Pharmacological ways to study pharmacological receptors

Gen. Pharmac., 1976, Vol. 7, pp. 87 to 91. Pergamon Press. Printed in Great Britain

PHARMACOLOGICAL WAYS TO STUDY PHARMACOLOGICAL RECEPTORS* Introductory remarks by M. ROCHA E SILVA Department of Pharmacology, Fac. Med. U.S.P. RibeirSo Preto, S~to Paulo, Brazil (Received 18 December 1975)

either action, and we know that many of the compounds, used in practical medicine, were obtained on the basis of an analysis of competitive and non-competitive action of antagonists upon receptors for acetylcholine, catecholamines and histamine, by strict pharmacological means, using the bioassay as a powerful tool, to screen compounds and deduce data about these mysterious and abstract structures that we call the pharmacological receptors (Clark, 1937). Take the immense literature on what has been called Autopharmacology by Dale (1953) and we can see that at the basis of physiology, pathology and psychology, thore stand methods of testing drugs with the biological preparations: the guinea-pig ileum, the uterus and duodenum of the rat, the rabbit and the cat, the striated muscle in the myoneural preparation, the blood pressure, the isolated heart and auricle, the lung, the gastrointestinal tract and the bioassays of the CNS effects of psychotropic drugs. This is to mention only the most used preparations in our laboratory, but we can amplify this list by mentioning in detail, the innumerable examples of bioassays used by pharmacologists (ROcha e Silva, 1972-1973). There is one reason to prefer the bioassay to the use of strictly biochemical methods. Take, for instance, the difference in labelling of an agonist either by radioactivity or by identifying it through its biological effect as a result of its interaction with the pharmacological receptor (Paton & Rang, 1965; Takagi & Ushida, 1970; Cuatrecasas et al., 1974). When we add a labelled antagonist and intend to study its fixation at the receptor site, we have no guarantee that the site of fixation actually is the specific receptor and I am sure that innumerable mistakes have been commited by simply assuming that an antagonist substance has been fixed upon the pharmacological receptor. However, when we demonstrate that the biological effect is being blocked, we don't have to mind whether the antagonist can be retained by other structures, and by knowing the apparent constant of equilibrium K~ we can even calculate a thermodynamic quantity, that one calls a "Free Energy Function".

FROM time to time we have to re-emphasize the importance of pharmacology, and the best way to do so is to try to define its scope. Nowadays, especially, this reappraisal of pharmacology is extremely useful because of the invasion of the field by distinguished biochemists who announce a new Golden Age by isolating receptors and relegating to a secondary activity those dealing with bioassays of drugs and agonists. To start this discussion, I would like to mention a phrase of Prof. Elliott, in Heffter's Handbuch, Vol. XXV, on bradykinin and related polypeptides, including kallidin and kallikrein, where he states that "in the time bradykinin was discovered, there was no biochemical method by which it could be identified" (Elliott, 1970). I would say, even today, 26 years after that event, it would be impossible to spot any active polypeptide by using pure chemical or biochemical methods. It is always possible to have blood extracts going through a Sephadex column and have, as usual, three peaks of ninhydrin stained material, but to know which is which, or which has any pharmacological or physiological activity we have to use a bioassay, as shown in Fig. 1, in which bradykinin was isolated in pure form at the Instituto Biol6gico of S~o Paulo, in 1955, and the results published in the Biochemical Journal, in 1956, in collaboration with Sylvia O. Andrade. From that publication, the impetus to clarify its chemical constitution and sequence, was given to the group of Prof. Elliott at the National Research Institute in London and to the group of Boissonnas, at Sandoz, in Basel. This history is now retold in the recently published Bradykinin Anthology (Rocha e Silva & Rothschild, 1974). This is an example of the cooperative work of biochemists, chemists and pharmacologists to clarify a field that still is giving interesting dividends. The other example I would like to give, and more related to the subject of this Symposium, is the enormous work done by medicinal chemists to develop the field of anti-cholinergic, sympatholytic and antihistaminic agents. An enormous mass of data has been offered to chemists to synthesize products with * Aided by grants from Funda~'?w de Amparo ~ Pesquisa do Estado de S?to Paulo (FAPESP) and Conselho Nacional de Desenvolvimento Cientifico e Tecnol6gico (CNPq).

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Fig. 1. Purification of bradykinin in a column of Amberlite IRC-50 (XE-64). Top: first passage through the Amberlite column. Bottom: the same material collected above was submitted to a new column of Amberlite. A specific activity of 5000 units per milligram of polypeptide (calculated as leucine) was obtained in both peaks of activity (top and bottom). Full lines: estimation of total amino acids by ninhydrin in the hydrolysates of the eluates. Dotted lines: biological activity (in units of bradykinin). (According to Andrade & Rocha e Silva, 1956).

that is solely concerned with the interaction of the antagonist with the pharmacological receptor, provided the apparent constant of equilibrium has been determined by purely biological means. [It is pertinent to recall that for any competitive antagonist: _-log Ki = pA2 the value of pA2 calculated in equilibrium (after a long time of contact) being related by an inverse log function to the constant of equilibrium antagonistreceptor.] We don't have to think of other possible sites of anchorage of the antagonist, as would generally be the case by using radioactive labelled antagonist (Rocha e Silva, 1966, 1969, 1975). The same would be stressed as regards the interaction of the agonist with the specific site of the receptor. Again, we have to consider the possibility of measuring affinity by pure pharmacological means. Again, if we use radioactive labelled agonists, we might commit the mistake of studying interaction of the agonist

with other unspecific structures, that might be called acceptors instead of receptors. This conforms to the idea that we might have silent receptors that might consume part of the strength of the agonist, but I must remind you that "silent receptors", if they exist at all, would not participate in the general computation of the affinity of the agonist or the antagonist toward the specific pharmacological receptors. I might make the usual joke that silent receptors are comparable to people that receive letters but do not answer them, and consequently it is extremely difficult to know of their existence, and besides they will not disturb in any way the exchange of correspondence with people who do answer letters. The consequence is that we may forget about the existence of silent receptors, at least in connection with the mechanism of functioning of the other receptors, the true ones, i.e. those that answer letters, when we stimulate them with the appropriate means of communication, that is, the ,chemical mediator for

Ways to study pharmacological receptors which receptors are made or have been constructed by the "architect" of living matter. In this connection, it appears extremely important that we define a parameter that has been called affinity. Usually we talk of a constant of affinity, that in the case of the antagonist is Ki or Ks which, as we have seen, is connected to the value of pA2 by an inverse logarithmic function. But the simple popularity of the index pA2, as defined by Schild, in 1947 indicates that affinity can be represented better by a logarithmic function, and still better by its inverse, pA: = - l o g K , where the value of the index increases with the value of the affinity, and we have the known plots in which pA2s for different agonists are compared in a sort of layout to indicate the relative potencies of the antagonists toward the different agonists. But it is also well known that for competitive antagonism the three constants of affinity K , the overall constant of affinity of the antagonist, and the constants K, of the agonist in the absence of the antagonist and K',, in the presence of a concentration (1) of the antagonist, are related through a single mathematical expression (Rocha e Silva, 1959): K',

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fl = K',/K,, and since (I) is known, we can easily calculate the constant of equilibrium or affinity K~ and therefore the pA2. If we take that expression in its logarithmic form:

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Fig. 2. Responses-log doses of histamine. A ( # ) control line, without antagonist; B, C and D, in the presence of dibenzyline (phenoxybenzamine) producing inhibitions reversed by increasing concentrations of histamine; E and F, lines corresponding to concentrations of dibenzyline that already affect the maximum response. The antagonism is fully competitive for lines A-D. Lines E and F indicate that larger concentrations of dibenzyline will affect the "number of available" receptors.

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log(fl- 1)=logI+pA2 where from pA2 can be deduced. I have to say that there are other methods for determination of pA2 such as the direct one devised by Schild (1947), and the one worked out by Arunlakshana & Schild (1959) and developed into a very elaborated method by Parker & Waud (1971) and Waud & Parker (1971). If I insist on making such points, although very elementary, it is because misunderstanding of them led distinguished pharmacologists to believe in an erroneous, "spare receptor" concept. Let me show clearly how this misleading concept was introduced in the literature about the action of a fl-haloalkylamine (dibenamine) with the histamine receptor (Nic' kerson, 1956; Furchgott, 1964; Barlow, 1964). We have to go back to some of the ways of representing the dose-response curves, as indicated in Figs. 2 and 3. The so-called competitive antagonism may be represented by either plot; the direct one, in which the concentration of the agonist is given in a log-scale in absissae (Fig. 2) or by inverting the data, and using the so-called "double reciprocal plot", as indicated in Fig. 3. Both representations are equivalent and either plot allows a measure of the affinity,~constant of the agonist; the first one, as the concentration of the

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M. ROCHA E SILVA

agonist needed to produce 50% of the maximum effect, and in the double reciprocal plot, as the slope of the normalized line K, in the absence (control), or K', in presence, of the antagonist. Now, if the antagonism is strictly competitive, the lines reaching a maximum (100%) will shift parallel to the abcissae axis (concentration) and if we prefer to use the double reciprocal plot, the lines will converge to the same point, at the ordinate axis, indicating the reciprocal of the maximum effect (1/Y,). In either case, we can prove that the same effect E, in the absence and presence of the antagonist, can be represented by an expression: X X' E = Y ' X ~ or E = Y m x , + K~ + K'n where X is the concentration producing the effect E, in the absence of, and X', the concentration needed to produce the same effect in the presence of, the antagonist. It is obvious, from the plots presented, that the constant of affinity, measured either way, will shift to higher values, indicating that the affinity has decreased in presence of the antagonist. But the introduction of the concept of spare receptor, was done by assuming that (a) dibenamine or fl-haloalkylamines in general, destroy receptors and therefore the maximum effect can only be obtained by inducing the agonist in higher concentrations to explore new pools of idle receptors. In some authorized papers it has been stated that no more than a fraction of 1% of the total receptors left, will be enough to induce a maximum effect (Furchgott, 1964; Barlow, 1964), or (b) fl-haloalkylamines occlude receptors for histamine, but the conclusion is always the same, that though knocked out by dibenamine, the remaining receptors (spare ones) will be able to produce the maximum effect (Parker & Waud, 1971 ; Thron, 1970). Either postulate will end up with the erroneous deduction that the constant of affinity K, of the agonist toward its receptors remains constant and therefore in the expression presented above, we may have the following situation: E=Ymx

X X' + Ka = X ' + Ko '

i.e. we have to apply an increased concentration, X' = qX, upon a structure that has the same affinity (K.) to produce the same response E. But this is the crux of the matter. When we add dibenamine, the obvious effect is to change K, that becomes K', > K,, i.e. the constant of affinity increased, and the affinity (pK.) decreased, and the only way to equalize the two expressions is by making K', K,

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which is a relationship typical or characteristic of competitive antagonism. Therefore, instead of destroying or occluding receptors, dibenamine, as any other competitive antagonist, in such concentrations

which still allow the maximum effect to be produced, decreases the affinity of the receptor for histamine in the proportion of K',/K, as shown in other situations of competitive antagonism. In continuation, we are going to show that (a) in a range of concentrations, dibenzyline will not destroy receptors, since they are always there, and can be recovered by a cold treatment as shown before (Rocha e Silva et al., 1972), and (b) the concept of spare receptors can be abandoned in face of a new formulation of the problem that we have called the Charni&e effect (Rocha e Silva, 1969, 1975). In the course of this Symposium the problem will be discussed in profundity by the distinguished biochemists and pharmacologists present here.

REFERENCES

ANDRADE S. 0. ~,~ ROCHA E SILVA M. (1956) Purification of bradykinin by ion exchange chromatography. Biochem. J. 64, 701-705. ARUNLAKSHANAO. & SCHILDH. O. (1959) Some quantitative uses of drug antagonism. Br. J. Pharmac. t4, 48-58. BARLOW R. B. (1964) Chemical Pharmacology. 2nd Edn, Methuen, London. CLARKA. J. (1937) General pharmacology. In Handb. Exp. Pharmacol. Vol. 4, pp. 1-228. Springer-Verlag, Berlin, (Reprinted 1970). CUATRECASASP., TELL G. P. E., S1CA V., PAR1KH ][. & CHANG K. J. (1974) Noradrenaline binding and the search for catecholamine receptors. Nature, Lond. 247. 92 97. DALE H. H. (1953) Adventures in Physiology. Pergamon Press, Oxford. ELLIOTT D. F. (1970) Discovery and characterization of bradykinin. In Handb. Exp. Pharmac. XXV, pp. 7-13, Springer-Verlag, Berlin. FURCHGOTT R. F. (1964) Receptor mechanisms. Ann. Rev. Pharmacol. 4, 21-50. NICKERSON M. (1956) Receptor occupancy and tissue response. Nature, Lond. 176. 697 698. PARKER R. B. & WAUD D. R. (1971) Pharmacological estimation of drug-receptor dissociation constants. Statistical evaluation. I Agonists. J. Pharmac. exp. Ther. 17% 13 24. PATON W. D. M. & RANG H. P. (1965) The uptake of atropine and related drugs by intestinal smooth muscle of the guinea-pig in relation to acetylcholine receptors. Proc. R. Soc. B. 163, 1-44. ROCHA E SILVAM. (1959) Concerning the theory of receptors in pharmacology. A rational method of estimation of pAx. Archs Int. Pharmacodyn. 118, 74-94. ROCHA E SILVA M. (1966) Action of histamine in smooth muscle. In Handb. Exp. Pharmak. Vol. XVIII/1 pp. 225 237. ROCHA E SILVA M. (1969) A thermodynamic approach to problems of drug interaction--I. The "Charni+re Theory". Eur. J. Pharmae. 6, 294-302. ROCHA E SELVAM. (1972-1973) Fundamentos de Farmacolo9ia e suas aplica¢6es fi Terap~utica. Cap. II, Edart, Silo Paulo.

Ways to study pharmacological receptors ROCHA E SILVA M. (1975) A thermodynamic approach to problems of drug antagonism: the microphysical model. In Concepts of Membranes in Regulation and Excitation. (Edited by ROCHA E SILVA M. & SUAREZ-KURTZ G.) Raven Press, New York. ROCHA E SILVA M., FERNANDES F. & ANTONIO A. (1972) Influence of temperature on recovery of inhibition by anti-histamines and fl-haloalkylamines toward histamine. Eur. J. Pharmac. 17, 333-340. ROCHA E SILVA M. & ROTHSCHILD HANNA A. (Editors) A Bradykinin Anthology, S.B.F.T.E. Hucitec, Sho Paulo.

91

SCHILD H. O. (1947) pA, a new scale for the measurement of drug antagonism. Br. J. Pharmac. 2, 189-200. TAKA61 K. & USnIDA M. (1970) Purification of histamine receptor in cat small intestinal muscle--I. Specificity of labeling histamine receptor with radioactive dibenamine. Jap. J. Pharmac. 20, 272-286. THRON C. D. (1970) Graphical and weighted regression analyses for the determination of agonist dissociation constants. J. Pharmac. exp. Ther. 175, 541-553. WAUD D, R. & PARKER R. B. (1971) Pharmacological estimation of drug-receptor dissociation constants--I. Competitive antagonists. J. Pharmac. exp. Ther. 177, 13-24.