Historical Background And Classical Receptor Theory

What is considered a "given" today, namely that drugs interact with specific binding sites on the cell membrane called receptors, is an extremely important tenet of pharmacology. As stated by Rang [1], it indeed was "Pharmacology's big idea." The main reason it is such an important cornerstone of pharmacology is that it introduces order into the apparent chaos of physiology. For example, a simple molecule such as epinephrine mediates a myriad of physiological processes. A large subset of these, namely cardiac chronotropy, inot-ropy, lusitropy, vascular relaxation, lacrimal, pancreatic, and salivary gland secretion, bronchiole, uterine and urinary bladder muscle relaxation, decreased stomach motility, skeletal muscle tremor, and melatonin synthesis are mediated by a small subset of membrane-bound proteins, specifically p1- and p2-adrenoceptors. This immediately puts order in the collection of physiological processes in that it gives them a common place to start, namely the interaction of epinephrine with the receptor. This order fits in well with the discipline of medicinal chemistry in that chemists have access to the processes for potential control.

At the turn of the twentieth century, different groups carried out research that caused them to postulate the existence of control points on cells that responded to chemicals, that is, receptors. For example, Paul Ehrlich (18541915) carried out studies on agent "606" (salvarsan) for syphilis. His work with dyes and bacteria led him to propose that there are "chemoreceptors" (actually a collection of "amboreceptors," "triceptors," and "polyceptors") on parasites, cancer cells, and microorganisms that could be exploited therapeutically [2]. In Cambridge, John Newport Langley (1852-1926) studied the drug jabo-randi (contains the alkaloid pilocarpine) and atropine and concluded that receptors were "switches" that received and generated signals and that these switches could be activated or blocked by specific molecules [2] . However, it is A.J. Clark (1885-1941) who is considered the father of modern receptor pharmacology. Clark was the first to suggest, from studies of acetylcholine and atropine, that a unimolecular interaction occurs between a drug and a "substance on the cell." As he put it [3] ...

... it is impossible to explain the remarkable effects observed except by assuming that drugs unite with receptors of a highly specific pattern..

In fact, it was Clark who described pharmacological phenomena in chemical terms [3, 4] , a concept readily accepted today, but quite heretical in Clark ' s time. The prevailing concepts guiding physiology at the turn of the century were rooted in homeopathic theories (i.e., a fundamental theory centered on the surface tension of the cell membrane) like the Arndt-Schulz Law and Weber-Fechner Law [2] , A generally accepted statement to describe physiologic phenomena was simply that "certain phenomena occur frequently."

One of Clark's most valuable contributions to pharmacology was the application of mathematical rules to the behavior of biological systems. Thus, the dose-response curve became the common currency of pharmacology, and its judicious use in the work of Clark and others built the framework for what had become known as "receptor theory," namely the application of simple thermodynamic rules to pharmacological systems.

An early example of mathematics applied to the study of receptors was provided by A.V. Hill, a student of Langley. He expressed the time course of contraction of frog rectus abdominus to the agonist nicotine (N) through an equilibrium concentration-response curve of the form:

where Y is contraction height, M is threshold, and k' , k are constants. While this work predated the routine use of binding isotherms to receptor work considerably, Hill lost interest in the approach, and it was left to Irving Langmuir, a chemist at General Electric Company in the United States, to devise an equation for the quantification of molecules binding to a surface, in particular, chemicals to metal filaments for light bulbs. Thus, the Langmuir adsorption isotherm quantifies the fraction of the substance bound to a surface (the pharmacological counterpart being receptor, denoted pA) by a molecule [A] as:

where KA is the ratio of what Langmuir referred to as the "rate of evaporation" of the substance away from the surface (pharmacologically, the rate of offset of the molecule from the receptor) divided by the "rate of condensation" of the molecule toward the surface (pharmacologically, the rate of onset toward the receptor). In pharmacological terms, the specific terminology for molecules that produced such activation is "agonist." While mechanistically, this equation is based on thermodynamic principles governing agonist binding to receptors, operationally, it also defines the universally observed relationship between agonists and the pharmacological responses they induce to tissues and cells. Thus, any observed receptor-mediated response in any tissue can be summarized by a form of the Langmuir isotherm where the fractional maximum given by Equation 1.2 is multiplied by the maximal response observed from the preparation (ResponseA = pA • Emax):

ResponseA (13)

where EC50 refers to the concentration of agonist A that produces half the maximal response to drug A. It should be noted that Equation 1.3 is written for a system demonstrating a Hill coefficient (in honor of A.V. Hill) of unity. If there is cooperativity in the system (either in the binding of the drug to the receptor [vide infra] or in the cellular processes that translate drug binding into cellular response), then Equation 1.3 becomes:

where n is the Hill coefficient for the dose-response curve. Figure 1.1 shows data from Clark (effect of acetylcholine on frog heart chronotropy) fit to the Langmuir adsorption isotherm (Eq. 1.3). Irrespective of mechanism, it can be seen that the curve shown in Fig. 1.1 concisely summarizes the data. Thus, the

Figure 1.1 (a) Alfred. J. Clark (1885-1941), Professor of Pharmacology at University College London and later Chairman of Pharmacology in Edinburgh. Clark applied chemical laws to biological phenomena and is regarded as the father of receptor pharmacology. (b) Clark's data showing responses of frog heart to acetylcholine. Data points are fit to the Langmuir adsorption isotherm (Eq. 1.3).

Figure 1.1 (a) Alfred. J. Clark (1885-1941), Professor of Pharmacology at University College London and later Chairman of Pharmacology in Edinburgh. Clark applied chemical laws to biological phenomena and is regarded as the father of receptor pharmacology. (b) Clark's data showing responses of frog heart to acetylcholine. Data points are fit to the Langmuir adsorption isotherm (Eq. 1.3).

16 data points are summarized by a shorthand that states acetylcholine produces a chronotropic effect in the frog heart that begins its effect at 50 nM, is half maximal at 2.8 |M, and is approximately maximum at 500 |M.

The adsorption isotherm furnished an extremely useful method of summarizing and handling dose-response data, but what was still required is a way to relate the observed data to the molecular mechanism of the drugs producing the effect. With no independent estimate of the affinity of the agonist he was using, Clark had to assume a one-to-one correspondence between the molecules of agonist he added to his preparation and the quanta of excitation those molecules gave to the tissue; that is, there was no provision for variance in the "power" of the molecules to induce tissue response. Ariens and Van Rossum, leading a germinal receptor group in Nijmegen, began the process of relating the molecular mechanism of drugs to the observed effects of drugs [5-7]. Thus was introduced the concept of "intrinsic activity," denoted a, as a scaling factor to accommodate the observation that not all agonists produce the maximal response of the preparation. Under these circumstances, Equation 1.4 becomes:

where, for a = 0.5, this would depict a drug that produced 50% of the tissue maximal response. Intrinsic activity became the first parameter designed to scale observed drug effect with the molecular "power" of an agonist to induce response. While this improved the correspondence between some observed effects of agonists, it was left to R.P. Stephenson, a pharmacologist working in Edinburgh, to extend this process to another level. Stephenson postulated that there was no reason to assume that tissue response was linked to agonist concentration in a linear manner (as was the requirement of the Clark and Ariens treatments). Instead, he postulated the existence of a theoretical parameter he called "stimulus," which is the result of the immediate interaction of the drug with the receptor [8]. This stimulus is imparted to the cell which then processes it in various ways, according to its needs, to yield tissue response. This loosely defined a function (referred to as the stimulus-response function) relating tissue excitation and response. Thus, tissue response was given as:

ResponseA = f

where e is a term efficacy (used to depict the power of the drug to produce response) and f is the stimulus-response mechanism. The important aspect of Equation 1.6 is that it allows the tissue response to be dissociated from receptor occupancy; experimental data would soon show the importance of that feature of the model.

Stephenson's concept of efficacy was required because of his observation that a series of related alkyltrimethylammonium compounds produced different maximal levels of guinea pig ileal contractions within a similar concentration range [8] . Since the agonist potencies indicated that the compounds had similar affinities for the receptor, Stephenson reasoned that another property of these molecules had to be operative to make them dissimilar in terms of producing muscle contraction; that was the property he termed "efficacy." This concept opened up a completely new way to look at tissue activation through receptors. Specifically, there were no constraints regarding the power of molecules to produce pharmacological response. Technically, powerful agonists could produce maximal tissue response by activating only a portion of the available receptors; the remaining portion would thus be described as being "spare" or not required for the production of maximal response. This offered maximal control to tissue systems since the cellular receptor density (i.e., varying proportions of spare receptors)

Log [Histamine] % Receptor Occupancy

Figure 1.2 Response of guinea pig ileum to contraction by histamine and the relationship between histamine receptor occupancy and tissue response. (a) Contractile responses of guinea pig ileum to histamine. Ordinates: percent maximal contraction to histamine (solid lines) or calculated receptor occupancy from Langmuir adsorption isotherm (Eq. 1.2) for dotted line. Abscissae: logarithm of molar concentrations of histamine. Data shown for control (n = 12) and after alkylation of a portion of the population of histamine receptors with SY-28 (Y-ethyl-Y-P-bromoethyl)-l'-naphylmethylamine) (200 nM exposure for 3min followed by 3h wash; n = 8). Data from Reference 11. (b) Calculated relationship between histamine receptor occupancy (dotted line in panel A) and control histamine response. This is the experimentally derived stimulus-response relationship between histamine and guinea pig ileum. Note the abscissal axis for panel B does not extend to complete receptor occupancy and that essentially 100% tissue response is obtained with an 18% histamine receptor occupancy.

Log [Histamine] % Receptor Occupancy

Figure 1.2 Response of guinea pig ileum to contraction by histamine and the relationship between histamine receptor occupancy and tissue response. (a) Contractile responses of guinea pig ileum to histamine. Ordinates: percent maximal contraction to histamine (solid lines) or calculated receptor occupancy from Langmuir adsorption isotherm (Eq. 1.2) for dotted line. Abscissae: logarithm of molar concentrations of histamine. Data shown for control (n = 12) and after alkylation of a portion of the population of histamine receptors with SY-28 (Y-ethyl-Y-P-bromoethyl)-l'-naphylmethylamine) (200 nM exposure for 3min followed by 3h wash; n = 8). Data from Reference 11. (b) Calculated relationship between histamine receptor occupancy (dotted line in panel A) and control histamine response. This is the experimentally derived stimulus-response relationship between histamine and guinea pig ileum. Note the abscissal axis for panel B does not extend to complete receptor occupancy and that essentially 100% tissue response is obtained with an 18% histamine receptor occupancy.

could be used by the tissue to control sensitivity to the hormone or neurotrans-mitter. Experimental evidence to support the existence of spare receptors came with the use of receptor alkylating agents (specifically, P-haloalkylamines) which could irreversibly block portions of the receptor population in any given tissue [9, 10]. It was observed that irreversible removal of large portions of receptor populations rendered tissues less sensitive to agonists but that these agonists still were capable of producing the maximal tissue response. This experimentally defined the shape of stimulus-response relationships as postulated by Stephenson; an example of the process used to do so is shown in Fig. 1.2. These data formed the concepts leading to the present model universally used to depict agonist response in tissues, namely the operational model.

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