Acta pharmacol. et toxicol. 1978,43 (II), 74-78
From the Department of Pharmacology, University of Uppsala, Box 573,751 23 Uppsala, Sweden
Methodological Aspects on Drug Receptor Binding Analysis BY Agneta Wahtstrom
Abstract: Although drug receptors occur in relatively low concentrations, they can be visualized by the use of appropriate radioindicators. In most cases the procedure is rapid and can reach a high degree of
accuracy. Specificity of the interaction is studied by competition analysis. The necessity of using several radioindicators to define a receptor population is emphasized. It may be possible to define isoreceptors and drugs with selectivity for one isoreceptor. Key-words: Drug receptor
-
receptor binding.
Some criteria must be settled before starting the discussion of receptor binding. The classical definition of a receptor involves a functional response. When the biochemical techniques have been applied to receptors, it is very often the receptor molecule which has been discussed, and the physiologic system as a whole has been left out. In this paper the relation between biologic potency and binding properties will be stressed. The main criterion for the receptor will be the specificity of the observed interaction in both functional and structural terms. There are also other criteria which help us to define a specific receptor, such as a) the receptor is situated only in the target tissue and is localized to a specific part of the target cell; b) there are agonists which bind to the receptor as well as competitive antagonists, but inactive substances d o not. In principle there are two different types of receptors, soluble receptors and membranebound receptors. The criteria discussed above should be fulfilled in both cases.
The primary binding process between the receptor and the ligand is probably similar although the receptor complex exerts its biologic effect according to two different principles. The activated membrane receptors induce the effect via cyclic nucleotides, while cytoplasmic receptors enter the cell nucleus where they interact with chromatin and affect the genome.
Radioindicators in receptor bitzding The receptor density is comparatively low for most receptors. In many cases it is well below the dissociation constant for the ligand in question. There are some guidelines for choosing the best properties of a labelled radioindicator when using it in a radioreceptor assay since the highest possible sensitivity is required. The labelled radioindicator ought to have a) a high specific radioactivity; b) reasonable receptor occupation compared to the concentration in the incubation mixture: c) as low non-receptor binding as possible.
80
T. HOKFELT ET AL
On the Methodology Hi5 tochemistry
The principles and technical outlines for the application of immunological principles in histochemistry were described more than 30 years ago by Coons and collaborators (see Coons 1958; see also Nairn 1969). It was possible to combine the specificity and sensitivity of the antigen-antibody reaction with the high resolution of the microscopic techniques. Two principles were devised, the direct and the indirect method. The direct technique involves labelling of the specific antibody with a fluorescent dye, e . g . fluorescein isothiocyanate (FITC), whereas in the indirect technique a secondary antibody is labelled. The latter modification appears t o be widely used by virtue of its higher sensitivity and the possibility of using one and the same labelled (secondary) antibody for several primary (specific) antisera. More recently, modifications of the Coons technique have been introduced. Horseradish peroxidase may be used as label, offering the advantage of analysing the tissue both in an ordinary light microscope and in the electron microscope (see Nakane 1971). The highly sensitive unlabelled antibody enzyme method of Sternberger and collaborators (see Sternberger 1974) has emerged as the method of choice in many laboratories for immunocytochemical studies both at the light microscopic and at the ultrastructural level. The routine procedure used in our laboratory for localizing peptides - and enzymes - at the light microscopic level is based on the indirect fluorescence technique of Coons and collaborators (see Coons 1958) and is as follows. The experimental animals are perfused via the vascular system (ascending aorta) with ice cold formalin (Pease 1962). After 30 min the tissues are dissected out, immersed in the same fixative for several hours, rinsed in phosphate buffer, cut on a cryostat (Dittes, Heidelberg, W . - Germany) and processed for the indirect immunofluorescence technique. The sections are incubated with the specific antiserum in a humid atmosphere at + 3 7 T for 30 min with the antisera diluted 1: 10- 1: 100 depending o n the characteristics of each antiserum, especially its affinity and tit-
er. Incubation may also b e carried out at a low temperature (+4"C) for a longer time (12-24 hrs), mostly allowing a considerably higher dilution of the antiserum. This procedure may result in less unspecific binding and in addition saves precious antiserum. After the first incubation the sections are rinsed and incubated with FITC conjugated sheep anti-rabbit antibodies (commercially available from Statens Bakteriologiska Laboratorium, SBL, Stockholm, Sweden) in a humid atmosphere at +37"C for 30 min, rinsed, mounted and examined in a fluorescence microscope. It is common to add a detergent such as Triton X-100 (Hartman et al. 1972) to the antisera with the aim of "solubilizing" membranes and enhancing the penetration of the antibodies into the sections. In spite of many advantageous characteristics of the immunocytochemical techniques there are several problems involved. Apart from technical difficulties such as poor penetration of the antibodies into the tissue, insufficient sensitivity and unspecific staining, the crucial and seemingly unsolvable problem is the possible occurrence of cross-reacting peptides. Although the antisera can be characterized with regard to cross-reactivity with known peptides, unknown peptides with an amino acid sequence similar to that of the antigen may be present in the tissue. The results obtained with immunocytochemistry must therefore be interpreted with caution and often descriptions such as "enkephalin-like immunoreactivity", "enkephalin immunoreactive material", etc., are used.
A ntisera
The bases for successful immunocytochemistry are "powerful" antisera, i . e . antisera with a high titre and, perhaps even more important, a high affinity (avidity) (see Sternberger 1974). Hardly any manipulations with the various steps in the staining procedure can compensate for an inferior quality of the antisera. This is important to realize, since, at least in our experience, the production of antisera is a hazardous enterprise with a yield of varying qualities. It is our experience that perhaps one o r two out of ten rabbits
76
AGNETA WAHLSTROM
Interpretative problems in binding studies
Applications of receptor binding analysis for flrr morphine receptor One application is the possibility of using the radioreceptor assay as a n adjunct to pharmacokinetic analysis. In table I it is obvious that etorphine, fentanyl and meperidine have the receptor affinity correlated to the analgesic activity. All of them are lipid soluble. Morphine, on the other hand, is water soluble, it conjugates fast and is excreted quickly. It does not enter the blood-brain barrier to the same extent a s the others. However, it shows about the same receptor affinity as fentanyl, and only if the receptor affinity data are compared with activity data after intraventricular injections the correlation is good. In pharmacokinetic analysis it is also of interest to study the precursor-active drug relationships. An example taken from the opiate field is heroin which only has a low affinity for opiate receptors (Terenius unpublished). Presumably it has to be deacylated to morphine before it becomes pharmacologically active.
A drug receptor is characterized by its high structural specificity. Critical points can arise when the binding of the radioindicator is characterized indirectly by the use of various drugs as competitors. An example is the studies of @-adrenergic receptors. The first investigators to approach them found that tritium-labelled catecholamines bound to whole cells o r microsomes from liver, heart and fat cells, with some specificity. But this interaction did not fulfil a number of criteria discussed above expected for the receptor (Cuatrecasas et d . , 1974). For instance, the binding was not functionally specific for catecholamine receptor interactions. The ligand was found to associate with a membrane catecholamine binding protein which may be related to an enzyme. This non-receptor binding curve was not a simple linear function of the ligand concentration. There are other examples, which can illustrate the problems of non-specific receptor binding. There is one example which shows that high-affinity sites may exist, although they are not related to the receptor binding. The steroids, progesterone and hydrocortisone both bind to the Isoreceptors and their study using plasma protein corticosteroid binding globulin radioindicators (CBG). Their dissociation constants are in the The word isoreceptor can be used to describe nanomolar range. Hydrocortisone has practical- chemically different receptor molecules activaly no affinity for the progestogen receptor, but ted by a group of functionally related ligands. the receptor has a dissociation constant of about The isoreceptors have specificity differences in 10-9M for progesterone (Wiest & Rao 1971). The the binding site. The classical example is the guinea-pig uterus o r human uterus have large muscarinic and the nicotinic receptors, u- and concentrations of the receptor, and such sam- P-receptors for the catecholamines and H I and ples present no significant problems if contami- H Zreceptors for histamine. Recently we outlined a general approach to nated by plasma. However, in rat uterus and breast cancer biopsies where receptor levels are study receptor heterogeneity (Terenius & very low difficulties arise. But if one adds an Wahlstrom 1976) in case a radioindicator with excess of hydrocortisone to saturate the plasma absolute specificity is not available. Therefore it protein binding site, it opens a possibility to is necessary to use several radioindicators and study the progestogen receptor (Terenius 1972). compare the effects of various competitors agaThere are also another way of solving the pro- inst each ofthem. The comparison must be done blem using a progestogen which does not bind to under identical conditions. We have designed a the plasma proteins as the radioindicator (Phili- set-up which allows us to do such an analysis. bert & Raynaud 1973). These examples show To avoid the introduced indicator error which is the caution which is necessary in interpreting a common to all titrations, the indicator solutions receptor role in a certain binding process. are identical pair-wise.
77
RECEPTOR BINDING ANALYSIS
+
>
Solution I [X*] [Y] Solution I1 [XI + [ y * j
a
0
the star signifying the radiolabelled agent, where
[X*l [Y*l
= =
+ cr
[XI [YI
The solutions are therefore identical and the receptor occupancy by the ligands will be identical, as well as the non-receptor binding. This system can be extended to three o r more radioindicators and competing solutions. Experiments with the different solutions are run in parallel under identical conditions and the competitors have the same serial dilution for each radioindicator. If the two radioindicators bind to the receptor in an identical way, the two competitors should give the same displacement curve. If the two indicators occupy different receptor be popu~ationsa difference in selectivity
depending On whether Or is used (Fig. 2 ) . In this type Of graph a competitor which displaces the two indicators Observed
as the
a a 0 CT L
k 0 u
U
0
s yo
OF CONTROL RADIOINDICATOR X
Fig. 2 Selectivity analysis graph. Competitors showing selectivity for sites preferentially occupied by one of the radioindicators may follow the curved lines. Competitors which show no selectivity for the sites labelled by the two radioindicators follow the straight line.
Table t
Relative receptor affinity and analgesic activity of narcotic analgesics
Substance
Analgesic activity Intraventricular Intravenous injection injection
Relative receptor affinity
Etorphine Fentanyl Meperidine Morphine
100 6 0.03 4
100 10 0.03 2
100 7
0.02 0.02
from Terenius, L., J. Pharrn. Pharmacol., 1974 b, 26, 146-148
Table I I
Receptor selectivity of opioid alkaloids and endogenous peptides Radioindicator Opioid agonists ( M ) Opioid antagonists (N) Opioid peptides (E)
* read vertically
Relative affinity*
Principle site occupied
N N
E
M
n
e
N
rn
n
E
E
M
78
AGNETA WAHLSTROM
equally will give a straight line, while a competitor with selectivity for one of the receptors gives a curved line. This analysis has been applied to the opiate receptors and evidence is found for a considerable heterogeneity. The opioid agonists and antagonists occupy different sites (Terenius & Wahlstrom 1976). Alkaloid antagonists show no selectivity, while the opioid agonists show very strong selectivity for the agonist-radioindicator. If the endogenous ligand Met-enkephalin is used as radioindicator one can find evidence that it interacts with a selective peptide receptor population. This is summarized in table I1 (Terenius 1977). It should be emphasized that this type of analysis cannot distinguish between cases where there are actually more than one receptor molecule o r where there are different modes of interaction for a single receptor molecule. In the case of opioids, there is evidence for the existence of different receptor molecules (Lord et d., 1977; Wahlstrom et al., 1976). This type of binding analysis might be very important when new drugs are under development, since selectivity of action always will be a goal in medicinal chemistry.
References Buller, R.E., W.T. Schrader& B.W. O’Malley: Steroids and the practical aspects of performing binding studies. J . Steroid. Biochem. 1976,7,321-326.
Changeux, J.P.: The cholinergic receptor protein from fish electric organ. In: Handbook of Psychophnrrnacology. Eds.: L.L. Iversen, S.D. lversen & S . H . Snyder. Plenum Press, New York and London 1975, 6,235-301. Cuatrecasas, P., G.P.E. Tell, V . Sica, J. Parikh & K.-J. Chang: Noradrenaline binding and the search for catecholamine receptors. Nature 1974, 247, 92-96. Levitzki, A.: The quantitative aspects of drug receptor interactions. J . Mol. Biol. 1975,97, 46-53. Lord, J.A.H., A.A. Waterfield, J. Hughes & H.W. Kosterlitz: Endogenous opioid peptides: multiple agonists and receptors. Nature 1977,267,495-499. Philibert, D. & J.-P. Raynaud: Progesterone binding in the immature mouse and rat uterus. Steroids 1973, 22, 89-98. Terenius, L.: A rapid assay of affinity for the narcotic receptor in rat brain: Application to methadone analogues. Acta pharmacol. et toxicol. 1974 a, 34, 88-91. Terenius, L.: Contribution of “receptor” affinity to analgesic potency. J . Pharm. Pharmacol. 1974 b, 26, 146-148. Terenius, L.: Opioid peptides and opiates differ in receptor selectivity. Psychoneuroendocrinc,loRy 1977,2,53-58. Terenius, L. & A. Wahlstrom: A method for site selectivity analysis applied to opiate receptors. Eirr. J . Pharmacol. 1976,40,241-248. Wahlstrom, A., M . Brandt, L. Moroder, E . Wiinsch, G. Lindeberg, U. Ragnarsson, L. Terenius & B. Hamprecht: Peptides related to 0-lipotropin with opioid activity. Effects on levels of adenosin 3’,5’-cyclic monophosphate in neuroblastoma x glioma hybrid cells. FEBS Lett. 1977, 77, 28-32. Wiest, W.G. & B.R. Rao: ProgFsterone binding proteins in rabbit uterus and human endometrium. In: Advances in Biosciences. Ed.: G . Raspe. Pergamon Press, Oxford 1971,7,251-266.
From the Department of Pharmacology, University of Uppsala, Box 573,751 23 Uppsala, Sweden
Methodological Aspects on Drug Receptor Binding Analysis BY Agneta Wahtstrom
Abstract: Although drug receptors occur in relatively low concentrations, they can be visualized by the use of appropriate radioindicators. In most cases the procedure is rapid and can reach a high degree of
accuracy. Specificity of the interaction is studied by competition analysis. The necessity of using several radioindicators to define a receptor population is emphasized. It may be possible to define isoreceptors and drugs with selectivity for one isoreceptor. Key-words: Drug receptor
-
receptor binding.
Some criteria must be settled before starting the discussion of receptor binding. The classical definition of a receptor involves a functional response. When the biochemical techniques have been applied to receptors, it is very often the receptor molecule which has been discussed, and the physiologic system as a whole has been left out. In this paper the relation between biologic potency and binding properties will be stressed. The main criterion for the receptor will be the specificity of the observed interaction in both functional and structural terms. There are also other criteria which help us to define a specific receptor, such as a) the receptor is situated only in the target tissue and is localized to a specific part of the target cell; b) there are agonists which bind to the receptor as well as competitive antagonists, but inactive substances d o not. In principle there are two different types of receptors, soluble receptors and membranebound receptors. The criteria discussed above should be fulfilled in both cases.
The primary binding process between the receptor and the ligand is probably similar although the receptor complex exerts its biologic effect according to two different principles. The activated membrane receptors induce the effect via cyclic nucleotides, while cytoplasmic receptors enter the cell nucleus where they interact with chromatin and affect the genome.
Radioindicators in receptor bitzding The receptor density is comparatively low for most receptors. In many cases it is well below the dissociation constant for the ligand in question. There are some guidelines for choosing the best properties of a labelled radioindicator when using it in a radioreceptor assay since the highest possible sensitivity is required. The labelled radioindicator ought to have a) a high specific radioactivity; b) reasonable receptor occupation compared to the concentration in the incubation mixture: c) as low non-receptor binding as possible.
80
T. HOKFELT ET AL
On the Methodology Hi5 tochemistry
The principles and technical outlines for the application of immunological principles in histochemistry were described more than 30 years ago by Coons and collaborators (see Coons 1958; see also Nairn 1969). It was possible to combine the specificity and sensitivity of the antigen-antibody reaction with the high resolution of the microscopic techniques. Two principles were devised, the direct and the indirect method. The direct technique involves labelling of the specific antibody with a fluorescent dye, e . g . fluorescein isothiocyanate (FITC), whereas in the indirect technique a secondary antibody is labelled. The latter modification appears t o be widely used by virtue of its higher sensitivity and the possibility of using one and the same labelled (secondary) antibody for several primary (specific) antisera. More recently, modifications of the Coons technique have been introduced. Horseradish peroxidase may be used as label, offering the advantage of analysing the tissue both in an ordinary light microscope and in the electron microscope (see Nakane 1971). The highly sensitive unlabelled antibody enzyme method of Sternberger and collaborators (see Sternberger 1974) has emerged as the method of choice in many laboratories for immunocytochemical studies both at the light microscopic and at the ultrastructural level. The routine procedure used in our laboratory for localizing peptides - and enzymes - at the light microscopic level is based on the indirect fluorescence technique of Coons and collaborators (see Coons 1958) and is as follows. The experimental animals are perfused via the vascular system (ascending aorta) with ice cold formalin (Pease 1962). After 30 min the tissues are dissected out, immersed in the same fixative for several hours, rinsed in phosphate buffer, cut on a cryostat (Dittes, Heidelberg, W . - Germany) and processed for the indirect immunofluorescence technique. The sections are incubated with the specific antiserum in a humid atmosphere at + 3 7 T for 30 min with the antisera diluted 1: 10- 1: 100 depending o n the characteristics of each antiserum, especially its affinity and tit-
er. Incubation may also b e carried out at a low temperature (+4"C) for a longer time (12-24 hrs), mostly allowing a considerably higher dilution of the antiserum. This procedure may result in less unspecific binding and in addition saves precious antiserum. After the first incubation the sections are rinsed and incubated with FITC conjugated sheep anti-rabbit antibodies (commercially available from Statens Bakteriologiska Laboratorium, SBL, Stockholm, Sweden) in a humid atmosphere at +37"C for 30 min, rinsed, mounted and examined in a fluorescence microscope. It is common to add a detergent such as Triton X-100 (Hartman et al. 1972) to the antisera with the aim of "solubilizing" membranes and enhancing the penetration of the antibodies into the sections. In spite of many advantageous characteristics of the immunocytochemical techniques there are several problems involved. Apart from technical difficulties such as poor penetration of the antibodies into the tissue, insufficient sensitivity and unspecific staining, the crucial and seemingly unsolvable problem is the possible occurrence of cross-reacting peptides. Although the antisera can be characterized with regard to cross-reactivity with known peptides, unknown peptides with an amino acid sequence similar to that of the antigen may be present in the tissue. The results obtained with immunocytochemistry must therefore be interpreted with caution and often descriptions such as "enkephalin-like immunoreactivity", "enkephalin immunoreactive material", etc., are used.
A ntisera
The bases for successful immunocytochemistry are "powerful" antisera, i . e . antisera with a high titre and, perhaps even more important, a high affinity (avidity) (see Sternberger 1974). Hardly any manipulations with the various steps in the staining procedure can compensate for an inferior quality of the antisera. This is important to realize, since, at least in our experience, the production of antisera is a hazardous enterprise with a yield of varying qualities. It is our experience that perhaps one o r two out of ten rabbits
76
AGNETA WAHLSTROM
Interpretative problems in binding studies
Applications of receptor binding analysis for flrr morphine receptor One application is the possibility of using the radioreceptor assay as a n adjunct to pharmacokinetic analysis. In table I it is obvious that etorphine, fentanyl and meperidine have the receptor affinity correlated to the analgesic activity. All of them are lipid soluble. Morphine, on the other hand, is water soluble, it conjugates fast and is excreted quickly. It does not enter the blood-brain barrier to the same extent a s the others. However, it shows about the same receptor affinity as fentanyl, and only if the receptor affinity data are compared with activity data after intraventricular injections the correlation is good. In pharmacokinetic analysis it is also of interest to study the precursor-active drug relationships. An example taken from the opiate field is heroin which only has a low affinity for opiate receptors (Terenius unpublished). Presumably it has to be deacylated to morphine before it becomes pharmacologically active.
A drug receptor is characterized by its high structural specificity. Critical points can arise when the binding of the radioindicator is characterized indirectly by the use of various drugs as competitors. An example is the studies of @-adrenergic receptors. The first investigators to approach them found that tritium-labelled catecholamines bound to whole cells o r microsomes from liver, heart and fat cells, with some specificity. But this interaction did not fulfil a number of criteria discussed above expected for the receptor (Cuatrecasas et d . , 1974). For instance, the binding was not functionally specific for catecholamine receptor interactions. The ligand was found to associate with a membrane catecholamine binding protein which may be related to an enzyme. This non-receptor binding curve was not a simple linear function of the ligand concentration. There are other examples, which can illustrate the problems of non-specific receptor binding. There is one example which shows that high-affinity sites may exist, although they are not related to the receptor binding. The steroids, progesterone and hydrocortisone both bind to the Isoreceptors and their study using plasma protein corticosteroid binding globulin radioindicators (CBG). Their dissociation constants are in the The word isoreceptor can be used to describe nanomolar range. Hydrocortisone has practical- chemically different receptor molecules activaly no affinity for the progestogen receptor, but ted by a group of functionally related ligands. the receptor has a dissociation constant of about The isoreceptors have specificity differences in 10-9M for progesterone (Wiest & Rao 1971). The the binding site. The classical example is the guinea-pig uterus o r human uterus have large muscarinic and the nicotinic receptors, u- and concentrations of the receptor, and such sam- P-receptors for the catecholamines and H I and ples present no significant problems if contami- H Zreceptors for histamine. Recently we outlined a general approach to nated by plasma. However, in rat uterus and breast cancer biopsies where receptor levels are study receptor heterogeneity (Terenius & very low difficulties arise. But if one adds an Wahlstrom 1976) in case a radioindicator with excess of hydrocortisone to saturate the plasma absolute specificity is not available. Therefore it protein binding site, it opens a possibility to is necessary to use several radioindicators and study the progestogen receptor (Terenius 1972). compare the effects of various competitors agaThere are also another way of solving the pro- inst each ofthem. The comparison must be done blem using a progestogen which does not bind to under identical conditions. We have designed a the plasma proteins as the radioindicator (Phili- set-up which allows us to do such an analysis. bert & Raynaud 1973). These examples show To avoid the introduced indicator error which is the caution which is necessary in interpreting a common to all titrations, the indicator solutions receptor role in a certain binding process. are identical pair-wise.
77
RECEPTOR BINDING ANALYSIS
+
>
Solution I [X*] [Y] Solution I1 [XI + [ y * j
a
0
the star signifying the radiolabelled agent, where
[X*l [Y*l
= =
+ cr
[XI [YI
The solutions are therefore identical and the receptor occupancy by the ligands will be identical, as well as the non-receptor binding. This system can be extended to three o r more radioindicators and competing solutions. Experiments with the different solutions are run in parallel under identical conditions and the competitors have the same serial dilution for each radioindicator. If the two radioindicators bind to the receptor in an identical way, the two competitors should give the same displacement curve. If the two indicators occupy different receptor be popu~ationsa difference in selectivity
depending On whether Or is used (Fig. 2 ) . In this type Of graph a competitor which displaces the two indicators Observed
as the
a a 0 CT L
k 0 u
U
0
s yo
OF CONTROL RADIOINDICATOR X
Fig. 2 Selectivity analysis graph. Competitors showing selectivity for sites preferentially occupied by one of the radioindicators may follow the curved lines. Competitors which show no selectivity for the sites labelled by the two radioindicators follow the straight line.
Table t
Relative receptor affinity and analgesic activity of narcotic analgesics
Substance
Analgesic activity Intraventricular Intravenous injection injection
Relative receptor affinity
Etorphine Fentanyl Meperidine Morphine
100 6 0.03 4
100 10 0.03 2
100 7
0.02 0.02
from Terenius, L., J. Pharrn. Pharmacol., 1974 b, 26, 146-148
Table I I
Receptor selectivity of opioid alkaloids and endogenous peptides Radioindicator Opioid agonists ( M ) Opioid antagonists (N) Opioid peptides (E)
* read vertically
Relative affinity*
Principle site occupied
N N
E
M
n
e
N
rn
n
E
E
M
78
AGNETA WAHLSTROM
equally will give a straight line, while a competitor with selectivity for one of the receptors gives a curved line. This analysis has been applied to the opiate receptors and evidence is found for a considerable heterogeneity. The opioid agonists and antagonists occupy different sites (Terenius & Wahlstrom 1976). Alkaloid antagonists show no selectivity, while the opioid agonists show very strong selectivity for the agonist-radioindicator. If the endogenous ligand Met-enkephalin is used as radioindicator one can find evidence that it interacts with a selective peptide receptor population. This is summarized in table I1 (Terenius 1977). It should be emphasized that this type of analysis cannot distinguish between cases where there are actually more than one receptor molecule o r where there are different modes of interaction for a single receptor molecule. In the case of opioids, there is evidence for the existence of different receptor molecules (Lord et d., 1977; Wahlstrom et al., 1976). This type of binding analysis might be very important when new drugs are under development, since selectivity of action always will be a goal in medicinal chemistry.
References Buller, R.E., W.T. Schrader& B.W. O’Malley: Steroids and the practical aspects of performing binding studies. J . Steroid. Biochem. 1976,7,321-326.
Changeux, J.P.: The cholinergic receptor protein from fish electric organ. In: Handbook of Psychophnrrnacology. Eds.: L.L. Iversen, S.D. lversen & S . H . Snyder. Plenum Press, New York and London 1975, 6,235-301. Cuatrecasas, P., G.P.E. Tell, V . Sica, J. Parikh & K.-J. Chang: Noradrenaline binding and the search for catecholamine receptors. Nature 1974, 247, 92-96. Levitzki, A.: The quantitative aspects of drug receptor interactions. J . Mol. Biol. 1975,97, 46-53. Lord, J.A.H., A.A. Waterfield, J. Hughes & H.W. Kosterlitz: Endogenous opioid peptides: multiple agonists and receptors. Nature 1977,267,495-499. Philibert, D. & J.-P. Raynaud: Progesterone binding in the immature mouse and rat uterus. Steroids 1973, 22, 89-98. Terenius, L.: A rapid assay of affinity for the narcotic receptor in rat brain: Application to methadone analogues. Acta pharmacol. et toxicol. 1974 a, 34, 88-91. Terenius, L.: Contribution of “receptor” affinity to analgesic potency. J . Pharm. Pharmacol. 1974 b, 26, 146-148. Terenius, L.: Opioid peptides and opiates differ in receptor selectivity. Psychoneuroendocrinc,loRy 1977,2,53-58. Terenius, L. & A. Wahlstrom: A method for site selectivity analysis applied to opiate receptors. Eirr. J . Pharmacol. 1976,40,241-248. Wahlstrom, A., M . Brandt, L. Moroder, E . Wiinsch, G. Lindeberg, U. Ragnarsson, L. Terenius & B. Hamprecht: Peptides related to 0-lipotropin with opioid activity. Effects on levels of adenosin 3’,5’-cyclic monophosphate in neuroblastoma x glioma hybrid cells. FEBS Lett. 1977, 77, 28-32. Wiest, W.G. & B.R. Rao: ProgFsterone binding proteins in rabbit uterus and human endometrium. In: Advances in Biosciences. Ed.: G . Raspe. Pergamon Press, Oxford 1971,7,251-266.
