ADRENAL CELLS IN TISSUE CULTURE .3. EFFECT OF ADRENOCORTICOTROPIN AND 3',5'-CYCLIC ADENOSINE MONOPHOSPHATE ON 11 BETA-HYDROXYLASE AND OTHER STEROIDOGENIC ENZYMES

Monolayer cultures of an adrenocorticotropin-responsive cell line from transplantable mouse adrenal tumors show an augmentation in maximum steroidogenic output in response to prolonged incubation with adrenocorticotropin and 3′,5′-cyclic adenosine monophosphate. This is associated with progressive increases in 11β hydroxylation of endogenously produced steroids. After 12-72-hr exposure to these agents, twofold or greater increases in the 11β hydroxylation of added [3H]pregnenolone or [3H]progesterone were found in stimulated cells. The continued presence of adrenocorticotropin was not required to elicit the stimulation. Adrenocorticotropin added de novo with the radioactive steroids did not stimulate the enzyme. In cells maintained in culture many months in the absence of adrenocorticotropin, as much as a tenfold stimulation could be obtained after 72-hr stimulation with adrenocorticotropin. Precise quantitation of this effect in the intact cell was obtained through the use of a sensitive radioactive assay. The properties of the enzyme in mitochondria obtained from these cultures were similar to those reported in bovine and rat adrenal systems. Reduced triphosphopyridine nucleotide was the optimum pyridine nucleotide cofactor, but Ca2+ (10 mM) was required to elicit the reaction. 11β Hydroxylation was also supported by pyruvate and a number of Krebs' cycle intermediates; the highest activity was obtained with isocitrate. Ca2+ inhibited 11β hydroxylation supported by Krebs' cycle intermediates. Cyanide was not inhibitory. Levels of stimulation comparable with that seen in the intact cells were obtained with mitochondria isolated from adrenocorticotropin-treated cells under all conditions which supported 11β hydroxylation. Adrenocorticotropin and 3′,5′cyclic adenosine monophosphate did not stimulate 3β-hydroxysteroid dehydrogenase or 20α-hydroxysteroid dehydrogenase. © 1969, American Chemical Society. All rights reserved.