Reemplazando los amortiguadores de vibración

▪ Competitive antagonists shift agonist dose-response curves to the right

As was the case for binding, reversible competitive antagonists produce parallel shifts to the right of plots of E (effect or response) versus log[A], where A is an agonist (Fig. 2.30). Such a relationship arises automatically from the equation:

Whatever the relationship between E (effect) and [AR], the inhibitory effect of a competitor (C), at any [A], can always be overcome or nullified by increasing [A] by the factor (1 + [C]/KC) which is the dose ratio (DR). In other words, inhibition is surmountable at any level of response and Emax is not reduced. There is an easy way to check whether experimental results agree with the theory. Since DR-1 = [C]/KC

it follows that:

where the latter relationship is known as the Schild plot (Fig. 2.31). In the Schild plot the intercept at log10 (DR-1) = 0 (where DR = 2) is the KC value. By analogy with pH notation, the negative log10 of KC is known as the pA2.

Figure 2.30 Dose-response curves for an agonist (A) in the presence of competitive antagonist (C), at different

concentrations such that [C]/KC equals 1, 10, 100 or 1000. Compare this figure with Fig. 2.7. Note that the x-axes are the same for both figures; however, the y-axis here is the ratio of the effects (E), produced by different concentrations of agonist, to the maximum effect (Emax).

Figure 2.31 Schild plot for a competitive antagonist (C). In this graph DR is the dose ratio, i.e. the value of A50 in the presence of a particular concentration of C divided by A50 in the absence of C. The data here are typical of those used to construct a Schild plot. The linear nature of this graph allows for easy estimation of KC and the slope. Note that a wide range of concentrations of C can be studied.

Sometimes the equation (DR-1) = [C]/KC does not hold for some agonists even though antagonists are clearly competitive in that they produce parallel shifts to the right. Deviations from the equation can be expected if the receptor has more than one binding site for agonist and the receptor is in its active form only when two or more agonist molecules are bound. An example of this situation is found with the nicotinic acetylcholine receptor in skeletal muscle.

▪ Not all antagonists are competitive

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Figure 2.32 This figure is analogous to Fig. 2.7 but in this case the antagonist is uncompetitive.

Not all drugs modify agonist binding in a competitive manner. The simplest reason for this is that a receptor contains more than one drug recognition site and that binding at one site alters the affinity at another. This type of interaction is known as an 'allosteric interaction'. It is characterized in saturation binding experiments by a half-maximal binding value, denoted by D50b (apparent KD), that can be varied continuously between two limits. At one extreme is the absence of the allosteric agent while at the other extreme virtually all receptors are bound to the allosteric agent.

When the antagonist binds in this manner it is said to be non-competitive.

Another theoretical scenario is one where the antagonist can bind only to the drug-receptor (DR) complex. This can occur only if the binding of D causes a change in conformation of the receptor (Fig. 2.32) such that it can now bind the antagonist. In such a case the antagonist may be termed an uncompetitive drug (U). Because of the dynamics of the interaction between drug (D), uncompetitive drug (U) and receptors, the apparent affinity of the receptor for drug D is increased in the presence of U. This essentially occurs because the form DR cannot exist and LUR must first

dissociate to form DR and U before breaking down to form free R. So, in effect, D is trapped in the DRU form.

▪ Non-competitive antagonists reduce Emax

When an antagonist (D) reduces Emax it is usually (and inaccurately) termed non-competitive (Fig. 2.33). Such antagonism results in marked deviations from the Schild plots for log (DR-1) versus log [D], seen with competitive antagonists. A good example of uncompetitive antagonism is ion channel blockade discussed earlier, in which the drug target is the ion channel that has been opened by an agonist.

Many clinically useful drugs are competitive antagonists for endogenous agonists such as neurohormones and autacoids, for example:

 Propranolol (β adrenoceptors).

 Haloperidol (dopamine receptors).

 Naloxone (opioid receptors).

 Phentolamine (α adrenoceptors).

 Cimetidine (histamine H2 receptors).

 Atropine (muscarinic receptors).

 Curare-like compounds (skeletal muscle nicotinic receptors).

Figure 2.33 Dose-response curves for an agonist (A) in the presence of different concentrations of a non-competitive antagonist (D). This graph is analogous to that for a competitive antagonist shown in Fig. 2.30. Note the rightward shift of the dose-response curve with increasing concentrations of D and the fall in maximum.

In each of these cases the nature of the antagonism and effectiveness of the compounds (and Kd values) were established using the methods outlined above.

When used clinically, the effects observed with a competitive antagonist actually represent inhibition of responses to an exogenous or endogenous agonist. An example is the depression of endogenous histamine-mediated acid

secretion in the stomach by cimetidine. Again, the dose-response curves are characterized by: (i) an Emax ; (ii) a dose at which effect is half maximum (usually termed EC50); and (iii) a slope parameter (h). As with dose-response curves for agonists, the exact form of such curves depends critically on the transduction system that links the active agonist-receptor complex (AR*) to the tissue response, since for competitive antagonists each concentration of antagonist corresponds to a reduction of [A]. Nevertheless, it can be shown that the EC50 is generally close to the dissociation constant, Kd multiplied by (1 + [A]/A50). That is, the EC50 is much the same, provided that the responses to the agonist are well below the maximum ([A]<A50). In general:

 Half-blockade of the agonist effect is associated with at least half-occupancy of receptors.

 Near-maximal effects of the competitive antagonist require that most receptors are bound by the drug.

This contrasts with what is found with most agonists, where near-maximal responses occur with a low receptor occupancy.

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When a competitive antagonist is used to block the actions of an endogenous agonist (e.g. a neurotransmitter), it is sometimes found that the EC50 is much more than the Kd. The simple explanation for this is that [A] at the receptor site is usually high relative to A₅₀. Alternatively, the phenomenon may arise because physiologically [A] is far from constant and equilibrium-type equations do not apply.

Dose-response curves for non-competitive antagonists representing inhibition of response to endogenous or exogenous agonist also resemble those shown in Figure 2.30. Since these agents can act by a variety of

mechanisms, the only generalization that can be made is that, in contrast to competitive antagonists, the EC₅₀ fails to rise in proportion to 1 + [A]/A50.

▪ The actions of irreversible antagonists persist

As indicated by their name, irreversible antagonists are characterized by an antagonism that persists despite removal of free antagonist. Generally, irreversible antagonism produced by such a drug is less if a high concentration of agonist is present during incubation with antagonist.

When irreversible antagonists were first developed, their effects on dose-response curves for agonists came as a surprise, since previously, pharmacologists had assumed that drug response was directly proportional to [AR]. With irreversible antagonists it became clear that such drugs produced irreversible inhibition of responses to agonists in a manner consistent with the loss of a proportion of available receptors. However, contrary to expectation, despite such losses there was often little or no change in Emax. Typical results are illustrated in Figure 2.34, where, at shorter incubation times with an irreversible antagonist, curves are at first shifted in parallel manner with increases in A50

values and no reduction of Emax, a pattern similar to that seen with competitive blockade. After a sufficient period of incubation, Emax is reduced while the shifts in A50 are also reduced. The explanation for this phenomenon is that maximal responses normally require the activation of only a small fraction of receptors. The existence of far more receptors than is needed to produce a maximum response is often referred to as 'receptor reserve'.

Receptor reserve implies that the A50 (the value of [A] for a half-maximum response) is less than KA ([A] for occupation of 50% of receptors). Thus the ratio KA/A50 is a reflection of receptor reserve and that reserve varies for agonists acting on the same receptor. Therefore, changes in receptor reserve among closely related analogs of an agonist provide yet another method of characterizing receptors. Receptor reserve is also influenced by the tissue itself; for example, activation of only a few of the available receptors may lead to maximal cellular or tissue responses due to marked amplification of receptor-activated signaling pathways.

Figure 2.34 Dose-response curves for an agonist (A) after increasing times of exposure to an irreversible antagonist. The more the curve is shifted to the right, the longer the tissue has been incubated with the irreversible antagonist.

The simplest and most generally accepted explanation for differences (of what is technically called 'intrinsic efficacy') between agonists is that only a particular conformation of the AR complex (say, AR*) is 'active' in the sense that it produces a response. Agonists with low intrinsic efficacy (A50 close to KA and little or no receptor reserve), which include partial agonists, have an AR that is seldom in the form AR*, while agonists with high intrinsic efficacy (A50

<<KA) have an AR mostly in the AR* form. Dose-response curves such as those shown in Figure 2.34 contain the information needed to calculate the KA of an agonist (not shown).

▪ A simple model for efficacy was proposed by Stephenson

The simple model of efficacy introduced by Stephenson is useful for understanding how results such as those in Figure 2.34 can arise. Suppose that AR produces a stimulus (S) which is proportional to [AR] and that response (E) is a function of S which is not limitless but saturates, then:

where e is a proportionality constant, called efficacy, that can vary from one agonist to another, and:

where E is effect and E'represents a hypothetical maximum that would occur if S could be made infinite. If agonists vary in the fraction (f) of AR that is in an active conformation (AR*), e will be proportional to f. Also, with e defined as above, it must be proportional to [Rt ] (or q[Rt ] if some receptors are made inoperable). Intrinsic efficacy is e/[Rt ].

On the basis of this model, agonists may be compared in terms of their relative e values, which are the same as relative intrinsic efficacies. The problem here is that the actual values of e obtained depend on the particular relationship assumed between E and S, which might or might not be true of any particular transduction system.

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▪ A partial agonist is an agonist of low intrinsic efficacy and e not much more than 1

From the above, a partial agonist can be seen simply as an agonist with low efficacy, with an e not much more than 1.

Experiments with irreversible antagonists have shown that, in agreement with this theory, partial agonists are

characterized by having A50 values close to KA values and no receptor reserve. As already pointed out, the low e of a partial agonist relative to a full agonist may be explained simply on the basis that with a partial agonist only a small fraction of the AR complexes are in the active conformation (AR*).

▪ When a full agonist is present at a high concentration, a partial agonist acts as an antagonist

Since a response to a partial agonist only occurs when receptor occupancy is high, it becomes apparent that partial agonists act as antagonists of full agonists. Therefore, a partial agonist can be used therapeutically to block the effect of an endogenous agonist (e.g. norepinephrine) while at the same time producing a steady low level of receptor activation in the absence of a full agonist. A classic example of partial agonism is seen with certain β blockers (e.g.

pindolol) that are partial agonists rather than competitive antagonists. Pindolol becomes an increasingly effective β blocker as sympathetic nerve activity on the heart increases. Thus when sympathetic activity on the heart is low, pindolol can increase heart rate, whereas the effect of sympathetic nerve activity on the heart is blocked by pindolol. A classic β-blocker drug never increases heart rate under any condition.

Agonism

 Agonism is the production of a molecular and cellular response to an interaction between a drug (agonist) and a receptor that activates the receptor. The intrinsic activity of a full agonist is defined as being equal to 1

 Partial agonism occurs when a drug interacts with a receptor to produce an average of less than 1 unit of molecular response. The average molecular intrinsic activity lies between 0 and 1

 Antagonism occurs when a drug interacts with a receptor to inhibit the action of an agonist. The molecular intrinsic activity is 0

 Inverse agonism occurs when a drug interacts with a receptor to reduce its resting level of molecular activity. The molecular intrinsic activity is -1

 Partial inverse agonism occurs when a drug interacts with a receptor to reduce the resting level of molecular activity. The molecular intrinsic activity lies between 0 and -1

▪ Drug responses change due to desensitization

Responses to drugs are often not fixed and constant over time, even though the concentrations of the drug at its receptor site may have reached steady-state values. In a variety of situations, responses to a drug may wane over time. Many factors lead to a loss in drug effects at an organ or system level, from progression of the disease being treated to physiologic adaptations. When the loss in responsiveness to a drug occurs at the level of the molecular target (e.g. the receptor) it is termed 'desensitization'. Many mechanisms have been found to contribute to

desensitization, operating at transcriptional, translational and protein levels of cellular regulation. These mechanisms may operate quickly (seconds to minutes) or relatively slowly, over the course of hours or days.

Mechanisms involved in rapidly developing desensitization have been extensively studied in molecular terms, especially for G protein-coupled receptors and particularly β adrenoceptors. At the cellular level, stimulation of β adrenoceptors with an agonist such as isoproterenol leads to activation of adenylyl cyclase and a brisk rise in intracellular concentrations of the second-messenger cAMP. However, in many cells, the capacity of isoproterenol to activate adenylyl cyclase declines with time, leading to a fall in cAMP concentrations in the cell. Phosphorylation of β adrenoceptors, association of these receptors with other proteins and changes in subcellular localization of the receptors may all contribute to the diminished ability of isoproterenol to activate cAMP accumulation.

Desensitization of β adrenoceptors (and other G protein-coupled receptors) can occur specifically due to the

phosphorylation of agonist-bound receptors by a G protein-coupled receptor kinase (GRK). GRKs constitute a family of kinases. GRK2, originally known as βARK kinase, was discovered on account of its capacity to phosphorylate agonist-occupied β adrenoceptors. Agonist occupancy of these receptors leads to binding of a GRK to the receptor and its phosphorylation. This mechanism has been termed 'homologous' desensitization since it specifically involves agonist-occupied receptors. After being phosphorylated, the receptors bind a member of the arrestin protein family, leading to steric hindrance of interaction between receptors and G proteins. The receptors may subsequently be sequestered away from the plasma membrane and move into the cytoplasm. Surprising new information suggests that the internalized receptors may contribute to novel mechanisms of β adrenoceptor signaling.

A second mechanism for receptor desensitization involves second-messenger feedback, which can lead to

desensitization of not only agonist-activated receptors but also different classes of receptors expressed in the same cell. This form of desensitization has been termed 'heterologous' desensitization, since the function of multiple types of receptors may simultaneously change after activation of just one receptor type. β adrenoceptors stimulate cAMP accumulation, which leads to activation of protein kinase A; the activated catalytic subunit of protein kinase A can phosphorylate not only β adrenoceptors, impairing their function, but also potentially a number of other receptors in the same cell with appropriate sites for phosphorylation by protein kinase A.

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Physiologic antagonism

▪ Physiologic antagonists oppose the actions of agonists by mechanisms independent of the agonist-receptor interaction

In an earlier section, antagonist drugs that inhibit the actions of agonists were considered to produce their inhibition by

acting in some manner on the same receptors as the agonists. However, drugs may also antagonize the actions of agonists by other mechanisms.

▪ Drugs can oppose responses to other drugs by acting on different molecular targets

It often happens that two drugs acting on different receptors have opposing actions at a tissue or organ level. When this occurs, the drugs can be considered to be physiologic or functional antagonists of one another, the former term being preferred. Obvious examples of such physiologic antagonism are epinephrine and acetylcholine, which respectively raise and lower heart rate, and glucagon and insulin, which respectively raise and lower blood glucose levels.

A more subtle example is antagonism of neuromuscular blockade due to a non-depolarizing neuromuscular blocking drug, such as pancuronium, by an anticholinesterase, such as neostigmine. The dose-response curve for

neuromuscular block versus dose of pancuronium is shifted by neostigmine in a non-competitive fashion. This occurs because the two drugs act on quite different molecular targets, the nicotinic receptor for pancuronium and the enzyme acetylcholinesterase for neostigmine. Blockade of the activity of the enzyme can do no more than double the height of end-plate potentials and, as a result, an anticholinesterase cannot reverse neuromuscular blockade due to an

excessive dose of pancuronium.

MEASURING RESPONSES AND PRACTICAL