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This Featured Protocol presents the full text of a cutting-edge Unit from Current Protocols in Pharmacology, including expert commentary sections with critical information designed to ensure the success of your experiments. 

From UNIT 1.4

Characterization of Opioid Receptors

Contributed by Robert N. DeHaven and Diane L. DeHaven-Hudkins
Adolor Corporation
Malvern, Pennsylvania

Copyright; 1998 John Wiley & Sons, Inc. All rights reserved.

This unit presents two convenient radioligand binding assay methods for opioid receptors. These are applicable to all three of the opioid receptors that have been cloned to date (the µ, k, and d receptors; see Table 1.4.1), and can be used with numerous commercially available radiolabeled ligands. Moreover, they can serve as reasonable starting points in the development of new assays. The two protocols detail a method for determining the binding of radioligand to cloned opioid receptors expressed on the surface of cultured cells (see Basic Protocol 1) and a similar method to study radioligand binding to receptors from tissue homogenates (see Alternate Protocol). Also included is a procedure for titrating inhibitors of opioid receptor-ligand binding (see Basic Protocol 2), along with guidelines for analysis of the resulting data (see Support Protocol).

NOTE: All protocols using live animals must first be reviewed and approved by an Institutional Animal Care and Use Committee (IACUC) or must conform to governmental regulations regarding the care and use of laboratory animals.

BASIC PROTOCOL 1

MEASUREMENT OF OPIOID RECEPTOR BINDING TO CLONED RECEPTORS IN MEMBRANES

This protocol describes the procedures for measuring binding of a radioligand to cloned opioid receptors expressed in cells. [3H]Diprenorphine, which is used as the radioligand, binds to cloned µ, d, or k receptors with high affinity.

Materials

Cell lines expressing the appropriate cloned human opioid receptor (e.g., CHO, HEK)
PBS (e.g., Life Technologies)
Tris assay buffer, pH 7.8 (see recipe), room temperature and 4°C
[3H]Diprenorphine (NEN Life Sciences)
Test compound(s)
100 µM naloxone or other unlabeled ligand to determine nonspecific binding
0.5% (w/v) polyethylenimine/0.1% (w/v) BSA in filter rinse buffer
Filter rinse buffer: e.g., 50 mM Tris·Cl, pH 7.8 (APPENDIX 2A), 4°C
Water-compatible scintillation cocktail (e.g., Beckman Ready-Solv)
Polytron homogenizer (Brinkmann)
Deep-well (1.0 ml) 96-well microtiter plates (Packard)
Filter-bottom (glass fiber filter) 96-well microtiter plates, glass fiber filter mats, or individual glass fiber filters (Whatman)
Cell harvester or vacuum filtration device appropriate for the filter style to be used (e.g., Skatron, Brandel, or Packard)
Additional reagents and equipment for Bradford or Lowry protein assay (APPENDIX 3A)

Prepare membranes for opioid receptor binding

  1. Remove growth medium from 500-cm2 culture plates containing confluent cells. Rinse cells twice in situ with PBS.

    2.  

     

    Quantities can be scaled up or down if desired. One person can generally harvest about 60 500-cm2 plates in a day, which yield 1 g of protein. 100 µg protein is required per assay .
     

  2. Add 10 ml room-temperature Tris assay buffer to each plate and scrape the cells from the plate using a plastic cell scraper.

    3.  
  3. Centrifuge cells in 12-ml aliquots 20 min in a swinging-bucket rotor at 2500 × g, 4°C. Resuspend each cell pellet in 20 ml Tris assay buffer, 4°C. Homogenize using a Polytron homogenizer.

    4.  

     

    Homogenization time may be optimized by examining cells microscopically, but ~30 sec at the lowest setting (1.0) should be sufficient.
     

  4. Centrifuge the homogenate 20 min at 48,000 × g, 4°C. Resuspend the pellets using a Polytron and pool them at a protein concentration of >1.0 mg/ml.

    5.  

     

    The resuspension volume will be learned by experience. However, the amount of protein can initially be estimated to be ~10% of the wet weight of the pellets.
     

  5. Determine protein concentration using the Bradford or Lowry method using BSA as a reference standard (APPENDIX 3A) and dilute the membranes to 1.0 mg protein per ml of Tris assay buffer. Store aliquots at or below -20°C (preferably ­80°C) until use or proceed directly to step 7.

    6.  

Measure opioid receptor binding

  1. Thaw the membrane preparation at room temperature or, if necessary, by gently heating to 37°C until the pellet is suspended.

    2.  

     

    Prolonged incubation can affect the integrity/stability of the receptors.

  2. Dilute the membrane preparation to a protein concentration twice the desired final assay protein concentration.

    3.  

     

    The final protein concentration must be determined experimentally for each preparation of cells, because even stably expressed receptors may be totally or partially lost. The final protein concentration should be selected such that specific receptor binding increases linearly with increasing protein concentration, total radioactivity bound is < 10% of added radioactivity, and the ratio of specific to nonspecific binding is optimized.

  3. Dilute the stock [3H]diprenorphine in Tris assay buffer to a concentration ten times the desired final concentration.

    4.  
  4. Prepare solutions of test compounds at ten times the desired final concentration.

    5.  
  5. Add 150 µl Tris assay buffer to each well of a deep-well microtiter plate.

    6.  
  6. Add 50 µl of the 10× test compound (from step 9) or buffer.

    7.  

     

    Naloxone (10 µM final concentration) is used to define nonspecific binding.

  7. Add 50 µl of the 10× [3H]diprenorphine ligand.

    8.  
  8. Add 250 µl membrane preparation (from step 7) to make a final assay volume of 500 µl in each well.

    9.  

     

    It is generally not practical to use an assay volume < 500 µl with tritiated opioid ligands. At smaller volumes, the concentration of radioligand needed may be higher than the Kd of the ligand and the applicable equations may be invalid. The minimum volume of addition is 50 µl, especially in the case of radioligand, because pipetting errors are unacceptably large when smaller volumes are added to plastic wells, which are hydrophobic.

    Although the order of addition is not important in an equilibrium assay, adding membranes last in a volume at least half of the final total volume provides good mixing of the reagents. This is necessary because it is not practical to mix assay solutions in 1-ml-capacity wells in microtiter plates. Nonspecific binding may be determined with any opioid ligand at 100 times its Kd value for that receptor to ensure saturation of the sites. Naloxone is generally used because it is inexpensive, and it inhibits only opioid receptor binding.

  9. Incubate the mixtures until binding has reached equilibrium.

    10.  

     

    For many opioid ligands, 1 hr at room temperature is sufficient to achieve equilibrium. However, this must be determined experimentally by measuring the binding at various times and choosing a time after which no further increase in binding occurs. Binding assays may also be carried out in an ice-water bath. This may be useful for less stable ligands (e.g., peptides) or to slow dissociation rates for rapidly equilibrating systems in which specific binding may be lost during the filter rinse (step 15).

  10. Soak the glass fiber filters in 0.5% polyethylenimine/0.1% BSA solution (see Table 1.4.4) while the reaction tubes or microtiter plates are incubating. Filter the assay mixtures, and then rinse the filters four times with 1 ml cold filter rinse buffer to remove unbound radioactivity.

    11.  

     

    The rinse conditions must be established experimentally. The goal is to remove excess radioligand trapped in or loosely bound to the membranes or filters without rinsing away specifically bound radioligand. This can be done by testing the number and volume of rinses to determine a point at which nonspecific binding is minimized and specific binding is unchanged.

  11. Place the filters in water-compatible scintillation cocktail or add the cocktail to filter plates, and determine radioactivity bound to the tissue by conventional scintillation spectroscopy.

    12.  

ALTERNATE PROTOCOL

MEASUREMENT OF OPIOID RECEPTOR BINDING IN TISSUE MEMBRANE HOMOGENATES

The procedures for measuring opioid binding to membranes derived from tissue are similar to those for cloned cells (see Basic Protocol 1). The following is a protocol for the binding of [3H]U­69,593 to k receptors prepared from guinea pig cerebellum. It can be adapted for the measurement of binding to µ receptors using [3H]D-Ala2-MePhe4-Gly-ol5-enkephalin ([3H]DAMGO) or d receptors using [3H]D-Ala2-D-Leu5-enkephalin ([3H]DADLE) as radioligands and for use with membranes prepared from rat forebrain. Whereas 10 µM U-50,488H is used in this protocol to define nonspecific binding to k receptors, naloxone, or a subtype-selective compound (see Tables 1.4.1 and 1.4.5) at a final concentration of 10 µM is used to define nonspecific binding to µ and d receptors.

Additional Materials (also see Basic Protocol 1)

Guinea pig
HEPES assay buffer (see recipe), ice cold
Radiolabeled ligand: [3H]U-69,593 (NEN Life Sciences)
Test compound
10 µM U-50,488H or other unlabeled opioid ligand to define nonspecific binding
Filter rinse buffer: 50 mM HEPES, pH 7.4, 4°C
Water-compatible scintillation cocktail (e.g., Beckman Ready-Solv)
Dissection instruments: operating scissors, bone rongeurs, bone cutting forceps, dissecting knife, microdissecting probes, dissecting plate.

Prepare membranes for kopioid receptor binding

  1. Sacrifice guinea pig by decapitation.

    2.  
  2. Remove brain and dissect out cerebellum.

    3.  
  3. Place cerebellum in 100 vol ice-cold HEPES assay buffer and homogenize 30 sec using a Polytron homogenizer at setting 7.

    4.  
  4. Centrifuge the homogenate 10 min at 48,000 × g, 4°C, in plastic tubes.

    5.  
  5. Resuspend the pellet in 100 vol HEPES assay buffer and incubate 15 min at 37°C with occasional stirring.

    6.  
  6. Centrifuge the homogenate 10 min at 48,000 × g, 4°C.

    7.  

     

    Steps 5 and 6 are important in order to denature and remove endogenous opioid peptides from the membrane preparation.

  7. Resuspend the pellet in HEPES assay buffer at the appropriate protein concentration (see Basic Protocol 1, steps 5 and 7). Store on ice until use (up to 2 hr).

    8.  

Measure k opioid receptor binding

  1. Dilute the [3H]U-69,593 ligand stock in HEPES assay buffer to a concentration ten times the desired final concentration.

    2.  
  2. Prepare solutions of test compounds at ten times the desired final concentration.

    3.  
  3. Add 150 µl assay buffer to each well of a deep-well microtiter plate.

    4.  
  4. Add 50 µl of the 10× test compound or buffer.

    5.  

     

    U-50,488H (10 µM) can be used to determine nonspecific binding.

  5. Add 50 µl of the 10× [3H]U-69,593 ligand (from step 8).

    6.  
  6. Add 250 µl membrane preparation (from step 7).

    7.  
  7. Incubate the mixtures until binding equilibrium is achieved.

    8.  

     

    For many ligands, 1 hr at room temperature is sufficient to achieve equilibrium. However, this must be determined experimentally by measuring the binding at various times and choosing a time after which no further increase in binding occurs. Binding assays may also be carried out in an ice-water bath. This may be useful for less stable ligands (e.g., peptides) to slow dissociation rates for rapidly equilibrating systems, in which specific binding may be lost during the filter rinse (step 15).

  8. Filter the assay mixtures through glass fiber filters on a vacuum filtration apparatus and rinse the filters four times with 1 ml per rinse of wash buffer.

    9.  

     

    Polyethylenimine soaking is not required in this assay.

  9. Place the filters in scintillation cocktail and determine membrane-bound radioactivity by liquid scintillation spectroscopy.

    10.  

BASIC PROTOCOL 2

TESTING INHIBITORS OF OPIOID RECEPTOR-LIGAND BINDING

The affinities (potencies) of compounds that bind to opioid receptors are determined by measuring their ability to inhibit binding of the radiolabeled ligand. The ability of a given concentration of an inhibitor to compete with the radiolabeled ligand for binding sites is dependent on both the affinity of the inhibitor for the receptor and the concentration of the radiolabeled ligand relative to its own affinity (Kd). Thus, in determining the affinity of an inhibitor it is necessary to take into account both the concentration and the affinity of the radiolabeled ligand.

Since computer programs perform nonlinear regression analysis using every point in a titration curve simultaneously, there is greater confidence when measuring a large number of concentrations of inhibitor than when measuring binding at a few concentrations of inhibitor with replicate measurements. In addition, determinations of total and residual binding must be very accurate for nonlinear curve-fitting routines. It is not strictly correct to refer to radioligand binding that remains at high concentrations of an inhibitor as nonspecific binding, since it may be bound to a site not inhibited by the test compound.

One explanation for such behavior may be that the inhibitor acts by allosterically modifying the receptor without necessarily binding to the same site as the radioligand. Nonlinear regression programs make no assumptions about how much binding may exist in the absence of test compound or whether the inhibition of binding by a test compound represents 100% inhibition of specific receptor binding. The best way to obtain accurate determinations of the binding in the absence of inhibitor and in the presence of large excess of inhibitor is by including several concentrations of test compound that do not inhibit binding at all and several that inhibit binding fully. These data points are very important to determine the goodness of fit of the data by nonlinear regression. A standard procedure is to test twelve concentrations of competitor at half-log intervals in the twelve wells of a single row in a 96-well microtiter plate (see Table 1.4.2).

  1. Prepare a stock solution of unlabeled test compound (competitor) at 100-fold the highest concentration to be tested.


    2. Most compounds are soluble in dimethyl sulfoxide which, at final concentrations up to 1%, has no effect on either total or nonspecific binding of opioid ligands. It is prudent to have previously determined the effects of common solvents on the binding of the particular ligand and receptor being tested or concomitantly run controls for the solvent.

  2. In a 96-well microtiter plate other than the assay plate, dilute the stock solution 1:10 in assay buffer into a well in column 12 of the plate.

    3.  
  3. Serially dilute this solution eleven times, combining 2.16 parts assay buffer with 1 part test compound solution (1/3.16), across the twelve wells in the row.

    4.  

     

    This results in a concentration range of nearly 6 orders of magnitude (see Table 1.4.2).

  4. Use a multichannel pipet to transfer 50-µl aliquots of these samples to the assay plate, resulting in an additional 10-fold dilution if the assay volume is 500 µl (see Basic Protocol 1, step 11).

    5.  

     

    Use 50 µl in Alternate Protocol step 11.

  5. Perform the binding assay (see Basic Protocol 1 for binding to cloned receptors; see Alternate Protocol for binding to receptors in tissue membrane homogenate).

    6.  
  6. Perform appropriate data analysis (see Support Protocol).

    7.  

SUPPORT PROTOCOL

DATA ANALYSIS

Curve-fitting programs such as GraphPad Prism (GraphPad Software) fit the data using nonlinear regression analysis of a four-parameter equation (DeLean et al., 1978) that determines the plateaus of the curve from the competition data rather than from separately determined total and nonspecific binding determinations. In fact, in a scale of the logarithms of concentrations of inhibitors it is not possible to include a value for zero inhibitor. Moreover, the preferred analysis requires no assumption that two inhibitors will block all of the radioligand binding to the same sites. This may be especially relevant in opioid receptor binding, where different chimeric receptor constructs yield different effects on the binding of ligands of various structural classes previously assumed to bind to a single site. Furthermore, Mansour et al. (1995a) have shown that the maximum displacement of [3H]EKC binding by certain peptides is less than the maximum displacement by naloxone or unlabeled ethylketocyclazocine. Accordingly, it should not be assumed that the maximum extent of inhibition of specific binding is the same for every competitor.

Analysis of these data using Prism yields the following data in the format shown in Table 1.4.3, where BOTTOM is the theoretical binding in the presence of an infinite dose of inhibitor, TOP is the binding in the absence of inhibitor, and the EC50 value is the concentration of inhibitor resulting in an extent of binding halfway between BOTTOM and TOP and therefore is a measure of the relative affinity of the inhibitor as illustrated in Figure 1.4.1.

REAGENTS AND SOLUTIONS

Use deionized, distilled water in all recipes and protocol steps. For common stock solutions, see APPENDIX 2A; for suppliers, see SUPPLIERS APPENDIX.

HEPES assay buffer

50 mM HEPES, pH 7.4 at room temperature
0.5 mg/liter aprotinin
200 mg/liter bacitracin
10 mg/liter leupeptin
10 mg/liter pepstatin A
Store at 4°C (stable at least one month)
The buffer can be stored overnight at room temperature to ensure equilibration for running the experiment the next day.

Tris assay buffer

50 mM Tris·Cl, pH 7.8 (APPENDIX 2A)
1.0 mM EGTA (free acid)
5.0 mM MgCl2
10 mg/liter leupeptin
10 mg/liter pepstatin A
200 mg/liter bacitracin
0.5 mg/liter aprotinin
Store at 4°C (stable at least 1 month)
Use of the sodium salt of EGTA and of NaOH to adjust the pH of the Tris buffer should be avoided because sodium inhibits agonist binding to opioid and other G protein-linked receptors in membrane preparations. The affinity of an agonist may be reduced to the point where binding can no longer be detected in a filtration assay. The protease inhibitors are needed to protect certain peptides that may be used as competitors or radiolabeled ligands. Since it is not practical to evaluate proteolysis for each peptide being investigated, it is best to include a variety of protease inhibitors in all binding assays. These protease inhibitors may also stabilize the receptor and should be included in all tubes in an assay.

The buffer can be stored overnight at room temperature.

COMMENTARY

Background Information

The role of endogenous opioids in the modulation of pain, and the efficacy of morphine and similar drugs in providing pain relief, underscores the importance of developing novel agents that interact with opioid receptor subtypes but lack the untoward side effects of constipation, respiratory depression, tolerance, and addiction generally associated with compounds active at these receptors. The opioid receptor was the first neurotransmitter receptor for which a radioligand binding filtration assay was described (Pert and Snyder, 1973; Terenius, 1973; Simon et al., 1973). From the 20 or more years of molecular pharmacology studies and in vivo and ex vivo pharmacology, a wide variety of radiolabeled ligands and other molecular tools are available to study opioid receptor binding and function. Although at least seven opioid receptors have been characterized pharmacologically in various tissue preparations, only three have been cloned (Table 1.4.1). These are termed µ, k, and d based on the relative affinities of morphine, ethylketocyclazocine, and the enkephalins for these sites (Simon, 1991). The recent cloning and expression of these receptors from both rodent and human provides a unique opportunity to study interactions of opioid and opioid-like compounds at the receptor level. Furthermore, the availability of opioid receptor clones obviates the need to use animals as a source of tissue and makes it possible to study a single opioid receptor subtype, eliminating the need to block binding to other opioid receptors when nonselective radioligands such as diprenorphine are used. The methods described in this unit are useful for both routine affinity determinations and high-throughput screening of compound libraries.

Scatchard analysis and kinetic measurements are reviewed in UNITS 1.2 & 1.3. Prior to conducting routine experiments with a new assay, saturation curves, association rates, and dissociation rates should be determined experimentally. The reader is referred to the work of Gillan and Kosterlitz (1982), Lahti et al. (1985), Mansour et al. (1995b), and Xue et al. (1994) as a starting point for comparison of literature values to experimental data.

Critical Parameters

The following checklist for validating a radioreceptor assay provides a summary of the general criteria that must be met to satisfy the assumptions of the equations used in calculating binding parameters.
  1. Is specific binding linear with respect to the amount of membrane protein in the assay?

    2.  
  2. Is total binding <10% of added radioligand?

    3.  
  3. Is there significant binding to the filters?

    4.  
  4. Does the filter wash protocol remove enough nonspecific binding to maximize the signal-to-noise ratio without decreasing specific binding?

    5.  
  5. Does a time course show that binding has reached equilibrium at every concentration of radioligand that may be used?

    6.  
  6. Is specific binding sensitive to pH in the neutral range? If so, it may be necessary to monitor pH of stored buffers or the effects of test compounds on pH of the assay mixtures.

    7.  
  7. What is the effect of temperature on equilibrium binding and on the time needed to reach equilibrium? One might test 4°C, room temperature, and 37°C to determine an incubation temperature with desirable kinetic parameters.

    8.  
When using tissue homogenates, a number of other factors should also be considered. Generally, homogenates of whole brain or brain regions are used, although binding to heart (Zhang et al., 1996), adrenal medulla (Castanas et al., 1985a,b), kidney (Dissanayake et al., 1991), and liver (Simantov et al., 1978) homogenates has also been described. Care must be taken to select an animal species and tissue known to contain a large number of endogenously expressed receptors, as significant interspecies differences in the numbers of µ, d, and k receptors have been reported (Clark et al., 1988). The effects of ions on binding should be evaluated separately for each receptor. In addition to the inhibitory effects of sodium on the binding of agonists (Kosterlitz et al., 1987), ions may have differential effects on the binding of radioligands to receptor subtypes (Paterson et al., 1986). For example, the divalent salts CaCl2, MgCl2, and MnCl2 inhibit binding to k receptors but potentiate the binding of [3H](2-D-penicillamine, 5-D-penicillamine) enkephalin (DPDPE) to d receptors (Paterson et al., 1986).

Troubleshooting

When assay results are not consistent with published data, the troubleshooting guide provided in Table 1.4.4 should be consulted for guidance on where to begin searching for problems with these protocols and how to correct them.

Anticipated Results

The affinities (Ki) observed for selected reference compounds at the cloned human µ, d, and k receptors using [3H]diprenorphine as ligand are listed in Table 1.4.5. Tables 1.4.2 and 1.4.3 provide experimentally determined values (respectively, for competition binding by three compounds to be cloned d opioid receptor and output after fitting that data) for comparison with those listed in Table 1.4.5. This type of comparison is useful in providing additional information for assay validation and in comparing the affinities of novel compounds to these reference agents.

Time Considerations

The amount of time required to perform a receptor binding assay for opioid receptors will depend on the type of experiment, the time to reach equilibrium for the individual assay, and the number of data points tested and analyzed. After assay validation is complete, typically at least 60 titration curves can be determined in a day, and for general screening at a single concentration of drug, at least 750 data points can be generated in a day when assays are performed and filtered using a 96-well format.

Literature Cited

Castanas, E., Bourhim, N., Giraud, P., Boudouresque, F., Cantau, P., and Oliver, C. 1985a. Interaction of opiates with opioid binding sites in the bovine adrenal medulla: I. Interaction with d and µ sites. J. Neurochem. 45:677-687.

Castanas, E., Bourhim, N., Giraud, P., Boudouresque, F., Cantau, P., and Oliver, C. 1985b. Interaction of opiates with opioid binding sites in the bovine adrenal medulla: II. Interaction with k sites. J. Neurochem. 45:688-699.

Clark, M.J., Carter, B.D., and Medzihradsky, F. 1988. Selectivity of ligand binding to opioid receptors in brain membranes from the rat, monkey and guinea pig. Eur. J. Pharmacol. 148:343-351.

DeLean, A., Munson, P.J., and Rodbard, D. 1978. Simultaneous analysis of families of sigmoidal curves: Application to bioassay, radioligand assay, and physiological dose-response curves. Am. J. Physiol. 235:E97-E102.

Dissanayake, V.U.K., Hughes, J., and Hunter, J.C. 1991. Opioid binding sites in the guinea pig and rat kidney: Radioligand homogenate binding and autoradiography. Mol. Pharmacol. 40:93-100.

Gacel, G., Dauge, V., Breuze, P., Delay-Goyet, P., and Roques, B.P. 1988. Development of conformationally constrained linear peptides exhibiting a high affinity and pronounced selectivity for opioid receptors. J. Med. Chem. 31:1891-1897.

Gillan, M.G.C. and Kosterlitz, H.W. 1982. Spectrum of the µ-, d- and k-binding sites in homogenates of rat brain. Br. J. Pharmacol. 77:461-469.

Kosterlitz, H.W., Paterson, S.J., Robson, L.E., and Traynor, J.R. 1987. Effects of cations on binding, in membrane suspensions, of various opioids at µ-sites of rabbit cerebellum and k-sites of guinea-pig cerebellum. Br. J. Pharmacol. 91:431-437.

Lahti, R.A., Mickelson, M.M., McCall, J.M., and VonVoigtlander, P.F. 1985. [3H]U-69593, a highly selective ligand for the opioid k receptor. Eur. J. Pharmacol. 109:281-284.

Mansour, A., Hoversten, M.T., Mansson, E., Bare, L., Watson, S.J., and Akil, H. 1995a. Apparent evidence of receptor subtypes: Receptor binding studies with the cloned rat and human k receptors. Analgesia 1:4-6.

Mansour, A., Hoversten, M.T., Taylor, L.P., Watson, S.J., and Akil, H. 1995b. The cloned µ, d, and k receptors and their endogenous ligands: Evidence for two opioid peptide recognition cores. Brain Res. 700:89-98.

Paterson, S.J., Robson, L.E., and Kosterlitz, H.W. 1986. Control by cations of opioid binding in guinea pig brain membranes. Proc. Natl. Acad. Sci. U.S.A. 83:6216-6220.

Pert, C.B. and Snyder, S.H. 1973. Opioid receptor: Demonstration in nervous tissue. Science 179:1011-1014.

Raynor, K., Kong, H., Chen, Y., Yasuda, K., Yu, L., Bell, G.I., and Reisine, T. 1994. Pharmacological characterization of the cloned k-, d-, and µ-opioid receptors. Mol. Pharmacol. 45:330-334.

Schiller, P.W., Weltrowska, G., Nguyen, T.M.-D., Wilkes, B.C., Chung, N.N., and Lemieux, C. 1993. TIPPy: A highly potent and stable pseudopeptide d opioid receptor antagonist with extraordinary d selectivity. J. Med. Chem. 36:3182-3187.

Simantov, R., Childers, S.R., and Snyder, S.H. 1978. [3H]Opioid binding: Anomalous properties in kidney and liver membranes. Mol. Pharmacol. 14:69-76.

Simon, E.J., Hiller, J.M., and Edelman, I. 1973. Stereospecific binding of the potent narcotic analgesic [3H]etorphine to rat brain homogenate. Proc. Natl. Acad. Sci. U.S.A. 70:1947-1949.

Simon, E.J. 1991. Opioid receptors and endogenous opioid peptides. Med. Res. Rev. 11:357-374.

Terenius, L. 1973. Characteristics of the "receptor" for narcotic analgesics in synaptic plasma membrane fraction from rat brain. Acta Pharmacol. Toxicol. 33:377-384.

Xue, J.-C., Chen, C., Zhu, J., Kunapuli, S., DeRiel, J.K., Yu, L., and Liu-Chen, L.-Y. 1994. Differential binding domains of peptide and non-peptide ligands in the cloned rat k opioid receptor. J. Biol. Chem. 269:30195-30199.

Zhang, W.-M., Jin, W.-Q., and Wong, T.M. 1996. Multiplicity of k opioid receptor binding in the rat cardiac sarcolemma. J. Mol. Cell. Cardiol. 28:1547-1554.

Key References

Knapp, R.J., Malatynska, E., Collins, N., Fang, L., Wang, J.Y., Hruby, V.J., Roeske, W.R., and Yamamura, H.I. 1995. Molecular biology and pharmacology of cloned opioid receptors. FASEB J. 9:516-525.

Raynor et al., 1994. See above.

Detailed description of binding to cloned opioid receptor subtypes, including an extensive evaluation of reference agents at each cloned receptor subtype.

Figure 1.4.1 [Back to text]

Competition curves for reference compounds generated from Prism software. Shown is data demonstrating inhibition of [3H]diprenorphine binding to the d receptor by competition with unlabeled diprenorphine, bremazocine, and levorphanol.

Table 1.4.1 Characteristics of Cloned Opioid Receptorsa
 
Receptor GenBank accession
number (human clone)		 Agonists Antagonists
µ  P35372 DAMGO, Sufentanil, PLO17  CTOP
d P41143  DPDPE, BUBU  Naltrindole, TIPPy
k P41145  U69,593, CI977, ICI 197067  Nor-binaltorphimine

a Abbreviations: BUBU, H-Tyr-D-(o-t-Bu)-Gly-Phe-Leu-Thr(o-t-Bu)-OH; CI977, [5R-5a,7a,8b]-N-methyl-N-[7-(1-pyrrolidinyl)1-oxaspiro[4,5]dec-8-yl]-4-benzofluranacetamide; CTOP, H-D-Phe-Cys-Tyr-D-Trp-Orn-Thr-Pen-Thr-NH2; DAMGO, (D-Ala2, N-Me-Phe4, glycinol5)-enkephalin; DPDPE, (D-Pen2, D-Pen5)-enkephalin; ICI 197067, (2S)-N-[2-(N-methyl-3,4-dichlorophenylacetamido)-3-methylbutyl]pyrrolidine HCl; PLO17, Tyr-Pro-MePhe, D-Pro-NH2; TIPPy, H-Tyr-Tic[CH2NH]-Phe-Phe-OH; U69,593, (5,7,8b)-N-methyl-N-[7-(1-pyrrolidinyl)1-oxaspiro[4,5]dec-8-yl]benzeneacetamide.

Table 1.4.2 Example of Titrations of Inhibitors of [3H]Diprenorphine Binding to Cloned d Opioid Receptorsa
 
[Inhibitor] log[inhibitor]
(M)		 dpm bound
    ±-Bremazocine Levorphanol Diprenorphine
3.16 pM ­11.5 2306    
10 pM ­11.0 2375    
31.6 pM ­10.5 2386 2435 2238
100 pM ­10.0 2312 2355 2234
316 pM ­9.5 2085 2359 1997
1.0 nM ­9.0 2014 2324 1437
3.16 nM ­8.5 1701 2088 997
10 nM ­8.0 1229 2192 608
31.6 nM ­7.5 846 1693 518
100 nM ­7.0 487 1270 409
316 nM ­6.5 425 926 427
1.0 µM ­6.0 391 570 388
3.16 µM ­5.5   475 350
10 µM ­5.0   390 357

a Source of receptor was human d receptor stably expressed in CHO cells; 60 µg protein was added to each assay well. Total dpm added to each well was 43,600 dpm (1.0 nM); total binding determined in the absence of inhibitor was 2684 dpm; and nonspecific binding determined in the presence of 10 µM naloxone was 371 dpm.

Table 1.4.3 Example of Data Output from Prism for EC50 Determinationsa


 
  (±) Bremazocine Levorphanol Diprenorphine
Variables      
BOTTOM 395.8 436.4 387.8
TOP 2307 2340 2328
LOG EC50 ­8.137 ­7.112 ­8.872
EC50 7.301e­009 7.721e­008 1.341e­009
Std. Error      
BOTTOM 46.21 56.32 17.37
TOP 33.31 40.04 33.14
LOG EC50  0.06027 0.07324 0.03602
95% Confidence Intervals      
BOTTOM 291.3 to 500.3 309.0 to 563.8 348.5 to 427.1
TOP 2232 to 2383 2249 to 2430 2253 to 2403
LOG EC50 ­8.273 to ­8.000 ­7.278 to ­6.947 ­8.954 to ­8.791
EC50 5.33e­9 to 9.99e­9 5.27e­8 to 1.13e­7 1.11e­9 to 1.61e­9
Goodness of Fit      
Degrees of Freedom 9 9 9
R2 0.9933 0.9901 0.9975
Absolute Sum of Squares 49,810 72,660 16,220
Sy.x 74.39 89.85 42.45

a Units of EC50 are molar (M). Exponent notation: 5.33e­8 = 5.33 × 10­8. Sy.x is the standard deviation of the residuals (vertical distances of the points from the line); its value is expressed in the same units as Y, in this case dpm.

Table 1.4.4 Troubleshooting Guide for Correcting Problems with a Receptor Binding Assay
 
Problem Possible cause Solution
No binding Receptor is absent or present at low concentration Try higher protein concentration and longer incubation
Specific binding has been washed away in the filter rinse step Check whether the expected affinity (Kd) of the ligand is at or below 50 nM; lower-affinity binding is difficult to measure in a filtration assay because specific binding can be removed by the filter wash
Ligand or receptor has degraded (has been metabolized by enzymes in the membrane prep or may be chemically unstable under the assay conditions used) Add inhibitor(s) of enzymes known or suspected to degrade ligand; these might include protease inhibitors, antioxidants, or inhibitors or enzymes known to metabolize ligand in vivo. Optimize assay conditions (e.g., reduce temperature).
Very high binding (>10% of total radioactivity added) Membrane concentration is too high Try a lower protein concentration
Ligand binding to the filters has occurred Peptide ligands or charged ligands often bind to glass fiber filters. For peptides or positively charged ligands, try presoaking the filters in 0.5% polyethyenimine (PEI) and 0.1% bovine serum albumin (BSA) in rinse buffer; for negatively charged ligands, try BSA alone on the filter or in the assay buffer. It is important to be aware that the ligand may bind to BSA, reducing the concentration of free ligand in the assay; hence, it may be better to presoak the filters in BSA rather than adding it to the assay.
Binding that is not linear with protein concentration Too much membrane prep has been used in the assay Try a lower protein concentration
Binding to the filters has been inhibited by protein or other components of the membranes  Filter binding may decrease as protein concentration in the assay is increased, resulting in both total and specific binding that does not seem to show good linearity with protein concentration. Try boiling a portion of a membrane prep to denature the receptor and then adding boiled membranes in decreasing amounts to the increasing amounts of receptor preparation so that the overall protein concentration is constant. It may not be necessary to continue this practice in routine assays if the binding is small or limited to nonspecific binding.
Ligand has been metabolized by enzyme(s) in the membrane preparation The rate of metabolism may increase as the protein concentration is increased. If a mechanism for the metabolism is known, add an appropriate enzyme inhibitor, if available. Alternatively, perform the assay on ice to reduce metabolism while retaining receptor binding.
Binding that reaches a peak but then declines over time Ligand has been metabolized by enzyme(s) in the membrane preparation See above (under No binding)
Receptor has degraded See above (under No binding)
Ligand has aggregated, precipitated, or bound to the container Try adding a small amount of a carefully tested solvent or detergent. The best solvents would probably have some polar character, e.g., DMSO or methanol. The effects of detergents on the receptor, membrane, or ligand are difficult to predict, so the optimal detergent must be determined empirically. Diprenorphine is a ligand of choice, especially when used in conjunction with cell ligands selectively transfected with a given receptor. This ligand is chemically very stable (see Commentary).

Table 1.4.5 Affinity Constants (Ki) of Reference Agents for the Cloned Human µ, d, and k Receptors


 
Compound Ki values (nM) Compound source
µ d k
b-Funaltrexamine 0.9 12 1.4 Tocris Cookson
Bremazocine 0.85 0.92 0.12 RBI
BUBUb 4.75 4.7 ND Bachem
CTOP 3.5 >10,000 >10,000 Bachem
DAMGO 14 >10,000 >10,000 Bachem
Dextrorphan >10,000 >10,000 >10,000 RBI
Diphenoxylate 54 310 >1,000 RBI
Diprenorphine 0.27 0.31 0.20 RBI
DPDPE >10,000 2.2 >10,000 Bachem
Fentanyl 13 310 900 RBI
GR89696 31 76 0.51 RBI
Levorphanol  2.3 8.6 7.7 RBI
Morphine 19 220 230 RBI
Nalbuphine 6.0 140 50 RBI
Naloxone 2.3 24 12 RBI
Naloxone benzoylhydrazone 0.54 4.1 1.0 RBI
Nor-binaltorphimine 51 7.2 0.042 RBI
SNC-80 >1,000 0.47 >1,000 Tocris Cookson
Spiradoline 150 >1,000 2.3 RBI
Sufentanylc 0.15 50 75 Janssend
TIPPya 3228 0.31 >1,000 McGill Universityd
U-50,488 >1,000 >1,000 4.2 RBI
U-69,593  720  >1,000 12  RBI

a Data from Schiller et al. (1993).
b Data from Gacel et al. (1988).
c Data from Raynor et al. (1994).
d Not commercially available.
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