Molecular Pharmacology, Vol 15, 588-597, Copyright © 1979 by the American Society for Pharmacology and Experimental Therapeutics

In Vitro and In Vivo Kinetic Analysis of the Interaction of a Norbornyl Derivative of Propranolol with -Adrenergic Receptors of Brain and C6 Glioma Cells; an Irreversible or Slowly Reversible Ligand

¹ Laboratoire de Physiologie Cellulaire, Collège de France, 11, Place Marcelin Berthelot, 75231 PARIS CEDEX 05

When injected into rats (30 mg/kg, i.p.), a norbornyl derivative of propranolol, 1-(2-exobicyclo[2,2,1]Hept-2-ylphenox)-3-[(1-methyl-ethylamino]-2-propanol (FM 24) blocked by 85% the cerebral cortex -adrenergic receptors measured on membranes prepared five hours after the treatment. The preparation of membranes included extensive washing and a 75 min incubation period at 37°. Injection of propranolol at the same dose did not modify the number of -adrenergic receptors in these membranes. When membranes were prepared 11 hours after a single injection of FM 24, 50% of -adrenergic receptors were still blocked; more than 24 hours were necessary for complete recovery. A detailed analysis of the FM 24 interaction with -adrenergic receptors was performed on -adrenergic receptors of C6 glioma cells. FM 24 behaves as a competitive antagonist of the [³H]-dihydroalprenolol binding and of the isoproterenol sensitive adenylate cyclase (K_I = 50 µM) when measured during a short period after starting the reaction. When measured after different incubation periods, FM 24 blocked the -adrenergic receptors in a mixed competitive and non-competitive manner. The non-competitive inhibition was time and concentration dependent. The time course of this non-competitive inhibition was delayed by the presence of the -adrenergic agonist isoproterenol. When membranes were washed, only the non-competitive inhibition of both [³H]-dihydroalprenolol binding and -adrenergic sensitive adenylate cyclase remained. It is proposed that the FM 24 formed a reversible complex with -adrenergic receptors which is slowly transformed to a more stable complex. An analysis of the dissociation of this more stable complex showed two components, one which dissociated within one hour and the other which did not dissociate even after 5 hours of incubation.

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ACKNOWLEDGMENTS We are indebted to Drs. Uzan and Le Fur for donation of FM 24, and interesting discussion, and would like to acknowledge the excellent technical assistance of Michelle Perez and Mina Cherif.