Nerve impulses elicit responses in smooth, cardiac, and skeletal muscles, exocrine glands, and postsynaptic neurons by liberating specific chemical neurotransmitters. The processes are presented in some detail because an understanding of the chemical mediation of nerve impulses provides the framework for our knowledge of the mechanism of action of drugs at these sites.
Historical Aspects. The earliest concrete proposal of a neurohumoral mechanism was made shortly after the turn of the twentieth century. Lewandowsky and Langley independently noted the similarity between the effects of injection of extracts of the adrenal gland and stimulation of sympathetic nerves. In 1905, T. R. Elliott, while a student with Langley at Cambridge, postulated that sympathetic nerve impulses release minute amounts of an epinephrine-like substance in immediate contact with effector cells. He considered this substance to be the chemical step in the process of transmission. He also noted that long after sympathetic nerves had degenerated, the effector organs still responded characteristically to the hormone of the adrenal medulla. Langley suggested that effector cells have excitatory and inhibitory "receptive substances" and that the response to epinephrine depended on which type of substance was present. In 1907, Dixon, impressed by the correspondence between the effects of the alkaloid muscarine and the responses to vagal stimulation, advanced the concept that the vagus nerve liberated a muscarine-like substance that acted as a chemical transmitter of its impulses. In the same year, Reid Hunt described the actions of ACh and other choline esters. In 1914, Dale investigated the pharmacological properties of ACh and other choline esters and distinguished its nicotine-like and muscarine-like actions. Intrigued with the remarkable fidelity with which this drug reproduced the responses to stimulation of parasympathetic nerves, he introduced the term parasympathomimetic to characterize its effects. Dale also noted the brief duration of action of this chemical and proposed that an esterase in the tissues rapidly splits ACh to acetic acid and choline, thereby terminating its action.
The studies of Loewi, begun in 1921, provided the first direct evidence for the chemical mediation of nerve impulses by the release of specific chemical agents. Loewi stimulated the vagus nerve of a perfused (donor) frog heart and allowed the perfusion fluid to come in contact with a second (recipient) frog heart used as a test object. The recipient frog heart was found to respond, after a short lag, in the same way as the donor heart. It thus was evident that a substance was liberated from the first organ that slowed the rate of the second. Loewi referred to this chemical substance as Vagusstoff ("vagus substance," "parasympathin"); subsequently, Loewi and Navratil presented evidence to identify it as ACh. Loewi also discovered that an accelerator substance similar to epinephrine and called Acceleranstoff was liberated into the perfusion fluid in summer, when the action of the sympathetic fibers in the frog's vagus, a mixed nerve, predominated over that of the inhibitory fibers. Feldberg and Krayer demonstrated in 1933 that the cardiac "vagus substance" also is ACh in mammals.
In addition to its role as the transmitter of most postganglionic parasympathetic fibers and of a few postganglionic sympathetic fibers, ACh has been shown to function as a neurotransmitter in three additional classes of nerves: preganglionic fibers of both the sympathetic and the parasympathetic systems, motor nerves to skeletal muscle, and certain neurons within the CNS.
In the same year as Loewi's discovery, Cannon and Uridil reported that stimulation of the sympathetic hepatic nerves resulted in the release of an epinephrine-like substance that increased blood pressure and heart rate. Subsequent experiments firmly established that this substance is the chemical mediator liberated by sympathetic nerve impulses at neuroeffector junctions. Cannon called this substance "sympathin." In many of its pharmacological and chemical properties, "sympathin" closely resembled epinephrine, but also differed in certain important respects. As early as 1910, Barger and Dale noted that the effects of sympathetic nerve stimulation were reproduced more closely by the injection of sympathomimetic primary amines than by that of epinephrine or other secondary amines. The possibility that demethylated epinephrine (norepinephrine) might be "sympathin" had been advanced repeatedly, but definitive evidence for its being the sympathetic nerve mediator was not obtained until specific assays were developed for the determination of sympathomimetic amines in extracts of tissues and body fluids. In 1946, von Euler found that the sympathomimetic substance in highly purified extracts of bovine splenic nerve resembled norepinephrine by all criteria used. Norepinephrine is the predominant sympathomimetic substance in the postganglionic sympathetic nerves of mammals and is the adrenergic mediator liberated by their stimulation. Norepinephrine, its immediate precursor dopamine, and epinephrine also are neurotransmitters in the CNS (see Chapter 14).
Evidence for Neurohumoral Transmission
The concept of neurohumoral transmission or chemical neurotransmission was developed primarily to explain observations relating to the transmission of impulses from postganglionic autonomic fibers to effector cells. Evidence supporting this concept includes:
demonstration of the presence of a physiologically active compound and its biosynthetic enzymes at appropriate sites
recovery of the compound from the perfusate of an innervated structure during periods of nerve stimulation but not (or in greatly reduced amounts) in the absence of stimulation
demonstration that the compound is capable of producing responses identical to responses to nerve stimulation
demonstration that the responses to nerve stimulation and to the administered compound are modified in the same manner by various drugs, usually competitive antagonists
While these criteria are applicable for most neurotransmitters, including norepinephrine and ACh, there are now exceptions to these general rules. For instance, NO has been found to be a neurotransmitter, in a few postganglionic parasympathetic nerves, in nonadrenergic, noncholinergic (NANC) neurons in the periphery, in the ENS, and in the CNS. However, NO is not stored in neurons and released by exocytosis. Rather, it is synthesized when needed and readily diffuses across membranes.
Chemical rather than electrogenic transmission at autonomic ganglia and the neuromuscular junction of skeletal muscle was not generally accepted for a considerable period because techniques were limited in time and chemical resolution. Techniques of intracellular recording and microiontophoretic application of drugs, as well as sensitive analytical assays, have overcome these limitations.
Neurotransmission in the peripheral and central nervous systems once was believed to conform to the hypothesis that each neuron contains only one transmitter substance. However, peptides such as enkephalin, substance P, neuropeptide Y, VIP, and somatostatin; purines such as ATP and adenosine; and small molecules such as NO have been found in nerve endings. These substances can depolarize or hyperpolarize nerve terminals or postsynaptic cells. Furthermore, results of histochemical, immunocytochemical, and autoradiographic studies have demonstrated that one or more of these substances is present in the same neurons that contain one of the classical biogenic amine neurotransmitters. For example, enkephalins are found in postganglionic sympathetic neurons and adrenal medullary chromaffin cells. VIP is localized selectively in peripheral cholinergic neurons that innervate exocrine glands, and neuropeptide Y is found in sympathetic nerve endings. These observations suggest that synaptic transmission in many instances may be mediated by the release of more than one neurotransmitter (see the next section).
Steps Involved in Neurotransmission
The sequence of events involved in neurotransmission is of particular importance because pharmacologically active agents modulate the individual steps.
The term conduction is reserved for the passage of an impulse along an axon or muscle fiber; transmission refers to the passage of an impulse across a synaptic or neuroeffector junction. With the exception of the local anesthetics, very few drugs modify axonal conduction in the doses employed therapeutically. Hence this process is described only briefly.
Axonal Conduction. At rest, the interior of the typical mammalian axon is ∼70 mV negative to the exterior. The resting potential is essentially a diffusion potential based chiefly on the 40 times higher concentration of K+ in the axoplasm as compared with the extracellular fluid and the relatively high permeability of the resting axonal membrane to K+. Na+ and Cl– are present in higher concentrations in the extracellular fluid than in the axoplasm, but the axonal membrane at rest is considerably less permeable to these ions; hence their contribution to the resting potential is small. These ionic gradients are maintained by an energy-dependent active transport mechanism, the Na+, K+-ATPase (Hille, 1992).
In response to depolarization to a threshold level, an action potential or nerve impulse is initiated at a local region of the membrane. The action potential consists of two phases. Following a small gating current resulting from depolarization inducing an open conformation of the channel, the initial phase is caused by a rapid increase in the permeability of Na+ through voltage- sensitive Na+ channels. The result is inward movement of Na+ and a rapid depolarization from the resting potential, which continues to a positive overshoot. The second phase results from the rapid inactivation of the Na+ channel and the delayed opening of a K+ channel, which permits outward movement of K+ to terminate the depolarization. Inactivation appears to involve a voltage-sensitive conformational change in which a hydrophobic peptide loop physically occludes the open channel at the cytoplasmic side. Although not important in axonal conduction, Ca2+ channels in other tissues (e.g., L-type Ca2+channels in heart) contribute to the action potential by prolonging depolarization by an inward movement of Ca2+. This influx of Ca2+ also serves as a stimulus to initiate intracellular events (Catterall, 2000; Hille, 1992), and Ca2+ influx is important in excitation-exocytosis coupling (transmitter release).
The transmembrane ionic currents produce local circuit currents around the axon. As a result of such localized changes in membrane potential, adjacent resting channels in the axon are activated, and excitation of an adjacent portion of the axonal membrane occurs. This brings about propagation of the action potential without decrement along the axon. The region that has undergone depolarization remains momentarily in a refractory state. In myelinated fibers, permeability changes occur only at the nodes of Ranvier, thus causing a rapidly progressing type of jumping, or saltatory, conduction.
The puffer fish poison, tetrodotoxin, and a close congener found in some shellfish, saxitoxin, selectively block axonal conduction; they do so by blocking the voltage-sensitive Na+ channel and preventing the increase in Na+ permeability associated with the rising phase of the action potential. In contrast, batrachotoxin, an extremely potent steroidal alkaloid secreted by a South American frog, produces paralysis through a selective increase in permeability of the Na+ channel, which induces a persistent depolarization. Scorpion toxins are peptides that also cause persistent depolarization, but they do so by inhibiting the inactivation process (Catterall, 2000). Na+ and Ca2+ channels are discussed in more detail in Chapters 11, Chapter 14, Chapter 20.
Junctional Transmission. The arrival of the action potential at the axonal terminals initiates a series of events that trigger transmission of an excitatory or inhibitory impulse across the synapse or neuroeffector junction. These events, diagrammed in Figure 8–3, are:
Steps involved in excitatory and inhibitory neurotransmission. 1. The nerve action potential (AP) consists of a transient self-propagated reversal of charge on the axonal membrane. (The internal potential Ei goes from a negative value, through zero potential, to a slightly positive value primarily through increases in Na+ permeability and then returns to resting values by an increase in K+ permeability.) When the AP arrives at the presynaptic terminal, it initiates release of the excitatory or inhibitory transmitter. Depolarization at the nerve ending and entry of Ca2+ initiate docking and then fusion of the synaptic vesicle with the membrane of the nerve ending. Docked and fused vesicles are shown. 2. Combination of the excitatory transmitter with postsynaptic receptors produces a localized depolarization, the excitatory postsynaptic potential (EPSP), through an increase in permeability to cations, most notably Na+. The inhibitory transmitter causes a selective increase in permeability to K+ or Cl–, resulting in a localized hyperpolarization, the inhibitory postsynaptic potential (IPSP). 3. The EPSP initiates a conducted AP in the postsynaptic neuron; this can be prevented, however, by the hyperpolarization induced by a concurrent IPSP. The transmitter is dissipated by enzymatic destruction, by reuptake into the presynaptic terminal or adjacent glial cells, or by diffusion. Depolarization of the postsynaptic membrane can permit Ca2+ entry if voltage-gated Ca2+ channels are present. (Reproduced with permission from Brunton L, Parker K, Blumenthal D, Buxton I (eds). Goodman & Gilman's Manual of Pharmacology and Therapeutics. New York: McGraw-Hill, 2008, p 94. Copyright © 2008 by The McGraw-Hill Companies, Inc. All rights reserved.)
Storage and release of the transmitter. The non-peptide (small molecule) neurotransmitters are largely synthesized in the region of the axonal terminals and stored there in synaptic vesicles. Peptide neurotransmitters (or precursor peptides) are found in large dense-core vesicles that are transported down the axon from their site of synthesis in the cell body. During the resting state, there is a continual slow release of isolated quanta of the transmitter; this produces electrical responses at the postjunctional membrane [miniature end-plate potentials (mepps)] that are associated with the maintenance of physiological responsiveness of the effector organ. A low level of spontaneous activity within the motor units of skeletal muscle is particularly important because skeletal muscle lacks inherent tone. The action potential causes the synchronous release of several hundred quanta of neurotransmitter. Depolarization of the axonal terminal triggers this process; a critical step in most nerve endings is the influx of Ca2+, which enters the axonal cytoplasm and promotes fusion between the axoplasmic membrane and those vesicles in close proximity to it (Meir et al., 1999). The contents of the vesicles, including enzymes and other proteins, then are discharged to the exterior by a process termed exocytosis. Synaptic vesicles may either fully exocytose with complete fusion and subsequent endocytosis or form a transient pore that closes after transmitter has escaped (Murthy and Stevens, 1998).
Synaptic vesicles are clustered in discrete areas underlying the presynaptic plasma membrane, termed active zones; they often are aligned with the tips of postsynaptic folds. Some 20-40 proteins, playing distinct roles as transporter or trafficking proteins, are found in the vesicle. Neurotransmitter transport into the vesicle is driven by an electrochemical gradient generated by the vacuolar proton pump.
The vesicle protein synaptobrevin (VAMP) assembles with the plasma membrane proteins SNAP-25 and syntaxin 1 to form a core complex that initiates or drives the vesicle–plasma membrane fusion process (Jahn et al., 2003). The submillisecond triggering of exocytosis by Ca2+ appears to be mediated by a separate family of proteins, the synaptotagmins. GTP-binding proteins of the Rab 3 family regulate the fusion. Several other regulatory proteins of less well-defined function—synapsin, synaptophysin, and synaptogyrin—also play a role in fusion and exocytosis, as do proteins such as RIM and neurexin that are found on the active zones of the plasma membrane. Many of the trafficking proteins are homologous to those used in vesicular transport in yeast.
An extensive variety of receptors has been identified on soma, dendrites, and axons of neurons, where they respond to neurotransmitters or modulators released from the same neuron or from adjacent neurons or cells (Miller, 1998; Westfall, 2004). Soma–dendritic receptors are those receptors located on or near the cell body and dendrites; when activated, they primarily modify functions of the soma–dendritic region such as protein synthesis and generation of action potentials. Presynaptic receptors are those presumed to be located on, in, or near axon terminals or varicosities; when activated, they modify functions of the terminal region such as synthesis and release of transmitters. Two main classes of presynaptic receptors have been identified on most neurons, including sympathetic and parasympathetic terminals. Heteroreceptors are presynaptic receptors that respond to neurotransmitters, neuromodulators, or neurohormones released from adjacent neurons or cells. For example, NE can influence the release of ACh from parasympathetic neurons by acting on α2A, α2B, and α2C receptors, whereas ACh can influence the release of NE from sympathetic neurons by acting on M2 and M4receptors (described later in the chapter). The other class of presynaptic receptors consists of autoreceptors, which are receptors located on or close to those axon terminals of a neuron through which the neuron's own transmitter can modify transmitter synthesis and release. For example, NE released from sympathetic neurons may interact with α2A and α2C receptors to inhibit neurally released NE. Similarly, ACh released from parasympathetic neurons may interact with M2 and M4 receptors to inhibit neurally released ACh.
Presynaptic nicotinic receptors enhance transmitter release in motor neurons (Bowman et al., 1990) and in a variety of other central and peripheral synapses (MacDermott et al., 1999).
Adenosine, dopamine (DA), glutamate, γ-aminobutyric acid (GABA), prostaglandins, and enkephalins have been shown to influence neurally mediated release of various neurotransmitters. The receptors for these agents exert their modulatory effects in part by altering the function of prejunctional ion channels (Miller, 1998; Tsien et al., 1988). A number of ion channels that directly control transmitter release are found in presynaptic terminals (Meir et al., 1999).
Combination of the transmitter with postjunctional receptors and production of the postjunctional potential. The transmitter diffuses across the synaptic or junctional cleft and combines with specialized receptors on the postjunctional membrane; this often results in a localized increase in the ionic permeability, or conductance, of the membrane. With certain exceptions (noted in the following discussion), one of three types of permeability change can occur:
a generalized increase in the permeability to cations (notably Na+ but occasionally Ca2+), resulting in a localized depolarization of the membrane, that is, an excitatory postsynaptic potential (EPSP)
a selective increase in permeability to anions, usually Cl–, resulting in stabilization or actual hyperpolarization of the membrane, which constitutes an inhibitory postsynaptic potential (IPSP)
an increased permeability to K+. Because the K+ gradient is directed out of the cell, hyperpolarization and stabilization of the membrane potential occur (an IPSP)
The potential changes associated with the EPSP and IPSP at most sites are the results of passive fluxes of ions down their concentration gradients. The changes in channel permeability that cause these potential changes are specifically regulated by the specialized postjunctional receptors for the neurotransmitter that initiates the response (see Figures 8–4, 8–5 11–5, and Chapter 14). These receptors may be clustered on the effector cell surface, as seen at the neuromuscular junctions of skeletal muscle and other discrete synapses, or distributed more uniformly, as observed in smooth muscle.
By using microelectrodes that form high-resistance seals on the surface of cells, electrical events associated with a single neurotransmitter-gated channel can be recorded (Hille, 1992). In the presence of an appropriate neurotransmitter, the channel opens rapidly to a high-conductance state, remains open for about a millisecond, and then closes. A short square-wave pulse of current is observed as a result of the channel's opening and closing. The summation of these microscopic events gives rise to the EPSP. The graded response to a neurotransmitter usually is related to the frequency of opening events rather than to the extent of opening or the duration of opening. High-conductance ligand-gated ion channels usually permit passage of Na+ or Cl–; K+ and Ca2+ are involved less frequently. The preceding ligand-gated channels belong to a large superfamily of ionotropic receptor proteins that includes the nicotinic, glutamate, and certain serotonin (5-HT3) and purine receptors, which conduct primarily Na+, cause depolarization, and are excitatory; and GABA and glycine receptors, which conduct Cl–, cause hyperpolarization, and are inhibitory. The nicotinic, GABA, glycine, and 5-HT3 receptors are closely related, whereas the glutamate and purinergic ionotropic receptors have distinct structures (Karlin and Akabas, 1995). Neurotransmitters also can modulate the permeability of K+ and Ca2+ channels indirectly. In these cases, the receptor and channel are separate proteins, and information is conveyed between them by G proteins. Other receptors for neurotransmitters act by influencing the synthesis of intracellular second messengers and do not necessarily cause a change in membrane potential. The most widely documented examples of receptor regulation of second-messenger systems are the activation or inhibition of adenylyl cyclase to modulate cellular cyclic AMP concentrations and the increase in cytosolic concentrations of Ca2+ that results from release of the ion from internal stores by inositol trisphosphate (see Chapter 3).
Initiation of postjunctional activity. If an EPSP exceeds a certain threshold value, it initiates a propagated action potential in a postsynaptic neuron or a muscle action potential in skeletal or cardiac muscle by activating voltage-sensitive channels in the immediate vicinity. In certain smooth muscle types in which propagated impulses are minimal, an EPSP may increase the rate of spontaneous depolarization, cause Ca2+ release, and enhance muscle tone; in gland cells, the EPSP initiates secretion through Ca2+ mobilization. An IPSP, which is found in neurons and smooth muscle but not in skeletal muscle, will tend to oppose excitatory potentials simultaneously initiated by other neuronal sources. Whether a propagated impulse or other response ensues depends on the summation of all the potentials.
Destruction or dissipation of the transmitter. When impulses can be transmitted across junctions at frequencies up to several hundred per second, there must be an efficient means of disposing of the transmitter following each impulse. At cholinergic synapses involved in rapid neurotransmission, high and localized concentrations of acetylcholinesterase (AChE) are available for this purpose. When AChE activity is inhibited, removal of the transmitter is accomplished principally by diffusion. Under these circumstances, the effects of released ACh are potentiated and prolonged (see Chapter 10).
Rapid termination of NE occurs by a combination of simple diffusion and reuptake by the axonal terminals of most of the released norepinephrine. Termination of the action of amino acid transmitters results from their active transport into neurons and surrounding glia. Peptide neurotransmitters are hydrolyzed by various peptidases and dissipated by diffusion; specific uptake mechanisms have not been demonstrated for these substances.
Non-electrogenic functions. The continual quantal release of neurotransmitters in amounts insufficient to elicit a postjunctional response probably is important in the transjunctional control of neurotransmitter action. The activity and turnover of enzymes involved in the synthesis and inactivation of neurotransmitters, the density of presynaptic and postsynaptic receptors, and other characteristics of synapses probably are controlled by trophic actions of neurotransmitters or other trophic factors released by the neuron or the target cells (Sanes and Lichtman, 1999).
A cholinergic neuroeffector junction showing features of the synthesis, storage, and release of acetylcholine (ACh) and receptors on which ACh acts. The synthesis of ACh in the varicosity depends on the uptake of choline viaa sodium-dependent carrier. This uptake can be blocked by hemicholinium. Choline and the acetyl moiety of acetyl coenzyme A, derived from mitochondria, form ACh, a process catalyzed by the enzyme choline acetyl transferase (ChAT). ACh is transported into the storage vesicle by another carrier that can be inhibited by vesamicol. ACh is stored in vesicles along with other potential cotransmitters (Co-T) such as ATP and VIP at certain neuroeffector junctions. Release of ACh and the Co-T occurs on depolarization of the varicosity, which allows the entry of Ca2+ through voltage-dependent Ca2+ channels. Elevated [Ca2+]in promotes fusion of the vesicular membrane with the cell membrane, and exocytosis of the transmitters occurs. This fusion process involves the interaction of specialized proteins associated with the vesicular membrane (VAMPs, vesicle-associated membrane proteins) and the membrane of the varicosity (SNAPs, synaptosome-associated proteins). The exocytotic release of ACh can be blocked by botulinum toxin. Once released, ACh can interact with the muscarinic receptors (M), which are GPCRs, or nicotinic receptors (N), which are ligand-gated ion channels, to produce the characteristic response of the effector. ACh also can act on presynaptic mAChRs or nAChRs to modify its own release. The action of ACh is terminated by metabolism to choline and acetate by acetylcholinesterase (AChE), which is associated with synaptic membranes.
The synthesis, storage, and release of ACh follow a similar life cycle in all cholinergic synapses, including those at skeletal neuromuscular junctions, preganglionic sympathetic and parasympathetic terminals, postganglionic parasympathetic varicosities, postganglionic sympathetic varicosities innervating sweat glands in the skin, and in the CNS. The neurochemical events that underlie cholinergic neurotransmission are summarized in Figure 8–4. Two enzymes, choline acetyltransferase and AChE, are involved in ACh synthesis and degradation, respectively.
Choline Acetyltransferase. Choline acetyltransferase catalyzes the final step in the synthesis of ACh—the acetylation of choline with acetyl coenzyme A (CoA) (Wu and Hersh, 1994). The primary structure of choline acetyltransferase is known from molecular cloning, and its immunocytochemical localization has proven useful for identification of cholinergic axons and nerve cell bodies.
Acetyl CoA for this reaction is derived from pyruvate via the multistep pyruvate dehydrogenase reaction or is synthesized by acetate thiokinase, which catalyzes the reaction of acetate with ATP to form an enzyme-bound acyladenylate (acetyl AMP). In the presence of CoA, transacetylation and synthesis of acetyl CoA proceed.
Choline acetyltransferase, like other protein constituents of the neuron, is synthesized within the perikaryon and then is transported along the length of the axon to its terminal. Axonal terminals contain a large number of mitochondria, where acetyl CoA is synthesized. Choline is taken up from the extracellular fluid into the axoplasm by active transport. The final step in the synthesis occurs within the cytoplasm, following which most of the ACh is sequestered within synaptic vesicles. Although moderately potent inhibitors of choline acetyltransferase exist, they have no therapeutic utility, in part because the uptake of choline is the rate-limiting step in ACh biosynthesis.
Choline and Choline Transport. The availability of choline is critical to the synthesis of acetylcholine and is provided from the diet as there is little de novo synthesis of choline in cholinergic neurons. Choline is also an essential component for the normal function of all cells, necessary for the structural integrity and signaling functions for cell membranes. Choline is taken up from the extracellular space by two transport systems: a ubiquitous low affinity, Na+-independent transport system that is inhibited by hemicholinium-3 with a Ki of ∼50 μM, and high affinity Na+- and Cl--dependent choline transport system that is also sensitive to inhibition by hemicholinium-3 (Ki= 10-100 nM). This second transport system is found predominantly in cholinergic neurons and is responsible for providing choline for ACh synthesis. Once ACh is released from cholinergic neurons following the arrival of action potentials, ACh is hydrolyzed by acetylcholine esterase (AChE) to acetate and choline. Choline is recycled after reuptake into the nerve terminal of cholinergic cells and reused for ACh synthesis. Under many circumstances this reuptake and availability of choline appear to serve as the rate limiting step in acetylcholine synthesis.
The gene for the high-affinity choline transporter (CHT1) has been cloned from a variety of species including human and it is homologous to the Na+-dependent glucose transport family. The location of CHT1 is in intracellular vesicular structures rather than the nerve terminal plasma membrane and co-localizes with synaptic vesicle markers such as vesicle-associated membrane protein 2 (VAMP2) and vesicular ACh transporter (VAChT). Although the details are incomplete there is evidence that in response to the cascade of events that culminate in transmitter release, there is also increased trafficking of CHT1 to the plasma membrane, where it functions to take up choline after hydrolysis of acetylcholine. Since choline transport is rate limiting for acetylcholine synthesis, increased availability of choline via its transport by CHT1 would favor an increase in ACh stores to maintain high levels of transmitter release during neuronal stimulation. This also suggests that the availability of CHT1 at the cell surface is dynamically regulated in a manner very similar to the regulation of the exocytosis of synaptic vesicles. The precise mechanisms involved in maintaining the distribution of CHT1 predominantly in intracellular vesicles rather than at the terminal surface like other neurotransmitter transporters are unclear (Ferguson and Blakely, 2004; Chaudhry et al. 2008).
Storage of Acetylcholine. Following the synthesis of acetylcholine, which takes place in the cytoplasm of the nerve terminal, ACh is transported into synaptic vesicles by VAChT using a proton electrochemical gradient to move ACh to the inside of the organelle. VAChT is thought to be a protein comprising 12 transmembrane domains with hydrophilic N- and C-termini in the cytoplasm. By sequence homology, VAChT appears to be a member of a family of transport proteins that includes two vesicular monoamine transporters. Transport of protons out of the vesicle is coupled to uptake of ACh into the vesicle and against a concentration gradient via the acetylcholine antiporter.
There appear to be two types of vesicles in cholinergic terminals: electron-lucent vesicles (40-50 nm in diameter) and dense-cored vesicles (80-150 nm). The core of the vesicles contains both ACh and ATP, at an estimated ratio of 10:1, which are dissolved in the fluid phase with metal ions (Ca2+ and Mg2+) and a proteoglycan called vesiculin. Vesiculin is negatively charged and is thought to sequester the Ca2+ or ACh. It is bound within the vesicle, with the protein moiety anchoring it to the vesicular membrane. In some cholinergic terminals there are peptides, such as VIP, that act as co-transmitters at some junctions. The peptides usually are located in the dense-cored vesicles. Vesicular membranes are rich in lipids, primarily cholesterol and phospholipids, as well as protein. The proteins include ATPase, which is ouabain-sensitive and thought to be involved in proton pumping and in vesicular inward transport of Ca2+. Other proteins include protein kinases (involved in phosphorylation mechanisms of Ca2+ uptake), calmodulin, atractyloside-binding protein (which acts as an ATP carrier), and synapsin (which is thought to be involved with exocytosis).
The vesicular transporter allows for the uptake of ACh into the vesicle, has considerable concentrating power, is saturable, and is ATPase-dependent. The process is inhibited by vesamicol (Figure 8–4). Inhibition by vesamicol is noncompetitive and reversible and does not affect the vesicular ATPase. The gene for choline acetyltransferase and the vesicular transporter are found at the same locus, with the transporter gene positioned in the first intron of the transferase gene. Hence a common promoter regulates the expression of both genes (Eiden, 1998).
Estimates of the ACh content of synaptic vesicles range from 1000 to over 50,000 molecules per vesicle, and it has been calculated that a single motor nerve terminal contains 300,000 or more vesicles. In addition, an uncertain but possibly significant amount of ACh is present in the extravesicular cytoplasm. Recording the electrical events associated with the opening of single channels at the motor end plate during continuous application of ACh has permitted estimation of the potential change induced by a single molecule of ACh (3 × 10−7 V); from such calculations it is evident that even the lower estimate of the ACh content per vesicle (1000 molecules) is sufficient to account for the magnitude of the mepps.
Release of Acetylcholine. Release of acetylcholine and co-transmitters (e.g., ATP and VIP or in some neurons, NO) occurs on depolarization of the nerve terminals and takes place by exocytosis. Depolarization of the terminals allows the entry of Ca2+ through voltage-gated Ca2+ channels. Elevated Ca2+ concentration promotes fusion of the vesicular membrane with the plasma membrane, allowing exocytosis to occur.
The molecular mechanisms involved in the release and regulation of release are not completely understood (Südhof, 2004). ACh, like other neurotransmitters, is stored in vesicles located at special release sites, close to presynaptic membranes and ready for release following the appropriate stimulus. The vesicles initially dock and are primed for release. A multiprotein complex appears to form and attach the vesicle to the plasma membrane close to other signaling elements. The complex involves proteins from the vesicular membrane and the presynaptic neuronal membrane, as well as other components that help link them together. Various synaptic proteins, including the plasma membrane protein syntaxin and synaptosomal protein 25 kDa (SNAP-25), and the vesicular membrane protein, synaptobrevin, form a complex. This complex interacts in an ATP-dependent manner with soluble N-ethylmalemide-sensitive fusion protein and soluble SNAPs. The ability of synaptobrevin, sytaxin, and SNAP-25 to bind SNAPs has led to their designation as SNAP regulators (SNARES). It has been hypothesized that most, if not all, intracellular fusion events are mediated by SNARE interactions. Important evidence supporting the involvement of SNARE proteins in transmitter release comes from the fact that botulinum neurotoxins and tetanus toxin, which block neurotransmitter release, proteolyze these three proteins (Südhof, 2004).
Two pools of acetylcholine appear to exist. One pool, the "depot" or "readily releasable" pool, consists of vesicles located near the plasma membrane of the nerve terminals; these vesicles contain newly synthesized transmitter. Depolarization of the terminals causes these vesicles to release ACh rapidly or readily. The other pool, the "reserve pool," seems to replenish the readily releasable pool and may be required to sustain ACh release during periods of prolonged or intense nerve stimulation.
Acetylcholinesterase (AChE). For ACh to serve as a neurotransmitter in the motor system and at other neuronal synapses, it must be removed or inactivated within the time limits imposed by the response characteristics of the synapse. At the neuromuscular junction, immediate removal is required to prevent lateral diffusion and sequential activation of adjacent receptors. Modern biophysical methods have revealed that the time required for hydrolysis of ACh at the neuromuscular junction is less than a millisecond. The Km of AChE for ACh is ∼50-100 μM. Choline has only 10–3 to 10–5 the potency of ACh at the neuromuscular junction.
While AChE is found in cholinergic neurons (dendrites, perikarya, and axons), it is distributed more widely than cholinergic synapses. It is highly concentrated at the postsynaptic end plate of the neuromuscular junction. Butyrylcholinesterase (BuChE; also known as pseudocholinesterase) is present in low abundance in glial or satellite cells but is virtually absent in neuronal elements of the central and peripheral nervous systems. BuChE is synthesized primarily in the liver and is found in liver and plasma; its likely physiological function is the hydrolysis of ingested esters from plant sources. AChE and BuChE typically are distinguished by the relative rates of ACh and butyrylcholine hydrolysis and by effects of selective inhibitors (Chapter 10). Almost all pharmacological effects of the anti-ChE agents are due to the inhibition of AChE, with the consequent accumulation of endogenous ACh in the vicinity of the nerve terminal. Distinct but single genes encode AChE and BuChE in mammals; the diversity of molecular structure of AChE arises from alternative mRNA processing (Taylor et al., 2000).
Numerous reports suggest that AChE plays other roles in addition to its classical function in terminating impulse transmission at cholinergic synapses. Non-classical functions of AChE might include hydrolysis of ACh in a non-synaptic context, action as an adhesion protein involved in synaptic development and maintenance, as a bone matrix protein, involvement in neurite outgrowth, and acceleration of the assembly of Aβ peptide into amyloid fibrils (Silman and Sussman, 2005).
Characteristics of Cholinergic Transmission at Various Sites. There are marked differences among various sites of cholinergic transmission with respect to architecture and fine structure, the distributions of AChE and receptors, and the temporal factors involved in normal function. In skeletal muscle, e.g., the junctional sites occupy a small, discrete portion of the surface of the individual fibers and are relatively isolated from those of adjacent fibers; in the superior cervical ganglion, ∼100,000 ganglion cells are packed within a volume of a few cubic millimeters, and both the presynaptic and postsynaptic neuronal processes form complex networks.
Skeletal Muscle. Stimulation of a motor nerve results in the release of ACh from perfused muscle; close intra-arterial injection of ACh produces muscular contraction similar to that elicited by stimulation of the motor nerve. The amount of ACh (10–17mol) required to elicit an end-plate potential (EPP) following its microiontophoretic application to the motor end plate of a rat diaphragm muscle fiber is equivalent to that recovered from each fiber following stimulation of the phrenic nerve.
The combination of ACh with nicotinic ACh receptors at the external surface of the postjunctional membrane induces an immediate, marked increase in cation permeability. On receptor activation by ACh, its intrinsic channel opens for ∼1 ms; during this interval, ∼50,000 Na+ ions traverse the channel. The channel-opening process is the basis for the localized depolarizing EPP within the end plate, which triggers the muscle AP. The latter, in turn, leads to contraction.
Following section and degeneration of the motor nerve to skeletal muscle or of the postganglionic fibers to autonomic effectors, there is a marked reduction in the threshold doses of the transmitters and of certain other drugs required to elicit a response; that is, denervation supersensitivity occurs. In skeletal muscle, this change is accompanied by a spread of the receptor molecules from the end-plate region to the adjacent portions of the sarcoplasmic membrane, which eventually involves the entire muscle surface. Embryonic muscle also exhibits this uniform sensitivity to ACh prior to innervation. Hence, innervation represses the expression of the receptor gene by the nuclei that lie in extrajunctional regions of the muscle fiber and directs the subsynaptic nuclei to express the structural and functional proteins of the synapse (Sanes and Lichtman, 1999).
Autonomic Effector Cells. Stimulation or inhibition of autonomic effector cells occurs on activation of muscarinic acetylcholine receptors (discussed later in the chapter). In this case, the effector is coupled to the receptor by a G protein (Chapter 3). In contrast to skeletal muscle and neurons, smooth muscle and the cardiac conduction system [sinoatrial (SA) node, atrium, atrioventricular (AV) node, and the His-Purkinje system] normally exhibit intrinsic activity, both electrical and mechanical, that is modulated but not initiated by nerve impulses.
In the basal condition, unitary smooth muscle exhibits waves of depolarization and/or spikes that are propagated from cell to cell at rates considerably slower than the AP of axons or skeletal muscle. The spikes apparently are initiated by rhythmic fluctuations in the membrane resting potential. Application of ACh (0.1 to 1 μM) to isolated intestinal muscle causes a decrease in the resting potential (i.e., the membrane potential becomes less negative) and an increase in the frequency of spike production, accompanied by a rise in tension. A primary action of ACh in initiating these effects through muscarinic receptors is probably partial depolarization of the cell membrane brought about by an increase in Na+ and, in some instances, Ca2+ conductance. ACh also can produce contraction of some smooth muscles when the membrane has been depolarized completely by high concentrations of K+, provided that Ca2+ is present. Hence, ACh stimulates ion fluxes across membranes and/or mobilizes intracellular Ca2+ to cause contraction.
In the heart, spontaneous depolarizations normally arise from the SA node. In the cardiac conduction system, particularly in the SA and AV nodes, stimulation of the cholinergic innervation or the direct application of ACh causes inhibition, associated with hyper-polarization of the membrane and a marked decrease in the rate of depolarization. These effects are due, at least in part, to a selective increase in permeability to K+ (Hille, 1992).
Autonomic Ganglia. The primary pathway of cholinergic transmission in autonomic ganglia is similar to that at the neuromuscular junction of skeletal muscle. Ganglion cells can be discharged by injecting very small amounts of ACh into the ganglion. The initial depolarization is the result of activation of nicotinic ACh receptors, which are ligand-gated cation channels with properties similar to those found at the neuromuscular junction. Several secondary transmitters or modulators either enhance or diminish the sensitivity of the postganglionic cell to ACh. This sensitivity appears to be related to the membrane potential of the postsynaptic nerve cell body or its dendritic branches. Ganglionic transmission is discussed in more detail in Chapter 11.
Prejunctional Sites. As described earlier, both cholinergic and adrenergic nerve terminal varicosities contain autoreceptors and heteroreceptors. ACh release therefore is subject to complex regulation by mediators, including ACh itself acting on M2 and M4 autoreceptors, and other transmitters (e.g., NE acting on α2A and α2C adrenergic receptors) (Philipp and Hein, 2004; Wess, 2004) or substances produced locally in tissues (e.g., NO). ACh-mediated inhibition of ACh release following activation of M2 and M4 autoreceptors is thought to represent a physiological negative-feedback control mechanism. At some neuroeffector junctions such as the myenteric plexus in the GI tract or the SA node in the heart, sympathetic and parasympathetic nerve terminals often lie juxtaposed to each other. The opposing effects of norepinephrine and ACh, therefore, result not only from the opposite effects of the two transmitters on the smooth muscle or cardiac cells but also from the inhibition of ACh release by NE or inhibition of NE release by ACh acting on heteroreceptors on parasympathetic or sympathetic terminals. The muscarinic autoreceptors and heteroreceptors also represent drug targets for both agonists and antagonists. Muscarinic agonists can inhibit the electrically induced release of ACh, whereas antagonists will enhance the evoked release of transmitter. The parasympathetic nerve terminal varicosities also may contain additional heteroreceptors that could respond by inhibition or enhancement of ACh release by locally formed autacoids, hormones, or administered drugs. In addition to α2A and α2C adrenergic receptors, other inhibitory heteroreceptors on parasympathetic terminals include adenosine A1receptors, histamine H3 receptors, and opioid receptors. Evidence also exists for β2-adrenergic facilitatory receptors.
Extraneuronal Sites. A large body of evidence now indicates that all elements of the cholinergic system including the enzyme ChAT, ACh synthesis, ACh release mechanisms, and both mAChRs and nAChRs, are functionally expressed independently of cholinergic innervation in numerous non-neuronal cells including those of humans. These non-neuronal cholinergic systems can both modify and control phenotypic cell functions such as proliferation, differentiation, formation of physical barriers, migration, and ion and water movements. The widespread synthesis of ACh in non-neuronal cells has changed the thinking that ACh acts only as a neurotransmitter. Each component of the cholinergic system in non-neuronal cells can be affected by pathophysiological conditions or secondary to disease states. For instance, blockade of mAChRs and nAChRs on non-neuronal cells can cause cellular dysfunction and cell death. Moreover, dysfunctions of non-neuronal cholinergic systems may be involved in the pathogenesis of diseases (e.g., inflammatory processes) (Kalamida et al., 2007; Wessler and Kirkpatrick, 2008).
Cholinergic Receptors and Signal Transduction
Sir Henry Dale noted that the various esters of choline elicited responses that were similar to those of either nicotine or muscarine depending on the pharmacological preparation. A similarity in response also was noted between muscarine and nerve stimulation in those organs innervated by the craniosacral divisions of the autonomic nervous system. Thus, Dale suggested that ACh or another ester of choline was a neurotransmitter in the autonomic nervous system; he also stated that the compound had dual actions, which he termed a "nicotine action" (nicotinic) and a "muscarine action" (muscarinic).
The capacities of tubocurarine and atropine to block nicotinic and muscarinic effects of ACh, respectively, provided further support for the proposal of two distinct types of cholinergic receptors. Although Dale had access only to crude plant alkaloids of then unknown structure from Amanita muscaria and Nicotiana tabacum, this classification remains as the primary subdivision of cholinergic receptors. Its utility has survived the discovery of several distinct subtypes of nicotinic and muscarinic receptors.
Although ACh and certain other compounds stimulate both muscarinic and nicotinic cholinergic receptors, several other agonists and antagonists are selective for one of the two major types of receptors. ACh is a flexible molecule, and indirect evidence suggests that the conformations of the neurotransmitter are distinct when it is bound to nicotinic or muscarinic cholinergic receptors.
Nicotinic receptors are ligand-gated ion channels whose activation always causes a rapid (millisecond) increase in cellular permeability to Na+ and Ca2+, depolarization, and excitation. By contrast, muscarinic receptors are G protein–coupled receptors (GPCRs). Responses to muscarinic agonists are slower; they may be either excitatory or inhibitory, and they are not necessarily linked to changes in ion permeability.
The primary structures of various species of nicotinic receptors (Changeux and Edelstein, 1998; Numa et al., 1983) and muscarinic receptors (Bonner, 1989; Caulfield and Birdsall, 1998) have been deduced from the sequences of their respective genes. That these two types of receptors belong to distinct families of proteins is not surprising, retrospectively, in view of their distinct differences in chemical specificity and function.
Subtypes of Nicotinic Acetylcholine Receptors. The nicotinic ACh receptors (nAChRs) are members of a superfamily of ligand-gated ion channels. The receptors exist at the skeletal neuromuscular junction, autonomic ganglia, adrenal medulla, the CNS and in non-neuronal tissues. The nAChRs are composed of five homologous subunits organized around a central pore (see Chaper 11). In general the nAChRs are further divided into two groups:
muscle type, found in vertebrate skeletal muscle, where they mediate transmission at the neuromuscular junction (NMJ)
neuronal type, found mainly throughout the peripheral nervous system, central nervous system, and also non-neuronal tissues
Both the mAChRs and nAChRs are the natural targets of ACh, synthesized, stored, and released from cholinergic neurons as well as numerous pharmacologically administered drugs (agonists and antagonists), including the alkaloids muscarine and nicotine.
cDNAs for 17 types of nAChRs subunits have been cloned from several species. These consist of α subunits (α1-α10) that compose the main ligand binding sites, β (β1-4), γ, δ, and ∊ subunits. The nAChRs have been further divided into two main types based on different binding properties to the toxin α-bungarotoxin (αβgtx): 1) the αβgtx binding nAChRs, which can be either homopentamers of α7, α8, or α9 subunits or heteropentamers (e.g., α2β∊[γ]δ); and 2) nAChRs that do not bind αβgtx. These contain the α2-α6 and β2-β4 subunits, exist only as heteropentamers, and bind agonists with high affinity (Albuquerque et al., 2009).
Muscle-Type nAChRs. In fetal muscle prior to innervation, in adult muscle after denervation, and in fish electric organs, the nAChR subunit stoichiometry is (α1)2β1γδ, whereas in adult muscle the γ subunit is replaced by ∊ to give the (α1)2β1∊δ stoichiometry (Table 8–2). The γ/∊ and δ subunits are involved together with the α1 subunits in forming the ligand-binding sites and in the maintenance of cooperative interactions between the α1 subunit. Different affinities to the two binding sites are conferred by the presence of different non α-subunits. Binding of ACh to the αγ and αδ sites is thought to induce a conformational change predominantly in the α1 subunits which interacts with the transmembrane region to cause channel opening.
Neuronal-Type nAChRs. Neuronal nAChRs are widely expressed in peripheral ganglia, the adrenal medulla, numerous areas of the brain, and non-neuronal cells such as epithelial cells and cells of the immune system. To date, nine α (α2-α10) and three β (β2-β4) subunit genes have been cloned. The α7-α10 subunits are found either as homopentamers (of five α7, α8, and α9 subunits) or as heteropentamers of α7, α8, and α9/α10. By contrast, the α2-α6 and β2-β4 subunits form heteropentamers usually with (αx)2(βy)3 stoichiometry. The α5 and β3 subunits do not appear to be able to form functional receptors when expressed alone or in paired combinations with α or β subunits, respectively (Kalamida et al., 2007).
The precise function of many of the neuronal nAChRs in the brain is not known; presumably, the considerable molecular diversity of the subunits can result in numerous nAChRs being formed with different physiological properties. There are few examples of neuronal nAChRs in the CNS mediating fast signaling propogation in a manner similar to that at the NMJ, and it is thought that they act more as synaptic modulators. Neuronal nAChRs are widely distributed in the CNS and are found at presynaptic, perisynaptic, and postsynaptic sites. At pre- and perisynaptic sites, nAChRs appear to act as autoreceptors or heteoreceptors to regulate the release of several neurotransmitters (ACh, DA, NE, glutamate, and 5-HT) at several diverse sites throughout the brain (Exley and Cragg, 2008). The synaptic release of a particular neurotransmitter can be regulated by different neuronal type nAChR subtypes in different CNS regions. For instance, DA release from striatal and thalamic dopamine neurons can be controlled by the α4β;2 subtype or both α4β2 and α6β2β3 subtypes, respectively. In contrast, glutametergic neurotransmission is regulated everywhere by α7 nAChRs (Kalamida et al., 2007).
Table 8-2Characteristics of Subtypes of Nicotinic Acetylcholine Receptors (nAChRs) ||Download (.pdf) Table 8-2 Characteristics of Subtypes of Nicotinic Acetylcholine Receptors (nAChRs)
|RECEPTOR (Primary Receptor Subtype)a ||MAIN SYNAPTIC LOCATION ||MEMBRANE RESPONSE ||MOLECULAR MECHANISM ||AGONISTS ||ANTAGONISTS |
|Skeletal Muscle (Nm) (α1)2β1∊δ adult (α1)2 β1γδ fetal ||Skeletal neuromuscular junction postjunctional) ||Excitatory; end-plate depolarization; skeletal muscle contraction ||Increased cation permeability (Na+; K+) ||ACh Nicotine Succinylcholine ||Atracurium Vecuroniumd-Tubocurarine Pancuronium α-Conotoxin α-Bungarotoxin |
|Peripheral Neuronal (Nn) (α3)2(β4)3 ||Autonomic ganglia; adrenal medulla ||Excitatory; depolarization; firing of postganglion neuron; depolarization and secretion of catecholamines ||Increased cation permeability (Na+; K+) ||ACh Nicotine Epibatidine Dimethylphenyl-piperazinium ||Trimethaphan Mecamylamine |
|Central Neuronal (CNS) (α4)2(β4)3 (α-btox-insensitive) ||CNS; pre- and postjunctional ||Pre- and post- synaptic excitation Prejunctional control of transmitter release ||Increased cation permeability (Na+; K+) ||Cytosine, epibatidine Anatoxin A ||Mecamylamine Dihydro-β-erythrodine Erysodine Lophotoxin |
|(α7)5(α-btox-sensitive) ||CNS; Pre- and post-synaptic ||Pre- and post-synaptic excitation Prejunctional control of transmitter release ||Increased permeability (Ca2+) ||Anatoxin A ||Methyllycaconitine α-Bungarotoxin α-Conotoxin ImI |
Subtypes of Muscarinic Receptors. In mammals, five distinct subtypes of muscarinic ACh receptors (mAChRs) have been identified, each produced by a different gene. Like the different forms of nicotinic receptors, these variants have distinct anatomic locations in the periphery and CNS and differing chemical specificities. The mAChRs are GPCRs (see Table 8–3 for characteristics of the mAChRs and Chapter 9 for further details).
Table 8-3Characteristics of Muscarinic Acetylcholine Receptor Subtypes (mAChRs) ||Download (.pdf) Table 8-3 Characteristics of Muscarinic Acetylcholine Receptor Subtypes (mAChRs)
|RECEPTOR ||SIZE; CHROMOSOME ||CELLULAR AND TISSUE LOCATIONa ||CELLULAR RESPONSEb ||FUNCTIONAL RESPONSEc ||DISEASE RELEVANCE |
|M1460 aa llq 12-13 ||CNS; Most abundant in cerebral cortex, hippocampus, striatum and thalamus Autonomic ganglia Glands (gastric and salivary) Enteric nerves ||Couples by Gq/11 Activation of PLC; ↑IP3 and ↑ DAG → ↑Ca2+ and PKC Depolarization and excitation (↑sEPSP) Activation of PLD2, PLA2; ↑AA ||Increased cognitive function (learning and memory) Increased seizure activity Decrease in dopamine release and locomotion Increase in depolarization of autonomic ganglia Increase in secretions ||Alzheimer's disease Cognitive dysfunction Schizophrenia |
|M2 ||466 aa 7q 35-36 ||Widely expressed in CNS, hind brain, thalamus, cerebral cortex, hippocampus, striatum, heart, smooth muscle, autonomic nerve terminals ||Couples by Gi/Go (PTX-sensitive) Inhibition of AC, ↓ cAMP Activation of inwardly rectifying K+ channels Inhibition of voltage-gated Ca2+ channels Hyperpolarization and inhibition ||Heart: SA node: slowed spontaneous depolarization; hyperpolarization, ↓ HR AV node: decrease in conduction velocity Atrium: ↓ refractory period, ↓ contraction Ventricle: slight ↓ contraction Smooth muscle: ↑ Contraction Peripheral nerves: Neural inhibition via autoreceptors and heteroreceptor ↓ Ganglionic transmission. CNS: Neural inhibition ↑ Tremors; hypothermia; analgesia ||Alzheimer's disease Cognitive dysfunction Pain |
|M3 ||590 aa 1q 43-44 ||Widely expressed in CNS (< than other mAChRs), cerebral cortex, hippocampus Abundant in smooth muscle and glands Heart ||Couples by Gq/11 Activation of PLC; ↑IP3 and ↑ DAG → ↑Ca2+ and PKC Depolarization and excitation (↑ sEPSP) Activation of PLD2, PLA2; ↑AA ||Smooth muscle ↑ contraction (predominant in some, e.g. bladder) Glands: ↑ secretion (predominant in salivary gland) Increases food intake, body weight fat deposits Inhibition of DA release Synthesis of NO ||Chronic obstructive pulmonary disease (COPD) Urinary incontinence Irritable bowel disease |
|M4 ||479 aa 11p 12-11.2 ||Preferentially expressed in CNS, particularly forebrain, also striatum, cerebral cortex, hippocampus ||Couples by Gi/G0 (PTX-sensitive) Inhibition of AC, ↓ cAMP Activation of inwardly rectifying K+ channels Inhibition of voltage-gated Ca2+ channels Hyperpolarization and inhibition ||Autoreceptor- and heteroreceptor-mediated inhibition of transmitter release in CNS and periphery. Analgesia; cataleptic activity Facilitation of DA release ||Parkinson disease Schizophrenia Neuropathic pain |
|M5 ||532 aa 15q 26 ||Substantia nigra Expressed in low levels in CNS and periphery Predominant mAchR in neurons in VTA and substantia nigra ||Couples by Gq/11 Activation of PLC; ↑IP3 and ↑DAG → ↑Ca2+ and PKC Depolarization and excitation (↑sEPSP) Activation of PLD2, PLA2; ↑ AA ||Mediator of dilation in cerebral arteries and arterioles (?) Facilitates DA release Augmentation of drug-seeking behavior and reward (e.g., opiates, cocaine) ||Drug dependence Parkinson disease Schizophrenia |
Different experimental approaches including immunohistochemical and mRNA hybridization studies have shown that mAChRs are present in virtually all organs, tissues, and cell types (Table 8–3). Although most cell types have multiple mAChR subtypes, certain subtypes often predominate in specific sites. For example, the M2 receptor is the predominant subtype in the heart and in CNS neurons is mostly located presynaptically, whereas the M3 receptor is the predominant subtype in the detrusor muscle of the bladder (Dhein et al., 2001; Fetscher et al., 2002) (see Table 8–3). M1, M4, and M5 receptors are richly expressed in the CNS, whereas the M2 and M4 receptor subtypes are widely distributed both in the CNS and peripheral tissue (Wess et al., 2007). M3 receptors are widely distributed in the periphery, and although they are also widely distributed in the CNS, they are at lower levels than other subtypes.
In the periphery, mAChRs mediate the classical muscarinic actions of ACh in organs and tissues innervated by parasympathetic nerves, although receptors may be present at sites that lack parasympathetic innervation (e.g., most blood vessels). In the CNS, mAChRs are involved in regulating a large number of cognitive, behavioral, sensory, motor, and autonomic functions. Owing to the lack of specific muscarinic agonists and antagonists that demonstrate selectivity for individual mAChRs and the fact that most organs and tissues express multiple mAChRs, it has been a challenge to assign specific pharmacological functions to distinct mAChRs. The development of gene-targeting techniques in mice has been very helpful in defining specific functions (Table 8–3) (Wess, 2004).
The basic functions of muscarinic cholinergic receptors are mediated by interactions with G proteins and by G protein–induced changes in the function of distinct member-bound effector molecules. The M1, M3, and M5 subtypes couple through the pertussis toxin–insensitive Gq/11responsible for stimulation of phospholipase C (PLC) activity. The immediate result is hydrolysis of membrane phosphatidylinositol 4,5 diphosphate to form inositol polyphosphates. Inositol trisphosphate (IP3) causes release of intracellular Ca2+ from the endoplasmic reticulum, with activation of Ca2+-dependent phenomena such as contraction of smooth muscle and secretion (Chapter 3). The second product of the PLC reaction, diacylglycerol, activates PKC (in conjunction with Ca2+ and phosphatidylserine). This arm of the pathway plays a role in the phosphorylation of numerous proteins, leading to various physiological responses. Activation of M1, M3, and M5 receptors can also cause the activation of phospholipase A2, leading to the release of arachidonic acid and consequent eicosanoid synthesis, resulting in autocrine/paracrine stimulation of adenylyl cyclase and an increase in cyclic AMP. These effects of M1, M3, and M5 mAChRs are generally secondary to elevations of intracellular Ca++ (Eglen, 2005).
Stimulation of M2 and M4 cholinergic receptors leads to interaction with other G proteins, (e.g., Gi and Go) with a resulting inhibition of adenylyl cyclase, leading to a decrease in cyclic AMP, activation of inwardly rectifying K+ channels, and inhibition of voltage-gated Ca2+ channels (van Koppen and Kaiser, 2003). The functional consequences of these effects are hyperpolarization and inhibition of excitable membranes. These are most clear in myocardium, where inhibition of adenylyl cyclase and activation of K+ conductances account for the negative inotropic and chronotropic effects of ACh. The specificity is not absolute, however, and depends on proper trafficking of the G protein subunits within the cell; consequently, heterologous systems may exhibit alternative interactions between mAChRs and G–protein coupled pathways (Nathanson, 2008). In addition, there are numerous reports suggesting the differential subcellular location of specific mAChR subtypes in a variety of cell types in the nervous system and in a variety of non-neuronal polarized cells.
Following activation by classical or allosteric agonists, mAChRs can be phosphorylated by a variety of receptor kinases and second-messenger regulated kinases; the phosphorylated mAChR subtypes then can interact with β-arrestin and possibly other adaptor proteins. As a result, mAChR signaling pathways may be differentially altered. Agonist activation of mAChRs also may induce receptor internalization and down-regulation (van Koppen and Kaiser, 2003). Muscarinic AChRs can also regulate other signal transduction pathways that have diverse effects on cell growth, survival, and physiology, such as the MAP kinase, phosphoinositide-3-kinase, RhoA, and Rac1 (Nathanson, 2008).
Changes in mAChR levels and activity have been implicated in the pathophysiology of numerous major diseases in the CNS and in the autonomic nervous system (Table 8–3). Phenotypic analysis of mAChR-mutant mice as well as the development of selective agonists and antagonists has led to a wealth of new information regarding the physiological and potential pathophysiological roles of the individual mAChR subtype (Langmead et al., 2008; Wess et al., 2007).
Under this general heading are norepinephrine (NE), the principal transmitter of most sympathetic postganglionic fibers and of certain tracts in the CNS; dopamine (DA), the predominant transmitter of the mammalian extrapyramidal system and of several mesocortical and mesolimbic neuronal pathways; and epinephrine, the major hormone of the adrenal medulla. Collectively, these three amines are called catecholamines.
A tremendous amount of information about catecholamines and related compounds has accumulated in recent years partly because of the importance of interactions between the endogenous catecholamines and many of the drugs used in the treatment of hypertension, mental disorders, and a variety of other conditions. The details of these interactions and of the pharmacology of the sympathomimetic amines themselves will be found in subsequent chapters. The basic physiological, biochemical, and pharmacological features are presented here.
Synthesis of Catecholamines. The steps in the synthesis of DA, NE (noradrenaline), and epinephrine (adrenaline) are shown in Figure 8–5. Tyrosine is sequentially 3-hydroxylated and decarboxylated to form dopamine. Dopamine isβ-hydroxylated to yield norepinephrine, which is N-methylated in chromaffin tissue to give epinephrine. The enzymes involved have been identified, cloned, and characterized (Nagatsu, 1991). Table 8–4 summarizes some of the important characteristics of the four enzymes. These enzymes are not completely specific; consequently, other endogenous substances, as well as certain drugs, are also substrates. For example, 5-hydroxytryptamine (5-HT, serotonin) can be produced from 5-hydroxy-L-tryptophan by aromatic L-amino acid decarboxylase (or dopa decarboxylase). Dopa decarboxylase also converts dopa into DA (Chapter 13) and methyldopa to α-methyldopamine, which in turn is converted by dopamine β-hydroxylase (DβH) to methylnorepinephrine.
Table 8-4Enzymes for Synthesis of Catecholamines ||Download (.pdf) Table 8-4 Enzymes for Synthesis of Catecholamines
|ENZYME ||OCCURRENCE ||SUBCELLULAR DISTRIBUTION ||COFACTOR REQUIREMENT ||SUBSTRATE SPECIFICITY ||COMMENTS |
|Tyrosine hydroxylase ||Widespread; sympathetic nerves ||Cytoplasmic ||Tetrahydrobiopterin, O2, Fe2+ ||Specific for l-tyrosine ||Rate limiting step. Inhibition can deplete NE |
|Aromatic l-amino acid decarboxylase ||Widespread; sympathetic nerves ||Cytoplasmic ||Pyridoxal phosphate ||Nonspecific ||Inhibition does not alter tissue NE and Epi appreciably |
|Dopamine β-hydroxylase ||Widespread; sympathetic nerves ||Synaptic vesicles ||Ascorbic acid, O2 (contains copper) ||Nonspecific ||Inhibition can decrease NE and Epi levels |
|Phenylethanolamine N-methyltransferase ||Largely in adrenal gland ||Cytoplasmic ||S-Adenosyl methionine (CH3 donor) ||Nonspecific ||Inhibition can decrease adrenal Epi/NE; regulated by glucocorticoids |
Steps in the enzymatic synthesis of dopamine, norepinephrine and epinephrine. The enzymes involved are shown in red; essential cofactors in italics. The final step occurs only in the adrenal medulla and in a few epinephrine-containing neuronal pathways in the brainstem.
The hydroxylation of tyrosine by tyrosine hydroxylase (TH) generally is regarded as the rate-limiting step in the biosynthesis of catecholamines (Zigmond et al., 1989); this enzyme is activated following stimulation of sympathetic nerves or the adrenal medulla. The enzyme is a substrate for PKA, PKC, and CaM kinase; phosphorylation is associated with increased hydroxylase activity. This is an important acute mechanism for increasing catecholamine synthesis in response to elevated nerve stimulation. In addition, there is a delayed increase in TH gene expression after nerve stimulation. This increased expression can occur at multiple levels of regulation, including transcription, RNA processing, regulation of RNA stability, translation, and enzyme stability (Kumer and Vrana, 1996). These mechanisms serve to maintain the content of catecholamines in response to increased transmitter release. In addition, TH is subject to feedback inhibition by catechol compounds, which allosterically modulate enzyme activity.
TH deficiency has been reported in humans and is characterized by generalized rigidity, hypokinesia, and low cerebrospinal fluid (CSF) levels of NE and DA metabolites homovanillic acid and 3-methoxy-4-hydroxyphenylethylene glycol (Wevers et al., 1999). TH knockout is embryonically lethal in mice, presumably because the loss of catecholamines results in altered cardiac function. Interestingly, residual levels of DA are present in these mice. It has been suggested that tyrosinase may be an alternate source for catecholamines, although tyrosinase-derived catecholamines are clearly not sufficient for survival (Carson and Robertson, 2002).
DβH deficiency in humans is characterized by orthostatic hypotension, ptosis of the eyelids, retrograde ejaculation, and elevated plasma levels of dopamine. In the case of DβH-deficient mice, there is ∼90% embryonic mortality (Carson and Robertson, 2002).
Our understanding of the cellular sites and mechanisms of synthesis, storage, and release of catecholamines derives from studies of sympathetically innervated organs and the adrenal medulla. Nearly all the NE content of innervated organs is confined to the postganglionic sympathetic fibers; it disappears within a few days after section of the nerves. In the adrenal medulla, catecholamines are stored in chromaffin granules (Aunis, 1998). These vesicles contain extremely high concentrations of catecholamines (∼21% dry weight), ascorbic acid, and ATP, as well as specific proteins such as chromogranins, DβH, and peptides including enkephalin and neuropeptide Y. Vasostatin-1, the N-terminal fragment of chromogranin A, has been found to have antibacterial and antifungal activity (Lugardon et al., 2000), as have other chromogranin A fragments such as chromofungin, vasostatin II, prochromacin, and chromacins I and II (Taupenot et al., 2003). Two types of storage vesicles are found in sympathetic nerve terminals: large dense-core vesicles corresponding to chromaffin granules; and small dense-core vesicles containing NE, ATP, and membrane-bound DβH.
The main features of the mechanisms of synthesis, storage, and release of catecholamines and their modifications by drugs are summarized in Figure 8–6. In the case of adrenergic neurons, the enzymes that participate in the formation of NE are synthesized in the cell bodies of the neurons and then are transported along the axons to their terminals. In the course of synthesis (Figure 8–6), the hydroxylation of tyrosine to dopa and the decarboxylation of dopa to dopamine take place in the cytoplasm. DA formed in the cytoplasm then is actively transported into the DβH-containing storage vesicles, where it is converted to NE. About 90% of the dopamine is converted to NE by DβH in sympathetic nerves; the remainder is metabolized. DA is converted by MAO to a aldehyde intermediate DOPAL, and then mainly converted to 3,4-dihydroxyphenyl acetic acid (DOPAC) by aldehyde dehydrogenase and to a minor extent 3,4-dihydroxyphenylethanol (DOPET) by aldehyde reductase. DOPAC is further converted to homovanillic acid (HVA) by o-methylation in non-neuronal sites.
An adrenergic neuroeffector junction showing features of the synthesis, storage, release, and receptors for norepinephrine (NE), the cotransmitters neuropeptide Y (NPY), and ATP. Tyrosine is transported into the varicosity and is converted to DOPA by tyrosine hydroxylase (TH) and DOPA to dopamine (DA) by the action of aromatic L-amino acid decarboxylase (AAADC). Dopamine is taken up into the vesicles of the varicosity by a transporter, VMAT2, that can be blocked by reserpine. Cytoplasmic NE also can be taken up by this transporter. Dopamine is converted to NE within the vesicle viathe action of dopamine-β-hydroxylase (DβH). NE is stored in vesicles along with other cotransmitters, NPY and ATP, depending on the particular neuroeffector junction. Release of the transmitters occurs upon depolarization of the varicosity, which allows entry of Ca2+ through voltage-dependent Ca2+ channels. Elevated levels of Ca2+ promote the fusion of the vesicular membrane with the membrane of the varicosity, with subsequent exocytosis of transmitters. This fusion process involves the interaction of specialized proteins associated with the vesicular membrane (VAMPs, vesicle-associated membrane proteins) and the membrane of the varicosity (SNAPs, synaptosome-associated proteins). In this schematic representation, NE, NPY, and ATP are stored in the same vesicles. Different populations of vesicles, however, may preferentially store different proportions of the cotransmitters. Once in the synapse, NE can interact with α and β adrenergic receptors to produce the characteristic response of the effector. The adrenergic receptors are GPCRs. α and βReceptors also can be located presynaptically where NE can either diminish (α2), or facilitate (β) its own release and that of the cotransmitters. The principal mechanism by which NE is cleared from the synapse is viaa cocaine-sensitive neuronal uptake transporter, NET. Once transported into the cytosol, NE can be re-stored in the vesicle or metabolized by monoamine oxidase (MAO). NPY produces its effects by activating NPY receptors, of which there are at least five types (YIthrough Y2). NPY receptors are GPCRs. NPY can modify its own release and that of the other transmitters viapresynaptic receptors of the Y2type. NPY is removed from the synapse by metabolic breakdown by peptidases. ATP produces its effects by activating P2X receptors or P2Y receptors. P2X receptors are ligand-gated ion channels; P2Y receptors are GPCRs. There are multiple subtypes of both P2X and P2Y receptors. As with the other cotransmitters, ATP can act prejunctionally to modify its own release viareceptors for ATP or viaits metabolic breakdown to adenosine that acts on P1 (adenosine) receptors. ATP is cleared from the synapse primarily by releasable nucleotidases (rNTPase) and by cell-fixed ectonucleotidases.
The adrenal medulla has two distinct catecholamine-containing cell types: those with NE and those with primarily epinephrine. The latter cell population contains the enzyme phenylethanolamine-N-methyltransferase (PNMT). In these cells, the NE formed in the granules leaves these structures, presumably by diffusion, and is methylated in the cytoplasm to epinephrine. Epinephrine then reenters the chromaffin granules, where it is stored until released. In adults, epinephrine accounts for ∼80% of the catecholamines of the adrenal medulla, with NE making up most of the remainder.
A major factor that controls the rate of synthesis of epinephrine, and hence the size of the store available for release from the adrenal medulla, is the level of glucocorticoids secreted by the adrenal cortex. The intra-adrenal portal vascular system carries the corticosteroids directly to the adrenal medullary chromaffin cells, where they induce the synthesis of PNMT (Figure 8–5). The activities of both TH and DβH also are increased in the adrenal medulla when the secretion of glucocorticoids is stimulated (Viskupic et al., 1994). Thus, any stress that persists sufficiently to evoke an enhanced secretion of corticotropin mobilizes the appropriate hormones of both the adrenal cortex (predominantly cortisol in humans) and medulla (epinephrine). This remarkable relationship is present only in certain mammals, including humans, in which the adrenal chromaffin cells are enveloped entirely by steroid-secreting cortical cells. In the dogfish, e.g., where the chromaffin cells and steroid-secreting cells are located in independent, noncontiguous glands, epinephrine is not formed. Nonetheless, there is evidence indicating that PMNT is expressed in mammalian tissues such as brain, heart, and lung, leading to extra-adrenal epinephrine synthesis (Kennedy et al., 1993).
In addition to de novo synthesis, NE stores in the terminal portions of the adrenergic fibers are also replenished by reuptake and restorage of NE following its release. At least two distinct carrier-mediated transport systems are involved: one across the axoplasmic membrane from the extracellular fluid to the cytoplasm (NET, the norepinephrine transporter, previously called uptake 1); and the other from the cytoplasm into the storage vesicles (VMAT2, the vesicular monoamine transporter) (Chandhry et al., 2008). A third transporter, ENT (the extraneuronal transporter, also called uptake 2), is responsible for the facilitated entry of catecholamines into non-neuronal cells.
For norepinephrine released by neurons, uptake by NET is more important than extraneuronal uptake and metabolism of norepinephrine. Sympathetic nerves as a whole remove ∼87% of released norepinephrine by NET, compared with 5% by extraneuronal uptake (ENT) and 8% by diffusion to the circulation. In contrast, clearance of circulating catecholamines is primarily by non-neuronal mechanisms, with liver and kidney accounting for over 60% of the clearance of circulating catecholamines. Because the VMAT2 has a much higher affinity for norepinephrine than does monoamine oxidase (MAO), over 70% of recaptured norepinephrine is sequestered into storage vesicles (Eisenhofer, 2001).
Storage of Catecholamines. Catecholamines are stored in vesicles, ensuring their regulated release; this storage decreases intraneuronal metabolism of these transmitters and their leakage outside the cell. The vesicular amine transporter (VMAT2) appears to be driven by pH and potential gradients that are established by an ATP-dependent proton translocase. For every molecule of amine taken up, two H+ ions are extruded (Brownstein and Hoffman, 1994). Monoamine transporters are relatively promiscuous and transport DA, NE, epinephrine, and 5-HT, e.g., as well as meta-iodobenzylguanidine, which can be used to image chromaffin cell tumors (Schuldiner, 1994). Reserpine inhibits monoamine transport into storage vesicles and ultimately leads to depletion of catecholamine from sympathetic nerve endings and in the brain. Several vesicular transport cDNAs have been cloned; these cDNAs reveal open reading frames predictive of proteins with 12 transmembrane domains (Chapter 5). Regulation of the expression of these various transporters may be important in the regulation of synaptic transmission (Varoqui and Erickson, 1997).
NET is Na+-dependent and is blocked selectively by a number of drugs, including cocaine and tricyclic antidepressants such as imipramine. This transporter has a high affinity for NE and a somewhat lower affinity for epinephrine (Table 8–5); the synthetic β adrenergic receptor agonist isoproterenol is not a substrate for this system.
Table 8-5Characteristics of Plasma Membrane Transporters for Endogenous Catecholamines ||Download (.pdf) Table 8-5 Characteristics of Plasma Membrane Transporters for Endogenous Catecholamines
|TYPE OF TRANSPORTER ||SUBSTRATE SPECIFICITY ||TISSUE ||REGION/CELL TYPE ||INHIBITORS |
|Neuronal || || || || |
|NET ||DA > NE > Epi || |
All sympathetically innervated tissue
Capillary endothelial cells
|DAT ||DA > NE > Epi || |
Parietal and endothelial cells
|Non-neuronal || || || || |
|OCT1 ||DA > Epi ≫ NE || |
Kidney (not human)
|OCT2 ||DA ≫ NE > Epi || |
Medullary proximal and distal tubules
Glial cells of DA-rich regions, some non-adrenergic neurons
|ENT (OCT 3) ||Epi ≫ NE > DA || |
Glial cells, others
Cortex, proximal and distal tubules
Syncytiotrophoblasts (basal membrane)
Photoreceptors, ganglion amacrine cells
There are actually two neuronal membrane transporters for catecholamines, the NE transporter (NET) mentioned above and the DA transporter (DAT); their characteristics are depicted in Table 8–5.
The NET is also present in the adrenal medulla, the liver, and the placenta, whereas the DAT is present in the stomach, pancreas, and kidney (Eisenhofer, 2001). NET and DAT are members of an extended family of biogenic amine and amino acid neurotransmitter transporters. Members share common structural motifs, particularly the putative 12-transmembrane helices. These plasma membrane transporters appear to have greater substrate specificity than do vesicular transporters and are targets for specific drugs such as cocaine (catecholamine transporters) and fluoxetine (serotonin transporter).
Certain sympathomimetic drugs (e.g., ephedrine and tyramine) produce some of their effects indirectly by displacing NE from the nerve terminals to the extracellular fluid, where it then acts at receptor sites of the effector cells. The mechanisms by which these drugs release NE from nerve endings are complex. All such agents are substrates for NET. As a result of their transport across the neuronal membrane and release into the axoplasm, they make carrier available at the inner surface of the membrane for the outward transport of NE ("facilitated exchange diffusion"). In addition, these amines are able to mobilize NE stored in the vesicles by competing for the vesicular uptake process. Reserpine, which depletes vesicular stores of NE, also inhibits this uptake mechanism, but in contrast with the indirect-acting sympathomimetic amines, it enters the adrenergic nerve ending by passive diffusion across the axonal membrane.
The actions of indirect-acting sympathomimetic amines are subject to tachyphylaxis. For example, repeated administration of tyramine results in rapidly decreasing effectiveness, whereas repeated administration of NE does not reduce effectiveness and, in fact, reverses the tachyphylaxis to tyramine. Although these phenomena have not been explained fully, several hypotheses have been proposed. One possible explanation is that the pool of neurotransmitter available for displacement by these drugs is small relative to the total amount stored in the sympathetic nerve ending. This pool is presumed to reside in close proximity to the plasma membrane, and the NE of such vesicles may be replaced by the less potent amine following repeated administration of the latter substance. In any case, neurotransmitter release by displacement is not associated with the release of DβH and does not require extracellular Ca2+; thus, it is presumed not to involve exocytosis.
There are also three extraneuronal transporters that handle a wide range of endogenous and exogenous substrates. The extraneuronal amine transporter (ENT), originally named uptake-2 and also designated OCT3, is an organic cation transporter. Relative to NET, ENT exhibits lower affinity for catecholamines, favors epinephrine over NE and DA, and shows a higher maximum rate of catecholamine uptake. The ENT is not Na+- dependent and displays a different profile of pharmacological inhibition. Other members of this family are the organic cation transporters OCT1 and OCT2 (Chapter 5). All three can transport catecholamines in addition to a wide variety of other organic acids, including 5-HT, histamine, choline, spermine, guanidine, and creatinine. The characteristics and location of the non-neuronal transporters are summarized in Table 8–5.
Release of Catecholamines. The full sequence of steps by which the nerve impulse effects the release of NE from sympathetic neurons is not known. In the adrenal medulla, the triggering event is the liberation of ACh by the preganglionic fibers and its interaction with nicotinic receptors on chromaffin cells to produce a localized depolarization; a subsequent step is the entrance of Ca2+ into these cells, which results in the extrusion by exocytosis of the granular contents, including epinephrine, ATP, some neuroactive peptides or their precursors, chromogranins, and DβH. Influx of Ca2+ likewise plays an essential role in coupling the nerve impulse, membrane depolarization, and opening of voltage-gated Ca2+ channels with the release of norepinephrine at sympathetic nerve terminals. Blockade of N-type Ca2+ channels leads to hypotension likely owing to inhibition of NE release. Ca2+-triggered secretion involves interaction of highly conserved molecular scaffolding proteins leading to docking of granules at the plasma membrane and ultimately leading to secretion (Aunis, 1998).
Reminiscent of the release of ACh at cholinergic terminals, various synaptic proteins, including the plasma membrane proteins syntaxin and SNAP-25, and the vesicle membrane protein synaptobrevin form a complex that interacts in an ATP-dependent manner with the soluble proteins N-ethylmaleimide-sensitive fusion protein (NSF) and soluble NSF attachment proteins (SNAPs). The ability of synaptobrevin, syntaxin, and SNAP-25 to bind SNAPs has led to their designation as SNAP receptors (SNAREs). It has been hypothesized that most, if not all, intracellular fusion events are mediated by SNARE interactions (Boehm and Kubista, 2002). As with cholinergic neurotransmission, important evidence supporting the involvement of SNARE proteins (e.g., SNAP-25, syntaxin, and synaptobrevin) in transmitter release comes from the fact that botulinum neurotoxins and tetanus toxin, which potently block neurotransmitter release, proteolyse these proteins.
Enhanced activity of the sympathetic nervous system is accompanied by an increased concentration of both DβH and chromogranins in the circulation, supporting the argument that the process of release following adrenergic nerve stimulation also involves exocytosis.
Adrenergic fibers can sustain the output of norepinephrine during prolonged periods of stimulation without exhausting their reserve supply, provided that synthesis and uptake of the transmitter are unimpaired. To meet increased needs for NE acute regulatory mechanisms are evoked involving activation of tyrosine hydroxylase and DβH (described earlier in the chapter).
Prejunctional Regulation of Norepinephrine Release. The release of the three sympathetic co-transmitters can be modulated by prejunctional autoreceptors and heteroreceptors. Following their release from sympathetic terminals, all three co-transmitters—NE, neuropeptide Y (NPY), and ATP—can feed back on prejunctional receptors to inhibit the release of each other (Westfall, 2004; Westfall et al., 2002). The most thoroughly studied have been prejunctional α2 adrenergic receptors. The α2Aand α2C adrenergic receptors are the principal prejunctional receptors that inhibit sympathetic neurotransmitter release, whereas the α2B adrenergic receptors also may inhibit transmitter release at selected sites. Antagonists of this receptor, in turn, can enhance the electrically evoked release of sympathetic neurotransmitter. NPY, acting on Y2 receptors, and ATP-derived adenosine, acting on P1 receptors, also can inhibit sympathetic neurotransmitter release. Numerous heteroreceptors on sympathetic nerve varicosities also inhibit the release of sympathetic neurotransmitters; these include: M2 and M4muscarinic, 5-HT, PGE2, histamine, enkephalin, and DA receptors. Enhancement of sympathetic neurotransmitter release can be produced by activation ofβ2adrenergic receptors, angiotensin AT2 receptors, and nACh receptors. All of these receptors can be targets for agonists and antagonists (Kubista and Boehm, 2006).
Termination of the Actions of Catecholamines. The actions of NE and epinephrine are terminated by:
reuptake into nerve terminals by NET
dilution by diffusion out of the junctional cleft and uptake at extraneuronal sites by ENT, OCT 1, and OCT 2
Following uptake, catecholamines can be metabolized (in neuronal and non-neuronal cells) or re-stored in vesicles (in neurons). Two enzymes are important in the initial steps of metabolic transformation of catecholamines—monoamine oxidase (MAO) and catechol-O-methyltransferase (COMT). In addition, catecholamines are metabolized by sulfotransferases (Dooley, 1998) (Chapter 6). However, termination of action by a powerful degradative enzymatic pathway, such as that provided by AChE at sites of cholinergic transmission, is absent from the adrenergic nervous system. The importance of neuronal reuptake of catecholamines is shown by observations that inhibitors of this process (e.g., cocaine and imipramine) potentiate the effects of the neurotransmitter; inhibitors of MAO and COMT have relatively little effect. However, MAO metabolizes transmitter that is released within the nerve terminal. COMT, particularly in the liver, plays a major role in the metabolism of endogenous circulating and administered catecholamines.
Both MAO and COMT are distributed widely throughout the body, including the brain; the highest concentrations of each are in the liver and the kidney. However, little or no COMT is found in sympathetic neurons. In the brain, there is also no significant COMT in presynaptic terminals, but it is found in some postsynaptic neurons and glial cells. In the kidney, COMT is localized in proximal tubular epithelial cells, where DA is synthesized, and is thought to exert local diuretic and natriuretic effects. The physiological substrates for COMT include l-dopa, all three endogenous catecholamines (DA, NE, and epinephrine), their hydroxylated metabolites, catecholestrogens, ascorbic acid, and dihydroxyindolic intermediates of melanin (Männistö and Kaakkola, 1999). There are distinct differences in the cytological locations of the two enzymes; MAO is associated chiefly with the outer surface of mitochondria, including those within the terminals of sympathetic or central noradrenergic neuronal fibers, whereas COMT is largely cytoplasmic except in the chromaffin cells of the adrenal medulla, where COMT is present as a membrane-bound form. These factors are of importance both in determining the primary metabolic pathways followed by catecholamines in various circumstances and in explaining the effects of certain drugs. Two different isozymes of MAO (MAO-A and MAO-B) are found in widely varying proportions in different cells in the CNS and in peripheral tissues. In the periphery, MAO-A is located in the syncytiotrophoblast layer of term placenta and liver, whereas MAO-B is located in platelets, lymphocytes, and liver. In the brain, MAO-A is located in all regions containing catecholamines, with the highest abundance in the locus ceruleus. MAO-B, on the other hand, is found primarily in regions that are known to synthesize and store 5-HT. MAO-B is most prominent in the nucleus raphe dorsalis but also in the posterior hypothalamus and in glial cells in regions known to contain nerve terminals. MAO-B is also present in osteocytes around blood vessels (Abell and Kwan, 2001). Selective inhibitors of these two isozymes are available (Chapter 15). Irreversible antagonists of MAO-A (e.g., phenelzine, tranylcypromine, and isocarboxazid) enhance the bioavailability of tyramine contained in many foods; tyramine-induced NE release from sympathetic neurons may lead to markedly increased blood pressure (hypertensive crisis). Selective MAO-B inhibitors (e.g., selegiline) or reversible MAO-A–selective inhibitors (e.g., moclobemide) are less likely to cause this potential interaction (Volz and Geiter, 1998; Wouters, 1998). MAO inhibitors are useful in the treatment of Parkinson's disease and mental depression (Chapters 15 and Chapter 22).
Inhibitors of MAO (e.g., pargyline and nialamide) can cause an increase in the concentration of NE, DA, and 5-HT in the brain and other tissues accompanied by a variety of pharmacological effects. No striking pharmacological action in the periphery can be attributed to the inhibition of COMT. However, the COMT inhibitors entacapone and tocapone have been found to be efficacious in the therapy of Parkinson's disease (Chong and Mersfelder, 2000) (Chapter 22).
There is ongoing passive leakage of catecholamines from vesicular storage granules of sympathetric neurons and adrenal medullary chromaffin cells. As a consequence, most metabolism of catecholamines takes place in the same cells where the amines are synthesized and stored. VMAT2 effectively sequesters ∼90% of the amines leaking into the cytoplasm back into storage vesicles; ∼10% escapes sequestration and is metabolized (Eisenhofer et al., 2004).
Sympathetic nerves contain MAO but not COMT, and this MAO catalyzes only the first step of a two-step reaction. MAO converts NE or epinephrine into a short-lived intermediate, DOPGAL, which undergoes further metabolism in a second step catalyzed by another group of enzymes forming more stable alcohol- or acid-deaminated metabolites. Aldehyde dehydrogenase metabolizes DOPGAL to 3,4-dihydroxymandelic acid (DOMA), while aldehyde reductase metabolized DOPGAL to 3,4-dihydroxyphenyl glycol (DOPEG). In addition to aldehyde reductase, a related enzyme, aldose reductase, can also reduce a catecholamine to its corresponding alcohol. This latter enzyme is present in sympathetic neurons and adrenal chromaffin cells. Under normal circumstances, DOMA is an insignificant metabolite of NE and epinephrine, with DOPEG being the main metabolite produced by deamination in sympathetic neurons and adrenal medullary chromaffin cells.
Once it leaves the sites of formation (sympathetic neurons, adrenal medulla), DOPEG is converted to 3-methyl, 4-hydroxy phenylglycol (MOPEG) by COMT. Therefore most MOPEG comes from the extraneuronal O-methylation of DOPEG produced in and diffusing rapidly from sympathetic neurons into the extracellular fluid. MOPEG is then converted to vanillylmandelic acid (VMA) by the sequential action of alcohol and aldehyde dehydrogenase. MOPEG is first converted to the unstable aldehyde metabolite MOPGAL and then to VMA, with VMA being the major end product of NE and epinephrine metabolism. Another route for the formation of VMA is conversion of NE and epinephrine into normetanephrine and metaneprhine, respectively, by COMT followed by deamination to MOPGAL and ultimately VMA. This is now thought to be only a minor pathway, as indicated by the size of the arrows on Figure 8–7.
In contrast to sympathetic neurons, adrenal medullary chromaffin cells contain both MAO and COMT. In chromaffin cells, the COMT is mainly present as the membrane-bound form of the enzyme in contrast to the form found in the cytoplasm of extra-neuronal tissue. This isoform of COMT has a higher affinity for catecholamines than does the soluble form found in most other tissues (e.g., liver and kidney). In adrenal medullary chromaffin cells, leakage of NE and epinephrine from storage vesicles leads to substantial intracellular production of the O-methylated metabolites normetanephrine and metanephrine. It is estimated that in humans, over 90% of circulating metanephrine and 25-40% of circulating normetanephrine are derived from catecholamines metabolized within adrenal chromaffin cells.
The sequence of cellular uptake and metabolism of catecholamines in extraneuronal tissues contributes very little (∼25%) to the total metabolism of endogenously produced NE in sympathetic neurons or the adrenal medulla. However, extraneuronal metabolism is an important mechanism for the clearance of circulating and exogenously administered catecholamines.
Steps in the metabolic disposition of catecholamines. Norepinephrine and epinephrine are first oxidatively deaminated to a short lived intermediate (DOPGAL) by monoamine oxidase (MAO). DOPGAL then undergoes further metabolism to more stable alcohol or acid deaminated metabolites. Aldehyde dehydrogenase (AD) metabolizes DOPGAL to 3,4-dihydroxymandelic acid (DOMA) while aldehyde reductase (AR) metabolizes DOPGAL to 3,4-dihydroxyphenyl glycol (DOPEG). Under normal circumstances DOMA is a minor metabolite with DOPEG being the major metabolite produced from norepinephrine and epinephrine. Once DOPEG leaves the major sites of its formation (sympathetic nerves; adrenal medulla), it is converted to 3-methoxy, 4-hydroxyphenylglycol (MOPEG) by catechol-0-methyl transferase (COMT). MOPEG is then converted to the unstable aldehyde (MOPGAL) by alcohol dehydrogenase (ADH) and finally to vanillyl mandelic acid (VMA) by aldehyde dehydrogenase. VMA is the major end product of norepinephrine and epinephrine metabolism. Another route for the formation of VMA is conversion of norepinephrine or epinephrine into normetanephrine or metanephrine by COMT either in the adreneal medulla or extraneuronal sites,with subsequent metabolism to MOPGAL and thence to VMA.
Classification of Adrenergic Receptors. Crucial to understanding the remarkably diverse effects of the catecholamines and related sympathomimetic agents is an understanding of the classification and properties of the different types of adrenergic receptors (or adrenoceptors). Elucidation of the characteristics of these receptors and the biochemical and physiological pathways they regulate has increased our understanding of the seemingly contradictory and variable effects of catecholamines on various organ systems. Although structurally related (discussed later), different receptors regulate distinct physiological processes by controlling the synthesis or release of a variety of second messengers (Table 8–6).
Table 8-6Characteristics for Adrenergic Receptor Subtypes ||Download (.pdf) Table 8-6 Characteristics for Adrenergic Receptor Subtypes
|SUBTYPEa ||GENE LOCATION IN HUMAN CHROMOSOME ||G-PROTEIN COUPLING ||PRINCIPLE EFFECTORS ||TISSUE LOCALIZATION ||SUBTYPE DOMINANT EFFECTSb |
|α1A ||8p21-p11.2 ||Gαq (α11/α14/α16) || |
↑ PLC, ↑PLA2
↑ Ca2+ channels
↑ Na+/H+ exchanger
Modulation of K+ channels
↑ MAPK Signaling
• Predominant receptor causing contraction of vascular smooth muscle
• Promotes cardiac growth and structure
• Vasoconstriction of large resistant arterioles in skeletal muscle
|α1B ||5q23-q32 ||Gαq (α11/α14/α16) || |
↑ PLC, ↑PLA2
↑ Ca2+ channels
Modulation of K+ channels
• Most abundant subtype in heart
• Promotes cardiac growth and structure
|α1D ||20p13 ||Gαq (α11/α14/α16) || |
↑ PLC, ↑PLA2
↑ Ca2+ channels
↑ Na+/H+ exchanger
Modulation of K+ channels
↑ MAPK Signaling
|• Predominant receptor causing vasoconstriction in aorta and coronary artery |
|α2A ||10q24-q26 || |
Gαo Family (αo1/αo2)
Inhibition of AC
• Predominant inhibitory receptor on sympathetic neurons
• Vasoconstriction of small procapillary vessels in skeletal muscle
|α2B ||2q12-q13 || |
Gαo Family (αo1/αo2)
Inhibition of AC
|• Predominant receptor mediating α2 vasoconstriction |
|α2c ||4p16 || |
Gαi Family (α11/α12/α13)
Gαo Family (αo1/αo2)
Inhibition of AC
• Predominant receptor modulating dopamine neurotransmission
• Predominant receptor inhibiting hormone release from adrenal medulla
|β1 ||10q240q26 ||Gαs || |
Activation of AC
Activation of L type Ca2+ channels
|• Predominant receptor in heart producing positive inotropic and chronotropic effects |
|β2c ||531-q32 ||Gαs || |
Activation of AC
↑ PKA activation
Activation of Ca2+ channels
Bronchial and GI smooth muscle
• Predominant receptor in smooth muscle relaxation
• Skeletal muscle hypertrophy
|β3c,d ||8p12-p11.2 ||Gαs || |
Activation of AC
Activation of Ca2+ channels
|• Predominant receptor producing metabolic effects |
Based on studies of the abilities of epinephrine, norepinephrine, and other related agonists to regulate various physiological processes, Ahlquist first proposed the existence of more than one adrenergic receptor. It was known that these drugs could cause either contraction or relaxation of smooth muscle depending on the site, the dose, and the agent chosen. For example, NE was known to have potent excitatory effects on smooth muscle and correspondingly low activity as an inhibitor; isoproterenol displayed the opposite pattern of activity. Epinephrine could both excite and inhibit smooth muscle. Thus, Ahlquist proposed the designations α and β for receptors on smooth muscle where catecholamines produce excitatory and inhibitory responses, respectively. An exception is the gut, which generally is relaxed by activation of either α or β receptors. The rank order of potency of agonists is isoproterenol > epinephrine ≥ norepinephrine for β adrenergic receptors and epinephrine ≥ norepinephrine ≫ isoproterenol for α adrenergic receptors (Table 8–3). This initial classification was corroborated by the finding that certain antagonists produce selective blockade of the effects of adrenergic nerve impulses and sympathomimetic agents atα receptors (e.g., phenoxybenzamine), whereas others produce selective β receptor blockade (e.g., propranolol).
β Receptors later were subdivided into β1 (e.g., those in the myocardium) and β2 (smooth muscle and most other sites) because epinephrine and norepinephrine essentially are equipotent at the former sites, whereas epinephrine is 10-50 times more potent than norepinephrine at the latter. Antagonists that discriminate between β1 and β2 receptors were subsequently developed (Chapter 12). A human gene that encodes a third β receptor (designated β3) has been isolated (Emorine et al., 1989). Since the β3 receptor is about tenfold more sensitive to norepinephrine than to epinephrine and is relatively resistant to blockade by antagonists such as propranolol, it may mediate responses to catecholamine at sites with "atypical" pharmacological characteristics (e.g., adipose tissue). Although the adipocytes are a major site of β3 adrenergic receptors, all three β adrenergic receptors are present in both white adipose tissue and brown adipose tissue. Animals treated with β3 receptor agonists exhibit a vigorous thermogenic response as well as lipolysis (Robidoux et al., 2004). Polymorphisms in the β3 receptor gene may be related to risk of obesity or type 2 diabetes in some populations (Arner and Hoffstedt, 1999). Also, there has been interest in the possibility that β3 receptor–selective agonists may be beneficial in treating these disorders (Weyer et al., 1999). The existence of a fourth type of βAR, β4AR, has been proposed. Despite intense efforts, β4AR, like α1LAR, has not been cloned. Current thinking is that the β4AR represents an affinity state of β1AR rather than a descrete receptor (Hieble, 2007).
There is also heterogeneity among α adrenergic receptors. The initial distinction was based on functional and anatomic considerations when it was realized that NE and other α-adrenergic receptors could profoundly inhibit the release of norepinephrine from neurons (Westfall, 1977) (Figure 8–6). Indeed, when sympathetic nerves are stimulated in the presence of certain α receptor antagonists, the amount of NE liberated by each nerve impulse increases markedly. This feedback-inhibitory effect of NE on its release from nerve terminals is mediated by α receptors that are pharmacologically distinct from the classical postsynaptic α receptors. Accordingly, these presynaptic α adrenergic receptors were designated α2, whereas the postsynaptic "excitatory" α receptors were designated α1 (Langer, 1997). Compounds such as clonidine are more potent agonists at α2 than at α1 receptors; by contrast, phenylephrine and methoxamine selectively activate postsynaptic α1 receptors. Although there is little evidence to suggest that α1 adrenergic receptors function presynaptically in the autonomic nervous system, it is now clear that α2 receptors also are present at postjunctional or nonjunctional sites in several tissues. For example, stimulation of postjunctional α2 receptors in the brain is associated with reduced sympathetic outflow from the CNS and appears to be responsible for a significant component of the antihypertensive effect of drugs such as clonidine (Chapter 12). Thus, the anatomic concept of prejunctional α2 and postjunctional α1 adrenergic receptors has been abandoned in favor of a pharmacological and functional classification (Tables 8–6 and 8–7).
Table 8-7Representative Agents Acting at Peripheral Cholinergic and Adrenergic Neuroeffector Junctions ||Download (.pdf) Table 8-7 Representative Agents Acting at Peripheral Cholinergic and Adrenergic Neuroeffector Junctions
|MECHANISM OF ACTION ||SYSTEM ||AGENTS ||EFFECT |
|1. Interference with synthesis of transmitter || |
Choline acetyl transferase inhibitors
α-Methyltyrosine (inhibition of tyrosine hydroxylase)
Minimal depletion of ACh
Depletion of NE
|2. Metabolic transformation by same pathway as precursor of transmitter ||Adrenergic ||Methyldopa ||Displacement of NE by α-methyl-NE, which is an α2 agonist, similar to clonidine, that reduces sympathetic outflow from CNS. |
|3. Blockade of transport system at nerve terminal membrane || |
Block of choline uptake with consequent depletion of ACh
Accumulation of NE at receptors
|4. Blockade of transport system of storage vesicle || |
Block of ACh storage
Destruction of NE by mitochondrial MAO, and depletion from adrenergic terminals
|5. Promotion of exocytosis or displacement of transmitter from storage sites || |
Cholinomimetic followed by anticholinergic
|6. Prevention of release of transmitter || |
|7. Mimicry of transmitter at postjunctional sites || |
Cholinergic Muscarinica Nicotinicb
Nicotine, epibatidine, cytisine
Terbutaline, albuterol metaproterenol
Selective α1 agonist
Sympathomimetic (periphery); reduced sympathetic outflow (CNS)
Non-selective α adrenomimetic
Selective cardiac stimulation (also activates α1 receptors)
Selective β2 receptor agonist (selective inhibition of smooth muscle contraction)
Nonselective β agonist
|8. Blockade of postsynaptic receptor || |
Prazosin, terazosin, doxasozin
PhenoxybenzamineNonselective α receptor blockade (irreversible)
Nonselective α receptor blockade (reversible)
Selective α1 receptor blockade (reversible)
α2 receptor blockade
Nonselective β receptor blockade
Selective β1 receptor blockade (cardiomyocytes; renal j-g cells)
Selective β2receptor blockade (smooth muscle)
|9. Inhibition of enzymatic breakdown of transmitter || |
Edrophonium, neostigmine, pyridostigmine
Nonselective MAO inhibitors Pargyline, nialamide
Selective MAO-B inhibitor Selegeline
Peripheral COMT inhibitor Entacapone
COMT inhibitor Tolcapone
Cholinomimetic (muscarinic sites)
Depolarization blockade (nicotinic sites)
Little direct effect on NE or sympathetic response; potentiation of tyramine
Adjunct in Parkinson disease
Adjunct in Parkinson disease
Cloning revealed additional heterogeneity of both α1 and α2adrenergic receptors (Bylund, 1992). There are three pharmacologically defined α1 receptors (α1A, α1B, and α1D) with distinct sequences and tissue distributions, and three cloned subtypes of α2 receptors (α2A, α2B, and α2C) (Table 8–6). A fourth type of α1 receptor, α1LAR, has been defined on the basis of a low affinity for a number of selective antagonists including prazosin (Hieble, 2007). This phenotype could be of physiological significance since the α1L profile has been identified in a variety of tissues across a number of different species, where it appears to regulate smooth muscle contractility in the vasculature and lower urinary tract. It also appears (at least in the mouse prostate smooth muscle) to be dependent upon expression of the α1AAR gene product. Moreover, an intact cellular environment is important for the manifestation of the α1LAR phenotype in vivo. Despite intense efforts, α12AR has not been cloned and it is doubtful that α1Lis a discrete AR (Hieble, 2007).
Owing to the lack of sufficiently subtype-selective ligands, the precise physiological function and therapeutic potential of the subtypes of adrenergic receptors have not been elucidated fully. Great advances in our understanding have been made through the use of genetic approaches using transgenic and receptor knockout experiments in mice (discussed later). These mouse models have been used to identify the particular receptor subtypes and pathophysiological relevance of individual adrenergic receptors subtypes (Philipp and Hein, 2004; Tanoue et al., 2002a, 2002b; Xiao et al., 2006).
Molecular Basis of Adrenergic Receptor Function. All of the adrenergic receptors are GPCRs that link to heterotrimeric G proteins. Each major type shows preference for a particular class of G proteins, i.e., α1 to Gq, α2 to Gi, and β to Gs (Table 8–6). The responses that follow activation of all types of adrenergic receptors result from G protein–mediated effects on the generation of second messengers and on the activity of ion channels, as discussed in Chapter 3. The pathways overlap broadly with those discussed for muscarinic acetylcholine receptors and are summarized in Table 8–6 (Drake et al., 2006; Park et al., 2008).
Structure of Adrenergic Receptors. Adrenergic receptors constitute a family of closely related proteins that are related both structurally and functionally to GPCRs for a wide variety of other hormones and neurotransmitters (see Chapter 3). Ligand binding, site-directed labeling, and mutagenesis have revealed that the conserved membrane-spanning regions are crucially involved in ligand binding (Hutchins, 1994; Strader et al.,1994). These regions appear to create a ligand-binding pocket analogous to that formed by the membrane-spanning regions of rhodopsin to accommodate the covalently attached chromophore, retinal, with molecular models placing catecholamines either horizontally (Strader et al., 1994) or perpendicularly (Hutchins, 1994) in the bilayer. The crystal structure of mammalian rhodopsin confirms a number of predictions about the structure of GPCRs (Palczewski et al., 2000).
β Adrenergic Receptors. The three β receptors share ∼60% amino acid sequence identity within the presumed membrane-spanning domains where the ligand-binding pocket for epinephrine and norepinephrine is found. Based on results of site-directed mutagenesis, individual amino acids in the β2 receptor that interact with each of the functional groups on the catecholamine agonist molecule have been identified.
β Receptors regulate numerous functional responses, including heart rate and contractility, smooth muscle relaxation, and multiple metabolic events in numerous tissues including adipose and hepatic cells and skeletal muscle (Lynch and Ryall, 2008) (Table 8–1). All three of the β receptor subtypes (β1, β2, and β3) couple to Gs and activate adenylyl cyclase (Table 8–7). However, recent data suggest differences in downstream signals and events activated by the three β receptors (Lefkowitz, 2000; Ma and Huang, 2002). Catecholamines promote β receptor feedback regulation, that is, desensitization and receptor down-regulation (Kohout and Lefkowitz, 2003). β Receptors differ in the extent to which they undergo such regulation, with the β2 receptor being the most susceptible. Stimulation of β adrenergic receptors leads to the accumulation of cyclic AMP, activation of the PKA, and altered function of numerous cellular proteins as a result of their phosphorylation (Chapter 3). In addition, Gs can enhance directly the activation of voltage-sensitive Ca2+ channels in the plasma membrane of skeletal and cardiac muscle.
Several reports demonstrate that β1, β2, and β3 receptors can differ in their intracellular signaling pathways and subcellular location (Brodde et al., 2006; Violin and Lefkowitz, 2007; Woo, et al., 2009). While the positive chronotropic effects of β1 receptor activation are clearly mediated by Gs in myocytes, dual coupling of β2 receptors to Gs and Gi occurs in myocytes from newborn mice. Stimulation of β2 receptors caused a transient increase in heart rate that is followed by a prolonged decrease. Following pretreatment with pertussis toxin, which prevents activation of Gi, the negative chronotropic effect of β2 activation is abolished. It is thought that these specific signaling properties of β receptor subtypes are linked to subtype-selective association with intracellular scaffolding and signaling proteins (Baillie and Houslay, 2005). β2 Receptors normally are confined to caveolae in cardiac myocyte membranes. The activation of PKA by cyclic AMP and the importance of compartmentation of components of the cyclic AMP pathway are discussed in Chapter 3. A representation of the general structure of adrenergic receptors is shown in Figure 8–8.
Subtypes of adrenergic receptors. All of the adrenergic receptors are heptaspanning GPCRs. A representative of each type is shown; each type has three subtypes: α1A, α1B, and α1D; α2A, α2B, and α2C; and β1, β2, and β3. The principle effector systems affected by α1, α2 and β receptors are depicted in Table 8–6. (Ψ) indicates a site for N-glycosylation. www indicates a site for thio-acetylation.
αAdrenergic Receptors. The deduced amino acid sequences from the three α1 receptor genes (α1A, α1B, and α1D) and three α2 receptor genes (α2A, α2B, and α2C) conform to the well-established GPCR paradigm (Bylund, 1992; Zhong and Minneman, 1999). The general structural features of αreceptors and their relation to the functions of ligand binding and G protein activation appear to agree with those set forth in Chapter 3 and earlier in this chapter for the β receptors. Within the membrane-spanning domains, the three α1 adrenergic receptors share ∼75% identity in amino acid residues, as do the three α2 receptors, but the α1 and α2 subtypes are no more similar than are the α and β subtypes (∼30-40%).
α2 Adrenergic Receptors. As shown in Table 8–6, α2 receptors couple to a variety of effectors (Aantaa et al., 1995; Bylund, 1992; Tan and Limbird, 2005). Inhibition of adenylyl cyclase activity was the first effect observed, but in some systems the enzyme actually is stimulated by α2 adrenergic receptors, either by Gi βγ subunits or by weak direct stimulation of Gs. The physiological significance of these latter processes is not currently clear. α2 Receptors activate G protein–gated K+ channels, resulting in membrane hyperpolarization. In some cases (e.g., cholinergic neurons in the myenteric plexus) this may be Ca2+-dependent, whereas in others (e.g., muscarinic ACh receptors in atrial myocytes) it results from direct interaction of βγ subunits with K+ channels. α2 Receptors also can inhibit voltage-gated Ca2+ channels; this is mediated by Go. Other second-messenger systems linked to α2 receptor activation include acceleration of Na+/H+ exchange, stimulation of PLCβ2 activity and arachidonic acid mobilization, increased phosphoinositide hydrolysis, and increased intracellular availability of Ca2+. The latter is involved in the smooth muscle–contracting effect of α2 adrenergic receptor agonists. In addition, the α2 receptors activate mitogen-activated protein kinases (MAPKs) likely by means of βγ subunits released from pertussis toxin–sensitive G proteins (Della Rocca et al., 1997; Richman and Regan, 1998). This and related pathways lead to activation of a variety of tyrosine kinase–mediated downstream events. These pathways are reminiscent of pathways activated by tyrosine kinase activities of growth factor receptors. Although α2 receptors may activate several different signaling pathways, the exact contribution of each to many physiological processes is not clear. The α2A receptor plays a major role in inhibiting NE release from sympathetic nerve endings and suppressing sympathetic outflow from the brain, leading to hypotension (Kable et al., 2000).
In the CNS, α2A receptors, which appear to be the most dominant adrenergic receptor, probably produce the antinociceptive effects, sedation, hypothermia, hypotension, and behavioral actions of α2 agonists (Lakhlani et al., 1997). The α2C receptor occurs in the ventral and dorsal striatum and hippocampus. It appears to modulate dopamine neurotransmission and various behavioral responses. The α2B receptor is the main receptor mediating α2-induced vasoconstriction, whereas the α2C receptor is the predominant receptor inhibiting the release of catecholamines from the adrenal medulla and modulating dopamine neurotransmission in the brain.
α1 Adrenergic Receptors. Stimulation of α1 receptors results in the regulation of multiple effector systems. A primary mode of signal transduction involves activation of the Gq-PLCβ-IP3-Ca2+ pathway and the activation of other Ca2+ and calmodulin sensitive pathways such as CaM kinases (Chapter 3). For example, α1 receptors regulate hepatic glycogenolysis in some animal species; this effect results from the activation of phosphorylase kinase by the mobilized Ca2+, aided by the inhibition of glycogen synthase caused by PKC-mediated phosphorylation. PKC phosphorylates many substrates, including membrane proteins such as channels, pumps, and ion-exchange proteins (e.g., Ca2+-transport ATPase). These effects presumably lead to regulation of various ion conductances.
α1 Receptor stimulation of PLA2 leads to the release of free arachidonate, which is then metabolized by the cyclooxygenase and lipoxygenase pathways to the bioactive prostaglandins and leukotrienes, respectively (Chapter 33). Stimulation of PLA2 activity by various agonists (including epinephrine acting at α1 receptors) is found in many tissues and cell lines, suggesting that this effector is physiologically important. PLD hydrolyzes phosphatidylcholine to yield phosphatidic acid (PA). Although PA itself may act as a second messenger by releasing Ca2+ from intracellular stores, it also is metabolized to the second messenger DAG. PLD is an effector for ADP-ribosylating factor (ARF), suggesting that PLD may play a role in membrane trafficking. Finally, some evidence in vascular smooth muscle suggests that α1 receptors are capable of regulating a Ca2+ channel by means of a G protein.
In most smooth muscles, the increased concentration of intracellular Ca2+ ultimately causes contraction as a result of activation of Ca2+-sensitive protein kinases such as the calmodulin-dependent myosin light-chain kinase; phosphorylation of the light chain of myosin is associated with the development of tension (see Chapter 3). In contrast, the increased concentration of intracellular Ca2+ that result from stimulation of α1 receptors in GI smooth muscle causes hyperpolarization and relaxation by activation of Ca2+-dependent K+ channels (McDonald et al., 1994).
As with α2 receptors, there is considerable evidence demonstrating that α1 receptors activate MAPKs and other kinases such as PI3 kinase leading to important effects on cell growth and proliferation (Dorn and Brown, 1999; Gutkind, 1998). For example, prolonged stimulation of α1 receptors promotes growth of cardiac myocytes and vascular smooth muscle cells. The α1A receptor is the predominant receptor causing vasoconstriction in many vascular beds, including the following arteries: mammary, mesenteric, splenic, hepatic, omental, renal, pulmonary, and epicardial coronary. It is also the predominant subtype in the vena cava and the saphenous and pulmonary veins (Michelotti et al., 2001). Together with the α1B receptor subtype, it promotes cardiac growth and structure. The α1B receptor subtype is the most abundant subtype in the heart, whereas the α1D receptor subtype is the predominant receptor causing vasoconstriction in the aorta. There is evidence to support the idea that α1B receptors mediate behaviors such as reaction to novelty and exploration and are involved in behavioral sensitizations and in the vulnerability to addiction (see Chapter 24).
Adrenergic Receptor Polymorphism. Numerous polymorphisms and slice variants of adrenergic receptors continue to be identified. Receptors α1A, α1B and β1D, β1, and β2 are polymorphic. Such polymorphisms in these adrenergic receptors could result in altered physiological responses to activation of the sympathetic nervous system, contribute to disease states and alter the responses to adrenergic agonists and/or antagonists (Brodde, 2008). Knowledge of the functional consequences of specific polymorphisms could theoretically result in the individualization of drug therapy based on a patient's genetic makeup and could explain marked inter-individual variability within the human population.
Localization of Adrenergic Receptors. Presynaptically located α2and β2 receptors fulfill important roles in the regulation of neurotransmitter release from sympathetic nerve endings. Presynaptic α2 receptors also may mediate inhibition of release of neurotransmitters other than norepinephrine in the central and peripheral nervous systems. Both α2 and β2 receptors are located at postsynaptic sites (Table 8–6), such as on many types of neurons in the brain. In peripheral tissues, postsynaptic α2 receptors are found in vascular and other smooth muscle cells (where they mediate contraction), adipocytes, and many types of secretory epithelial cells (intestinal, renal, endocrine). Postsynaptic β2 receptors can be found in the myocardium (where they mediate contraction) as well as on vascular and other smooth muscle cells (where they mediate relaxation) and skeletal muscle (where they can mediate hypertrophy). Indeed, most normal human cell types express β2receptors. Both α2 and β2 receptors may be situated at sites that are relatively remote from nerve terminals releasing NE. Such extrajunctional receptors typically are found on vascular smooth muscle cells and blood elements (platelets and leukocytes) and may be activated preferentially by circulating catecholamines, particularly epinephrine.
In contrast, α1 and β1 receptors appear to be located mainly in the immediate vicinity of sympathetic adrenergic nerve terminals in peripheral target organs, strategically placed to be activated during stimulation of these nerves. These receptors also are distributed widely in the mammalian brain (Table 8–6).
The cellular distributions of the three α1 and three α2 receptor subtypes still are incompletely understood. In situ hybridization of receptor mRNA and receptor subtype-specific antibodies indicates that α2A receptors in the brain may be both pre- and postsynaptic. These findings and other studies indicate that this receptor subtype functions as a presynaptic autoreceptor in central noradrenergic neurons (Aantaa et al., 1995; Lakhlani et al., 1997). Using similar approaches, α1A mRNA was found to be the dominant subtype message expressed in prostatic smooth muscle (Walden et al., 1997).
Refractoriness to Catecholamines. Exposure of catecholamine-sensitive cells and tissues to adrenergic agonists causes a progressive diminution in their capacity to respond to such agents. This phenomenon, variously termed refractoriness, desensitization, or tachyphylaxis, can limit the therapeutic efficacy and duration of action of catecholamines and other agents (Chapter 3). The mechanisms are incompletely understood. They have been studied most extensively in cells that synthesize cyclic AMP in response to β2 receptor agonists.
Multiple mechanisms are involved in desensitization, including rapid events such as receptor phosphorylation by both G-protein receptor kinases (GRKs) and by signaling kinases such as PKA and PKC, receptor sequestration, uncoupling from G proteins, and activation of specific cyclic nucleotide phosphodiesterases. More slowly occurring events also are seen, such as receptor endocytosis, which decreases receptor number. An understanding of the mechanisms involved in regulation of GPCR desensitization has developed over the last few years (Kohout and Lefkowitz, 2003; Violin and Lefkowitz, 2007). Such regulation is very complex and exceeds the simplistic model of GPCR phosphorylation by GRKs followed by arrestin binding and uncoupling of G-protein signaling. It is known that GRK activities are extensively regulated by numerous interactions with and modifications by other proteins. β-Arrestin, now recognized as a scaffolding protein, can physically interrupt signaling to the G proteins as well as to further enhance GPCR desensitization by causing translocation of cytosolic proteins to the receptor (e.g., phosphodiesterase and cSrc). These, in turn, can turn off signaling at its source by degrading cyclic AMP or phosphorylating GRK2 to enhance its activity toward the receptor (DeFea, 2008; DeWire et al., 2007; Hanyaloglu and VonZastrow, 2008).