Module 3 Pharmacodynamics PDF

Module 3 Pharmacodynamics I hope that each student feels comfortable with the mechanisms involved in pharmacokinetics, which includes absorption, distribution, biotransformation, and excretion of a drug. The next important sub-specialty in pharmacology is pharmacodynamics. This term covers the event...

Module 3 Pharmacodynamics I hope that each student feels comfortable with the mechanisms involved in pharmacokinetics, which includes absorption, distribution, biotransformation, and excretion of a drug. The next important sub-specialty in pharmacology is pharmacodynamics. This term covers the events involving the action of a drug at the site of the target. Included in this topic are the study of: the mechanism of action of drugs the therapeutic and toxic effects the relationship between the chemical structure of a drug and its activity Some of the important terms that we will study in this section may seem very familiar to you due to drug advertising. Unfortunately, pharmaceutical companies have been known to use these terms in confusing or even misleading ways. In this module you will begin to understand the molecular workings of a drug at the site that it binds in the body, usually a receptor. Why would a member of the Health Professions team need to know the specifics of the molecular interactions between a drug and its receptor? The answer has two parts. First, this section only provides a brief appreciation of the concepts of molecular pharmacology – the reality is much more complicated. Secondly, the understanding of all Drug pharmacological principles starts at the interaction of the drug with its cellular receptor. Without an Receptor understanding of that interaction, you will lack the ability to categorize drugs using their receptor information. You will be reduced to memorizing the negative side effects of a drug, rather than deducing them from your cellular Cell understanding of physiology and pharmacology. Thus, this course will spend a great deal of time Figure 1: The drug (red circle) binds to its receptor discussing the mechanisms that on the cell surface and a number of intracellular govern pharmacology, rather than molecules are activated. In this case, they finally making you memorize drug actions act on the DNA in the nucleus to induce genetic and side effects. changes. 1 Figure 1 illustrates the binding of a drug to its receptor. Once a drug enters the body, it must bind to something, typically a protein on the surface of a cell (a receptor), in order to have an effect. There are some exceptions to this model that will be discussed later. In this cartoon the drug diffuses in the extracellular space toward the surface of the cell. When it comes close enough to the receptor, it will bind to it. Such an event typically will induce a change in the receptor molecule. For example, the receptor may undergo a structural or conformational (shape) change or it may become phosphorylated (a phosphorous moiety is added to the receptor molecule). In any case, an intracellular cascade of events will follow involving so called downstream molecules (downstream of the binding of the drug with wits target receptor) – other proteins will Ca2+ Ca2+ become phosphorylated, different Ca2+ Ca2+ proteins will bind to each other, or Ca2+ new molecules will be formed. The Ca2+ Ca2+ cellular end point will be the final effect that the drug was intended to Ca2+ Contraction have. In the example shown in Ca2+ figure 1, the drug initiates several Ca2+ Ca2+ Ca 2+ intracellular steps (a cascade that Ca2+ Ca2+ Ca2+ Ca2+ involves downstream molecules) before reaching its cellular end point – the last molecule of the cascade binding to DNA in the nucleus. Ca2+ Ca2+ Ca2+ Ca2+ Ca2+ Let’s walk through a typical drug Ca2+ Ca2+ signaling cascade (figure 2). Calcium is an important signal Ca2+ Relaxation molecule in a variety of cells, but it is particularly important in muscle Ca2+ cells. Calcium is required in order for actin and myosin to interact, which is necessary for muscle cell contraction. Contraction of the smooth muscle cells found within arteries means that the diameter of Figure 2: Upper panel: When calcium enters the the artery will narrow. Such cell, it binds to the contractile molecules causing narrowing is particularly hazardous them to contract and the cell to shorten. In vascular for coronary arteries that supply smooth muscle cells this decreases the diameter of blood to the heart, because a the vessel wall. Lower panel: When a drug blocks decrease in the vessel diameter will the ability of calcium to enter through the channel also mean a decrease in the blood (calcium channel blocker), then the contractile flow to the heart. molecules can’t contract and the cell relaxes. Blocking the entry of calcium into muscle cells is one way to reduce the amount of contraction in an artery. Thus, a class of drugs commonly prescribed for patients with heart disease is the calcium channel blocker. 2 Calcium channel blockers (shown as red bars in the lower panel of figure 2) bind to voltage- gated calcium channels (the channels are the receptors) on the surface of the coronary artery smooth muscle cells (triangles, figure 2). By inhibiting the entry of calcium into the cell, the drug blocks the interaction of the contractile elements (actin and myosin, shown in blue and red). By halting contraction, the cell is actually encouraged to relax, thus increasing the diameter of the blood vessels, decreasing local blood pressure, and increasing the flow of blood to that region. Before going further, it will be helpful to have a full understanding of the terms involved in pharmacodynamics. The next section will define and describe common pharmacodynamic terms. Efficacy is defined as the ability of a drug to initiate a response subsequent % Relaxation to binding to its target. Figure 3 shows a typical dose/response curve with the Y axis indicating the cellular effect of the drug (relaxation). In other words after the calcium channel blocker bound to the voltage-gated calcium channel, how effective was it in 0 0.01 0.1 1.0 10 100 nM blocking contraction of the cell. That would be Drug Dose the percentage of relaxation achieved with Figure 3: Illustration of the effect of increasing the dose of the blocking of the calcium calcium channel blocker on the amount of relaxation measured channels. Typically, in the blood vessels. when a drug is in a low concentration in the extracellular space, it has little effect on the neighboring cells. The concentration of the drug must be high enough to increase the chances of a drug molecule interacting with its receptor. Thus, at a low dose the drug is below its therapeutic dose. As the drug concentration is increased to a threshold level, the effect of the drug can be measured. Typically there is a linear portion of the relationship, in which an increase in the drug concentration increases the amount of relaxation linearly. As the dose of the drug increases, the effect will plateau, which means that there is no further increment in the response even if larger doses are added. One simple way to think of this phenomenon mechanistically is that all of the voltage-gated calcium channels have a drug molecule (blockers) bound to them. Thus, the dose response curve is saturated. 3 Saturation of all of the receptors with bound drug is not the only way to reach a plateau in the dose response curve. Another mechanism for reaching the plateau of a dose response relationship occurs when downstream molecules are the limiting steps. For example, an intracellular molecule may be phosphorylated after the drug binds to the receptor. In some cases the rate of phosphorylation of B that protein may be low. So that even with high doses of the drug, the Effect A downstream events cannot occur any faster and so the measured response is the same. When comparing two drugs, the drug that reached the 0 0.01 0.1 1.0 10 100 nM greatest magnitude on the Y axis of the Drug Dose dose response Figure 4: Two drugs have different dose/response relationships. curve is the drug that is considered the most efficacious. This comparison is limited to the effect of the drugs after they have reached their receptors. Figure 4 compares two drugs with different efficacies. Clearly Drug B has the greater efficacy. However, efficacy does not take into Dose/Response Relationship consideration the pharmacokinetic properties discussed in Module 2 of 100 this course. A drug that is more % Maximal Effect effective when at the site of the cell, may never reach its target in levels that are relevant, due to poor pharmacokinetic properties such as 50 poor absorption. Thus, a pharmaceutical company may correctly make the claim that their drug is more effective than the competitor’s, but effectiveness in 0 pharmaceutical terms may not be the Concentration of drug most important parameter when Figure 5: While efficacy is measured on the Y choosing a drug. Efficacy can be axis, potency is measured on the x axis of the measured in a variety of ways. dose/response curve as shown here. 4 In a controlled research study, Drug A may be very effective. However, in the real world, the patient may not take the drug exactly as prescribed. So what is the real-world efficacy is described by “use effectiveness”. Use efficacy indicates how two drugs may compare in the general population. Niaspan, an extended release form of niacin, is commonly prescribed to increase the levels of high density lipoproteins (HDLs). However, it can cause flushing of the face, can interrupt sleep, and should not be taken with alcohol. So frequently people do not take it daily, as prescribed. They take it when they are home, not taking any alcohol and early in the afternoon. This may decrease the number of days of the week that they actually take the drug. So how often a person takes the drug during the week will change the use efficacy of the drug. Potency is the dose of the drug that produces a given response. A more potent drug requires a lower dosage to produce the same effect. Potency is a term that is commonly misunderstood by the public. Many consumers think that the more potent drug is always better. That may not be the case. One of the characteristics of drugs is the 50% effect level. That is the dose required to produce 50% of the maximal effect. The drug with the lower dose required to produce 50% of the maximal effect is the more potent drug. Thus, while efficacy was measured as the greatest magnitude on the Y axis, potency is measured as the lowest value for a given effect on the X axis (figure 5). The red line on figure 4 shows the 50% effect level for Drug A (blue line). It has a lower dose value (X axis) than drug B. Thus drug A may be considered more potent, even though it is less efficacious. Both potency and efficacy are terms used by drug companies to imply that their drug will take away your heartburn, relieve your runny nose, or decrease your blood pressure better than the competitor’s drug. However, these terms can be used misleadingly by the drug companies. A company may talk about their wonderfully Dose/Response Relationship efficacious drug and use data about what the drug does 100 when it contacts the cell responding Effect receptor. However, if the drug is mostly bound up in the fat tissue, potency and efficacy have little relevance, because Maximal 50 little of the drug has made it to of people the target tissue. ED50 %% The effect of a drug can be 0 discussed at many levels. Until now we have limited our Concentration of drug discussion about the effect of Figure 6: At the point where the drug provides a positive a drug to the cellular response. effect to 50% of treated people, the drug concentration is However, the effectiveness of termed the ED50. a drug can also be measured 5 in an entire tissue. For example, we can measure the effect of a drug on the heart beat. Drug effectiveness can be measured in terms of an entire population. The drug concentration can be plotted on the X axis while the Y axis is the percentage of people that were helped by the drug. How one defines "helped by the drug" is up to the person creating the graph. For example, if one is studying the effect of pain medication on headaches, one could plot the percentage of people whose headaches were completely relieved by the medication. In contrast, one could define the population of people responding to the drug as those that reported that their pain was decreased by 50%. It is important when reading the information distributed by pharmaceutical companies to carefully determine how that company defined a positive response to the drug. The dose at which 50% of the people report that they had a positive response to the drug is called the effective dose 50 or the ED50 (figure 6). Another term for the same value is the therapeutic dose 50 or the TD50. Unlike the cellular response which can be graded (the cell relaxed by 20%), the response measured in this graph is all or none. A person either responded to the drug, or they did not. For example, an ED50 graph plotting the effect of an anti-spasticity drug would not inform the reader as to how much the spasticity was reduced, only that 50% of the people given the drug reported that their spasticity decreased by some amount defined by the researcher doing the study. LD 50 Just as one can plot the Dose/Response Relationship positive response that a population had to a drug, one can plot the 100 negative effects and even determine the lethal dose of a drug. The median toxic dose is shown on the Y axis of figure 7. The toxic dose is also called % death the lethal dose (LD). For obvious 50 ethical reasons, the lethal dose of a drug cannot be determined using human studies. These studies must be completed first using in vitro assays such as cell culture in which the dose that kills individual cells is 0 determined. Based on results from in Concentration of drug vitro tests, pharmacologists develop studies using animals of different Figure 7: When the Y axis is converted to % of species to check for toxic effects. animals dying as a result of the drug, an LD50 can The LD50 level is the dose of drug be calculated. that causes death in 50% of the animals tested. In the past, experiments were designed sometimes using hundreds of mice to methodically determine the dose causing 50% of the animals to die. Today, mathematical modeling and better in vitro assays have greatly reduced the number of animals that must be tested in order to determine the safe prescription level for humans so that frequently only a few animals are required. However, it is important to note that in the majority of cases the LD50 for the cell culture experiments do not match the LD50 of the animal studies. Thus, 6 one can’t replace the animal studies with computer modeling or cell culture work. There is simply no replacement for animal testing in order to continue to make new life-saving drugs and protect the public. Therapeutic Index. The ratio of the LD50/ED50 is called the therapeutic index (TI). The therapeutic index is calculated as the dose required to produce toxic effects divided by the dose required to achieve the desired therapeutic effects. The larger the number, the safer the drug. All drugs, even substances found normally in the body, are toxic at high enough doses. Hopefully it takes a much larger dose to invoke a toxic response than it does to cause a beneficial effect. Acetaminophen has a TI of 27, while Valium has a TI of 3. For us to be good consumers of pharmaceutical products, the therapeutic index should be an important parameter in terms of choosing one type of drug over another. This number explains why people rarely use acetaminophen for suicide attempts. Surprisingly, TI is a term the public rarely hears about from pharmaceutical companies or even from their pharmacist. Affinity The terms and definitions discussed above imply that a drug Drug binds to its receptor and stays bound until the response is complete or the drug is degraded. That is not the correct model for most drug/receptor interactions. Most drugs bind and are released from the receptor quickly. Membrane Receptor Within milliseconds another drug molecule may bind to the same receptor. Thus over a period of a Figure 8: The positive charge of the drug attracts second, some drug molecules are it to the negative charge of the receptor. jumping on and off their receptors hundreds of times. This rate of association and dissociation affects the amount of drug that may be required to activate the receptors. The attraction and binding of a drug to a receptor is called the association, while the removal of the drug from the receptor (or unbinding) is called the dissociation. Both association and dissociation of a drug with its receptor determine the affinity that the drug has for the receptor. Association (Ka) and dissociation (Kd) values are governed by the law of mass action. Now I am asking you to think back to your undergraduate chemistry classes. The properties that draw a drug molecule to a receptor and then lead to binding it may include electrical forces (charge on the molecules), other physical forces such as Van der Waals forces or hydrophobic forces, and simply the size and shape of the molecules (how they fit together is designated as a steric effect). In figure 8, the drug binding site of the receptor is surrounded by negative charges while the drug has many positive charges. These two molecules will be attracted to one another because opposite charges attract to each other. The fact that their shapes allow them to come into close proximity with each other (steric effect), indicates that they will have a high affinity for one another. A drug with a high affinity binds readily to the receptor, even if the concentration of the drug is low. The actual Ka and Kd values cannot be determined from the functional dose/response 7 curves that we have reviewed previously. It must be calculated biochemically by actually measuring the amount of binding of the two molecules. Association and dissociation values only indicate the percentage of receptors that are bound to a drug; they do not predict the amount of activity after binding. Thus, Ka and Kd are not good indicators of drug efficacy. Selectivity describes how much affinity an agonist has for one receptor versus another. A natural neurotransmitter in the body, acetylcholine binds to a muscarinic receptor that has different subtypes (m1-m5). When Membrane acetylcholine binds to the m2 receptor subtype in the heart, Figure 9: Specific (solid arrow) and non-specific (hatched arrow) it causes a change binding is shown. in the heart rate. When acetylcholine binds to another subtype of the muscarinic receptors, the m3 receptor in coronary artery smooth muscle cells, it causes them to contract thus reducing the blood flow to the heart. If a pharmacologist attempted to make a derivative of acetylcholine to control heart rate, they would want to design a drug that had higher affinity for the m2 receptor than for the m3 receptor. The effect would be a drug with greater selectivity for the heart receptor than Membrane for the coronary artery receptor. Selectivity is a relative term since no Figure 10: The agonist is shown in red, attempting to bind to its drug is completely selective receptor. However, an antagonist (white) has already selective. At a high bound to the receptor, and is blocking the ability of the agonist to enough concentration, bind. a drug will bind to something that it was 8 not designed to bind to, this is called nonselective binding. In figure 9, two drugs that obviously fit best with specific receptors are shown. The red drug would prefer the red receptor and the white drug the white receptor. However, if there is a lot of white drug around and all the white receptors are already bound to white drugs, then the excess white drug would likely bind a few red receptors (crosshatch line), indicating non-specific binding. Agonist versus antagonist A ligand is any compound that binds to a receptor. Once bound, it may activate the receptor and start the downstream cascade, or it may simply block other drugs from binding to the receptor. These two options define the difference between an agonist and an antagonist. A ligand that is an agonist binds to the receptor and activates it, while a ligand that is an antagonist will inhibit the receptor from becoming activated, either by binding and not allowing other compounds to reach the receptor, or by turning the receptor off via a chemical modification. A good example of an antagonist is the calcium channel blocker we discussed earlier for arterial smooth muscle cells. The channel blocker binds to the calcium channel and does not allow calcium to enter the cell through that channel. This section defining pharmacological terms has introduced at least eleven new designations that the reader must know. Now we will examine the actual interaction properties of drugs with respect to their receptors. RECEPTORS At this point we have discussed the drug to receptor interaction in great detail. The actual receptors involved have been left a mystery. There are several places within a cell where receptors can be found. The classic receptor is found on the cell membrane (plasma membrane receptor) so that drugs traveling in the blood stream or in the extracellular space can bind easily to a portion of the receptor protruding from the membrane. Membrane receptors make up the largest known category of receptors in the body. In this fashion the drug itself does not have to enter the cell to have an effect. Receptors located within the cell (intracellular receptors) require that the drug enters the cell to have an effect. For example, receptors found inside the cell whose target is to alter gene activity in the nucleus, can only be activated by drugs that can pass through the plasma membrane of the cell. Only after binding to the drug the receptor can enter the nucleus and do its job on the target gene. Table 1 provides a partial list of receptors according to their location and activity. Table 1. List of ligands for different types of cellular receptors. Plasma membrane receptors Intracellular receptors Adrenergic ligands Caffeine GABA Testosterone Acetylcholine Estrogen Dopamine Glucocorticoids 9 Histamine Progesterone Serotonin Vitamin D Adenosine Thyroid Hormone Glycine Angiotensin Plasma membrane receptors Cell surface receptors are often linked to second messenger molecules within the cell. The drug (being the first messenger) binds to the receptor, which when activated will stimulate or inhibit some type of intracellular effector (such as an enzyme) that will lead to production of a second messenger that would mediate a change in cell function. Thus, when a plasma membrane receptor is activated pharmacologists say that a "signaling cascade" has been initiated. In figure 1 there are three intermediates in the signaling cascade from the point where the drug (red circle) binds to the receptor to the alteration in gene function when a molecule binds to the DNA. There are several types of receptors that reside within the plasma membrane. We will briefly review the major categories. G-protein coupled receptors The G-protein coupled receptors [also called G-protein-linked receptors or G-protein receptors] were the first class of receptors to be identified and are still the largest category. The "G" stands for guanosine triphosphate (GTP), a molecule similar to a better known adenosine triphosphate (ATP). When activated G-proteins bind GTP. Over 100 different compounds within the body are known to activate G-protein receptors. Table 2 below shows just a few of the G-protein receptors known to reside in the human body. In order to help classify them, they are listed according to the category of a ligand (neurotransmitter, hormone etc.) that bind to them. When a G-protein receptor is activated it binds to a G-protein, which sits attached to the membrane. Presently, over 25 different G-proteins have been identified. The target effect of the receptor will depend on which G-protein is activated. Table 2. G-protein coupled receptors grouped as per ligand type. Small molecule Peptide Sensory Adrenergic Endorphin Rhodopsin Muscarinic Cholecystokinin Olfactory Dopamine Bradykinin Histamine Substance P Serotonin Thrombin Adenosine Angiotensin II Leukotrienes Vasopressin Prostacyclin Parathyroid hormone Thromboxane Enkephalin 10  G-proteins act as signal transducers and amplifiers. For example a signaling cascade in the eye uses a rhodopsin receptor (a G- protein receptor), which amplifies the signal 100,000 fold from activation of the receptor to the final steps. Amplification of a signal can occur at each step in the cascade. In figure 11 three G-protein receptors (red) are activated by a bound drug (green). G- Figure 11: The drug (green) binds to its G-protein coupled proteins (blue) bind to the receptor (red), which causes receptor binding to the G- activated receptors, while protein (blue) leading to activation of the G-protein. The other G-proteins have activated G-protein binds to the next downstream protein already bound to the (gold). Since each receptor can bind and activate more than receptor, been activated, one G-protein, there is amplification in the system. In this and moved away from the example, only three active receptors turned on 13 G-proteins. receptor (blue with gold circles). These activated G-proteins can then act on a number of other molecules in the cell. So in this Specificity illustration binding of three molecules of drug to three receptors m2 activated 13 m3 downstream Gq molecules. A single drug Gi molecule acting on a single receptor initiated a cascade that can be amplified m3 m2 by hundreds of times. Gq Gi This system also provides flexibility. The exact mechanism Figure 12: While the m3 type of muscarinic G-protein receptors prefers that determines to bind to the Gq protein, it can bind to the Gi protein, if no Gq is present. Similarly, m2 receptors would bind to Gq if Gi is not available. 11 which G-protein will be activated depends on several factors. First, there is some specificity between the receptor and its G-protein. An example is the acetylcholine receptor discussed previously with its m2 and m3 receptor types. The m3 muscarinic receptor will tend to bind a nearby G-protein of the Gq class rather than other G-proteins. The muscarinic receptor of the m2 subclass will tend to bind G-proteins of the Gi category. Thus, there is specificity within the signaling cascade based on the type of receptor activated and the type of nearby G- proteins. However, if an m3 receptor is in a cell that has little Gq protein, then it can bind to another category of G-protein, like the Gi group (figure 12). So flexibility can be built into the system, depending on which G-proteins and receptors are expressed in given cells. The receptors that are linked with G-proteins have several common characteristics. First, one end of the receptor is in the extracellular space. In most cases, this region is involved in the drug binding to the receptor. Another common feature is that the receptor crosses the membrane of the cell seven times. This crossing is so consistent with the G-protein receptors that they are often termed the seven transmembrane domain receptors. The intracellular loops of the receptor along with the tail that sticks into the cell make up the intracellular portion of the receptor that binds to the G-proteins when activated (see schematic representation of the receptor parts in figure 13). When G-proteins are activated (and this happens when a ligand binds to a corresponding G- protein receptor), they initiate a cascade of events. Each step can be a site for disease when one of the steps has gone wrong. The disease processes involving G-protein signaling will be discussed later. In addition, each step in the cascade can be a site for pharmacological interventions. We have already discussed the binding of the receptor to the G-protein and its subsequent activation. The subsequent events will depend on which G-protein has been activated. We will review just one of the hundreds of possible outcomes following G-protein activation. Figure 13 shows an Drug example of G-protein receptor activation leading to the binding of a Gq subtype of G- protein. This protein, still anchored in the membrane, binds to PLC PIP2 and activates phospholipase C G protein (PLC), which converts GTP phosphatidylinositol Ca2+ biphosphate (PIP2) into IP3 Ca2+ inositol trisphosphate Ca2+ (IP3). IP3 diffuses away Ca2+ from the membrane Figure 13: The drug binds to the G-protein receptor (shown with and binds to an IP3 7 trans-membrane domains) activating the G-protein. The G- receptor that can be protein activates the phospholipase C (PLC), which turns PIP2 into IP3. IP3 binds to a receptor on the endoplasmic reticulum (pink) and triggers release of calcium. 12 found on intracellular structures such as the endoplasmic reticulum or the nuclear membrane. Once IP3 binds to its receptor, it opens an ion channel that allows calcium to exit the intracellular store within the endoplasmic reticulum, and calcium rushes into the cytoplasm of the cell. Depending on the type of cell that is involved, an increase in calcium may cause a cell to contract (muscle), to secrete (exocrine cell), or to release its neurotransmitter (neuron). You are certainly not expected to memorize each step in this G-protein signaling pathway. This example is provided only to illustrate how many different steps there are in a single signaling cascade. Again, each step, from the receptor being activated, to the release of calcium, is a possible site for the cause of a disease if anything goes wrong. Additionally, each step in the cascade is a site for possible pharmacological intervention to correct such a disease. One examples of a disease involving a G-protein is acromegaly in which a G-protein is overactive and induces too much secretion of certain hormones causing overgrowth of the bones and joints. G-protein receptors have been identified as being involved in several disease processes. As this course progresses through the semester, we will point out diseases that are G-protein linked, but for now we will highlight two disorders that are classic G-protein-linked diseases. Whooping cough is an illness that involves the Gi (“i” stands for inhibitory) subclass of G- proteins. The pertussis toxin, which is the cause of whooping cough, enters the cell and chemically alters the Gi subunit so that it cannot interact with and inhibit its downstream target, and the signaling cascade stops there. This blockage of the Gi pathway in the lungs results in an increase of secretions in the bronchial tubes that are difficult to clear, resulting in the child with a whooping-like cough. Likewise cholera is a disease of G-proteins. The cholera toxin also enters the cell and chemically alters a G-protein, this time it is the Gs (“s” stands for stimulatory) protein. In contrast to the effect of whooping cough, which turns off the signaling pathway, cholera toxin effectively keeps the Gs signaling pathway turned "on" all the time. Thus, the receptor is by- passed, as it does not have to be activated in order for the Gs protein to send its message onto the downstream molecules in the pathway. No drug or endogenous molecule bound to the receptor is necessary to activate the pathway in this disease. In fact, the receptor is NOT turned on. However, the Gs protein has activated its downstream signaling cascade. This increase in the Gs activity causes an increase in water secretion in the intestines leading to diarrhea associated with cholera. Tyrosine kinase receptors 13 We now leave the G-protein receptors for a different kind of receptor. Growth factors frequently utilize a type of plasma membrane receptor called a receptor tyrosine kinase, for the transmittal of the signal into the cell. This family of receptors has several subclasses that have common features. Most are single proteins that cross the membrane once in contrast to the G-protein receptor that crosses the membrane seven times. There are some exceptions to this general shape. The insulin receptor is a member of the tyrosine kinase family of receptors, but it crosses the membrane two times. In figure 14 a schematic Insulin Receptor is shown of the insulin receptor Platelet-Derived Growth Factor Receptor and the platelet-derived growth factor receptor. All of the Figure 14: Schematic of insulin and platelet-derived tyrosine kinase receptors have growth factor receptors. a site in the intracellular portion of the protein that contains at least one tyrosine amino acid (shown as yellow blocks in figures 14 and 15). This tyrosine residue is phosphorylated by a kinase activity when the receptor is activated, thus giving rise to the name of the receptors. (Kinases are enzymes that catalyze phosphorylation of proteins.) Typically binding of the growth factor or drug activates the tyrosine kinase receptor causing phosphorylation of the receptor itself. Subsequently, the receptor will phosphorylate downstream molecules, thus initiating the signaling cascade. Binding sites for ligands (in this case insulin or platelet-derived growth factor) are shown as blue blocks in the figure. In the case of the growth ? hormone receptor, once a Cell Growth and Division hormone or other ligand binds to the receptor, it activates an adapter protein shown as a Ras MAP kinase Transcription question mark because in some Factor cases the identity of the protein has not been determined at this Figure 15: When the platelet-derived growth factor time. In other cases a variety of receptor is activated a number of intracellular molecules proteins can serve in this role. are subsequently activated including Ras, MAP kinase, The next step in the cascade is and specific transcription factors that control cell growth the activation of a small protein and division. called Ras, which activates 14 mitogen-activated protein (MAP) kinase and finally induces transcription of certain genes that lead to cell growth and division. Perturbations in the normal growth factor signaling cascade can result in cancerous transformations (growth out of control results in tumor formation). In some tumors the problem lies in the tyrosine kinase receptor itself. The receptor may undergo a mutation that causes it to be turned on constantly. In this case the receptor will initiate the signaling cascade without the presence of growth factors (the ligands). Other tumors secrete too much growth hormone so that the receptors are over-stimulated. Approximately 30% of all human cancers involve a mutation in the Ras protein that results in constitutively dividing cells leading to uncontrolled cell division. Channel receptors In some cases the same protein that acts as the receptor may also be the end point of the cascade. In other words, there is little or no signaling cascade. The receptor is activated by a drug, which alters the receptor’s shape or activity in some manner, and that same receptor molecule changes a function within the cell. Ion channels are the best example of proteins that act both as the receptor and the endpoint. A molecule will bind to the ion channel receptor, activating it. Na+ Activation of the ion channel ACh portion of the molecule Na+ opens a pore through the center allowing ions such as Na + potassium, calcium or sodium to cross the membrane. These receptors act more quickly than the other receptors that require several steps before they have an effect (i.e. the G- Na+ protein-linked receptors). One of the best studied examples of a one-step Na + receptor is the nicotinic acetylcholine receptor shown in figure 16. The Figure 16: Acetylcholine (red) binds to its receptor (blue) ligand (acetylcholine) binds which causes a change in the shape of the receptor to the nicotinic receptor, allowing sodium to travel through the center of the receptor and sodium passes through and across the cell membrane into the cell. the center of the receptor thus entering the cell. An increase in intracellular sodium causes depolarization of the skeletal muscle leading to contraction. Receptors that act as ion channels are needed in locations in the body where an immediate reaction is required by the cells. Quick responses are typical of the nervous system. Thus, 15 many receptor channels are found in neurons. These channels normally bind neurotransmitters released by upstream neurons. When we study drugs that act on the nervous system, many will be binding to ion channels such as the nicotinic receptor. They include the ion channel receptors of the neurotransmitters GABA, glutamate, and glycine. Intracellular receptors Unlike neurotransmitters that induce a quick response, hormones tend to induce a slower acting response that may change the cellular activity over the course of days. While the action of a hormone is slower to occur, it is typically much longer lasting than neurotransmitters. Hormones may bind to receptors that lie within the plasma membrane as described previously for the growth hormones binding to receptor tyrosine kinases. Contrastingly, the hormone may act on sites within the cytoplasm, the nucleus, or possibly in other organelles. An important factor that determines the absorption of a drug, as well as its distribution and elimination, is the cell membrane. The cell membrane is composed of a bimolecular layer of phospholipids with the hydrophilic heads facing the outer surfaces and the carbon chains pointing toward the interior and creating a continuous hydrophobic band within the ] membrane. The membrane maintains relative impermeability to highly water-soluble compounds. Yet lipid-soluble substances penetrate the membrane by dissolving within the lipid. In order for any drug to bind to an intracellular receptor it must either be lipid soluble so that it can diffuse through the membrane, or it must have a specific transporter within the plasma membrane that carries the drug molecule into the cell. Caffeine is a perfect example of a drug that can quickly diffuse through the plasma membrane without the need of a transporter. Caffeine binds to a receptor found on the surface of the endoplasmic T reticulum. T Nuclear receptors Some hormones that can pass through the plasma membrane may be targeted DHT to the nucleus (estrogen, DHT testosterone, DNA DHT glucocorticoid, progesterone, vitamin D, thyroid hormone) and activate or inactivate genes. Nucleus The response from nuclear receptors is usually slower Figure 17: Testosterone (T) diffuses through the plasma than that mediated by membrane, into the cell where it is converted to DHT, and cytoplasmic second then binds to its intracellular receptor (blue) and transported messengers. There is also into the nucleus. The activated receptor binds to DNA and turns on genes important in cell growth. 16 a more graded response to increasing hormone concentrations rather than amplification scheme seen in the G-protein signaling cascade, because each hormone-receptor complex interacts with one molecule of DNA. Thus, hormonal effects are usually not an all-or-none phenomena. Once inside the nucleus, the drug binds to its nuclear receptor. A molecule destined to interact with DNA typically contains regions called zinc fingers. Zinc fingers are loops of amino acids held together in the shape of fingers by interactions with the ion zinc. The zinc finger is the portion of the nuclear receptor that will interact with the DNA either activating or inhibiting transcription of a particular portion of the cell’s DNA, a gene. Many of the receptors that make their way to the nucleus are originally found in their inactivated form in the cytosol (i.e. estrogen receptors). These are not actually categorized as nuclear receptors, they are intracellular receptors. Once the receptor is activated (bound to a drug) it translocates to the nucleus. Figure 17 shows the hormone testosterone (T) passively diffusing into the cell where it is converted by 5a Reductase to DHT, which then binds to the cytosolic testosterone receptor. Once the receptor is activated it moves to the nucleus by being transported through the nuclear membrane. There the activated receptor binds to a site on the DNA to stimulate expression of a particular gene. Abnormal nuclear receptor activity can lead to several pathological findings. For example, males with testicular feminization syndrome have testes that secrete testosterone normally, but the cytosolic testosterone receptor is absent from the cell. Thus, testosterone is in the system, but cannot exert its effect without a receptor. Since all mammals are female by default, the affected males have secondary female characteristics such as enlarged breasts. Mutant nuclear receptors for thyroid hormone lead to hypothyroidism, because the cells cannot respond to normal levels of the hormone when it is released. Receptor regulation As discussed earlier, many receptors and downstream molecules within the signaling cascade have several forms or subtypes. While they may work in similar manners, slight changes in the amino acid sequence may alter drug binding, effector stimulation, or distribution of the receptor. In addition, cells have other mechanisms providing methods of regulating the receptor/drug interaction. 17  Desensitization is one manner in which cells can control the receptor/drug interactions. Desensitization describes a rapid decrease in responsiveness of a cell to a drug. Using P a variety of P techniques the cell changes the 1 2 3 receptor so that it Figure 18: 1) The drug advances towards the receptor. 2) The drug will not bind the and receptor bind. 3) The binding of the drug causes the drug with such a intracellular portion of the receptor to be phosphorylated (P). high affinity, or has less activity once stimulated. One technique that cells use to induce such changes is to phosphorylate the receptor thus blocking the activity of this receptor (figure 18). In practical terms the receptor will be less sensitive to the drug whether it is the endogenous stimulant such as a neurotransmitter, or if it is the exogenous drug. The beta adrenergic receptor found in the heart is phosphorylated after activation. Once phosphorylated, it cannot be activated again. This means that even if another drug molecule is in the vicinity of the receptor, the receptor cannot be activated. It is in an inactivated conformation. With time, the inactivation usually ends and the receptor becomes active again. Down regulation is a slower process and Figure 19: The activated receptor (with the blue drug bound) involves reduction stimulates the membrane to pinch off taking some receptors with the of the number of membrane. In this way, the number of receptors on the cell surface receptors on the is decreased. membrane. Once the agonist binds to 18 the receptor and its action has been induced, the receptor is internalized into the cytoplasm of the cell where it may be recycled or completely degraded (Figure 19). The decrease in the number of receptors on the cell’s surface may remain for days after the agonist is gone. Receptors may be internalized into vesicles and stored until they can be reinserted into the plasma membrane or they can be degraded and new receptors produced to take their place. SUMMARY The interaction between the receptor and the agonist is at the heart of all understanding involving the mechanism of action for drugs. The specificity of a drug is the molecular basis for the side effects any particular drug will have. In other words, the more specific a drug for the target tissue, the fewer side effects it will have. Down regulation of receptors resulting in fewer receptors on the surface of the cell, partially explains why addicting drugs require higher and higher doses to elicit the same response (it is because there are fewer and fewer receptors present for the drug to act on). With this basic knowledge of the pharmacodynamics in hand, drugs can be classified into the types of receptors they act on, thus reducing the need for a lot of rote memorization in pharmacology. What’s next? Now we have completed the section of the course focusing on the basic principles of pharmacology from pharmacodynamics to pharmacokinetics. From this point forward in the course, each module will describe drugs for illnesses for physiological systems (like the the immune system or neurological system). 19