Radioligand Binding to Quantify Adrenergic Receptor Expression in the Heart

Abstract

β-adrenergic receptors regulate cardiac function in the healthy and failing heart. Expression of β-adrenergic receptors is decreased in heart failure due to the chronic overactivation of the sympathetic nervous system, which contributes to declines in cardiac function and disease progression. Furthermore, therapies that prevent β-adrenergic receptor downregulation or restore β-adrenergic receptor levels are beneficial making the determination of cardiac β-adrenergic receptor expression an important consideration in the heart. While quantitative RT-PCR can provide an indication of β-adrenergic receptor density and subtype expression, mRNA does not always correlate with functional protein levels. Additionally, β-adrenergic receptor antibodies lack specificity making immunoblotting and other antibody-based techniques unreliable. Radioligand binding assays were developed over 50 years ago and remain the gold standard for quantifying β-adrenergic receptor densities in biological samples. This technique capitalizes on the binding of high affinity, highly specific ligands to receptors and can give quantifiable levels of receptor expression. Furthermore, competition assays using subtype-selective antagonists generate binding profiles and can differentiate β-adrenergic receptor subtype expression in cardiac tissue. This article focuses on the quantification of β-adrenergic receptors in the heart using saturation and competition radioligand binding techniques to quantify β-adrenergic receptor density and ligand affinities in cardiac membranes.

INTRODUCTION:

Heart failure is the leading cause of death in the United States and worldwide resulting in tremendous morbidity, mortality and economic burden (Roth et al., 2020; Tsao et al., 2022). While heart failure encompasses a number of conditions with varying etiologies, it shares a common outcome of the heart being unable to pump enough blood to meet the body’s needs. In order to compensate for this decline in cardiac function, several neurohormonal mechanisms are activated to maintain cardiac output. A primary neurohormonal mechanism activated to restore cardiac output is the sympathetic nervous system. Activation of the sympathetic nervous system results in the release of the catecholamines epinephrine and norepinephrine, which activate adrenergic receptors to mediate physiological responses.

Adrenergic receptors belong to the G protein-coupled receptor superfamily of receptors (GPCRs). Adrenergic receptors are divided into three main types based on the signaling pathways they activate, α1-, α2- and β-adrenergic receptors, which are further divided into 9 subtypes (α1A, α1B, α1D, α2A, α2B, α2C, β1, β2, β3). Variations in adrenergic receptor subtype expression between cell types and tissues partially accounts for differential responses to catecholamine stimulation. Of particular importance in the context of the heart, β-adrenergic receptors are the predominant adrenergic receptor type present, with α1-adrenergic receptors being expressed at lower levels. There are large differences in adrenergic receptor expression between cell types of the heart, with β1-adrenergic receptors being highly expressed in cardiomyocytes whereas β2- and β3-adrenergic receptors have low or no expression in cardiomyocytes and high expression on non-myocyte populations such as fibroblasts, endothelial cells and immune cells (Myagmar et al., 2017).

During heart failure, there is a chronic activation of the sympathetic nervous system as the body’s attempt to compensate for declines in the heart’s capacity to pump blood and maintain adequate function. This occurs primarily through the highly expressed β1-adrenergic receptor subtype on cardiomyocytes which, acting through canonical Gαs signaling mechanisms and PKA activation, can positively regulate inotropy and chronotropy. However, this overactivation of the sympathetic nervous system and subsequent prolonged stimulation of cardiac adrenergic receptors results in downregulation of cardiomyocyte β1-adrenergic receptors through GRK2 and β-arrestin-dependent mechanisms. Thus, decreased β-adrenergic receptor levels represent a hallmark of heart failure and therapies that preserve or restore β-adrenergic receptor expression are beneficial, making β-adrenergic receptor density an important readout for cardiac function and heart failure progression (de Lucia, Eguchi, & Koch, 2018). Additionally, β-blockers are a main treatment for numerous cardiac conditions including compensated, congestive heart failure, arrhythmias and angina. Of particular note, in compensated congestive heart failure, use of β-blockers restores β1-adrenergic receptor density on cardiomyocytes, which improves contractile function of the heart (de Lucia, Eguchi, & Koch, 2018). G protein-coupled receptors are notoriously difficult to generate specific antibodies for, hindering efforts at protein quantification (Michel, Wieland, & Tsujimoto, 2009). This includes antibodies for adrenergic receptors, which have been shown in several independent studies from different research groups to be non-specific (Bohmer, Pfeiffer, & Gericke, 2014; Hamdani & van der Velden, 2009; Jensen, Swigart, & Simpson, 2009; Pradidarcheep et al., 2009). Due to the unreliability of antibodies, radioligand binding remains the gold standard for detecting G protein-coupled receptor protein expression in cells and tissues. Receptor-ligand binding assays are a classical pharmacological tool used to monitor the properties of receptor-ligand interactions and remain the gold standard method in receptor research (Flanagan, 2016). This relatively simple tool can be used to determine key characteristics of a receptor system including the affinity of compounds for a given receptor, changes in receptor number and receptor subcellular localization.

Ligand binding assays rely on the binding of a ligand, such as an antagonist or agonist, to a molecule of interest, including a receptor, and a detection method to quantify binding. In the case of radioligand binding assays, ligands have a radioisotope incorporation, commonly tritium (³H) or iodine-125 (¹²⁵I), that can be detected using liquid scintillation or gamma counting, respectively. Classic radioligand binding assays use a filtration-based system where the receptor of interest is incubated with an appropriate radioligand and the reaction is terminated by filtration to remove the unbound radioligand. The receptor bound radioligand is then quantified. For membrane bound receptors, such as GPCRs, this generally involves membrane isolation however, whole cell or tissue slide assays can also be performed. There are three main types of radioligand binding experiments, which provide different information about the ligand-receptor interaction of interest. Saturation binding is used to determine the affinity of the radioligand for the receptor and the binding site density. These types of experiments are useful for determining the β-adrenergic receptor density in the heart. Competition binding is used to quantify the affinity of a competing, unlabeled ligand. By using subtype selective ligands, relative β-adrenergic receptor subtype expression can be identified using this method. Kinetic experiments determine the forward and reverse rate constants for radioligand binding. While these experiments are useful for determining binding properties of ligands, they will not be discussed further.

CAUTION:

Radioactive substances including iodine-125 (¹²⁵I) are health hazards. Follow all appropriate guidelines and regulations for the use and handling of radioactive substances including proper personal protective gear, monitoring of surface contamination by Geiger-Mueller detector with NaI scintillation probe and swipe surveys, appropriate shielding and dosimeter monitoring when working with ¹²⁵I-cyanopindolol (¹²⁵I-CYP).

STRATEGIC PLANNING:

Radioligand binding requires a receptor source, traditionally membranes isolated from cells or tissue. The following protocol is suitable for membrane isolation from fresh or flash frozen heart tissues. Following membrane isolation, it is recommended to determine the optimal membrane concentration for subsequent saturation and competition binding experiments. This will determine the lowest amount of membrane necessary to give the largest specific binding (Figure 4). However, radioligand binding for β-adrenergic receptors in the heart is frequently performed and this step can be omitted. Membrane concentrations determined experimentally or from literature references are subsequently used for saturation binding experiments to determine the B_max, which represents the β-adrenergic receptor density (Figure 5). Further competition binding experiments can be performed to determine β-adrenergic receptor subtype expression based on binding characteristics of subtype-selective ligands (Figure 6).

Figure 4.

Representative data generated from binding to determine optimal protein concentration. Total and non-specific binding reactions are performed with constant amounts of ¹²⁵I-CYP and the non-radiolabeled antagonist propranolol, while membrane concentrations are varying. Total binding is depicted by the grey line whereas non-specific binding is shown in black. Specific binding, equal to total binding minus the non-specific binding, is greatest at concentrations higher than 10 μg (outlined in red). Optimal membrane concentration for saturation and competition binding is the lowest membrane concentration that has a large degree of specific binding, which is 10-20 μg.

Figure 5.

Representative data generated from saturation binding experiments. A. Total and non-specific binding reactions are performed with constant amounts of membrane and the non-radiolabeled antagonist propranolol and varying concentrations of ¹²⁵I-CYP. Total binding is depicted by the grey line whereas non-specific binding is shown in black. Specific binding, equal to total binding minus the non-specific binding is shown in red. B. From the specific binding curve, the B_max, which represents maximal binding, can be determined and is the β-adrenergic receptor density for the sample. The K_d can also be determined and is a measure of the ligand’s affinity for the receptor.

Figure 6.

Representative data generated from competition binding experiments. Varying concentrations of the β1-adrenergic receptor selective antagonist, GCP 12177A, competes for ¹²⁵I-CYP binding sites on membranes isolated from heart. The % of ¹²⁵I-CYP inhibition results in a two-site binding curve due to the presence of high affinity β1-adrenergic receptor binding sites and a low affinity β2-adrenergic receptor binding sites on cardiac membranes.

BASIC PROTOCOL 1: Radioligand Binding to Quantify Adrenergic Receptor Expression in the Heart.

Traditional radioligand binding experiments quantify GPCR expression on membranes, thus requiring a membrane isolation of your desired cell or tissue type. This step is performed using a cellular or tissue lysis protocol followed by differential centrifugation. Alternatively, whole cell binding can be performed on isolated cell. Following sample preparation, it is beneficial to determine the optimal sample amount for running subsequent binding assay. This is performed using a set amount of radioligand and non-radiolabeled ligand to label receptors with increasing concentrations of protein (Figure 4). This will allow the researcher to determine the concentration of protein that gives the largest degree of separation between the specific and non-specific binding as well as the lowest protein concentration that gives this optimal specific binding without using excessive protein concentrations and wasting sample. From this experiment, the optimal protein concentration is determined for subsequent saturation or competition binding experiments.

Saturation binding provides useful information regarding the number of receptors expressed on the researcher’s sample of interest and the dissociation constant (K_d) of the ligand (Figure 5). This type of binding experiment involves using a constant amount of protein and non-radiolabeled ligand while varying the amount of radioligand. The concentration of non-radiolabeled ligand should be in excess of the radiolabeled ligand in order to prevent specific binding of the radioligand and allow the researcher to calculate specific binding of a ligand for a given receptor. Radioligand concentrations should span a range of several points below and above the K_d of the ligand in order to accurately quantify the K_d of the given ligand. Furthermore, higher concentrations of the radioligand should reach a plateau or the point where all receptor binding sites become occupied by the ligand in order to accurately calculate a B_max, which represents the receptor density for a given sample.

Competition binding uses a fixed concentration of protein and radioligand while varying non-radiolabeled ligand amounts to inhibit the binding of the radioligand (Figure 6). This type of experiment will also provide a dissociation constant (K_d), that is often converted into an affinity (K_i) of a ligand using the Cheng-Prusoff transformation. This is particularly useful when subtype-selective inhibitors for receptors are used and can help differentiate receptor isoforms that cannot be differentiated using a non-selective radioligand.

Once equilibrium is reached for binding reactions, reactions are collected using a filtration manifold system such as a Brandel Harvester that collects the samples on filter paper. Subsequent washing removes all unbound ligands leaving the radioligand bound receptor on the filter paper, which can be collected and analyzed by scintillation or gamma counting. Figure 1 shows a general protocol outline where membranes are isolated from heart tissue. These membranes contain the receptor of interest, β-adrenergic receptors, that are then incubated in the presence of radioligand, ¹²⁵I-CYP, with or without the non-radiolabeled antagonist, propranolol. Membranes incubated with ¹²⁵I-CYP represent the total counts whereas propranolol blocks ¹²⁵I-CYP binding to the receptor, giving the non-specific binding. Upon binding equilibrium, samples are collected using a Brandel Harvester, which accumulates the reaction on filters. Washing of the filter removes all unbound ligand leaving the radioligand bound to receptor complex on the filter to collect for analysis by gamma counting.

Figure 1.

General protocol schematic for saturation binding experiments. Isolated membranes are generated from the heart and serve as a receptor source for binding studies. Binding reactions are performed for total binding, with the radioligand ¹²⁵I-CYP alone and non-specific Binding using ¹²⁵I-CYP plus an excess of non-radiolabeled ligand propranolol. Incubation of reactions allows binding to equilibrate and membranes are collected using a Brandel Harvester for analysis by gamma counting.

Materials:

Tris pH 7.4 stock solution (see recipe)

Ethylenediaminetetraacetic acid (EDTA) stock solution (see recipe)

1000x Aprotinin stock solution (see recipe)

1000x Leupeptin stock solution (see recipe)

Lysis buffer (see recipe)

Protein assay (Pierce Coomassie Plus (Bradford) Assay Kit cat # 23236 or equivalent)

Glycerol (optional)

Binding buffer (see recipe)

Magnesium chloride (MgCl₂) stock solution (see recipe)

Methanol (e.g. Fisher Scientific cat # A452SK-4)

5x Propranolol stock solution (see recipe)

Iodo-(±)-cyanopindolol [¹²⁵I] (¹²⁵I-CYP; Perkin Elmer cat # NEX174)

0.1% Bovine serum albumin (BSA) solution (see recipe)

Wash buffer (see recipe)

CGP 20712A (Tocris cat # C231)

ICI 118,551 (Sigma-Aldrich cat # I127)

Tissue homogenizer (e.g. Next Advance Bullet Blender Bead Homogenizer or equivalent)

1.7 mL microcentrifuge tubes (e.g. Corning Costar microcentrifuge tubes with snap cap cat # CLS3620 or equivalent)

Refrigerated microcentrifuge (e.g. Eppendorf Microcentrifuge 5430R or equivalent)

12 x 75 mm borosilicate glass test tubes (e.g. Thomas Scientific cat # TT-1505 or equivalent)

12 x 75 mm polypropylene assay tubes (e.g. Thomas Scientific cat # 1177J54 or equivalent)

Shaking water bath (e.g. Shel Lab Shaking Water Bath Model SWB-27 or equivalent)

Filtration Manifold System (e.g. Brandel Harvester Model M-48 or equivalent)

Whatman GF/C Filter Paper (Whatman cat # FP-205 or equivalent)

Gamma Counter (e.g. Perkin Elmer Wizard 2 1-Detector Gamma Counter or equivalent)

Protocol:

Membrane Preparation:

All steps should be carried out with cold buffers and performed at 4°C to minimize protein degradation.

1. Homogenize hearts, in 1-2 mL of cold lysis buffer using a Next Advance Bullet Blender Bead Homogenizer on setting 3 for 5 min

   Excised hearts can be immediately used or flash frozen and stored at −80°C until use. Using 1 mL of lysis buffer per ¼ mouse heart provides enough protein for one competition assay (24 samples).

2. Incubate samples on ice for 15 min.

3. Centrifuge samples in 1.7 mL microcentrifuge tubes in a microcentrifuge at 2000 rpm for 5 minutes.

   This step removes nuclei, mitochondria and other cellular debris.

4. Transfer supernatant to fresh, high-speed centrifuge tube.

5. Centrifuge sample at 30,000 x g for 25 minutes.

6. Resuspend pellet in binding buffer by pipetting up and down.

   Avoid vortexing membranes, which can cause degradation. 200 μL of buffer works well for ¼ mouse heart. Include 10% glycerol in binding buffer if storing membranes for future use.

7. Quantify protein concentration using a protein assay.

8. Samples can be used immediately or stored with 10% glycerol at −80°C until use.

   Store samples in single use aliquots to minimize repeated freeze/thawing of membranes which can result in sample degradation.

Radioligand Binding to Determine Protein Amount:

All steps should be carried out with cold buffers and performed at 4°C to minimize protein degradation. Before starting, warm water bath (or incubator) to 37°C, make buffers and chill buffers to 4°C.

1. On ice, set up reaction tubes (4 mL; 12 x 75 mm polypropylene assay tubes) in a binding rack on ice performing all reactions in duplicate.

   Most Brandel Harvesters are for 24 or 48 samples. All spaces must contain a tube regardless of the number of samples. Reactions can be performed at various volumes by adjusting the stock concentrations so the final reaction concentrations are equivalent to those outlined in this protocol. The following protocol will be for a 250 μL reaction volume.

2. Dilute membranes from the “Membrane Preparation” to the desired reaction concentration in ice cold Binding Buffer for a final volume of 150 μL per reaction.

   Perform each concentration in duplicate for both Total Binding and Non-Specific Binding reactions. Thus, it is recommended to make 750 μL of membrane per condition to ensure adequate volumes of sample. Remaining diluted membrane can be analyzed by protein assay to ensure the expected concentration following dilution. For heart samples, 25 μg of protein is commonly used for radioligand binding experiments so performing a protein curve with concentrations of 0, 2.5, 5, 10, 25, 50 μg of membrane per reaction is a suggested range.

3. Add 50 μL of Binding Buffer to all Total Binding reactions.

   Total Binding reactions will include 150 μL membrane, 50 μL Binding Buffer and 50 μL ¹²⁵I-CYP for a total reaction volume of 250 μL.

4. Add 50 μL of 5x Propranolol Stock Solution to all Non-Specific Binding Tubes.

   Non-Specific Binding reactions will include 150 μL membrane, 50 μL Propranolol and 50 μL ¹²⁵I-CYP for a total reaction volume of 250 μL.

5. Add 150 μL of diluted membranes from Step 2 to the appropriate tubes.

6. Dilute ¹²⁵I-CYP to a 1 x 10⁻⁹ M stock in Binding Buffer.

   Since ¹²⁵I labeled compounds lose activity rapidly, the concentration of ¹²⁵I-CYP received from the vendor needs to be calculated daily using the following equation: (0.5^(x/t1/2)) (0.1 Ci/L) / 2.2x10⁶ Ci/mol=concentration in the stock vial with t1/2 being the half-life of the radioligand, which is 60 days for ¹²⁵I, x being the days past the initial concentration, 0.1 Ci/L being the stock concentration at x=0 and 2.2x10⁶ Ci/mol being the specific activity of the radioligand.

7. Add 50 μL of ¹²⁵I-CYP (1 x 10⁻⁹ M stock solution) to all tubes (200 pM final reaction concentration).

8. Briefly vortex all tubes to mix the reaction

9. Incubate samples at 37°C for 1 h in a shaking water bath.

   This allows for the reaction to reach equilibrium.

10. Wash the harvester 5x by filling a rack of empty tubes with ~1 mL of cold Wash Buffer and aspirating the Wash Buffer.

    This ensures the buffer in the harvester lines is clean and fresh prior to experimentation.

11. Prepare GC/F filters by soaking them in 0.1 % BSA solution and placing them on the harvester.

    0.1 % BSA reduces non-specific binding to the filters. When placing the GC/F filters on the harvester, ensure that all of the mesh is covered. This is necessary to generate an appropriate vacuum to aspirate the reaction and buffer.

12. Using a Brandel Harvester, aspirate the reaction.

13. Wash the reaction tubes 5x with ~ 1 mL cold Wash Buffer.

    Ensuring that the Wash Buffer is cold and performing the washes rapidly will prevent dissociation of the radioligand-receptor complex.

14. Using forceps and only touching the outer edge, place filter circles into prelabeled gamma counter tubes (12 x 75 mm polypropylene assay tubes or equivalent).

    Ensure filter circles are at the bottom of the gamma counter tubes prior to reading.

15. Count samples on a gamma counter using the appropriate program for ¹²⁵I.

    Include 50 μL of the ¹²⁵I-CYP stock in triplicate to confirm final concentration.

16. Wash harvester with ~2 L water and end with a 10% ethanol wash.

Saturation Binding:

All steps should be carried out with cold buffers and performed at 4°C to minimize protein degradation. Before starting, warm water bath (or incubator) to 37°C, make buffers and chill buffers to 4°C. Figure 2 depicts an example of an experimental layout for saturation binding experiments.

• 1.
  On ice, set up reaction tubes (4 mL; 12 x 75 mm polypropylene assay tubes) in a binding rack on ice performing all reactions in duplicate.

  Most Brandel Harvesters are for 24 or 48 samples. All spaces must contain a tube regardless of the number of samples. Reactions can be performed at various volumes by adjusting the stock concentrations so the final reaction concentrations are equivalent to those outlined in this protocol, but the following protocol will be for a 250 μL reaction volume.

• 2.
  Dilute membranes from the “Membrane Preparation” to the desired reaction concentration in ice cold Binding Buffer for a final volume of 150 μL per reaction.

  Perform each concentration in duplicate for both Total Binding and Non-Specific Binding reactions. Remaining diluted membrane can be analyzed by protein assay to ensure the expected concentration following dilution. For heart samples, 25 μg of membrane is commonly used for radioligand binding experiments however, optimal membrane concentration determined from the “Radioligand Binding to Determine Protein Amount” protocol above is recommended.

• 3.
  Add 50 μL of Binding Buffer to all Total Binding reactions.

  Total Binding reactions reflect the amount of ¹²⁵I-CYP bound to the receptor in a given reaction. Total Binding reactions will include 150 μL membrane, 50 μL Binding Buffer and 50 μL ¹²⁵I-CYP for a total reaction volume of 250 μL.

• 4.
  Add 50 μL of 5x Propranolol Stock Solution to all Non-Specific Binding Tubes.

  Non-Specific Binding reactions include a non-radiolabeled ligand, in this case propranolol, in excess of the K_d, to compete with ¹²⁵I-CYP for receptor binding sites. This is used to determine how much radioligand is non-specifically binding to the membrane of interest and from total and non-specific binding reactions, the amount of specific binding can be determined for a sample. It is preferable to choose a ligand that is chemically different from the radioligand to prevent similar non-specific interactions as well as it is best to have a high affinity and specificity for the receptor of interest. Additionally, antagonists are desirable over agonists due to potential changes in receptor confirmation and levels that can occur with agonist stimulation. Non-Specific Binding reactions will include 150 μL membrane, 50 μL Propranolol and 50 μL ¹²⁵I-CYP for a total reaction volume of 250 μL.

• 5.Add 150 μL of diluted membranes from Step 2 to all tubes.
• 6.
  Dilute ¹²⁵I-CYP to a 5 x 10⁻¹¹, 1 x 10⁻¹⁰, 2.5 x 10⁻¹⁰, 5 x 10⁻¹⁰, 2.5 x 10⁻⁹ M stocks in Binding Buffer.

  Perform each concentration in duplicate for both Total Binding and Non-Specific Binding reactions. Thus, it is recommended to make at least 400 μL per stock concentration to ensure adequate volumes for each reaction condition and extra to perform total counts in triplicate. Since ¹²⁵I labeled compounds lose activity rapidly, the concentration of ¹²⁵I-CYP received from the vendor needs to be calculated daily using the following equation: (0.5^(x/t1/2)) (0.1 Ci/L) / 2.2x10⁶ Ci/mol=concentration in the pig with t1/2 being the half-life of the radioligand, which is 60 days for ¹²⁵I, x being the days past the initial concentration, 0.1 Ci/L being the stock concentration at x=0 and 2.2x10⁶ Ci/mol being the specific activity of the radioligand. ¹²⁵I-CYP amounts for saturation binding reflect concentrations surrounding the expected K_d for ¹²⁵I-CYP binding to β-adrenergic receptors in cardiac tissue.

• 17.Add 50 μL of ¹²⁵I-CYP (1 x 10⁻⁹ M stock solution) to all tubes (200 pM final reaction concentration).
• 7.Briefly vortex all tubes to mix the reaction.
• 8.
  Incubate samples at 37°C for 1 h in a shaking water bath.

  This allows for the reaction to reach equilibrium.

• 9.
  Wash the harvester 5x by filling a rack of empty tubes with ~1 mL of cold Wash Buffer and aspirating the Wash Buffer.

  This ensures the buffer in the harvester lines is clean and fresh prior to experimentation.

• 10.
  Prepare GC/F filters by soaking them in 0.1 % BSA solution and placing them on the harvester.

  0.1 % BSA reduces non-specific binding to the filters. When placing the GC/F filters on the harvester, ensure that all of the mesh is covered. This is necessary to generate an appropriate vacuum to aspirate the reaction and buffer.

• 11.Using a Brandel Harvester, aspirate the reaction.
• 12.
  Wash the reaction tubes 5x with ~ 1 mL cold Wash Buffer.

  Ensuring that the Wash Buffer is cold and performing the washes rapidly will prevent dissociation of the radioligand-receptor complex.

• 13.
  Using forceps and only touching the outer edge, place filter circles into prelabeled gamma counter tubes (12 x 75 mm polypropylene assay tubes or equivalent).

  Ensure filter circles are at the bottom of the gamma counter tubes prior to reading.

• 14.
  Count samples on a gamma counter using the appropriate program for ¹²⁵I.

  Include 50 μL of the ¹²⁵I-CYP stock in triplicate to confirm final concentration. Alternatively, scintillation counting can be used to detect beta counts.

• 15.Wash harvester with ~2 L water and end with a 10% ethanol wash.

Figure 2.

Example layout for saturation binding experiments. A. Total and non-specific reactions are performed in duplicate with increasing concentrations of ¹²⁵I-CYP (10⁻¹¹ to 5 x 10⁻¹⁰ M). B. Total reaction components include membrane, which contains the receptor of interest and a selective, high-affinity radioligand, ¹²⁵I-CYP. C. Non-specific reactions include membrane and radioligand plus the addition of a selective, high-affinity non-radiolabeled ligand, propranolol. Concentrations of propranolol exceed those of ¹²⁵I-CYP, preventing ¹²⁵I-CYP binding and allow for the determination of non-specific ¹²⁵I-CYP binding. From Total and Non-Specific Binding results, the degree of specific ¹²⁵I-CYP binding can be calculated.

Saturation Binding Analysis:

Gamma counter readings are given in counts per minute (CPM), but values are generally expressed as fmol/mg protein. Thus, DPM readings need to be converted into a molar value.

1. Convert CPM to disintegrations per minute (DPM):

   DPM = CPM/E where E = efficiency of counting

   Efficiency of counting is gamma counter dependent and needs to be determined for the individual instrument being used.

2. Convert DPM to molar (mol):

   DPM * (1 Ci/2.22x10¹² DPM) * (1 mol/2.2x10⁶ Ci) = mol of radiochemical

   1 Ci = 2.2x10¹² DPM is a conversion factor whereas 2.2x10⁶ Ci/mol is the specific activity of ¹²⁵I-CYP.

3. Calculate molarity by dividing by the reaction volume:

   Mol/0.00025 L = M of radiochemical

4. Calculate Specific Binding:

   Total Binding – Non-specific Binding (with propranolol) = Specific Binding

5. Express as fmol/mg protein (Figure 5):

   M of radiochemical * 1x10¹⁵ fmol/mol * (1/amount protein from protein assay) = specific binding in fmol/mg protein

6. Also calculate amount of radioligand per reaction from total counts.

   This should equal the expected concentration in a given reaction.

Competition Binding:

All steps should be carried out with cold buffers and performed at 4°C to minimize protein degradation. Before starting, warm water bath (or incubator) to 37°C, make buffers and chill buffers to 4°C. Figure 3 depicts an example layout of competition binding experiments for the β1-adrenergic receptor subtype selective antagonist CGP 120712A.

1. On ice, set up reaction tubes (4 mL; 12 x 75 mm polypropylene assay tubes) in a binding rack on ice performing all reactions in duplicate.

   Most Brandel Harvesters are for 24 or 48 samples. All spaces must contain a tube regardless of the number of samples. Reactions can be performed at various volumes by adjusting the stock concentrations so the final reaction concentrations are equivalent to those outlined in this protocol, but the following protocol will be for a 250 μL reaction volume.

2. Dilute membranes from the “Membrane Preparation” to the desired reaction concentration in ice cold Binding Buffer for a final volume of 150 μL per reaction.

   Perform each concentration in duplicate. Remaining diluted membrane can be analyzed by protein assay to ensure the expected concentration following dilution. For heart samples, 25 μg of membrane is commonly used for radioligand binding experiments however, optimal membrane concentration determined from the “Radioligand Binding to Determine Protein Amount” protocol above is recommended.

3. Prepare 5x stock solutions of ICI 118,551, propranolol and CGP 120712A.

   For competition binding experiments, a 12-point curve of varying ligand concentrations is recommended. The concentration range should span from no inhibition of ¹²⁵I-CYP binding to total inhibition of ¹²⁵I-CYP binding with a particular focus in the expected K_i range. For ICI 118,551, propranolol and CGP 120712A, a recommended concentration range of 10⁻⁴ to 10⁻¹² M (5x stock of 5x10⁻⁴ to 5x10⁻¹² M) is a good starting point.

4. Add 50 μL of antagonist Stock Solution to the appropriate tubes.

5. Add 150 μL of diluted membranes from Step 2 to all tubes.

6. Dilute ¹²⁵I-CYP to a 1 x 10⁻⁹ M stock in Binding Buffer.

   Since ¹²⁵I labeled compounds lose activity rapidly, the concentration of ¹²⁵I-CYP received from the vendor needs to be calculated daily using the following equation: (0.5^(x/t1/2)) (0.1 Ci/L) / 2.2x10⁶ Ci/mol=concentration in the pig with t1/2 being the half-life of the radioligand, which is 60 days for ¹²⁵I, x being the days past the initial concentration, 0.1 Ci/L being the stock concentration at x=0 and 2.2x10⁶ Ci/mol being the specific activity of the radioligand.

7. Add 50 μL of ¹²⁵I-CYP stock solutions to all tubes (200 pM final concentration).

8. Briefly vortex all tubes to mix the reaction

9. Incubate samples at 37°C for 1 h in a shaking water bath.

   This allows for the reaction to reach equilibrium.

10. Wash the harvester 5x by filling a rack of empty tubes with ~1 mL of cold Wash Buffer and aspirating the Wash Buffer.

    This ensures the buffer in the harvester lines is clean and fresh prior to experimentation.

11. Prepare GC/F filters by soaking them in 0.1% BSA solution and placing them on the harvester.

    0.1% BSA reduces non-specific binding to the filters. When placing the GC/F filters on the harvester, ensure that all of the mesh is covered. This is necessary to generate an appropriate vacuum to aspirate the reaction and buffer.

12. Using a Brandel harvester, aspirate the reaction.

13. Wash the reaction tubes 5x with ~ 1 mL cold Wash Buffer.

    Ensuring that the Wash Buffer is cold and performing the washes rapidly will prevent dissociation of the radioligand-receptor complex.

14. Using forceps and only touching the outer edge, place filter circles into prelabeled gamma counter tubes (12 x 75 mm polypropylene assay tubes or equivalent).

    Ensure filter circles are at the bottom of the gamma counter tubes prior to reading.

15. Count samples on a gamma counter using the appropriate program for ¹²⁵I.

    Include 50 μL of the ¹²⁵I-CYP stock in triplicate to confirm final concentration.

16. Wash harvester with ~2 L water and end with a 10% ethanol wash.

Figure 3.

Example layout for competition binding experiments. Serial dilutions of non-radiolabeled ligand, CGP 20712A, a β1-adrenergic receptor selective antagonist are performed for final reaction concentrations ranging from 10⁻⁴ to 10⁻¹² M. Membrane and radioligand concentrations are kept constant and increasing concentrations of CGP 20712A inhibit ¹²⁵I-CYP. Similarly, competition binding using the β2-adrenergic receptor selective antagonist ICI 118,551 and propranolol are performed.

Competition Binding Analysis:

1. Calculate Specific Binding:

   Total Binding - Non-specific Binding (with propranolol) = Specific Binding

2. Plot specific binding as a percentage of total binding against the log concentration of the competing ligand (Figure 6).

REAGENTS AND SOLUTIONS

Use deionized, distilled water in all recipes and protocol steps.

Tris pH 7.4 stock solution, 1 M

Dissolve 6.0 g Tris in 50 mL of Milli-Q water. pH with hydrochloric acid (HCl) and sodium hydroxide (NaOH) to 7.4. Store at room temperature.

Ethylenediaminetetraacetic acid stock solution, 0.5 M

Dissolve 73.06 g of ethylenediaminetetraacetic acid (EDTA) in 500 mL of Milli-Q water. Store at room temperature.

Aprotinin stock solution, 1000x

Dissolve 1 mg Aprotinin in 1 mL Milli-Q water. Store at 4°C.

Leupeptin stock solution, 1000x

Dissolve 1 mg Leupeptin in 1 mL Milli-Q water. Store at 4°C.

Magnesium Chloride stock solution, 1 M

Dissolve 47.6 g of magnesium chloride (anhydrous; MgCl₂) in 500 mL Milli-Q water. Store at room temperature.

Propranolol stock solution, 5x

Dissolve 1 mg (±)-propranolol hydrochloride (Sigma-Aldrich cat # P0884) in 338 μL methanol to generate a 10⁻² M stock. Dilute 10 μL of 10⁻² M stock in 1990 μL Binding Buffer to generate a 5x (50 μM) stock. Make fresh propranolol stock immediately prior to performing experiments and store at 4°C during use.

Bovine Serum Albumin solution, 0.1%

Dissolve 1 g Bovine Serum Albumin (BSA) in 1 L Milli-Q water to generate a 0.1% solution.

Lysis Buffer

500 μL 1 M Tris pH 7.4 stock (25 mM final concentration)

200 μL 0.5 M EDTA stock (5 mM final concentration)

20 μL 1000 x Aprotinin stock (1 x (1 μg/μL) final concentration)

20 μL 1000 x Leupeptin stock (1 x (1 μg/μL) final concentration)

Add 19.26 mL Milli-Q water for a final volume of 20 mL. Make fresh immediately prior to performing experiments and store at 4°C during use.

Binding Buffer

1875 μL 1 M Tris pH 7.4 stock (75 mM final concentration)

100 μL 0.5 M EDTA stock (2 mM final concentration)

312.5 μL 1 M MgCl₂ stock (12.5 mM final concentration)

25 μL 1000 x Aprotinin stock (1 x (1 μg/μL) final concentration)

25 μL 1000 x Leupeptin stock (1 x (1 μg/μL) final concentration)

Add 22.638 mL Milli-Q water for a final volume of 25 mL. Make fresh immediately prior to performing experiments and store at 4°C during use. 25 mL of Binding Buffer is sufficient for 1 rack (48 tubes) of reactions.

Wash Buffer

20 mL 1 M Tris pH 7.4 stock (10 mM final concentration)

40 mL 0.5 M EDTA stock (10 mM final concentration)

Add 1940 mL Milli-Q water for a final volume of 2 L. Chill to 4°C prior to performing experiments. 2 L of Wash Buffer is sufficient for 1 rack (48 tubes) of reactions. An additional 2 L is needed for cleaning the harvester prior to and following harvesting samples.

COMMENTARY:

Background Information:

Radioligand binding assays were first developed over 50 years ago and have remained a gold standard method in receptor research (Maguire, Kuc, & Davenport, 2012). While alternative techniques have been developed to measure receptor density and ligand affinity, radioligand binding remains a widely used method due to its sensitivity and ability to quantitate ligand binding. Molecular techniques to quantitate receptor expression, such as quantitative RT-PCR can be ambiguous since they are detecting mRNA, which does not always correlate with protein expression. Furthermore, antibody based techniques such as immunoblotting have been notoriously unreliable for measuring GPCR expression due to the lack of specificity of GPCR antibodies (Michel et al., 2009). This is particularly true of adrenergic receptors where there have been multiple independent reports that the available antibodies lack specificity (Bohmer et al., 2014; Hamdani & van der Velden, 2009; Jensen et al., 2009; Pradidarcheep et al., 2009). However, measuring β-adrenergic receptor expression, particularly in the heart, is an important indicator of pathophysiology and cardiac function. In response to decreased cardiac output, the sympathetic nervous system is activated as the body attempts to compensate for this decline in cardiac function. However, chronic activation of the sympathetic nervous system, which occurs in heart failure, downregulates β1-adrenergic receptors on cardiomyocytes, potentiating disease progression (de Lucia et al., 2018). Therapeutic strategies that preserve or restore cardiac β-adrenergic receptor have been found to be beneficial, making β-adrenergic receptor density an important readout for cardiac function and heart failure progression. This is true for β-blockers, a common therapy for compensated congestive heart failure, which restore β1-adrenergic receptor density on cardiomyocytes and improve contractile dysfunction (de Lucia et al., 2018).

Saturation binding experiments are the most useful binding experiment to determine changes in β-adrenergic receptor density or receptor number. These experiments involved increasing concentrations of a radioligand while holding the amount of receptor constant. As the concentration of the radioligand increases, an increased amount binds to the receptor until the point of saturation occurs and no more radioligand can bind. When this data is plotted with the bound radioligand on the Y-axis and concentration of the radioligand on the X-axis, a hyperbolic association occurs. From this saturation curve the maximal binding (B_max) can be determined (Figure 5). The B_max represents the density of the receptor in the sample being studied. Saturation curves also estimates the dissociation constant (K_d) of the radioligand, which is inverse of the affinity of the ligand and defined as the concentration of ligand that occupies 50% of the binding sites. While saturation binding experiments are the primary method through which receptor density is determined, competition experiments can provide useful information regarding receptor subtype expression and ligand affinities.

In competition experiments, the varying amounts of non-radiolabeled compound is incubated with constant quantities of radioligand and receptor. The amount of bound radioligand is plotted versus the concentration of unlabeled ligand using a logarithmic scale generating a sigmoidal curve (Figure 6). From the competition curve, the concentration of an unlabeled drug required to inhibit specific binding of the radioligand by 50% (IC₅₀) value can be estimated and the affinity of that compound (K_i). By using subtype selective ligands, relative levels of β1 and β2-adrenergic receptor subtypes can be determined. This is important due to the differential effects of β1 and β2-adrenergic receptors in the heart (de Lucia et al., 2018).

While radioligand binding is a relatively simple yet powerful tool, it is not without its disadvantages, primarily, the safety and regulatory concerns of working with radioactivity. Washing steps have the potential to cause dissociation of the receptor-ligand complex and also generate a large amount of radioactive waste. The latter problem may be circumvented using centrifugation-based techniques such as thin layer gel filtration chromatography methods, which may also minimize protein-ligand dissociation due to decreased dilution effects. Additionally, non-radioactive receptor binding assays have also been developed as a way to reduce the use of radioactive ligands. Most commonly used are fluorescence-based assays however, these assays tend to be less sensitive than their radioactive counterparts due to fluorescent signals being prone to artifacts such as autofluorescence, quenching and inner-filter effects.

Critical Parameters:

Choice of receptor source

While isolated membranes are a common receptor source for radioligand binding, alternative sources including whole cells and tissue slices may also be used. When generating a receptor source, caution must be taken to prevent disruption of cell surface receptors. Enzymatic digestion or cell scraping can damage membrane components, including receptors, and alter results. Compartmentalization of receptors can be examined using differential centrifugation protocols to identify membrane localization of receptors.

Choice of radioligand

When selecting radioligands for use, it is important to consider the specific activity of the radioligand with high specific activities being best suited for ligand binding assays. It is also desirable to have low non-specific binding and high selectivity. Many ligands are labeled with ³H or ¹²⁵I with advantages and disadvantages associated with both. ¹²⁵I-labeled ligands often have high specific activity and emit both beta and gamma energy, making them detectible by scintillation or gamma detection. Detection using a gamma counter eliminates the need for liquid scintillation and reduces the amount of waste generated and costs. However, the stability of ¹²⁵I-labeled ligands is lower (60 day half-life) than ³H-labeled compounds (12.4 year half-life) making their window of use less. Contrarily, ³H-labeled ligands will only emit beta energy and less energy compared with ¹²⁵I-labeled compounds, making scintillation detection necessary. However, ³H-labeled ligands are more stable and can be used over an extended period of time. ³H-labeled ligands have a lower specific activity which decreases detectability. Furthermore, since ¹²⁵I-labeled ligands are gamma emitters, they pose an increased health risk in comparison to ³H-labeled ligands and require additional protective measures such as shielding and dosimeter monitoring. However, this also means that they can be readily detected using a Geiger-Mueller detector with NaI scintillation probe, making surface contamination readily detectible whereas ³H use necessitates swipe tests. While the protocol outlined above uses ¹²⁵I-CYP for a radioligand, alternatively, the tritiated radioligands (−)-CGP-12177, [5,7-³H] (Perkin Elmer, cat # NET1061) and Dihydroalprenolol hydrochloride, Levo-[Ring, Propyl-³H(N)] (DHA; Perkin Elmer, cat # NET720) are radiolabeled β1- and β2-adrenergic receptor selective antagonists that can be used.

Choice of ligand for determining non-specific binding

When choosing a ligand for determining non-specific binding, it is preferable to choose a ligand that is different than the radioligand. Since each ligand binds to a receptor in a different manner, choosing structurally different ligands increases the specificity of binding by preventing both the radioligand and non-radiolabeled ligand from binding to the same non-specific sites. Additionally, it is optimal to choose a ligand that has a high affinity for the receptor of interest and a low affinity for non-specific binding sties. While both agonists and antagonists are used in radioligand binding experiments, it is preferable to use antagonists whenever possible. Agonists can activate the receptor, initiating downstream events including receptor desensitization, which can confound results.

Ligand concentrations

When performing radioligand binding experiments, it must be remembered that no ligand is completely selective for a given receptor or receptor subtype. Each ligand has a unique pharmacological profile and the preferred ligand for binding assays should bind selectively to the receptor of interest in the assay conditions used. The methods outlined in this protocol us a Tris-based buffer system however, binding of ligands to most receptors, including β-adrenergic receptors, occurs in a multitude of buffers. Hepes-based buffers are also commonly used in radioligand binding studies, but differing buffers may alter ligand binding affinities and equilibrium kinetics, which should be considered when designing and comparing experiments. For saturation binding experiments, it is important to choose radioligand concentrations that are several points higher and lower than the K_d, while higher points should start to plateau (Figure 5). This ensures accurate determinations of K_d and B_max. Furthermore, concentrations of the non-radiolabeled ligand should be in excess of the K_d in order to block the radioligand from binding to nearly all binding sites. For competition experiments, radioligand concentrations should be in the range of the K_d, which allows for the non-radiolabeled ligand to inhibit binding. Concentrations of the non-radiolabeled ligand should plateau at both the top and the bottom of the inhibition curve (Figure 6).

Troubleshooting:

Table 1 describes some commonly encountered problems associated with the protocol described in this article along with potential causes and ways to overcome these problems.

Table 1.

Troubleshooting Guide for Radioligand Binding Experiments

Problem	Possible Cause	Solution
No Specific Binding	Receptor is not expressed    Receptor is degraded    Inappropriate ligand concentrations (Values are beyond the Bmax or below the KD)	Ensure that the receptor is expressed in the tissue of interest    Perform all experiments at 4°C. Include the proteinase inhibitors aprotinin and leupeptin in all buffers    Optimize radioligand and non-radioligand concentrations.
Multisite binding	Inappropriate ligand concentrations    Multiple receptor isoforms    Minimally selective ligand	Optimize radioligand and non-radioligand concentrations.    Perform a partial F-test analysis to determine if one-site or multi-site binding is occur and use the appropriate curve fit analysis.    Choose high affinity, highly selective ligands for use in experiments
Poorly fit curves	Ligand-receptor dissociation    Membrane degradation    Equilibrium is not reached	Ensure buffers are cold, filtration and washing is rapid    Ensure everything is kept cold, minimize freeze thawing of samples, include the proteinase inhibitors aprotinin and leupeptin in all buffers    Optimize incubation times and temperatures
Inaccurate total counts	Radioligand binding to tubes    Inaccurate pipetting	Use low-binding tubes for reagents and reaction preparations    Use proper pipetting technique for performing all experiments

Statistical Analysis:

Saturation binding curves are obtained by plotting the specific binding against the concentration of the radioligand. A non-linear curve fit is performed and from that, the B_max and K_d can be determined (Figure 5).

Competition binding curves are obtained by plotting specific binding as a percentage of total binding against the log concentration of the competing ligand (Figure 6). The partial F-test can be used to differentiate a single binding site from multiple populations of binding sites. In the instance of a single binding site, a one-site curve fit is optimal whereas a two-site fit is used when there are multiple binding sites. From the curve fit, K_i values can be determined.

Understanding Results:

Expected results for “Radioligand Binding to Determine Protein Concentration” are depicted in Figure 4. ¹²⁵I-CYP binding is enhanced with increasing amounts of sample in the total binding reactions. Inclusion of propranolol in the reaction to compete for ¹²⁵I-CYP binding sites generates a decreased ¹²⁵I-CYP binding curve compared with the total binding at a given protein concentration. The difference between the total binding and non-specific binding curves is the specific binding. At lower protein concentrations, the difference between total and non-specific binding is less than at higher protein concentrations. However, this increase in specific binding that occurs reaches a point where it plateaus and the change in specific binding observed at higher points is negligible. Choosing the minimal concentration of membrane where there is maximal specific binding for subsequent experiments ensures the most accurate binding results while conserving on sample.

The expected results for “Saturation Binding Experiments” are depicted in Figure 5. Increasing ¹²⁵I-CYP binding is observed in the total binding reactions with increasing concentrations of ¹²⁵I-CYP that flattens as the receptor becomes saturated at higher concentrations. Inclusion of propranolol in the reaction to compete for ¹²⁵I-CYP binding sites has decreased ¹²⁵I-CYP binding compared with the total binding at a given ¹²⁵I-CYP concentration and represents the non-specific binding curve. Specific binding is calculated as the total binding minus the non-specific binding. Similar to the total binding, specific binding is saturable and plateaus at higher concentrations of ¹²⁵I-CYP. From this graph, the B_max can be determined and represents the receptor density. The K_d can also be identified, which is inverse of the affinity of the ligand and defined as the concentration of ligand that occupies 50% of the binding sites. An ideal radioligand will have a high affinity, low K_d for the receptor of interest.

The expected results for “Competition Binding Experiments” are depicted in Figure 6. Increasing amounts of CGP 12177A inhibit ¹²⁵I-CYP binding. The generated curve is biphasic suggesting two binding sites. Since CGP 12177A is a β1-adrenergic receptor selective antagonist, it binds to β1-adrenergic receptors with a high affinity and β2-adrenergic receptors with a lower affinity. Conversely, ICI 118,551 is a β2-adrenergic receptor selective antagonist and binds to β2-adrenergic receptors with a high affinity and β1-adrenergic receptors with a lower affinity. Thus, an ICI 118,551 competition curve would similarly have a biphasic curve with the proportion of high affinity binding sites and low affinity binding sites being the reverse of CGP 12177A. Contrarily, since propranolol is a non-selective β-adrenergic receptor antagonist, a steeply sloped competition curve would be expected, indicating a high affinity, one-site binding.

Time Considerations:

The outlined experiments can be performed in a relatively short amount of time. Membrane preparations can be performed in 1-2 h and samples can be immediately used for binding experiments or stored in 10% glycerol at −80°C for 1 month. Binding experiments can be performed in 2-3 h with an additional 1-2 h for gamma counting and data analysis.

DATA AVAILABILITY STATEMENT:

Data sharing not applicable to this article as no datasets were generated or analyzed during the current study