Improved Models for Pharmacological Null Experiments: Calculation of Drug Efficacy at Recombinant D1A Dopamine Receptors Stably Expressed in Clonal Cell Lines

doi:10.1016/0028-3908(96)84625-9

Neuropharmacology

Volume 35, Issue 5, May 1996, Pages 549-570

https://doi.org/10.1016/0028-3908(96)84625-9 Get rights and content

Abstract

Modern drug discovery demands accurate knowledge of the drug properties of affinity and efficacy at specific receptor proteins. Furthermore, drugs with well defined properties make better tools with which to explore and understand receptor regulation. The use of clonal cell lines stably expressing a given recombinant receptor may provide a highly useful model in which drug effects may be studied on one receptor subtype at a time. The present report was designed to evaluate the utility of a general method in which a clonal cell line stably expressing a recombinant D1A dopamine receptor was used as a model system for studying drug actions by null models. The null model for receptor occlusion (to calculate agonist K_a) and the null model for relative efficacy (to calculate test agonist affinity and ε_r) were evaluated in these studies. To initiate these studies, rat C6 glioma cells that do not normally express DA receptors have been modified by stable transfection with the primate D1A DA receptor [Machida et al., 1992(Molec. Pharmacol. 41: 652–659)] to a density of ≈ 200 fmol/mg protein. The recombinant receptors show robust stimulation of cAMP in the stably transfected C6 cells.

Calculation of agonist K_a from dose-response data requires that a portion of the cell's receptors be occluded in the absence of changes in post-receptor events leading to the response. Receptor reserve is typically reduced by alkylation, thereby lowering maximal response. Unfortunately, most of the currently available alkylating agents are not selective either for a particular receptor or for receptors vs other proteins within a signaling pathway. Short-term agonist treatment offers a possible complement to the use of non-selective or poorly characterized alkylating drugs for reducing maximum response in appropriate cell systems.

The null method of receptor occlusion was used to determine the K_a for dopamine when maximum response was decreased by alkylation vs short-term agonist treatment. Direct non-linear curve fitting was used to analyze the data. In addition to DA, two other compounds were used to reduce receptor reserve to validate the method: fenoldopam (relatively high efficacy) and SKF38393 (low efficacy). Analyses indicated that the affinity of DA was similar whether calculated by alkylation (1.1 ± 0.58 μM), 75 min DA treatment (0.57 ± 0.16 μM) or 45 min treatment with DA (0.86 ± 0.11 μM). Short-term agonist treatment experiments using multiple concentrations of DA, fenoldopam, or SKF38393 to decrease receptor reserve provided additional support for the validity of the K_a determinations using this procedure.

Other experiments were conducted according to the null model for relative efficacy in which the affinity for DA is calculated by comparing the DA response before and after receptor occlusion, and the affinity and relative intrinsic efficacy of the test agonist are determined as a function of its actions relative to DA. We used the following four test drugs: + Br-APB, a novel agent with potential dopamine agonist properties, and three high-affinity DA agonists, fenoldopam, R-(−)-apomorphine (APO), and SKF38393. Intrinsic efficacy values relative to that of DA (1.0) were as follows: fenoldopam, 0.46 ± 0.11; APO, 0.19 ± 0.13; SKF38393, 0.07 ± 0.01; and +Br-APB, 0.26 ± 0.40. The agonist affinities (K_a) were: fenoldopam, 0.018 ± 0.008 μM; APO, 0.80 ± 0.18 μM; SKF38393, 0.16 ± 0.04 μM; Br-APB, 0.43 ± 0.29 μM; and DA, 0.58 ± 0.17 μM. EC₅₀/K_a ratios were consistent with relative intrinsic efficacies and K_a values were similar to K_L values reported for membrane binding studies.

Finally, Monte Carlo simulations were conducted to determine the precision of the parameter estimates. These simulations of 1000 experiments each, using the empirical variance of ≈ 3% of the maximum response, indicated a precision for the DA K_a of ≈ 16%, for the independently determined fenoldopam K_a of ≈ 24%, and of the fenoldopam ε_r of ≈ 16% of the parameter values. To our knowledge, this represents the first report of agonist affinity plus relative intrinsic efficacy measurement at any recombinant receptor. The present results also provide the first report of the precision of these important drug parameter measurements using Monte Carlo methods. Together, these results suggest that clonal cell lines which stably express a recombinant receptor of interest may offer a unique system with which to determine essential drug properties. The method is able to accommodate agonists with very high potency and intrinsic activity, extremely low relative intrinsic efficacy, and can determine whether or not a drug is behaving as expected for a classical partial agonist. Copyright © 1996 Elsevier Science Ltd

Section snippets

Null models

Under the null model assumptions, a reduction in receptor number produces a linear shift along the log dose axis in the agonist dose-response curve relative to control (Parker and Waud, 1971; Ruffolo, 1982). This shift is also termed the dose-ratio, because the degree of curve shift along the log dose axis equals the ratio of agonist doses producing the same response in the control and occluded curves. The shift is directly related to the K_a of the agonist and the proportion of receptors

Receptor occlusion experiments

Possible endogenous confounding receptors. DA binds to both β-adrenergic and 5-HT receptors, although with much lower affinity than to DA receptors. Serotonin produces no effect, either by itself, or on forskolin-stimulated cAMP accumulation in C6 cells (data not shown), suggesting an absence of 5-HT receptors linked to adenylate cyclase in this system. To determine if endogenous 5-HT2 receptors could indirectly affect cAMP production in C6 cells, a further experiment was conducted using 10 μM

DISCUSSION

In the present report, we have attempted to provide a theoretical rationale (see below) and empirical validation of a short-term agonist treatment protocol as a complement to the use of alkylating agents for calculating DA K_a in an appropriate cell system. Initially, we took advantage of the endogenous β2-adrenergic receptors of C6 cells to establish that the DA pretreatment did not alter the cAMP accumulation response to isoproterenol. This suggested that, under our pretreatment conditions, DA

CONCLUSIONS

A major underlying hypothesis in this work is that knowledge of the structure-activity relationships for agonist affinity and relative intrinsic efficacy may yield improved design of agents used in the treatment of disease. Results from the present studies have demonstrated the feasibility of calculating agonist K_a and relative intrinsic efficacy even for weak partial D1A agonists (such as SKF38393 and R-(−)-APO) using second messenger measurements in intact clonal cells. Clearly, a perfectly

Acknowledgements

The authors gratefully acknowledge the excellent technical assistance of Mr Scott Burns, Ms Michele Bergeron and Ms Julienne Rhee, and Dr Rueben Gonzales for his many helpful comments and suggestions. Our special thanks go also to Dr Kim Neve for generously providing the C6-D1A cells used for these experiments and to Dr Curt Machida for conducting the cloning and expression work which made that line possible. This work was supported in part by a grant from the NIH (RR08579). Chun Mak and Melva

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Improved Models for Pharmacological Null Experiments: Calculation of Drug Efficacy at Recombinant D1A Dopamine Receptors Stably Expressed in Clonal Cell Lines

Abstract

Section snippets

Null models

Receptor occlusion experiments

DISCUSSION

CONCLUSIONS

Acknowledgements

J. Biol. Chem.

Neurochem. Int.

TIPS

J. Biol. Chem.

Life Sci.

J. Biol. Chem.

TIPS

Life Sci.

J. Biol. Chem.

3D QSAR for intrinsic activity of 5-HT1A receptor ligands by the method of comparative molecular field analysis

J. Comp. Chem.

Densensitization of DA1 dopamine receptors coupled to adenylyl cyclase in opossum kidney cells

Am. J. Physiol.

β-Adrenergic receptor kinase: identification of a novel protein kinase that phosphorylates the agonist-occupied form of the receptor

Proc. Natn. Acad. Sci. U.S.A.

Removal of phosphorylation sites from the β2-adrenergic receptor delays onset of agonist-promoted desensitization

Nature

Molecular diversity of the dopamine receptors

A. Rev. Pharmacol. Toxicol.

Contributions of conserved serine residues to the interactions of ligands with dopamine D2 receptors

J. Neurochem.

Molecular cloning and expression of the gene for a human D1 dopamine receptor

Nature

Simultaneous analysis of families of sigmoidal curves: application to bioassay, radioligand assay and physiological dose-response curves

Am. J. Physiol.

Estimation of apparent agonist affinity constants using desensitization of the ileal muscarinic receptor

J. Pharmacol. Exp. Ther.

Estimation of the affinities of allosteric ligands using radioligand binding and pharmacological null methods

Molec. Pharmacol.

Dopamine agonist-induced elevation of striatal acetylcholine: relationship between receptor occupancy and response in normal and denervated rat striatum

Molec. Pharmacol.

Recent advances in the molecular biology of dopamine receptors

A. Rev. Neurosci.

Fenoldopam is a partial agonist at dopamine-1 (DA1) receptors in LLC-PK1 cells

J. Pharmacol. Exp. Ther.

Phenoxybenzamine treatment differentiates dopaminergic 3H-ligand binding sites in bovine caudate membranes

Molec. Pharmacol.

Potency of D1 receptor affinity ligands with alkylating moieties

Soc. Neurosci. Abstr.

A small region of the β-adrenergic receptor is selectively involved in its rapid regulation

Proc. Natn. Acad. Sci. U.S.A.

Affinities of full agonists for cardiac β-adrenoceptors calculated by use of in vitro desensitization

Naunyn-Schmiedeberg's Arch. Pharmacol.

N-Ethylmaleimide inactivates adenylate cyclase-coupled but not presynaptic striatal D2 receptor systems

Eur. J. Pharmacol.

D1A dopamine receptor stimulation inhibits Na+/K+-ATPase activity through protein kinase A

Molec. Pharmacol.

A new method for estimation of agonist dissociation constants (Ka): Directly fitting the postinactivation concentration-response curve to a nested hyperbolic equation

J. Pharmacol. Exp. Ther.

D1A dopamine receptor stimulation inhibits Na⁺/K⁺-ATPase activity through protein kinase A

A new method for estimation of agonist dissociation constants (K_a): Directly fitting the postinactivation concentration-response curve to a nested hyperbolic equation