Chapter 3: Pharmacodynamics: Molecular Mechanisms of Drug Action

Pharmacodynamics is the study of the biochemical and physiological effects of drugs and their mechanisms of action. Understanding pharmacodynamics can provide the basis for the rational therapeutic use of a drug and the design of new and superior therapeutic agents. Simply stated, pharmacodynamics refers to the effects of a drug on the body. In contrast, the effects of the body on the actions of a drug are pharmacokinetic processes (Chapter 2), and include absorption, distribution, metabolism, and excretion of drugs (often referred to collectively as ADME). Many adverse effects of drugs and drug toxicities can be anticipated by understanding a drug's mechanism(s) of action, its pharmacokinetics, and its interactions with other drugs. Thus, both the pharmacodynamic properties of a drug and its pharmacokinetics contribute to safe and successful therapy. The effects of many drugs, both salutory and deleterious, may differ widely from patient to patient due to genetic differences that alter the pharmacokinetics and the pharmacodynamics of a given drug. This aspect of pharmacology is termed pharmacogenetics and is covered in Chapter 7.

The effects of most drugs result from their interaction with macromolecular components of the organism. These interactions alter the function of the pertinent component and initiate the biochemical and physiological changes that are characteristic of the response to the drug. The term drug receptor or drug target denotes the cellular macromolecule or macromolecular complex with which the drug interacts to elicit a cellular response, i.e., a change in cell function. Drugs commonly alter the rate or magnitude of an intrinsic cellular response rather than create new responses. Drug receptors are often located on the surface of cells, but may also be located in specific intracellular compartments such as the nucleus. Many drugs also interact with acceptors (e.g., serum albumin) within the body. Acceptors are entities that do not directly cause any change in biochemical or physiological response. However, interactions of drugs with acceptors such as serum albumin can alter the pharmacokinetics of a drug's actions.

From a numerical standpoint, proteins form the most important class of drug receptors. Examples include the receptors for hormones, growth factors, transcription factors, and neurotransmitters; the enzymes of crucial metabolic or regulatory pathways (e.g., dihydrofolate reductase, acetylcholinesterase, and cyclic nucleotide phosphodiesterases); proteins involved in transport processes (e.g., Na⁺,K⁺-ATPase); secreted glycoproteins (e.g., Wnts); and structural proteins (e.g., tubulin). Specific binding of drugs to other cellular constituents such as DNA is also exploited for therapeutic purposes. For example, nucleic acids are particularly important drug receptors for certain cancer chemotherapeutic agents and antiviral drugs.

Physiological Receptors

A major group of drug receptors consists of proteins that normally serve as receptors for endogenous regulatory ligands. These drug targets are termed physiological receptors. Many drugs act on physiological receptors and are particularly selective because physiological receptors have evolved to recognize and respond to individual signaling molecules with great selectivity. Drugs that bind to physiological receptors and mimic the regulatory effects of the endogenous signaling compounds are termed agonists. If the drug binds to the same recognition site as the endogenous agonist (the primary or orthosteric site on the receptor) the drug is said to be a primary agonist. Allosteric (allotopic) agonists bind to a different region on the receptor referred to as an allosteric or allotopic site. Drugs that block or reduce the action of an agonist are termed antagonists. Antagonism most commonly results from competition with an agonist for the same or overlapping site on the receptor (a syntopic interaction), but can also occur by interacting with other sites on the receptor (allosteric antagonism), by combining with the agonist (chemical antagonism), or by functional antagonism by indirectly inhibiting the cellular or physiological effects of the agonist. Agents that are only partly as effective as agonists regardless of the concentration employed are termed partial agonists. Many receptors exhibit some constitutive activity in the absence of a regulatory ligand; drugs that stabilize such receptors in an inactive conformation are termed inverse agonists (Figure 3–1) (Kenakin, 2004; Milligan, 2003). Note that partial agonists and inverse agonists that interact syntopically with a full agonist will behave as competitive antagonists.

Drug Specificity

The strength of the reversible interaction between a drug and its receptor, as measured by the dissociation constant, is defined as the affinity of one for the other. Both the affinity of a drug for its receptor and its intrinsic activity are determined by its chemical structure. The chemical structure of a drug also contributes to the drug's specificity. A drug that interacts with a single type of receptor that is expressed on only a limited number of differentiated cells will exhibit high specificity. An example of such a drug is ranitidine, an H₂ receptor antagonist used to treat ulcers (Chapter 45). If, however, a receptor is expressed ubiquitously on a variety of cells throughout the body, drugs acting on such a widely expressed receptor will exhibit widespread effects, and could produce serious side effects or toxicities if the receptor serves important functions in multiple tissues.

There are numerous examples of drugs that work through a discrete action, but have effects throughout the body. These include the inotropic drug digoxin, which inhibits the ubiquitously expressed enzyme Na⁺,K⁺-ATPase (Chapter 28), and the antifolate anticancer drugs such as methotrexate that inhibit dihydrofolate reductase, an enzyme required by all cells for the synthesis of purines and thymidylate (Chapters 60 and 61). The Na⁺ channel blocker lidocaine has effects in peripheral nerves, the heart, and the central nervous system (CNS) because Na⁺ channels are highly expressed in all these tissues (Chapters 20 and 29). Lidocaine has local anesthetic effects when administered locally to prevent or relieve pain, but can also have cardiac and CNS effects if it reaches the systemic circulation. Even if the primary action of a drug is localized, as might be the case with injected lidocaine, the subsequent physiological effects of the drug may be widespread. One example would be immunosuppressant drugs (Chapter 35) that specifically inhibit cells of the immune system; their use is limited by the risk of opportunistic systemic infections. Other examples of drugs that act locally but have global effects are diuretics (Chapter 25) that act on cells in the kidney to alter serum electrolytes such as K⁺. However, the hypokalemia that a diuretic such as furosemide can cause can significantly increase the risk of skeletal muscle cramps and cardiac arrhythmias.

Many clinically important drugs exhibit a broad (low) specificity because the drug is able to interact with multiple receptors in different tissues. Such broad specificity might enhance the clinical utility of a drug, but also contribute to a spectrum of adverse side effects due to off-target interactions. One example of a drug that interacts with multiple receptors is amiodarone, an agent used to treat cardiac arrhythmias. In cardiac muscle, amiodarone inhibits Na⁺, Ca²⁺, and K⁺ channels, and noncompetitively inhibits β adrenergic receptors (Table 29–3). All of these drug-receptor interactions may contribute to its therapeutic efficacy and widespread use to treat many forms of arrhythmia. However, amiodarone also has a number of serious toxicities, some of which are due to the drug's structural similarity to thyroid hormone and its ability to interact with nuclear thyroid receptors. Amiodarone's salutary effects and toxicities may also be mediated through interactions with receptors that are poorly characterized or unknown.

Structure-Activity Relationships and Drug Design

A significant number of clinically useful drugs were developed in an era when drug discovery primarily involved screening compounds for their capacity to elicit salutary effects in patients or an animal disease model such as the spontaneously hypertensive rat or seizure-prone mouse. The receptors responsible for the clinical effects of many of these drugs have yet to be identified, although significant efforts are devoted toward identifying their mechanisms of action. Conversely, sequencing of the entire human genome has identified novel genes related by sequence to known receptors, but because the endogenous and exogenous ligands for these putative receptors are still unknown, these are referred to as orphan receptors. Orphan receptors are still found in the G–protein coupled receptor and nuclear hormone receptor families. Detailed knowledge of a drug's molecular target(s) can inform the development of new drugs with greater efficacy and lower toxicities. With the advent of transgenic animal models, it is now feasible to develop animal models that can test hypotheses regarding the possible physiological effects of altering a specific receptor's function and to predict the effects of receptor antagonists and agonists by genetically altering the receptor's expression and function. The methods and rationale currently used by the pharmaceutical industry to design and invent new drugs is covered fully in Chapter 1 and outlined briefly below.

Receptor occupancy theory assumes that response emanates from a receptor occupied by a drug, a concept that has its basis in the law of mass action. The basic currency of receptor pharmacology is the dose-response (or concentration-response) curve, a depiction of the observed effect of a drug as a function of its concentration in the receptor compartment. Figure 3–2 shows a typical dose-response curve; it reaches a maximal asymptotic value when the drug occupies all the receptor sites.

Affinity, Efficacy, and Potency. In general, the drug-receptor interaction is characterized by (1) binding of drug to receptor and (2) generation of a response in a biological system, as illustrated in Equation 3-1 where the drug or ligand is denoted as L and the inactive receptor as R. The first reaction, the reversible formation of the ligand-receptor complex LR, is governed by the chemical property of affinity.

(Equation 3-1)

where LR* is produced in proportion to [LR] and leads to a response. This simple relationship illustrates the reliance of the affinity of the ligand (L) with receptor (R) on both the forward or association rate (k₊₁) and the reverse or dissociation rate (k₋₁). At any given time, the concentration of ligand-receptor complex [LR] is equal to the product of k₊₁[L][R], the rate of formation of the bi-molecular complex LR, minus the product k₋₁[LR], the rate dissociation of LR into L and R. At equilibrium (i.e., when δ[LR]/δt = 0), k₊₁[L][R] = k₋₁[LR]. The equilibrium dissociation constant (K_D) is then described by ratio of the off and on rate constants (k₋₁/k₊₁).

Thus, at equilibrium,

(Equation 3-2)

The affinity constant or equilibrium association constant (K_A) is the reciprocal of the equilibrium dissociation constant (i.e., K_A = 1/K_D); thus a high-affinity drug has a low K_D and will bind a greater number of a particular receptor at a low concentration than a low-affinity drug. As a practical matter, the affinity of a drug is influenced most often by changes in its off-rate (k₋₁) rather than its on-rate (k₊₁).

Equation 3-2 permits us to write an expression of the fractional occupancy (f) of receptors by agonist:

(Equation 3-3)

This can be expressed in terms of K_A (or K_D) and [L]:

(Equation 3-4)

This relationship illustrates that when the concentration of drug equals the K_D (or 1/K_A), f = 0.5, that is, the drug will occupy 50% of the receptors. Note that this relationship describes only receptor occupancy, not the eventual response that is often amplified by the cell. Many signaling systems reach a full biological response with only a fraction of receptors occupied (described later). Potency is defined by example in Figure 3–3. Basically, when two drugs produce equivalent responses, the drug whose dose-response curve (plotted as in Figure 3-3A) lies to the left of the other (i.e., the concentration producing a half-maximal effect [EC₅₀] is smallest) is said to be the more potent.

Response to Drugs. The second reaction shown in Equation 3-1 is the reversible formation of the active ligand-receptor complex, LR*. The ability of a drug to activate a receptor and generate a cellular response is a reflection of its efficacy. Historically, efficacy has been treated as a proportionality constant that quantifies the extent of functional change imparted to a receptor-mediated response system on binding a drug. Thus, a drug with high efficacy may be a full agonist, eliciting, at some concentration, a full response. A drug with a lower efficacy at the same receptor may not elicit a full response at any dose (Figure 3–1). When it is possible to describe the relative efficacy of drugs at a particular receptor, a drug with a low intrinsic efficacy will be a partial agonist. A drug that binds to a receptor and exhibits zero efficacy is an antagonist. When the response of an agonist is measured in a simple biological system, the apparent dissociation constant, K_app, is a macroscopic equilibrium constant that reflects both the ligand binding equilibrium and the subsequent equilibrium that results in the formation of the active receptor LR*.

Quantifying Agonism. When the relative potency of two agonists of equal efficacy is measured in the same biological system, and downstream signaling events are the same for both drugs, the comparison yields a relative measure of the affinity and efficacy of the two agonists (Figure 3–3). It is convenient to describe agonist response by determining the half-maximally effective concentration (EC₅₀) for producing a given effect. Thus, measuring agonist potency by comparison of EC₅₀ values is one method of measuring the capability of different agonists to induce a response in a test system and for predicting comparable activity in another. Another method of estimating agonist activity is to compare maximal asymptotes in systems where the agonists do not produce maximal response (Figure 3–3B). The advantage of using maxima is that this property depends solely on efficacy, whereas drug potency is a mixed function of both affinity and efficacy.

Quantifying Antagonism. Characteristic patterns of antagonism are associated with certain mechanisms of blockade of receptors. One is straightforward competitive antagonism, whereby a drug with affinity for a receptor but lacking intrinsic efficacy competes with the agonist for the primary binding site on the receptor (Ariens, 1954; Gaddum, 1957). The characteristic pattern of such antagonism is the concentration-dependent production of a parallel shift to the right of the agonist dose-response curve with no change in the maximal response (Figure 3–4A). The magnitude of the rightward shift of the curve depends on the concentration of the antagonist and its affinity for the receptor (Schild, 1957).

A partial agonist similarly can compete with a "full" agonist for binding to the receptor. However, increasing concentrations of a partial agonist will inhibit response to a finite level characteristic of the drug's intrinsic efficacy; a competitive antagonist will reduce the response to zero. Partial agonists thus can be used therapeutically to buffer a response by inhibiting excessive receptor stimulation without totally abolishing receptor stimulation (for instance, pindolol, a β antagonist with slight intrinsic agonist activity, will prevent overstimulation of the heart by blocking effects of endogenous catecholamines but will assure slight receptor stimulation in patients overly sensitive to the negative inotropic and negative chronotropic effects of β blockade).

An antagonist may dissociate so slowly from the receptor that its action is exceedingly prolonged, as with the opiate partial agonist buprenorphine and the Ca²⁺ channel blocker amlodipine. In the presence of a slowly dissociating antagonist, the maximal response to the agonist will be depressed at some antagonist concentrations (Figure 3–4B). Operationally, this is referred to as noncompetitive antagonism, although the molecular mechanism of action really cannot be inferred unequivocally from the effect. An antagonist may also interact irreversibly (covalently) with a receptor, as do the α adrenergic antagonist phenoxybenzamine and the acetylcholinesterase inhibitor DFP (diisopropylfluorophosphate), to produce relatively irreversible effects. An irreversible antagonist competing for the same binding site as the agonist can produce the pattern of antagonism shown in Figure 3–4B. Noncompetitive antagonism can also be produced by another type of drug, referred to as an allosteric or allotopic antagonist. This type of drug produces its effect by binding to a site on the receptor distinct from that of the primary agonist, thereby changing the affinity of the receptor for the agonist. In the case of an allosteric antagonist, the affinity of the receptor for the agonist is decreased by the antagonist (Figure 3–4C). In contrast, a drug binding at an allosteric site could potentiate the effects of primary agonists (Figure 3–4D); such a drug would be referred to as an allosteric agonist or co-agonist (May et al., 2007).

Individuals vary in the magnitude of their response to the same concentration of a single drug or to similar drugs, and a given individual may not always respond in the same way to the same drug concentration. Attempts have been made to define and measure individual "sensitivity" (or "resistance") to drugs in the clinical setting, and progress has been made in understanding some of the determinants of sensitivity to drugs that act at specific receptors. Drug responsiveness may change because of disease or because of previous drug administration. Receptors are dynamic, and their concentration and function may be up- or down-regulated by endogenous and exogenous factors.

Data on the correlation of drug levels with efficacy and toxicity must be interpreted in the context of the pharmacodynamic variability in the population (e.g., genetics, age, disease, and the presence of co-administered drugs). The variability in pharmacodynamic response in the population may be analyzed by constructing a quantal concentration-effect curve (Figure 3–5A). The dose of a drug required to produce a specified effect in 50% of the population is the median effective dose (ED₅₀, Figure 3–5A). In preclinical studies of drugs, the median lethal dose (LD₅₀) is determined in experimental animals (Figure 3-5B). The LD₅₀/ED₅₀ ratio is an indication of the therapeutic index, which is a statement of how selective the drug is in producing its desired effects versus its adverse effects. A similar term, the therapeutic window, is the range of steady-state concentrations of drug that provides therapeutic efficacy with minimal toxicity (Figure 3–6). In clinical studies, the dose, or preferably the concentration, of a drug required to produce toxic effects can be compared with the concentration required for therapeutic effects in the population to evaluate the clinical therapeutic index. Since pharmacodynamic variation in the population may be marked, the concentration or dose of drug required to produce a therapeutic effect in most of the population usually will overlap the concentration required to produce toxicity in some of the population, even though the drug's therapeutic index in an individual patient may be large. Also, the concentration-percent curves for efficacy and toxicity need not be parallel, adding yet another complexity to determination of the therapeutic index in patients. Finally, no drug produces a single effect, and the therapeutic index for a drug will vary depending on the effect being measured.

Factors Modifying Drug Action. Many factors can influence the therapeutic efficacy and safety of a drug in an individual patient. These same factors give rise to inter-individual variability in the dose required to obtain optimal therapeutic effect with minimal adverse effects. Some of the factors that contribute to the wide patient-to-patient variability in the dose required for optimal therapy observed with many drugs are shown in Figure 3–7. Therapeutic success and safety result from integrating evidence of efficacy and safety with knowledge of the individual factors that determine response in a given patient. Determinants of inter-individual variation in response to drugs that are due to pharmacokinetics include disease-related alterations such as impaired renal and liver clearance due to renal and hepatic disease, circulatory failure, altered drug binding to plasma proteins, impaired GI absorption, and pharmacokinetic drug interactions. The effects of these factors on variability of drug pharmacokinetics are described more thoroughly in Chapters 2 and 5, 6, 7.

Receptors That Affect Concentrations of Endogenous Ligands

A large number of drugs act by altering the synthesis, storage, release, transport, or metabolism of endogenous ligands such as neurotransmitters, hormones, and other intercellular mediators. For instance, there are many examples of drugs that act on neuroeffector junctions by altering neurotransmitter synthesis, storage of neurotransmitter in vesicles, release of neurotransmitters into the synaptic cleft, and subsequent removal of the neurotransmitter from the synaptic cleft by hydrolysis or transport into the pre-synaptic or post-synaptic neuron. The effects of these drugs can either enhance or diminish the effects of the neurotransmitter in order to achieve the desired therapeutic effect. For instance, some of the drugs acting on adrenergic neurotransmission (Chapters 8 and 12) include α-methyltyrosine (inhibits synthesis of norepinephrine (NE)), cocaine (blocks NE reuptake), amphetamine (promotes NE release), and selegeline (inhibits NE breakdown). There are similar examples for other neurotransmitter systems including acetylcholine (ACh; Chapters 8 and 10), dopamine (DA), and serotonin (5HT; Chapters 13, 14, 15). Drugs that affect the synthesis of circulating mediators such as vasoactive peptides (e.g., angiotensin-converting enzyme inhibitors; Chapter 26) and lipid-derived autocoids (e.g., cyclooxygenase inhibitors; Chapter 33) are also widely used in the treatment of hypertension, inflammation, myocardial ischemia, and other disease states.

Receptors That Regulate the Ionic Milieu

A relatively small number of drugs act by affecting the ionic millieu of blood, urine, and the GI tract. The receptors for these drugs are ion pumps and transporters, many of which are expressed only in specialized cells of the kidney and GI system. Drug effects on many of these receptors can have effects throughout the body due to changes in blood electrolytes and pH. For instance, most of the diuretics (e.g., furosemide, chlorothiazide, amiloride) act by directly affecting ion pumps and transporters in epithelial cells of the nephron that increase the movement of Na⁺ into the urine, or by altering the expression of ion pumps in these cells (e.g., aldosterone). Chapter 25 provides a detailed description of the mechanisms of action of diuretic drugs. Another therapeutically important target is the H⁺,K⁺-ATPase (proton pump) of gastric parietal cells. Irreversible inhibition of this proton pump by drugs such as esomeprazole reduces gastric acid secretion by 80-95% (Chapter 45) and is a mainstay of therepy for peptic ulcer.

Cellular Pathways Activated by Physiological Receptors

Signal Transduction Pathways. Physiological receptors have at least two major functions, ligand binding and message propagation (i.e., signaling). These functions imply the existence of at least two functional domains within the receptor: a ligand-binding domain and an effector domain. The structure and function of these domains in many families of receptors have been deduced from high-resolution crystal structures of the receptor proteins and/or by analysis of the behavior of intentionally mutated receptors. Many drugs target the extracelluar ligand-binding domain of physiological receptors. Examples include the widely used β adrenergic antagonists. However, drugs can affect the receptor by targeting either domain, as in the case of anticancer drugs used to target the epidermal growth factor receptor (EGFR; Chapters 60, 61, 62). Cetuximab is a monoclonal antibody that targets the extracellular ligand-binding domain of the EGFR and inhibits epidermal growth factor (EGF) signaling, whereas the small molecule drugs gefitinib and erlotinib bind the intracellular effector domain and block the protein tyrosine kinase activity of the activated EGFR.

The regulatory actions of a receptor may be exerted directly on its cellular target(s), on effector protein(s), or may be conveyed by intermediary cellular signaling molecules called transducers. The receptor, its cellular target, and any intermediary molecules are referred to as a receptor-effector system or signal transduction pathway. Frequently, the proximal cellular effector protein is not the ultimate physiological target but rather is an enzyme, ion channel, or transport protein that creates, moves, or degrades a small molecule (e.g., a cyclic nucleotide, inositol trisphosphate, or NO) or ion (e.g., Ca²⁺) termed a second messenger. If the effector is an ion channel or ion pump, the effect of ligand binding can be a change in membrane potential that alters the excitability of the cell. Second messengers can diffuse in the proximity of their synthesis or release and convey information to a variety of targets, which may integrate multiple signals. Even though these second messengers originally were thought of as freely diffusible molecules within the cell, imaging studies show that their diffusion and intracellular actions are constrained by compartmentation—selective localization of receptor-transducer-effector-signal termination complexes—established by protein-lipid and protein-protein interactions (Baillie, 2009). All cells express multiple forms of proteins designed to localize signaling pathways by protein-protein interactions; these proteins are termed scaffolds or anchoring proteins. Examples of scaffold molecules include the AKAPs (A-kinase anchoring proteins) that bind the regulatory subunit of the cyclic AMP dependent protein kinase (PKA) near its substrate(s) in various subcellular compartments (Carnegie et al., 2009).

Signal Integration and Amplification. Receptors and their associated effector and transducer proteins also act as integrators of information as they coordinate signals from multiple ligands with each other and with the differentiated activity of the target cell. For example, signal transduction systems regulated by changes in cyclic AMP (cAMP) and intracellular Ca²⁺ are integrated in many excitable tissues. In cardiac myocytes, an increase in cellular cAMP caused by activation of β adrenergic receptors enhances cardiac contractility by augmenting the rate and amount of Ca²⁺ delivered to the contractile apparatus; thus, cAMP and Ca²⁺ are positive contractile signals in cardiac myocytes. By contrast, cAMP and Ca²⁺ produce opposing effects on the contractility of smooth muscle cells: as usual, Ca²⁺ is a contractile signal, however, activation of β adrenergic receptors on these cells activates the cAMP-PKA pathway, which leads to relaxation through the phosphorylation of proteins that mediate Ca²⁺ signaling, such as myosin light chain kinase and ion channels that hyperpolarize the cell membrane. Thus, the distinct patterns of integration of signal transduction systems within target cells can lead to a variety of pharmacodynamic effects that result from functional interactions downstream from the receptors. These functional interactions can be synergistic, additive, or antagonistic.

Another important property of physiological receptors is their capacity to significantly amplify a physiological signal. Neurotransmitters, hormones, and other extracellular ligands are often present at the ligand-binding domain of a receptor in very low concentrations (nM to μM levels). However, the effector domain or the signal transduction pathway often contains enzymes and enzyme cascades to catalytically amplify the intended signal. In this regard, the description of receptor occupancy-cellular response in Equation 3-1 is an oversimplification. The ability of virtually all receptors to amplify physiological signals makes them excellent targets for natural ligands and drugs. When, e.g., a single agonist molecule binds to a receptor that is an ion channel, hundreds of thousands to millions of ions flow through the channel every second. Similarly, the binding of a single photon to cis-retinal in the photoreceptor rhodpsin is eventually amplified ~1 × 10⁶-fold. In the case of nuclear receptors, a single steroid hormone molecule binding to its receptor initiates the transcription of many copies of specific mRNAs, which in turn can give rise to multiple copies of a single protein.

Structural and Functional Families of Physiological Receptors

Receptors for physiological regulatory molecules can be assigned to functional families whose members share similar molecular structures and biochemical mechanisms with common features. Table 3–1 outlines six major families of receptors with examples of their physiological ligands, signal transduction systems, and drugs that affect these systems. The basic structure of their ligand-binding domains, effector domains, and how agonist binding influences the regulatory activity of the receptor is well understood for each of these signal transduction systems. The relatively small number of biochemical mechanisms and structural formats used for cellular signaling is fundamental to the ways in which target cells integrate signals from multiple receptors to produce additive, sequential, synergistic, or mutually inhibitory responses.

G Protein–Coupled Receptors (GCPRs)

Receptors and G Proteins. GPCRs span the plasma membrane as a bundle of seven α-helices (Palczewski et al., 2000) (Figure 3–8). Humans express over 800 GPCRs that make up the third largest family of genes in humans, with roughly half of these GPCRs dedicated to sensory perception (smell, taste, and vision). The remaining receptors regulate an impressive number of physiological functions including nerve activity, tension of smooth muscle, metabolism, rate and force of cardiac contraction, and the secretion of most glands in the body. Included among the ligands for GPCRs are neurotransmitters such as ACh, biogenic amines such as NE, all eicosanoids and other lipid signaling molecules, peptide hormones, opioids, amino acids such as GABA, and many other peptide and protein ligands. GPCRs are important regulators of nerve activity in the CNS and are the receptors for the neurotransmitters of the peripheral autonomic nervous system. For example, ACh released by the parasympathetic nervous system regulates the functions of glands and smooth muscle through its action on muscarinic receptors. NE released by the sympathetic nervous system interacts with α and β adrenergic receptors to regulate cardiac function and the tone of vascular smooth muscle (Chapters 8, 9, 10, 11, 12). Because of their number and physiological importance, GPCRs are the targets for many drugs; perhaps half of all non-antibiotic prescription drugs act at these receptors.

Receptor Subtypes. There are multiple receptor subtypes within families of receptors. Ligand-binding studies with multiple chemical entities initially identified receptor subtypes; molecular cloning has greatly accelerated the discovery and definition of additional receptor subtypes; their expression as recombinant proteins has facilitated the discovery of subtype-selective drugs. Distinct but related receptors may display distinctive patterns of selectivity among agonist or antagonist ligands. When selective ligands are not known, the receptors are more commonly referred to as isoforms rather than as subtypes. The distinction between classes and subtypes of receptors, however, is often arbitrary or historical. The α₁, α₂, and β adrenergic receptors differ from each other both in ligand selectivity and in coupling to G proteins (G_q, G_i, and G_s, respectively), yet α and β are considered receptor classes and α₁ and α₂ are considered subtypes. The α_1A, α_1B, and α_1C receptor isoforms differ little in their biochemical properties, although their tissue distributions are distinct. The β₁, β₂, and β₃ adrenergic receptor subtypes exhibit differences in both tissue distribution and regulation by phosphorylation by G–protein receptor kinases (GRKs) and PKA.

Pharmacological differences among receptor subtypes are exploited therapeutically through the development and use of receptor-selective drugs. Such drugs may be used to elicit different responses from a single tissue when receptor subtypes initiate different intracellular signals, or they may serve to differentially modulate different cells or tissues that express one or another receptor subtype. For example, β₂ adrenergic agonists such as terbutaline are used for bronchodilation in the treatment of asthma in the hope of minimizing cardiac side effects caused by stimulation of the β₁ adrenergic receptor (Chapter 12). Conversely, the use of β₁-selective antagonists minimizes the chance of bronchoconstriction in patients being treated for hypertension or angina (Chapters 12 and 27). Increasing the selectivity of a drug among tissues or among responses elicited from a single tissue may determine whether the drug's therapeutic benefits outweigh its unwanted effects.

G Proteins. GPCRs couple to a family of heterotrimeric GTP-binding regulatory proteins termed G proteins. G proteins are signal transducers that convey the information that agonist is bound to the receptor from the receptor to one or more effector proteins (Gilman, 1987). G–protein-regulated effectors include enzymes such as adenylyl cyclase, phospholipase C, cyclic GMP phosphodiesterase (PDE6), and membrane ion channels selective for Ca²⁺ and K⁺ (Table 3–1, Figure 3–8). The G protein heterotrimer is composed of a guanine nucleotide-binding α subunit, which confers specific recognition to both receptors and effectors, and an associated dimer of β and γ subunits that helps confer membrane localization of the G protein heterorimer by prenylation of the γ subunit. In the basal state of the receptor-heterotrimer complex, the α subunit contains bound GDP and the α-GDP:βγ complex is bound to the unliganded receptor (Figure 3-8). The G protein family is comprised of 23 α subunits (which are the products of 17 genes), 7 β subunits, and 12 γ subunits. The α subunits fall into four families (G_s, G_i, G_q, and G_12/13) which are responsible for coupling GPCRs to relatively distinct effectors. The G_s α subunit uniformly activates adenylyl cyclase; the G_i α subunit can inhibit certain isoforms of adenylyl cyclase; the G_q α subunit activates all forms of phospholipase Cβ; and the G_12/13 α subunits couple to guanine nucleotide exchange factors (GEFs), such as p115RhoGEF for the small GTP-binding proteins Rho and Rac. The signaling specificity of the large number of possible βγ combinations is not yet clear; nonetheless, it is known that K⁺ channels, Ca²⁺ channels, and PI-3 kinase (PI3K) are some of the effectors of free βγ dimer (Figure 3–8).

G Protein Activation. When an agonist binds to a GPCR, there is a conformational change in the receptor that is transmitted from the ligand-binding pocket to the second and third intracellular loops of the receptor which couple to the G protein heterotrimer. This conformational change causes the α subunit to exchange its bound GDP for GTP (Figure 3-8). Binding of GTP activates the α subunit and causes it to release both the βγ dimer and the receptor, and both the GTP-bound α subunit and the βγ heterodimer become active signaling molecules (Gilman, 1987). The interaction of the agonist-bound GPCR with the G protein is transient; following activation of one G protein, the receptor is freed to interact with other G proteins. Depending on the nature of the α subunit, the active, GTP-bound form binds to and regulates effectors such as adenylyl cyclase (via G_s α) or phospholipase Cβ (via G_q α). The βγ subunit can regulate many effectors including ion channels and enzymes such as PI3-K (Figure 3–8). The G protein remains active until the GTP bound to the α subunit is hydrolyzed to GDP. The α subunit has a slow intrinsic rate of GTP hydrolysis that is modulated by a family of proteins termed regulators of G protein signaling (RGSs). The RGS proteins greatly accelerate the hydrolysis of GTP and are potentially attractive drug targets (Ross and Wilkie, 2000). Once the GDP bound to the α subunit is hydrolyzed to GDP, the βγ subunit and receptor recombine to form the inactive receptor-G protein heterotrimer basal complex that can be reactivated by another ligand-binding event (Figure 3–8).

Second Messengers

Cyclic AMP. Cyclic AMP is synthesized by adenylyl cyclase under the control of many GPCRs; stimulation is mediated by the G_s α subunit, inhibition by the G_i α subunit. The cyclic AMP pathway provides a good basis for understanding the architecture and regulation of many second messenger signaling systems (for an overview of cyclic nucleotide action, see Beavo and Brunton, 2002).

There are nine membrane-bound isoforms of adenylyl cyclase (AC) and one soluble isoform found in mammals (Hanoune and Defer, 2001). The membrane-bound ACs are glycoproteins of ~120 kDa with considerable sequence homology: a small cytoplasmic domain; two hydrophobic transmembrane domains, each with six membrane-spanning helices; and two large cytoplasmic domains. Membrane-bound ACs exhibit basal enzymatic activity that is modulated by binding of GTP-liganded α subunits of the stimulatory and inhibitory G proteins (G_s and G_i). Numerous other regulatory interactions are possible, and these enzymes are catalogued based on their structural homology and their distinct regulation by G protein α and βγ subunits, Ca²⁺, protein kinases, and the actions of the diterpene forskolin. Cyclic AMP generated by adenylyl cyclases has three major targets in most cells, the cyclic AMP dependent protein kinase (PKA), cAMP-regulated guanine nucleotide exchange factors termed EPACs (exchange factors directly activated by cAMP), and via PKA phosphorylation, a transcription factor termed CREB (cAMP response element binding protein). In cells with specialized functions, cAMP can have additional targets such as cyclic nucleotide-gated ion channels (Wahl-Schott and Biel, 2009), cyclic nucleotide-regulated phosphodiesterases (PDEs), and several ABC transporters (MRP4 and MRP5) for which it is a substrate (see Chapter 7).

PKA. The best understood target of cyclic AMP is the PKA holoenzyme consisting of two catalytic (C) subunits reversibly bound to a regulatory (R) subunit dimer to form a heterotetramer complex (R₂C₂). At low concentrations of cAMP, the R subunits inhibit the C subunits; thus the holoenzyme is inactive. When AC is activated and cAMP concentrations are increased, four cyclic AMP molecules bind to the R₂C₂ complex, two to each R subunit, causing a conformational change in the R subunits that lowers their affinity for the C subunits, causing their activation. The active C subunits phosphorylate serine and threonine residues on specific protein substrates.

Cyclic AMP–Regulated Guanine Nucleotide Exchange Factors (GEFs). The small GTP-binding proteins are monomeric GTPases and key regulators of cell function. The small GTPases operate as binary switches that exist in GTP- or GDP-liganded conformations. They integrate extracellular signals from membrane receptors with cytoskeletal changes and activation of diverse signaling pathways, regulating such processes as phagocytosis, progression through the cell cycle, cell adhesion, gene expression, and apoptosis (Etienne-Manneville and Hall, 2002). A number of extracellular stimuli signal to the small GTPases directly or through second messengers such as cyclic AMP.

PKG. Stimulation of receptors that raise intracellular cyclic GMP concentrations (Figure 3-11) leads to the activation of the cyclic GMP-dependent protein kinase (PKG) that phosphorylates some of the same substrates as PKA and some that are PKG-specific. In some tissues, PKG can also be activated by cAMP. Unlike the heterotetramer (R₂C₂) structure of the PKA holoenzyme, the catalytic domain and cyclic nucleotide-binding domains of PKG are expressed as a single polypeptide, which dimerizes to form the PKG holoenzyme.

PDEs. Cyclic nucleotide phosphodiesterases form another family of important signaling proteins whose activities are regulated via the rate of gene transcription as well as by second messengers (cyclic nucleotides or Ca²⁺) and interactions with other signaling proteins such as β arrestin and protein kinases. PDEs hydrolyze the cyclic 3′,5′-phosphodiester bond in cAMP and cGMP, thereby terminating their action.

Other Second Messengers

Ca²⁺. Calcium is an important messenger in all cells and can regulate diverse responses including gene expression, contraction, secretion, metabolism, and electrical activity. Ca²⁺ can enter the cell through Ca²⁺ channels in the plasma membrane (See "Ion Channels", below) or be released by hormones or growth factors from intracellular stores. In keeping with its role as a signal, the basal Ca²⁺ level in cells is maintained in the 100 n range by membrane Ca²⁺ pumps that extrude Ca²⁺ to the extracellular space and a sarcoplasmic reticulum (SR) Ca²⁺-ATPase (SERCA) in the membrane of the endoplasmic reticulum (ER) that accumulates Ca²⁺ into its storage site in the ER/SR.

Hormones and growth factors release Ca²⁺ from its intracellular storage site, the ER, via a signaling pathway that begins with activation of phospholipase C at the plasma membrane, of which there are two primary forms, PLCβ and PLCγ. GPCRs that couple to G_q or G_i activate PLCβ by activating the G protein α subunit (Figure 3–8) and releasing the βγ dimer. Both the active, G_q-GTP bound α subunit and the βγ dimer can activate certain isoforms of PLCβ. PLCγ isoforms are activated by tyrosine phosphorylation, including phosphorylation by receptor and non-receptor tyrosine kinases. For example, growth factor receptors such as the epidermal growth factor receptor (EGFR) are receptor tyrosine kinases (RTKs) that autophosphorylate on tyrosine residues upon binding their cognate growth factor. The phosphotyrosine formed on the cytoplasmic domain of the RTK is a binding site for signaling proteins that contain SH2 domains, such as PLCγ. Once recruited to the RTK's SH2 domain, PLCγ is phosphorylated/activated by the RTK.

Ion Channels

The lipid bilayer of the plasma membrane is impermeable to anions and cations, yet changes in the flux of ions across the plasma membrane are critical regulatory events in both excitable and non-excitable cells. To establish and maintain the electrochemical gradients required to maintain a membrane potential, all cells express ion transporters for Na⁺, K⁺, Ca²⁺, and Cl⁻. For example, the Na⁺,K⁺-ATPase pump expends cellular ATP to pump Na⁺ out of the cell and K⁺ into the cell. The electrochemical gradients thus established are used by excitable tissues such as nerve and muscle to generate and transmit electrical impulses, by non-excitable cells to trigger biochemical and secretory events, and by all cells to support a variety of secondary symport and antiport processes (Chapter 5).

Passive ion fluxes down cellular electrochemical gradients are regulated by a large family of ion channels located in the membrane. Humans express ~232 distinct ion channels to precisely regulate the flow of Na⁺, K⁺, Ca²⁺, and Cl⁻ across the cell membrane (Jegla et al., 2009). Because of their roles as regulators of cell function, these proteins are important drug targets. The diverse ion channel family can be divided into subfamilies based on the mechanisms that open the channels, their architecture, and the ions they conduct. They can also be classified as voltage-activated, ligand-activated, store-activated, stretch-activated, and temperature-activated channels. Examples of channels that are major drug targets are detailed next.

Voltage-Gated Channels. Humans express multiple isoforms of voltage-gated channels for Na⁺, K⁺, Ca²⁺, and Cl⁻ ions. In nerve and muscle cells, voltage-gated Na⁺ channels are responsible for the generation of robust action potentials that depolarize the membrane from its resting potential of −70 mV up to a potential of +20 mV within a few milliseconds. These Na⁺ channels are composed of three subunits, a pore-forming α subunit and two regulatory β subunits. The α subunit is a 260 kDa protein containing four domains that form a Na⁺ ion-selective pore by arranging into a pseudo-tetramer shape. The β subunits are ~36 kDa proteins that span the membrane once (Figure 3–9A). Each domain of the α subunit contains six membrane-spanning helices (S1-S6) with an extracellular loop between S5 and S6, termed the pore-forming or P loop; the P loop dips back into the pore and, combined with residues from the corresponding P loops from the other domains, provides a selectivity filter for the Na⁺ ion. Four other helices surrounding the pore (one S4 helix from each of the domains) contain a set of charged amino acids that form the voltage sensor and cause a conformational change in the pore at more positive voltages leading to opening of the pore and depolarization of the membrane (Purves and McNamara, 2008). The voltage-activated Na⁺ channels in pain neurons are targets for local anesthetics such as lidocaine and tetracaine, which block the pore, inhibit depolarization, and thus block the sensation of pain. They are also the targets of the naturally occurring marine toxins, tetrodotoxin and saxitoxin (Chapter 20). Voltage-activated Na⁺ channels are also important targets of many drugs used to treat cardiac arrhythmias (Chapter 29).

Ligand-Gated Channels. Channels activated by the binding of a ligand to a specific site in the channel protein have a diverse architecture and set of ligands. Major ligand-gated channels in the nervous system are those that respond to excitatory neurotransmitters such as acetylcholine or glutamate (or agonists such as AMPA and NMDA) and inhibitory neurotransmitters such as glycine or γ-aminobutyric acid (GABA) (Purves and McNamara, 2008). Activation of these channels is responsible for the majority of synaptic transmission by neurons both in the CNS and in the periphery (Chapters 8, 11, 14). In addition, there are a variety of more specialized ion channels that are activated by intracellular small molecules, and are structurally distinct from conventional ligand-gated ion channels. These include ion channels that are formally members of the K_v family, such as the hyperpolarization and cAMP-gated (HCN) channel expressed in the heart (Wahl-Schott and Biel, 2009) that is responsible for the slow depolarization seen in phase 4 of AV and SA nodal cell action potentials (Chapter 29), and the cyclic nucleotide-gated (CNG) channels important for vision (Chapter 64). The intracellular small molecule category of ion channels also includes the IP₃-sensitive Ca²⁺ channel responsible for release of Ca²⁺ from the ER and the sulfonylurea "receptor" (SUR1) that associates with the K_ir6.2 channel to regulate the ATP-dependent K⁺ channel (K_ATP) in pancreatic beta cells. The K_ATP channel is the target of oral hypoglycemic drugs such as sulfonylureas and meglitinides that stimulate insulin release from pancreatic β cells and are used to treat type 2 diabetes (Chapter 43). Other specialized channels include the 5-HT₃-regulated channel expressed on afferent vagal nerves that stimulates emesis. Ondansetron is an important antagonist of the 5-HT₃-gated channel used to inhibit emesis caused by drugs or disease (Chapter 46).

TRP Channels. The transient receptor potential (TRP) channels comprise a superfamily of ubiquitously expressed ion channels that is remarkable in its diversity and domain strucutre (Clapham 2003; Venkatachalam and Montell, 2007). Although the TRP channels are not presently targets of approved drugs, there is significant interest in developing drugs that can alter the function of these ion channels because of their roles in various sensory phenomena such as pain, temperature, osmolarity, touch, olfaction, vision, and hearing. Because these channels contain multiple domains, they can act as signal integrators and most can be activated by multiple mechanisms. There are 27 TRP channel genes in humans, representing six different TRP channel families. TRP channels contain six membrane-spanning segments and the functional ion channels consist of tetrameric complexes. Closely related TRP channels can form heterotetramers. The TRP channels are cation channels, but as with other heteromultimeric ion channels, the subunit composition of the multimeric channels can prescribe a number of important channel characteristics, including ion selectivity and activation properties. The intracellular domains of TRP channels can include ankyrin domains, protein kinase domains, and ADP-ribose pyrophosphatase domains. Mutations in TRP channels are known to cause several disease including hypomagnesemia and hypocalcemia, and various renal disorders and neurodegenerative diseases.

Transmembrane Receptors Linked to Intracellular Enzymes

Mammalian cells express a diverse group of physiological membrane receptors with extracellular ligand-binding domains and an intrinsic enzymatic activity on the cytoplasmic surface of the cell. These molecules include the receptor tyrosine kinases (RTKs) such as the epidermal growth factor (EGF) and insulin receptors, which contain intrinsic tyrosine kinases in the cytoplasmic domain of the receptor; tyrosine kinase-associated receptors without enzymatic activity, such as the receptors for γ-interferon, which recruit the cytoplasmic Janus tyrosine kinases (JAKs); receptor serine-threonine kinases such as the TGF-β receptor; and receptors linked to other enzyme activities such as the receptors for natriuretic pepides, which have a cytoplasmic guanylate cyclase activity that produces a soluble second messenger, cyclic GMP (see the next section). Receptors responsible for innate immunity, the Toll-like receptors and those for tumor necrosis factors (TNF-α), have many features in common with the JAK-STAT receptors.

Receptor Tyrosine Kinases. The receptor tyrosine kinases include receptors for hormones such as insulin, for multiple growth factors such EGF, platelet-derived growth factor (PDGF), nerve growth factor (NGF), fibroblast growth factor (FGF), vascular endothelial growth factor (VEGF), and ephrins. With the exception of the insulin receptor, which has α and β chains (Chapter 43), these molecules consist of single polypeptide chains with large, cysteine-rich extracellular domains, short transmembrane domains, and an intracellular region containing one (or in some cases two) protein tyrosine kinase domains. Activation of growth factor receptors leads to cell survival, cell proliferation, and differentiation. Activation of the ephrin receptors leads to neuronal angiogenesis, axonal migration, and guidance (Ferguson, 2008; Hubbard and Till, 2000).

JAK-STAT Receptor Pathway. Cells express a family of receptors for cytokines such as γ-interferon and hormones like growth hormone and prolactin, which signal to the nucleus by a more direct manner than the receptor tyrosine kinases. These receptors have no intrinsic enzymatic activity, rather the intracellular domain binds a separate, intracellular tryosine kinase termed a Janus kinase (JAK). Upon the dimerization induced by ligand binding, JAKs phosphorylate other proteins termed signal transducers and activators of transcription (STATs), which translocate to the nucleus and regulate transcription (Figure 3–10B). The entire pathway is termed the JAK-STAT pathway (Gough et al., 2008; Wang et al., 2009). There are four JAKs and six STATs in mammals which, depending on the cell type and signal, combine differently to activate gene transcription. For example, prolactin appears to use JAK1, JAK2, and STAT5 to stimulate milk production.

Receptor Serine-Threonine Kinases. Protein ligands such as TGF-β activate a family of receptors that are analogous to the receptor tyrosine kinases except that they have a serine/threonine kinase domain in the cytoplasmic region of the protein. There are two isoforms of the monomeric receptor protein, type I (7 forms) and type II (5 forms). In the basal state, these proteins exist as monomers; upon binding an agonist ligand, they dimerize, leading to phosphorylation of the kinase domain of the type I monomer, which activates the receptor. The activated receptor then phosphorylates a gene regulatory protein termed a Smad. There are multiple Smads in cells; once phosphorylated by the activated receptor on a serine residue, Smad dissociates from the receptor, migrates to the nucleus, associates with transcription factors and regulates genes leading to morphogenesis and transformation. There are also inhibitory Smads (the Smad6 or Smad7 isoforms) that compete with the phosphorylated Smads to terminate signaling.

Toll-Like Receptors. Signaling related to the innate immune system is carried out by a family of over ten single membrane-spanning receptors termed Toll-like receptors (TLR), which are highly expressed in hematopoeitic cells. In a single polypeptide chain, these receptors contain a large extracellular ligand-binding domain, a short membrane-spanning domain, and a cytoplasmic region termed the TIR domain that lacks intrinsic enzymatic activity. Ligands for TLR are comprised of a multitude of pathogen products including lipids, peptidoglycans, lipopeptides, and viruses. Activation of these receptors produces an inflammatory response to the pathogenic microorganisms. As with all single membrane-spanning receptors, the first step in activation of TLR by ligands is dimerization, which in turn causes signaling proteins to bind to the receptor to form a signaling complex.

TNF-α Receptors. The mechanism of action of tumor necrosis factor α (TNF-α) signaling to the NF-κB transcription factors is very similar to that used by Toll-like receptors in that the intracellular domain of the receptor has no enzymatic activity. The TNF-α receptor is another single membrane-spanning receptor with an extracellular ligand-binding domain, a transmembrane domain, and a cytoplasmic domain termed the death domain.

Receptors That Stimulate Synthesis of Cyclic GMP

The signaling pathways that regulate the synthesis of cyclic GMP in cells include hormonal regulation of transmembrane guanylate cyclases such as the atrial natriuretic peptide receptor (ANP) and the activation of soluble forms of guanylate cyclase by nitric oxide (NO). The downstream effects of cyclic GMP are carried out by multiple isoforms of PKG, cyclic GMP-gated ion channels, and cyclic GMP-modulated phosphodiesterases that degrade cyclic AMP (described later).

Natriuretic Peptide Receptors. The class of membrane receptors with intrinsic enzymatic activity includes the receptors for three small peptide ligands released from cells in cardiac tissues and the vascular system. These peptides are atrial natriuretic peptide (ANP), which is released from atrial storage granules following expansion of intravascular volume or stimulation with pressor hormones; brain natriuretic peptide (BNP), which (in spite of its name) is synthesized and released in large amounts from ventricular tissue in response to volume overload; and C-type natriuretic peptide (CNP), which is synthesized in the brain and endothelial cells. Like BNP, CNP is not stored in granules; rather, its synthesis and release are increased by growth factors and sheer stress on vascular endothelial cells (Potter et al., 2009). The major physiological effects of these hormones are to decrease blood pressure (ANP, BNP), to reduce cardiac hypertrophy and fibrosis (BNP), and to stimulate long bone growth (CNP).

The transmembrane receptors for ANP, BNP, and CNP are ligand-activated guanylate cyclases. The ANP and BNP receptors contain a ~450 amino acid extracellular domain that binds the peptide, a short 20 amino acid transmembrane domain, and large intracellular domains that contain a kinase homology region, a dimerization domain, and a C-terminal guanylate cyclase domain. Phosphorylation of serine residues in the kinase domain is important for activity; dephosphorylation of these residues leads to desensitization of the receptor. Ligand binding brings the juxtamembrane regions together and stimulates guanylate cyclase activity (Figure 3–11).

NO Synthase and Soluble Guanylate Cyclase. Nitric oxide (NO) is a unique signal, a very labile gas produced locally in cells by the enzyme nitric oxide synthase (NOS); the resulting NO is able to markedly stimulate the soluble form of guanylate cyclase to produce cyclic GMP. There are three forms of nitric oxide synthase, neuronal NOS (nNOS or NOS1), endothelial NOS (eNOS or NOS3), and inducible NOS (iNOS or NOS2). All three forms of this enzyme are widely expressed but are especially important in the cardiovascular system, where they are found in myocytes, vascular smooth muscle cells, endothelial cells, hematopoietic cells, and platelets.

In humans, nuclear hormone receptors comprise a superfamily of 48 receptors that respond to a diverse set of ligands. The receptor proteins are transcription factors able to regulate the expression of genes controlling numerous physiological processes such as reproduction, development, and metabolism. Well-known members of the family include receptors for circulating steroid hormones such as androgens, estrogens, glucocorticoids, thyroid hormone, and vitamin D. Other members of the family are receptors for a diverse group of fatty acids, bile acids, lipids, and lipid metabolites. The latter receptors function as sensors for the metabolic state of the cell and respond to changes in locally available molecules (McEwan, 2009). Examples include a number of nuclear receptors that are important in inducing drug metabolizing enzymes, such as the retinoic acid receptor (RXR); the liver X receptor (LXR—the ligand is 22-OH cholesterol); the farnesoid X receptor (FXR—the ligand is chenodeoxycholic acid); and the peroxisome proliferator-activated receptors (PPARs α, β, and γ; 15 deoxy prostaglandin J2 is one possible ligand for PPARγ; the cholesterol-lowering fibrates bind to and regulate PPARγ). In the inactive state, receptors for steroids such as glucocorticoids reside in the cytoplasm and translocate to the nucleus upon binding ligand. Other members of the family such as the LXR and FXR receptors reside in the nucleus and are activated by changes in the concentration of hydrophobic lipid molecules.

The maintenance of many organs requires the continuous renewal of cells. Examples include mucosal cells lining the intestine and a variety of cells in the immune system including T-cells and neutrophils. Cell renewal requires a balance between survival and expansion of the cell population, or cell death and removal. The process by which cells are genetically programmed for death is termed apoptosis. Apoptosis is a highly regulated program of biochemical reactions that leads to cell rounding, shrinking of the cytoplasm, condensation of the nucleus and nuclear material, and changes in the cell membrane that eventually lead to presentation of phosphatidylserine on the outer surface of the cell. Phosphatidylserine is recognized as a sign of apoptosis by macrophages, which engulf and phagocytize the dying cell. Notably, during this process the membrane of the apoptotic cell remains intact and the cell does not release its cytoplasm or nuclear material. Thus, unlike necrotic cell death, the apoptotic process does not initiate an inflammatory response. Understanding the pathways regulating apoptosis is important because apoptosis plays an important role in normal cells and because alterations in apoptotic pathways are implicated in a variety of diseases such as cancer, and neurodegenerative and autoimmune diseases (Bremer et al., 2006; Ghavami et al., 2009). Thus, maintaining or restoring normal apoptotic pathways is the goal of major drug development efforts to treat diseases that involve dysregulated apoptotic pathways (Fesik, 2005) and selectively stimulating apoptotic pathways could be useful in removing unwanted cells.

Two major signaling pathways induce apoptosis. Apoptosis can be initiated by external signals that have features in common with those used by ligands such as TNF-α or by an internal pathway activated by DNA damage, improperly folded proteins, or withdrawal of cell survival factors (Figure 3-13). Regardless of the mode of initiation, the apoptotic program is carried out by a large family of cysteine proteases termed caspases. The caspases are highly specific cytoplasmic proteases that are inactive in normal cells but become activated by apoptotic signals (Danial and Korsmeyer, 2004; Ghobrial et al., 2005; Strasser et al., 2000).

Receptors not only initiate regulation of biochemical events and physiological function but also are themselves subject to many regulatory and homeostatic controls. These controls include regulation of the synthesis and degradation of the receptor, covalent modification, association with other regulatory proteins, and relocalization within the cell. Transducer and effector proteins are regulated similarly. Modulatory inputs may come from other receptors, directly or indirectly, and receptors are almost always subject to feedback regulation by their own signaling outputs.

Continued stimulation of cells with agonists generally results in a state of desensitization (also referred to as adaptation, refractoriness, or down-regulation) such that the effect that follows continued or subsequent exposure to the same concentration of drug is diminished. This phenomenon, called tachyphylaxis, occurs rapidly and is important therapeutically; an example is attenuated response to the repeated use of β adrenergic receptor agonists as bronchodilators for the treatment of asthma (Chapters 12 and 36).

Desensitization can result from temporary inaccessibility of the receptor to agonist or from fewer receptors being synthesized and available at the cell surface (e.g., down-regulation of receptor number). Phosphorylation of GPCR receptors by specific GPCR kinases (GRKs) plays a key role in triggering rapid desensitization. Phosphorylation of agonist-occupied GPCRs by GRKs facilitates the binding of cytosolic proteins termed arrestins to the receptor, resulting in the uncoupling of G protein from the receptor (Moore et al., 2007). The β-arrestins recruit proteins, such as PDE4, that limit cyclic AMP signaling, and clathrin and β2-adaptin, that promote sequestration of receptor from the membrane (internalization), thereby providing a scaffold that permits additional signaling steps.

Conversely, supersensitivity to agonists also frequently follows chronic reduction of receptor stimulation. Such situations can result, e.g., following withdrawal from prolonged receptor blockade (e.g., the long-term administration of β adrenergic receptor antagonists such as metoprolol) or in the case where chronic denervation of a preganglionic fiber induces an increase in neurotransmitter release per pulse, indicating postganglionic neuronal supersensitivity. Supersensitivity can be the result of tissue response to pathological conditions, such as occurs in cardiac ischemia due to the synthesis and recruitment of new receptors to the surface of the myocyte.

It seems likely that effects of several classes of CNS-active agents depend on similar agonist- and antagonist-induced changes in receptor-effector systems (Chapters 14, 15, 18).

It is instructive to examine the pharmacodynamic interactions of physiological ligands and drugs that can occur in the context of a pathophysiological setting. Consider the vascular wall of an arteriole (Figure 3–14). Several cell types interact at this site, including vascular smooth muscle cells (SMCs), endothelial cells (ECs), platelets, and post-ganglionic sympathetic neurons. A variety of physiological receptors and ligands are represented, including ligands that cause SMCs to contract (angiotensin II [AngII], norepinephrine [NE]) and relax (nitric oxide [NO], B-type natriuretic peptide [BNP], and epinephrine), as well as ligands that alter SMC gene expression (platelet-derived growth factor [PDGF], AngII, NE, and eicosanoids). The intracellular second messengers Ca²⁺, cAMP, and cGMP are also shown.

AngII has both acute and chronic effects on SMC. Interaction of AngII with AT₁ receptors (AT₁-R) causes the formation of the second messenger IP₃ through the action of the AT₁-R with the G_q-PLC-IP₃ pathway. IP₃ causes the release of Ca²⁺ from the endoplasmic reticulum; the Ca²⁺ binds and activates calmodulin and its target protein, myosin light chain kinase (MLCK). The activation of MLCK results in the phosphorylation of myosin, leading to smooth muscle cell contraction. Activation of the sympathetic nervous system also regulates SMC tone through release of NE from post-ganglionic sympathetic neurons impinging on SMCs. NE binds α₁ adrenergic receptors that couple to the G_q-PLC-IP₃ pathway, causing an increase in intracellular Ca²⁺ and, as a result, contraction, an effect that is additive to that of AngII. The contraction of SMCs is opposed by several physiological mediators that promote relaxation, including NO, BNP, and epinephrine. NO is formed in ECs by the action of two NO synthase enzymes, eNOS and iNOS. The NO formed in ECs diffuses into SMCs, and activates the soluble form of guanylate cyclase (sGC), which catalyzes the formation of cyclic GMP from GTP. The increase in cyclic GMP activates PKG, which phosphorylates protein substrates in SMCs that reduce intracellular concentrations of Ca²⁺ by several different mechanisms including reducing entry of extracellular Ca²⁺ through L-type voltage-gated Ca²⁺ channels. Intracellular concentrations of cyclic GMP are also increased by activation of the transmembrane BNP receptor (BNP-R), whose guanylate cyclase activity is increased when BNP binds. BNP is released from cardiac muscle in response to increased filling pressures. The contractile state of the arteriole is thus regulated acutely by a variety of physiological mediators working through a number of signal transduction pathways. In a patient with hypertension, SMC tone in an arteriole may be elevated above normal due to one or more changes in endogenous ligands or signaling pathways. These include elevated circulating concentrations of AngII, increased activity of the sympathetic nervous system, and decreased NO production by endothelial cells. Pharmacotherapy might include the use of one or more drugs to block or counteract the acute pathological changes in blood pressure as well as to prevent long-term changes in vessel wall structure due to stimulation of SMC proliferation and alterations in SMC gene expression.

Drugs commonly used to treat hypertension include β₁ antagonists to reduce secretion of renin (the rate-limiting first step in AngII synthesis), a direct renin inhibitor (aliskiren) to block the rate-limiting step in AngII production, angiotensin-converting enzyme (ACE) inhibitors (e.g., enalapril) to reduce the concentrations of circulating AngII, AT₁ receptor blockers (e.g., losartan) to block AngII binding to AT₁ receptors on SMCs, α₁ adrenergic blockers to block NE binding to SMCs, sodium nitroprusside to increase the quantities of NO produced, or a Ca²⁺ channel blocker (e.g., nifedipine) to block Ca²⁺ entry into SMCs. β₁ antagonists would also block the baroreceptor reflex increase in heart rate and blood pressure elicited by a drop in blood pressure induced by the therapy. ACE inhibitors also inhibit the degradation of a vasodilating peptide, bradykinin (Chapter 26). Thus, the choices and mechanisms are complex, and the appropriate therapy in a given patient depends on many considerations, including the diagnosed causes of hypertension in the patient, possible side effects of the drug, efficacy in a given patient, and cost.

Some of the mediators that cause coronary vasoconstriction and hypertension, such as AngII and NE, can also have chronic effects on the vascular wall through mechanisms involving alterations in SMC gene expression. These effects on gene expression can alter the biochemical and physiological properties of the SMC by stimulating hypertrophy, proliferation, and synthesis of proteins that remodel the extracellular matrix. The pathways involved in these chronic effects include many of the same pathways used by growth factor receptors, such as PDGF which can also be involved in the vascular wall remodeling that occurs in neointimal hyperplasia associated with coronary artery restenosis. One of the beneficial effects of using ACE inhibitors and AT₁ receptor blockers in the treatment of hypertension is their ability to prevent the long-term pathological remodeling of the vascular wall that results from chronic activation of AT₁ receptors by AngII.