Action of Feedback Regulator on Adenylate Cyclase

A factor [the feedback regulator (FR)] formed by adipocytes after the stimulation of a cAMP raising hormone has been found to be a potent inhibitor of membrane-bound adenylate cyclase [EC 4.6.1.1; ATP pyrophosphate-lyase (cyclizing)]. In a standard assay system using rat adipocyte plasma membrane as the source of adenylate cyclase, the FR inhibited adenylate cyclase by lowering the Vmaxwithout affecting the apparent Kmfor ATP (0.3-0.6 mM). The apparent Kafor epinephrine (5-6 μ M) was also not affected by FR. The inhibitory action of FR was partially countered by Mg2+ions. An increase in phosphorylation of plasma membrane was observed when FR was present in the incubation system. The concentration required for a 50% inhibition was four times higher when adenosine 5′-(β ,γ -imino)triphosphate [AMP-P(NH)P] replaced ATP as the substrate for adenylate cyclase, implying that adenylate cyclase was inactivated by phosphorylation caused by FR. Increase in FR inhibition obtained by adding low concentrations of adenosine 5′-(α ,β -methylene)triphosphate or ATP to AMP-(NH)P as the substrate supports this view. The inhibitory action was reversible. These results are consistent with the previously reported phenomena that (1) the unresponsiveness of adipocytes after hormone stimulation is due to the formation of FR, and (2) the recovery of responsiveness of the stimulated cells by washing the cells with regular buffer medium is a result of the removal of FR. The hormone-initiated biphasic curve of cAMP levels in adipocytes is believed to be due to the negative feedback action of FR on adenylate cyclase. The mechanism of action of FR on inhibition of adenylate cyclase appears to be related to the phosphorylation of certain membrane components.