A= High Receptor Reserve B=Medium Receptor reserve C=No Receptor Reserve

Applications to Therapeutics

Few drugs interact with one and only one receptor. Such a drug would be said to be specific, that is producing effects by specifically interacting with a single receptor. Most drugs interact with several receptors and thus have the capability to produce distinctly different pharmacologic effects. Some of these effects could be beneficial, some could be toxic. Such a drug would be said to be a selective. The factors that determine which particular effect of a drug will be observed are the affinity and intrinsic activity of a drug. 

To illustrate this point consider the following example. A drug is capable of producing actions at 2 distinct receptors. At each of these receptors, the ligand has a different affinity as well as pharmacologic effect.

Receptor System # 1:

K_D = 0.4, intrinsic activity 1.0,effect- lowering of systemic arterial blood pressure.

Receptor System # 2:

K_D = 40.0, intrinsic activity 1.0,effect- lethal ventricular arrhythmias.

Thus, this drug could either be a highly beneficial therapeutic agent or a lethal poison. An overwhelming majority of drugs used in clinical practice produce their therapeutic effects due to interactions at multiple pharmacologic receptors.

This also illustrates that whether the drug will be beneficial or poisonous depends on the skill and knowledge of the individual prescribing the agent.

The Therapeutic Index

The therapeutic index is the ratio of the ED50 of a drug to produce a toxic effect to the ED50 to produce a therapeutic effect. For the drug example above, the ED50 for the beneficial effect of blood pressure lowering is 0.4 nM while the ED50 for toxicity is 40 nM. Therefore, the therapeutic index will be:

Advanced Concepts Regarding Partial Agonists

Partial agonists have lower intrinsic activities than full agonists but values greater than competitive antagonists. At certain concentrations partial agonists actually can be antagonists. This information is especially relevant to understanding the pharmacology of opiates.

1. Integrate pharmacodynamic principles to aid in the understanding of adrenergic receptors and the actions of drugs on these receptors.
   
2. Understand the criteria upon which alpha and beta receptors are defined.
   
3. Understand the second messenger systems utilized by alpha and beta receptors and how activation of these receptors leads to a change in physiologic function.
   
4. Understand the effects of alpha and beta receptor activation on the heart and blood vessels.
   
5. Understand the effects of isoproterenol, epinephrine and norepinephrine on the cardiovascular system.

Isoproterenol - Isuprel

Epinephrine - Adrenalin

Norepinephrine- Levophed

The adrenergic receptors which subserve the responses of the sympathetic nervous system have been divided into two discrete subtypes: alpha adrenergic receptors (alpha receptors) and beta adrenergic receptors (beta receptors). The classification of these receptors, and indeed receptors in general, is based on the interaction of agonists and antagonists with the receptors.

	PUTATIVE STRUCTURE OF ADRENERGIC RECEPTORS
PROPOSED BINDING OF NE TO THE BETA RECEPTOR

Beta receptors have been further subdivided into beta₁ and beta₂ receptors. It should be pointed out that beta₃ and beta₄ receptors have recently been isolated, cloned and characterized. The beta₃ receptor may be involved in regulating the metabolism of fatty acids. This receptor could be the site of antiobesity drugs in the future. The functions of the beta₄ receptor remains to be discovered. For the purposes of this material we will focus on the beta₁ and beta₂ receptors only. The classification of beta receptors is based on the interaction of a series of drugs with these receptors. The ability of epinephrine, norepinephrine and isoproterenol to increase the force of myocardial contraction was examined and the dose-response curves shown below were obtained. Equilibrium dissociation constants for these ligands were ISO, 80 nm, E, 800 nM, and NE, 1000 nM. Thus, the rank order of affinities for the beta receptor in the heart is ISO>E>NE. A beta receptor with these characteristics is referred to as a beta₁ receptor. The equilibrium dissociation constant is often used as a "finger print" to identify a receptor. Regardless of its location, the receptor will interact in the same manner with ligands and have the same dissociation constants for agonists and antagonists. The logic above is a standard approach to differentiate receptors. The interaction of agonists and antagonists with receptor systems often indicates heterogeneity within the main receptor population. These different receptors are referred to as receptor subtypes. Receptor subtypes are routinely exploited in drug development to make ligands that interact selectively with one subtype in preference to another. Specific examples of this principle are presented in these lectures and throughout the course.

 

The ability of the same compounds to produce bronchodilation was examined and a different set of dose response curves and equilibrium dissociation constants were obtained. The dissociation constants were ISO, 80 nm, E, 800 nM, and NE, 10,000 nM. Notice how the ability to active the beta receptors is dependent on the structure of the drugs under study. Clearly then the receptor in the lung is different from that in the heart and is referred to as a beta₂ receptor.

Most tissues express multiple receptors. However, the receptor mainly utilized by the sympathetic nervous system to affect myocardial function in the heart is the beta₁ receptor; while in vascular and nonvascular smooth muscle it is the beta₂ receptor.

1. Agonist binds to the myocardial beta₁-adrenergic receptor. This receptor is a typical G-protein coupled receptor.
   

2. In the unstimulated state the G-protein is complexed with GDP.
   

3. The receptor promotes exchange of GTP for GDP and release of Gα/GTP.
   

4. The G"/GTP complex activates adenylate cyclase.
   

5. Intracellular cAMP increases and activates cAMP dependent protein kinase (PKA).
   

6. PKA phosphorylates the Ca²⁺ channel promoting Ca²⁺ influx.
   

7. Intracellular Ca²⁺ increases activating the contractile proteins.
   

8. PKA phosphorylates the sarcoplasmic reticulum leading to an increase in Ca²⁺ uptake and release.
   

9. PKA phosphorylates troponin changing its calcium binding kinetics
   

10. Prolonged stimulation can lead to receptor down regulation via PKA and other protein kinases which phosphorylate the receptor. The other protein kinases which are involved in phosphorylation are referred to as G-protein coupled receptor kinases or GRKS. These phosphorylation steps lead to internalization of the receptor.

Effect of Beta Receptor Activation on the Heart:

Activation of the beta₁ receptor leads to increases in contractile force and heart rate. The increase is myocardial contraction is a result of activation of those beta receptors associated with the atria and ventricle (especially the ventricles) while the increases in rate of contraction are due to activation of those receptors associated with the SA and AV nodes as well as the His-Purkinjie system.

1. Increase slope of phase 4 spontaneous depolarization
   

2. Increase in maximal rate of phase 0 depolarization
   

3. Increase conduction velocity
   

4. Decrease refractory period

These electrophysiologic factors contribute to the orderly, rhythmic electrical activity that assures the efficient contractile activity of the heart. In response to beta receptor activation, these parameters increase and the heart beats at a faster rate. However, excess stimulation of the beta receptor by catecholamines can enhance these variables to such an extent that arrhythmias can occur. Rhythm disturbances are a major concern with drugs that activate the beta₁ receptor. Drugs to be covered that have a tendency to generate arrhythmias include epinephrine, isoproterenol, norepinephrine, dopamine and dobutamine.

Effect of Beta₂ Receptor Activation on Smooth Muscle

The beta₂ receptor associated with smooth muscle also utilizes the cAMP signaling system. However, the results of receptor mediated increases in cAMP levels in smooth muscle are different than those occurring in cardiac muscle. Therefore, the consequences of PKA phosphorylation of key structures in smooth muscle lead to relaxation.  There is also evidence that the beta/gamma subunit of the G-protein contributes to signaling in beta₂ receptor systems.

Structures Phosphorylated in Smooth Muscle

    1)    sarcolemma - Decrease Ca²⁺ influx

    2)     sarcoplasmic reticulum - Enhance Ca²⁺ uptake 

    3)       decrease actin-myosin interactions - muscle relaxation

The net result of these activities is to inhibit calcium pathways in smooth muscle leading to relaxation.

Regulation of Receptor Function

Continuous exposure of an agonist results in a phenomenon referred to as desensitization.  The same concentration of agonist becomes less effective at producing the same level of effect.  When this desensitization occurs very rapidly, it is referred to as tachyphylaxis. Recent evidence has suggested potential mechanisms by which the process tachyphylaxis occur. The receptor becomes phosphorylated in the third cytoplasmic loop and c-terminal tail.  The phosphorylated receptor is less efficient at activation the G-protein and also exhibits lower affinity for agonists.  The receptors can also be removed from and sequestered away from the cell surface.  These events indicate that the second messengers not only regulate intracellular processes but are also capable of regulating the receptor systems which generate them.

If the ability of isoproterenol, epinephrine and norepinephrine to produce constriction of vascular smooth muscle is studied, the following dose-response curves and equilibrium dissociation constants were obtain E, 5 uM, NE, 6, uM and ISO, 1000 uM. You should begin to understand the reasons why the receptor causing vasoconstriction MUST be different from that causing cardiac contraction or broncodilation.

The receptor that produces vasoconstriction is referred to as an alpha receptor (affinity rankings of E ≥ NE >>>ISO). Observe how the structure of each drug affects that ability of these ligands to activate the alpha receptor. The concentration of isoproterenol necessary to activate alpha receptors is so large that isoproterenol can be thought of as a pure beta receptor agonist.

Alpha receptors have also been subdivided into alpha₁ and alpha₂ receptors. Epinephrine and norepinephrine have equal affinity at both alpha₁ and alpha₂ receptors. However, other drugs were found to have higher affinity for one receptor over another. These differences in affinity were the evidence used to subclassify the receptors into alpha₁ and alpha₂. More recently, three subtypes of the alpha₁-receptor, the alpha_1A, alpha_1B and alpha_1D have been isolated, cloned and characterized. Similarly, 3 subtypes of the alpha₂-receptor, the alpha_2A, the alpha_2B and the alpha_2C have also been identified. There is little doubt that these receptor subtypes subserve different physiologic functions. Pharmacologic agents are being developed which have the potential to selectively activate or block one of these receptors in preference to another. However, for the purposes of this course, it is necessary to remember only the alpha₁ and alpha₂ receptors.

Alpha₁ and alpha₂ receptors exist postsynaptically. Like the beta receptor, these receptors are G-protein coupled receptors, thus they activate cellular signaling subsequent to interaction with a G-protein. Activation of these receptors on vascular smooth muscle leads to vasoconstriction. The mechanism linking the alpha₂ receptor to contraction is not well understood.

Mechanism of Alpha1 Receptor Activation of Smooth Muscle Contraction

In the case of the postjunctional alpha₁ receptor, the inositol phosphate/diacylglycerol signaling pathway is activated by receptor occupancy.

1. Agonist binds to the vascular smooth muscle alpha₁-receptor. The receptor is a typical G-protein coupled receptor with 7 membrane spanning regions.
   

2. In the unstimulated state the G-protein is complexed with GDP.
   

3. The receptor promotes exchange of GTP for GDP and release of Gα/GTP.
   

4. The G-protein activates phospholipase C leading to an increase in the intracellular second messengers, inositol triphosphate (IP3) and diacylglycerol (DAG).
   

5. IP3 binds to specific sites on the SR and stimulates the release of intracellular Ca²⁺.
   

6. Ca²⁺ influx is activated.
   

7. Like the beta-receptors, alpha receptors can also be desensitized and down regulated via phosphorylation of the receptor. However,  the alpha₁ receptors are much more resistant to desensitization and down regulation than are the beta receptors.

Alpha₂ receptors exist presynaptically. Activation of these receptors inhibits the release of norepinephrine. The mechanism of this regulatory involves the stimulation of a G-protein gated K⁺ channel leading to membrane hyperpolarization.

Norepinephrine acts at presynaptic alpha₂ receptors to inhibit its own release.

Effect of Catecholamines on Vascular Smooth Muscle Epinephrine

Associated with vascular smooth muscle are a large number of alpha₁ receptors relative to beta₂ receptors. However, epinephrine has a higher affinity for the beta₂ receptor relative to the alpha₁ receptor. Activation of the beta₂ receptor would produce vasodilation while activation of the alpha₁ receptor would result in vasoconstriction. Recall from the lectures on drug-receptor interactions the magnitude of effect is dependent on the degree of receptor occupancy. Therefore, the effect of epinephrine on smooth muscle is dependent on its relative affinity for alpha₁ and beta₂ receptors and its concentration. At low doses, epinephrine can selectively stimulate beta₂ receptors producing muscle relaxation and a decrease in peripheral resistance. However, once epinephrine concentrations are reached which bind to the alpha₁ receptor, vasoconstriction will occur. The two effects (smooth muscle relaxation and contraction) will oppose one another. However, as the concentration of epinephrine increases, the predominant effect will be vasoconstriction.

Effects of Norepinephrine and Isoproterenol on Smooth Muscle

Recall that norepinephrine in physiologically relevant concentrations has little affinity for beta₂ receptors. Therefore, it will stimulate only alpha₁ receptors producing an increase in peripheral vascular resistance. In contrast, the lack of activity at the alpha₁-receptor means that isoproterenol will produce only a beta₂-receptor mediated vasodilation.

Other Cardiovascular Functions

Alpha₁ receptors also exist on the myocardium. These receptors increase force without affecting rate. The role of these receptors in physiologic regulation of myocardial performance or as a site of drug action is unclear.

Oral dosing of norepinephrine, epinephrine and isoproterenol is not possible due to rapid metabolism in gastrointestinal mucosa and liver. Therefore, these agents are given I.V., I.M., topically and in aerosol sprays. There is very limited clinical use of norepinephrine. Epinephrine is often used in combination with local anesthetic agents to prolong the duration of anesthetic action. This also reduces the toxicity of the local anesthetic by limiting its defusion away from the injection site. Epinephrine can also be used to in surgical procedures to reduce blood loss. A major concern with using pressors is the effect on systemic arterial pressure. Clinical studies have shown that epinephrine blood levels increase following its intraoral administration. There is also the risk that epinephrine could increase heart rate. The risk of these increases is dependent on characteristics of the patient. For example, hypertensive patients or those ischemic heart disease or patients taking other drugs that affect sympathetic nervous system function are at higher risk than patients without these conditions. Epinephrine is used in the treatment of various shock syndromes and in emergency situations related to bronchial asthma. Isoproterenol is also used in the acute management of airways dysfunction to produce broncodilation.

Effects On The Cardiovascular System

For the drugs listed below, indicate how the drugs would affect (increase, decrease, no changes) heart rate, contractile force, total peripheral resistance (TPR) and systemic arterial blood pressure. Recall the equations below. Remember also that the effectors in the cardiovascular system (brain, kidney, heart and blood vessels) are all involved in the integrated regulation of blood pressure.

Blood pressure = Cardiac Output xTPR
Cardiac output = Stroke Volume x Heart Rate
Blood pressure = (Stroke volume x Heart rate) x Total peripheral vascular resistance

Be sure to make an attempt at answering the question BEFORE you click on the answer.

Learning Objectives

1. Understand the potential sites of action for sympathomimetics and sympatholytics.
   
2. Understand the pharmacologic actions and therapeutic effects of dopamine.
   
3. Understand how the pharmacodynamic actions of dopamine illustrate the properties of a drug that interacts with multiple receptors.
   
4. Know that there are beta₂ agonists, their mechanisms of action and therapeutic uses.
   
5. Know the mechanism of action and cardiovascular effects of amphetamine and cocaine.
   
6. Know the effects and therapeutic uses of drugs that can activate the alpha1 adrenergic receptors.

Key Drugs*

Amphetamine-Adderall

Albuterol - Ventolin - 13^th leading prescription drug in the US in 2003- source- rxlist.com

Cocaine

Dopamine - Intropin

Methylphenidate - Ritalin - 102nd leading prescription drug in the US in 2003- source- rxlist.com

Phenylephrine - Neosynephrine

Sympathomimetics: are synthetic analogs of naturally occurring catecholamines that bind to beta or alpha receptors and mimic the actions of the endogenous neurotransmitters. These agents can be divided into direct and indirect acting sympathomimetics.

Sympatholytics: are synthetic analogs which bind to beta or alpha receptors and block the actions of endogenous neurotransmitters or other sympathomimetics.

In addition to interacting with receptors, adrenergic agonists and antagonists can interact at sites on the nerve terminal to produce sympathomimetic or sympatholytic effects. These potential sites are indicated by the numbers. A majority of drugs are direct acting agonists or antagonists. Only a small number of drugs work through the other listed mechanisms.

1. Direct acting agonists or antagonists can act at postsynaptic receptors.
   

2. Indirect acting agonists release neurotransmitters from presynaptic nerve terminals to produce a sympathomimetic effect.
   

3. Drugs such as Guanethidine can inhibit the Ca²⁺-dependent release of norepinephrine and thus have a sympatholytic effect.
   

4. Drugs such as Reserpine cause the destruction of storage granules, and as a result, depletion of the synaptic terminal of norepinephrine which is also a sympatholytic action.
   

5. Blockade of monoamine transporters by drugs such as cocaine and amphetamine produce a sympathomimetic effect. Transporters for norepinphrine as well as other monoamines are the site of action for tricyclic antidepressants and serotonin reuptake inhibitors.
   

6. Inhibition of monoamine oxidase by drugs such as Tranylcypromine.

 

SYMPATHOMIMETICS ACTING AT BETA RECEPTOR SYSTEMS EXAMPLES: Dopamine Beta2 agonists DOPAMINE-An illustration of the actions of a drug that activates multiple receptors Dopamine has a complex pharmacology. It can activate at least 4 different receptors: the beta1, dopamine1 (DA1), alpha1 and alpha2.  DA1 receptors exist in the renal vascular bed. Activation of these receptors produces a decrease in renal vascular resistance and an increase in renal blood flow. Activation of the beta1 receptor increases the force of myocardial contraction. Dopamine has a very unusual action on the heart in that it selectively increases the force of myocardial contraction without a significant effect on heart rate. However, high doses of dopamine, like all catecholamines which activate the beta1 system, can induce rhythm disturbances.

low doses: the DA1 receptors will be activated moderate doses:the beta1 receptors will be activated high doses:- the alpha receptors will be activated

The diagram below shows the effect of dopamine on systemic arterial blood pressure, contractile force and heart rate. Initially blood pressure decreases (why?) After this decrease in blood pressure, contractile force increases (via what mechanism?) Note that there is a selective effect on contractile force as heart rate does not increase until higher concentrations are used. Finally, systemic arterial blood pressure increases (what receptors mediate this effect?)

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The Consequences of Multiple Receptor Activation in the Treatment of Congestive Heart Failure

Dopamine can be used to treat congestive heart failure and cardiogenic shock. In congestive heart failure, the heart is not able to eject blood efficiently. As a result there is a decrease in cardiac output which triggers a series of compensatory actions. These include fluid retention, vasoconstriction, an increase in peripheral vascular resistance, an increase in the levels of circulating catecholamines and tissue hypoxia. Dopamine is used because it has the potential to improve these negative circulatory events. For example, by inducing renal vasodilation (via DA₁ receptors), blood flow to the kidney is improved and urine output increases. By increasing the force of myocardial contraction the cardiac output is increased. Dopamine can be used in home health care to improve congestive heart failure.

 

Receptor	Result of Receptor Activation	Contribution to Therapeutic Effect
DA1	The renal vasodilation will improve renal blood flow and increase GFR	This will increase urine output and decrease fluid retention and edema.
beta1	Produces a positive inotropic effect	Increase in cardiac output. This is beneficial in CHF.
alpha1 and alpha2	Vasoconstriction is not a desired effect.	This will counteract the beneficial effects on renal blood flow. In addition, increases in TPR will negatively affect cardiac output.

Pharmacokinetics of Dopamine

Similar to epinephrine and norepinephrine, dopamine has a short plasma half life. It can only be used intravenously in constant or intermittent infusions.

Dobutamine 

The effects of dobutamine on the cardiovascular system are summarized below

1. Activates myocardial beta₁ receptors to increase the force of myocardial contraction
   

2. Little effect on heart rate at therapeutic doses - high doses can induce arrhythmias
   

3. Causes a decrease in blood pressure and TPR
   

4. Does not activate dopamine receptors

It is interesting to note that dobutamine used clinically is a racemic mixture of (+) and (-) isomers. These individual isomers have different pharmacologic properties:

1. (+) Dobutamine is a beta₁ and beta₂ agonist.
   

2. (-) Dobutamine is an alpha₁ agonist.
   

3. The observed clinical profile is due to a combination of these pharmacological effects.

The use of isomers as a single drug product is very common. Most isomeric pairs have the same activity. Dobutamine is an unusual example of a pair of isomers that have distinctly different activities.

Uses and Pharmacokinetics

Dobutamine is used in similar situations as dopamine; namely, the short term treatment of cardiogenic shock and congestive heart failure. Its use is associated with a decrease in LV filling pressure. Like dopamine, dobutamine is not orally active and must be given intravenously and can be used in home health care.

Selective Beta₂ Agonists

These agents have a higher affinity for beta₂ receptors when compared to beta₁. These agents activate cellular processes by increasing cAMP levels as discussed in the lecture on Adrenergic Receptors.

Clinical Applications of Selective Beta₂ Agonists

These agents are used in situations that call for the relaxation of smooth muscle, specifically the smooth muscle associated with airways or uterus such as:

• Bronchial asthma

• Chronic bronchitis

• Emphysema

• Premature labor-tocolytics-Ritodrine

 

ALPHA₁ AGONISTS

Direct Acting Agents

These are agents which directly active the alpha₁ -adrenergic receptor. They are less potent than the endogenous agonists epinephrine or norepinephrine. However, because of structural modifications they are orally active and have longer plasma half-lives. There are 2 structural classes of alpha₁ agonists the phenylethylamines which are close structural analogs of epinephrine and norepinephrine and the structurally unrelated imidazolines. The major action of these agents is to produce alpha₁-receptor mediated vasoconstriction.

Phenylethylamines	Imidazolines
Phenylephrine
Pseudoephedrine	Oxymetazoline
Methoxamine	Naphazoline
Metaraminol	Tetrahydrozoline
Ephedrine

Clinical Uses of Agents that Activate the Alpha₁-Adrenergic Receptor

1) Hemorrhage control - previously discussed for epinephrine.

2) With local anesthetics - previously discussed for epinephrine. Levonordefrin (methylnorepinephrine) is also used for this purpose.

3) Hypotension - metaraminol, methoxamine

4) Ophthalmic preparations - to induce mydrasis (phenylephrine), to decrease intraocular pressure (apraclonidine, an alpha₂ agonist) and topically for symptomatic relief of irritation (many of the above agents).

5) Cough and cold preparations and nasal decongestants - Many of the above phenethylamines and imidazolines.

6) Alpha₁- adrenergic receptor agonists used to be used to slow heart rate in patients with atrial tachycardia - Can you reason why this would be so?

Indirect Acting Agents

These agents require the presence of endogenous monoamine neurotransmitters (norepinephrine, epinephrine, dopamine, serotonin) to produce their effects. Indirect acting agonists work at the nerve terminal to promote the release and/or block the reuptake of endogenous neurotransmitters. These agents have little activity if these neurotransmitters are depleted. Cocaine and amphetamine interact with cell surface monoamine transporters for dopamine (DAT), serotonin (SERT) and norepipephrine (NET). These transporters are expressed peripherally and in specific brain loci and are the site of action of psychostimulants and antidepressant drugs.

 

Cocaine:  Blocks reuptake of monoamines into nerve endings. Cocaine also has local anesthetic activity.

 

Amphetamine: Promotes the release of monoamines from nerve endings from the terminal cytoplasm. There is only a limited amount of neurotransmitter in this pool. Amphetamine also blocks the reuptake of monoamines. Several structural analogs of amphetamine and "amphetamine like" agents are available for clinical use. These include:

Dexamphetamine (the resolved and more potent d-isomer of amphetamine)

Hydroxyamphetamine

Methamphetamine

Methylphenidate

An important site of action of these drugs is in the CNS. These agents produce a feeling of well being and euphoria. Cocaine and amphetamine have a significant abuse potential because of these mood enhancing effects. Tachyphylaxis or tolerance to the stimulating actions of these agents can develop. These agents produce an increase in systemic arterial blood pressure. Heart rate can either decrease or increase depending on the levels of the drug. Drug toxicity effects multiple organ systems and can result in arrhythmias, hypertension, psychosis and convulsions. The local anesthetic activity of cocaine can also contribute to rhythm disturbances.

Clinical Therapeutics of CNS Stimulants

1) Because of its local anesthetic activity, cocaine has some limited uses as a oral, nasal and ophthalmic local anesthetic.

2) Appetite suppression - amphetamine and analogs

3) Narcolepsy - methylphenidate, amphetamine analogs

4) Attention deficient disorder with hyperactivity (ADHD) - methylphenidate, amphetamine and analogs

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Antagonists

Antagonists exhibit affinity for the receptor but do not have intrinsic activity at the receptor. An antagonist that binds to the receptor in a reversible mass-action manner is referred to as a competitive antagonist. Because the antagonist does not have intrinsic activity, once it binds to the receptor, it blocks binding of agonists to the receptor. A key point about competitive antagonists is that like agonists, they bind in a reversible manner. This has important implications regarding the effect competitive antagonists have on the configuration of the dose-response curve of agonists. Because competitive antagonists bind in a reversible manner, agonists, if given in high concentrations, can displace the antagonist from the receptor and the agonist can then produce its effect. The antagonist action can, in effect, be surmounted. Because the antagonist can be completely displaced, the agonist is still able to produce the same maximal effect observed prior to antagonist treatment. However, because higher agonist concentrations were necessary to displace the antagonist, the agonist dose-response curve is shifted to the right in the presence of a competitive antagonist. This can be illustrated with two equilibrium equations:

The antagonist [B] and agonist [D] are competing for the same limited number of receptors [R]. The drug that binds to the receptor in the highest concentration will be determined by two factors.

These factors are the affinities of the agonist and antagonist for the receptor and their relative concentrations. In the presence of a competitive antagonist equation #2 is modified as follows:

 

 

 

Where [B] is the concentration of antagonist and Kb is the affinity exhibited by the antagonist for the receptor. Inspection of this equation will reveal that the affinity of the agonist, Kd, is modified by the term (1+[B]/Kb). If the concentration of antagonist [B] is large in relation to its affinity Kb, the term (1+[B]/Kb) will be large. Therefore, the major effect of an antagonist is to shift the dose-response curve for an agonist to the right. The dose-response curve obtained in the presence of a competitive antagonist is parallel to the dose-response curve obtained in the absence of antagonist. If the Kb is small and the concentration high, the antagonist will have a more pronounced effect than if the Kb is large and the antagonist concentration is small. This also points out that large concentrations of the agonist can overcome the actions of a competitive antagonist. Assume that the agonist, D, and the antagonist, B, have equal affinity for the receptor. If the concentration of D is much larger than B, the value of E/Emax will not be significantly decreased by the presence of the antagonist. This again illustrates that the actions of the competitive antagonist can be surmounted by the agonist.

Prazosin is a competitive antagonist of the action of agonist PE

 

To summarize, the key features of a competitive antagonist are:

1. Reversible binding to the receptor.
2. The blockade can be overcome by increasing the agonist concentration.
3. The maximal response of the agonist is not decreased.
4. The agonist dose-response curve in the presence of a competitive antagonist is displaced to the right parallel to the curve in the absence of agonist.

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Irreversible Receptor Antagonists

Another type of antagonist is referred to as an irreversible receptor antagonist. The properties of irreversible antagonists are markedly different from competitive antagonists. Irreversible receptor antagonists are chemically reactive compounds. These ligands first bind to the receptor. Following this binding step, the ligand then reacts with the functional groups of the receptor. The consequence of this chemical reaction is that the ligand becomes covalently bound to the receptor. Because a chemical bond is formed, an irreversible ligand does not freely dissociate from the receptor. It remains attached to the receptor for a long period of time. The synthesis of new receptor protein may be required to generate a receptor free of an irreversible blocker. Because the ligand is covalently bound to the receptor, the binding of agonists, and hence their pharmacologic activity, are blocked. Unlike competitive antagonists, the blocking activity of irreversible receptor antagonists can not be overcome by increasing the agonist concentration. The antagonism therefore cannot be overcome by increasing the agonist concentration. Recall, that the effect of an agonist is proportional to the active drug-receptor complexes formed. Because an irreversible receptor antagonist reduces the total number of active receptors, [RT], the maximal pharmacologic effect Emax is also decreased. The reduction in maximal agonist reponse is the hallmark of irreversible antagonists. The shape of the dose-response curve is also altered because of this decrease in maximal effect. The dose-response is shifted to the right and the maximal response is depressed.

To summarize, the properties of irreversible receptor blockers are:

1. Chemically reactive compound, therefore covalently binds with the receptor
2. The receptor is irreversibly inactivated and the blockade can not be overcome with increasing agonist concentration.
3. Shifts the agonist dose-response curve to the right and depresses maximal responsiveness.

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Learning Objectives

1. Understand how activation of α₂ receptors decreases sympathetic outflow and cause hypotension.
   
2. Understand the pharmacologic properties and therapeutic uses for prazosin-like drugs.
   
3. Understand the pharmacologic properties of propranolol and atenolol and the therapeutic uses of beta-adrenergic receptor blockers.

 

Key Drugs*

Atenolol - Tenormin and various trade names - 4^th leading prescription drug in the US in 2003- source- rxlist.com

Clonidine - Minipres, various trade names

Propranolol - Inderal - various trade names

Terazosin - Hytrin

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Alpha₂ Agonists As Sympatholytics

• Clonidine

• Methyldopa

• Guanabenz

• Guanfacine

 

Actions

1. These agents stimulate alpha₂ receptors in the nucleus tractus solitarius (NTS) to decrease sympathetic outflow to the heart and blood vessels.
   

2. This results in a decrease in peripheral vascular resistance.
   

3. Clonidine, guanfacine and guanabenz are active drugs that are direct alpha₂ receptor agonists.
   

4. Methyldopa is a prodrug which must first be taken up into the nerve terminal and converted to "-methylnorepinephrine. This is the same synthetic pathway that converts dopa to norepinephrine.
   

5. Clonidine is the most widely used drug of this class. It can be given orally or in patch form. Side effects include drowsiness. This is a common occurrence for drugs that work in the CNS. Another prominent side effect of clonidine is presistent dry mouth. Clonidine has many other uses including opiate withdrawal, nicotine withdrawal, vascular headaches, diabetic diarrhea, glaucoma, ulcerative colitis and Tourette's syndrome. The other drugs are second-line agents used in the treatment of hypertension. Methyldopa has the particularly troublesome side effect of inducing hemolytic anemia.

 

SELECTIVE AND NONSELECTIVE ALPHA₁-ANTAGONISTS

Prazosin and analogs(doxazosin, terazosin, trimazosin) - Selective, competitive antagonists

Tamsulosin- Selective, competitive antagonist

Phentolamine-Nonselective, competitive antagonist

Phenoxybenzamine-Irreversible receptor antagonist

Effects of Prazosin and Analogs on the Cardiovascular System:

1. Prazosin and its analogs are selective alpha1-receptor blockers used to treat hypertension. These agents have similar cardiovascular actions, differing only in pharmacokinetic parameters. Doxazosin, trimazosin and terazosin are more widely used than prazosin.
   

2. These agent relax the smooth muscle associated with arteries and veins.

3. This results in a decrease in systemic arterial blood pressure due to a decrease in peripheral vascular resistance and venous return.

4. The reduction in arterial blood pressure does not result in a significant increase in heart rate.

5. Treatment with these drugs can result in fluid retention as a response to the lowering of blood pressure. Thus the drugs can be prescribed with a diuretic in the treatment of hypertension.

6. May have beneficial effects on lipid profiles by increasing HDL cholesterol and decreasing LDL cholesterol.

7. The effectiveness of this class of drugs for the treatment of hypertension was recently called into question by results from the Antihypertensive and Lipid Lowering Treatment to Prevent Heart Attack Trial (ALLHAT). The ALLHAT study showed that patients taking doxazosin were 25 % more like to have "cardiovascular events" and twice as likely to be hostipalized for heart failure than patients taking the thiazide diuretic, chlorthalidone.

 

Actions in Benign Prostatic Hypertrophy

1. Prazosin and related analogs also relax the smooth muscle associated with the bladder neck and prostate.
   

2. Tamsulosin has a similar action and is also used to treat BPH. Tamsulosin is an example of a drug that is selective for one of the subtypes of the alpha1-adrenergic receptors. This ligand selectively blocks the alpha1A-receptor. The alpha1A-receptor is involved in regulating the smooth muscle tone associated with the prostate. Therefore, tamsulosin selectively antagonizes the receptor subtype involved in regulating prostate function. Tamsulosin is less likely than the prazosin analogs to cause hypotension.

 

Side Effects

Postural hypotension and first dose syncope, which occur with greater frequency with the prazosin analogs than with tamsulosin.

 

 

Phentolamine

 

1. Nonselective competitive α ₁ and α₂ blocker.

2. Used to treat pheochromocytoma.

 

Phenoxybenzamine

1. Irreversible  α ₁ and α₂  receptor antagonist.

2. Used to treat pheochromocytoma.

 

BETA ADRENERGIC RECEPTOR BLOCKERS

1. These drugs are competitive antagonists of the beta adrenergic receptor

2. The beta blockers used in clinical therapeutics are either selective for the beta₁ receptor or nonselective beta₁ and beta₂ antagonists.

 

SELECTIVE AND NONSELECTIVE BETA BLOCKERS

 

Cardiovascular Effects of the Beta Blockers

Cardiovascular Effects and Clinical Uses of the Beta Blockers

The beta1-adrenergic receptor associated with the heart increases the force and rate of myocardial contraction. Beta antagonists block the ability of the sympathetic nervous system to increase the contractile force and the rate of contraction. The release of renin from the kidney is also regulated by the beta1-receptor. By blocking renin secretion beta1 blockers reduce the formation and hence the biological activity of angiotensin II. Beta1-receptor antagonists decrease blood pressure. While the mechanisms underlying this effect are not completely understood, they certainly involve a decrease in cardiac output and heart rate as well as decreasing angiotensin II levels. This reduction in blood pressure makes the beta blockers useful in the treatment of hypertension. Beta blockers are also useful in treating ischemic heart disease. This is because two major determinants of myocardial oxygen consumption are the force and rate of myocardial contraction which are diminished by this class of drugs. Beta blockers are also given following a myocardial infarction to prevent reinfarction. As will be discussed in the lectures on heart failure, certain beta blockers, specifically, metoprolol, bisoprolol and carvedilol, can be used to treat congestive heart failure. Certain arrhythmias are due to excess stimulation of the beta1-receptors. Thus beta blockers are useful in treating supraventricular tachyarrhythmias. There are many indications for beta blockers unrelated to cardiovascular therapeutics.

 

Propranolol - the Prototype Beta Blocker

1) Propranolol is a nonselective beta blocker

2) It was the first clinically approved beta blocker and the standard to which newer drugs have been compared.

Disadvantages of Nonselective Beta Blockers

A major disadvantage of nonselective beta blockers is the fact that they will block beta₂ receptors associated with airway or vascular smooth muscle. This unwanted action can exacerbate airway diseases (asthma, emphysema, chronic bronchitis) or peripheral vascular disease (Raynaud’s Disease). To overcome this disadvantage, "selective" beta₁ blockers have been developed. These agents have the ability to preferentially block beta₁ receptors. However, this selectivity is only relative and in higher doses selective antagonists will also block beta₂ receptors.

Intrinsic Sympathomimetic Activity of Certain Beta Blockers

Certain beta blockers actually have a modest degree of agonist activity. In other words these agents are partial agonists with low intrinsic activity. This is referred to as intrinsic sympathomimetic activity or ISA. These drugs may have a lesser effect on resting heart rate or cardiac output than compounds without ISA.

Membrane Stabilizing Activity

This refers to the ability of some of the beta blockers to also block sodium channels. As a result nerve cells become less excitable, hence the term "membrane stabilizing." In the case of beta blockers this membrane stabilizing activity does not contribute to its therapeutic action. However, you will often find this term used to describe the beta blockers.

Endocrine Effects

Beta blockers should be used with caution in patients with diabetes. In fact, nonselective beta blockers are contraindicated in diabetic patients. This is because catecholamines utilize the beta₂ receptor to promote glycogenolysis and mobilize glucose. This effect would be blocked by non-selective beta blockers. In addition all beta blockers mask the tachycardia associated with hypoglycemia. As a result the diabetic patient is deprived of one of the earliest physiologic responses to hypoglycemia.

Side Effects

The beta blockers have a variety of side effects. These include sedation, fatigue, and impairment of mental function. Hypotension and bradycardia can occur. These agents increase triglycerides and decrease HDL cholesterol. The effects on glucose metabolism has been discussed above. Nonselective beta blockers exacerbate peripheral vascular disease and airway dysfunction.

Labetalol, Carvedilol

These ligands block alpha₁ receptors as well as beta₁ and beta₂ receptors. Labetalol is used to treat hypertension. The side effect profile is what would be expected of a drug that blocks both alpha₁ and beta receptors. These include orthostatic hypotension, sedation, fatigue and other affects attributed to the blockade of beta receptors. In addition to treating hypertension, several recent clinical trials have shown carvedilol to be very effective in treating congestive heart failure. There are several proposed mechanisms underlying this effectiveness. Blockade of the beta₁ receptor appears to be more relevant than alpha₁ receptor blockade. This results in an improvement in left ventricular function. One pathophysiology of heart failure is that the heart increases dimensions. These increases result in a hypertrophied heart with decreased contractile performance. Carvedilol reverses these changes. Furthermore, carvedilol as antioxidant and antiproliferative activity. The extent to which these actions contribute to therapeutic efficacy in not clear.

Reserpine - Guanethidine

These drugs are not widely prescribed.

 

Reserpine

1. Depletes catecholamines from nerve endings in CNS and periphery.
   

2. Interferes with the vesicular storage of norepinephrine and other neurotransmitters.
   

3. This results in an inhibition of both alpha and beta receptor dependent events.
   

4. Reserpine produces hypotension due to decreased peripheral vascular resistance and cardiac output.
   

5. This drug can produce a variety of unpleasant CNS side effects such as insomnia, sedation and depression.

 

Guanethidine

1. Blocks the Ca²⁺ dependent release of catecholamines from nerve endings.
   

2. Long term use of Guanethidine depletes catecholamines from nerve terminals.
   

3. Does not interfere with central neurotransmitter storage or function.
   

4. Produces hypotension and bradycardia.

 

Uses of Reserpine and Guanethidine

1. Hypertension

 

Monoamine Oxidase Inhibitors

1. Inhibit monoamine oxidase.

2. Produce hypotension.

3. Can precipitate a hypertensive crisis.

 

Uses

1. Hypertension

2. Depression

 

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Reasons for the Nonlinear Relationship Between Receptor Occupancy and Physiologic Response

To understand how the relationship between occupancy and response can be non linear let us analyze the components which contribute to the response.

G-Protein Coupled Receptors

G-protein coupled receptors are a large family of receptors that serve as the site of action for many drugs. The name reflects the fact that the activity of these receptors is regulated by interaction with guanine nucleotide regulatory proteins (hence G-proteins). Despite major differences in the physiologic responses they activate and the variety of second messengers involved, the structure of all G-protein coupled receptors is similar. G-protein coupled receptors have a single polypeptide chain which passes through the cell membrane seven times. This arrangement results in the formation of loops on both the extracellular and intracellular sides of the membrane. Seven clusters of hydrophobic amino acids make up the membrane spanning domains of the receptor. The membrane spanning regions also form a binding pocket with which agonists and antagonists interact. The intracellular loops are thought to be necessary for the interaction with G-proteins and second messenger systems.

The G-protein Regulatory Cycle

In cellular signaling pathways involving G-proteins, the receptor/agonist complex does not interact directly with the enzyme which generates the second messenger. Rather, an intermediate or transducing protein couples the receptor to the second messenger generating system. This is the role of the G-protein . There is not a single G-protein, but a family of G-proteins which functions to regulate second messenger systems. G-proteins consist of three subunits: alpha, beta and gamma. In the resting state the receptor is not occupied by an agonist and the G-protein exists as trimer of the alpha, beta and gamma subunits with GDP bound to the alpha subunit. In this state, G-proteins are poor activators of intracellular signaling systems. Agonist binding to the receptor promotes the dissociation of the GDP and binding of GTP. GTP binding promotes the dissociation of the alpha subunit from the beta and gamma subunits. It is the GTP bound alpha subunit that activates effector enzyme systems. The alpha subunit is also a GTPase and is thus able to hydrolyze the GTP. The hydrolysis of GTP to GDP deactivates the alpha subunit and terminates the activation effector systems. The alpha subunit/GDP complex is then re-associated with the beta and gamma subunits to complete the regulatory cycle. The G-protein heterotrimer is again available for interaction with a receptor and activation of second messenger generating systems. Therefore, the rate at which the GTP is hydrolyzed regulates the time the G-protein is active. The longer the G-protein is active, the more second messenger can be generated.

 

 

 

 

 

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In a responding system which has a linear relationship between occupancy and physiologic response, there is a direct proportionality between the degree of receptor activation and the generation of second messengers. While this is difficult to conceptualize, it can be thought of as a small amount of receptor occupancy producing a small increase in the level of the second messenger. This small amount of second messenger activates a small increase in physiologic response. In the more realistic nonlinear occupancy versus response system, a small degree of receptor occupancy generates a large increase in second messenger levels which in turn generate an even larger physiologic response. The signal is amplified at every step of the signal transduction process. In this fashion, then, a small degree of receptor occupancy leads to a large physiologic response. Consider the following example. In a given beta-receptor system, 50,000 cAMP molecules are needed to yield a full response. In a linear response relationship, 50,000 receptors would have to be occupied to give a full response. However, in a nonlinear system, only 100 would be required to achieve a full response.

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Inverse Agonists

Traditionally, G-protein coupled receptors were thought to be inactive and that agonist occupation was required to allow the receptor to assume an active conformation. Recently, though, it has been suggested that the receptor can be active without the presence of agonist. The term for this is constitutive activity. Constitutively active receptors are thought to be coupled to second messenger pathways in the absence of agonists.

 

This has led to the postulate that in addition to traditional agonists, drugs can function as inverse agonists. Inverse agonists bind to constitutively active receptors and shift the equilibrium to the formation of the inactive conformer. In this system an inverse agonist would actually reverse receptor activity. The concept of inverse agonism has added a level of complexity to our thinking of drug action. As the diagram below illustrates, the spectrum of drug activity can range from a full agonist to a full inverse agonist.

The relevance of constitutively active receptors and inverse agonists to normal physiology and pathophysiology has not been established. That being stated, the concept of a constitutively active receptor does offer insights which could help to explain pathophysiologic conditions. If the process of disease induced the expression of a constitutively active receptor, the receptor would no longer be under the influence of the sympathetic nervous system. This could occur in hypertension with a constitutively active GPCR being expressed in any number of areas including the brain, kidneys or peripheral blood vessels. In this scenario, drugs with inverse agonist properties could prove to be safe, rational therapeutics.

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