Apparent capacity of cardiac muscarinic receptors for different radiolabeled antagonists6
Introduction
Compounds radiolabeled to a high specific activity are uniquely suited for studies on the binding properties of receptors and other macromolecules. Their high sensitivity permits measurements at very low levels of labeled material, extending to attomole quantities in the case of some nuclides. Also, the measured signal depends only on the specific radioactivity and the efficiency of the counting process. The former is known in advance, at least in principle, and the latter usually can be measured to a high degree of precision. Since the specific signal is invariant, the amount of labeled compound can be determined readily in absolute units.
Tritium is a particularly useful nuclide owing to its comparatively long half-life and its specific activity of 28.8 Ci/matom, which is well matched to the levels of receptor found in many preparations. Isotopic purity approaches 100% in many commercial products, and the substitution of tritium for hydrogen generally appears to have little or no discernible effect on the properties of the ligand. This equivalence is often assumed, but in many cases it can be tested by comparing the binding of the radioligand with that of the unlabeled analogue. The synthesis of tritiated compounds generally yields a mixture of two or more products differing in the distribution of tritium and hydrogen atoms within the molecule. Since the labeled and unlabeled compounds are pharmacologically indistinguishable, the effective specific activity can be calculated as a weighted average based on the specific activities and relative amounts of the different species.
The binding pattern defined by graded concentrations of a radioligand at thermodynamic equilibrium is sensitive to radiochemical and chemical impurities [1], [2], [3], [4], [5]. While the radiochemical purity typically is stated by the manufacturer, chemical purity tends to have a lower profile. It has been pointed out, however, that disregarding an unlabeled contaminant is akin to overestimating the specific activity of the radioligand [3], [4], [5]. The concentration of the radioligand therefore is underestimated throughout, and that, in turn, leads to artifactually low values of both the capacity and the potency (i.e. ec50) in a population of identical and mutually independent sites.
In studies on cardiac muscarinic receptors, batches of the muscarinic antagonist N-[3H]methylscopolamine were found to contain appreciable quantities of the unlabeled precursor scopolamine; similarly, batches of N-[3H]methylquinuclidinylbenzilate were found to contain unlabeled quinuclidinylbenzilate. In the present report, we compare the binding of contaminated N-[3H]methylscopolamine, N-[3H]methylscopolamine devoid of scopolamine, and [3H]quinuclidinylbenzilate to muscarinic receptors from porcine atria. A competition between N-[3H]methylscopolamine and contaminating scopolamine can account for results that otherwise are anomalous in terms of mutually independent sites. Possible alternatives to the notion of a contaminated radioligand are considered and, in some cases, ruled out. Strategies are employed to estimate the amount of the impurity and to determine the effective specific activity of an impure product according to a model that seems to fit. The results suggest that chemical impurities have contributed to various effects used routinely to monitor the properties of muscarinic and other G protein-linked receptors. A preliminary report of this work has appeared elsewhere [6].
Section snippets
Muscarinic ligands
N-[3H]Methylscopolamine was obtained as the chloride salt from NEN Life Science Products, Inc. (lot 2924211, 84.0 Ci/mmol; lot 3144205, 84 Ci/mmol; lot 3167197, 84.5 Ci/mmol; lot 3318049, 82.0 Ci/mmol; lot 3273247, 82.0 Ci/mmol; lot 3406009, 83.5 Ci/mmol) and as the bromide salt from Amersham Pharmacia Biotech (batch 27, 78.3 Ci/mmol). N-[3H]Methylquinuclidinylbenzilate was obtained as the chloride salt from NEN (lot 3248957, 83.5 Ci/mmol; lot 3329162, 83.5 Ci/mmol; lot 3329351, 83.5 Ci/mmol;
Results
Data on the binding properties of G protein-linked receptors typically are analyzed in terms of Scheme 1, either implicitly as a sum of hyperbolic terms or explicitly according to Eq. 3 or equivalent expressions. In studies at graded concentrations of the radioligand, designated below as L1, unlabeled compounds generally are assumed to be absent except when added to define the level of non-specific binding (i.e. [Li]/Kij ≫ [L1]/K1j).
If non-radiolabeled ligands are present, their effect depends
Discussion
Various preparations from porcine atria have revealed a marked difference in the apparent capacity of muscarinic receptors for the antagonists N-[3H]methylscopolamine and [3H]quinuclidinylbenzilate. Such differences have been noted previously, and they typically are attributed to heterogeneity among the receptors per se or to their differential localization within the preparation. Neither explanation seems plausible for the present data, which are inconsistent with the underlying premise of
Acknowledgements
We are grateful to the managers and staff of Quality Meat Packers Ltd. and the New York Pork and Food Exchange (Toronto) for supplies of porcine atria and for their cooperation in allowing immediate access to fresh tissue. We thank Pauline Bennett, Mario Maniscalco, Steven Souza, and Dr. Ron Vander-Mallie of NEN Life Science Products, Inc. for numerous helpful discussions, for their cooperation in various matters related to the radioligands used in this investigation, including the provision of
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Abbreviations: ABT, 3-(2′-aminobenzhydryloxy)tropane; ECL, enhanced chemiluminescence; NMS, N-methylscopolamine; PMSF, phenylmethylsulfonyl fluoride; and QNB, l-quinuclidinylbenzilate.