Mechanism of action of spironolactone on cortisol production by guinea pig adrenocortical cells.

Studies were done to determine the mechanism(s) of action of spironolactone (SL) and of its deacetylated metabolite, 7 alpha-thio-SL, to inhibit cortisol secretion by guinea pig adrenocortical cells in vitro. Preincubation of cells at 37 degrees C with SL or with 7 alpha-thio-SL caused a time-dependent decline in subsequent ACTH-stimulated cortisol secretion. In the absence of a preincubation, neither compound affected cortisol production, indicating the need for production of an active metabolite. When the 17 alpha-hydroxylase inhibitor, SU-10'603, was included during the preincubation period, neither SL nor 7 alpha-thio-SL decreased cortisol secretion, indicating the involvement of the 17 alpha-hydroxylase in the activation of both compounds. By contrast, neither the 11 beta-hydroxylase inhibitor, metyrapone, nor the cholesterol sidechain cleavage inhibitor, aminoglutethimide, diminished the effects of SL or of 7 alpha-thio-SL on cortisol secretion. Preincubation of cells with SL or 7 alpha-thio-SL also decreased the conversion of exogenous progesterone to cortisol, but did not affect cortisol production from the 17 alpha-hydroxylated substrates, 17 alpha-hydroxyprogesterone and 11-deoxycortisol, suggesting that only 17 alpha-hydroxylation was impaired. In addition, there was a decline in 17 alpha-hydroxylase activity in microsomes isolated from cells preincubated with SL or with 7 alpha-thio-SL, but no change in microsomal 21-hydroxylase or in mitochondrial 11 beta-hydroxylase and cholesterol sidechain cleavage activities. The results indicate that the direct effects of SL and of 7 alpha-thio-SL on the adrenal cortex to decrease cortisol production result from the selective inhibition of 17 alpha-hydroxylation. Since 17 alpha-hydroxylase activity is apparently required for the activation of both compounds, suicide inhibition of the enzyme may be the mechanism of action.

Spironolactone inhibition of cortisol production by guinea pig adrenocortical cells.

Prior investigations with adrenal subcellular fractions demonstrated that the diuretic, spironolactone (SL), was converted to a reactive metabolite by adrenal microsomes, resulting in the degradation of microsomal cytochrome(s) P-450. Studies were done to evaluate the effects of SL and 7 alpha-thio-SL, a putative intermediate in the activation pathway, on cortisol production by intact guinea pig adrenocortical cells. Preincubation of adrenal cells with SL or 7 alpha-thio-SL caused time-dependent and concentration-dependent decreases in subsequent ACTH-stimulated cortisol production. 7 alpha-Thio-SL was a far more potent inhibitor than SL. In the absence of a preincubation period, neither SL nor 7 alpha-thio-SL affected cortisol production. The results indicate that the effects of SL on adrenal microsomal cytochrome(s) P-450 compromise steroid synthesis by intact adrenal cells and lend support to the hypothesis that metabolism of the drug is required for the inhibition of steroidogenesis.