1. Cholinergic transmission Lecture 8

2. Learning outcome: Muscarinic and nicotinic actions of acetylcholine Acetylcholine receptors Physiology of cholinergic transmission Electrical events in transmission at cholinergic synapses Effects of drugs on cholinergic transmission Drugs affecting autonomic ganglia Neuromuscular-blocking drugs Drugs acting presynaptically Drugs that enhance cholinergic transmission

3. Muscarinic and nicotinic actions of acetylcholine The discovery of the pharmacological action of acetylcholine arose from work on adrenal glands. Adrenal extracts were known to produce a rise in blood pressure owing to their content of adrenaline. Reid hunt found that after adrenaline had been removed from such extracts, they produce a fall in blood pressure instead of a rise. He attributed the fall to their content of choline, but later concluded that more potent derivative of choline must be responsible. With taveau he tested a number of choline derivatives and discovered that acetylcholine was some 100,000 times more active than choline in lowering the rabbits blood pressure.

4. In the study of the pharmacological actions of acetylcholine carried out in 1914,Dale distinguished two types of activity, which he designated as muscarinic and nicotinic The muscarinic actions of acetylcholine are those that can be reproduced by injection of muscarine,the active principle of the poisonous mushroom amanita muscuaria, and can be abolished by small doses of atropine (the poisonous constituents of deadly nightshade) On the whole, muscarinic actions corresponds to those of parasympathetic stimulation. After the muscarinic effects have been blocked by atropine, larger doses of acetylcholine produce another set of effect, closely similar to those of nicotine. They include:

5.  stimulation of all autonomic ganglia  stimulation of voluntary muscle  secretion of adrenaline from the adrenal medulla Acetylcholine receptors Nicotinic Receptor fall into three main classes, the muscle, ganglionic and CNS types; they are typically of ligand - gated ion channels Muscle receptors are confined to the skeletal neuromuscular junction; ganglionic receptors are responsible for transmission at the sympathetic and parasympathetic ganglia; CNS – type receptors are widespread in the brain.

7. Muscarinic Receptors: Gene cloning has revealed five distinct types of muscurinic receptor, but only four have been distinguished functionally and pharmacologically. M 1 – receptors (‘neural’) are found mainly on CNS and peripheral neurons and gastric parietal cells. They mediate excitatory effects, for example the slow muscarinic excitation mediated by acetylcholine in sympathetic ganglia and central neurons. This excitation is produced by decrease in K+ conductance, which causes membrane depolarization. Deficiency of this kind of acetylcholine – mediated effect in the brain is possibly associated with dementia. M 1 – receptors are also involved in the increase of gastric acid secretion following vagal stimulation

8. M 2 – receptors (‘cardiac’) occur in the heart, and also on the presynaptic terminals of peripheral and central neurons. They exert inhibitory effects, mainly by increasing K + conductance and by inhibiting calcium channels. M 2 – receptor activation is responsible for vagal inhibition of the heart, as well as presynaptic inhibition in the CNS and periphery. M 3 – receptor ( ‘glandular/smooth muscle’) produce mainly excitatory effects, i.e. stimulation of glandular secretions ( salivary, bronchial, sweat, etc) and contraction of visceral smooth muscle.

9. M 3 – receptors also mediate relaxation (mainly vascular) of smooth muscle, which results from the release of nitric oxide from neighboring endothelial cells. M 1 -, M 2 -, and M 3 - receptors occur also in specific locations in CNS. M 4 – and M 5 - receptors are largely confined to the CNS, and their functional role is not well understood. Muscarinic receptors all belong to the family of G- protein coupled receptors. The odd- numbered members of the group (M 1,M 3, M 5 ) act through the inositol phosphate pathway, while the even – numbered receptors (M 2,M 4 ) act by inhibiting adenylate cyclase and thus reducing intracellular cAMP

11. Acetylcholine synthesis and release Acetylcholine is synthesized within the nerve terminal from choline, which is taken up into the nerve terminal by a specific carrier, similar to the that which operates for many transmitters. Free choline within the nerve terminal is acetylated by a cytosolic enzyme, choline acetyltransferase, which transfers the acetyl group from acetyl- CoA. The rate – limiting process in acetylcholine synthesis appears to be choline transport, the activity of which is regulated according to the rate at which acetylcholine is being released.

12. Cholinesterase is present in the present in the presynaptic nerve terminals and acetylcholine is continually being hydrolyzed and resynthesised. Most of the acetylcholine synthesized, however, is packaged into synaptic vesicles, in which its concentration is very high, from where release occurs by exocytosis, triggered by Ca 2+ entry into the nerve terminal Accumulation of acetylcholine is coupled to the large electrochemical gradient for H + that exists between intracellular organelles and cytosol; it is blocked selectively by experimental drug Vesamicol

14. After the synthesis of Acetylcholine in the nerve terminals they are being released to postsynaptic cleft to interact with the receptors. Some of it succumbs on the way to hydrolysis by acetylcholinesterase (AChE), an enzyme which is bound to the basement membrane that lies between the pre- and postsynaptic membrane. Presynaptic Modulation :

15. Drugs affecting muscarinic receptors Muscarinic agonist Muscarinic agonist, as a group, are often referred to as parasympathomimetics because the main effects that they produce in the whole animal resemble those of parasympathetic stimulation. effects of muscarinic agonist  Cardiovascular effects  Smooth muscle  Sweating, lacrimation, salivation and bronchial secretion

16.  Effects on the eye  Central effects. Muscarinic antagonist Muscarinic receptor antagonists are often referred to as parasympatholytic because they selectively block the effects of parasympathetic nerve activity. All of them are competitively antagonists.

17. effects of muscarinic antagonists (atropine)  Inhibition of secretions  Effects on heart rate  Effects on the eye  Effects GI tract  Effects on other smooth muscle  Effects on the CNS

18. Neuromuscular – blocking drugs Drugs can block neuromuscular transmission either by acting presynaptically, to inhibit acetylcholine synthesis or release, or by acting postsynaptically. Clinically, neuromuscular block is used only as an adjunct to anesthesia, it is not for therapeutic intervention. The drugs that are used all work by interfering with the postsynaptic action of acetylcholine. They fall into two categories:

19. Non- depolarizing blocking agents (the majority), which act by blocking acetylcholine receptors (and,in some cases, also by blocking ion channels.) Depolarizing blocking agents causes paralysis by blocking neuromuscular transmission, rather than by abolishing nerve conduction or muscle contractility. E.g. of non-depolarizing drugs are tubocurarine, pancuronium, vecuronium, atracurium and gallamine. Mechanism of action Non-depolarizing agents all act as competitive antagonists at the acetylcholine receptors of the endplate and largely accounts for their action.

20. The drugs also block ion channels, some non – blocking agents also appear to block presynaptic autoreceptors and thus inhibit the release of acetylcholine during repetitive stimulation of the motor nerve. Effects of non-depolarizing neuromuscular-blocking agents are mainly a result of motor paralysis, though some of the drugs also produce clinically significant autonomic effects. Unwanted effects: the main side-effects of tubocurarine is a fall in arterial pressure, chiefly a result of ganglion block and release of histamine from mast cells which also give rise to bronchospasm in sensitive individuals.

21. Depolarizing blocking agents, which are agonists at acetylcholine receptors. They cause paralysis without appreciable ganglion – blocking activity. Drugs acting presynaptically. Drugs that inhibit acetylcholine synthesis The rate- limiting process in acetylcholine synthesis appear to be the transport of choline into the nerve terminals, and drugs (e.g. hemicholinium and triethylcholine) that inhibit acetylcholine synthesis do so by blocking this step. Hemicholine acts as a competitive inhibitor of choline uptake but is not appreciably taken up itself.

22. Triethylcholine, as well as inhibiting choline uptake, is itself transported and acetylated within the terminals, forming acetyltriethylcholine. This is stored in place of acetylcholine and released as false transmitter, but it has no depolarising effect on the postsynaptic membrane. Drugs that inhibit acetylcholine release Acetylcholine release by nerve impulse involves the entry of Ca 2+ into the nerve terminal; the increase in intracellular Ca 2+ concentrations stimulates exocytosis and increases the rate of quantal release.

23. Agents that inhibit Ca 2+ entry include Mg 2+ and various aminoglycoside antibiotics (e.g. streptomycin and neomycin) Botulinum toxin and β- bungarotoxin,act specifically to inhibit acetylcholine release. Peptidase and phospholipase activity. Drugs that enhances cholinergic transmission. Drugs that enhance cholinergic transmission act either by inhibiting cholinesterase or by increasing acetylcholine release. Distribution and function of cholinesterase. Two major distinct types of cholinesterase namely acetylcholinesterase (AChE) and butyrylcholinesterase (BChE)

24. AChE and BChE are closely related in molecular structure but differing in their distribution, substrate specificity and functions. Both consists of globular catalytic subunits which constitute the soluble form found in plasma (BChE) and cerebrospinal fluid (AChE). AChE is bound to basement membrane in the synaptic cleft at cholinergic synapses, where it function to hydrolyse the released transmitter. The soluble form of AChE is also present in cholinergic nerve terminals, where it seems to have a role in regulating the free Ach concentration from which it may be secreted.

25. BChE has wide spread distribution, being found in the tissue such as liver, skin, brain and GI smooth muscle, as well as in soluble form in the plasma. It hydrolysis butyrylcholine more rapidly than acetylcholine as well as other esters such as procaine,suxamethonium and propanidid.