Cholinergic Neurons: Neurotransmission & Synthesis

• Drugs affecting the autonomic nervous system (ANS) are divided into two groups based on the type of neuron involved.
• Acetylcholine (ACh) is used as a neurotransmitter by preganglionic fibers terminating in the adrenal medulla.
• ACh is also used by autonomic ganglia (both parasympathetic and sympathetic) and postganglionic fibers of the parasympathetic division.
• The postganglionic sympathetic division of sweat glands uses acetylcholine.
• Cholinergic neurons innervate muscles of the somatic system and play a significant role in the central nervous system (CNS).

Neurotransmission at Cholinergic Neurons

• Neurotransmission in cholinergic neurons involves six sequential steps: synthesis, storage, release, receptor binding, degradation, and recycling.

Synthesis of Acetylcholine

• Choline is transported into the cholinergic neuron via an energy-dependent carrier system that cotransports sodium.
• This carrier system can be inhibited by hemicholinium.
• Choline, carrying a permanent positive charge due to its quaternary nitrogen, cannot diffuse through the membrane.
• The uptake of choline is the rate-limiting step in ACh synthesis.
• Choline acetyltransferase catalyzes the reaction of choline with acetyl coenzyme A (CoA) to form ACh in the cytosol.

Storage of Acetylcholine in Vesicles

• ACh is packaged into presynaptic vesicles through active transport.
• Mature vesicles contain ACh, adenosine triphosphate (ATP), and proteoglycan.
• Co-transmission, where synaptic vesicles contain a primary neurotransmitter (ACh) and a co-transmitter (ATP), is common in autonomic neurons.
• ATP can increase or decrease the effect of the primary neurotransmitter.

Release of Acetylcholine

• An action potential triggers voltage-sensitive sodium channels, leading to the opening of voltage-sensitive calcium channels on the presynaptic membrane.
• The increase in intracellular calcium promotes the fusion of synaptic vesicles with the cell membrane, releasing their contents into the synaptic space.
• Botulinum toxin can block this release.
• Black widow spider venom causes all the ACh stored in synaptic vesicles to be emptied into the synaptic gap.

Binding to the Receptor

• ACh diffuses across the synaptic space and binds to postsynaptic receptors on the target cell.
• ACh can bind to presynaptic receptors on the neuron that released it or to other targeted presynaptic receptors.
• Postsynaptic cholinergic receptors are divided into two classes: muscarinic and nicotinic.
• Receptor binding initiates a biologic response within the cell, like a nerve impulse or enzyme activation, mediated by second messenger molecules.

Degradation of Acetylcholine

• Acetylcholinesterase (AChE) rapidly cleaves ACh into choline and acetate in the synaptic cleft, terminating the signal at the postjunctional effector site.

Recycling of Choline

• Choline is recaptured by a sodium-coupled, high-affinity uptake system that transports it back into the neuron.
• The recaptured choline can then be acetylated into ACh.

Cholinergic Receptors (Cholinoceptors)

• Cholinoceptors are divided into two families: muscarinic and nicotinic receptors.
• These families are distinguished by their differing affinities for agents that mimic the action of ACh (cholinomimetic agents).

Muscarinic Receptors

• Muscarinic receptors are G protein-coupled receptors (metabotropic receptors).
• They bind ACh and muscarine, an alkaloid found in poisonous mushrooms.
• Muscarinic receptors have a weak affinity for nicotine.
• There are five subclasses of muscarinic receptors (M1-M5), but only M1, M2, and M3 have been functionally characterized.

Locations of Muscarinic Receptors

• Muscarinic receptors are found on autonomic effector organs like the heart, smooth muscle, brain, and exocrine glands.
• All five subtypes are found on neurons.
• M1 receptors are on gastric parietal cells.
• M2 receptors are on cardiac cells and smooth muscle.
• M3 receptors are on the bladder, exocrine glands, and smooth muscle.

Mechanisms of Acetylcholine Signal Transduction

• When M1 or M3 receptors are activated, they interact with a G protein (Gq), which activates phospholipase C.
• Phospholipase C activation leads to the production of inositol-1,4,5-trisphosphate (IP3) and diacylglycerol (DAG), both second messengers.
• IP3 causes an increase in intracellular calcium.
• Calcium stimulates or inhibits enzymes or causes hyperpolarization, secretion, or contraction.
• Diacylglycerol activates protein kinase C, which phosphorylates numerous proteins within the cell.
• Activation of the M2 subtype on cardiac muscle stimulates a G protein (Gi) that inhibits adenylyl cyclase and increases K+ conductance.
• This results in a decrease in heart rate and force of contraction.

Muscarinic Agonists

• Pilocarpine is a nonselective muscarinic agonist used to treat xerostomia and glaucoma.
• Efforts are underway to develop muscarinic agonists and antagonists directed against specific receptor subtypes.
• M1 receptor agonists are being investigated for Alzheimer's disease treatment.
• M3 receptor antagonists are for chronic obstructive pulmonary disease treatment.

Nicotinic Receptors

• These receptors bind ACh and nicotine but have a weak affinity for muscarine.
• The nicotinic receptor is composed of five subunits and functions as a ligand-gated ion channel.
• The binding of two ACh molecules allows the entry of sodium ions, resulting in the depolarization of the effector cell.
• Low concentrations of nicotine stimulate the receptor; high concentrations block it.
• Nicotinic receptors are in the CNS, adrenal medulla, autonomic ganglia, and the neuromuscular junction (NMJ) in skeletal muscles.
• NMJ receptors are designated NM, while others are NN.
• Ganglionic receptors are selectively blocked by mecamylamine, while NMJ receptors are blocked by atracurium.

Direct-Acting Cholinergic Agonists

• Cholinergic agonists mimic ACh effects by directly binding to cholinoceptors (muscarinic or nicotinic).
• These agents include endogenous choline esters (ACh and synthetic esters like carbachol and bethanechol) and naturally occurring alkaloids (nicotine and pilocarpine).
• Direct-acting cholinergic drugs have a longer duration of action than ACh.
• Pilocarpine and bethanechol preferentially bind to muscarinic receptors and are called muscarinic agents.
• Direct-acting agonists show little specificity, limiting their clinical usefulness.

Acetylcholine

• Acetylcholine is a quaternary ammonium compound that cannot penetrate membranes.
• It is the neurotransmitter of parasympathetic, somatic nerves, and autonomic ganglia.
• It has limited therapeutic importance due to its multiplicity of actions and rapid inactivation by cholinesterases.
• ACh has both muscarinic and nicotinic activity, including decreasing heart rate and cardiac output, which mimics vagal stimulation.
• Intravenous ACh causes a brief decrease in cardiac rate (negative chronotropy) and stroke volume.

Blood Pressure Effects

• Injection of ACh causes vasodilation and lowering of blood pressure through an indirect mechanism.
• ACh activates M3 receptors on endothelial cells, leading to nitric oxide production from arginine.
• Nitric oxide diffuses to vascular smooth muscle cells, stimulating protein kinase G production and smooth muscle relaxation via phosphodiesterase-3 inhibition.
• Vascular cholinergic receptors have no known function in the absence of administered cholinergic agents.
• Atropine blocks these muscarinic receptors, preventing ACh from producing vasodilation.

Other Actions

• In the GI tract, acetylcholine increases salivary and gastric acid secretion and stimulates intestinal secretions and motility.
• It enhances bronchiolar secretions and causes bronchoconstriction.
• Methacholine, a direct-acting cholinergic agonist, is used in asthma diagnosis due to its bronchoconstricting properties.
• In the genitourinary system, ACh increases the tone of the detrusor muscle, causing urination.
• In the eye, ACh stimulates ciliary muscle contraction for near vision and constricts the pupillae sphincter muscle (miosis).
• An ACh 1% solution is used during ophthalmic surgery to produce miosis.

Bethanechol

• Bethanechol is an unsubstituted carbamoyl ester structurally related to ACh.
• It is not hydrolyzed by AChE due to esterification, but it is inactivated by other esterases.
• Bethanechol lacks nicotinic actions but has strong muscarinic activity.
• Its major actions are on the smooth musculature of the bladder and GI tract, with a 1-hour duration of action.
• Bethanechol directly stimulates muscarinic receptors, increasing intestinal motility and stimulating the detrusor muscle.
• The trigone and sphincter muscles relax, producing urination.
• It treats the atonic bladder, especially with post-partum or post-operative, non-obstructive urinary retention.

Adverse Effects of Bethanechol

• Bethanechol causes generalized cholinergic stimulation including sweating, salivation, flushing, decreased blood pressure, nausea, abdominal pain, diarrhea, and bronchospasm.
• Atropine sulfate may be administered to overcome severe cardiovascular or bronchoconstrictor responses.

Carbachol

• Carbachol has both muscarinic and nicotinic actions.
• It is an ester of carbamic acid and a poor substrate for AChE.
• It is biotransformed by other esterases at a slow rate.
• It has profound effects on cardiovascular and GI systems due to its ganglion-stimulating activity.
• It can cause epinephrine release from the adrenal medulla via nicotinic action.
• Locally instilled into the eye to mimic ACh effects, causing miosis and a spasm of accommodation.
• This causes the vision to become fixed at some distance.
• It's high potency and receptor nonselectivity limits therapeutic use except in the eye.
• It is a miotic agent to treat glaucoma by causing pupillary contraction and decreases intraocular pressure.
• Side effects are limited because it lacks systemic penetration.

Pilocarpine

• A tertiary amine alkaloid, stable to hydrolysis by AChE.
• It is less potent than ACh but is uncharged and can penetrate the CNS.
• It exhibits muscarinic activity and is primarily used in ophthalmology.
• Pilocarpine produces rapid miosis, contraction of the ciliary muscle, and spasm of accommodation.
• One of the most potent stimulators of secretions(sweat, tears, and saliva).
• Its application is limited because of lack of selectivity.
• Used to treat glaucoma and is the drug of choice for emergency lowering of intraocular pressure.
• It opens the Schlemm canal which causes an immediate drop in intraocular pressure by inceasing drainage; it starts in a few minutes, lasts 4 to 8 hours, and can be repeated.
• Topical carbonic anhydrase inhibitors and β-adrenergic blockers effectively treat glaucoma.
• The miotic action reverses mydriasis due to atropine.

The Drug Pilocarpine Treats

• This drug may promote salivation in xerostomia patients resulting from head and neck irradiation.
• It alleviates Sjogren syndrome, which is characterized by dry mouth and lack of tears.
• Pilocarpine tablets together with cevimeline, a cholinergic drug also has the drawback of being nonspecific and treats sjrogens syndrome.
• Blurred vision, night blindness, and brow ache are some of the adverse effects which mark Pilocarpine usage.
• Poisoning is categorized by the exaggeration of various parasympathetic effects, including profuse sweating (diaphoresis) and salivation.
• Effects are caused by eating mushrooms and containing muscarine, parenteral atropine is used to counteract against Pilocarpine

Indirect Acting Cholinergic Agonists

• These consist of anticholinesterase agents (Reversible), AChE is an enzyme which cleaves ACh to acetate and choline and ends it's actions from the post and presynaptically region in a the nerve terminal that is membrane-bound.
• Inhibitors of AChE help indirect provision of cholinergic action by the prevention of ACh degradation, this accumulating ACh space results in response in all the cholinergic receptors, like both muscarinic and nicotinic receptors of the ANS.
• In order to accomplish the stated goal, the following should be the mechanism and classification in reversible AChE inhibitors.

Edrophonium

• The AChE prototype in short acting.
• Edrophonium attaches reversibly to the AChE's center therefore preventing hydrolysis of ACh and is rapidly absorbed with little action time because of renal elimination.
• This quarternary amine helps to limite the effects towards the periphery
• It is utilized in diagnosing mysthenia gravis and an autoimmune deficiency.
• A quick increase in muscle strength comes about becaus of Edrophonium which is administered to patients with mysthenia gravis.

Physostigmine

• Found from plants and a substrate for AChE and forms a relatively stable intermediate
• Wide range of effects and stimulates muscarinic and nicotinic sides of ANS but also nicotinic receptors. Contraction of GI smooth muscles, miosis and hypotension can be the result of Muscarinic. Skeletal muscle twitches, fasciculations and paralysis (at high doses) are the effects of Nicotinic stimulation. action is about takes about 30 minutes for the stimualtion, Physostigmine can stimulate the cholinergic sites.

Effects of Physostigmine

• Treats atropine to overdoses the anticholinergic effects.
• High doses effect the occurence of bradycardia.
• inhibition of AChE may cause accumulation in skeletal muscle and lead to paralysis.

Neostigmine

• it inhibits AChE
• It is is a Qauternary nitrogen, and is not absorp from the GI.
• stimulate greater effect on skeletal muscle to stimulate the contractility.
• Has limited action time with 30 minute usage.
• It stimulated bladder and GI tract as an antidote and manages symptoms.
• Such effcts inclue decreases in blood and can cause bronchospasm.

Pyridostigmine

• Treats the chronic effects of myasthenia gravis with inermediate action .
• Adverse effects are similar to those made.

Tacrine, donepezil, rivastigmine, galantamine

• Treats Alzeihmer symtoms but doesn't stop it's track to cognitive declination.
• Gl distress causes the adverse effects.

Synthetic organophosphate

• The capacity to covalently bond to achieve the long lasting ACh level and toxicity caused by the military
• Related to parathion and malathion for pesticide usages.

Echothiophate Mechanism

• action is to covalently bind to phosphate group.
• Requires synthesis of new molecules which produces in increase in ACH
• The loss may cause new actions.
• Causes generizal cholinergic stimulation and paralysis of motor function that leads to breathing difficulties and the increased pressure causes glaucoma.