PCTH AUTONOMIC NERVOUS SYSTEM (ANS) Brief overview of anatomy and function of the ANS:

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1 1 PCTH AUTONOMIC NERVOUS SYSTEM (ANS) MJAW Sept 2014 Please note: This is an extensive set of notes for a first exposure to pharmacology. Not all handouts will be so voluminous. The intent is to make you read and think about pharmacology. Memorizing so many drug names in Pharmacology is intimating and it is not possible to know them all, but it is best to know those in bold type. Brief overview of anatomy and function of the ANS: The peripheral nervous system is outside the Central Nervous System (brain and spinal cord). It consists of the autonomic nervous system (ANS) and the motor (somatic) nervous system. The ultimate control of both systems lies within the CNS in command and control centres. However, while the ANS has considerable autonomy, CNS imperatives drive the motor (skeletal muscle) nervous system. Thus one consciously walks, talks, etc. as a result of central commands from the CNS but digestion, heart rate, etc. are not so directly controlled. The ANS serves to regulate the internal organs and function of the body, while the motor system serves skeletal muscle (locomotion) functions. Basic anatomy of the autonomic nervous system (ANS) Anatomically the peripheral ANS is comprised of three (3) anatomic and functional divisions: sympathetic, parasympathetic and enteric. The basic pattern for both sympathetic(s) and parasympathetic sections (PS) of the ANS consists of pre-ganglionic neurons arising from the CNS (where there are ANS centres) that travel to peripheral ganglia from where postganglionic nerves innervate target organs. The peripheral parasympathetic CNS outflows are to: o Cranial nerves (cranial nerves III, VII, IX and X) o Sacral nerves Parasympathetic ganglia usually lie close to, or within, the target organ Sympathetic nerves leave the CNS via thoracic and lumbar spinal roots to synapse at sympathetic ganglia which form two (2) paravertebral chains, plus some midline ganglia The enteric nerves arise from neurons in the intramural plexi of the gastrointestinal tract. This enteric system receives input from both sympathetic and parasympathetic nerves, but can act autonomously to control motor and secretion functions of the intestine. The following diagrams outline the peripheral ANS both structure and function. The two major systems (PS and S), to a degree, act as counterparts to each other. Thus, parasympathetic nerve activity slows heart rate while sympathetic activity increases heart rate. However, such simplification cannot be taken too far. For example, via effects on heart and blood vessels, sympathetic nerve activity increases blood pressure whereas parasympathetic nerve activity has little or no effect on blood pressure. An accelerator/brake analogy has been used for the roles of the PS and S, but this is overly simplistic. Each and every organ in the body has different degrees of sympathetic and parasympathetic innervation. The pharmacology of the ANS mainly concerns drugs acting on efferent side of the ANS since fewer drugs influence ANS afferent activity going to the CNS, or the ANS centres within the CNS. For the greater part, most ANS drugs either mimic, accentuate, or block responses to efferent ANS activity.

2 2 The role of the ANS is to maintain homeostasis and ensure that internal organs respond to the requirements of the body. The following Diagrams (1-3) indicate which organs are innervated by the ANS. For convenience in Diagram 1 para-sympathetic activity is on the left and sympathetic on the right. Diagram 1 An incomplete overview of the ANS Diagram 2 provides more details including the location of ganglia for both parasympathetic (left) and sympathetic (right) pathways and more information as to organ responses to PS and S nerve activity. Note: parasympathetic ganglia are located close to, on, or in the organ they innervate, whereas the sympathetic ganglia are mainly located in close proximity to the spinal column and, most importantly communicate with each other particularly via the paravertebral sympathetic chain that lies just outside the spinal column, but within the thorax. This anatomical pattern allows for an overall coordinated activation of the sympathetic system, as opposed to the directed and localized activation of single organs by parasympathetic nerves. Diagram 2 ANS more detail

3 3 To complete your boredom quotient Diagram 3 further amplifies the anatomical and functional differences between the PS and S sections of the ANS but, this time, with the parasympathetic section on the right. Diagram 3 better illustrates the peripheral wiring of the ANS and the central role of the parasympathtic vagus nerve in controlling internal organs. Note: the role of the cranial nerves in eye, lacrymal and salivary glands in the head, and the sacral nerves in the lower part of gastrointestinal and urinary organs. This diagram exemplifies the wiring differences in the the two sections inasmuch that most PS ganglia lie on or within the organ they innervate. On the otherhand, the diagram also shows the important paravertebral sympathetic ganglion chain which forms a network with few external way stations such as the coeliac and superior and inferior mesenteric ganglia. The left side diagrams in insets illustrate how sympathetic nerves innervate blood vessels and skin. Sweating is an important function of the sympathetic nervous system as well as control of body hair and skin blood vessels. When we are hot we sweat, body hair stands upright and skin flushes - all driven by sympathetic activation BUT, as seen later, sweating in particular involves post ganglionic cholinergic activity.

4 4 ANS:- Neurotransmitter Molecules Transmission from nerve endings to nerves and from nerves to effector tissue in the CNS, ganglia and at post neuronal effector sites is chemical via neurotransmitters Acetylcholine (Ach) is the principal transmitter molecule at ganglia and at post-ganglionic parasympathetic nerve endings. Norepinephrine NA or NE (noradrenaline) is the principal neurotransmitter at post-ganglionic sympathetic nerve endings. There are a few special exceptions to the above: sympathetic nerves to sweat glands release ACh the adrenal medulla (which is a sympathetic ganglion) releases mainly epinephrine (adrenaline).

5 5 Cholinergic refers to sites at which acetylcholine is the primary neurotransmitter. Adrenergic refers to sites at which norepinephrine is the primary neurotransmitter. Overall summary of the physiological function of the ANS The autonomic nervous system controls: smooth muscle (visceral and vascular) activity; exocrine (and some endocrine) secretions; rate and force of the heart; certain metabolic processes (e.g. glucose utilization) Sympathetic and parasympathetic systems sometimes have opposing actions in certain situations (e.g. control of the heart rate, gastrointestinal smooth muscle); but not in others (e.g. salivary glands, ciliary muscle of the eye) Sympathetic activity increases in stress (fight, fright, flight responses) whereas parasympathetic activity predominates during satiation and repose. Both systems exert continuous physiological control of specific organs under normal conditions. In addition to the cholinergic and adrenergic ANS some parts of the ANS are Non Adrenergic Non Cholinergic (NANC). The relative importance of the NANC system in physiology and pathology is still being resolved. A simplified?? table of actions of the ANS Organ PSymp Symp Importance Outcome Heart Atria Bradycardia Tachycardia P=PS Ventricle No effect Positive force S>>>PS Cardiac modulation AV node Slowed transmission Increased Equal Blood vessel Very little Constrict S>>>>PS Raise blood pressure Lung PS>S Bronchial muscle Contract (usually limited) Relax (usually limited) PS>>S Bronchial muscle tone Bronchial glands Secretions increased Drying of secretions Eye Narrow iris Dilate pupil PS>>>S Dominant effect Contracts lens PS Accommodation of vision Nose Reduces patency Increases patency S>>PS Salivary glands Watery secretions Thick mucoid secretions PS>>S Dry mouth if PS blocked Tear glands Tears PS Dry eyes if PS blocked Adrenal glands Nicotinic ganglion PS (celiac ganglion) Release of adrenaline Kidney Liver Pancreas Large intestine Stimulates gall bladder Glycogenolysis S liver PS gall bladder Contracts Relaxes rectum Contracts rectum PS controls defecation Small intestine Contracts Relaxes PS>>>S Enteric nerves important Stomach Acid release Slows digestion PS>>>S Spleen Contracts S Bladder Contracts Relaxes PS>>S PS controls

6 6 micturition Genitalia Male tumescence Detumescence PS for up, S for down Involves NO ANS Peripheral Neurotransmitter molecules The principal transmitter at post-ganglionic parasympathetic endings is acetylcholine (ACh) and also at all ganglia. Norepinephrine - NE (noradrenaline NA) is the major transmitter in post ganglionic sympathetic nerve endings. Preganglionic neurons of both PS and S sections are cholinergic and ganglionic transmission is primarily via stimulation of post synaptic ganglionic nicotinic ACh receptors on post synaptic neurones (some muscarinic receptors are also present in ganglia) Postganglionic parasympathetic nerve endings are cholinergic with ACh acting on muscarinic receptors (MChR) located on the effector organs. Postganglionic sympathetic neurons are mainly noradrenergic A few special sympathetic nerves are cholinergic (e.g. sweat glands) and ACh acts on muscarinic ACh receptors Transmitters other than noradrenaline and acetylcholine (NANC transmitters) also occur in the autonomic system. The main ones are nitric oxide (NO) and VIP (parasympathetic), ATP and NPY (sympathetic). Others, such as 5-HT, GABA and dopamine, also play a role. Co-transmission involving co-release of more than one transmitter is a common phenomenon Some definitions used in Pharmacology CHOLINERIC TRANSMISSION AGONISTS such as neurotransmitters, hormones, natural or synthetic molecules that activate (i.e. agonize) the receptors to which they bind, and produce a functional response. RECEPTORS - are drug targets, some of which are plasma membrane proteins, to which drugs bind, and subsequently do, or do not, produce a functional action. ANTAGONISTS bind selectively to a receptor without activating it thereby denying agonist access to their receptor binding site thus they act as blockers. PARTIAL AGONISTS Stimulate a receptor inefficiently, but will produce a functional response when the receptor is not occupied by a full agonist and reduce the actions of a full agonist INVERSE AGONISTS reduce activity in receptors that are spontaneously active in absence of agonist. NON/UN COMPETITIVE ANTAGONISTS occupy receptors at or near the agonist binding site and do so without agonizing them. Thus they antagonize agonists in a selective receptor specific manner. They may act reversibly or irreversibly. ALLOSTERIC MODULATORS do not bind to an agonist recognition site but their binding at an allosteric site modulates receptor activity. Receptors agonized (stimulated) by ACh (known as cholinoceptors or cholinergic receptors) exist as various subtypes, whose presence and density varies with tissue type. There are two major types: MUSCARINIC and NICOTINIC. Various subtypes of each of these two main types have been identified molecularly and are associated with different tissues. Drugs are invented (discovered) to selectively target such sub-types so as to produce tissue specific functional actions.

7 7 Muscarinic Receptors: M1, M2, M3, (M4&5) are GPCRs (G Protein Coupled Receptors) found peripherally, and in the CNS. Nicotinic Receptors The two main peripheral types of nicotinic receptors are those on skeletal muscle (Nm) and those on nervous tissue such as ganglia (Nn). Both are an essential functional part of ACh-gated ion channels which, when opened by ACh, have selective permeability to both Na and K ions. Their opening produces depolarization of the post synaptic (post junctional) membrane. There are further sub- types of Nicotinic receptors within the CNS. Synthesis and Metabolism of ACh Acetylcholine is synthesized in cholinergic nerve endings from the amino alcohol - choline, and the carboxylic acid - acetic acid, both common in tissues. The enzyme choline acetylase forms the ester, acetylcholine. Synthesized acetylcholine is taken into, and stored, in vesicles in cholinergic nerve endings. Vesicles are mobilized by calcium ions entering via voltage-opened Ca ++ channels (N type calcium channels) as a result of a sodium dependent action potential at the nerve terminal. The mobilized vesicle fuses with the pre-synaptic nerve membrane and releases ACh into the synaptic cleft where it diffuses to post-synaptic (post-junctional) cholinergic receptor. Released acetylcholine is rapidly hydrolyzed by acetylcholinesterase into choline and acetate, thereby terminating its action. Acetate is free to participate in intermediate metabolism while choline is re-cycled - taken up into the pre-synaptic nerve ending. Drugs are available to interfere with the whole process, from ACh synthesis, to vesicle storage, to Ca ++ dependent release, to choline re-uptake. However, not many of such drugs have therapeutic utility. Vesamicol is a reversible blocker of the intracellular transporter responsible for ensuring storage of ACh into vesicles. N type calcium channel blockers (ω-conotoxins, Ziconotide, Caroverine and others) prevent opening of neuronal calcium channels. Such blockers interfere with neuronal release of all transmitters that rely on calcium for activation of their vesicles. This ubiquitous action on all nerve endings limits their utility. Hemicholinium blocks the uptake of choline into the nerve ending Botulinus toxin (from the anaerobe bacterium Clostridium botulinum) depletes nerve endings of ACh). It is a two-chain polypeptide whose light chain is a protease that disables a fusion proteins (SNAP-25) that prevents vesicle functioning. Function is restored slowly by synthesis of new vesicles, rather than by re-filling. Of the above drugs, only botulinus toxin (botox) is routinely used therapeutically (in neuromuscular junction pharmacology, skeletal muscle dystonias and cosmetics). MUSCARINIC AGONISTS The discovery in plants, fungi, and later in animals, of chemicals that had parasympathetic-like actions was central to the analysis of cholinergic actions and analysis of the ANS. One fungal chemical was muscarine (Amanita muscaria). This has actions that mimicked stimulation of the parasympathetic system. Nicotine from plants, including tobacco (genus Nicotiana), stimulates nicotinic receptors in ganglia resulting in the stimulation of the peripheral parts of the ANS. Muscarinic agonists are of limited therapeutic use in medicine. Interestingly atropine from the plant family Solanaceae (nightshade) was found to block the actions of muscarine (a muscarinic blocker) helped define muscarinic receptors.

8 8 MUSCARINIC AGONIST & their receptors (MAChR: M1-5) All muscarinic receptors are GPCRs (G protein coupled receptors): M1,3 &5 via Gq/11 family; M2 & 4 through Gi and Go when receptor is agonized so as to: - Activate phospholipase C to form inositol triphosphate (IP3) and diacylglycerol (DAG) which are intracellular second messengers - inhibit adenylate cyclase to reduce intracellular camp - inhibit opening of calcium channels - activate potassium channels to generate an inward potassium current - ikmach MAChRs- are present on effector tissue at cholinergic post ganglionic nerve endings, and sympathetic sweat glands. They also occur in ganglion where they are of lesser importance than nicotinic receptors Subtypes of muscarinic receptors Note: There is a lack of really selective (100:1 or more) agonists, and/or antagonists, for the subtypes of M receptors, and a lack of clarity regarding their individual functional importance. Different muscarinic agonists have different profiles of action in intact animals due to different potencies on the various sub-types of muscarinic receptors, but we have still to fully elucidate the role of each the sub-types. The following is an approximate summary. M1 on neuronal tissue in ANS ganglia, CNS, exocrine glands - involve Gq, IP3 and DAG to elevate Ca ++, produces "slow" excitation in ganglia. Agonists that are partially selective:- McN A- 343, muscarine. Antagonists, again are only partially selective:- atropine, oxybutynin, ipratropium, mamba toxin muscarinic toxin 7, pirenzepine. M2 on cardiac tissue (PS nerves innervate atria, atrioventricular and sinus nodes, but not ventricles) - decrease heart rate and reduced atrial contraction via Gi (decrease in camp) and K channels. Inhibit synaptic transmission. Generally inhibitory effects. No really selective agonists (maybe bethanechol), nor antagonists (dimetindene, otenzepad) M3 increases glandular secretion (e.g. sweat glands), lungs, contract: gut, bronchial and blood vessel smooth muscles. However also relax blood vessel smooth muscles via releasing the relaxant nitric oxide (NO) from endothelium of blood vessels. Few, if any selective agonists carbachol, oxotremorine, pilocarpine (in eye) or antagonists (tolterodine, oxybutynin, ipratropium, darifenacin, tiotropium). M4 in CNS and acts as a regulatory auto receptor inhibits adenyl cyclase- regulatory role in CNS dopamine transmission. Agonist not so selective (oxotremerine, carbachol), antagonists (tropicamide some 2-5x), AFDX-384, dicycloverine) M5 down regulates camp and protein kinase A. No really selective agonists (milameline, sabcomeline) nor antagonists (VU compounds)

9 9 All M receptors equally stimulated by the agonist ACh and blocked by the general muscarinic receptor antagonist - ATROPINE MUSCARINIC/nicotinic AGONISTS Many are acetylcholine (ACh) analogs. The relationships between chemical structure and actions (agonists or antagonist) are known as Structure Activity Relationships (SAR). Acetylcholine: Stimulates all types of cholinoceptors (nicotinic and muscarinic) Muscarine: Agonist at muscarinic receptors only. Methacholine (MCh) stimulates only muscarinic receptors. SAR Critical chemical centres in acetylcholine are the positive Nitrogen and the carbonyl oxygen (-C=O). Those with an ester (acid + alcohol) linkage, such as ACh and methacholine MCh (methacholine = acetylbetamethylacetylcholine) are metabolized by acetylcholinesterase (AChE). It might be surprising that very small changes in acetylcholine s structure produces methacholine and loss of nicotinic activity. Knowledge of SAR potentially allows one to invent selective agonists and antagonists, but this has not been totally successful with muscarinic receptors, despite our long knowledge of the cholinergic system. This is a pity since the above list of receptors should provide an avenue to new drugs with specificity for specific disease via selective action (agonist or antagonist) on sub-types of muscarinic receptors. Therapeutic uses of Muscarinic Agonists (see above for details regarding sub types of the M receptor) There are two pharmacological ways of way of mimicking the activity of the PS system: 1) by muscarinic agonists, or 2) by anticholinesterase drugs. The therapeutic value of activation are relatively limited, although there was intense interest in the use of muscarinic agonists to treat symptoms of Alzheimer s disease. M1 receptors play a role in cognition and may have a potential therapeutic role. Classic agonists are methacholine, bethanecholine and carbamoylcholine (carbachol). Bethanecholine is muscarinic receptor selective, methacholine is more selective for muscarinic versus nicotinic, carbachol more nicotinic Pilocarpine, a M3 agonists, has been used in glaucoma as has aceclidine. Other uses include dry mouth in diseases such as Sjögren's syndrome. Arecoline is a natural alkaloid fund in betel nut which if chewed aids digestion and gastrointestinal function.

10 10 CHOLINESTERASE INHIBITORS Cholinesterase are enzymes in tissue and plasma. They have their own characteristic profiles with respect to factors such as: specificity, substrate site, inhibitors, and types of inhibition. The main type - acetylcholinesterase has high selectivity for ACh and is present at cholinergic junctions and synapses. It has clearly defined substrate sites with acctive sites which recognize: 1) N + and 2), the ester linkage of acetylcholine. Anticholinesterase drugs can bind to either site, or both, and act as reversible or irreversible inhibitors. Non-specific plasma and tissue esterases breakdown esters and lack selectivity for ACh. ACTIONS OF CHOLINESTERASE INHIBITORS The actions of reversible cholinesterase inhibitors are principally due to inhibition of acetylcholinesterase and resulting accumulation of ACh at cholinergic synapses resulting in excess muscarinic and nicotinic receptor stimulation. In addition, some have additional direct agonist effects on the receptor. Irreversible inhibitors such as the organophosphate compounds can have long term toxic actions that are not due to inhibition of cholinesterases. Such inhibitors are lipid soluble, enter the brain, and have profound effects at CNS cholinergic sites. Inhibitors with a charged nitrogen (N + ) in their structure do not penetrate the CNS to a significant degree. Chemical types of Cholinesterase Inhibitors 1. Amino alcohols (edrophonium) bind reversibly at N + site and are shorter acting. 2. Carbamates (neostigmine) are longer lasting and bind at both sites 3. Organophosphates (diisopropyl flurophosphate DFP) bind irreversibly at the esteratic site Edrophonium is very short acting and can be used to diagnose myasthenia gravis (see later) increases muscle strength shortly after i.v. administration of edrophonium is a positive test for myasthenia gravis. Also used to test the adequacy of the treatment of myasthenia gravis with a longer acting drug (e.g. pyridostigmine) where if edrophonium increases muscle strength more intensive anticholinesterse treatment is required. Organophosphates covalently phosphorylate enzyme at the ester site and, unless quickly reversed, this action becomes irreversible. Used as insecticide and poison gases (vapours) e.g. chlorpyrifos, malathion and diisofluorophosphonate (nerve gas) with over 100 organophosphate & 20 carbamate insecticides available that cause many cases of poisoning in agriculture. The toxicity of such compounds includes CNS effects, e.g. convulsions, GI, respiratory, urinary effects stimulatory, like direct-acting agents, cardiovascular effects - both sympathetic & parasympathetic stimulation, fibrillation of skeletal muscle fibers, depolarizing blockade and muscle paralysis. Symptoms of poisoning: Salivation, Lacrimation, Urination, Defecation, Gastric Emptying - hence acronym, SLUDGE Treatment of poisoning with cholinesterase inhibitors. In addition to general supportive medical care, specific treatment includes atropine (or atropinic drugs) to block muscarinic receptors in the periphery and brain. Atropine is most important in blocking excess stimulation of muscarinic receptors in the periphery and in the CNS. Recent exposure (hours) to organophosphates can be reversed by oximes such as PAM2 and DAM. Reversible anticholinesterase inhibitors have been used for prophylaxis via protection of the active site of the enzyme from organophosphates. The military around the World have antidote/treatment packs involving all the above

11 11 drugs; these sometimes include a reversible anticholinesterase to deny the irreversible anticholinesterase access to active sites on the enzyme. MUSCARINIC ANTAGONISTS The original definitions of drugs as being antagonists were based upon the ability of certain drugs (derived from plants and herbs) to selectively antagonize (inhibit/prevent) the actions of the ANS, neurotransmitters, and their analogues. The most simplistic view of an antagonist for a particular type of receptor is a drug which occupies the agonist recognition site without agonizing (activating) the receptor. Thus antagonists "deny" agonists access to their receptor to prevent endogenous or exogenous agonists from binding. This model is analogous to the substrate/inhibitor models for enzymes. This simplistic view works well in many cases. Conventionally, by Mass Action kinetics, the reaction is as follows:- A+R = AR which, after suitable transduction, gives a response I+R = IR which gives no response Where A is an agonist, and I antagonist (inhibitor) By forming the IR complex, a sufficient concentration of I will prevent A having an action by competition for R, while a high enough concentration of A will prevent I forming IR by competition for R: thus, we have Competitive Antagonism. ATROPINE is the prototypical muscarinic receptor competitive antagonist Atropine, from a plant Atropa belladonna - extracts widened the pupils and so "beautified" medieval Italian ladies. Other muscarinic antagonists are found in other plants, e.g. Datura stramonium. Many synthetic atropinics have been made in attempts to try and achieve selectivity for subtypes of muscarinic receptors. The primary action of muscarinic receptor antagonists is to block responses due to activation of peripheral parasympathetic nerves or cholinergic muscarinic nerves in the CNS although some atropinics have other, ancillary, pharmacological actions. The formula for atropine fits the SAR for muscarinic receptor agonists and antagonists. Note: atropine is larger than acetylcholine, but parts of its structure (N and ester linkage) mimic elements of ACh. It binds to the ACh binding site on the muscarinic receptor, but fails to activate it. Note: it has a greater lipophilic nature (phenyl ring) than acetylcholine. THERAPEUTIC USE OF COMPETITIVE MUSCARINIC ANTAGONISTS

12 12 Many similar drugs have been found, or synthesized, following the discovery of atropine. Most of their actions are principally a reflection of blockade of muscarinic cholinoceptors and the action of endogenous ACh. However, some have other (extra) pharmacological actions. As a class, competitive antimuscarinics will have in vivo actions only when there is parasympathetic activity while any other pharmacological actions could be a possible bonus for certain drugs of this class. It is also important to recognize that other classes of drugs can have competitive muscarinic antagonist actions. One useful aide memoire for the functional actions of atropine, and by implication parasympathetic cholinergic importance, are the symptoms of Atropine Poisoning Dry as a bone - no sweating Blind as a bat no ocular accommodation Mad as a hatter CNS symptoms. In the past the use of mercury by hatters resulted in CNS toxicity hence the expression. Red as a beet a result of vasodilation and no sweating - in children atropine poisoning produces dangerous hyperpyrexia Atropinic drugs include: Alkaloids (naturally occurring) atropine, scopolamine (hyoscine) Tertiary amines dicyclomine, benztropine Quaternary amines ipratropium Examples of the use of this class of drugs in medicine: CARDIOVASCULAR - atropine and related drugs to reverse sinus node bradycardia. Heart rate is under continuous sympathetic and parasympathetic activity. At rest, and when asleep, parasympathetic nerves are dominant, whereas during exercise the sympathetic nerves are most important. OPTHALMIC - tropicamide or cyclopentolate to dilate pupils. Control of the muscles of the iris and lens is dominated by parasympathetic nerves therefore their blockade with a muscarinic antagonist will dilate the pupil and paralyze accommodation for near vision (making reading difficult). Used for their ophthalmic actions and eye examinations, but have a possible side effect of glaucoma. LUNGS atropinics can produce bronchodilation in some asthmatics: ipratropium or similar drugs by inhalation. Such drugs are helpful for some asthmatics, and in chronic obstructive pulmonary disease (COPD) since some bronchoconstriction and hyper-secretion in asthma and COPD is dependent upon muscarinic receptor stimulation. MOTION SICKNESS can be alleviated with hyoscine (scopolamine) acting via a CNS action. The CNS mechanism(s) responsible for this effect has not been fully elucidated. Hyoscine has been used as a so-called truth serum to produce CNS confusion and thereby reveal truths. PARKINSONISM - benzhexol and benztropine to limit movement disorders due to other classes of AntiParkinson drugs via a CNS actions. GASTOINTESTINAL CONDITIONS - e.g. with dicycloverine to facilitate G.I. endoscopy and in hyperirritable bowel conditions,. Their old use in peptic ulcer has been discontinued.

13 13 ANAESTHESIA atropinics are sometimes used to reduce troublesome secretions which are normally due to parasympathetic stimulation, or to prevent bradycardia. Despite our current knowledge of the different sub types of muscarinic receptors relatively little success has met attempts to synthesize muscarinic antagonists with selectivity for the different M receptor subtypes. It is an area which has still to be fully explored. Examples: Pirenzipine: selective (more potent one) M1 (5 times)> M2 20 times> M3. Gallamine: some selectivity for M2 Others have some relative selectivity for M3 (HHSD) and M2 (AF-DX116) Mamba toxins (from snakes) for M1 and M4 As an example of ANS pharmacology consider the pharmacology of the Eye The eye is mainly controlled by parasympathetic nerves and cholinergic innervation: sympathetic nerves have a minor role. Thus, muscarinic antagonists can have marked actions in the eye - dilate the pupil and prevent accommodation. By interfering with drainage of the eye such drugs can cause or exacerbate glaucoma. The extraocular skeletal muscle fibres that control movement of the eye are skeletal muscles but, very unusually, have multiple skeletal motor nerve innervation on each muscle fibre. Glaucoma (increased intraocular pressure that can destroy the eye s function) is a mismatch of secretion of ocular fluids, and their drainage. Drugs for glaucoma can increase drainage, or reduce formation of fluid. Drugs and Glaucoma Muscarinic antagonists can exacerbate glaucoma. Muscarinic agonists and anticholinesterases can alleviate, glaucoma. Various other drugs can be useful in glaucoma, including beta blockers which appear to decrease secretion, as well as prostaglandins. Nicotinic Cholinergic Transmission There are various types of nicotinic receptors in the periphery and the CNS. There are two main families: nicotinic skeletal muscle and nicotinic ganglionic cholinoceptors (see Google for the biochemical characteristics of nicotinic receptors, location, etc.) Nicotinic receptors (NAChR) are found on the: Post junctional membranes of the skeletal neuromuscular endplate on a 1:1 relationship with acetylcholinesterase enzyme. Skeletal muscle spindle (afferent physiological receptor in skeletal muscle-extrafusal fibres). At the ganglion of the sympathetic, parasympathetic and enteric ANS Within CNS at various synaptic sites - both pre and post synaptic Functionally there are at least two main peripheral types of nicotinic receptors (NAChR): skeletal muscle and ganglionic (with at least two different CNS types). NAChR is a ligand-gated ion channel where 2 molecules of the neurotransmitter ACh bind so as to open the channel. NAChR receptors in skeletal muscle are generally "protected" from overstimulation with ACh by adjacent AChE molecules. In the neuromuscular junction ACh released from the nerve generally only binds once to a NAChR before it is hydrolyzed by acetylcholine into choline and acetate.

14 14 Structure of NAChR (Nicotinic receptor): Transmembrane spanning proteins (ligand-gated channel) are "gated" by ACh to open to allow trans-membrane movement of ions - Na+ (Inwards) and K+ (Outwards) resulting in depolarization of the post-synaptic end plate membrane. Different subtypes of AChR nicotinic receptor are composed of alpha and beta components in various proportions. In the skeletal muscle form of the nicotinic receptor 2 (two) ACh molecules have to bind to two recognition sites in order to open the channel (co-operativity). Ganglionic nicotinic receptors Agonists: ACh, CCh, nicotine (after which the receptor is named), lobeline and DMPP All "agonize" receptor but high and continuous occupancy desensitizes the receptor to the action of further ACh this results in so-called desensitizing block. Nicotine first activates receptor, and then blocks it. This type of block also occurs in the skeletal muscle receptor. Antagonists (ganglion blockers): Hexamethonium (C6), trimethaphan Therapeutic Uses: Nowadays rarely, if ever, used. For an understanding of the effects of nicotinic ganglionic blockade (and incidentally which aspects of the autonomic nervous system are functional important in normal life) see "hexamethonium man" as described by Paton. W. D. M. Paton, Pharm. Rev. 6, 59 (1954) Hexamethonium man: is a pink complexioned person, except when he has stood for a long time, when he may get pale and faint. His handshake is warm and dry. He is a placid and relaxed companion; for instance he may laugh, but he can t cry because the tears cannot come. Your rudest story will not make him blush, and the most unpleasant circumstances will fail to make him pale. His socks and his shirt stay very clean and sweet. He wears corsets(!!) and may, if you meet him out, be rather fidgety (corsets to compress his splanchnic vascular pool, fidgety to keep the venous return going from his legs). He dislikes speaking much unless helped with something to moisten his dry mouth and throat. He is long-sighted and easily blinded by bright light. The redness of his eyeballs may suggest irregular habits and in fact his head is rather weak. But he always behaves as a gentlemen and never belches or hiccups. He tends to get cold, and keeps well wrapped up. But his health is good; he does not have chilblains and those diseases of modern civilization, hypertension and peptic ulcers, pass him by. He is thin because his appetite is modest; he never feels hunger pains and his stomach never rumbles. He gets rather constipated so his intake of liquid paraffin is high. As old age comes on he will suffer from retention of urine and impotence, but frequency, precipitancy, and strangury will not worry him. One is uncertain how he will end, but perhaps if he is not careful, by eating less and less and getting colder and colder, he will sink into a symptomless, hypoglycemic coma and die, as was proposed for the universe, a sort of entropy death. Skeletal muscle nicotinic receptors (Neuromuscular-Skeletal Muscle Junction)

15 15 Action potentials travelling down motor nerve axons (nerve impulse) reach the nerve ending and open N-type calcium channel allowing Ca ++ entry into the nerve ending and this entry facilitates mobilization of ACh vesicles, their binding to nerve ending membrane, and release of ACh. ACh binds to the NAChR (nicotinic skeletal muscle receptor) which opens to increase permeability to K + and Na + so producing depolarization at the endplate region. The depolarization, if large enough, will trigger an action potential at the plasma membrane of the surrounding skeletal muscle fibre. The muscle action potential spreads across the rest of the muscle to induce contraction via triggering Ca ++ entry into the cell, and releasing Ca ++ onto the actin-myosin contractile elements. The effector tissue (neuromuscular endplate) is a matrix of NAChR and acetylcholinesterase (AChE) molecules closely packed together. As a result, any free ACh is quickly inactivated. A low level of spontaneous (resting release) of ACh vesicles always occurs. Each of such spontaneously released vesicles causes a miniature end plate potential, but no activation of the skeletal muscle. In most skeletal muscles in most mammalian skeletal muscle each muscle fibre has one single end plate on which a single motor nerve axon terminates. A few special skeletal muscles have multiple end plates with multiple axonal innervations. This exceptional pattern of multiple innervation in man is found in the extra-ocular muscles of the eye, and on muscle spindles (part of the skeletal muscle feedback control system embedded in muscles). The reserve for transmission from nerve to skeletal muscle is high - up to 10 times more ACh released than is necessary to elicit an action potential in the muscle cell. There is also an excess of NAChRs on the end plate itself. The extent of this receptor reserve is apparent in the rare autoimmune disease, myasthenia gravis, where over 90% of the end plate nicotinic receptors have to be inactivated by endogenous autoimmune antibodies before neuromuscular transmission is impaired.

16 16 Myasthenia gravis is rare ( cases per million, mostly females). If antibodies reduce functional receptors by more than 90%, muscle weakness occurs first apparent in certain muscle groups (ptosis of eyelid). Diagnosis includes detection of NAChR antibodies as well as functionally by a short-lived increase in muscular strength after i.v. injection of an ultra-short acting anticholinesterase, edrophonium (a provocative test). Treatment of myasthenia gravis includes immunosuppressive drugs, thymectomy, and longer-acting anticholinesterases (together with atropine). NmAChR Agonists: Classically include ACh, CCh (carbamycholine) and succinylcholine. Succinylcholine is ester of a dicarboxylic acid (succinic acid) with two acetates. Succinylcholine "agonizes" the NAChR, but at high enough concentrations producing continuous receptor occupancy and desensitizes them resulting in a depolarizing block (which may be followed by a desensitizing block). Antagonists (neuromuscular blockers): The classic (first available) neuromuscular blocker was d-tubocurarine (from a S. American arrow poison) followed by decamethonium (C10 - the first synthetic compound) and succinylcholine. Many synthetic curare-like molecules have since been invented. Pharmacology of Neuromuscular Blocking drugs. The major use of neuromuscular blockers drugs is in anaesthesia/surgery. A short acting blocker will produce paralysis of the laryngeal muscles allowing easy and rapid endotracheal intubation. In surgery, skeletal muscle relaxation reduces the needs for high concentrations of anaesthetic drugs. Anaesthestic drugs at high concentrations abolish most reflex skeletal muscle movements but carry a higher risk. Available neuromuscular blockers all produce skeletal muscle paralysis, but none are ideal. Ease of reversal of blockade with anticholinesterases is important, as is a lack of interactions with other drugs at the level of drug metabolism, and elsewhere. Other than in anaesthesia, such blockers are also used selectively to alleviate certain spastic skeletal muscle conditions.

17 17 Succinylcholine (suxamethonium) - a special historical case The depolarizing n.m. blocker succinylcholine is normally very short acting via rapid hydrolysis due to plasma esterases. A rare genetic absence of cholinesterases results in a long duration of action (3-7 hours versus 3-7 minutes). Given i.v. it produces very rapid and normally short-lived relaxation hence its use for endotracheal intubation. However, it can cause skeletal muscle spasm and muscle fasciculation - especially in extra ocular muscles with subsequent muscle aches. Serum potassium rises as a result of muscle damage due to muscle fasciculation. There is no real antidote for this drug. Non depolarizing neuromuscular blockers (competitive antagonists at NAChR) D-tubocurarine, was isolated from curare, a South American dart poison. Classic experiments (C. Bernard, 1800s) identified the neuromuscular junction as its site of action. However, decades passed before it was used in medicine. Since then newer curare drugs have been invented since d- tubocurarine s pharmacology is not ideal ; it releases histamine, and can lower blood pressure (at high concentrations it blocks nicotinic receptors on ganglia). Synthetic curare-like drugs come from two chemical classes: 1) steroid derivatives; 2) isoquinoline derivatives (e.g. d-tubocurarine). Drugs in both classes have a nitrogen that is charged at physiological ph; therefore they are poorly orally absorbed (curare was used as an arrow poison to obtain monkeys for food). Steroid derivatives with the ending curonium include pancuronium, vecuronium, and rocuronium. They are similar in their clinical actions and in pharmacology, except rocuronium and vecuronium which are shorter acting. Pancuronium, the oldest, has more side effects. Isoquinolone derivatives: with the ending curium include atacurium, mivacurium and doxacurium. Mivacurium is short acting and broken down by plasma esterases. Uses: intravenous to produce neuromuscular blockade to aid surgery rarely in any other conditions. Reversal of neuromuscular blockade Neuromuscular blockade due to competitive neuromuscular blockers can be reversed (within limits) by intravenous anticholinesterase drugs, such as neostigmine and pyridostigmine. These block cholinesterase and thereby raise ACh concentrations in the junctional cleft. The elevated ACh activates the NAChRs that are not occupied by the neuromuscular blocking drug. However, neuromuscular blockade in vivo is a dynamic situation and direct competition for a NAChR receptor site between the elevated ACh and competitive blockers is not so important. Anticholinesterase drugs will elevate acetylcholine levels throughout the body to a sufficient level as to produce muscarinic effects and an atropinic drug will block such unwanted muscarinic effects. Excessive neuromuscular blockade with the competitive non-depolarizing drugs cannot easily be reversed by an anticholinesterase since the junctional cleft concentration of ACh cannot be raised sufficiently by AChE inhibitors to functionally compete with the blocker and thereby activate NACHRs. Sugammadex is a drug that complexes with steroidal neuromuscular blockers thereby inactivating them.

18 18 Assessment of neuromuscular blockade: is with peripheral nerve stimulation while recording the resulting muscle contractions using trains of 4 stimuli, double pulses, or post tetanic stimulation. Other Drugs Acting on Skeletal Muscle Activity Spasmolytic drugs act on spinal cord reflexes to reduce inappropriate skeletal muscle activity. They include diazepam and baclofen. Izanidine is an imidazoline derivative that relaxes by way of central (CNS) mechanisms. Dantrolene which acts on excitation-contraction mechanisms in skeletal muscle is somewhat selective in reducing inappropriate skeletal muscle activity. It is used in the inherited condition, malignant hyperthermia, a condition triggered by some general anaesthetics and succinylcholine where poor control of intracellular calcium concentrations brings about excessive activation of skeletal muscle activity and metabolism. Botulinum toxin (a protein) directly depletes cholinergic nerves of ACh. Once ACh is depleted with botulinus toxin it takes months for the nerve ending to recover. Botulinus toxin (botox) given locally can produce local skeletal muscle blockade via such depletion and therefore is used therapeutically to block inappropriate muscle contractions in various motor nerve and muscle diseases (e.g. strabismus). Non-therapeutic use of botulinus toxin ( botox ) is cosmetic, used to remove the lines of worry, age, too much sun, booze and tobacco. Such lines are due to underlying contraction of fine facial skeletal muscles. Botox provides that smoother] look, but of course repeated treatments are necessary (see above). More importantly botulinus toxin is also used therapeutically to treat muscle spasms, upper motor neuron syndrome, excessive sweating (hyperhydrosis due to parasympathetic cholinergic nerves) and cervical dystonia. Botulinus protein has two forms, A & B, both produced naturally by an anaerobic bacterium (Clostridium botulinum) and can occur in poorly canned and preserved foods. It is absorbed orally and is responsible for occasional poisoning. In such potentially lethal poisoning the patient has to be artificially ventilated until the skeletal motor nerve endings regain their function. MjaW2014