The Classification of Drugs and Drug Receptors in Isolated Tissues
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0031-6997/84/3603-0165$02.00/0 PHARMACOLOGICAL REVIEWS Vol. 36, No.3 Copyright © 1984 by The American Society for Pharmacology and Experimental Therapeutics Printed in U.S.A. The Classification of Drugs and Drug Receptors in Isolated Tissues TERRY P. KENAKIN Department of Pharmacology, The Welkome Research Laboratories, Burroughs Welkome Co., Research Triangle Park, North Carolina I. Introduction 165 A. Isolated tissue and binding studies 166 II. Factors in the choice of isolated tissues 167 A. Animals 167 B. Preservation of tissue viability 167 C. Some isolated tissues from animals and man 168 D. Methods of tissue preparation 168 E. Measurement of tissue responses 172 F. Sources of variation in tissues 172 1. Heterogeneous receptor distribution 172 2. Animal maturity 173 G. Comparisons between isolated tissues 173 Downloaded from III. Equilibrium conditions in isolated tissues 173 A. Chemical degradation of drugs 174 B. Release of endogenous substances 174 C. Removal of drugs by tissues 176 1. Diffusion into isolated tissues 176 2. Drug removal processes in isolated tissues 177 by guest on June 7, 2018 3. Consequences of uptake inhibition in isolated tissues 179 IV. Quantification of responses of agonists 183 A. Dose-response curves 183 B. Drug receptor theory 184 C. The relationship between stimulus and response 185 1. Tissue response as a function of stimulus 185 2. Receptor density 188 V. Methods of drug receptor classification 188 A. Agonist potency ratios 188 B. Selective agonism 189 C. Measurement of agonist affinity and relative efficacy 192 1. Agonist affinity 192 2. Agonist efficacy 195 3. Experimental manipulation of receptor number and efficiency of stimulus response coupling 196 D. Competitive Antagonism 200 1. The Schild regression 200 2. Other methods to calculate PKB 207 VI. Operational concepts in receptor classification 207 VII. Relevance to new drugs 208 VIII. Conclusions 209 “We pharmacologists must acquire a knowledge of the toots which we use. “ R. BUCHHEIM, 1849 (324) I. Introduction able in the definition of drug action and the design of THE MAJOR premise of this review is that isolated more effective therapeutic agents for man. In this con- tissues can be used efctively to obtain information text, the bias of this paper will be pharmacological in about drugs and drug receptors which transcends species that receptors will be used to gain information about and function. This information, in turn, should be valu- drugs rather than the more physiological bias of drugs 165 166 KENAKIN used to gain information about receptors. This bias is binding studies have been noted as well. For example, reflected in the still very timely statement made by the binding Kd values for some fl2-adrenoceptor agonists Buchheim 135 years ago. do not correlate well with the relative potencies of these The information about drugs obtained from isolated drugs in isolated tissues (413). Similarly, although the tissues becomes useful for classification purposes which, selectivities of antagonists for fl- and fl2-adrenoceptors it is hoped, leads to general statements about structure as measured in binding and isolated tissue studies cor- and activity. The essentially circular nature of the drug relate well, the correlation for agonists is poor (71). This and drug receptor classification process should be kept is most likely because pharmacological agonist activity in mind. New receptor types or subtypes are discovered depends upon affinity and intrinsic efficacy, the latter only after the discovery of new selective drugs. Then new parameter being essentially inaccessible in binding stud- drugs are classified by their interactions with these new ies. Agonist activity in broken cell preparations can be receptors. It will be another bias of this paper that drugs measured (for example, fl-adrenoceptor agonists on ad- probably have more than one activity and are selective enylyl cyclase) but the lack of amplification which is rather than specific. This is a parsimonious view in present in tissues makes detection of all but quite pow- receptor terms since it seeks to explain selectivity or lack erful agonists not possible biochemically. The amplifi- of it in terms of multiple properties of drugs rather than cation processes inherent in the stimulus-response mech- multiple subtypes of receptors. anisms of isolated tissues make isolated tissues much The use of isolated tissues to classify drugs has its more suitable for prediction of agonist activity in vivo. drawbacks in the dependence of this process on previous Ingenious methods have been applied in binding stud- classifications. For example, in the early subclassifica- ies to differentiate between agonists and antagonists tion process for 9-adrenoceptors, the guinea pig trachea which theoretically could lead to quantification of rela- was classified as containing 92-athenoceptors and tra- tive efficacy biochemically. Thus the differential effects cheal-selective f3-agonists were accorded the correspond- of sodium ion on opiate receptor binding (516), the ing label of 132-adrenoceptor selective. The advent of data differences in free energy of binding (691) or effects of which suggest that trachea contains both j3- and 32- GTP and GpNHpp (439, 699) on fl-adrenoceptor binding adrenoceptors necessarily must bring into question the and the differential effects of GABA on benzodiazepine original f32-selective classification of these agonists. Un- binding (586) all offer unique approaches to this problem. fortunately, there is usually a time lag between the One apparent advantage binding studies have over classification of the drugs and the reclassification of the isolated tissue studies is the ability to measure receptor tissue and the possibly erroneously classified agonists density. Care must be taken, however, in interpreting may be used to classify other tissues incorrectly. The these estimates of receptor density. The difficulty comes potential for a baroque web of conflicting classification in predicting the relevance of the actual proportions of data for tissues and drugs in this process is obvious. heterogeneous receptor populations to pharmacological Some of these problems may be avoided if tissue selec- responses; i.e., will a ratio of8O:20 f3- to 32-adrenoceptors tivity is not assumed to be receptor selectivity. in a tissue translate to a more - than fi2-adrenoceptor In this paper, an attempt will be made to review some profile in terms of pharmacological responses? Actually of the null methods available to measure the strictly the probability of direct correspondence is low in view of drug-receptor-related parameters of affinity and intrinsic the coupling processes involved between receptor acti- efficacy, and more importantly, the internal checks in vation and tissue response. For example, Homburger and these methods to detect receptor heterogeneity. The ma- coworkers (325) have noted that even though f- and /32- jor advantage of isolated tissue experimentation is the adrenoceptors coexist on single C6 cloned glioma cells, potential to directly measure relative efficacy of agonists the coupling of the two receptors to adenyl cyclase is not (235). In this regard, this technique holds advantages of equal efficiency. The effects of receptors coupling on over biochemical binding techniques which primarily tissue responsiveness can be demonstrated in ontoge- yield estimates of affinity. netic studies. For example, although /3-adrenoceptors can A. Isolated Ttssue and Binding Studies be detected in fetal mouse hearts in binding studies, no Currently there is controversy over the significance of responses to isoproterenol can be elicited until later in the similarities and differences between binding and the cycle of tissue development (709). A study by Hoff- isolated tissue data. Unfortunately, there are all too few mann and coworkers (318) has shown that although the laboratories that do both techniques and critically com- state of oestrous in female rats greatly affected the pare the results (159). There are studies that show esti- relative number of a-adrenoceptor and /3-adrenoceptor mates of affinity of drugs in isolated tissue and binding subtypes, the changes in pharmacological responses did studies to be equivalent for a variety of receptors includ- not coincide with the receptor changes measured in the ing muscarinic receptors (52, 53, 235, 612, 712), f-adre- binding studies. In view of the influence of receptor noceptors (458, 459, 699), and a-adrenoceptors (320, coupling on drug responses, a theoretically more com- 606). However, differences between isolated tissue and plete approach to the study of the mechanism of action DRUGS AND DRUG RECEPTORS IN ISOLATED TISSUES 167 of drugs would incorporate both binding and isolated seen that complete delivery of dissolved oxygen to the tissue studies. cells of a given isolated tissue depends upon the partial . pressure of oxygen in the organ bath medium (Po2 bath), H. Factors in the Choice of Isolated Tissues the thickness of the tissue, and the rate of oxygen con- There are many factors to consider when choosing an sumption of the tissue (Vo2). Thus, for a flat isolated isolated tissue system for pharmacological experiments. tissue preparation (122): Numerous techniques have been identified to detect and eliminate obstacles to the attainment of the primary PO2(walI) Vo2 (T.X - X2) (1) =1- requisites for isolated tissues, namely uniformity and PO2(h) 1205D PO2(b5th) stability. where PO2(5ll) is the partial pressure of oxygen