Desensitization in rat parotid to beta-adrenergic agonists and counteracting effects of forskolin are conserved in membrane and detergent-solubilized adenylate cyclase catalyst activity.

Cyclic AMP accumulation in rat parotid slices is only transiently stimulated by isoproterenol (Harper, J.F. and Brooker, G. Molec. Pharmacol. 13:1048-1059, 1977); the progressive loss of isoproterenol effect is termed desensitization. In this report we show that desensitized cyclic AMP accumulation is associated with desensitization of adenylate cyclase in subsequently prepared membranes and in adenylate cyclase that has been detergent-solubilized from desensitized membranes. Adenylate cyclase in membranes made from isoproterenol-desensitized tissue is desensitized to both the stimulating effects of isoproterenol with 6 mM MgCl2 and of forskolin with 30 mM MnCl2. We have previously determined (Harper, J.F. J. Cyclic Nucleo. Prot. Phosphoryl. Res. 9:401-414, 1984) that cyclic AMP accumulation desensitized to isoproterenol is rapidly counteracted by 1 microM forskolin but not 0.1 microM forskolin. Similarly, if 1 microM forskolin was included in the desensitizing incubation with isoproterenol then adenylate cyclase subsequently prepared was not desensitized. Development of desensitized adenylate cyclase was only partially affected by 0.1 microM forskolin. Desensitization is counteracted by forskolin only on intact cells. Once tissue is homogenized, desensitized adenylate cyclase does not respond as well to forskolin as does control adenylate cyclase. The site of desensitization appears to be at or near the adenylate cyclase catalytic unit. Desensitization of adenylate cyclase catalytic activity remains demonstrable after membranes are solubilized with CHAPS. The adenylate cyclase activity remaining in the supernatant following solubilization of desensitized membranes is depressed to nearly the same extent as found in the membranes. Further, desensitized adenylate cyclase in membrane preparations and after solubilization is desensitized to stimulatory effects of forskolin with 30 mM MnCl2, a condition under which forskolin is probably acting directly on the adenylate cyclase catalytic unit. Desensitization appears not to be dependent on activity of the inhibitory guanine nucleotide regulatory protein (Gi), since pertussis toxin is without effect on desensitization of cyclic AMP accumulation to isoproterenol.