Molecular Pharmacology, Vol 13, 314-329, Copyright © 1977 by the American Society for Pharmacology and Experimental Therapeutics

Use of (-)-[³H]Dihydroalprenolol to Study Beta Adrenergic Receptor-Adenylate Cyclase Coupling in C6 Glioma Cells: Role of 5'-Guanylylimidodiphosphate

¹ Laboratoire de Physiologic Cellulaire, Collège de France, 75231 Paris Cedex 05, France

(-)-[³H]Dihydroalprenolol, a potent beta adrenergic antagonist, was used as a ligand to characterize the beta adrenergic receptors coupled with adenylate cyclase in C6 glioma cell membranes. Binding of (-)-[³H]dihydroalprenolol was Saturable with 0.47 ± 0.03 pmole/mg of protein (N = 4) and occurred on a single category of specific binding sites. C6 glioma cells contained approximately 10,000 sites/cell. The dissociation binding constant of (-)-[³H]dihydroalprenolol was 5.7 ± 0.9 nM (N = 5), and its apparent inhibition constant for the (-)-isoproterenol-activated adenylate cyclase was 2.9 ± 0.5 nM (N = 4). The association of (-)-[³H]dihydroalprenolol with its binding sites was very fast, less than 1 min at 30°, a ligand concentration of 45 nM. Dissociation was a first-order reaction, with a rate constant of 0.35 min^-1. An association rate constant of 6 x 10⁷ M^-1 min^-1 was computed. Activation of the adenylate cyclase by (-)-isoproterenol (0.1 mM) was instantaneous. After prior saturation of the binding sites with (-)-[³H]dihydroalprenolol, activation of the adenylate cyclase by (-)-isoproterenol was slow and limited by the dissociation of (-)-[³H]dihydroalprenolol from its binding sites. Beta adrenergic agonists competed for (-)-[³H]dihydroalprenolol binding sites and activated the adenylate cyclase with an effectiveness typical of beta, adrenergic specificity, since the order of potency was (-)-isoproterenol > (-)-norepinephrine epinephrine. Whatever the agonist considered, there was a 3-fold difference between the agonist dissociation constants determined by analyzing the competitive displacement of (-)-[³H]dihydroalprenolol from binding sites, and the apparent affinity (K_A app) for adenylate cyclase activation. This difference indicates a hyperbolic relationship between receptor occupancy and adenylate cyclase activation. Complete activation of the enzyme requires full occupation of the specific binding sites. Beta blocking agents (but not alpha blocking agents or neuroleptics) inhibited (-)-[³H]dihydroalprenolol binding and the activated adenylate cyclase in the same order of potency. The ratio of the antagonist dissociation constants for binding (K_D) to their apparent inhibition constants (K_i app) for adenylate cyclase was about 2 for (-)-[³H]dihydroalprenolol and all the other beta antagonists tested. Like adenylate cyclase activation, binding was stereospecific, since (+)-propranolol required concentrations about 30 times higher than the (-) isomer to inhibit binding or adenylate cyclase stimulation half-maximally. These equilibrium and rate study experiments suggest that (-)-[³H]dihydroalprenolol binding sites have all the features expected of beta adrenergic receptors functionally coupled with the adenylate cyclase. The GTP analogue 5'-guanylylimidodiphosphate [Gpp(NH)p] stimulated the basal adenylate cyclase catalytic state but reduced the maximal velocity of the agonist-activated state. The effect of Gpp(NH)p on these two adenylate cyclase states was irreversible. Gpp(NH)p reduced the affinity of (-)-isoproterenol for the beta receptor but increased its apparent affinity for adenylate cyclase activation. Gpp(NH)p therefore increased the efficiency of coupling between receptor occupancy and adenylate cyclase stimulation. It had no effect on the binding of (-)-alprenolol to the beta receptor. The following working hypothesis is proposed to explain these results: by modifying the equilibrium between the different states of the beta receptor-adenylate cyclase system, Gpp(NH)p changes the characteristics of the beta receptor when the receptor is "coupled" with the adenylate cyclase as a result of its interaction with an agonist.

Note:
ACKNOWLEDGMENTS We are indebted to Doctor P. Benda for providing us C6 glioma cells and for his advice on cell culture, M. Perez for excellent technical assistance, C. Roy for many stimulating discussions, and Miss M. Dreyfus for her help in the preparation of the manuscript.

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