Basic Pharmacology of the Direct Acting Cholinoceptor Stimulants

BASIC PHARMACOLOGY OF THE DIRECT ACTING CHOLINOCEPTOR STIMULANTS

The direct-acting cholinomimetic drugs can be divided on the basis of chemical structure into esters of choline (including acetyl-choline) and alkaloids (such as muscarine and nicotine). Many of these drugs have effects on both receptors; acetylcholine is typical. A few of them are highly selective for the muscarinic or for the nicotinic receptor. However, none of the clinically useful drugs is selective for receptor subtypes in either class.

Chemistry & Pharmacokinetics

A. Structure

Four important choline esters that have been studied extensively are shown in Figure 7–2. Their permanently charged quaternary ammonium group renders them relatively insoluble in lipids. Many naturally occurring and synthetic cholinomimetic drugs that are not choline esters have been identified; a few of these are shown in Figure 7–3. The muscarinic receptor is strongly stereo-selective: (S)-bethanechol is almost 1000 times more potent than (R)-bethanechol.

B. Absorption, Distribution, and Metabolism

Choline esters are poorly absorbed and poorly distributed into the central nervous system because they are hydrophilic. Although all are hydrolyzed in the gastrointestinal tract (and less active by the oral route), they differ markedly in their susceptibility to hydrolysis by cholinesterase. Acetylcholine is very rapidly hydrolyzed ; large amounts must be infused intravenously to achieve concentrations sufficient to produce detectable effects. A large intravenous bolus injection has a brief effect, typically 5–20 seconds, whereas intramuscular and subcutaneous injections pro-duce only local effects. Methacholine is more resistant to hydroly-sis, and the carbamic acid esters carbachol and bethanechol are still more resistant to hydrolysis by cholinesterase and have corre-spondingly longer durations of action. The β-methyl group (methacholine, bethanechol) reduces the potency of these drugs at nicotinic receptors (Table 7–2).

The tertiary natural cholinomimetic alkaloids (pilocarpine, nicotine, lobeline; Figure 7–3) are well absorbed from most sites of administration. Nicotine, a liquid, is sufficiently lipid-soluble to be absorbed across the skin. Muscarine, a quaternary amine, is less completely absorbed from the gastrointestinal tract than the tertiary amines but is nevertheless toxic when ingested—eg, in certain mushrooms—and it even enters the brain. Lobeline is a plant derivative similar to nicotine. These amines are excreted chiefly by the kidneys. Acidification of the urine accelerates clear-ance of the tertiary amines .

Pharmacodynamics

A. Mechanism of Action

Activation of the parasympathetic nervous system modifies organ function by two major mechanisms. First, acetylcholine released from parasympathetic nerves activates muscarinic receptors on effector cells to alter organ function directly.

Second, acetylcholine released from parasympathetic nerves interacts with muscarinic receptors on nerve terminals to inhibit the release of their neu-rotransmitter. By this mechanism, acetylcholine release and circu-lating muscarinic agonists indirectly alter organ function by modulating the effects of the parasympathetic and sympathetic nervous systems and perhaps nonadrenergic, noncholinergic (NANC) systems.

As indicated, muscarinic receptor subtypes have been characterized by binding studies and cloned. Several cellular events occur when muscarinic receptors are activated, one or more of which might serve as second messengers for muscarinic activa-tion. All muscarinic receptors appear to be of the G protein-coupled type (Table 7–1). Muscarinic agonist binding activates the inositol trisphosphate (IP₃), diacylglycerol (DAG) cascade. Some evidence implicates DAG in the opening of smooth muscle calcium channels; IP₃ releases calcium from endoplasmic and sarcoplasmic reticulum. Muscarinic agonists also increase cellular cGMP concentrations. Activation of muscarinic receptors also increases potassium flux across cardiac cell membranes (Figure 7–4A) and decreases it in ganglion and smooth muscle cells. This effect is mediated by the binding of an activated G protein βγ subunit directly to the channel. Finally, muscarinic receptor activation in some tissues (eg, heart, intestine) inhibits adenylyl cyclase activity. Moreover, muscarinic agonists attenuate the activation of adenylyl cyclase and modulate the increase in cAMP levels induced by hormones such as catecholamines. These muscarinic effects on cAMP generation reduce the physiologic response of the organ to stimulatory hormones.

The mechanism of nicotinic receptor activation has been stud-ied in great detail, taking advantage of three factors: (1) the recep-tor is present in extremely high concentration in the membranes of the electric organs of electric fish; (2) α-bungarotoxin, a com-ponent of certain snake venoms, binds tightly to the receptors and is readily labeled as a marker for isolation procedures; and receptor activation results in easily measured electrical and ionic changes in the cells involved. The nicotinic receptor in muscle tissues is a pentamer of four types of glycoprotein sub-units (one monomer occurs twice) with a total molecular weight of about 250,000 (Figure 7–4B). The neuronal nicotinic recep-tor consists of α and β subunits only (Table 7–1). Each subunit has four transmembrane segments. The nicotinic receptor has two agonist binding sites at the interfaces formed by the two α subunits and two adjacent subunits (β, γ, ε). Agonist binding to the receptor sites causes a conformational change in the protein (channel opening) that allows sodium and potassium ions to dif-fuse rapidly down their concentration gradients (calcium ions may also carry charge through the nicotinic receptor ion channel). Binding of an agonist molecule by one of the two receptor sites only modestly increases the probability of channel opening; simultaneous binding of agonist by both of the receptor sites greatly enhances opening probability. 

Nicotinic receptor activation causes depolarization of the nerve cell or neuromuscular end plate membrane. In skeletal muscle, the depolarization initiates an action potential that propagates across the muscle membrane and causes contraction (Figure 7–4B).

Prolonged agonist occupancy of the nicotinic receptor abolishes the effector response; that is, the postganglionic neuron stops firing (ganglionic effect), and the skeletal muscle cell relaxes (neuromus-cular end plate effect). Furthermore, the continued presence of the nicotinic agonist prevents electrical recovery of the postjunctional membrane. Thus, a state of “depolarizing blockade” occurs initially during persistent agonist occupancy of the receptor. Continued agonist occupancy is associated with return of membrane voltage to the resting level. The receptor becomes desensitized to agonist, and this state is refractory to reversal by other agonists.

B. Organ System Effects

Most of the direct organ system effects of muscarinic cholinoceptor stimulants are readily predicted from knowledge of the effects of parasympathetic nerve stimulation (see Table 6–3) and the distri-bution of muscarinic receptors. Effects of a typical agent such as acetylcholine are listed in Table 7–3. The effects of nicotinic ago-nists are similarly predictable from knowledge of the physiology of the autonomic ganglia and skeletal muscle motor end plate.

1.     Eye—Muscarinic agonists instilled into the conjunctival saccause contraction of the smooth muscle of the iris sphincter (result-ing in miosis) and of the ciliary muscle (resulting in accommoda-tion). As a result, the iris is pulled away from the angle of the anterior chamber, and the trabecular meshwork at the base of the ciliary muscle is opened. Both effects facilitate aqueous humor outflow into the canal of Schlemm, which drains the anterior chamber.

2.     Cardiovascular system—The primary cardiovasculareffects of muscarinic agonists are reduction in peripheral vascular resistance and changes in heart rate. The direct effects listed in Table 7–3 are modified by important homeostatic reflexes, as described and depicted in Figure 6–7. Intravenous infusions of minimally effective doses of acetylcholine in humans (eg, 20–50 mcg/min) cause vasodilation, resulting in a reduction in blood pressure, often accompanied by a reflex increase in heart rate. Larger doses of acetylcholine produce bradycardia and decrease atrioventricular node conduction velocity in addition to hypotension.

The direct cardiac actions of muscarinic stimulants include the following: (1) an increase in a potassium current (I_K(ACh)) in the cells of the sinoatrial and atrioventricular nodes, in Purkinje cells,

and also in atrial and ventricular muscle cells; (2) a decrease in the slow inward calcium current (I_Ca) in heart cells; and (3) a reduc-tion in the hyperpolarization-activated current (I_f ) that underlies diastolic depolarization (Figure 7–4A). All these actions are medi-ated by M₂ receptors and contribute to slowing the pacemaker rate. Effects (1) and (2) cause hyperpolarization, reduce action potential duration, and decrease the contractility of atrial and ventricular cells. Predictably, knockout of M₂ receptors eliminates the bradycardic effect of vagal stimulation and the negative chro-notropic effect of carbachol on sinoatrial rate.

The direct slowing of sinoatrial rate and atrioventricular con-duction that is produced by muscarinic agonists is often opposed by reflex sympathetic discharge, elicited by the decrease in blood pressure (see Figure 6–7). The resultant sympathetic-parasympathetic interaction is complex because muscarinic modulation of sympa-thetic influences occurs by inhibition of norepinephrine release and by postjunctional cellular effects. Muscarinic receptors that are present on postganglionic parasympathetic nerve terminals allow neurally released acetylcholine to inhibit its own secretion. The neuronal muscarinic receptors need not be the same subtype as found on effector cells. Therefore, the net effect on heart rate depends on local concentrations of the agonist in the heart and in the vessels and on the level of reflex responsiveness

Parasympathetic innervation of the ventricles is much less extensive than that of the atria; activation of ventricular muscar-inic receptors causes much less physiologic effect than that seen in atria. However, the effects of muscarinic agonists on ventricular function are clearly evident during sympathetic nerve stimulation because of muscarinic modulation of sympathetic effects (“accen-tuated antagonism”).

In the intact organism, intravascular injection of muscarinic agonists produces marked vasodilation. However, earlier studies of isolated blood vessels often showed a contractile response to these agents. It is now known that acetylcholine-induced vasodi-lation arises from activation of M₃ receptors and requires the presence of intact endothelium (Figure 7–5). Muscarinic ago-nists release endothelium-derived relaxing factor (EDRF), iden-tified as nitric oxide (NO), from the endothelial cells. The NO diffuses to adjacent vascular smooth muscle, where it activates guanylyl cyclase and increases cGMP, resulting in relaxation (see Figure 12–2). Isolated vessels prepared with the endothelium preserved consistently reproduce the vasodilation seen in the intact organism. The relaxing effect of acetylcholine was maximal at 3 × 10⁻⁷ M (Figure 7–5). This effect was eliminated in the absence of endothelium, and acetylcholine, at concentrations greater than 10⁻⁷ M, then caused contraction. This results from a direct effect of acetylcholine on vascular smooth muscle in which activation of M ₃ receptors stimulates IP₃ production and releases intracellular calcium.

Parasympathetic nerves can regulate arteriolar tone in vascular beds in thoracic and abdominal visceral organs. Acetylcholine released from postganglionic parasympathetic nerves relaxes coro-nary arteriolar smooth muscle via the NO/cGMP pathway in humans as described above. Damage to the endothelium, as occurs with atherosclerosis, eliminates this action, and acetylcho-line is then able to contract arterial smooth muscle and produce vasoconstriction. Parasympathetic nerve stimulation also causes vasodilation in cerebral blood vessels; however, the effect often appears as a result of NO released either from NANC (nitrergic) neurons or as a cotransmitter from cholinergic nerves. The relative contributions of cholinergic and NANC neurons to the vascular effects of parasympathetic nerve stimulation are not known for most viscera. Skeletal muscle receives sympathetic cholinergic vasodilator nerves, but the view that acetylcholine causes vasodila-tion in this vascular bed has not been verified experimentally. Nitric oxide, rather than acetylcholine, may be released from these neurons. However, this vascular bed responds to exogenous cho-line esters because of the presence of M₃ receptors on endothelial and smooth muscle cells.

The cardiovascular effects of all the choline esters are similar to those of acetylcholine—the main difference being in their potency and duration of action. Because of the resistance of methacholine, carbachol, and bethanechol to acetylcholinesterase, lower doses given intravenously are sufficient to produce effects similar to those of acetylcholine, and the duration of action of these syn-thetic choline esters is longer. The cardiovascular effects of most of the cholinomimetic natural alkaloids and the synthetic analogs are also generally similar to those of acetylcholine.

Pilocarpine is an interesting exception to the above statement. If given intravenously (an experimental exercise), it may produce hypertension after a brief initial hypotensive response. The longer-lasting hypertensive effect can be traced to sympathetic ganglionic discharge caused by activation of postganglionic cell membrane M₁ receptors, which close K⁺ channels and elicit slow excitatory (depolarizing) postsynaptic potentials. This effect, like the hypotensive effect, can be blocked by atropine, an antimuscarinic drug.

3. Respiratory system—Muscarinic stimulants contract thesmooth muscle of the bronchial tree. In addition, the glands of the tracheobronchial mucosa are stimulated to secrete. This combina-tion of effects can occasionally cause symptoms, especially in individuals with asthma. The bronchoconstriction caused by muscarinic agonists is eliminated in knockout animals in which the M₃ receptor has been mutated.

4. Gastrointestinal tract—Administration of muscarinic ago-nists, as in parasympathetic nervous system stimulation, increases the secretory and motor activity of the gut. The salivary and gas-tric glands are strongly stimulated; the pancreas and small intesti-nal glands are stimulated less so. Peristaltic activity is increased throughout the gut, and most sphincters are relaxed. Stimulation of contraction in this organ system involves depolarization of the smooth muscle cell membrane and increased calcium influx. 

Muscarinic agonists do not cause contraction of the ileum in mutant mice lacking M₂ and M₃ receptors. The M₃ receptor is required for direct activation of smooth muscle contraction, whereas the M₂ receptor reduces cAMP formation and relaxation caused by sympathomimetic drugs.

5. Genitourinary tract— Muscarinic agonists stimulate thedetrusor muscle and relax the trigone and sphincter muscles of the bladder, thus promoting voiding. The function of M₂ and M₃ receptors in the urinary bladder appears to be the same as in intes-tinal smooth muscle. The human uterus is not notably sensitive to muscarinic agonists.

6. Miscellaneous secretory glands—Muscarinic agonistsstimulate secretion by thermoregulatory sweat, lacrimal, and nasopharyngeal glands. 

7. Central nervous system—The central nervous system con-tains both muscarinic and nicotinic receptors, the brain being relatively richer in muscarinic sites and the spinal cord containing a preponderance of nicotinic sites. 

All five muscarinic receptor subtypes have been detected in the central nervous system. The roles of M₁ through M₃ have been analyzed by means of experiments in knockout mice. The M₁ subtype is richly expressed in brain areas involved in cognition. Knockout of M₁ receptors was associated with impaired neuronal plasticity in the forebrain, and pilocarpine did not induce seizures in M₁ mutant mice. The central nervous system effects of the synthetic muscarinic agonist oxotremorine (tremor, hypothermia,and antinociception) were lacking in mice with homozygously mutated M₂ receptors. Animals lacking M₃ receptors, especially those in the hypothalamus, had reduced appetite and diminished body fat mass.

In spite of the smaller ratio of nicotinic to muscarinic recep-tors, nicotine and lobeline (Figure 7–3) have important effects on the brain stem and cortex. Activation of nicotinic receptors occurs at presynaptic and postsynaptic loci. Presynaptic nicotinic recep-tors allow acetylcholine and nicotine to regulate the release of several neurotransmitters (glutamate, serotonin, GABA, dopamine, and norepinephrine). Acetylcholine regulates norepineph-rine release via α3β4 nicotinic receptors in the hippocampus and inhibits acetylcholine release from neurons in the hippocampus and cortex. The α4β2 oligomer is the most abundant nicotinic receptor in the brain. Chronic exposure to nicotine has a dual effect at nicotinic receptors: activation (depolarization) followed by desensitization. The former effect is associated with greater release of dopamine in the mesolimbic system. This effect is thought to contribute to the mild alerting action and the addictive property of nicotine absorbed from tobacco. When the β2 sub-units are deleted in reconstitution experiments, acetylcholine binding is reduced, as is the release of dopamine. The later desen-sitization of the nicotinic receptor is accompanied by increased high-affinity agonist binding and an upregulation of nicotinic binding sites, especially those of the α4β2 oligomer. Sustained desensitization may contribute to the benefits of nicotine replace-ment therapy in smoking cessation regimens. In high concentra-tions, nicotine induces tremor, emesis, and stimulation of the respiratory center. At still higher levels, nicotine causes convul-sions, which may terminate in fatal coma. The lethal effects on the central nervous system and the fact that nicotine is readily absorbed form the basis for the use of nicotine as an insecticide.

8. Peripheral nervous system—Autonomic ganglia areimportant sites of nicotinic synaptic action. The nicotinic agents shown in Figure 7–3 cause marked activation of these nicotinic receptors and initiate action potentials in postganglionic neurons (see Figure 6–8). Nicotine itself has a somewhat greater affinity for neuronal than for skeletal muscle nicotinic receptors. The action is the same on both parasympathetic and sympathetic ganglia. The initial response therefore often resembles simultaneous discharge of both the parasympathetic and the sympathetic nervous systems. In the case of the cardiovascular system, the effects of nicotine are chiefly sympathomimetic. Dramatic hypertension is produced by parenteral injection of nicotine; sympathetic tachycardia may alternate with a bradycardia mediated by vagal discharge. In the gastrointestinal and urinary tracts, the effects are largely parasym-pathomimetic: nausea, vomiting, diarrhea, and voiding of urine are commonly observed. Prolonged exposure may result in depo-larizing blockade of the ganglia. 

Neuronal nicotinic receptors are present on sensory nerve endings—especially afferent nerves in coronary arteries and the carotid and aortic bodies as well as on the glomus cells of the lat-ter. Activation of these receptors by nicotinic stimulants and of muscarinic receptors on glomus cells by muscarinic stimulants elicits complex medullary responses, including respiratory altera-tions and vagal discharge.

9. Neuromuscular junction—The nicotinic receptors on theneuromuscular end plate apparatus are similar but not identical to the receptors in the autonomic ganglia (Table 7–1). Both types respond to acetylcholine and nicotine. (However, as noted, the receptors differ in their structural requirements for nicotinic blocking drugs.) When a nicotinic agonist is applied directly (by iontophoresis or by intra-arterial injection), an immediate depolarization of the end plate results, caused by an increase in permeability to sodium and potassium ions (Figure 7–4). The contractile response varies from disorganized fascicula-tions of independent motor units to a strong contraction of the entire muscle depending on the synchronization of depolariza-tion of end plates throughout the muscle. Depolarizing nicotinic agents that are not rapidly hydrolyzed (like nicotine itself ) cause rapid development of depolarization blockade; transmission blockade persists even when the membrane has repolarized. This latter phase of block is manifested as flaccid paralysis in the case of skeletal muscle.