a result near vision is impaired.  Effects on other smooth muscles: relaxation of smooth muscle (bronchi, biliary tract and urinary tract)

6/10/ Effects of Muscarinic antagonists: Main Effects of Atropine (cont)  Effects on the GIT: inhibition of gastric acid secretion (e.g. pirenzepine).  CNS effects : atropine produces mainly excitatory effects on the CNS. At low doses, this causes mild restlessness; higher doses cause agitation and disorientation ; depressant including amnesia, with hyoscine), including anti-emetic effect and anti-parkinsonian effect.

6/10/ Clinical Uses of Muscarinic Antagonists 1) Cardiovascular (CVS) – treatment of sinus bradycardia (e.g. after myocardial infarction) (I.V. atropine). 2) Ophthalmic-To dilate pupil (e.g. tropicamide eye drops (short-acting) & cyclopentolate eye drops (long-acting).

6/10/ Clinical Uses of Muscarinic Antagonists (cont.) 3. Neurological -prevention of motion sickness e.g hyoscine (orally or transdermally) -Parkinsonism, especially to counteract movement disorders caused by antipsychotic drugs (benzhexol, benztropine).

6/10/ Clinical Uses of Muscarinic Antagonists (cont.) 4. Respiratory – Asthma (ipratropium by inhalation) 5. anaesthetic pre-medication- to dry secretions e.g. atropine, hyoscine ( current anaesthetics are relatively non-irritant, so this use is now less important)

6/10/ Clinical Uses of Muscarinic Antagonists (cont.) 6. G.I.T. – to treat peptic ulcer disease by suppressing gastric acid secretion e.g. pirenzepine (M1- selective antagonist). This is used less since the introduction of histamine H2-antagonists and proton pump inhibitors - To facilitate endoscopy and g.i.t radiology by relaxing g.i.t smooth muscle (Antispasmodic action e.g. hyoscine butylbromide).

6/10/ DRUGS AFFECTING AUTONOMIC GANGLIA GANGLION STIMULANTS: Most nicotinic receptor agonists affect both ganglionic and motor end plate receptors, but some show selectivity. Include: Nicotine, lobeline, Epibatidine, suxamethonium and decamethonium.

6/10/ Ganglion Stimulants (cont..) Both sympathetic and parasympathetic ganglia are stimulated, so effects are complex; including tachycardia and increase of BP, variable effects on G.I.T. motility and secretions, increased bronchial, salivary and sweat secretions. Additional effects result from stimulation of other neuronal structures, including sensory an noradregergic nerve terminals

6/10/ GANGLION-BLOCKING DRUGS Include: hexamethonium, trimetaphan, tubocurarine, pancuronium, atracurium, vecuronium. Block all autonomic ganglia and enteric ganglia.

6/10/ Ganglion-Blocking Drugs… Main effects are: Hypotension and loss of cardiovascular reflexes; inhibition of secretions; GIT paralysis and impaired micturition. Clinically obsolete, except for occasional use of trimethaphan to produce controlled hypotension in anaesthesia

6/10/ NEURO-MUSCULAR BLOCKING DRUGS These drugs can block neuromuscular transmission either by acting presynaptically, to inhibit Ach synthesis or release, or by acting postsynaptically.

6/10/ Two categories of Neuromuscular- Blocking Drugs: Non depolarising blocking drugs (majority) which act by blocking Ach receptors (and in some cases, also by blocking ion channels) Depolarising blocking drugs, which are agonists at ACh receptors.

6/10/ Non-depolarizing Blockers Include: Tubocurarine, pancuronium, vecuronium, atracurium, gallamine, mivacurium. all are quarternary ammonium compounds which are poorly absorbed but generally rapidly excreted and do not cross the placenta.

6/10/ Non-depolarizing Blockers (cont.) - Act as competitive antagonists at the nicotinic receptor and differ mainly in duration of action.

6/10/ Non-depolarizing Blockers (cont.) Effects: Extrinsic eye muscle (causing double vision), small muscles of the face, limbs and pharynx (causing difficult in swallowing) are the first to be affected while the respiratory muscles are the last to be affected and the first to recover.

6/10/ Non-depolarizing Blockers (cont.) Main Side Effects: fall in BP as a result of ganglion block; histamine release from mast cells giving rise to bronchospasm in sensitive individuals

6/10/ Non-depolarizing Blockers (cont.) Pharmacokinetics: - these drugs are mostly metabolized by the liver or excreted unchanged in urine, except atracurium which hydrolyses spontaneously in plasma and mivacurium hydrolysed by plasma cholinesterase.

6/10/ DEPOLARIZING BLOCKING DRUGS Include: suxamethonium act on the motor end plate just like Ach (i.e. it is an agonist that increases the cation permeability of the end plate).

6/10/ Depolarising Blocking Drugs (cont) Suxamethonium is hydrolysed by plasma cholinesterase and is normally very short acting; but may cause long lasting paralysis in a small group of congenitally cholinesterase deficient individuals. Side effects: bradycardia, cardiac dysrhythmias due to K+ release (esp. in burned or injured patients), increased intraocular pressure, prolonged paralysis, malignant hyperthermia (rare).

6/10/ Difference of Non-Depolarising & Depolarising Blocking Drugs Non-depolarising block is reversible by anticholinesterase drugs, while depolarising block is not. Non-depolarising blockers do not produce fasciculations and post operative muscle pain as do depolarising agents.

6/10/ Drugs That Inhibit Cholinesterase 3 main groups of anticholinestrases:  Short-acting e.g. edrophonium  medium-acting e.g. neostigmine, pyridostigmine, physostigmine  irreversible anticholinesterases e.g. dyflos, ecothiopate and parathion.

6/10/ Short-acting anticholinesterases Edrophonium – a quartenary ammonium compound that binds to the anionic site of the enzyme only. The ionic bond formed is readily reversible. - Action of drug is very brief Used for diagnostic purposes (myasthenia gravis).

6/10/ Medium-duration anti- cholinesterases These drugs posses strongly basic groups which bind to the anionic site, These drugs are all carbamyl, as opposed to acetyl, esters. Carbamylated enzyme does not hydrolyse easily (hence long-duration of action).

6/10/ Irreversible anticholinesterases These are pentavalent phosphorus compounds containing a labile group e.g. Fluoride (in dyflos) or an organic group (in parathion & ecothiopate). This group is released, leaving the residue of the molecule attached covalently through the phosphorus atom to the serine-OH group of the enzyme.

6/10/ Effects of anticholinesterase drugs Are due mainly to enhancement of cholinergic transmission at cholinergic autonomic synapses and at the NMJ. Those that cross the BBB (e.g. physostigmine, organophosphates) also have marked CNS effects.

6/10/ Effects of anticholinesterase drugs… Autonomic effects include bradycardia, hypotension, excessive secretions, bronchoconstriction, g.i.t. hypermotility, decrease of intraocular pressure Effects on neuromuscular junction causes muscle fasciculation and increased twitch tension and can produce depolarisation block.

6/10/ DRUGS THAT ENHANCE CHOLINERGIC TRANSMISSION Do so by inhibiting cholinesterase (main group) or by increasing acetylcholine release.

6/10/ There are two main forms of cholinesterase:- - acetylcholinesterase (AChE) which is mainly membrane-bound, relatively specific for Ach and responsible for rapid Ach hydrolysis at cholinergic synapses. - Butyrylcholinesterase (BChE) or pseudocholinesterase, which is relatively non-selective and occurs in plasma and may tissues.

6/10/ Cholinesterase reactivation Pralidoxime reactivates the enzyme by bringing an oxime group into close proximity with the phosphorylated esteratic site. This group is a strong nucleophile and attracts the phosphate group from the serine – OH group of the enzyme. - has to be given early to be effective.

6/10/ Clinical uses of anticholinesterases In anaesthesia to reverse the action of non- depolarising neuromuscular blocking drugs at the end of an operation (neostigmine) In the treatment of myasthenia gravis (neostigmine and pyridostigmine) In the treatment of glaucoma (ecothiopate eye drops).

6/10/ Reference Pharmacology text book by Rang & Dale

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