[47]
RADIOLIGAND
BINDING
ASSAY FOR
LTDa R E C E P T O R
421
the receptor binding level, since only agonists inhibition curves (against [3H]ICI 198,615) are shifted to lower affinity by stable GTP analogs) 8 However, one has to bear in mind that although these binding assays are highly efficient, rapid, and possess high capacity to test antagonist potency and mechanism, they do not provide broad pharmacological information as do functional receptor assays that utilize viable tissues. Some of the more notable examples are the phosphodiesterase inhibitory properties of LY 171,883 (Ref. 7) and the thromboxane A2 inhibitory activity of FPL 55,712 (Ref. 19) that may enhance antibronchospastic properties in viable tissues or animal models, leading to an apparent overestimation of their potency. An additional important discrepancy is the ability of all LTD4 antagonists to inhibit LTC4 and LTD4 contractile activity on human lung equally well. z°'21 This is in contrast to functional3 and ligand binding ~7 experiments that demonstrate distinct binding sites for LTC4 and LTD4 (and ICI 198,615). Acknowledgments Mr. R. C. Falcone and Ms. C. A. Catanese are gratefully acknowledged for conducting these experiments and Dr. R. D. Krell for reviewing this manuscript. is C. A. Catanese, R. C. Falcone, and D. Aharony, J. Pharmacol. Exp. Ther. 251, 846 (1989). 19 A. F. Welton, W. C. Hope, L. D. Tobias, and J. G. Hamilton, Biochem. Pharmacol. 30, 1378 (1981). 2o C. K. Buckner, R. D. Krell, R. B. Laravuso, D. B. Coursin, P. R. Bernstein, andJ. A. Will, J. Pharmacol. Exp. Ther. 237, 558 (1986). 21 C. K. Buckner, R. Saban, W. L. Castleman, and J. A. Will, Ann. N. Y. Acad. Sci. 524, 181 (1988).
[47] S u l f i d o p e p t i d e L e u k o t r i e n e R e c e p t o r B i n d i n g Assays By SEYMOURMONG Introduction The sulfidopeptide leukotriene receptor binding assay has been used extensively in antagonist compound screening, ~ in the identification of C. D. Perchonock, I. Uzinkas, M. E. McCarthy, K. F. Erhard, J. G. Gleason, M, A. Wasserman, R. M. Muccitelli, J. F. Devan, S. S. Tucker, L. M. Vickery, T. Kirchner, B. M. Weichman, S. Mong, M. O. Scott, G. Chi-Rosso, H.-L. Wu, S. T. Crooke, and J. F. Newton, J. Med. Chem. 29, 1442 (1986).
METHODS IN ENZYMOLOGY, VOL. 187
Copyright © 1990 by Academic Press, Inc. All rights of reproduction in any form reserved.
422
PHARMACOLOGY: ANTAGONIST/SYNTHESIS INHIBITORS
[47]
antagonists from fermentation broths and natural products, 2 and also for the quantification of sulfidopeptide leukotrienes in biological samples. Sulfidopeptide leukotrienes are highly lipophilic and tend to interact nonspecifically with plastics, paper, test tubes, glass, and other nonreceptor sites. The radiolabeled agonists are metabolically unstable and sensitive to chemical degradation. These are the major factors to be carefully considered when setting up radioligand binding systems to study the receptors and the mechanisms of receptor regulation. Recent advancement of the synthesis of potent receptor antagonists has alleviated some of the vexing difficulties. When appropriately controlled and validated, the radioligand binding assay has proved to be extremely useful. Methods
Cell Culture Any type of media or serum for culturing is sufficient, as long as the cells are in good condition. Cells derived from primary cultures tend to dedifferentiate quickly and lose their leukotriene receptors. 3 Cells are harvested, washed, and resuspended in binding assays. Chemicals used in culturing the cells, such as mercaptoethanol, retinoic acid, vitamins, dimethyl sulfoxide, serum protein, and fetal calf serum should be washed away, unless proved to be necessary. These agents tend to interact with radioligands causing problems. Whole cell binding is only recommended when radiolabeled high-affinity antagonists (e.g., [3H]ICI-198615) are used. Radiolabeled agonist ([3H]LTC4, [3H]LTD4, and [3H]LTE4) binding usually is influenced by processes such as desensitization, i.e., receptor uncoupling or receptor internalization, and hence the data derived from such studies are difficult to evaluate.
Membrane Preparation Tissues or cells can be preserved by quick freezing, usually by submersion in liquid nitrogen, and stored in an ultra-low temperature ( - 7 0 ° to - 8 0 °) freezer for up to 1 year. Primary cultured smooth muscle cells, cell lines, lungs, hearts, and tracheas, from guinea pig, sheep, monkey, or humans have been stored in this manner and have subsequently provided usable membrane preparations. Human lungs, from 18-hr postmortem 2 Z. Tian, M. N. Chang, M. Sandrino, L. Huang, J. Pan, B. Arison, J. Smith, and Y. K. T. Lain, Phytochemistry 26, 2361 (1987). 3 S. Mong, J. Miller, H.-L. Wu, and S. T. Crooke, J. Pharmacol. Exp. Ther. 244, 508 (1988).
[47]
RADIOLIGAND BINDING ASSAY FOR
LTD4 RECEPTOR
423
traffic accident victims, have been preserved this way for up to 8 months and still yielded usable membranes for ligand binding studies. Tissues (10 g) are minced into 50-500 mm 3 blocks and transferred to 80 ml homogenization buffer [0.25 M sucrose in 50 mM PIPES buffer, pH 6.5, containing the following protease inhibitors; phenylmethylsulfonyl fluoride (0.5 mM), bacitracin (100/zg/ml), benzamidine (100/xM), and soybean trypsin inhibitor (100/~g/ml)]. Cells are directly resuspended in homogenization buffer at a concentration 108-109/ml. The pH of the buffer is adjusted with either HCI or KOH. The tissue (or cell) is broken with a Polytron homogenizer at a setting of 6-7, for 40 to 100 sec with 10-sec pulses. The homogenate is then centrifuged (1000 g) for 10 min at 4° to remove the tissue clumps and unbroken cells. The supernatant can be centrifuged at 30,000 g for 20 min at 4° to yield crude membrane pellets for binding studies. A better way to enrich the receptor-containing plasma membrane preparation 3 is to layer the supernatant (25 ml) from the first centrifugation (1000 g) on a 10-ml 40% sucrose cushion in a Beckman SW-27 nitrocellulose centrifuge tube. The centrifuge tubes are spun at 100,000 g for 90 min at 4°. The membranes at the 10/40% sucrose interface are collected, pooled, diluted with an equal volume (10 mM) of Tris buffer (pH 7.4), and pelleted by centrifugation at 150,000 g for 30 min at 4°. The pellets contain plasma membrane-enriched fractions that can be used directly or quickly frozen and stored. It is advisable to use liquid nitrogen to freeze the membrane pellets and avoid repeated cycles of freezethawing. Short-term storage (24-48 hr, 0°) of guinea pig lung membranes in protease-containing buffer is associated with gradual loss (10-15% per day) of the receptor binding activity.
Radioligand Binding Conditions Association and Dissociation of Radiolabeled Agonist and Antagonist. Binding of radiolabeled agonists ([3H]LTD4 or [3H]LTE4) is usually regulated by cations and pH. Therefore, each of these factors must be determined independently to minimize nonspecific binding and to avoid metabolic conversion of the radioligands. When these factors and the conditions are well-defined, one can then plan for extensive saturation, competition, and screening experiments. In our laboratory, for kinetic experiments, [3H]LTD4 (or [3H]LTE4) is added to an incubation mixture that contains 20 mM PIPES buffer (pH 6.5), 10 mM cysteine, 10 mM glycine, 5 mM CaCI2, 5 mM MgC12,0.5 riM [3H]LTD4 (or [3H]LTE4) and 1000 /~g/ml of membrane protein in a volume of 1 ml. The mixture is incubated at room temperature (22°) from zero to 60 min in Nunc Minisorb tubes (Gibco, Grand Island, NY). Aliquots (100/zl) are retrieved at varying
424
PHARMACOLOGY: ANTAGONIST/SYNTHESIS INHIBITORS
[47]
time points and analyzed for membrane-associated radioligand binding. A separate incubation mixture is set up similarly, except that the unlabeled homologous ligand (LTD4 or LTE4) is also included in the mixture at 0.5/,~M, to determine the nonspecific binding. Bound radioligand is separated from the unbound ligands by filtration and washing. The aliquots are diluted in a reservoir containing ice-cold washing buffer (10 mM Tris-HC1, pH 7.5), and filtered through Whatman GF/C filter discs. The filter discs are rapidly washed four times with a total of 20 ml of the washing buffer (0°) in less than 15 sec. It is important to keep the washing buffer cold and the process brief. Under these conditions, approximately 99% of the receptorassociated radioligand remains tightly bound, even with additional washing with 30 ml of ice-cold buffer. Total and nonspecific binding are defined as binding in the absence or presence of the excess cold ligand, respectively. The specific binding component is defined as the difference between total and nonspecific binding of the radioligand. Binding of the radiolabeled high-affinity antagonist ([3H]ICI-198615) can be performed similarly. The nonspecific binding is determined in the presence of the unlabeled homologous ligand (ICI- 198615). To determine if the radioligand binding is reversible, a small volume (2- 3/zl) of a high concentration (10 raM) of unlabeled agonist or antagonist homologous ligand is added singly, or premixed with other agents (e.g., GTP or NaC1), which could affect the association or dissociation rates of the radioligand, and added to the incubation mixtures at the steady-state of binding. Aliquots of the incubation mixture are retrieved and analyzed for membrane-bound radioactivity. Equilibrium Saturation Binding of Radioligand. The assumption that radioligand binding to the specific sites is at a equilibrium state is usually determined by the manifestation of a stable, steady-state, reversible binding of the radioligands and the lack of extensive bioconversion or chemical degradation of the radioligand, during the time course of the binding study. Therefore, the exact conditions employed in the saturation binding should be determined from the data derived from the kinetic experiments. Using guinea pig lung membranes, the incubation mixture containing 20 mM PIPES buffer (pH 6.5), 5 mM cysteine, 5 mM glycine, 5 mM CaCI2, 5 mM MgCI2, membrane protein (10-30/.~g/ml), and varying concentrations of radioligand in Nunc Minisorb test tubes are prepared in triplicate and incubated for 60 min at 22°. The concentration range of the radioligand and the volume of the incubation mixture can be adjusted based on the expected density of the receptor and the binding affinity of the radioligand to the receptor. It is also important not to add nonspecific binding protein, such as bovine serum albumin, to the incubation mixture. Glass test tubes and exogenous protein tend to interfere with radioligand-receptor interac-
[47]
RADIOLIGAND BINDING ASSAY FOR LTD4 RECEPTOR
425
tion, especially in the subnanomolar concentration range of the radioligands used. Competition ofRadiolabeled Ligands. The exact conditions of radioligand competition experiments are dictated by the results obtained from saturation experiments. With guinea pig lung membranes, 0.5 nM [3H]LTD4, 0.8 riM [3H]LTE4 or 0.3 nM [3H]ICI-198615 (approximately 3to 4-fold of the respective KDs) are used to label 80-90% of the available sites in the membranes. Ca 2+ , Mgz+ , cysteine, and glycine (5-10 raM) are included when [3H]LTDa is used to prevent metabolic conversion of [3H]LTD4. Cysteine and glycine are omitted when [3H]LTE4 or [3H]ICI198615 is employed. The membranes are added to the incubation mixtures containing 20 mM PIPES buffer (pH 6.5), the radiolabeled ligand, and varying concentrations of the competing ligands in a volume of 0.5 ml in triplicate and incubated at 22° for 60 rain. Data Calculation. The radioactivity (cpm) determined from liquid scintillation spectrometry can be converted to dpm and then to fmol or pmol of the respective radioligand. Quenching of the radioactivity must be carefully determined and corrected. To determine the dissociation constant (KD) and the maximum binding density (Bronx), the Scafit program, originally described by Delean et al. (1980), 4 can be used to analyze the saturation data. The LIGAND or Superfit programs can also be used and are readily accessible in most research institutions. Results and Discussion
Association and Dissociation o f Radioligand Binding to Membranes There are two major objectives in studying the kinetics of radioligand binding: (1) to establish the steady-state of radioligand receptor binding, and (2) to optimize the specific binding and minimize the metabolic conversion or degradation of the radioligand. The results in Fig. 1A illustrate the kinetics of [3H]LTD4 binding to guinea pig lung membranes. Note that binding of [3H]LTD4 to guinea pig lung membrane reaches a plateau 20 min after the reaction is initiated. Prolonged incubation (greater than 2 hr) or in the absence of cysteine and glycine results in a decrease of the level of specific binding5 suggesting a loss of the receptors or [3H]LTD4 due to conversion to [3H]LTE4. The level of specific binding of [3H]LTD4 is decreased when EDTA (1-10 raM), Na + (1-100 raM), or GppNHp 4 A. Delean, J. M. Stadel, and R. J. Lefkowitz, J. Biol. Chem. 255, 7108 (1980). 5 S. Mong, H.-L. Wu, J. M. Stadel, M. A. Clark, and S. T. Crooke, Eur. J. Pharmacol. 29, 102 (1984).
426
[47]
PHARMACOLOGY; ANTAGONIST/SYNTHESIS INHIBITORS
0
7
B
6
-
A
5
X
E "0
4
Z O Z C0
ok
3
d
~2 "T" 1
| 10
I 20
I 30
I 40
I 50
I 60
TIME (MINUTE) o~ 100
co 0 o~ LJ-
60
0 n LLJ "1- o9
40
RADIOLIGAND
BINDING
ASSAY FOR
LTDa R E C E P T O R
421
the receptor binding level, since only agonists inhibition curves (against [3H]ICI 198,615) are shifted to lower affinity by stable GTP analogs) 8 However, one has to bear in mind that although these binding assays are highly efficient, rapid, and possess high capacity to test antagonist potency and mechanism, they do not provide broad pharmacological information as do functional receptor assays that utilize viable tissues. Some of the more notable examples are the phosphodiesterase inhibitory properties of LY 171,883 (Ref. 7) and the thromboxane A2 inhibitory activity of FPL 55,712 (Ref. 19) that may enhance antibronchospastic properties in viable tissues or animal models, leading to an apparent overestimation of their potency. An additional important discrepancy is the ability of all LTD4 antagonists to inhibit LTC4 and LTD4 contractile activity on human lung equally well. z°'21 This is in contrast to functional3 and ligand binding ~7 experiments that demonstrate distinct binding sites for LTC4 and LTD4 (and ICI 198,615). Acknowledgments Mr. R. C. Falcone and Ms. C. A. Catanese are gratefully acknowledged for conducting these experiments and Dr. R. D. Krell for reviewing this manuscript. is C. A. Catanese, R. C. Falcone, and D. Aharony, J. Pharmacol. Exp. Ther. 251, 846 (1989). 19 A. F. Welton, W. C. Hope, L. D. Tobias, and J. G. Hamilton, Biochem. Pharmacol. 30, 1378 (1981). 2o C. K. Buckner, R. D. Krell, R. B. Laravuso, D. B. Coursin, P. R. Bernstein, andJ. A. Will, J. Pharmacol. Exp. Ther. 237, 558 (1986). 21 C. K. Buckner, R. Saban, W. L. Castleman, and J. A. Will, Ann. N. Y. Acad. Sci. 524, 181 (1988).
[47] S u l f i d o p e p t i d e L e u k o t r i e n e R e c e p t o r B i n d i n g Assays By SEYMOURMONG Introduction The sulfidopeptide leukotriene receptor binding assay has been used extensively in antagonist compound screening, ~ in the identification of C. D. Perchonock, I. Uzinkas, M. E. McCarthy, K. F. Erhard, J. G. Gleason, M, A. Wasserman, R. M. Muccitelli, J. F. Devan, S. S. Tucker, L. M. Vickery, T. Kirchner, B. M. Weichman, S. Mong, M. O. Scott, G. Chi-Rosso, H.-L. Wu, S. T. Crooke, and J. F. Newton, J. Med. Chem. 29, 1442 (1986).
METHODS IN ENZYMOLOGY, VOL. 187
Copyright © 1990 by Academic Press, Inc. All rights of reproduction in any form reserved.
422
PHARMACOLOGY: ANTAGONIST/SYNTHESIS INHIBITORS
[47]
antagonists from fermentation broths and natural products, 2 and also for the quantification of sulfidopeptide leukotrienes in biological samples. Sulfidopeptide leukotrienes are highly lipophilic and tend to interact nonspecifically with plastics, paper, test tubes, glass, and other nonreceptor sites. The radiolabeled agonists are metabolically unstable and sensitive to chemical degradation. These are the major factors to be carefully considered when setting up radioligand binding systems to study the receptors and the mechanisms of receptor regulation. Recent advancement of the synthesis of potent receptor antagonists has alleviated some of the vexing difficulties. When appropriately controlled and validated, the radioligand binding assay has proved to be extremely useful. Methods
Cell Culture Any type of media or serum for culturing is sufficient, as long as the cells are in good condition. Cells derived from primary cultures tend to dedifferentiate quickly and lose their leukotriene receptors. 3 Cells are harvested, washed, and resuspended in binding assays. Chemicals used in culturing the cells, such as mercaptoethanol, retinoic acid, vitamins, dimethyl sulfoxide, serum protein, and fetal calf serum should be washed away, unless proved to be necessary. These agents tend to interact with radioligands causing problems. Whole cell binding is only recommended when radiolabeled high-affinity antagonists (e.g., [3H]ICI-198615) are used. Radiolabeled agonist ([3H]LTC4, [3H]LTD4, and [3H]LTE4) binding usually is influenced by processes such as desensitization, i.e., receptor uncoupling or receptor internalization, and hence the data derived from such studies are difficult to evaluate.
Membrane Preparation Tissues or cells can be preserved by quick freezing, usually by submersion in liquid nitrogen, and stored in an ultra-low temperature ( - 7 0 ° to - 8 0 °) freezer for up to 1 year. Primary cultured smooth muscle cells, cell lines, lungs, hearts, and tracheas, from guinea pig, sheep, monkey, or humans have been stored in this manner and have subsequently provided usable membrane preparations. Human lungs, from 18-hr postmortem 2 Z. Tian, M. N. Chang, M. Sandrino, L. Huang, J. Pan, B. Arison, J. Smith, and Y. K. T. Lain, Phytochemistry 26, 2361 (1987). 3 S. Mong, J. Miller, H.-L. Wu, and S. T. Crooke, J. Pharmacol. Exp. Ther. 244, 508 (1988).
[47]
RADIOLIGAND BINDING ASSAY FOR
LTD4 RECEPTOR
423
traffic accident victims, have been preserved this way for up to 8 months and still yielded usable membranes for ligand binding studies. Tissues (10 g) are minced into 50-500 mm 3 blocks and transferred to 80 ml homogenization buffer [0.25 M sucrose in 50 mM PIPES buffer, pH 6.5, containing the following protease inhibitors; phenylmethylsulfonyl fluoride (0.5 mM), bacitracin (100/zg/ml), benzamidine (100/xM), and soybean trypsin inhibitor (100/~g/ml)]. Cells are directly resuspended in homogenization buffer at a concentration 108-109/ml. The pH of the buffer is adjusted with either HCI or KOH. The tissue (or cell) is broken with a Polytron homogenizer at a setting of 6-7, for 40 to 100 sec with 10-sec pulses. The homogenate is then centrifuged (1000 g) for 10 min at 4° to remove the tissue clumps and unbroken cells. The supernatant can be centrifuged at 30,000 g for 20 min at 4° to yield crude membrane pellets for binding studies. A better way to enrich the receptor-containing plasma membrane preparation 3 is to layer the supernatant (25 ml) from the first centrifugation (1000 g) on a 10-ml 40% sucrose cushion in a Beckman SW-27 nitrocellulose centrifuge tube. The centrifuge tubes are spun at 100,000 g for 90 min at 4°. The membranes at the 10/40% sucrose interface are collected, pooled, diluted with an equal volume (10 mM) of Tris buffer (pH 7.4), and pelleted by centrifugation at 150,000 g for 30 min at 4°. The pellets contain plasma membrane-enriched fractions that can be used directly or quickly frozen and stored. It is advisable to use liquid nitrogen to freeze the membrane pellets and avoid repeated cycles of freezethawing. Short-term storage (24-48 hr, 0°) of guinea pig lung membranes in protease-containing buffer is associated with gradual loss (10-15% per day) of the receptor binding activity.
Radioligand Binding Conditions Association and Dissociation of Radiolabeled Agonist and Antagonist. Binding of radiolabeled agonists ([3H]LTD4 or [3H]LTE4) is usually regulated by cations and pH. Therefore, each of these factors must be determined independently to minimize nonspecific binding and to avoid metabolic conversion of the radioligands. When these factors and the conditions are well-defined, one can then plan for extensive saturation, competition, and screening experiments. In our laboratory, for kinetic experiments, [3H]LTD4 (or [3H]LTE4) is added to an incubation mixture that contains 20 mM PIPES buffer (pH 6.5), 10 mM cysteine, 10 mM glycine, 5 mM CaCI2, 5 mM MgC12,0.5 riM [3H]LTD4 (or [3H]LTE4) and 1000 /~g/ml of membrane protein in a volume of 1 ml. The mixture is incubated at room temperature (22°) from zero to 60 min in Nunc Minisorb tubes (Gibco, Grand Island, NY). Aliquots (100/zl) are retrieved at varying
424
PHARMACOLOGY: ANTAGONIST/SYNTHESIS INHIBITORS
[47]
time points and analyzed for membrane-associated radioligand binding. A separate incubation mixture is set up similarly, except that the unlabeled homologous ligand (LTD4 or LTE4) is also included in the mixture at 0.5/,~M, to determine the nonspecific binding. Bound radioligand is separated from the unbound ligands by filtration and washing. The aliquots are diluted in a reservoir containing ice-cold washing buffer (10 mM Tris-HC1, pH 7.5), and filtered through Whatman GF/C filter discs. The filter discs are rapidly washed four times with a total of 20 ml of the washing buffer (0°) in less than 15 sec. It is important to keep the washing buffer cold and the process brief. Under these conditions, approximately 99% of the receptorassociated radioligand remains tightly bound, even with additional washing with 30 ml of ice-cold buffer. Total and nonspecific binding are defined as binding in the absence or presence of the excess cold ligand, respectively. The specific binding component is defined as the difference between total and nonspecific binding of the radioligand. Binding of the radiolabeled high-affinity antagonist ([3H]ICI-198615) can be performed similarly. The nonspecific binding is determined in the presence of the unlabeled homologous ligand (ICI- 198615). To determine if the radioligand binding is reversible, a small volume (2- 3/zl) of a high concentration (10 raM) of unlabeled agonist or antagonist homologous ligand is added singly, or premixed with other agents (e.g., GTP or NaC1), which could affect the association or dissociation rates of the radioligand, and added to the incubation mixtures at the steady-state of binding. Aliquots of the incubation mixture are retrieved and analyzed for membrane-bound radioactivity. Equilibrium Saturation Binding of Radioligand. The assumption that radioligand binding to the specific sites is at a equilibrium state is usually determined by the manifestation of a stable, steady-state, reversible binding of the radioligands and the lack of extensive bioconversion or chemical degradation of the radioligand, during the time course of the binding study. Therefore, the exact conditions employed in the saturation binding should be determined from the data derived from the kinetic experiments. Using guinea pig lung membranes, the incubation mixture containing 20 mM PIPES buffer (pH 6.5), 5 mM cysteine, 5 mM glycine, 5 mM CaCI2, 5 mM MgCI2, membrane protein (10-30/.~g/ml), and varying concentrations of radioligand in Nunc Minisorb test tubes are prepared in triplicate and incubated for 60 min at 22°. The concentration range of the radioligand and the volume of the incubation mixture can be adjusted based on the expected density of the receptor and the binding affinity of the radioligand to the receptor. It is also important not to add nonspecific binding protein, such as bovine serum albumin, to the incubation mixture. Glass test tubes and exogenous protein tend to interfere with radioligand-receptor interac-
[47]
RADIOLIGAND BINDING ASSAY FOR LTD4 RECEPTOR
425
tion, especially in the subnanomolar concentration range of the radioligands used. Competition ofRadiolabeled Ligands. The exact conditions of radioligand competition experiments are dictated by the results obtained from saturation experiments. With guinea pig lung membranes, 0.5 nM [3H]LTD4, 0.8 riM [3H]LTE4 or 0.3 nM [3H]ICI-198615 (approximately 3to 4-fold of the respective KDs) are used to label 80-90% of the available sites in the membranes. Ca 2+ , Mgz+ , cysteine, and glycine (5-10 raM) are included when [3H]LTDa is used to prevent metabolic conversion of [3H]LTD4. Cysteine and glycine are omitted when [3H]LTE4 or [3H]ICI198615 is employed. The membranes are added to the incubation mixtures containing 20 mM PIPES buffer (pH 6.5), the radiolabeled ligand, and varying concentrations of the competing ligands in a volume of 0.5 ml in triplicate and incubated at 22° for 60 rain. Data Calculation. The radioactivity (cpm) determined from liquid scintillation spectrometry can be converted to dpm and then to fmol or pmol of the respective radioligand. Quenching of the radioactivity must be carefully determined and corrected. To determine the dissociation constant (KD) and the maximum binding density (Bronx), the Scafit program, originally described by Delean et al. (1980), 4 can be used to analyze the saturation data. The LIGAND or Superfit programs can also be used and are readily accessible in most research institutions. Results and Discussion
Association and Dissociation o f Radioligand Binding to Membranes There are two major objectives in studying the kinetics of radioligand binding: (1) to establish the steady-state of radioligand receptor binding, and (2) to optimize the specific binding and minimize the metabolic conversion or degradation of the radioligand. The results in Fig. 1A illustrate the kinetics of [3H]LTD4 binding to guinea pig lung membranes. Note that binding of [3H]LTD4 to guinea pig lung membrane reaches a plateau 20 min after the reaction is initiated. Prolonged incubation (greater than 2 hr) or in the absence of cysteine and glycine results in a decrease of the level of specific binding5 suggesting a loss of the receptors or [3H]LTD4 due to conversion to [3H]LTE4. The level of specific binding of [3H]LTD4 is decreased when EDTA (1-10 raM), Na + (1-100 raM), or GppNHp 4 A. Delean, J. M. Stadel, and R. J. Lefkowitz, J. Biol. Chem. 255, 7108 (1980). 5 S. Mong, H.-L. Wu, J. M. Stadel, M. A. Clark, and S. T. Crooke, Eur. J. Pharmacol. 29, 102 (1984).
426
[47]
PHARMACOLOGY; ANTAGONIST/SYNTHESIS INHIBITORS
0
7
B
6
-
A
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X
E "0
4
Z O Z C0
ok
3
d
~2 "T" 1
| 10
I 20
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I 40
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I 60
TIME (MINUTE) o~ 100
co 0 o~ LJ-
60
0 n LLJ "1- o9
40
