Molecular Pharmacology, Vol 12, 362-372, Copyright © 1976 by the American Society for Pharmacology and Experimental Therapeutics

Cell Density and Receptor-Adenylate Cyclase Relationships in the C-6 Astrocytoma Cell

¹ Departments of Pharmacology and Biochemistry, Albert Einstein College of Medicine, Bronx, New York 10461

The maximum stimulation at saturating concentrations of epinephrine (V_max) of catecholamine-sensitive adenylate cyclase of rat C-6 astrocytoma cells in culture is significantly enhanced when the cells achieve confluence. This relationship is apparent whether measured as increased levels of adenosine cyclic 3',5'-monophosphate (cAMP) in intact cells, without or with serum present, or as ATP-dependent cAMP formation by cell homogenates. Corresponding to this increase, the adenylate cyclase activity stimulated by fluoride increased with increased cell density, as was also noted for cAMP phosphodiesterase. When intact cells in the presence of serum are exposed to epinephrine for 2 min, the epinephrine concentration giving half-maximal stimulation (K_m) increases from 100 to 600 nM as cell cultures grow from low density to confluence; similarly, an increase in the dissociation constant K_b for the antagonist propranolol, from 1 to 10 nM, was found as cell density increased. The attribution of those trends to intercellular interaction rather than to total cell number per plate or to manipulation of cells at the time of subculture is supported by the absence of such trends in nonhomogeneously dispersed cell culture preparations. Thus, under these conditions of cell culture and investigation, the effective affinity for catecholamine or antagonist decreases as a function of cell density. It was further noted, however, that in the absence of serum the K_m for epinephrine did not change with cell density, and after cells had been incubated with propranolol for 45 min the apparent K_b was 1 nM, independent of cell density. It is concluded that the increase in V_max, with increasing cell density is not associated with any inherent change in the affinity or sensitivity of the catecholamine receptor-cyclase complex, but may be related to an increase in total catalytic activity.

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ACKNOWLEDGMENTS We gratefully acknowledge the technical assistance of Mrs. Elaine Keehn. We are indebted to Dr. S. Seifter and Mr. D. L. Prinstein for reviewing the manuscript.