Maintenance of whole cell isoproterenol and forskolin responsiveness in adenylate cyclase of permeabilized cells.

Abstract

Digitonin-permeabilized monolayers of C6-2B rat astrocytoma cells exhibit adenylate cyclase activity in the presence of exogenously added ATP. The adenylate cyclase retains the qualitative and quantitative characteristics of hormone stimulated cyclic AMP accumulation in whole cells including GTP dependency and 100 fold stimulation by isoproterenol. Forskolin increased enzymatic activity in the absence of added GTP, however forskolin efficacy and potency was enhanced by GTP. Low non-efficacious concentrations of forskolin, without added GTP, supported isoproterenol-stimulated cyclase activity. The GTP-stimulated isoproterenol response was potentiated by forskolin. Forskolin support of isoproterenol stimulated cyclase in the absence of GTP raises the possibility that forskolin can act independently of GTP in coupling receptors to cyclase catalytic units and/or that forskolin could increase the efficacy and potency of GTP in the coupling reaction. Permeabilization of C6-2B and other cultured cells yields a preparation of adenylate cyclase which retains the enzyme in a state which closely approximates its activity in the native membrane--a system which could prove useful in studies of the regulation of adenylate cyclase in vivo.