Drugs affecting the autonomic nervous system (ANS) are divided into two groups according to the type of neuron involved in their mechanism of action. The cholinergic drugs, which are described in this and the following chapter, act on receptors that are activated by acetylcholine (ACh), whereas the adrenergic drugs (discussed in Chapters 6 and 7) act on receptors stimulated by norepinephrine or epinephrine. Cholinergic and adrenergic drugs both act by either stimulating or blocking receptors of the ANS. Figure 4.1 summarizes the cholinergic agonists discussed in this chapter.
Figure 4.1. Summary of cholinergic agonists.
The Cholinergic Neuron
The preganglionic fibers terminating in the adrenal medulla, the autonomic ganglia (both parasympathetic and sympathetic), and the postganglionic fibers of the parasympathetic division use ACh as a neurotransmitter (Figure 4.2). Also the postganglionic sympathetic division of sweat glands use acetylcholine. In addition, cholinergic neurons innervate the muscles of the somatic system and also play an important role in the central nervous system (CNS). [Note: Patients with Alzheimer disease have a significant loss of cholinergic neurons in the temporal lobe and entorhinal cortex. Most of the drugs available to treat the disease are acetylcholinesterase (AChE) inhibitors (see p. 108).]
Figure 4.2. Sites of actions of cholinergic agonists in the autonomic and somatic nervous systems.
Neurotransmission at cholinergic neurons
Neurotransmission in cholinergic neurons involves six sequential steps: 1) synthesis, 2) storage, 3) release, 4) binding of ACh to a receptor, 5) degradation of the neurotransmitter in the synaptic cleft (that is, the space between the nerve endings and adjacent receptors located on nerves or effector organs), and 6) recycling of choline and acetate (Figure 4.3).
Figure 4.3. Synthesis and release of acetylcholine from the cholinergic neuron.
AcCoA = acetyl coenzyme A.
Synthesis of acetylcholine
Choline is transported from the extracellular fluid into the cytoplasm of the cholinergic neuron by an energy-dependent carrier system that cotransports sodium and can be inhibited by the drug hemicholinium. [Note: Choline has a quaternary nitrogen and carries a permanent positive charge, and, thus, 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 (an ester) in the cytosol. Acetyl CoA is derived from the mitochondria and is produced by the pyruvate oxidation and fatty acid oxidation.
Storage of acetylcholine in vesicles
ACh is packaged and stored into presynaptic vesicles by an active transport process coupled to the efflux of protons. The mature vesicle contains not only ACh but also adenosine triphosphate (ATP) and proteoglycan. [Note: ATP has been suggested to be a cotransmitter acting at prejunctional purinergic receptors to inhibit the release of ACh or norepinephrine.] Cotransmission from autonomic neurons is the rule rather than the exception. This means that most synaptic vesicles will contain the primary neurotransmitter (here, ACh) as well as a cotransmitter that will increase or decrease the effect of the primary neurotransmitter. The neurotransmitters in vesicles appear as beadlike structures, known as varicosities, along the nerve terminal of the presynaptic neuron.
Release of acetylcholine
When an action potential propagated by voltage-sensitive sodium channels arrives at a nerve ending, voltage-sensitive calcium channels on the presynaptic membrane open, causing an increase in the concentration of intracellular calcium. Elevated calcium levels promote the fusion of synaptic vesicles with the cell membrane and the release of their contents into the synaptic space. This release can be blocked by botulinum toxin. In contrast, the toxin in black widow spider venom causes all the ACh stored in synaptic vesicles to empty into the synaptic gap.
Binding to the receptor
ACh released from the synaptic vesicles diffuses across the synaptic space and binds to either of two postsynaptic receptors on the target cell, to presynaptic receptors in the membrane of the neuron that released the ACh, or to other targeted presynaptic receptors. The postsynaptic cholinergic receptors on the surface of the effector organs are divided into two classes: muscarinic and nicotinic (see Figure 4.2 and p. 146). Binding to a receptor leads to a biologic response within the cell, such as the initiation of a nerve impulse in a postganglionic fiber or activation of specific enzymes in effector cells, as mediated by second-messenger molecules (see p. 29).
Degradation of acetylcholine
The signal at the postjunctional effector site is rapidly terminated, because AChE cleaves ACh to choline and acetate in the synaptic cleft (see Figure 4.3). [Note: Butyrylcholinesterase, sometimes called pseudocholinesterase, is found in the plasma but does not play a significant role in the termination of ACh’s effect in the synapse.]
Recycling of choline
Choline may be recaptured by a sodium-coupled, high-affinity uptake system that transports the molecule back into the neuron. There, it is acetylated into ACh that is stored until released by a subsequent action potential.
Cholinergic Receptors (Cholinoceptors)
Two families of cholinoceptors, designated muscarinic and nicotinic receptors, can be distinguished from each other on the basis of their different affinities for agents that mimic the action of ACh (cholinomimetic agents or parasympathomimetics).
Muscarinic receptors belong to the class of G protein–coupled receptors. These receptors, in addition to binding ACh, also recognize muscarine, an alkaloid that is present in certain poisonous mushrooms. By contrast, the muscarinic receptors show only a weak affinity for nicotine (Figure 4.4A). Binding studies and specific inhibitors, as well as cDNA characterization, have distinguished five subclasses of muscarinic receptors: M1, M2, M3, M4, and M5. Although five muscarinic receptors have been identified by gene cloning, only M1, M2, and M3 receptors have been functionally characterized.
Figure 4.4. Types of cholinergic receptors.
Locations of muscarinic receptors
These receptors have been found on ganglia of the peripheral nervous system and on the autonomic effector organs, such as the heart, smooth muscle, brain, and exocrine glands (see Figure 3.3). Specifically, although all five subtypes have been found on neurons, M1 receptors are also found on gastric parietal cells, M2 receptors on cardiac cells and smooth muscle, and M3 receptors on the bladder, exocrine glands, and smooth muscle. [Note: Drugs with muscarinic actions preferentially stimulate muscarinic receptors on these tissues, but at high concentration they may show some activity at nicotinic receptors.]
Mechanisms of acetylcholine signal transduction
A number of different molecular mechanisms transmit the signal generated by ACh occupation of the receptor. For example, when the M1 or M3 receptors are activated, the receptor undergoes a conformational change and interacts with a G protein, designated Gq, that in turn activates phospholipase C.1 This leads to the hydrolysis of phosphatidylinositol-(4,5)-bisphosphate to yield diacylglycerol and inositol (1,4,5)-trisphosphate. Both inositol (1,4,5)-trisphosphate and diacylglycerol are second messengers. Inositol (1,4,5)-trisphosphate causes an increase in intracellular Ca2+ (see Figure 3.10C).
This cation can then interact to stimulate or inhibit enzymes or to cause hyperpolarization, secretion, or contraction. Diacylglycerol activates protein kinase C. This enzyme phosphorylates numerous proteins within the cell. In contrast, activation of the M2 subtype on the cardiac muscle stimulates a G protein, designated Gi, which inhibits adenylyl cyclase2 and increases K+ conductance (see Figure 3.10B). The heart responds with a decrease in rate and force of contraction.
Muscarinic agonists and antagonists
Attempts are currently underway to develop muscarinic agonists and antagonists that are directed against specific receptor subtypes. For example, pirenzepine, a tricyclic anticholinergic drug, has a greater selectivity for inhibiting M1 muscarinic receptors, such as in the gastric mucosa. At therapeutic doses, pirenzepine does not cause many of the side effects seen with the non-subtype-specific drugs; however, it does produce a reflex tachycardia on rapid infusion due to blockade of M2 receptors in the heart.
Therefore, the usefulness of pirenzepine as an alternative to proton pump inhibitors in the treatment of gastric and duodenal ulcers is questionable. Darifenacin is a competitive muscarinic receptor antagonist with a greater affinity for the M3 receptor than for the other muscarinic receptors. The drug is used in the treatment of overactive bladder. [Note: At present, no clinically important agents interact solely with the M4 and M5 receptors.]
These receptors, in addition to binding ACh, also recognize nicotine but show only a weak affinity for muscarine (see Figure 4.4B). The nicotinic receptor is composed of five subunits and it functions as a ligand-gated ion channel (see Figure 3.10A). 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, and at high concentration blocks the receptor.
Nicotinic receptors are located in the CNS, adrenal medulla, autonomic ganglia, and the neuromuscular junction (NMJ). Those at the NMJ are sometimes designated NM, and the others, NN. The nicotinic receptors of autonomic ganglia differ from those of the NMJ. For example, ganglionic receptors are selectively blocked by hexamethonium, whereas NMJ receptors are specifically blocked by tubocurarine.
Direct-Acting Cholinergic Agonists
Cholinergic agonists (also known as parasympathomimetics) mimic the effects of ACh by binding directly to cholinoceptors. These agents may be broadly classified into two groups: choline esters, which include ACh, and synthetic esters of choline, such as carbachol and bethanechol. Naturally occurring alkaloids, such as pilocarpine, constitute the second group (Figure 4.5). All of the direct-acting cholinergic drugs have longer durations of action than ACh.
Some of the more therapeutically useful drugs (pilocarpine and bethanechol) preferentially bind to muscarinic receptors and are sometimes referred to as muscarinic agents. [Note: Muscarinic receptors are located primarily, but not exclusively, at the neuroeffector junction of the parasympathetic nervous system.] However, as a group, the direct-acting agonists show little specificity in their actions, which limits their clinical usefulness.
Figure 4.5. Comparison of the structures of some cholinergic agonists.
Acetylcholine [ah-see-teel-KOE-leen] is a quaternary ammonium compound that cannot penetrate membranes. Although it is the neurotransmitter of parasympathetic and somatic nerves as well as autonomic ganglia, it lacks therapeutic importance because of its multiplicity of actions (leading to diffuse effects) and its rapid inactivation by the cholinesterases. ACh has both muscarinic and nicotinic activity. Its actions include:
Decrease in heart rate and cardiac output
The actions of ACh on the heart mimic the effects of vagal stimulation. For example, if injected intravenously, ACh produces a brief decrease in cardiac rate (negative chronotropy) and stroke volume as a result of a reduction in the rate of firing at the sinoatrial (SA) node. [Note: It should be remembered that normal vagal activity regulates the heart by the release of ACh at the SA node.]
Decrease in blood pressure
Injection of ACh causes vasodilation and lowering of blood pressure by an indirect mechanism of action. ACh activates M3 receptors found on endothelial cells lining the smooth muscles of blood vessels. This results in the production of nitric oxide from arginine.3 [Note: nitric oxide (NO) is also known as endothelium-derived relaxing factor.] (See p. 363 for more detail on NO.) NO then diffuses to vascular smooth muscle cells to stimulate protein kinase G production, leading to hyperpolarization and smooth muscle relaxation via phosphodisterase-3 inhibition. In the absence of administered cholinergic agents, the vascular receptors have no known function, because ACh is never released into the blood in any significant quantities. Atropine blocks these muscarinic receptors and prevents ACh from producing vasodilation.
In the gastrointestinal (GI) tract, acetylcholine increases salivary secretion and stimulates intestinal secretions and motility. It also enhances bronchiolar secretions. In the genitourinary tract, ACh increases the tone of the detrusor urinae muscle, causing expulsion of urine. In the eye, ACh is involved in stimulating 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.
Bethanechol [be-THAN-e-kole] is an unsubstituted carbamoyl ester, structurally related to ACh, in which the acetate is replaced by carbamate, and the choline is methylated (see Figure 4.5). Hence, it is not hydrolyzed by AChE (due to the esterification of carbamic acid), although it is inactivated through hydrolysis by other esterases. It lacks nicotinic actions (due to the addition of the methyl group) but does have strong muscarinic activity. Its major actions are on the smooth musculature of the bladder and GI tract. It has about a 1-hour duration of action.
Bethanechol directly stimulates muscarinic receptors, causing increased intestinal motility and tone. It also stimulates the detrusor muscle of the bladder, whereas the trigone and sphincter are relaxed. These effects increase voiding pressure and decrease bladder capacity to cause expulsion of urine.
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 well as megacolon.
Bethanechol causes the effects of generalized cholinergic stimulation (Figure 4.6). These include sweating, salivation, flushing, decreased blood pressure, nausea, abdominal pain, diarrhea, and bronchospasm. Atropine sulfate may be administered to overcome severe cardiovascular or bronchoconstrictor responses to this agent.
Figure 4.6. Some adverse effects observed with cholinergic agonists.
Carbachol [KAR-ba-kole] has both muscarinic as well as nicotinic actions. It lacks the methyl group present in bethanechol (see Figure 4.5). Like bethanechol, carbachol is an ester of carbamic acid and a poor substrate for AChE (see Figure 4.5). It is biotransformed by other esterases but at a much slower rate.
Carbachol 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. It 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.
Because of its high potency, receptor nonselectivity, and relatively long duration of action, carbachol is rarely used therapeutically except in the eye as a miotic agent to treat glaucoma by causing pupillary contraction and a decrease in intraocular pressure. Onset of action for miosis is 10 to 20 minutes. Intraocular pressure is reduced for 4 to 8 hours.
At doses used ophthalmologically, little or no side effects occur due to lack of systemic penetration (quaternary amine).