The stimulation by adrenocorticotropin of the phosphorylation of adrenal inhibitor-1: a possible role in steroidogenesis.

Adrenocorticotropin (ACTH) acts via protein kinase A and the putative phosphorylation of a regulatory protein(s). We have examined a role in this process for inhibitor-1 which, following phosphorylation by protein kinase A, inhibits a phosphoprotein phosphatase activity. In the tissues we have examined inhibitor-1 was found primarily in the cytosol (90%) with the rest in the mitochondrial pellet. The highest concentration was in the adrenal cortex. Using adrenal cortex slices, the stimulation of steroidogenesis by ACTH and dibutyryl cAMP is paralleled by a corresponding increase in the phosphorylation of inhibitor-1 and this is not affected by inhibitors of protein synthesis which inhibit the steroidogenic response. The increase in the phosphorylation of inhibitor-1 occurs in the cytosol, while that in the mitochondrial pellet is not affected. Exogenous phosphorylated inhibitor-1, however, was found to inhibit phosphoprotein phosphatase activity in the mitochondrial pellet. The results suggest that the ACTH-induced increase in phosphorylated inhibitor-1 in the cytosol can affect susceptible phosphoprotein phosphatase activity both in the cytosol and the mitochondrial pellet and, hence, the level of phosphorylation of regulatory protein(s) involved in steroidogenesis.

A regulation of the level of phosphorylated phospholamban by inhibitor-1 in rat heart preparations in vitro.

Inhibitor-1 following phosphorylation by protein kinase A inhibits phosphoprotein phosphatase-1. We have found that in the rat heart inhibitor-1 is present only in the cytosolic fraction and that its phosphorylation in ventricular slices was increased by isoproterenol but not by isoproterenol and propranolol together. Cardiac microsomal phosphoprotein phosphatase activity, with added phosphorylase a as the substrate, was inhibited 33% by phosphorylated inhibitor-1. Phosphorylated inhibitor-1 decreased the dephosphorylation by exogenous phosphoprotein phosphatase-1 of phospholamban present in the sarcoplasmic reticulum membranes. These results suggest an interaction of cytoplasmic inhibitor-1 with either cytoplasmic or membrane-bound phosphoprotein phosphatase-1 with a subsequent effect on the level of phosphorylated phospholamban and the probable involvement of this interaction in the cardiac response to beta-adrenergic hormones.

The stimulation by calcium and its inhibition by ADP of cholesterol side-chain cleavage activity in adrenal mitochondria.

Cholesterol side-chain cleavage activity (cholesterol SCC) in a mitochondrial preparation is increased by calcium in a sigmoidal manner. A 5-10-fold increase is obtained and an effect may be seen at 20 microM CaCl2. ADP inhibits the stimulation by calcium with a shift of the sigmoid curve to the right and 10 microM ADP results in a 4-fold increase in the amount of CaCl2 required to obtain one-half the maximal stimulation value. The inhibition is specific for ADP and inhibition by ATP is due to the formation of ADP. The characteristics of the calcium-ADP modulation are such that a suitable ADP-inhibited level of cholesterol sidechain cleavage activity will be stimulated by a given increment of calcium to a greater extent than in the absence of the added ADP. Steroid 11 beta-hydroxylation is also stimulated in a sigmoidal manner by calcium and this stimulation is inhibited by ADP. The 11 beta-hydroxylation, however, is less sensitive to calcium and ADP so that changes in cholesterol SCC are obtained at concentration of calcium and ADP where minimal effects on 11 beta-hydroxylation are found. Calmodulin-like activity is present in the mitochondrial preparation. No evidence, however, for a role for calmodulin in the calcium-ADP effects could be obtained, but the possibility of its involvement cannot be excluded. The calcium-ADP modulations are of a magnitude and take place at sufficiently low concentrations to suggest a physiological role in the regulation of mitochondrial steroidogenesis.

Studies on the stimulation by Ca2+ and the inhibition by ADP of steroid 11beta-hydroxylation in adrenal mitochondria.

The Ca2+ stimulation and ADP inhibition of the steroid 11beta-hydroxylation enzyme system in adrenal mitochondria have been investigated. The Ca2+-stimulation is competitively inhibited by Sr2+. Nucleoside monophosphates were not inhibitory and among the nucleoside diphosphates and triphosphates only ADP, CDP and ATP were inhibitory with ADP being more inhibitory than the other nucleotides. None of the non-inhibitory nucleotides could reverse the ADP inhibition. ADP appears to inhibit the Ca2+-stimulation since the activity in the absence of Ca2+ was not inhibited by ADP. Ca2% and ADP did not affect the binding of deoxycorticosterone to the mitochondrial preparation. Ca2+ stimulated the reduction of cytochrome P-450 by NADPH and this was inhibited by ADP. These effects were independent of the presence of deoxycorticosterone. Neither Ca2+ nor ADP affected the reduction of adrenodoxin by NADPH. The inhibition by ADP with respect to Ca2+ or to deoxycorticosterone is non-competitive. The Ca2+ stimulation with respect to deoxycorticosterone is uncompetitive. Preincubation of the mitochondrial preparation with Ca2+ decreases the inhibition by ADP and preincubation with ADP increases its inhibitory effects. These data have been interpreted to indicate that Ca2+ and ADP influence the relationship between adrenodoxin and cytochrome P-450 so that a complex of increased activity in the reduction of cytochrome P-450 is formed in the presence of Ca2+ and a less active complex is formed in the presence of Ca2+ plus ADP.

The inhibition by ADP of NADPH-supported adrenal steroid 11beta-hydroxylation.

The 11beta-hydroxylation of deoxycorticosterone to form corticosterone in adrenal mitochondria has been found to be inhibited by ADP and ATP, with ADP being the more inhibitory of the two. The evidence suggests that the ADP directly affects the enzyme system.

Some characteristics of adrenal steroidogenesis and their possible relationships to the action of the adrenocorticotropic hormone.

PIP: An attempt to define in quantitative terms the characteristics of the biphasic rate curve for pregnenolone synthesis in cell-free systems from the adrenal using male Sprague-Dawley rats is reported. When adrenocorticotropic hormone (ACTH) was used 2 units of .2 ml of .9% saline were injected ip 15 minutes before killing the rats. The effect of ACTH on adrenal steroidogenesis is in the stimulation of the rate of conversion of cholesterol to pregnenolone. This reaction sequence is thought to occur in the mitochondria. Methods of preparing subcellular fractions are described. Incubation of pregnenolone with mitochondria for 20 minutes at 20 degree C resulted in a 70% disappearance of the pregnenolone. This loss does not occur if the mitochondria are boiled, indicating an enzymatic process. The rate of pregnenolone synthesis characteristically shows a biphasic curve with a rapid primary rate and a slower secondary rate. ACTH administration in vivo increased both rates but the percentage increase was greater for the secondary rate. In addition an increase in the duration of the primary rate resulted. Different explanations are offered for these characteristics. Pregnenolone may act as an inhibitor of its own synthesis from cholesterol but not from 20alpha-hydroxycholesterol. Substances that cause mitochondria to swell may stimulate pregnenolone synthesis. Another theory proposes that the limiting ACTH-sensitive step is the rate at which mitochondrial cholesterol is transported to or binds to the cholesterol side-chain cleavage enzyme. The possible role of an inhibitor in the regulation of steroidogenesis is indicated. Data are consistent with the observation that the transition from the primary rate to the slower secondary rate shows the accumulation of an inhibitory substance. The action of ACTH would then be to modify the structure of the cholesterol side-chain cleavage enzyme so that there is a decreased susceptibility of the enzyme to the inhibitor.^ieng

Concerning the subcellular distribution of 3beta-hydroxysteroid dehydrogenase/isomerase in the rat adrenal.

The distribution of 3beta-hydroxysteroid dehydrogenase/isomerase in the subcellular fractions of rat adrenal gland has been determined. Based on the total activity found, 55% was in the microsomal fraction, 10% in the heavy-mitochondrial fraction, 3% in the light-mitochondrial fraction and 26% in the fraction consisting of cell-debris plus nuclei. Ninety-five percent of the total activity was recovered in the fractions. Approximately half the activity in the heavy-mitochondrial fraction could be accounted for by microsomal contamination.

Mitochondrial protein synthesis and the stimulation of steroidogenesis by cyclic adenosine 3',5'-monophosphate in isolated rat adrenal cells.

The stimulation by cyclic AMP of steroidogenesis in rat adrenal cells isolated by trypsin treatment was inhibited by D-threo-chloramphenicol and by its L-threo-isomer. The former is an inhibitor of mitochondrial protein synthesis while the latter is not. Both substances, at concentrations which inhibit steroidogenesis, inhibit amino acid incorporation into the proteins of microsomes. Inhibition in other subcellular fractions also occurs depending on the isomer and its concentration. In no case was there a preferential inhibition of amino acid incorporation into mitochondrial proteins. Carbomycin, another inhibitor of mitochondrial protein synthesis, gave similar results. In addition, subfractionation of mitochondria in these experiments revealed no preferential inhibition of amino acid incorporation into the proteins of either the soluble of membrane fractions of this organelle. The above results were obtained at several concentrations of the inhibitors when only partial inhibition of steroidogenesis was present. Both isomers of chloramphenicol inhibited steroidogenesis in a cell-free system to an extent equal to that found with cyclic AMP-stimulated steroidogenesis in intact cells. It is concluded that these inhibitors of mitochondrial protein synthesis have multiple metabolic effects in adrenal cells.