Pharmacodynamics: Receptors and Concentration-Response Relationships

SITES OF DRUG ACTION

For most drugs, the site of action is a specific macromolecule, generally termed a receptor or a drug target, which may be a membrane protein, a cytoplasmic or extracellular enzyme, or a nucleic acid. A drug may show organ or tissue selectivity as a consequence of selective tissue expression of the drug target. For example, the action of the proton pump inhibitor esomeprazole occurs specifically in the parietal cells that line the gastric pits of the stomach because that is where its target, the potassium/hydrogen adenosine triphosphatase (K⁺/H⁺-ATPase), is expressed. Although the actions of a few drug types, such as osmotic diuretics (see Chapter 21), may not involve receptors as they are usually defined, the concept of receptors as sites of drug action is critical to understanding pharmacology.

Receptors (i.e., drug targets) fall into many classes, but two types predominate:

The former molecules could be considered generalized and the latter specialized receptors. Generalized receptors can include biological molecules with any function, including enzymes, lipids, or nucleic acids. The earlier example of the parietal cell K⁺/H⁺-ATPase is an example of this type of drug target. Specialized receptors include molecules like ion channels and proteins in the plasma membrane, designed to detect chemical signals and initiate a cellular response via activation of signal transduction pathways. The biological function of these molecules is to respond to neurotransmitters, hormones, cytokines, and autocoids and convey information to the cell, resulting in an altered cellular response. These types of receptors are the primary targets of most drugs in clinical use.

The concept of receptors was first proposed more than a century ago by the German chemist Paul Ehrlich, who was trying to develop specific drugs to treat parasitic infections. He proposed the idea of specific “side chains” on cells that would interact with a drug, based on mutually complementary structures. Each cell would have particular characteristics to recognize particular molecules. He proposed that a drug binds to a receptor much like a key fits into a lock.

This lock and key hypothesis is still relevant to how we understand receptors today. It emphasizes the idea that the drug and receptor must be structurally complementary to recognize each other and initiate an effect.

The specificity of such interaction raises the concept of molecular recognition. Drug receptors or targets must have molecular domains that are spatially and energetically favorable for binding specific drug molecules. It is not surprising that most receptors are proteins, because proteins undergo folding to form three-dimensional structures that could easily be envisioned to complement the structures of drug molecules. Enzymes are also reasonably common drug targets, although they fall under the generalized receptor class discussed.

The vast majority of drugs are small molecules with molecular weights below 500 to 800. These molecules interact with their protein targets via a number of different chemical bonds. The principal types of chemical bonds are depicted in Figure 1-1. These bonds apply to the interactions between drugs and classical receptors. Covalent bonds require considerable energy to break and are classified as irreversible when formed in drug-receptor complexes. Ionic bonds are also strong but may be reversed by a change in pH. Most drug-receptor interactions involve multiple weak bonds.

FIGURE 1–1 Types of chemical bonds and attractive forces between molecules that are pertinent to the interaction of drugs with their active sites.

AGONISTS AND ANTAGONISTS

Molecules that bind to receptors may have two major effects on the conformation of the receptor molecule. Agonists will bind to the receptor and activate it, like a key will fit into a lock and turn it. Activation of the receptor by agonist binding initiates a conformational change in the receptor and activation of one or more downstream signaling pathways. An example of the action of an agonist is provided by the effect of acetylcholine on the nicotinic cholinergic receptor at the neuromuscular junction. When acetylcholine binds to its binding sites on the external surface of this receptor, the channel opens and allows Na⁺ to flow down its electrochemical gradient and depolarize the muscle cell.

Antagonists are drugs that bind to the receptor but do not have the unique structural features necessary to activate it. In the lock and key analogy, antagonists can fit in the lock but cannot open it. Like agonists, antagonists fit into a specific binding site within the receptor but lack the proper structural features to initiate a conformational change leading to receptor activation. However, because they occupy the binding site of the receptor, antagonists inhibit activation by agonists. An example of antagonists is the class of neuromuscular blocking drugs used in the operating room to relax skeletal muscles during surgery. These drugs are analogs of curare, the active molecule in plant extracts used as arrow poisons by Native South Americans and studied by early European explorers. Curare is an antagonist at nicotinic cholinergic receptors at the neuromuscular junction and blocks the ability of acetylcholine or similar agonists to activate this receptor. This blockade inhibits muscle depolarization and causes paralysis of skeletal muscle, including the diaphragm and intercostal muscles needed for respiration. Several modern analogs of curare are available and used routinely during general anesthesia for relaxing muscle tone in patients undergoing surgery (see Chapter 12).

A third class of drugs that interact with receptors are allosteric modulators. These compounds bind to a site on the receptor distinct from that which normally binds agonist, called an allosteric site. Occupation of this site can either increase or decrease the response to the natural agonist, depending on whether it is a positive or negative modulator. Because allosteric modulators bind to sites different from where agonists bind, interactions between agonists and allosteric modulators are not competitive. The binding sites for agonists, antagonists, and allosteric modulators are depicted in Figure 1-2.

FIGURE 1–2 Major features of receptors depicting binding sites for agonists, antagonist, and allosteric modulators. Receptors embedded in cell membranes generally extend further on both the extracellular and intracellular sides. Attached to these proteins on the extracellular side are carbohydrate (glycosylation) chains. Shown are binding sites on the extracellular side for binding two molecules of an endogenous transmitter (dark blue symbols) to activate the transmembrane receptor. Agonists and antagonists compete with the endogenous transmitter for binding sites. Allosteric agonists (activators) or antagonists enhance or block the signal, respectively, by binding to allosteric sites that influence (wavy line) signal transmission. Other drugs can block signal transmission within the membrane or at intracellular signal reception points. The arrows indicate the direction of communication to the other side of the membrane.

RECEPTORS

Receptors are a primary focus for investigating the mechanisms by which drugs act. With the sequencing of the human genome, the structures and varieties of most receptors have now been identified. This advance has revealed many new receptors that could be potential drug targets for further pharmaceutical development. The major features of receptors are listed in Box 1-1. Three major concepts are illuminated by the concept of drug receptors.

The first is the quantitative relationship between drug concentration and the subsequent physiological response. This response is determined primarily by the affinity of the drug for the receptor, which is a measure of the binding constant of the drug for the receptor protein. A high affinity means that a low concentration of drug is needed to occupy receptor sites, whereas a low affinity means that much higher concentrations of drug are needed. The concentration-response curve is also influenced by the number of receptors available for binding. In general, more receptors can produce a greater response, although this is not always the case.

The second key concept is that receptors and their distribution in the tissues of the body are responsible for the specificity of drug action. The size, shape, and charge of a receptor determine its affinity for binding any of the vast array of chemically different hormones, neurotransmitters, or drug molecules it may encounter. If the structure of the drug changes even slightly, the type of receptor the drug binds to will also often change. Drug binding to receptors often exhibits stereoselectivity, in which stereoisomers of a drug that are chemically identical, but have different orientations around a single bond, can have very different affinities. For example, the L-isomer of narcotic analgesics is approximately 1000 times more potent than the D-isomer, which is essentially inactive for pain relief (see Chapter 36). The presence or absence of a single hydroxyl group, methyl group, or other apparently minor structural change can also dramatically alter the affinity of a drug for a receptor.

Receptors also explain the key concept of pharmacological antagonists, which prevent agonist activation by binding to a receptor. Administration of an antagonist will block tonic or stimulated activity of endogenous neurotransmitters and hormones, thus interfering with their normal physiological functions. An example is propranolol, which, by antagonizing β₁ adrenergic receptors, prevents the normal increase in heart rate associated with activation of the sympathetic nervous system (see Chapter 11).

Specialized receptors are usually involved in the normal regulation of cell function by hormones, neurotransmitters, growth factors, steroids, and autocoids. Although they are often found on the cell surface, where they are easily accessible to hydrophilic messengers, many hormone receptors are located inside the cell, and ligands for these molecules easily cross the cell membrane (see Part V). An example of an intracellular receptor is the glucocorticoid receptor (see Chapter 39). For most receptor types, multiple distinct subtypes can cause similar or distinct responses. This diversity of receptors and responses provides new targets for drug development.

LIGAND-RECEPTOR INTERACTIONS

In most cases, a drug (D) binds to a receptor (R) in a reversible bimolecular reaction described as:

(1-1)

Occupancy of the receptor by the drug may or may not alter its conformation. Antagonists participate only in the first equilibrium as they bind to the receptor and occupy the binding site. Agonists, on the other hand, have the appropriate structural features to force the bound receptor into an active conformation (DR*). Therefore agonists participate in both equilibria, binding to the receptor and initiating a conformational change. This conformational change leads to a series of events causing a cellular response. It is important to remember that the DR complex is usually reversible for both agonists and antagonists.

RECEPTOR SUPERFAMILIES

Four major superfamilies of receptors are involved in signal transduction, representing the targets of clinically useful drugs. These include ligand-gated ion channels (LGICs), G-protein–coupled receptors (GPCRs), receptor tyrosine kinase (RTKs), and nuclear hormone receptors. Table 1-1 contains a list of receptors in these classes that are important in the actions of several therapeutically useful drugs.

TABLE 1–1 Examples of Specialized Receptors

ACTH, Adrenocorticotrophic hormone; EGF, epidermal growth factor; Epi, epinephrine; NGF, nerve growth factor; NE, norepinephrine.

LGICs are most important in the central and peripheral nervous systems, excitable tissues such as the heart, and the neuromuscular junction. They include nicotinic cholinergic receptors (Fig. 1-3) at the neuromuscular junction, many of the γ-aminobutyric acid (GABA) and glutamate receptors in the brain, and one type of serotonin receptor. These receptors are responsible for fast synaptic transmission, where release of a transmitter causes an electrical effect on the postsynaptic neuron by opening a specific ion channel and leading to a change in membrane potential. LGICs are complex proteins composed of four or five subunits, and the specific subunit combinations differ at different sites in the body, allowing for selectivity of effects.

FIGURE 1–3 Crystal structure of the nicotinic cholinergic receptor. Binding sites for acetylcholine are shown as asterisks, with the gating portions shown with arrows.

Rights were not granted to include this figure in electronic media. Please refer to the printed book.

From Unwin N. The Croonian Lecture 2000. Nicotinic acetylcholine receptor and the structural basis of fast synaptic transmission. Philos Trans R Soc Lond B Biol Sci 2000;355:1813–1829.

For example, the subunits of the nicotinic receptor at the neuromuscular junction differ from those of the nicotinic receptor at autonomic ganglia. As a consequence, although the responses to acetylcholine and ion gating properties of these two channels are similar, these receptors are activated and antagonized by different drugs. This property allows selective blockade of the neuromuscular junction by drugs that do not block the channel at autonomic ganglia (see Chapter 12). Typically, LGICs have two binding sites for agonist and binding sites for allosteric modulators, which increase or decrease the ability of the transmitter to open the channel. One important class of allosteric modulators is the benzodiazepines, which are used extensively for the treatment of anxiety and sleep disorders (see Chapter 31). These drugs bind to γ-aminobutyric acid type A (GABA_A) receptors, which are ligand-gated chloride ion channels that are activated by the inhibitory neurotransmitter GABA. Benzodiazepines have no effect on channel opening by themselves, but their binding dramatically increases the ability of GABA to open the channel.

GPCRs are probably the most important class of receptors in pharmacology, because most currently marketed drugs target this receptor superfamily. GPCRs are much simpler than ligand-gated ion channels, being usually composed of a single subunit that contains seven transmembrane spanning domains. They are thought to have a single binding site, and as yet, there are only a few allosteric modulators for this class of receptors. GPCRs activate signals by inducing a conformational change that activates a large family of G-proteins to regulate signaling pathways (Fig. 1-4). These events regulate a host of important cellular functions (Box 1-2). GPCRs represent the largest protein family in the human genome, accounting for approximately 2% of all human genes. Approximately half of these receptors are olfactory receptors for detecting odorants; most of the remaining GPCRs respond to neurotransmitters, hormones, autocoids, and cytokines.

FIGURE 1–4 Structure of GPCRs and signaling molecules involved in regulation of adenylyl cyclase. Binding of the ligand to the stimulatory receptor (left) produces a conformation change that is transmitted to the α subunit of G_s. This activates G_s by exchanging bound guanosine 5’-diphosphate (GDP) with guanosine 5’-triphosphate (GTP) to give active α_s. G_s dissociates, with active α_s activating the adenylyl cyclase. The βγ subunits are released and freed for other signaling functions. Activation of an inhibitory receptor (right) causes GDP–GTP exchange on the α_i subunit, which can inhibit the adenylyl cyclase.

RTKs contain an extracellular ligand binding domain, one transmembrane spanning segment, and an intracellular tyrosine kinase domain. Binding of a ligand to the extracellular domain causes dimerization of the receptor and stimulates a tyrosine kinase activity within the intracellular domain. The best examples of these receptors are the growth factor receptors such as epidermal growth factor or nerve growth factor receptors (Fig. 1-5). When growth factors bind to these receptors, they cause tyrosine phosphorylation of the receptor, other proteins, or both, which leads to activation of a large number of cellular pathways. Cytokine receptors are part of this subfamily because they are structurally very similar. However, instead of intrinsic enzymatic activity within the receptor molecule, they have docking sites where tyrosine kinase enzymes bind (Fig. 1-6). These receptors are activated by a variety of molecules such as erythropoietin, interleukins, and growth hormone. RTKs are an increasingly important target for drugs to treat neoplastic diseases, where cell growth is uncontrolled (see Chapters 53 and 54).

FIGURE 1–5 Pathways used by growth factors, stressors, and ultraviolet radiation to regulate cell function. A, Example of the mechanism used by growth factors (GF) to activate mitogen-activated protein kinases (MAP kinase). Binding of the GF induces dimerization of the receptor, thereby activating its kinase, which leads to phosphorylation of the receptor (P) on tyrosine residues. This creates binding sites for SH₂ domains of multiple signaling proteins (PI 3 kinase, Grb₂, PLC γ, and an SH₂-containing tyrosine phosphatase [SH₂ PTPase] are shown). The interaction between Grb₂ and the mSos protein activates ras, leading to activation of the MAP kinase cascade. B, The similarity of the protein kinase cascades used by growth factors, stressors, and ultraviolet radiation, leading to the activation of MAP kinase and two related kinases, the Jun N-terminal kinase (JNK) and p38 MAP kinase; rac, a low molecular weight G-protein in the ras superfamily; MEKK1 and MEKK3, two MEK kinases analogous in function to raf-1 but with differing substrate specificities; SEK-1, a kinase analogous to MEK that phosphorylates and activates JNK.

FIGURE 1–6 The pathways used by growth hormone, interferons, and cytokines to regulate nuclear events. The two isoforms of the receptor, α and β are shown, with the Janus kinase (JAK) bound to the β form. The cytoplasmic signal transducers and activators of transcription (STAT) proteins are shown as ovals. Activation of the receptor by growth hormone (GH) or cytokines, such as interleukins (IL), leads to dimerization and phosphorylation of the JAK and STAT proteins on tyrosine residues. The STAT proteins translocate to the nucleus and activate transcription of certain genes.

Nuclear hormone receptors are located in the cytosol, and unlike the other receptor superfamilies that are activated on the cell surface, these receptors bind their ligand in the cytoplasm and translocate to the nucleus. Intracellular receptors respond to highly hydrophobic compounds that easily cross cell membranes, including various classes of steroids, but there are also nuclear receptors for other compounds, such as retinoic acid. These receptors are usually ligand-activated transcription factors that, when bound by ligand, dimerize, enter the nucleus, and bind directly to specific DNA recognition sequences and increase or decrease transcription of particular genes (Fig. 1-7).

FIGURE 1–7 Members of the steroid receptor family bind to DNA at the hormone response element (HRE) and facilitate (or inhibit) formation of active transcription complexes at the promoter. Binding of the hormone (H) to the ligand-binding domain (LBD) causes translocation of the protein to the nucleus, dimerization, and the formation of a complex of proteins with the DNA-binding domain (DBD) binding to the HRE and activating the promoter.

OTHER TARGETS

In contrast to the four classes of specialized receptors that have been honed by evolution to provide the selectivity and specificity required for intercellular communication, there are also intracellular enzymes that provide drug targets with excellent specificity. Two notable examples include esomeprazole, which inhibits the parietal cell H⁺/K⁺-ATPase and is very useful for treatment of gastric hyperacidity (see Chapter 18), and imatinib mesylate, which inhibits the abl tryrosine kinase and is useful in the treatment of leukemias (see Chapter 55). Neither the H⁺/K⁺ -ATPase nor the abl tyrosine kinase are specialized receptors for transmembrane signaling, but both are central to control of a primary function in the cells in which they are expressed. Thus both of these drugs act with excellent specificity.

RECEPTOR CLASSIFICATION

For each type of hormone, neurotransmitter, growth factor, or autocoid, there is at least one specific receptor. Although it was first thought that there was a single receptor for each messenger, it is now clear that in most cases there is a family of receptors with multiple subtypes. Some of these families are quite large. For example, 14 known receptors respond specifically to serotonin, 9 to epinephrine (Epi) and norepinephrine (NE), more than 20 to acetylcholine, and 25 to 30 to glutamate. Although the evolutionary pressure leading to the continued existence of so many receptors is not understood, it is clear that there can be complexity, redundancy, and multiplicity in the effects of a single agonist. Therefore understanding the tissue distribution and biology of these different receptor isoforms is an important goal that will allow development of specific drugs for each novel target. This strategy will provide opportunities for obtaining specific therapeutic responses without unwanted side effects.

Receptors are commonly named after the natural agonist that activates them. For example, acetylcholine acts through cholinergic receptors; Epi (adrenaline) and NE (noradrenaline) act through adrenergic receptors; and serotonin acts through serotonergic receptors. Receptor activation is very specific, and there is little cross-reactivity between natural compounds and other receptors. For instance, acetylcholine binds only to cholinergic receptors and does not bind to adrenergic receptors or members of other receptor families. This is true for essentially all transmitters and hormones. However, a transmitter like dopamine (the immediate precursor of NE), which has its own family of dopaminergic receptors, also binds with low affinity to adrenergic receptors as a consequence of its structural similarity to NE. This is advantageous, because dopamine is used clinically to stimulate β₁ adrenergic receptors in cardiac failure (see Chapter 11).

As mentioned, each receptor family typically contains multiple subtypes that may be characterized pharmacologically by the use of selective agonists, antagonists, or both. For example, there are two major subfamilies of cholinergic receptors, nicotinic and muscarinic. Nicotinic cholinergic receptors are selectively activated by the agonist nicotine and are selectively blocked by drugs like curare. In contrast, muscarinic cholinergic receptors are selectively activated by the agonist muscarine and are selectively blocked by the antagonist atropine (see Chapter 10). Nicotine and curare have essentially no effect on muscarinic cholinergic receptors, and muscarine and atropine have essentially no effect on nicotinic cholinergic receptors. Both nicotinic and muscarinic cholinergic receptor subfamilies, like that for most other neurotransmitters, consist of multiple subtypes. These are discussed in subsequent chapters.

The situation is complicated because multiple receptor subtypes for one transmitter can coexist on a single cell, raising the possibility that one transmitter can deliver multiple messages to the same cell. These messages may be opposing, complementary, or independent. For example, various combinations of adrenergic receptors can be present on the same cell. The β₁ adrenergic receptor activates adenylyl cyclase through a G-protein (G_s). Because the α₂ adrenergic receptor inhibits adenylyl cyclase through a different G-protein (G_i), mutually antagonistic signals will be generated by the presence of both subtypes in response to the same neurotransmitter, NE. In a like manner, additive signals can be generated by the presence of the β₂ adrenergic receptor, which also activates adenylyl cyclase through G_s, or independent signals can be generated by the presence of the α₁ adrenergic receptor, which activates phospholipase C (Fig. 1-8). Overall, the response of a cell to a single transmitter (or a drug that mimics a neurotransmitter) depends on the types and relative proportions of receptor subtypes present in the cell.

FIGURE 1–8 Activation of multiple receptors by a single transmitter: effects on signal transduction. Coactivation of more than one receptor subtype for NE can result in second-messenger responses, which are opposing, additive, or independent. G-proteins shown for stimulatory (G_s), inhibitory (G_i), and phospholipase (G_q). NE, Norepinephrine; AC, adenylyl cyclase; ATP, adenosine triphosphate; PLC, phospholipase C; PIP₂, phosphatidylinositol 4,5-bisphosphate; IP₃, inositol 1,4,5-trisphosphate; DAG, 1,2-diacylglycerol.

CONCENTRATION-RESPONSE RELATIONSHIPS

Binding of a drug to a receptor is a reversible bimolecular interaction, as described in Equation 1-1. This equation follows the law of mass action, which states that at equilibrium, the product of the active masses on one side of the equation divided by the product of active masses on the other side of the equation is a constant. Therefore concentrations of both drug and receptor are important in determining the extent of receptor occupation and subsequent tissue response.

Quantification of the amount of drug necessary to produce a given response is referred to as a concentration–response relationship. Practically, one rarely knows the concentration of drug at the active site, so it is usually necessary to work with dose-response relationships. The dose of a drug is simply the amount administered (e.g., 10 mg), whereas the concentration is the amount per unit volume (e.g., mg/ml). To achieve similar concentrations in patients, it is often necessary to adjust the dose based on patient size, weight, and other factors (see Chapter 3).

Dose-response curves are usually assumed to be at equilibrium, or steady-state, when the rate of drug influx equals the rate of drug efflux, although this is an ideal that is not often achieved in practice. Once the drug reaches its receptors, many responses are graded; that is, they vary from minimum to maximum response. Most concentration- and dose-response curves are plotted on log scales rather than linear scales to make it easier to compare drug potencies; the log scale yields an S-shaped curve, as shown (Fig. 1-9).

FIGURE 1–9 Concentration-response curve for receptor occupancy. A, Arithmetic scale. B, Logarithmic concentration scale. K_D (app) is the concentration of drug occupying half of the available receptor pool.

Quantal responses are all-or-none responses to a drug. For example, after the administration of a hypnotic drug, a patient is either asleep or not. Construction of dose-response curves for quantal responses requires the use of populations of subjects who are characterized by interindividual variability. A few subjects demonstrate an initial response at a low dose, most subjects demonstrate an initial response at an intermediate dose, and a few subjects demonstrate an initial response at a high dose (Fig. 1-10, A), resulting in a bell-shaped “Gaussian” distribution of sensitivity. Quantal dose-response curves are often plotted in a cumulative manner, comparing the dose of drug on the x-axis with the cumulative percentage of subjects responding to that dose on the y-axis (Fig. 1-10, B).

FIGURE 1–10 Quantal effects. Typical set of data after administration of increasing doses of drug to a group of subjects and observation of minimum dose at which each subject responds. Data shown are for 100 subjects. Mean (μ) and median dose is 3.0 mg/kg; standard deviation (σ) is 0.8 mg/kg. A, Results plotted as histogram (bar graph) showing number responding at each dose; smooth curve is normal distribution function calculated for μ of 3.0 and σ of 0.8. B, Data from A replotted as a cumulative percentage responding versus dose with dose shown in B-1 on arithmetic scale (as in A) and in B-2 on a logarithmic scale. ED (effective dose) values are shown for doses at which 10%, 50%, or 95% of subjects respond.

Occupation of a receptor by a drug is derived from the mass action law (see Equation 1-1) and is:

(1-2)

where R_T represents the total number of receptors and K_D is the equilibrium dissociation constant (or affinity constant) of the drug for the receptor. Therefore the proportion of drug bound, relative to the maximum proportion that could be bound, is equal to the concentration of drug divided by the concentration of drug plus its affinity constant. It is important to note that [DR]/[R_T] describes the proportion of receptors bound, or fractional occupancy, which ranges from zero when no drug is bound to one when all receptors are occupied by drug. This equation can be used to calculate what proportion (not actual number) of receptors will be occupied at a particular concentration of drug and demonstrates that fractional occupancy depends only on the concentration of drug and its affinity constant, not on total receptor number.

The K_D value is a fixed parameter describing the affinity of a drug for the receptor binding site. From Equation 1-2, it is clear that when K_D equals [D], half of the receptors are occupied; thus K_D is the concentration of drug that achieves half maximal saturation of the receptor population. This means that drugs with a high K_D (low affinity) will require a high concentration for occupancy, whereas drugs with a low K_D (high affinity) require lower concentrations. K_D is also equal to the ratio of the rate of dissociation of the [DR] complex to its rate of formation. Thus the K_D of a drug is a reflection of the structural affinity of the drug and its receptor, that is, how quickly the drug binds to the receptor and how long it stays bound. Every drug/receptor combination will have a characteristic K_D, although these values can differ by many orders of magnitude. For example, glutamate has approximately millimolar affinity for its receptors, whereas some β adrenergic antagonists have nanomolar affinities for their receptors.

ANTAGONISTS

There are two major classes of antagonists, competitive and noncompetitive. Most antagonists are competitive antagonists. These drugs compete with agonists for the same binding site on a given receptor. If the receptor is occupied by a competitive antagonist, then agonist binding to the receptor is reduced. Likewise, if the receptor is bound by an agonist, antagonist binding will be diminished. When present alone, each drug will occupy the receptors in a concentration-dependent manner as described in Equation 1-2. However, when both drugs are present and competing for the same binding site, the equation describing agonist occupancy is:

(1-3)

where D represents agonist, B represents antagonist, and K_D and K_B represent their relative affinity constants. This equation demonstrates that in the presence of a competitive antagonist, the apparent affinity (K_D) of the agonist [D] for the receptor is altered by the factor 1 + [B]/K_B. As the concentration of the antagonist increases, more agonist is required to cause an effect. Therefore a competitive antagonist reduces the response to the agonist. However, if the concentration of the agonist is increased, it can overcome the receptor blockade caused by the competitive antagonist. In other words, blockade by competitive antagonists is surmountable by increasing the concentration of agonist. With two drugs competing for the same binding site, the drug with the higher concentration relative to its affinity constant will dominate. It is important to remember that the key factor is the ratio of the drug concentration relative to its affinity constant, not simply drug concentration.

Equation 1-3 also demonstrates the very important point that the presence of a competitive antagonist causes a shift to the right in the agonist dose-response curve (decreased apparent K_D) but no change in the shape of the curve. This parallel rightward shift is diagnostic of competitive antagonists and means that every portion of the dose-response curve is shifted by exactly the same amount (Fig. 1-11). The magnitude of rightward shift is dependent on the concentration of antagonist, which is variable, divided by its affinity constant, which is fixed. Therefore, if the concentration of drug is known, measuring the magnitude of the rightward shift allows direct calculation of the K_B for the antagonist. This is very important because the affinity constant is essentially a molecular description of how well a drug binds to a particular receptor and is constant for any drug/receptor pair. Thus comparison of affinity constants for antagonists at receptors in different tissues is the best way to determine whether they are the same or different, making antagonists extremely useful in subclassifying receptors.

FIGURE 1–11 Competitive antagonism; both the agonist (AG) and the antagonist (ANT) compete and bind reversibly to the same receptor site. The presence of the competitive antagonist causes a parallel shift to the right in the concentration-response curve for the agonist.

The second, less common type of antagonist is the noncompetitive antagonist. There are a number of different noncompetitive antagonists, but most drugs in this class are irreversible alkylating agents. An example of this class of drug is phenoxybenzamine, a drug that acts predominantly on α₁ adrenergic receptors and is used to mitigate the effects of catecholamines secreted by adrenal tumors (see Chapter 11). Drugs of this class contain highly reactive groups, and when they bind to receptors, they form covalent bonds, occupying the binding site in an essentially irreversible, nonsurmountable manner. Because they decrease the number of available receptors, noncompetitive antagonists will usually decrease the maximum response to an agonist without affecting its EC₅₀ (concentration causing half-maximal effect). An advantage of a noncompetitive antagonist is its long-lasting effect. Because the drug binds a receptor irreversibly, the drug effect lasts until new receptors are synthesized.

PARTIAL AGONISTS

As discussed, agonists can participate in both equilibria shown in Equation 1-1, binding to and activating receptors to cause a conformational change. Partial agonists have a dual activity, that is, they act partially as agonists and partially as antagonists. When bound to their receptors, partial agonists are only partly able to shift the receptor to its activated conformational state. Efficacy is the proportion of receptors that are forced into their active conformation when occupied by a particular drug. It is used to describe the maximal effect of partial agonists in causing a receptor conformational change and can range from 0 to 1. Drugs with full efficacy are called “full agonists,” drugs with some efficacy are “partial agonists,” whereas drugs with zero efficacy are “antagonists.”

Partial agonists can also partially inhibit the response to full agonists acting at the same receptor type (Fig. 1-12). If both full and partial agonists are present, as the concentration of partial agonist is increased, more receptors will be occupied by the partial agonist. This will cause a decrease in response, because some of the receptors will no longer be activated. At very high concentrations of partial agonist relative to its affinity constant, all of the receptors will be occupied by partial agonist, and the full agonist becomes essentially irrelevant. Therefore a diagnostic feature of a partial agonist is that it inhibits the action of a full agonist to the level of its own maximal effect.

FIGURE 1–12 Partial agonists, such as the nasal decongestant oxymetazoline, give maximal responses that are lower than those of full agonists, such as Epi, in vascular smooth muscle. At high enough concentrations of oxymetazoline, the effect of Epi is reduced to the level of activity of oxymetazoline alone.

SIGNAL AMPLIFICATION—SPARE RECEPTORS

In some cases the response elicited by a drug is proportional to the fraction of receptors occupied. More commonly, a maximal response can be achieved when only a small fraction of receptors are occupied by an agonist. This phenomenon defines the concept of spare receptors, or a receptor reserve. The reason for this behavior is that there are several intervening amplification steps downstream from the initial receptor-triggering event. If there were a 1:1 stochiometry between GPCR activation and G-protein stimulation, for example, the existence of 10,000 receptors and only 1000 G-proteins in a particular cell would result in only 10% of receptors needing to be activated to cause a full response. Further receptor occupancy would not result in an increase in the magnitude of response. When the signaling pathways involve amplification steps, the EC₅₀ for an agonist may be much lower than the concentration needed to cause half maximal receptor occupation (K_D).

Spare receptors are important in all-or-none responses, where it is especially important that activation does not fail (e.g., the neuromuscular junction or the heart). The presence of spare receptors shifts the agonist dose-response curve to the left of the K_D for binding of agonist to receptor, and the degree of shift is proportional to the proportion of spare receptors (Fig. 1-13). Thus spare receptors make a tissue more sensitive to an agonist without changing its affinity for the receptor. Because the existence of spare receptors is fairly common, the EC₅₀ for an agonist is usually not equal to its K_D. For example, the β₁ adrenergic receptor has the same chemical and physical properties in every tissue in which it is expressed. However, the EC₅₀ for a particular drug in activating responses mediated by this receptor can vary by orders of magnitude in different tissues, depending on the degree of receptor reserve.

FIGURE 1–13 Logarithmic concentration–response curves for a single agonist acting on the same receptor subtype in tissues with different proportions of spare receptors (A, B, C, and D) and eliciting muscle contraction in vitro. Note that all tissues show the same maximum response to drug (intrinsic activity). The agonist shows its highest potency (lowest EC₅₀) at the tissue with greatest proportion of spare receptors (A) and its lowest potency at the tissue with the lowest proportion of spare receptors (D).

This means that if an agonist has a different EC₅₀ in two different tissues, one cannot conclude that the receptors in one tissue are different from the receptors in the other tissue, because there is no predictable relationship between EC₅₀ and K_D for a particular agonist/receptor combination. Spare receptors are also responsible for tissue specific actions of agonists. Because the presence of spare receptors increases the potency of an agonist, tissues with a high proportion of spare receptors will respond to agonists at lower concentrations, even if they contain exactly the same receptor subtypes.

Spare receptors also complicate the analysis of partial agonists. If a drug is a partial agonist in one tissue, it may be a full agonist in another tissue, which has a higher proportion of spare receptors. In the GPCR example described, where only 10% of the receptors must be activated to cause a full response, a weak partial agonist may activate this 10% and appear to be a full agonist. Because of this problem, a different term, intrinsic activity, is used to describe the ability of a tissue to respond to agonist stimulation. Efficacy, which is the ability of the agonist to cause the receptor to assume an active conformation, is analogous to K_D in that both are constant for a given drug/receptor pair. It is an intrinsic property that depends on the structural complementarity of the drug and the receptor molecules. Intrinsic activity, however, is highly context dependent. It varies in different tissues because of the presence of different proportions of spare receptors and downstream amplification mechanisms. A drug can be a partial agonist in efficacy, but a full agonist in intrinsic activity when spare receptors are present.

RECEPTOR DESENSITIZATION AND SUPERSENSITIVITY

The response of any cell to hormones or neurotransmitters is tightly regulated and can vary depending on other stimuli impinging on the cell. Very often, the number of receptors in the membrane of a cell or responsiveness of the receptors themselves is regulated. One hormone can sensitize a cell to the effects of another hormone, and more commonly, when a cell is continuously exposed to stimulation by a transmitter or hormone, it may become desensitized. An example of this phenomenon is the loss of the ability of inhaled β₂ adrenergic agonists to dilate the bronchi of asthmatic patients after repeated use of the drug (see Chapter 16). A hormone or agonist can affect the way a cell responds to itself (homologous effects) or how it responds to other hormones (heterologous effects). As an example of the latter phenomenon, exposure of a cell to estrogen sensitizes many cells to the effects of progesterone.

Changes in receptor binding affinity and signaling efficiency often occur rapidly. Receptor phosphorylation of serines, threonines, or tyrosines is a common mechanism of regulating responsiveness. Phosphorylation can rapidly change affinity or signaling efficiency and can also target a receptor for internalization and degradation.

The mechanisms involved in homologous and heterologous desensitization of β₂ adrenergic receptors are well understood (Fig. 1-14). Receptor phosphorylation on serine-threonine residues by three different protein kinases plays a role in the loss of responsiveness. These include β adrenergic receptor kinase (β-ARK), cAMP-dependent protein kinase, and protein kinase C. Phosphorylation of the β adrenergic receptor inhibits its ability to interact with G-proteins and subsequently leads to its sequestration or internalization in a compartment where it cannot interact with extracellular hormone. β-ARK is particularly important in homologous desensitization. It is capable only of phosphorylating the active, agonist-bound form of the receptor.

FIGURE 1–14 Phosphorylation is important in receptor desensitization. Pathways of stimulation of β-ARK and cAMP-dependent protein kinase in homologous desensitization, and cAMP-dependent protein kinase and protein kinase C (PKC) in heterologous desensitization by α₁ adrenergic receptor (α₁ rec) and prostaglandin PGE receptor (PGE rec). AC, Adenylyl cyclase; DAG, diacylglycerol; ER, endoplasmic reticulum; G, G- protein; IP₃, inositol trisphosphate; p, phosphorylated state.

Other protein kinases, such as cAMP-dependent protein kinase, may also prefer the agonist-bound form of the receptor but not to the same extent as β-ARK. Although β-ARK was originally described as a kinase specific for the β adrenergic receptor, its specificity is not unique, in that a large family of related protein kinases has been discovered. Between them, they can phosphorylate many different G-protein–coupled receptors in their agonist-bound state. Phosphorylation inhibits the ability of the receptor to interact with G proteins and subsequently leads to its sequestration or internalization in a compartment where it cannot interact with extracellular hormone.

In the absence of hormone, most receptors are not localized to particular regions of the cell membrane. When a hormone binds, receptors rapidly migrate to coated pits. These are specialized invaginations of the membrane surrounded by an electron-dense cage formed by the protein clathrin; this is where receptor-mediated endocytosis occurs. Segments of membranes within coated pits rapidly pinch off to form intracellular vesicles rich in receptor-ligand complexes (Fig. 1-15). Vesicles then fuse with tubular-reticular structures. In most cases dissociated hormone is incorporated into vesicles that fuse with lysosomes, with the hormone then degraded by lysosomal enzymes. Dissociated receptor recirculates to the cell surface. However, a fraction of internalized hormone may also be recirculated to the cell surface along with receptor, and then released. This process is termed retroendocytosis. Free receptors may recirculate to the cell surface or may be sequestered temporarily in an intracellular membrane compartment. Alternatively, receptor may be transported to lysosomes, where it is also degraded. The latter two cases result in a net decrease in cell receptor number.

FIGURE 1–15 Pathways of receptor internalization and recycling. CURL, Compartment of uncoupling of receptor and ligand.

Finally, it is important to realize that the number of receptors in the plasma membrane of cells is not static (Fig. 1-16). Receptor number may be increased or decreased under the influence of hormonal mechanisms. Altered receptor number attributable to internalization or degradation has an intermediate time course, whereas an altered rate of receptor synthesis occurs more slowly. Receptors may also be up regulated, and this phenomenon can result in receptor supersensitivity. Up regulation can occur after exposure of the receptor to an antagonist, or inhibition of transmitter synthesis or release. In addition, other hormones can increase receptor number. For example, excessive production of thyroid hormone can increase the synthesis of β adrenergic receptors in cardiac tissue, leading to some of the signs and symptoms of Graves’ disease (see Chapter 42). Thus the number of cell-surface receptors, and thereby hormone sensitivity, can be continuously regulated. This property of receptor biology can be exploited therapeutically. For example, during the third trimester of pregnancy, under the influence of nuclear hormones, the number of β₂ adrenergic receptors on uterine smooth muscle is dramatically increased, allowing the use of selective β₂ adrenergic agonists, like terbutaline, to delay premature labor (see Chapter 11).

FIGURE 1–16 Long-term treatment with agonists or antagonists can alter postsynaptic receptor density or responsiveness.