Naglaa F. El- Orabi , Ph D, M Sc, B SC Department of Pharmacology & Toxicology,

1. PHL 315Pharmacology I Part II Naglaa F. El-Orabi, Ph D, M Sc, B SC Department of Pharmacology & Toxicology, College of Pharmacy, King Saud University, Riyadh, KSA

2. Cholinergic Transmission

3. References “ Rang & Dale’s Pharmacology” 6th ed., Rang HP, Dale MM, Ritter JM, Moore PK, eds. Elsevier Science, 2007. Chapter 10, page 144

4. Cholinergic transmission • An important traditional classification of autonomic nerves is based on the primary neurotransmitter molecules released from their terminals into adrenergic and cholinergic. • A large number of peripheral ANS fibers synthesize and release Ach; they are cholinergic fibers. • These include allpreganglionic efferent autonomic fibers(both sympathetic and parasympathetic). • In addition, most parasympatheticand somesympathetic(to sweat glands) postganglionic fibers are cholinergic. • Somatic (non-autonomic) motor fibers (NMJs) to skeletal muscle (motor end plates). • Ach is also very important central NT.

5. Parasympathetic nervous syste (PSNS)Anatomy • PSNS originates from cranio-sacral parts of spinal cord. • Cranial outflow originate from cranial nerve nuclei in the brain stem. Preganglionic fibers run via: • Oculomotor nerve (III) • Facial nerve (VII) • Glossopharyngeal nerve (IX) • Vagus nerve (X) • These nerve fibers innervate organs of the head & neck (eye, nasal mucosa, salivary glands,…), thorax & upper abdomen ( heart, respiratory system, esophagus, stomach, pancreas, liver, small intestine and upper half of the large intestine).

6. PSNS anatomy (cont.) • Sacral outflow originate from visceral motor region of spinal cord (S2-S4). Preganglionic fibers run via pelvic nerves. • These nerve fibers innervate organs of the pelvis and lower abdomen (lower half of large intestine, the rectum, urinary and reproductive systems) • PSNS does not innervate most of blood vessels, sweat glands, adrenal medulla and arrectorpili muscles.

8. PSNS anatomy (cont.) • Parasympathetic pathway • Brain areas (hypothalamus & brain stem) • Cranial or sacral outflow • Relatively long pre-ganglionic neurons to terminal or intramural ganglia (in walls of viscera, close to effector organs) • Short post-ganglionic neurons.

9. PSNS Neurotransmitters • All Parasympathetic nerve fiber (both preganglionic postganglionic) are cholinergic, that is, they work by releasing AChneurotransmitter. • Acetylcholine is synthesized in the cytoplasm of neuronal cells from acetyl-CoAand cholinethrough the catalytic action of the enzyme cholineacetyltransferase (ChAT). • Acetylcholine is then transported into the storage vesicle. Release of transmitter occurs when voltage-sensitive calcium channels in the terminal membrane are opened, allowing an influx of Ca2+. The resulting increase in intracellular Ca2+ causes fusion of vesicles with the surface membrane and exocytotic expulsion of acetylcholine into the junctional cleft and interact with postsynaptic receptors.

10. PSNS neurotransmitters (cont.) • Acetylcholine's action is terminated by metabolic degradation by the enzyme acetylcholinesterase (AChE). AChE splits ACh into choline and acetate, neither of which has significant transmitter effect, and thereby terminates the action of the transmitter . Most cholinergic synapses are richly supplied with AChEs; the half-life of ACh in the synapse is very short (1-2 milliseconds). • AChE is also found in other tissues, eg, RBCs. Another cholinesterase with a lower specificity for ACh, butyrylcholinesterase (pseudocholinesterase) , is found in blood plasma, liver, glia, and many other tissues. Little or no acetylcholine escapes into the circulation. Any acetylcholine that reaches the circulation is immediately inactivated by plasma esterases.

11. ChAT ACETYLCHOLINE VAT Na+- CHT

12. Cholinergic receptors • Cholinergic receptors have two major families; nicotinic (nAChR) and muscarinic (mAChR) receptors. • Ach acts as specific agonist for both receptor subclass • In contrast, because of their unique configurations, • Nicotine and Muscarine are selective for the cholinergic receptor subtypes whose structure complements their own.

13. (a) Muscarinic receptors • mAChRs are G-protein-coupled receptors causing: • activation of PLC (hence ↑IP3, DAG as 2nd messengers) • inhibition of adenylylcyclase (↓cAMP) • activation or inhibition of ion (K+ & Ca2+ ) channels . • mAChRs mediate ACh effects at postganglionic parasympathetic synapses (mainly heart, smooth muscle, glands), and contribute to ganglionic excitation. They occur in many parts of the CNS. • Five main types of mAChR occur (M1-5). • All mAChRs are activated by acetylcholine and blocked by atropine. There are also subtype-selective agonists and antagonists.

14. Muscarinic receptors (cont.)

15. (b) Nicotinic receptors • nAChRs are directly coupled to cation channels (like Na+/K+ channels), and mediate fast excitatory synaptic transmission at the neuromuscular junction (Skeletal muscles), autonomic ganglia, and various sites in CNS.) • Muscle (Nm) and neuronal (Nn) nAChRs differ in their molecular structure and pharmacology. • mAChRs and nAChRs occur presynaptically as well as postsynaptically, and function to regulate transmitter release.

16. Nicotinic receptors (cont.)

17. (α1)2β1δγ

18. Physiological actions of muscarinic stimulation

19. Physiological actions of muscarinic stimulation (cont.)

20. Modifying Autonomic Nervous System Function • Parasympathomimetics = Cholinoceptor stimulants: bind to acetylcholine receptors (Muscarinic & Nicotinic) and stimulates them or enhance cholinergic transmission by other mechanism: • Muscarinic agonists (stimulants) • Anticholinesterases and other drugs that enhance cholinergic transmission. • Ganglion-stimulating drugs • Parasympatholytics (cholinergic antagonists – anticholinergic drugs): bind to acetylcholine receptors and reduce the effects of parasympathetic stimulation by preventing endogenous acetylcholine from binding to them: • Muscarinic antagonists • ganglion-blocking drugs • Neuromuscular-blocking drugs

21. ChAT ChAT ACETYLCHOLINE ACETYLCHOLINE VAT VAT Na+- CHT Na+- CHT Muscarinic antagonists

22. Parasympathomimetics(Muscarinic receptor stimulants)

23. Direct cholinoceptor stimulants • Choline esters • e.g. Acetylcholine, Methacholine,Carbachol and Bethanechol • Colinomimetic alkaloids • e.g., Muscarine, Oxotremorine and Pilocarpine • Many of these muscarinic agonists are used as experemintal tools. • Use of muscarinic receptor agonists, is contraindicated in patients with asthma, coronary insufficiency and peptic ulcers

24. Direct cholinoceptor stimulants (cont.)1- Bethanechol(Urecholine®) • Selectively stimulates muscarinic receptors (with further selectivity for M3 receptors) • Unlike acetylcholine, bethanechol is not hydrolyzed by cholinesterase and will therefore have a long duration of action • Clinicaluses: • To assist bladder emptying in non-obstructive urinary retentionresulting from general anesthetic or diabetic neuropathy of the bladder • To treat gastroparesis(delayed gastric emptying), because it stimulates GI motility and secretion • To stimulate salivary gland secretion in patients with xerostomia(dry mouth, nasal passages, and throat)

25. Bethanechol (cont.) • Side Effects associated with bethanechol therapy: • Abdominal cramps or discomfort • Nausea and diarrhea • Excessive salivation • Hypotension and bradycardia • Urinary urgency • Bronchial constriction and asthmatic attacks

26. Direct cholinoceptor stimulants (cont.)1- Pilocarpine (Salagen®) • Indications: It is more commonly used than bethanechol to induce salivation, and also for various purposes in ophthalmology. • Treatment of primary or acute glaucoma and also to lower IOP prior to surgery for acute glaucoma by local instillation in the form of eye drops. • 2. Treatment of symptoms of dry mouth from salivary gland hypofunction caused by radiotherapy for cancer of the head and neck (xerostomia )

27. Pilocarpine(cont.) • Side Effects associated with pilocarpine therapy: • Most of them are related to its non-selective action as a muscarinic receptor agonist • Excessive sweating • Excessive salivation • Bronchospasm and increased bronchial mucus secretion • Bradycardia, hypotension • Nausea and diarrhea • It may result in miosis when used chronically as an eye drop

28. Muscarinic effects on the eye Normal: Ciliary Muscle Relaxed Suspensory Ligaments Under Tension Lens is Flattened Focus on Distant Objects Accommodation: Ciliary Muscle Contracts Reduced Tension on Suspensory Ligaments Lens becomes Round Focus on Near Objects

29. Muscarinic effects on the eye (cont.) • The smooth muscles of the iris: • The sphincter muscle is innervated by M3 receptors. Its contraction under the influence of muscarinic agonist (e.g. pilocarpine) results in miosis, and its blockade by muscarinic antagonist (e.g. atropine) results in mydriasis. • On the other hand, the radial muscle is innervated by α-1 receptor. Its contraction by an agonist results in mydriasis and its blockade results in miosis. Sphincter muscle Radial muscle

30. Glaucoma • Glaucoma is an eye disorder in which the optic nerve suffers damage, permanently impacting vision in the affected eye(s) and progressing to complete blindness if untreated. • It is often associated with increased pressure of the aqueous humourin the eye (Intraocular pressure “IOP”). • The aqueous humour is a thick watery substance filling the space between the lens and the cornea. It is rich in amino acid, glucose, antioxidants , and immunoglobulins. Its main role to maintains IOP and keep the eyes slightly distended. In addition to providing nutrition and protection for the occular tissues • Aqueous humour is secreted into the posterior chamber by the ciliary body epithelium, it flows in through the pupil to the anterior chamber, and then to drain out of the eye via Schlemm's canal into the veins of the orbit.

31. Glaucoma (cont.)

32. Parasympathomimetics(Muscarinic receptor stimulants)

33. Indirect cholinoceptor stimulants • Drugs that enhance cholinergic transmission act either by inhibiting cholinesterase or by increasing ACh release. • Example of drugs that enhance cholinergic transmission via increase of Ach release: - Aminopyridines, which block K+ channels and thus prolong the action potential in the presynaptic nerve terminal. - This drug are not selective for cholinergic nerves but increase the evoked release of many different transmitters, so have too many unwanted effects to be useful in treating neuromuscular disorders.

34. Cholinesterase inhibitors • Indirect-acting agents produce their primary effects by inhibiting acetylcholinesterase, which hydrolyzes acetylcholine to choline and acetic acid by forming a complex with acetylcholinesterase enzyme .By inhibiting acetylcholinesterase, the indirect-acting drugs increase the endogenous ACh concentration in synaptic clefts and neuroeffector junctions. The excess ACh, in turn, stimulates cholinergic receptors to evoke increased responses. These drugs act primarily where ACh is physiologically released and are thus amplifiers of endogenous ACh. • Some cholinesterase inhibitors also inhibit butyrylcholinesterase (pseudocholinesterase).

35. Cholinesterase inhibitors (Cont’d) • The inhibitory effect of anticholinesterases may be: • Reversible: as that produced by edrophonium, pyridostigmine, physostigmine (eserin) or neostigmine • Irreversible: such as echothiophate and malathion (orgonophosphorus compounds).

36. Cholinesterase inhibitors (Cont’d)

37. Cholinesterase inhibitors (Cont’d) Pharmacological effects:

38. Cholinesterase inhibitors (Cont’d) Therapeutic uses:

39. Cholinesterase inhibitors (Cont’d) Toxicity: Acute toxicity (cholinergic crisis): Treated by atropine and pralidoxime A- miosis, nausea, vomiting, diarrhea, salivation, sweating, lacrimationcutaneousvasodilation, and bronchial constriction and excessive urination B-These manifestations are followed by: central stimulation, which cause convulsions and may progress to coma and respiratory arrest; skeletal muscle paralysis hypertension and cardiac arrhythmias.

40. Ganglion Stimulants • Autonomic ganglia (both sympathetic and parasympathetic) and neuromuscular junctions contain nAChRs (Nn and Nm respictively). Most nAChR agonists affect both ganglionic and neuromuscular junction receptors. • Nicotine (at low conc), lobeline, Tetramethylammonium (TMA) and dimethylphenylpiperazinium (DMPP) affect ganglionic receptors preferentially.

41. Ganglion Stimulants (cont.) • Nicotine and lobeline are tertiary amines found in the leaves of tobacco and lobelia plants • Nicotine is the most commonly encountered nicotinic agonist • It has Biphasic action on ganglionicnAChR • Stimulates at low doses • Stimulates then blocks at high doses • Nicotine works in both PNS and CNS . • One of the most toxic effects is the dependence-producing psychoactive compounds overall

42. Ganglion Stimulants (cont.) Pharmacological actions of nicotine: Nicotine has a complex effect in autonomic ganglia A- At low dosages it stimulates ganglionicnAChRs ( causing marked activation and initiation of action potentials in postganglionic neurons) thus enhancing both sympathetic and parasympathetic neurotransmission The initial response therefore often resembles simultaneous discharge of both the parasympathetic and the sympathetic nervous systems - Regarding CVS, the effects of nicotine are chiefly sympathomimetics (increased HR, force of contraction and vasoconstriction)

43. Ganglion Stimulants (cont.) - In the GI, glands and urinary tracts, the effects are largely parasympathomimetic (Increased tone, motility and secretions of the GIT, increased bronchial, salivary and sweat secretions, and also urinary outflow). B- As nicotine dosages increase, nicotine possesses some antagonist effect at nicotinic receptors. Prolonged exposure results in depolarizing blockade of the ganglia (initial increase then decrease in HR, vasodilation )

44. Ganglion Stimulants (cont.) • Nicotine have the ability to cross the BBB and affects CNS (especially brainstem and cortex) • It cause initial stimulation followed by depression upon increasing the dose. • Nicotine may induce tremor, vomting, and stimulation of the respiratory center. At still higher levels, nicotine causes convulsions, which may terminate in fatal coma • Nicotine is one of the most dependence-producing drugs.

45. Ganglion Stimulants (cont.) • Most of ganglion stimulants are not used clinically, but only as experimental tools. • Only nicotine is used clinically in the form of transdermal patches, gums, SL tablets which is used as an aid to smoking cessation.

46. Anticholinergic drugs Muscarinic blockers Nicotinic blockers Ganglionic blockers Non-selective Neuromuscular blockers Selective

47. 1- Muscarinic blockers Parasympatholytics • Muscarinic receptor antagonists • Muscarinic receptor antagonists are competitive antagonists whose chemical structures usually contain ester and basic groups in the same relationship as ACh. • Two main subgroups of muscarinic antagonists arerecognized: • 1- Naturally occurring (non-selective) compounds: most of these compounds are alkaloids found in solanaceous plants like Atropa belladonnaand Daturastramonium, e.g. atropine, hyoscine (scopolamine). • 2- Synthetic (more selective) derivatives of atropine:like Ipratropium(broncheal muscles), Cyclopentolate(eye), Oxybutynin (urinary bladder) , and Pirenzepine(M1-selective).

48. 1-Atropine • Atropine is an alkaloid derived from the plant • Atropa belladonna. • It is act as non selective competitive inhibitor of Ach on muscarinic receptors both peripherally and centrally. Pharmacokinetics: • It is a tertiary ammonium, lipid-soluble compound that is readily absorbed from the GIT or conjunctival sac and cross BBB. • Metabolized in the liver, excreted in urine. • It Has short duration of action on most organs except eye

49. Atropine (cont.) Pharmacological effects: Effects on CNS • Atropine produces mainly excitatory effects on the CNS. • At low doses, this causes mild restlessness. • higher doses cause agitation and disorientation. • In atropine poisoning, marked excitement , irritability, hyperactivity and a hyperthermia. These central effects are the result of blocking mAChRs in the brain, and they are opposed by anticholinesterase drugs such as physostigmine, which is an effective antidote to atropine poisoning.

50. Atropine pharmacological effects (cont.) • Atropine also affect the extrapyramidalsystem, reducing the involuntary movement and rigidity of patients with Parkinson's disease and counteracting the extrapyramidal side effects of many antipsychotic drugs.