Cholinergic Agonists

Cholinergic Agonists

I. Overview

• Drugs affecting the autonomic nervous system (ANS) are divided into two groups based on the type of receptors involved:

  • Cholinergic drugs: Act on receptors activated by acetylcholine (ACh).

  • Adrenergic drugs: Act on receptors stimulated by norepinephrine or epinephrine.

• Cholinergic and adrenergic drugs can either stimulate or block receptors of the ANS.

• Direct-acting cholinergic agonists: Acetylcholine, Bethanechol, Carbachol, Cevimeline, Methacholine, Pilocarpine, Nicotine (NIC)

• Indirect-acting agonists: Donepezil, Edrophonium, Galantamine, Neostigmine, Physostigmine, Pyridostigmine, Rivastigmine

• Irreversible indirect-acting agonists: Echothiophate

• Acetylcholinesterase reactivators: Pralidoxime

II. The Cholinergic Neuron

• ACh is used as a neurotransmitter by:

  • Preganglionic fibers terminating in the adrenal medulla.

  • Autonomic ganglia (both parasympathetic and sympathetic).

  • Postganglionic fibers of the parasympathetic division.

  • Postganglionic sympathetic division of sweat glands.

• Cholinergic neurons also innervate muscles of the somatic system and play a role in the central nervous system (CNS).

A. Neurotransmission at Cholinergic Neurons

• Neurotransmission involves six steps:

  1. Synthesis of ACh.

  2. Storage.

  3. Release.

  4. Binding of ACh to the receptor.

  5. Degradation of ACh in the synaptic cleft.

  6. Recycling of choline.

1. Synthesis of Acetylcholine

• Choline is transported into the cholinergic neuron via an energy-dependent carrier system that cotransports sodium.

  • Choline has a quaternary nitrogen, carrying a permanent positive charge, and cannot diffuse through the membrane.

• 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.

2. Storage of Acetylcholine in Vesicles

• ACh is packaged and stored into presynaptic vesicles by an active transport process.

• Mature vesicles contain ACh, adenosine triphosphate (ATP) (a cotransmitter), and proteoglycan.

• Cotransmission from autonomic neurons is common, with synaptic vesicles containing both a primary neurotransmitter (ACh) and a cotransmitter (ATP).

3. Release of Acetylcholine

• When an action potential arrives at a nerve ending, voltage-sensitive calcium channels open, increasing intracellular calcium concentration.

• Elevated calcium levels promote fusion of synaptic vesicles with the presynaptic membrane, releasing vesicular contents into the synaptic space.

• Botulinum toxin blocks this release.

• Black widow spider venom causes all ACh stored in synaptic vesicles to empty into the synaptic gap.

4. Binding To the Receptor

• ACh diffuses across the synaptic space and binds to:

  • Postsynaptic receptors on the target cell.

  • Presynaptic receptors on the membrane of the neuron that released ACh.

  • Other targeted presynaptic receptors.

• Postsynaptic cholinergic receptors are divided into two classes: muscarinic and nicotinic.

• Binding to a receptor leads to a biologic response, such as:

  • Initiation of a nerve impulse in a postganglionic fiber.

  • Activation of specific enzymes in effector cells, mediated by secondary messengers.

5. Degradation of Acetylcholine

• The signal at the postjunctional effector site is rapidly terminated by acetylcholinesterase (AChE).

• AChE cleaves ACh to choline and acetate in the synaptic cleft.

6. Recycling of Choline

• Choline is recaptured by a sodium-coupled, high-affinity uptake system that transports it back into the neuron.

• There, it is acetylated into ACh.

III. Cholinergic Receptors (Cholinoceptors)

• Two families of cholinoceptors:

  • Muscarinic receptors.

  • Nicotinic receptors.

• Distinguished by different affinities for cholinomimetic agents (agents that mimic the action of ACh).

A. Muscarinic Receptors

• Muscarinic receptors are G protein-coupled receptors (metabotropic receptors).

• They bind ACh and muscarine (an alkaloid in poisonous mushrooms).

• They show weak affinity for nicotine.

• Five subclasses of muscarinic receptors, but only $M<em>1$, $M</em>2$, and $M_3$ are functionally characterized.

1. Location of Muscarinic Receptors

• Found on autonomic effector organs, such as:

  • Heart.

  • Smooth muscle.

  • Brain.

  • Exocrine glands.

• All five subtypes are found on neurons.

• $M_1$ receptors are also found on gastric parietal cells.

• $M_2$ receptors are also found on cardiac cells and smooth muscle.

• $M_3$ receptors are also found on the lung, bladder, exocrine glands, and smooth muscle.

• Drugs with muscarinic actions preferentially stimulate muscarinic receptors on these tissues, but at high concentrations, they may show some activity at nicotinic receptors.

2. Mechanism of Acetylcholine Signal Transduction

• Different molecular mechanisms transmit the signal generated by ACh occupation of the receptor.

• Activation of $M<em>1$ or $M</em>3$ receptors:

  • Receptor undergoes a conformational change and interacts with a G protein that activates phospholipase C.

  • Leads to production of second messengers inositol-1,4,5-trisphosphate ($IP_3$) and diacylglycerol (DAG).

  • $IP_3$ causes an increase in intracellular $Ca^{2+}$, which stimulates or inhibits enzymes or causes hyperpolarization, secretion, or contraction.

  • DAG activates protein kinase C, an enzyme that phosphorylates numerous proteins within the cell.

• Activation of the $M_2$ subtype on the cardiac muscle:

  • Stimulates a G protein that inhibits adenylyl cyclase and increases $K^+$ conductance.

  • The heart responds by decreasing both the rate and force of contraction.

3. Muscarinic Agonists

• Act directly by binding and activating the muscarinic receptors or indirectly by inhibiting acetylcholinesterase (AChE), which breaks down ACh.

B. Nicotinic Receptors

• Recognize nicotine but show weak affinity for muscarine.

• Composed of five subunits and functions as a ligand-gated ion channel (ionotropic receptor).

• Binding of two ACh molecules elicits a conformational change that allows the entry of sodium ions, resulting in the depolarization of the effector cell.

• Nicotine at low concentration stimulates the receptor, whereas nicotine at high concentration blocks the receptor.

• Located in the CNS, the adrenal medulla, autonomic ganglia, and the neuromuscular junction (NMJ) in skeletal muscles.

  • Those at the NMJ are sometimes designated $N<em>M$, and the others, $N</em>N$.

• Nicotinic receptors of autonomic ganglia differ from those of the NMJ.

  • Ganglionic receptors are selectively blocked by mecamylamine.

  • NMJ receptors are specifically blocked by neuromuscular blocking drugs like atracurium.

IV. Direct-Acting Cholinergic Agonists

• Mimic the effects of ACh by binding directly to cholinoceptors (muscarinic or nicotinic).

• Broadly classified into two groups:

  1. Choline esters: Include endogenous ACh and synthetic esters of choline, such as carbachol and bethanechol.

  2. Naturally occurring alkaloids: Such as nicotine and pilocarpine and their synthetic analogs (cevimeline).

• All direct-acting cholinergic drugs have a longer duration of action than ACh.

• The therapeutically useful drugs (pilocarpine and bethanechol) preferentially bind to muscarinic receptors and are referred to as muscarinic agents.

• As a group, the direct-acting agonists manifest poor specificity in their actions, which limits clinical usefulness.

A. Acetylcholine

• A quaternary ammonium compound that cannot penetrate membranes because of its positive charge.

• It is the neurotransmitter of parasympathetic and somatic nerves as well as autonomic ganglia.

• Lacks therapeutic importance because of its multiplicity of actions (leading to diffuse effects) and its rapid inactivation by the cholinesterases.

• Maintains both muscarinic and nicotinic activity.

1. Decrease in Heart Rate and Cardiac Output

• The actions of ACh on the heart mimic the effects of vagal stimulation.

• If injected intravenously, ACh acts on $M_2$ receptors to produce a brief decrease in cardiac rate (bradycardia) and, subsequently, cardiac output resulting from a reduction in the rate of firing at the sinoatrial (SA) node.

• Normal vagal activity regulates the heart by the release of ACh at the SA node.

2. Decrease in Blood Pressure

• Injection of ACh causes vasodilation and lowering of blood pressure by an indirect mechanism of action.

• ACh activates $M_3$ receptors found on endothelial cells lining the smooth muscles of blood vessels.

• This results in the production of nitric oxide from arginine.

• Nitric oxide then diffuses to vascular smooth muscle cells to stimulate protein kinase G production, leading to hyperpolarization and smooth muscle relaxation via phosphodiesterase-3 inhibition.

• Vascular cholinergic receptors have no known function in the absence of administered cholinergic agents, because ACh is never released into the blood in significant quantities.

• Atropine blocks these muscarinic receptors and prevents ACh from producing vasodilation.

3. Other Actions

• In the gastrointestinal (GI) tract, acetylcholine increases salivary secretion, increases gastric acid secretion, and stimulates intestinal secretions and motility.

• It also enhances bronchiolar secretions and causes bronchoconstriction.

  • Methacholine, a direct-acting cholinergic agonist, is used to assist in the diagnosis of asthma due to its bronchoconstricting properties.

• In the genitourinary tract, ACh increases the tone of the detrusor muscle, causing urination.

• In the eye, ACh is involved in stimulation of ciliary muscle contraction for near vision and in the constriction of the pupillae sphincter muscle, causing miosis (marked constriction of the pupil).

• ACh (1% solution) is instilled into the anterior chamber of the eye to produce miosis during ophthalmic surgery.

B. Bethanechol

• An unsubstituted carbamoyl ester, structurally related to ACh.

• Not hydrolyzed by AChE due to the esterification of carbamic acid, although it is inactivated through hydrolysis by other esterases.

• Lacks nicotinic actions (due to the addition of the methyl group) but does have strong muscarinic activity.

• Major actions are on the smooth musculature of the bladder and GI tract.

• Has about a 1-hour duration of action.

1. Actions

• Directly stimulates muscarinic receptors, causing increased intestinal motility and tone.

• Stimulates the detrusor muscle of the bladder, whereas the trigone and sphincter muscles are relaxed.

• These effects stimulate urination.

2. Therapeutic Uses

• In urologic treatment, bethanechol is used to stimulate the atonic bladder, particularly in postpartum or postoperative, nonobstructive urinary retention.

• Bethanechol may also be used to treat neurogenic atony as manifested in clinical pathology such as toxic megacolon.

3. Adverse Effects

• Can cause generalized cholinergic stimulation, with sweating, salivation, flushing, decreased blood pressure (with reflex tachycardia), nausea, abdominal pain, diarrhea, and bronchospasm.

• Atropine sulfate may be administered to overcome severe cardiovascular or bronchoconstrictor responses to this agent.

C. Carbachol (Carbamylcholine)

• Has both muscarinic and nicotinic actions.

• An ester of carbamic acid and a poor substrate for AChE.

• Biotransformed by other esterases, but at a much slower rate.

1. Actions

• Has profound effects on both the cardiovascular and GI systems because of its ganglion-stimulating activity, and it may first stimulate and then depress these systems.

• Can cause release of epinephrine from the adrenal medulla by its nicotinic action.

• Locally instilled into the eye, it mimics the effects of ACh, causing miosis and a spasm of accommodation in which the ciliary muscle of the eye remains in a constant state of contraction.

• The vision becomes fixed at some particular distance, making it impossible to focus.

2. Therapeutic Uses

• Rarely used because of its high potency, receptor nonselectivity, and relatively long duration of action.

• Intraocular use provides miosis for eye surgery and lowers intraocular pressure in the treatment of glaucoma.

3. Adverse Effects

• With ophthalmologic use, few adverse effects occur due to lack of systemic absorption (quaternary amine-positively charged).

D. Pilocarpine

• The alkaloid pilocarpine is a tertiary amine and does not undergo hydrolysis by AChE.

• Compared with ACh and its derivatives, it is far less potent but is uncharged and can penetrate the CNS at therapeutic doses.

• Exhibits muscarinic activity and is used primarily in ophthalmology.

1. Actions

• Applied topically to the eye, pilocarpine produces rapid miosis, contraction of the ciliary muscle, and spasm of accommodation.

• One of the most potent stimulators of secretions such as sweat, tears, and saliva, but its use for producing these effects has been limited due to its lack of selectivity.

2. Therapeutic Uses

• Used to treat glaucoma and is the drug of choice to reduce intraocular pressure emergently for both open-angle and angle-closure glaucoma.

• Extremely effective in opening the trabecular meshwork around the Schlemm canal, causing an immediate drop in intraocular pressure because of the increased drainage of aqueous humor.

• This action occurs within a few minutes, lasts 4 to 8 hours, and can be repeated.

• Topical carbonic anhydrase inhibitors, such as dorzolamide and β-adrenergic blockers such as timolol, are effective in treating glaucoma but are not used to reduce intraocular pressure emergently.

• The miotic action of pilocarpine is also useful in reversing mydriasis due to atropine administration.

• Facilitates salivation in patients with xerostomia resulting from irradiation of the head and neck.

• Sjögren syndrome, which is characterized by dry mouth and lack of tears, is treated with oral pilocarpine tablets or cevimeline, a cholinergic drug that also has the drawback of being nonspecific.

3. Adverse Effects

• Can cause blurred vision, night blindness, and brow ache.

• Poisoning with this agent is characterized by exaggeration of various parasympathetic effects, including profuse sweating (diaphoresis) and salivation.

• The effects are similar to those produced by consumption of mushrooms of the genus Inocybe, which contain muscarine.

• Parenteral atropine, at doses high enough to cross the blood-brain barrier, is administered to counteract the toxicity of pilocarpine.

V. Indirect-Acting Cholinergic Agonists: Anticholinesterase Agents (Reversible)

AChE is an enzyme that specifically cleaves ACh to acetate and choline and, thus, terminates its actions.
It is located both pre- and postsynaptically in the nerve terminal where it is membrane bound.
Inhibitors of AChE (anticholinesterase agents or cholinesterase inhibitors) indirectly enhance cholinergic action by preventing the degradation of ACh.
This results in accumulation of ACh in the synaptic space.
Therefore, these drugs can provoke a response at all cholinoceptors, including both muscarinic and nicotinic receptors of the ANS, as well as at the NMJ and in the brain.
The reversible AChE inhibitors can be broadly classified as either short-acting or intermediate-acting agents.

A. Edrophonium

• The prototype short-acting AChE inhibitor.

• Binds reversibly to the active center of AChE, preventing hydrolysis of ACh.

• Has a short duration of action of 10 to 20 minutes due to rapid renal elimination.

• A quaternary amine, and its actions are limited to the periphery because its polarity prevents crossing the blood-brain barrier.

• Historically was used in the diagnosis of myasthenia gravis, an autoimmune disease caused by antibodies to the nicotinic receptor at the NMJ.

• Intravenous injection leads to a rapid increase in muscle strength in patients with myasthenia gravis by increasing the local amounts of ACh available to bind to the NMJ.

• Due to the availability of other agents and improved diagnostic techniques for myasthenia gravis, edrophonium has been removed from the market.

B. Physostigmine

• A nitrogenous carbamic acid ester found naturally in plants and is a tertiary amine.

• A substrate for AChE, and it forms a relatively stable carbamoylated intermediate with the enzyme, which then becomes reversibly inactivated.

• The result is potentiation of cholinergic activity throughout the body.

1. Actions

• Has a wide range of effects and stimulates not only the muscarinic and nicotinic sites of the ANS but also the nicotinic receptors of the NMJ.

• Muscarinic stimulation can cause contraction of GI smooth muscles, miosis, bradycardia, and hypotension.

• Nicotinic stimulation can cause skeletal muscle twitches, fasciculations, and skeletal muscle paralysis (at higher doses).

• Its duration of action is about 30 minutes to 2 hours, and it is considered an intermediate-acting agent.

• The tertiary amine structure permits physostigmine to enter and stimulate the cholinergic sites in the CNS.

2. Therapeutic Uses

• Used in the treatment of overdoses of drugs with anticholinergic actions, such as atropine.

• Due to its ability to enter the CNS, it is able to reverse the unwanted CNS effects of anticholinergics.

3. Adverse Effects

• High doses may lead to convulsions.

• Bradycardia and a decrease in cardiac output may also occur.

• Inhibition of AChE at the NMJ leads to the accumulation of ACh, and theoretically, the resulting continuous NMJ depolarization may result in paralysis of skeletal muscle.

• However, these effects are rarely seen with therapeutic doses.

C. Neostigmine

• A synthetic compound that is also a carbamic acid ester, and it reversibly inhibits AChE in a manner similar to physostigmine.

1. Actions

• Unlike physostigmine, neostigmine has a quaternary nitrogen.

• Therefore, it is more polar, poorly absorbed from the GI tract, and unable to enter the CNS.

• Its effect on skeletal muscle is greater than physostigmine, and it can stimulate contractility before paralyzing any muscular activity.

• Has an intermediate duration of action, usually 30 minutes to 2 hours.

2. Therapeutic Uses

• Used to stimulate the bladder and GI tract and as a reversal agent for the paralytic activity of competitive neuromuscular-blocking agents.

• Also used to manage symptoms of myasthenia gravis.

3. Adverse Effects

• Include those of generalized cholinergic stimulation, such as salivation, flushing, decreased blood pressure, nausea, abdominal pain, diarrhea, and bronchospasm.

• Does not cause CNS side effects and is not used to overcome toxicity of central-acting antimuscarinic agents such as atropine.

• Contraindicated when intestinal or urinary bladder obstruction is present.

D. Pyridostigmine

• Another cholinesterase inhibitor used in the chronic management of myasthenia gravis.

• Its duration of action is intermediate (3-6 hours) but longer than that of neostigmine.

• Adverse effects are similar to those of neostigmine.

E. Tacrine, Donepezil, Rivastigmine, and Galantamine

• Patients with Alzheimer disease (AD) have a deficiency of cholinergic neurons, and therefore, lower levels of ACh are present in the CNS.

• This observation led to the development of anticholinesterases as possible therapies for the deterioration of cognitive function.

• Tacrine, the first agent in this category, has been replaced by others because of its hepatotoxicity.

• Despite the ability of donepezil, rivastigmine, and galantamine to delay the progression of AD, none can stop its progression.

• GI distress is their primary adverse effect

VI. Indirect-Acting Cholinergic Agonists: Anticholinesterase Agents (Irreversible)

• A number of synthetic organophosphate compounds have the ability to bind covalently to AChE.

• The result is a long-lasting increase in ACh at all sites where it is released.

• Many of these drugs are extremely toxic and were developed by the military as nerve agents for chemical warfare.

• Related compounds, such as parathion and malathion, are used as insecticides.

A. Echothiophate

1. Mechanism of Action

• An organophosphate that covalently binds via its phosphate group at the active site of AChE.

• Once this occurs, the enzyme is permanently inactivated, and restoration of AChE activity requires the synthesis of new enzyme molecules.

• Following covalent modification of AChE, the phosphorylated enzyme slowly releases one of its ethyl groups.

• The loss of an alkyl group, which is called "aging," makes it impossible for chemical reactivators, such as pralidoxime, to break the bond between the remaining drug and the enzyme.

2. Actions

• Include generalized cholinergic stimulation, paralysis of motor function (causing breathing difficulties), and convulsions.

• Produces intense miosis and intraocular pressure falls from the facilitation of outflow of aqueous humor.

• Atropine in high dosages can reverse many of the peripheral and some of the central muscarinic effects if administered quickly enough.

3. Therapeutic Uses

• Historically, a topical ophthalmic solution was used for the treatment of open-angle glaucoma.

• However, due to its side effect profile, which includes the risk of developing cataracts, echothiophate was rarely used and is no longer available.

VII. Toxicology of Anticholinesterase Agents

• Irreversible AChE inhibitors (mostly organophosphate compounds) are commonly used as agricultural insecticides in the United States, which has led to numerous cases of accidental poisoning with these agents.

• In addition, they are frequently used for suicidal and homicidal purposes.

• Organophosphate nerve gases such as sarin are used as agents of warfare and chemical terrorism.

• Toxicity with these agents is manifested as nicotinic and muscarinic signs and symptoms (cholinergic crisis).

• Depending on the agent, the effects can be just peripheral or can affect the whole body.

A. Reactivation of Acetylcholinesterase

• Pralidoxime (2-PAM) can reactivate inhibited AChE.

• However, it is unable to penetrate into the CNS and therefore is not useful in treating the CNS effects of organophosphates.

• The presence of a charged group allows it to approach an anionic site on the enzyme, where it essentially displaces the phosphate group of the organophosphate and regenerates the enzyme.

• If given before aging of the alkylated enzyme occurs, it can reverse both muscarinic and nicotinic peripheral effects of organophosphates but not the CNS effects.

• With the newer nerve agents that produce aging of the enzyme complex within seconds, pralidoxime is less effective.

• In addition, it cannot overcome toxicity of reversible AChE inhibitors (for example, physostigmine).

B. Other Treatments

• Atropine is administered to prevent muscarinic side effects.

• Such effects include increased bronchial and salivary secretion, bronchoconstriction, and bradycardia.

• Diazepam also administered to reduce the persistent convulsions caused by these agents.

• General supportive measures, such as maintenance of patent airway, oxygen supply, and artificial respiration, may be necessary as well.