Leyla C. Ramirez

The role of ACTH in determining the metabolic pathways of deoxycorticosterone by newborn rat adrenal cells in primary culture

The metabolism of deoxycorticosterone (DOC) by newborn rat adrenal cells in primary culture at various times after culture, with and without ACTH, was studied. After 5 days in culture before addition of ACTH, the main products of the metabolism of DOC were corticosterone and 18-hydroxy-11-deoxycorticosterone in a 2:1 ratio. Smaller amounts of 20 alpha-dihydrocorticosterone and 18-hydroxycorticosterone were also found. No reduced metabolites of DOC were detected. Without ACTH the conversion of DOC to corticosterone and 18-hydroxyDOC declined rapidly. After 13 days in culture, this conversion accounted for only half the metabolites. The reductive metabolism of DOC which yields products reduced at 20 alpha and/or 3 alpha/beta and 5 alpha accounted for the other half. When ACTH (22 mU/ml) was added to the culture daily for several weeks, the primary metabolism of DOC remained that of 11 beta- and 18-hydroxylation yielding corticosterone and 18-hydroxyDOC. A minor reductive metabolism was found. Both cultures produced 6 beta-hydroxyDOC. These results demonstrate that ACTH is needed to maintain the efficiency of the 11 beta/18-hydroxylating system. They also show that ACTH controls the type of metabolism predominant in the rat adrenal cell and may be responsible for the balance between the biosynthesis of glucocorticoids and their reductive catabolism in the fasciculata zone of the adrenal gland.

Bioconversion of 16α-hydroxyprogesterone to 16α-hydroxylated corticosteroids by newborn rat adrenal cells in primary culture

Abstract The bioconversion of 2α-hydroxyprogesterone into 2-hydroxylated steroids was accomplished using newborn rat adrenal cells in primary culture. The products were purified using column and thin-layer chromatography, and identified by GC-MS. They resulted principally from the enzymatic reactions of 21-hydroxylation, 11β-hydroxylation, reduction of 20-oxo and 3-oxo groups, and epimerization of the substrate. In addition, minor metabolites resulted from 18-hydroxylation, 6β-hydroxylation and reduction of the 3-oxo-4-ene group. The identification of these compounds allowed us to conclude that the metabolism of 2α-hydroxyprogesterone is similar to that of progesterone in this cellular system. Assuming that the 2β-epimers of the different metabolites arose principally from the transformation of 2β-hydroxyprogesterone, the specificity of the various enzyme systems seems to be similar for both epimers except in the case of the 11β-hydroxylation where the reaction appears stereospecific for the 2β-epimer. The 2α-hydroxyl group on ring A seems to favor the reduction of the 3-oxo group and it does this stereospecifically to the 3β-structure. The epimerization of the substrate, which is most likely enzymatically induced, is the first example of steroid epimerization reported in the adrenal. This is a practical preparative method for synthesizing a variety of steroids hydroxylated at C-2 from a single substrate and could be adjusted to the production of important quantities of 2-hydroxylated metabolites of corticosteroids.

18-Hydroxylase activity in the Y1 adrenal cell line

18-Hydroxylase activity, reported here for the first time in the mouse adrenal tumor cell line (Y1), was expressed in the metabolism of 11-deoxycorticosterone (DOC) and corticosterone (B). Detected after 24 h of incubation, it was more evident after 48 h and produced mostly 18-hydroxy-20 alpha-DHB from these exogenous substrates. However, 18-hydroxylation was quantitatively less significant than the metabolism of 20 alpha-reduction and 11 beta-hydroxylation (of DOC). The latter is also the predominant metabolism of progesterone in this cell line, during the conversion of cholesterol from the serum-supplemented culture media. The cytochrome P-450 11 beta activity of the Y1 cells is similar to that of the mouse in vivo which catalyzes the production of an 11 beta 18-dihydroxylated metabolite as the principal 18-hydroxylated steroid. It is different from that of other species, such as the rat and the bovine, both in terms of the ratio of 11 beta- to 18-hydroxylated metabolites and of the structure of these metabolites.

18-Hydroxylation in the Y-1 adrenal cell line: Response to acth and to culture conditions

The 18-hydroxylation of deoxycorticosterone in the Y-1 adrenal cell line was studied under various incubation and cell culture conditions and compared to 11 beta-hydroxylation. Repeated incubation of the substrate increased both 18- and 11 beta-hydroxylation in the Y-1 cells. Furthermore, both 18- and 11 beta-hydroxylation were increased with increased serum concentration and prolonged incubation time. While the increase in 11 beta-hydroxylation seemed to be independent of the type of serum, 18-hydroxylation was much more important in cells cultured in fetal or newborn calf serum supplemented medium than in those cultured in horse serum supplemented medium. As expected, ACTH treatment increased 11 beta-hydroxylation; however, it decreased 18-hydroxylation. The different regulation of these two hydroxylating pathways by ACTH, point to a heterogeneity of the cytochrome P-450(11) beta of the Y-1 cell line.

Evidence of formation of isomeric methoximes from 20-oxosteroids

The formation and gas chromatographic behavior of syn- and anti-isomers in position 20 of the methoxime-trimethylsilyl (MO-TMS) derivatives of many 20-oxo and 3,20-dioxo-21-hydroxysteroids is reported. The existence of such isomers was established from the gas chromatographic (GC) and mass spectrometric analysis of the MO-TMS derivatives of 3 alpha,21-dihydroxy-5 beta-pregnan-20-one and its 17 alpha-epimer. The degree of separation during GC analysis of the syn- and anti-isomers in position 20, as well as those in position 3, is associated to the position of additional hydroxyl groups on the steroid ring. These data are very important for the location of oxygenated substituents such as 2 alpha/2 beta, 6 alpha/6 beta, 11 beta, 16 alpha, 17 alpha, 18, 19 or 21-hydroxyl groups during structural studies of 20-oxo and 3,20-dioxosteroids.

Aldosterone synthase activity in the Y-1 adrenal cell line

The Y-1 adrenal cell line was shown to produce 20 alpha-dihydroaldosterone from deoxycorticosterone. This compound was identified by GC-MS by comparison with the previously synthesized reference compound. Two other 18-hydroxylated metabolites were identified as 11 beta,18-dihydroxy-20 alpha-dihydroprogesterone from endogenous cholesterol and 18-hydroxy-20 alpha-dihydro-11-dehydrocorticosterone from DOC. The conditions necessary for the synthesis of these compounds are culturing in 20% serum-supplemented medium and repeated incubations with the substrate. The production of 11 beta-hydroxylated steroids and that of 18-oxygenated steroids is stimulated differently by ACTH and angiotensin II suggesting the expression of two different enzymes, cytochrome P-450(11)beta and cytochrome P-450aldo. The Y-1 cell line can secrete either 11 beta-hydroxylated steroids characteristic of the glucocorticoid pathway or 18-oxygenated steroids characteristic of the mineralocorticoid pathway, which in vivo are generally produced in two different zones of the adrenal cortex. This cell line should be an interesting model for the study of the molecular mechanisms regulating the expression of these two enzymes involved in the final steps of the steroidogenic pathways.

19-Hydroxylated steroids, new metabolites produced by the Y1 adrenal cell line.

The expression of 19-hydroxylase activity in the Y1 adrenal cell line is reported here for the first time. Two new metabolites from the incubation of deoxycorticosterone (DOC) with these cells, 19-hydroxy-20 alpha-dihydroDOC and 19-hydroxy-20 alpha-dihydrocorticosterone, have been identified. The most important of the two is the 11 beta,19-dihydroxylated metabolite, which is produced in smaller amounts than 18-hydroxy-20 alpha-dihydrocorticosterone. A third 19-hydroxylated metabolite was identified as 19-hydroxy-20 alpha-dihydroprogesterone, produced from the cholesterol in the serum supplemented medium. These results show that the cytochrome P-450(11)beta of this cell line expresses 19-hydroxylase activity in addition to 11 beta- and 18-hydroxylase activities, as do those of other species.

Biosynthesis of (20S)-20α-reduced steroids simultaneously with corticosteroids in primary cultures of newborn rat adrenocortical cells stimulated by ACTH

Newborn rat adrenal cells in primary culture produce corticosteroid hormones and (20S)-20 alpha-reduced progesterone metabolites in amounts which depend on ACTH concentrations and stimulation time. Eight (20S)-20 alpha-reduced progesterone metabolites, including 18-hydroxy-(20S)-20 alpha-dihydroprogesterone, were identified by comparison of their data in high performance liquid chromatography and in gas chromatography-mass spectrometry to those of existing or newly synthesized reference steroids. Quantitative studies of individual steroid biosynthesis were also performed using high performance liquid chromatography and gas chromatography. Several experiments were made without ACTH and with different concentrations of ACTH for periods of more than 3 weeks. The importance of the two main steroidogenic pathways, corticosteroid biosynthesis and progesterone reductive metabolism was modified by ACTH stimulation of the cultured cells. The progesterone reductive metabolism, important without ACTH and in the first days of ACTH stimulation, was decreased by 6.6 mU of ACTH/ml or higher concentrations but remained active throughout the life span of the stimulated cell cultures.

Chromatographic and mass spectrometric characteristics of 20-dihydroaldosterone

The 20 alpha-reduced derivative of aldosterone, 20 alpha-dihydroaldosterone, was needed as reference compound in order to continue the studies on 18-hydroxylation in the Y-1 adrenal cell line. It was obtained by reduction of aldosterone with sodium borohydride. Analysis of the products of the reaction as methoxime trimethylsilyl (MO-TMS) derivatives by gas chromatography (GC) and GC-mass spectrometry (GC-MS) showed three possible forms of the compound. Their identification was confirmed by comparison with the products obtained by stereospecific reduction of aldosterone using 3 alpha,20 beta-hydroxysteroid dehydrogenase. Chromatographic behavior and mass spectra are given for the three forms of 20 alpha-dihydroaldosterone as the MO-TMS derivatives; that is, the 18-aldehyde, the 18,11 beta-hemiacetal, and the 11 beta:18,18:20 alpha-acetal. The possible origin of these different forms is discussed as a function of these results and of the results obtained by complementary analysis on high-performance liquid chromatography.

Regulation of 11 beta/18-steroid hydroxylation in newborn rat adrenal cells in primary culture.

In newborn rat adrenal cells in primary culture, the level of activity of the 11 beta/18-steroid hydroxylase system involved in the last step of the corticosteroid biosynthesis is increased by ACTH. A parallel study of 11 beta- and 18-hydroxylation showed the same apparent Km values (64 microM) for both hydroxylations. The Vmax values differed: 11.5 micrograms/10(6) cells/h for corticosterone and 6.9 micrograms/10(6) cells/h for 18-hydroxyDOC. A dose response study of the ACTH effect, measured by the bioconversion of deoxycorticosterone to corticosterone and 18-hydroxyDOC, showed maximum hydroxylation with a dose of 2.2 mU of ACTH/ml. Addition of ACTH after several weeks in culture produced a smaller increase in 11 beta/18-hydroxylation. Removal of ACTH after several weeks of treatment produced an immediate decrease in corticosteroid production; readdition of ACTH produced an increase to the previous level in the case of the 22 mU/ml dose, but not in the case of the 2.2 mU/ml dose. The use of actinomycin D demonstrated that ACTH affects mainly the biosynthesis of protein which must be renewed approximately every 24 h. Finally, the effect of pretreatment or co-treatment with various concentrations of the end products of the reaction showed no inhibition or destruction of the 11 beta/18-hydroxylating enzyme system. Therefore, the regulation of the 11 beta/18-steroid hydroxylase system in these cell cultures seems to be accomplished through the induction by ACTH of the transcription involved in the biosynthesis of cytochrome P450(11) beta and the amount of available precursor furnished by endogenous steroidogenesis.