Abstract

Catecholamine-induced desensitization was compared in two strains of rat glioma C6 cells [low passage (C6LP) and high passage (C6HP)]. When exposed to isoproterenol, C6LP cells accumulated high levels of cyclic AMP (3-4 nmoles/mg of protein), whereas C6HP cells did not. After prolonged exposure to isoproterenol, both C6LP and C6HP cells exhibited a diminished response when rechallenged with the agonist. The desensitization process was both time- and dose-dependent and similar parameters were observed for C6LP and C6HP cells. C6HP cells exposed to isoproterenol in the presence of a phosphodiesterase inhibitor initially accumulated large amounts of cyclic AMP, but the inhibitor did not alter the time course of agonist-induced desensitization. In addition, the inhibitor, which by itself elevated cyclic AMP levels more than did isoproterenol by itself in C6HP cells, did not induce a refractory state. Agonist-induced loss in beta-adrenergic receptors was much slower than the rate of desensitization. Membranes from desensitized cells, however, exhibited an apparent lower affinity for agonist as measured by agonist displacement of labeled antagonist binding. A substantial loss of isoproterenol-stimulated adenylate cyclase activity in membranes prepared from agonist-treated C6LP and C6HP cells was observed without a significant loss in NaF-stimulated activity. Isoproterenol-treated C6HP cells remained completely responsive to cholera atoxin. Desensitized C6LP cells exhibited a reduced response to the toxin, which was partially overcome by the phosphodiesterase inhibitor. Activation of cyclase, however, was not reduced in either C6LP or HP cells treated with agonist and then toxin. Furthermore, cyclase was activated to the same extent when membranes from control or desensitized cells were incubated with the A₁ subunit of cholera toxin and NAD. We conclude that the large accumulation of cyclic AMP in C6LP cells exposed to beta-agonists does not mediate the subsequent desensitization process. Instead, the initial stages of catecholamine-induced desensitization in both C6LP and C6HP cells appear to represent a specific uncoupling of the beta-adrenergic receptor from a functional regulatory component of adenylate cyclase.