Radioligands

There should be specific, saturable binding to the receptor, accompanied by pharmacological characteristics appropriate to the functional effects, demonstrable using a radioactive, eg, tritium or iodine-125, ligand to label the receptor. Radioligand binding assays (1,6) have become a significant means by which to identify and characterize receptors and enzymes (see Immunoassays Radioactive tracers). Isolation of the receptor or expression of the receptor in another cell, eg, an oocyte can be used to confirm the existence of a discrete entity. 

CB, was identified by radioligand binding techniques and subsequently cloned. A second receptor subclass, CB2, has been identified in human spleen and also has been cloned (34). Table 5 Hsts cannabinoid receptor ligands. 

Direct quantitation of receptor concentrations and dmg—receptor interactions is possible by a variety of techniques, including fluorescence, nmr, and radioligand binding. The last is particularly versatile and has been appHed both to sophisticated receptor quantitation and to dmg screening and discovery protocols (50,51). The use of high specific activity, frequendy pH]- or p lj-labeled, dmgs bound to cmde or purified cellular materials, to whole cells, or to tissue shces, permits the determination not only of dmg—receptor saturation curves, but also of the receptor number, dmg affinity, and association and dissociation kinetics either direcdy or by competition. Complete theoretical and experimental details are available (50,51). 

FIGURE 3.3 Displacement of prebound radioligand [A ] by non-radioactive concentrations of [A]. Curve for a= 1 denotes no cooperativity in binding (i.e., formation of the receptor dimer does not lead to a change in the affinity of the receptor for either [A] or [A ]). The curve a= 10 indicates a system whereby formation of the receptor dimer leads to a tenfold increase in the affinity for both [A ] and [A], In this case, it can be seen that addition on the nonradioactive ligand [A] actually leads to an increase in the amount of radioligand [A ] bound before a decrease at higher concentrations of [A], For this simulation [A ]/Kd = 0.1. 

Radioligand binding to receptor dimers demonstrating co-operative behavior (3.13.1)... 

Radioligand Binding to Receptor Dimers Demonstrating Cooperative Behavior... 

It is assumed that receptor dimers can form in the cell membrane (two [R] species to form one [R-R] species). Radioligand [A ] can bind to the receptor [R] to form radioactive complexes [A R], [A R — AR], and [A R — A R], It is also assumed that there is an allosteric interaction between... 

The radioactive signal (denoted p) is produced from the receptor species bound to radioligand [A ] ... 

Assuming that all interactions of the species are possible, the system consists of the receptor CCR5 [R], radioligand CD4 [CD]), viral coat protein gpl20 [gp], and potential displacing ligand [B] ... 

Estimating the Bmax value is technically difficult since it basically is an exercise in estimating an effect at infinite drug concentration. Therefore, the accuracy of the estimate of Bmax is proportional to the maximal levels of radioligand that can be used in the experiment. The attainment of saturation binding can be deceiving when the ordinates are plotted on a linear scale, as they are in Figure 4.2. 

In practice, there will be a limited number of ligands available that are chemically traceable (i.e., radioactive, fluorescent). Therefore, the bulk of radioligand experiments designed to quantify ligand affinity are done in a displacement mode whereby a ligand is used to displace or otherwise affect the binding of a traceable ligand. 

FIGURE 4.11 Relationship between the observed IC50 for allosteric antagonists and the amount of radioligand present in the assay according to Equation 4.13. Dotted line shows relationship for a competitive antagonist. 

FIGURE 4.12 Effect of alcuronium on the binding of [3H] methyl-QNB (filled circles) and [3H] atropine (open circles) on muscarinic receptors. Ordinates are percentage of initial radioligand binding. Alcuronium decreases the binding of [3H] methyl-QNB and increases the binding of [3H] atropine. Data redrawn from [7]. 

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