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Strategies in neuropeptide receptor binding research
Neuroscience &BiobehavioralReviews, Vol. 9, pp. 413-420, 1985. ©AnkhoInternational Inc. Printed in the U.S.A.
0149-7634/85$3.00 + .00
Strategies in Neuropeptide Receptor Binding Research RI~MI Q U I R I O N * A N D P I E R R E T T E G A U D R E A U - t
*Douglas Hospital Research Centre, 6875 LaSalle Blvd., Verdun, Quebec Canada H4H 1R3 tLaboratory o f Neuroendocrinology, HOpital Notre-Dame, Montreal, Quebec Canada R e c e i v e d 10 D e c e m b e r 1984 QUIRION, R. AND P. GAUDREAU. Strategies in neuropeptide receptor binding research. NEUROSCI BIOBEHAV REV 9(3) 413-420, 1985.--Strategies and general approaches used in neuropeptide receptor binding assays are described. Special attention is given to the nature of the ligand, its physical and chemical stability and the demonstration of an appropriate ligand selectivity pattern. Examples are given to illustrate critical aspects of neuropeptide receptor binding assays. Strong correlation between binding and bioassay data is also stressed. Peptides
Receptor
Binding assays
Enzyme degradation
Bioassays
insure proper analysis of data. In the following sections, strategies used to characterize peptide receptor binding sites will be covered.
IN the past two decades, receptor binding assays have become highly popular. They are currently used not only in pharmacology and biochemistry but als0 in cell biology, psychology, physiology and clinical sciences. In fact, receptor binding techniques are seen as a must in modern laboratories dealing with drug mechanisms of action. The characterization of opioid receptors [70, 98, 107] and subsequent discovery of enkephalins [35] greatly stimulated research on neuropeptides and their respective receptor sub-types. As for drugs and "classical" neurotransmitters, neuropeptide receptor binding assays have to meet various criteria. First, binding sites should be saturable and of high affinity (Kd, low nanomolar range) and low capacity (Bmax, fmol/mg protein). Generally, there is a very limited number of receptors on most tissue membranes. Thus, it should be possible to readily saturate the number of displaceable binding sites. Saturation is indicated when the amount of displaceable binding becomes constant as a function of ligand concentrations. These sites must also possess a ligand selectivity pattern similar to those reported for various bioassays, electrophysiological and/or behavioral assays. It is certainly the most important criterium to fulfill before suggesting that binding sites under study are relevant to active in vivo receptor sites [74,80]. If it is not possible to correlate the structure-activity results from bioassays with receptor binding data, careful controls (e.g., binding to filters, sequestration sites) must be done to exclude artifacts. Even then, hypothesis on the existence of yet another class of receptors must be put forward with a great deal of caution. A second important aspect of receptor binding research concerns the nature of the probes. The ligand must be of high specific activity (>15 Ci/mmol, if tritium) and possesses high affinity for binding sites. Preferably, it should be chemically stable and resistant to enzymatic degradation. This last criterium is met by various ligands (e.g., opiates, benzodiazepines, antidepressants) but not by most radiolabelled peptides. Thus, neuropeptide receptor binding research presents specific problems that must be resolved to
Nature of the Isotope Almost all neuropeptide binding assays are performed with ligands labelled with either tritium (3H) or radioactive iodine (1~I). The choice of either one is mainly dependent on the peptide involved. Since the density of binding sites for most peptides is quite low (fmol/mg protein) an iodinated ligand is likely to give better results because of its high specific activity (up to 2000 Ci/mmol). If the peptide contains tyrosine or histidine residues, it is possible to label it by direct iodination using various approaches such as chloramine T. However, it is not always possible to use such iodinated peptide for receptor binding study; particularly when binding capacity and/or biological activity depend upon an intact phenolic ring [16, 23, 77, 78, 84-86, 87, 104]. In addition to the relatively large size of the iodine which can significantly alter the binding properties of the molecule, other chemical alterations of the peptides, such as, oxidations of methionyl and trypthophyl residues may happen when a direct iodination method is used (e.g., chloramine T). A much milder approach consists in the use of the Bolton Hunter method [7] in which the 125I-Nhydroxysuccinimidyl 3-(4-hydroxyphenyl) proprionate acylates specifically N ~ and N • amino groups. The advantage of this method is that any peptides with a free N-terminal amino group can be labelled. However, if N • amino groups are important for the activity of the molecule, temporary protection of this function must be considered during the BoltonHunter reaction. In general, tritium-labelled compounds, prepared by either exchange reaction or chemical synthesis, are used by a large number of scientists since they are commercially available. Moreover, the half-life of 3H being much longer than ~25I, it is sometimes preferable to use a 3H-labelled ligand which insures long-term availability.
413
QUIRION AND GAUDREAU
414
TABLE l APPROACHESUSED TO DECREASELIGANDBINDINGTO FILTERSIN VARIOUSASSAYS Ligand [3H]Substance P [3H]Dynorphin (1-17) [3H]Dynorpbin (1-9) [3H]Dynorphin (1-8) [3H]D-Ala2, D-Leu'~enkephalin [~H]Neurotensin [125I]Neurotensin [125I]Angiotensin [~I]Somatostatin [:~H]Bradykinin
Treatment 0.1% polyethyleneimine 0.4% BSA* plus 0.1% polylysine 1.0% polyethyleneimine 0.1% polyethyleneimine t-amyl alcohol saturated water Incubation buffer 2% BSA Incubation buffer 1% BSA 0.1% polyethyleneimine
Reference 8,42,65,76 116 89 75 45 38,39 41 108 1 50
*BSA=bovine serum albumin.
High Purity of the Ligand The highest purity (>99%) of the ligand should be sought. To insure that the radiolabelled ligand is pure, a high pressure liquid chromatography (HPLC) analysis should be done prior to the binding assay. An impure radiolabelled peptide could generate artifacts. First, biphasic Scatchard plots can be obtained suggesting the existence of multiple classes of sites when, in fact, only one type of site is present. Impure tracers generally give lower affinity and number of sites values than in reality. Curvilinear Scatchard plots are likely to be due to the increasing proportion of denatured ligand (or other impurities) present in the incubation medium when increasing concentrations of the tracer. Second, Hill coefficients smaller than unity that can be indicative of complex interaction between the ligands and its binding sites (negative and positive cooperativity) may only be due to impure ligands. Finally, poor specific to non-specific binding ratio will certainly be observed. Thus, great care should be taken to insure high purity of the labelled ligand.
Physico-Chemical Properties of the Ligand Most peptides are positively or negatively charged at physiological pH. Consequently, they have a tendency to "bind" to inert materials such as glass, plastic, etc. Many of them are also highly hydrophobic and are likely to be sequestrated into lipids of membrane preparations. To minimize problems such as binding to glass and/or plastic tubes, it is important to coat these materials with either serum albumin, polylysine, bacitracin or silicone. It has been shown that such coating markedly reduced the absorption of substance P [103], opioid peptides [44] and calcitonin [57] to tubes and syringes. It is believed that positive charges present on these molecules neutralize the negative charges of the silica which in turn, markedly decrease the adhesion of positively charged peptides to glass. Another major problem encountered in receptor assays is related to binding of peptides to the filters used in the rapid filtration technique. Binding of labelled ligands to filters should always be tested in a first series of experiments. If it is found that an important fraction (more than 20-25%) of the ligand is bound to filters, various approaches should be used to resolve this problem. As a first step, various types of filters could be tested (GF/B, GF/C, GF/F, Millipore, etc.) to choose those that appear to be the most appropriate. Then, soaking the filters in serum albumin, polylysine or polyethyleneimine solutions usually markedly decreased ligand bind-
ing to filters (Table 1). However, if this approach is not satisfactory, control experiments (no membranes) should be run in all assays and binding values substracted from those obtained in presence of membrane preparations. Centrifugation assays should also be considered if filter binding remains important after coating. This technique is particularly useful if the dissociation rate of the tracer from its binding sites is extremely rapid (less than few seconds). With this technique. it is possible to stop the reaction in 2-3 seconds. However, the non-specific binding is generally greater in the centrifugation assay due to the trapping of tracer in the pellet. Similar approaches could be used for in vitro section Omding techniques [34, 79, 117]. For example, if ligand binds to gelatin-coated slides, it is possible to add polyethyleneimine to the gelatin solution or to the pre-incubation buffer. It generally markedly reduces binding to coated slides.
Chemical and Biological Stability of the Ligand In receptor binding assays of non-peptide drugs and neurotransmitters, the chemical and biological stability of the ligand is generally not a major problem. However, it is probably the most critical aspect of any peptide binding assays. Many neuropeptides must be protected against oxidation that will likely reduce their binding ability. Such peptides (e.g., cholecystokinin) usually contain methionine and tryptophan in their sequence. Protection against oxidation is achieved by storing and incubating the radiolabelled ligand in a mixture containing either dihydrothreitol or/3-mercaptoethanol as anti-oxydants [24, 25, 103, 120]. Care must also be taken during manipulation of stocks of ligand. They should be divided in small aliquots, flushed under nitrogen or argon and subsequently stored at -80°C. Under these conditions, we have been able to keep stocks of [3HI substance P and [3H] cholecystokinin for many weeks without major loss of activity [24, 25, 83, 97]. Another problem encountered in peptide binding assays is the marked sensitivity of certain ligands to enzymatic degradation. It is possible to verify the integrity of the ligand after tissue incubation by analytical HPLC and/or bioassays. For example, it has been shown that without protection, substance P [42] or dynorphin (1-9) [89] are very rapidly degraded by membrane preparations. Lower incubation temperatures and mixture of enzyme inhibitors are usually used to reduce enzyme activity in membrane preparations. Table 2 described some "cocktails" of inhibitors used in various neuropeptide binding assays. The most widely used enzyme inhibitor is certainly bacitracin, a non-selective protease in-
NEUROPEPTIDE RECEPTOR BINDING
415
TABLE 2 MIXTURE OF PROTEASE INH1BITORS AND ANTIOXYDANTS USED IN VARIOUS NEUROPEPTIDE RECEPTORBINDINGASSAYS Peptide ACTH Angiotensin
Bombesin Bradykinin
Calcitonin Cholecystokinin
Eledoisin Glucagon GnRH analogues
Insulin y-MSH Neurotensin Opioid peptides Dynorphin (1-17) Dynorphin (1-9) Dynorphin (1-8) ]3-endorphin Enkephalin derivatives Secretin Substance P
Somatostatin
TRH Vasopressin
VIP
Protease Inhibitors and Antioxydants Bacitracin PMSF and DTT Bacitracin, PMSF and aprotinin Bacitracin Bacitracin Bacitracin, DTT and captopril None Bacitracin None Bacitracin Bacitracin and DTT Bacitracin None None Bacitracin DTT None Bacitracin Lima bean trypsin inhibitor None Bacitracin None Bestatin, captopril and dipeptides Bacitracin, chymostatin and leupeptin Bacitracin None Bacitracin Bacitracin PMSF and aprotinin None Bacitracin Chymostatin Bacitracin, PMSF and chloromercuribenzoate Bacitracin, chymostatin leupeptin None Bacitracin Bacitracin and ethylmercurithiosalicylate Bacitracin, aprotinin and PMSF None Bacitracin None Bacitracin Bacitracin and aprotinin Leupeptin, pepstatin and benzamidine Bacitracin Bacitracin and aprotinin
References 9 5,14,26,31,99 55 56,59,69,113 92 37,50 21,52 57,63,88 73 33,93,94 24,25,36,120 3 6,90,102 12,51,54 13,43,68 72 32 119 67 11,38,39,41,53,110 27,82,118 116 89 75 28,47 10,15,18,40,45,62,114 2,81 22 61 58 4,109,112 46 30 8,42,65,76,83,97 91 17,105 1
100,101 64 60,96 29,48,95 111,115 19 66 20 106
Abbreviations used: ACTH, adrenocorticotropic hormone; DTT, dithiothreitol; GIP, gastric inhibitory peptide: GnRH, gonadotropin releasing hormone; y-MSH, y-melanocyte stimulating hormone; PMSF, phenylmethylsulfonylfluoride; TRH, thyrotropin-releasing hormone and VIP, vasoactive intestinal polypeptide.
416
QUIRION AND GAUDREAU TABLE 3 RELATIVE POTENCIES OF SUBSTANCE P (SP), ITS FRAGMENTS AND HOMOLOGUES 1N VARIOUS ASSAYS Binding Assay* Peptide
[:3H]SP
Substance P Substance P (3-11) Substance P (5-11) Substance P (7-11) Substance P---COOH Physalaemin Eledoisin Kassinin
Bioassay$
['e~I]SP
['25I]Physalaemin
MCCt
DCA
RA
100 7.6 1.9 0.01 0.01 14 2.6 1.3
100 -0.6 0.01 0.005 125 1.0 --
100 16.8 3.3 0.01 inactive 18 0.9 --
100 -20 0.01 -100 33 15
100 70 30 --100 40 18
100 3.1 0.3 0.02 0.01 16 0.8 0.07
*[aH]SP, [225I]SP and ['25I]Physalaemin binding data are from [76,83], [97] and 1112] respectively, tMCC means mouse mesencephalic cell culture. Data are from [4]. SDCA and RA means dog carotid artery and rabbit aorta, respectively. Data are derived from [86].
TABLE 4 RELATIVE POTENCIES OF NEUROTENSIN (NT), ITS FRAGMENTS AND ANALOGUES IN VARIOUS ASSAYS Binding Assay Peptide Neurotensin Neurotensin (8-13) Neurotensin (%13) [D-Argg]-NT [Trp"]-NT [Phelq-NT Xenopsin
Bioassay$
[ZH]NT*
['z'~I][Trp' qNT
NT 29~
RSS
RPV
GPI
100 39 8 0.5 91 20 96
100 -1 -100 8 .
100 120 1.3 0.24 -4
100 69 11 0.65 130 16
100 94 2.4 0.8 115 9.4
100 20 3 4 14 33 55
.
.
.
*[3H]NT and ['2'~I][Trp"]NT binding data are from [82] and [53] respectively, t i l T 29 is a cell line derived from human colon. Data drived from [39]. SRSS, RPV and GPI means rat stomach strip, rat portal vein and guinea pig illeum. Data derived from [77, 78, 87], [11] and [39] respectively.
hibitor. H o w e v e r , in m a n y cases, it is not enough and more selective agents such as chymostatin (serine protease blocker), leupeptin (leucine-aminopeptidase blocker), captopril (angiotensin converting e n z y m e inhibitor) and thiorphan (enkephalinase inhibitor) must be used (Table 2). Substance P is a good e x a m p l e illustrating the importance of the use of an appropriate mixture of e n z y m e inhibitors in o r d e r to insure stability of the radiolabelled ligand [42,83]. L e e et al. [42] have shown that in the p r e s e n c e o f bacitracin, chymostatin and leupeptin, substance P degradation by salivary gland m e m b r a n e s was almost fully inhibited. H o w ever, they have also reported that thiolpeptidase inhibitors such as N-ethylmaleimide and p-chloromercuriphenylsulfate markedly inhibit substance P binding. The same phenome n o n is also often o b s e r v e d with bacitracin if used in high concentrations. Thus, certain protease inhibitors are able to protect the ligand against e n z y m a t i c degradation but at the same time inhibit its binding to r e c e p t o r binding sites. This possibility should be kept in mind while trying to design the m o s t appropriate mixture of protease inhibitors.
Dynorphin is also highly sensitive to degradation. Recently, R o b s o n et al. [89] have shown that a mixture of bestatin, captopril and dipeptides such as leucyl-leucine and arginyl-arginine must be used to protect [aH] dynorphin (1-9) against e n z y m a t i c degradation. Thus, each ligand appears to be differentially sensitive to proteolytic degradation and the most appropriate mixture of e n z y m e inhibitors must be determined e a c h time. Finally, the availability of radiolabelled peptide analogues containing D-amino acids at cleavage sites usually markedly increase their resistance to e n z y m e degradation and facilitate the characterization of r e c e p t o r binding sites [1, 18, 71].
Ligand Selectivity Pattern After the elaboration o f appropriate handling and incubation conditions that insure the stability o f peptide ligands, it is possible to obtain data on the pharmacological nature (affinity, n u m b e r of sites) of the r e c e p t o r binding sites. H o w e v e r , the most important criterium to fulfill the claim that
NEUROPEPTIDE RECEPTOR BINDING
417
binding sites are relevant to the r e c e p t o r u n d e r study is the d e m o n s t r a t i o n o f an appropriate ligand selectivity pattern [74,80]. If the same r e c e p t o r is present in various systems, the structure-activity relationship should be similar in binding assays and various bioassays such as in vitro isolated preparations, and electrophysiological or behavioral tests. E x a m p l e s of these types of correlation are reported in Table 3 and Table 4. It has been d e m o n s t r a t e d that three different substance P iigands s h o w similar ligand selectivity pattern in all binding assays that correlated well with bioassay data (Table 3). Similar results have been obtained for neurotensin (Table 4). This strongly suggests that such binding assay data are relevant to the p h a r m a c o l o g y o f the neuropeptide r e c e p t o r under study. Without appropriate ligand selectivity pattern, it is not possible to s h o w that specific binding sites for a given peptide are relevant to its effects. T h e s e sites could only be related to storage sites, carrier proteins or o t h e r sequestration sites present in m e m branes.
usually chemically and/or biologically unstable. H o w e v e r , under appropriate conditions, binding assays are powerful means to study possible m e c h a n i s m s of action o f neuropeptides. Such assays provide a rapid m e t h o d to screen for and isolate new e n d o g e n o u s peptides and study their structureactivity relationships. M o r e o v e r , r a d i o r e c e p t o r binding assays are also currently used in clinics to monitor neurotransmitters and drug levels in biological fluids. It is e x p e c t e d that neuropeptide r a d i o r e c e p t o r assays could also be used for clinical purposes in the near future. Finally, the availability of stable synthetic analogues of most neuropeptides should greatly facilitate the characterization o f neuropeptide receptors and eventually lead to their complete purification. Synthetic neuropeptide r e c e p t o r s could then be used to study the precise m e c h a n i s m o f interactions between ligands and binding sites. This could certainly help to design analogues with super agonist and antagonist properties that could be clinically useful. ACKNOWLEDGEMENTS
Conclusions
In s u m m a r y , neuropeptide r e c e p t o r binding studies are c h a r a c t e r i z e d principally by the nature o f the ligand which is
R6mi Quirion is a scholar of the "Fonds de la recherche en Sant6 du Quebec." The expert secretarial assistance of Mrs. Joan Currie and Mrs. Denise Robertson is acknowledged.
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QUIRION AND GAUDREAU
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NEUROPEPTIDE
RECEPTOR BINDING
66. Pearlmutter, A. F., M. G. Costantini and B. Loeser. Characterization of 3H-AVP binding sites in particulate preparations of rat brain. Peptides 4: 335-341, 1983. 67. Pedersen, R. C. and A. C. Browne. Lys-T3-melanotropin binds with high affinity to the rat adrenal cortex. Endocrinology 112: 1279-1287, 1983. 68. Perrin, M. H., J. E. Rivier and W. W. Vale. Radioligand assay for gonadotropin-releasing hormone: relative potencies of agonists and antagonists. Endocrinology 106:128%1296, 1980. 69. Pert, A., T. W. Moody, C. B. Pert, L. A. Dewald and J. Rivier. Bombesin: receptor distribution in brain and effects on nociception and locomotor activity. Brain Res 193: 209-220, 1980. 70. Pert, C. B. and S. H. Snyder. Opiate receptor: Demonstration in nervous tissue. Science 179:1011-1014, 1973. 71. Pert, C. B., A. Pert, J. K. Chang and B. T. W. Fong. [DAla~]-Met-enkephalinamide: a potent long lasting synthetic pentapeptide analgesic. Science 194: 330-332, 1976. 72. Pieper, D. R., J. S. Richards and J. C. Marshall. Ovarian gonadotropin-releasing hormone (GnRH) receptors: characterization, distribution and induction by GnRH. Endocrinology 108:1148-1155, 1981. 73. Praissman, M., P. A. Martinez, C. F. Saladino, J. M. Berkowitz, A. W. Steggles and J. A. Finkelstein. Characterization of cholecystokinin binding sites in rat cerebral cortex using a 125I-CCK-8 probe resistant to degradation. J Neurochem 40: 1406-1413, 1983. 74. Quirion, R. Bioassays in modern peptide research. Peptides 3: 223-230, 1982. 75. Quirion, R. and C. Pilapil. Opioid receptor binding profile of [3H] dynorphin A (1-8) in rat and guinea pig brain. Eur J Pharmacol 99: 361-363, 1984. 76. Quirion, R. and C. Pilapil. Comparative potencies of substance P, substance K and neuromedin K on brain 3H-substance P receptor. Neuropeptide 4: 325-330, 1984. 77. Quirion, R., D. Regoli, F. Rioux and S. St-Pierre. The stimulatory effects of neurotensin and related peptides in rat stomach strips and guinea pig atria. Br J Pharmacol 68: 83-91, 1980. 78. Quirion, R., D. Regoli, F. Rioux and S. St-Pierre. Structureactivity studies with neurotensin: analysis of position 9, 10, 11. Br J Pharmacol 69: 68%692, 1980. 79. Quirion, R., R. P. Hammer, Jr., M. Herkenham and C. B. Pert. Phencyclidine (angel dust)/ty "opiate" receptor: Its visualization by tritium-sensitive film. Proc Natl Acad Sci USA 78: 5881-5885, 1981. 80. Quirion, R., W. D. Bowen, M. Herkenham and C. B. Pert. Visualization and solubilization of rat brain opiate receptors with a " k a p p a " ligand selectivity pattern. Cell Mol Neurobiol 2: 333-345, 1982. 81. Quirion, R., J. M. Zajac, J. L. Morgat and B. P. Roques. Autoradiographic distribution of mu and delta opiate receptors in rat brain using highly selective ligands. Life Sci 33: 227-230, 1983. 82. Quirion, R., P. Gaudreau, S. St-Pierre, F. Rioux and C. B. Pert. Autoradiographic distribution of [3H] neurotensin receptors in rat brain: Visualization by tritium-sensitive film, Peptides 3: 757-763, 1982. 83. Quirion, R., C. W. Shults, T. W. Moody, C. Pert, T. N. Chase and T. L. O'Donohue. Autoradiographic distribution of substance P receptors in rat central nervous system. Nature 303: 714-716, 1983. 84. Regoli, D. and J. Barabe. Pharmacology of bradykinin and related kinins. Pharmacol Rev 32: 1-46, 1980. 85. Regoli, D., W. K. Park and F. Rioux. Pharmacology of angiotensin. Pharmacol Rev 26: 6%123, 1974. 86. Regoli, D., J. Mizrahi, P. D'OrleansoJuste and E. Escher. Receptors for substance P. II. Classification by agonist fragments and homologues. Eur J Pharmacol 97: 171-177, 1984.
419
87. Rioux, R., R. Quirion, D. Regoli, M. A. Leblanc and S. StPierre. Pharmacological characterization of neurotensin receptors in the rat portal vein using analogues and fragments of neurotensin. Eur J Pharmacol 66: 273-279, 1980. 88. Rizzo, A. J. and D. Goltzman. Calcitonin receptors in the central nervous system of the rat. Endocrinology 108: 1672-1677, 1981. 89. Robson, L. E., M. G. C. Gillan, A. T. McKnight and H. W. Kosterlitz. [3H]-Dynorphin A (1-9): binding characteristics and degradation profile in brain homogenates. Life Sci 33: 283-286, 1983. 90. Rodbell, M., H. M. J. Krans, S. L. Pohl and L. Birnbauner. The glucagon-sensitive adenyl cyclase system in plasma membranes of rat liver. III. Binding of glucagon: Method of assay and specificity. J Biol Chem 246: 1861-1871, 1971. 91. Rorstad, O. P., A. Schronbrunn and J. B. Martin. Somatostatin radioreceptor assay: development and application to the measurement of somatostatin-like activity in the rat central nervous system. Can J Physiol Pharmacol 61: 532-537, 1983. 92. Roscher, A. A., V. C. Manganiello, C. L. Jelsima and J. Moss. Receptors for bradykinin in intact cultured human fibroblasts. J Clin Invest 72: 626-635, 1983. 93. Saito, A., I. D. Goldfine and J. A. Williams. Characterization of receptors for cholecystokinin and related peptides in mouse cerebral cortex. J Neurochem 37: 483-490, 1981. 94. Saito, A., H. Sankaran, I. R. Goldfine and J. R. Williams. Cholecystokinin receptors in the brain: characterization and distribution. Science 208:1155-1156, 1980. 95. Schiffrin, E. L. and J. Genest. 3H-vasopressin binding to the rat mesemteric artery. Endocrinology 113: 40%411, 1983. 96. Sherif, N. A., N. S. Pilotte and D. R. Burt. Biochemical and autoradiographic studies of TRH receptors in sections of rabbit spinal cord. Biochem Biophys Res Comrnun 116: 66%674, 1983. 97. Shults, C. W., R. Quirion, B. Chronwall, T. N. Chase and T. L. O'Donohue. A comparison of the anatomical distribution of substance P and substance P receptors in the rat central nervous system. Peptides 5: 1097-1128, 1984. 98. Simon, E. J., J. M. Hiller and I. Edelman. Stereospecific binding of the potent narcotic analgesic [3H]etorphine to rat brain homogenate. Proc Natl Acad Sci USA 70: 1947-1949, 1973. 99. Sirett, N. E., A. S. McLean, J. J. Bray and J. I. Hubbard. Distribution of angiotensin II receptors in rat brain. Brain Res 122: 29%312, 1977. 100. Srikant, C. B. and Y. C. Patel. Receptor binding of somatostatin-28 is tissue specific. Nature 294: 25%260, 1981. 101. Srikant, C. B. and Y. C. Patel. Somatostatin receptors: identification and characterization in rat brain membranes. Proc Natl Acad Sci USA 78: 3930-3934, 1981. 102. Srikant, C. B., D. Freeman, K. McCorkle and R. H. Unger. Binding and biological activity of glucagon in liver cell membranes of chronically hyperglucagonemic rats. J Biol Chem 252: 7434-7436, 1977. 103. Stewart, J. M. Precautions in the use of substance P. In: Substance P-Dublin 1983, edited by P. Skrabanek and D. Powell. Dublin: Boole Press, 1983, pp. 4-5. 104. St-Pierre, S., R. Quirion, R. Kerouac, F. Jolicoeur and F. Rioux. Neurotensin. Peptides Proteins Rev 2: 83-171, 1984. 105. Taparel, D., J. P. Estere, C. H. Susini, N. Vaysse, D. Bales, G. Berthon, E. Wirnsch and A. Ribet. Binding of somatostatin to guinea pig pancreatic membranes: regulation by ions. Biochem Biophys Res Commun 115: 827-833, 1983. 106. Taylor, D. and C. B. Pert. Vasoactive intestinal polypeptide: specific binding to rat brain membranes. Proc Natl Acad Sci USA 76: 660-664, 1979. 107. Terenius, L. Characteristics of the receptor for narcotic analgesics in synaptic membrane fractions from rat brain. Acta Pharmacol Toxicol (Copenh) 33: 377-384, 1973.
420 108. Tonnaer, J. A. D. M., G. M. H. Engels, K. Voshart, V. M. Wiegant and W. De Jong. Binding of angiotensins to rat brain tissue: Structure activity relationship. Brain Res Bull 10: 295300, 1983. 109. Torrens, Y., J. C. Beaujouan, A. Viger and J. Glowinski. Properties of a 125Isubstance P derivative binding to synaptosomes from various brain structures and the spinal cord of the rat. Naunyn Schmeidebergs Arch Pharmacol 324: 134-139, 1983. 110. Uhl, G. R., J. P. Bennett and S. H. Snyder. Neurotensin, a central nervous system peptide: apparent receptor binding in brain membranes. Brain Res 130: 299-313, 1977. 111. Van Leeuwen, F. W. and P. Walters. Light microscopic autoradiographic localization of [3H] arginine-vasopressin binding sites in the rat brain and kidney. Neurosci Lett 41: 61-66, 1983. 112. Wolf, S. S., T. W. Moody, R. Quirion and T. L. O'Donohue. Biochemical characterization and autoradiographic localization of central substance P receptors using 1251-physalaemin. Brain Res, 332: 29%307, 1985. 113. Wolf, S. S., T. W. Moody, T. L. O'Donohue, M. A. Zarbin and M. J. Kuhar. Autoradiographic visualization of rat brain binding sites for bombesin-like peptides. Eur J Pharmacol 87: 163164, 1983.
QU1RION AND GAUDREAU 114. Wood, P. L., S. E. Charleson, D. Lane and R. L. Hudgin. Multiple opiate receptors: Differential binding of mu, kappa, and delta agonists. Neuropharmacology 20: 1215-1220, 1981. 115. Yamamura, H. 1., K. W. Gee, R. E. Brinton. T. P. Davis, M. Hadley and J. K. Wamsley. Light microscopic autoradiographic visualization of [3H]-arginine vasopressin binding siles in the rat brain. Life Sci 32: 1919-1924, 1983. 116. Young, E., J. M. Walker, R. Houghten and H. Akil. [3H]dynorphin binding to guinea pig and rat brain. Lilt" S¢i 33: 287-290, 1983. 117. Young, W. S., 111 and M. J. Kuhar. A new method for receptor in rat brain: ['~H]opioid receptors in rat brain. Brain Res 179: 255-270, 1979. 118. Young, W. S. and M. J. Kuhar. Neurotensin receptor localization by light microscopic autoradiography in rat brain. Brait~ Res 206: 273-285, 1981. 119. Young, W. S., M. J. Kuhar, J. Roth and M. J. Brownstein. Radiohistochemical localization of insulin receptors in the adult and developing rat brain. Neuropeptides h 15-22, 1980. 120. Zarbin, M. A., R. B. Innis, J. K. Wamsley, S. H. Snyder and M. J. Kuhar. Autoradiographic localization of cholecystokinin receptors in rodent brain. J Neurosci 3: 877-906, 1983.
0149-7634/85$3.00 + .00
Strategies in Neuropeptide Receptor Binding Research RI~MI Q U I R I O N * A N D P I E R R E T T E G A U D R E A U - t
*Douglas Hospital Research Centre, 6875 LaSalle Blvd., Verdun, Quebec Canada H4H 1R3 tLaboratory o f Neuroendocrinology, HOpital Notre-Dame, Montreal, Quebec Canada R e c e i v e d 10 D e c e m b e r 1984 QUIRION, R. AND P. GAUDREAU. Strategies in neuropeptide receptor binding research. NEUROSCI BIOBEHAV REV 9(3) 413-420, 1985.--Strategies and general approaches used in neuropeptide receptor binding assays are described. Special attention is given to the nature of the ligand, its physical and chemical stability and the demonstration of an appropriate ligand selectivity pattern. Examples are given to illustrate critical aspects of neuropeptide receptor binding assays. Strong correlation between binding and bioassay data is also stressed. Peptides
Receptor
Binding assays
Enzyme degradation
Bioassays
insure proper analysis of data. In the following sections, strategies used to characterize peptide receptor binding sites will be covered.
IN the past two decades, receptor binding assays have become highly popular. They are currently used not only in pharmacology and biochemistry but als0 in cell biology, psychology, physiology and clinical sciences. In fact, receptor binding techniques are seen as a must in modern laboratories dealing with drug mechanisms of action. The characterization of opioid receptors [70, 98, 107] and subsequent discovery of enkephalins [35] greatly stimulated research on neuropeptides and their respective receptor sub-types. As for drugs and "classical" neurotransmitters, neuropeptide receptor binding assays have to meet various criteria. First, binding sites should be saturable and of high affinity (Kd, low nanomolar range) and low capacity (Bmax, fmol/mg protein). Generally, there is a very limited number of receptors on most tissue membranes. Thus, it should be possible to readily saturate the number of displaceable binding sites. Saturation is indicated when the amount of displaceable binding becomes constant as a function of ligand concentrations. These sites must also possess a ligand selectivity pattern similar to those reported for various bioassays, electrophysiological and/or behavioral assays. It is certainly the most important criterium to fulfill before suggesting that binding sites under study are relevant to active in vivo receptor sites [74,80]. If it is not possible to correlate the structure-activity results from bioassays with receptor binding data, careful controls (e.g., binding to filters, sequestration sites) must be done to exclude artifacts. Even then, hypothesis on the existence of yet another class of receptors must be put forward with a great deal of caution. A second important aspect of receptor binding research concerns the nature of the probes. The ligand must be of high specific activity (>15 Ci/mmol, if tritium) and possesses high affinity for binding sites. Preferably, it should be chemically stable and resistant to enzymatic degradation. This last criterium is met by various ligands (e.g., opiates, benzodiazepines, antidepressants) but not by most radiolabelled peptides. Thus, neuropeptide receptor binding research presents specific problems that must be resolved to
Nature of the Isotope Almost all neuropeptide binding assays are performed with ligands labelled with either tritium (3H) or radioactive iodine (1~I). The choice of either one is mainly dependent on the peptide involved. Since the density of binding sites for most peptides is quite low (fmol/mg protein) an iodinated ligand is likely to give better results because of its high specific activity (up to 2000 Ci/mmol). If the peptide contains tyrosine or histidine residues, it is possible to label it by direct iodination using various approaches such as chloramine T. However, it is not always possible to use such iodinated peptide for receptor binding study; particularly when binding capacity and/or biological activity depend upon an intact phenolic ring [16, 23, 77, 78, 84-86, 87, 104]. In addition to the relatively large size of the iodine which can significantly alter the binding properties of the molecule, other chemical alterations of the peptides, such as, oxidations of methionyl and trypthophyl residues may happen when a direct iodination method is used (e.g., chloramine T). A much milder approach consists in the use of the Bolton Hunter method [7] in which the 125I-Nhydroxysuccinimidyl 3-(4-hydroxyphenyl) proprionate acylates specifically N ~ and N • amino groups. The advantage of this method is that any peptides with a free N-terminal amino group can be labelled. However, if N • amino groups are important for the activity of the molecule, temporary protection of this function must be considered during the BoltonHunter reaction. In general, tritium-labelled compounds, prepared by either exchange reaction or chemical synthesis, are used by a large number of scientists since they are commercially available. Moreover, the half-life of 3H being much longer than ~25I, it is sometimes preferable to use a 3H-labelled ligand which insures long-term availability.
413
QUIRION AND GAUDREAU
414
TABLE l APPROACHESUSED TO DECREASELIGANDBINDINGTO FILTERSIN VARIOUSASSAYS Ligand [3H]Substance P [3H]Dynorphin (1-17) [3H]Dynorpbin (1-9) [3H]Dynorphin (1-8) [3H]D-Ala2, D-Leu'~enkephalin [~H]Neurotensin [125I]Neurotensin [125I]Angiotensin [~I]Somatostatin [:~H]Bradykinin
Treatment 0.1% polyethyleneimine 0.4% BSA* plus 0.1% polylysine 1.0% polyethyleneimine 0.1% polyethyleneimine t-amyl alcohol saturated water Incubation buffer 2% BSA Incubation buffer 1% BSA 0.1% polyethyleneimine
Reference 8,42,65,76 116 89 75 45 38,39 41 108 1 50
*BSA=bovine serum albumin.
High Purity of the Ligand The highest purity (>99%) of the ligand should be sought. To insure that the radiolabelled ligand is pure, a high pressure liquid chromatography (HPLC) analysis should be done prior to the binding assay. An impure radiolabelled peptide could generate artifacts. First, biphasic Scatchard plots can be obtained suggesting the existence of multiple classes of sites when, in fact, only one type of site is present. Impure tracers generally give lower affinity and number of sites values than in reality. Curvilinear Scatchard plots are likely to be due to the increasing proportion of denatured ligand (or other impurities) present in the incubation medium when increasing concentrations of the tracer. Second, Hill coefficients smaller than unity that can be indicative of complex interaction between the ligands and its binding sites (negative and positive cooperativity) may only be due to impure ligands. Finally, poor specific to non-specific binding ratio will certainly be observed. Thus, great care should be taken to insure high purity of the labelled ligand.
Physico-Chemical Properties of the Ligand Most peptides are positively or negatively charged at physiological pH. Consequently, they have a tendency to "bind" to inert materials such as glass, plastic, etc. Many of them are also highly hydrophobic and are likely to be sequestrated into lipids of membrane preparations. To minimize problems such as binding to glass and/or plastic tubes, it is important to coat these materials with either serum albumin, polylysine, bacitracin or silicone. It has been shown that such coating markedly reduced the absorption of substance P [103], opioid peptides [44] and calcitonin [57] to tubes and syringes. It is believed that positive charges present on these molecules neutralize the negative charges of the silica which in turn, markedly decrease the adhesion of positively charged peptides to glass. Another major problem encountered in receptor assays is related to binding of peptides to the filters used in the rapid filtration technique. Binding of labelled ligands to filters should always be tested in a first series of experiments. If it is found that an important fraction (more than 20-25%) of the ligand is bound to filters, various approaches should be used to resolve this problem. As a first step, various types of filters could be tested (GF/B, GF/C, GF/F, Millipore, etc.) to choose those that appear to be the most appropriate. Then, soaking the filters in serum albumin, polylysine or polyethyleneimine solutions usually markedly decreased ligand bind-
ing to filters (Table 1). However, if this approach is not satisfactory, control experiments (no membranes) should be run in all assays and binding values substracted from those obtained in presence of membrane preparations. Centrifugation assays should also be considered if filter binding remains important after coating. This technique is particularly useful if the dissociation rate of the tracer from its binding sites is extremely rapid (less than few seconds). With this technique. it is possible to stop the reaction in 2-3 seconds. However, the non-specific binding is generally greater in the centrifugation assay due to the trapping of tracer in the pellet. Similar approaches could be used for in vitro section Omding techniques [34, 79, 117]. For example, if ligand binds to gelatin-coated slides, it is possible to add polyethyleneimine to the gelatin solution or to the pre-incubation buffer. It generally markedly reduces binding to coated slides.
Chemical and Biological Stability of the Ligand In receptor binding assays of non-peptide drugs and neurotransmitters, the chemical and biological stability of the ligand is generally not a major problem. However, it is probably the most critical aspect of any peptide binding assays. Many neuropeptides must be protected against oxidation that will likely reduce their binding ability. Such peptides (e.g., cholecystokinin) usually contain methionine and tryptophan in their sequence. Protection against oxidation is achieved by storing and incubating the radiolabelled ligand in a mixture containing either dihydrothreitol or/3-mercaptoethanol as anti-oxydants [24, 25, 103, 120]. Care must also be taken during manipulation of stocks of ligand. They should be divided in small aliquots, flushed under nitrogen or argon and subsequently stored at -80°C. Under these conditions, we have been able to keep stocks of [3HI substance P and [3H] cholecystokinin for many weeks without major loss of activity [24, 25, 83, 97]. Another problem encountered in peptide binding assays is the marked sensitivity of certain ligands to enzymatic degradation. It is possible to verify the integrity of the ligand after tissue incubation by analytical HPLC and/or bioassays. For example, it has been shown that without protection, substance P [42] or dynorphin (1-9) [89] are very rapidly degraded by membrane preparations. Lower incubation temperatures and mixture of enzyme inhibitors are usually used to reduce enzyme activity in membrane preparations. Table 2 described some "cocktails" of inhibitors used in various neuropeptide binding assays. The most widely used enzyme inhibitor is certainly bacitracin, a non-selective protease in-
NEUROPEPTIDE RECEPTOR BINDING
415
TABLE 2 MIXTURE OF PROTEASE INH1BITORS AND ANTIOXYDANTS USED IN VARIOUS NEUROPEPTIDE RECEPTORBINDINGASSAYS Peptide ACTH Angiotensin
Bombesin Bradykinin
Calcitonin Cholecystokinin
Eledoisin Glucagon GnRH analogues
Insulin y-MSH Neurotensin Opioid peptides Dynorphin (1-17) Dynorphin (1-9) Dynorphin (1-8) ]3-endorphin Enkephalin derivatives Secretin Substance P
Somatostatin
TRH Vasopressin
VIP
Protease Inhibitors and Antioxydants Bacitracin PMSF and DTT Bacitracin, PMSF and aprotinin Bacitracin Bacitracin Bacitracin, DTT and captopril None Bacitracin None Bacitracin Bacitracin and DTT Bacitracin None None Bacitracin DTT None Bacitracin Lima bean trypsin inhibitor None Bacitracin None Bestatin, captopril and dipeptides Bacitracin, chymostatin and leupeptin Bacitracin None Bacitracin Bacitracin PMSF and aprotinin None Bacitracin Chymostatin Bacitracin, PMSF and chloromercuribenzoate Bacitracin, chymostatin leupeptin None Bacitracin Bacitracin and ethylmercurithiosalicylate Bacitracin, aprotinin and PMSF None Bacitracin None Bacitracin Bacitracin and aprotinin Leupeptin, pepstatin and benzamidine Bacitracin Bacitracin and aprotinin
References 9 5,14,26,31,99 55 56,59,69,113 92 37,50 21,52 57,63,88 73 33,93,94 24,25,36,120 3 6,90,102 12,51,54 13,43,68 72 32 119 67 11,38,39,41,53,110 27,82,118 116 89 75 28,47 10,15,18,40,45,62,114 2,81 22 61 58 4,109,112 46 30 8,42,65,76,83,97 91 17,105 1
100,101 64 60,96 29,48,95 111,115 19 66 20 106
Abbreviations used: ACTH, adrenocorticotropic hormone; DTT, dithiothreitol; GIP, gastric inhibitory peptide: GnRH, gonadotropin releasing hormone; y-MSH, y-melanocyte stimulating hormone; PMSF, phenylmethylsulfonylfluoride; TRH, thyrotropin-releasing hormone and VIP, vasoactive intestinal polypeptide.
416
QUIRION AND GAUDREAU TABLE 3 RELATIVE POTENCIES OF SUBSTANCE P (SP), ITS FRAGMENTS AND HOMOLOGUES 1N VARIOUS ASSAYS Binding Assay* Peptide
[:3H]SP
Substance P Substance P (3-11) Substance P (5-11) Substance P (7-11) Substance P---COOH Physalaemin Eledoisin Kassinin
Bioassay$
['e~I]SP
['25I]Physalaemin
MCCt
DCA
RA
100 7.6 1.9 0.01 0.01 14 2.6 1.3
100 -0.6 0.01 0.005 125 1.0 --
100 16.8 3.3 0.01 inactive 18 0.9 --
100 -20 0.01 -100 33 15
100 70 30 --100 40 18
100 3.1 0.3 0.02 0.01 16 0.8 0.07
*[aH]SP, [225I]SP and ['25I]Physalaemin binding data are from [76,83], [97] and 1112] respectively, tMCC means mouse mesencephalic cell culture. Data are from [4]. SDCA and RA means dog carotid artery and rabbit aorta, respectively. Data are derived from [86].
TABLE 4 RELATIVE POTENCIES OF NEUROTENSIN (NT), ITS FRAGMENTS AND ANALOGUES IN VARIOUS ASSAYS Binding Assay Peptide Neurotensin Neurotensin (8-13) Neurotensin (%13) [D-Argg]-NT [Trp"]-NT [Phelq-NT Xenopsin
Bioassay$
[ZH]NT*
['z'~I][Trp' qNT
NT 29~
RSS
RPV
GPI
100 39 8 0.5 91 20 96
100 -1 -100 8 .
100 120 1.3 0.24 -4
100 69 11 0.65 130 16
100 94 2.4 0.8 115 9.4
100 20 3 4 14 33 55
.
.
.
*[3H]NT and ['2'~I][Trp"]NT binding data are from [82] and [53] respectively, t i l T 29 is a cell line derived from human colon. Data drived from [39]. SRSS, RPV and GPI means rat stomach strip, rat portal vein and guinea pig illeum. Data derived from [77, 78, 87], [11] and [39] respectively.
hibitor. H o w e v e r , in m a n y cases, it is not enough and more selective agents such as chymostatin (serine protease blocker), leupeptin (leucine-aminopeptidase blocker), captopril (angiotensin converting e n z y m e inhibitor) and thiorphan (enkephalinase inhibitor) must be used (Table 2). Substance P is a good e x a m p l e illustrating the importance of the use of an appropriate mixture of e n z y m e inhibitors in o r d e r to insure stability of the radiolabelled ligand [42,83]. L e e et al. [42] have shown that in the p r e s e n c e o f bacitracin, chymostatin and leupeptin, substance P degradation by salivary gland m e m b r a n e s was almost fully inhibited. H o w ever, they have also reported that thiolpeptidase inhibitors such as N-ethylmaleimide and p-chloromercuriphenylsulfate markedly inhibit substance P binding. The same phenome n o n is also often o b s e r v e d with bacitracin if used in high concentrations. Thus, certain protease inhibitors are able to protect the ligand against e n z y m a t i c degradation but at the same time inhibit its binding to r e c e p t o r binding sites. This possibility should be kept in mind while trying to design the m o s t appropriate mixture of protease inhibitors.
Dynorphin is also highly sensitive to degradation. Recently, R o b s o n et al. [89] have shown that a mixture of bestatin, captopril and dipeptides such as leucyl-leucine and arginyl-arginine must be used to protect [aH] dynorphin (1-9) against e n z y m a t i c degradation. Thus, each ligand appears to be differentially sensitive to proteolytic degradation and the most appropriate mixture of e n z y m e inhibitors must be determined e a c h time. Finally, the availability of radiolabelled peptide analogues containing D-amino acids at cleavage sites usually markedly increase their resistance to e n z y m e degradation and facilitate the characterization of r e c e p t o r binding sites [1, 18, 71].
Ligand Selectivity Pattern After the elaboration o f appropriate handling and incubation conditions that insure the stability o f peptide ligands, it is possible to obtain data on the pharmacological nature (affinity, n u m b e r of sites) of the r e c e p t o r binding sites. H o w e v e r , the most important criterium to fulfill the claim that
NEUROPEPTIDE RECEPTOR BINDING
417
binding sites are relevant to the r e c e p t o r u n d e r study is the d e m o n s t r a t i o n o f an appropriate ligand selectivity pattern [74,80]. If the same r e c e p t o r is present in various systems, the structure-activity relationship should be similar in binding assays and various bioassays such as in vitro isolated preparations, and electrophysiological or behavioral tests. E x a m p l e s of these types of correlation are reported in Table 3 and Table 4. It has been d e m o n s t r a t e d that three different substance P iigands s h o w similar ligand selectivity pattern in all binding assays that correlated well with bioassay data (Table 3). Similar results have been obtained for neurotensin (Table 4). This strongly suggests that such binding assay data are relevant to the p h a r m a c o l o g y o f the neuropeptide r e c e p t o r under study. Without appropriate ligand selectivity pattern, it is not possible to s h o w that specific binding sites for a given peptide are relevant to its effects. T h e s e sites could only be related to storage sites, carrier proteins or o t h e r sequestration sites present in m e m branes.
usually chemically and/or biologically unstable. H o w e v e r , under appropriate conditions, binding assays are powerful means to study possible m e c h a n i s m s of action o f neuropeptides. Such assays provide a rapid m e t h o d to screen for and isolate new e n d o g e n o u s peptides and study their structureactivity relationships. M o r e o v e r , r a d i o r e c e p t o r binding assays are also currently used in clinics to monitor neurotransmitters and drug levels in biological fluids. It is e x p e c t e d that neuropeptide r a d i o r e c e p t o r assays could also be used for clinical purposes in the near future. Finally, the availability of stable synthetic analogues of most neuropeptides should greatly facilitate the characterization o f neuropeptide receptors and eventually lead to their complete purification. Synthetic neuropeptide r e c e p t o r s could then be used to study the precise m e c h a n i s m o f interactions between ligands and binding sites. This could certainly help to design analogues with super agonist and antagonist properties that could be clinically useful. ACKNOWLEDGEMENTS
Conclusions
In s u m m a r y , neuropeptide r e c e p t o r binding studies are c h a r a c t e r i z e d principally by the nature o f the ligand which is
R6mi Quirion is a scholar of the "Fonds de la recherche en Sant6 du Quebec." The expert secretarial assistance of Mrs. Joan Currie and Mrs. Denise Robertson is acknowledged.
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