Autonomic Pharmacology: Cholinergic DrugsPresenter: Marc Imhotep Cray, M.D.Professor PharmacologyRecommended Reading:Cholinergic DrugsTutorial Worth Visiting:Cholinergic ANSClinical:E-Medicine ArticlesMyasthenia Gravis9/30/20092Online Reference ResourceIVMS Online Textbook Series BRS Pharmacology, 4th EditionGary C. Rosenfeld PhD;DavidS. Loose PhDFull Text OnlineStudent ResourcesPassword Protected, for Enrolled Students Only9/30/20093From IAU Online Textbook SeriesPharm. BookFigure 2-1 Organization of the autonomic nervous system.9/30/20094Cholinergic Biosynthesis Acetylcolineis formed from two precursors: choline:which is derived from dietary and intraneuronal sources acetyl coenzyme:which is made from glucose in the mitochondria of neuronsAcetylcholineis synthesized from choline and acetyl-CoA by the enzyme choline acetyl transferase (ChAT)to form acetylcholine, which is immediately stored in small vesicular compartments closely attached to the cytoplasmic side of presynaptic membranes. ChATis a selective markerfor cholinergic neurons9/30/20095Cholinergic Biosynthesis1) Synthesisof acetylcholine (ACh) from acetyl CoA and choline 2) Storageof ACh in synaptic vesicles3) Releaseof ACh ( fusion of synaptic vesicle with presysnaptic membrane and release of ACh into the synapse) 4) Actionof ACh by binding to and activating receptors (nicotinic in autonomic gangliaand neuromuscular junctionand, muscarinic in many sites) 5) Inactivationby enzymatic breakdown of ACh by acetylcholinesterase (AChE) located in the synapse.ACh is degradedin the synaptic cleft by acetylcholinesteraseto choline and acetate 9/30/20096Cholinergic Agents-Direct Acting and Indirect Acting Choline EstersAcetylcholine Bethanechol (Urecholine) Carbachol Methacholine (Provocholine) AlkaloidsMuscarine Pilocarpine (Pilocar)There are threemain typesof cholinesterase:Short-acting:edrophonium medium-acting:neostigmine (2-4h), pyridostigmine (3-6h) physostigmine irreversible:organophosphates, dyflos, ecothiopate Agents-Direct ActingIndirect Acting9/30/20097Spectrum of Action of Choline Esters Location of cholinergic synapses mainly determine the spectrum of action of acetycholine and choline estersCholinergic Synaptic Sitesautonomic effector sites: innervated by post-ganglionic parasympathetic fibers some CNS synapses autonomic ganglia and the adrenal medulla skeletal muscle motor endplates (motor nerves) 9/30/20098Spectrum of Action of Choline Esters(2) Cholinergic influences are prominent in many organ systems:Choline EsterSensitivity to ACHECardiovascularGastrointestinalUrinary BladderEye (Topical)Atropine SensitiveActivity at Nicotinic SitesAcetylcholineMethacholineCarbacholNoBethanecholNo? ?No9/30/20099Spectrum of Action of Choline Esters(3)Cholinergic Receptors:Cholinergic refers to responses in various systems to the natural transmitter molecule Acetycholine (ACh)If one looks at a set of responses where ACh is the normal transmitter, observation has shown that thosesame responses are differently sensitive to the extrinisic molecules Nicotine and MuscarineNicotinecomes from tobacco, Muscarinecomes from certain mushroomsSee: NS The Reception and Transmission of Extracellular InformationReceptors-A Brief Note9/30/200910Spectrum of Action of Choline Esters(4)Based on the different sensitivities shown above, Cholinergic receptors are subclassified into two categories,Nicotinicand Muscarinic, named for the extrinsic compounds that stimulate only that category. 9/30/200911Spectrum of Action of Choline Esters(5)NicotinicReceptorsStimulated by ACh and nicotine, not stimulated by muscarine. Found at all ganglionic synapses. Also found at neuromuscular junctions Blocked by hexamethonium. 9/30/200912Spectrum of Action of Choline Esters(6) NicotinicReceptorsThe physiological responses to stimulation and block are complex since both sympathetic and parasympathetic systems are affected The final response of any one organ system depends on which system has a stronger tonic influence EXAMPLE:Under normal circumstances, the heart receives more parasympathetic influence than sympatheticGanglionic blockade would lower parasympathetic influence more than sympathetic, and thus heart rate would increase 9/30/200913Spectrum of Action of Choline Esters(6) MuscarinicReceptorsStimulated by ACh and muscarine, not stimulated by nicotineFound at target organs when ACh is released by post-ganglionic neurons (all of parasympathetic, and some sympathetic) Stimulated selectively by Muscarine and Bethanechol etc. Blocked by Atropine9/30/200914Spectrum of Action of Choline Esters(7) MuscarinicReceptorsStimulation causes: Increased sweating Decreased heart rate Decreased blood pressure due to decreased cardiac output Bronchoconstriction and increased bronchosecretion. Contraction of the pupils, and contraction of ciliary body for near vision Tearing and salivation Increased motility and secretions of the GI system. Urination and defecation Engorgement of genitalia 9/30/200915Cholinergic Receptors: Subtypes, Tissues, Responses and Molecular MechanismsMuscarinic Receptor Coupling MechanismsFive types of cholinergic receptors have been identified by molecular cloning methods. The five muscarinic receptor subtypes, M1 -M5, are associated with specific anatomical sites For example:M1 -ganglia; secretory glandsM2 -myocardium, smooth muscleM3 , M4 :smooth muscle, secretory glands9/30/200916Cholinergic Receptors: Subtypes, Tissues, Responses and Molecular MechanismsNicotinic Muscle ReceptorAntagonistsTissueResponsesMolecular AspectsTubocurarinealpha-bungarotoxinNeuromuscular JunctionMembrane Depolarization leading to muscle contractionNicotinic (muscle) receptor's cation ion channel opening9/30/200917Cholinergic Receptors: Subtypes, Tissues, Responses and Molecular Mechanisms(2)Nicotinic Neuronal ReceptorAntagonistsTissueResponsesMolecular AspectsMecamylamine (Inversine)Autonomic GangliaDepolarization: postsynaptic cell activationNicotinic (muscle) receptor's cation ion channel openingAdrenal MedullaCatecholamine secretionCNSunknown9/30/200918Cholinergic Receptors: Subtypes, Tissues, Responses and Molecular Mechanisms(3)Muscarinic Type M1AntagonistTissueResponsesMolecular AspectsAtropinePirenzepine (more selective)Autonomic GangliaDepolarization (late EPSP)Stimulation of Phospholipase C (PLC): activation of inositol-1,4,5 triphosphate (IP3) and diacylglycerol (DAG) leading to increased cytosolic Ca2+CNSUnknown9/30/200919Cholinergic Receptors: Subtypes, Tissues, Responses and Molecular Mechanisms(4)Muscarinic Type M2Tissue (Heart)ResponsesMolecular AspectsSA nodedecreased phase 4 depolarization; hyperpolarizationK+ channel activation through ß-gamma Gisubunits;Gi-mediated inhibition of adenylyl cyclase which decreases intracellular Ca2+levels.(Gican inhibit directly Ca2+channel opening)Atriumdecreased contractility; decreased AP durationAV nodedecreased conduction velocityVentricledecreased contractility9/30/200920Signal Transduction:Comparison of Muscarinic and Nicotinc ReceptorsNicotinic ReceptorsLigand-gated ion channels Agonist effects blocked by tubocurarine Receptor activation results in:rapid increases of Na+ and Ca2+ conductance deplorization excitation Subtypes based on differing subunit composition: Muscle and Neuronal Classification Discussed Above 9/30/200921Signal Transduction:Comparison of Muscarinic and Nicotinc ReceptorsMuscarinic ReceptorsG-protein coupled receptor system Slower responses Agonist effects blocked by atropine At least five receptor subtypes have been described by molecular cloning9/30/200922Muscarinic Receptors: Second Messenger SystemsActivation of IP3, DAG cascadeDAG may activate smooth muscle Ca2+ channels IP3 releases Ca2+ from endoplasmic and sarcoplasmic reticulum Increase in cGMP Increase in intracellular K+ by cGMP-K+ channel binding inhibition of adenylyl cyclase activity (heart) 9/30/200923Muscarinic Receptors: Second Messenger Systems(2)9/30/200924Direct vs. Indirect-Acting CholinomimeticsA direct-acting cholinomimeticdrug produces its pharmacological effect by receptor activationAn indirect-acting druginhibitsacetylcholinesterase, thereby increasing endogenous acetylcholine levels, resulting in increased cholinergic response. 9/30/200925Pharmacological Effects of Cholinomimetics1)Vasodilation This effect is mediated by muscarinic receptor activation and is especially prominent in the salivary gland and intestines9/30/200926Pharmacological Effects of Cholinomimetics(2)Vasodilation cont.The vascular response is due to endothelial cell nitric oxide (NO)release following agonist interactions with endothelial muscarinic receptor Increased NO activates guanylate cyclase which increases cyclic GMP concentrations 9/30/200927Pharmacological Effects of Cholinomimetics(3)Vasodilation cont.Subsequent activation of a Ca2+ ion pump reduces intracellular Ca2+ Reduction in intracellular Ca2+ causes vascular smooth muscle relaxation Ca2+ complexes with calmodulin activating light-chain myosin kinase Increased cGMP promotes dephosphorylation of myosin light-chains. Smooth-muscle myosin must be phosphorylated in order to interact with actin and cause muscle contraction. 9/30/200928Nitric Oxide (NO) and VasodilitationSchematic below from: http://www.nature.com/nature/journal/v396/n6708/fig_tab/396213a0_F1.html9/30/200929Pharmacological Effects of Cholinomimetics(4)2)Negative chronotropic effect (Decrease in heart rate)Decreases phase 4 (diastolic depolarization)As a result, it takes longer for the membrane potential to reach threshold. Mediated by M2muscarinic receptors 9/30/200930Pharmacological Effects of Cholinomimetics(5)3) Decreased SA nodal and AV nodal conduction velocityExcessive vagal tone may induce bradyarrhythmiasincluding partial or total heart block (impulses cannot pass through the AV node to drive the ventricular rate; in this case, the idioventricular or intrinsic ventricular rate must maintain adequate cardiac output) Transmission through the AV node is especially dependent on Ca2+ currents.ACh decreases calcium currents in the atrioventricular node9/30/200931Pharmacological Effects of Cholinomimetics(6)4) Negative inotropism (decreased myocardial contractility)more prominent in atrial than ventricular tissue. due to a decrease in Ca2+ inward current in the ventricle, adrenergic tone dominates;at higher levels of sympathetic tone, a reduction in contractility due to muscarinic stimulation is noted. Muscarinic stimulation reduces the response to norepinephrine by opposing increases in cAMP in addition to reducing norepinephrine release from adrenergic terminals 9/30/200932Clinical Uses Gastrointestinal & GenitourinaryBethanechol (Urecholine)GI smooth muscle stimulantpostoperative abdominal distention paralytic ileus esophageal reflux; promotes increased esophageal motility (other drugs are more effective, e.g. dopamine antagonist (metoclopramide) or serotonin agonists (cisapride) 9/30/200933Clinical Uses(2)Urinary bladder stimulantpost-operative; post-partum urinary retention alternative to pilocarpine to treat diminished salivation secondary e.g. to radiation Carbachol not used due to more prominent nicotinic receptor activation Methacholine used for diagnostic purposes.testing for bronchial hyperreactivity and asthma9/30/200934Clinical Uses(3)Opthalmological UsesAcetylcholine and Carbachol may be used for intraocular use as a miotic in surgery Carbachol may be used also in treatment of glaucoma.Pilocarpine is used in management of glaucoma and has become the standard initial drug for treating the open-angle form. Sequential adminstration of atropine (mydriatic) and pilocarpine (miotic) is used to break iris-lens adhesions. 9/30/200935Adverse Effects: Muscarinic AgonistsAdverse Effects: Muscarinic Agonistssalivation diaphoresis colic GI hyperactivity headache loss of accommodation 9/30/200936Major contraindication to the use of muscarinic agonists Asthma:Choline esters (muscarinic agonists) can produce bronchoconstriction. In the predisposed patient, an asthmatic attack may be induced. Hyperthyroidism:Choline esters (muscarinic agonists) can induce atrial fibrillation in hyperthyroid patients. Peptic ulcer:Choline esters (muscarinic agonists), by increasing gastric acid secretion, may exacerbate ulcer symptoms. Coronary vascular disease:Choline esters (muscarinic agonists), as a result of their hypotensive effects, can further compromise coronary blood flow. 9/30/200937Indirect-acting Cholinomimetic DrugsAcetylcholinesterase InhibitorsThere are three classesof anticholinesterase agents 1.Reversible, Short-Acting Anticholinesterases 2.CarbamylatingAgents: Intermediate-Duration Acetylcholinesterase Inhibitors3.PhosphorylatingAgents: Long-Duration Acetylcholinesterase Inhibitors 9/30/200938Reversible, Short-Acting Anticholinesterases1) edrophonium (Tensilon)and2) tacrine (Cognex), associate with the choline binding domainThe short duration of edrophonium (Tensilon) action is due to its binding reversibility and rapid renal clearance. Tacrine (Cognex), being more lipophillic, has a longer duration. 9/30/200939Carbamylating Agents: Intermediate-Duration Acetylcholinesterase InhibitorsPhysostigmineNeostigmineare acetylcholinesterase inhibitors that form a moderately stable carbamyl-enzyme derivative The carbamyl-ester linkage is hydrolyzed by the esterase, but much more slowly compared to acetylcholine. As a result, enzyme inhibition by these drugs last about 3 -4 h (t ½ = 15 -30 min). Neostigminepossesses a quaternary nitrogen and thus has a permanent positive chargeBy contrast, physostigmineis a tertiary amine9/30/200940Phosphorylating Agents: Long-Duration Acetylcholinesterase InhibitorsOrganophosphate acetylcholinesterase inhibitors, such as diisopropyl fluorophosphate (DFP)form stable phosphorylated serine derivatives. For DFP the enzyme effectively does not regenerate following inhibition. 9/30/200941Phosphorylating Agents: Long-Duration Acetylcholinesterase Inhibitors(2)Furthermore, in the case of DFP, the loss, termed "aging", of an isopropyl group, further stabilizes the phosphylated enzymeThe application of the terms "reversible" and "irreversible" depends on the duration of enzyme inhibitionrather than strictly based on mechanism 9/30/200942Organophosphate poisoning ParathionParathion, a low volatility and aqueous-stable, organophosphate is used as an agriculural insecticide. Parathion is converted to paraoxon by mixed function oxidases. Both the parent compound and its metabolite are effective acetylcholinesterase inhibitors (P=S to P=O). Parathionprobably is the most commoncause of accidental organophosphate poisoning and deathThe phosphothioate structure is present in other common insecticides: dimpylate, fenthion, and chlorpyrifos. 9/30/200943Tx of Organophosphate poisoning-PralidoxinePralidoxineis a cholinesterase activatorIt is used as an antidote to organophosphates poisoningUnfortunately, pralidoxinedoes not cross the blood brain barrier to treat the central effects of organophosphate poisoning.It has to be given very early after poisoning as within a few hours the phosphorylated enzyme undergoes a change (aging) that renders it no longer susceptible to reactivation9/30/200944Clinical applications of anticholinesterases They are also used in cases of overdose with either the muscarinic antagonist, atropine, or muscle relaxants (nicotinic antagonists)Pralidoxineis a cholinesterase activator.organophosphates poisoning9/30/200945Opthalmological Uses of Anticholinesterase Drugs When applied to the conjunctiva, acetylcholinesterase inhibitors produce:constriction of the pupillary sphincter muscle (miosis) contraction of the ciliary muscle (paralysis of accommodation or loss of far vision). Loss of accommodation disappears first, while the miotic effect is longer lasting. During miosis, elevated intraocular pressure (glaucoma) declines due to enhanced flow of aqueous humor. In glaucoma, elevation of intraocular pressure can cause damage to the optic disc and blindness. 9/30/200946Gastrointestinal and Urinary BladderNeostigmine is the anticholinesterase agent of choice for treatment of paralytic ileus or urinary bladder atony. Direct acting cholinomimetic drugs are also useful. 9/30/200947Myasthenia Gravis See Clinical: E-Medicine ArticleMyasthenia GravisMyasthenia Gravis appears to be caused by the binding of anti-nicotinic receptor antibodies to the nicotinic cholinergic receptor. Binding studies using snake alpha-neurotoxins determined a 70% to 90% reduction of nicotinic receptors per motor endplate in myasthenic patients 9/30/200948Myasthenia Gravis(2)Receptor number is reduced by:increased receptor turnover (rapid endocytosis) blockade of the receptor binding domain antibody damage of postsynaptic muscle membrane9/30/200949Myasthenia Gravis(3)Anticholinesterase, edrophonium (Tensilon), is useful in differential diagnosis for myasthenia gravis.In this use, edrophonium (Tensilon) with its rapid onset (30 s) and short duration (5 min) may cause an increase in muscle strength. 9/30/200950Myasthenia Gravis(4)This change is due to the transient increase in acetylcholine concentration at the end plate. Edrophonium (Tensilon)may also be used to differentiate between muscle weakness due to excessive acetylcholine (cholinergic crisis) and inadequate drug dosing. Anticholinesterase drugs provide9/30/200951Antimuscarinic Effects on Organ SystemsCentral Nervous SystemEffects of Antimuscarinic AgentsIn normal doses, atropine produces little CNS effect.In toxic doses, CNS excitation results in restlessness, hallucinations, and disorientation. At very high doses, atropine can lead to CNS depression which causes circulatory and respiratory collapse. By contrast, scopolamine at normal therapeutic doses causes CNS depression, including drowsiness, fatigue and amnesia. 9/30/200952Antimuscarinic Effects on Organ SystemsCentral Nervous SystemEffects of Antimuscarinic Agents cont.Scopolamine also may produce euphoria, a basis for some abuse potential. Scopolamine may exhibit more CNS activity than atropine because scopolamine crosses the blood brain barrier more readily. Scopolamine (transdermal) is effective in preventing motion sickness. Antimuscarinics are used clinically as preanesthetic medication to reduce vagal effects secondary to visceral manipulation during surgery. Antimuscarinics with L-DOPA are used in Parkinson's disease. Extrapyramidal effects induced by some antipsychotic drugs may be treated with antimuscarinic agents. 9/30/200953Antimuscarinic Effects on Organ SystemsAutonomic Ganglia and Autonomic Nerve TerminalsThe primary cholinergic receptor class at autonomic ganglia is nicotinic; however, muscarinic M1-cholinergic receptors are also present. Muscarinic M1-ganglionic cholinergic receptor activation produce a slow EPSP that may have a modulatory role.Muscarinic receptors are also located at adrenergic and cholinergic presynaptic sites where their activation reduces transmitter release.Blockade of these presynaptic receptors increase transmitter release.9/30/200954OpthalmologicalMuscarinic receptor antagonists block parasympathetic responses of the ciliary muscle and iris sphincter muscle, resulting in paralysis of accommodation (cycloplegia) and mydriasis (pupillary dilation). Mydriasis results in photophobia, whereas cycloplegia fixes the lens for far vision only (near objects appear blurred). Antimuscarinic Effects on Organ Systems9/30/200955Opthalmological cont.Systemic atropine at usual doses does not produce significant ophthalmic effect. By contrast, systemic scopolamine results in both mydriasis and cycloplegia. Note that sympathomimetic-induced mydriasis occurs without loss of accommodation. Atropine-like drugs can increase intraocular pressure, sometimes dangerously, in patients with narrow-angle glaucoma. Increases in intraocular pressure is not typical in wide-angle glaucoma.Antimuscarinic Effects on Organ Systems9/30/200956Antimuscarinic Effects on Organ SystemsCardiovascular SystemAntagonistTissue (Heart)ResponsesMolecular AspectsThe dominant effect of atropine or other antimuscarinic drug administration is an increase in heart rate. This effect is mediated by M2-receptor blockade thereby blunting cardiac vagal tone.atropineSA nodedecreased phase 4 depolarization; hyperpolarizationK+ channel activation (hyperpolarizing) through ß-gamma Gisubunits*;Gi-mediated inhibition of adenylyl cyclase* (negative inotropism)(Gican inhibit directly Ca2+channel opening)Atriumdecreased contractility; decreased AP durationAV nodedecreased conduction velocityVentricledecreased contractilityMuscarinic Type M2