Inhibition of bovine cytochrome P-450(11 beta) by 18-unsaturated progesterone derivatives.

The last step of aldosterone biosynthesis, an 11 beta-hydroxylation followed by two 18-hydroxylations, are catalyzed, in the bovine system, by the same enzyme, the cytochrome P-450(11 beta) (deoxycorticosterone (DOC)-->corticosterone-->18-hydroxycorticosterone-->aldosterone). The 11 beta- and 18-hydroxylase activities were studied separately with a reconstituted enzymic system, using 11-deoxy[14C]corticosterone and [3H]corticosterone, respectively, as substrates. The inhibition of 11 beta-hydroxylase activity by corticosterone was competitive (Ki = 60 microM) showing that transformation of both substrates occurs at the same site. Double-label/double-substrate experiments, using an equimolar mixture of 11-deoxy[14C]corticosterone and [3H]corticosterone, suggested that 18-hydroxycorticosterone is directly formed from 11-deoxycorticosterone without the intermediate corticosterone leaving the enzyme. Inhibitions by 18-vinylprogesterone and 18-ethynylprogesterone, potent inhibitors of aldosterone biosynthesis [Viger, A., Coustal, S., Pérard, S., Piffeteau, A. & Marquet, A. (1989) J. Steroid Biochem. 33, 119-124], were characterized for both activities (11 beta- and 18-hydroxylase). The value of reversible Ki for the 18-hydroxylation (Ki = 5 microM for 18-vinylprogesterone and 30 microM for 18-ethynylprogesterone) is lower than that for the 11 beta-hydroxylation (30 microM and 100-150 microM, respectively); the former inhibitor is stronger than the latter for both steps. The binding of substrates and inhibitors to the active site was also examined by difference absorption spectroscopy. 18-Vinylprogesterone gave rise to a type I spectrum with a Ks value of 35 microM close to that of progesterone, while 18-ethynylprogesterone showed a reverse type I spectrum with a much higher Ks value (140 microM). Based on these results, a hypothetical model, involving a conformational change of the enzyme for the second step, is proposed.

Nitric oxide formation during the cytochrome P-450-dependent reductive metabolism of 18-nitro-oxyandrostenedione.

18-Nitro-oxyandrostenedione (18-ONO2A), a potential mechanism-based inhibitor of the last steps of aldosterone biosynthesis, is well recognized by different cytochrome P-450s, which are able to metabolize it reductively into nitric oxide (NO) and 18-hydroxyandrostenedione. Rat liver microsomal P-450s are able to carry out this reaction with increased efficiency under anaerobic conditions. P-450 3A isozymes induced upon treatment of rats with dexamethasone or troleandomycin were best able to bind and metabolize 18-ONO2A. This reaction was shown to occur in the presence of dioxygen as well, suggesting that it may be of physiological relevance. The formation of NO was detected as a transient P-450-Fe(II)NO complex by UV-visible and EPR spectroscopy. In addition, steroidogenic tissues containing cytochrome P-450s such as bovine adrenal mitochondria or human placental microsomes also were capable of binding and metabolizing 18-ONO2A as judged by the formation of an Fe(II)NO complex. This recognition of a steroid nitrate, a potential antialdosterone and its subsequent metabolism under reductive conditions to generate NO both in hepatic and steroidogenic tissues, can be of pharmacological interest, because NO has been demonstrated to modulate steroidogenesis in addition to other processes such as vascular relaxation, neurotransmission or cytostasis. A nitrate derivative of a steroid could perhaps act as a vectorized NO precursor in which the steroid moiety is targeted specifically to steroid receptors or steroidogenic tissues, thus leading to localized NO liberation.

An improved synthesis of 18-norandrost-4-ene-3,17-dione.

We describe the synthesis of 13 beta- and 13 alpha-H-18-nor-androst-4-ene-3,17-dione (1a and 1b) from 18-hydroxyprogesterone (18-->20) hemiketal, via the 18-acetoxy-17 beta-hydroxyandrost-4-en-3-one formed by a modified Baeyer-Villiger reaction. Saponification of 18-acetoxyandrost-4-ene-3,17-dione with sonication, then retroaldolization in the presence of a formaldehyde trap, methone, afforded the mixture of 1a and 1b with 80% yield in a "one-pot" procedure and at room temperature. This yield was greatly improved, compared with the already published procedure.

Renal action of progesterone and 18-substituted derivatives.

The recently synthesized progesterone (P) derivatives, 18-vinylprogesterone (18VP) and 18-ethynylprogesterone (18EP), are potent inhibitors of aldosterone synthesis by adrenal glands. To evaluate the potential interest of these compounds as antihypertensive drugs, we determined whether they also interact with renal mineralocorticosteroid receptors (MR) in kidney and, if so, whether they mimic or antagonize aldosterone action. For this purpose, we evaluated the potency of 18VP and 18EP 1) to displace [3H]aldosterone binding in cytosolic fractions of kidney from adrenalectomized rats and 2) to interfere with aldosterone-induced stimulation of Na(+)-K(+)-ATPase in the collecting tubule of adrenalectomized rats. The properties of 18VP and 18EP were compared with those of their precursor progesterone and of the antimineralocorticosteroid spironolactone. The binding of [3H]aldosterone was restricted to cytosolic MR by presaturating glucocorticosteroid receptor with RU 38486. All compounds tested displaced [3H]aldosterone binding with the following efficiency: spironolactone greater than aldosterone greater than P greater than 18VP greater than 18EP; apparent Kd varied between 0.66 and 16.4 nM. Spironolactone, P, and 18VP antagonized aldosterone-induced stimulation of Na(+)-K(+)-ATPase in the collecting tubule, whereas 18EP mimicked the mineralocorticosteroid action. The different steroids tested altered Na(+)-K(+)-ATPase stimulation and aldosterone binding with the same order of potency.

18-Substituted progesterone derivatives as inhibitors of aldosterone biosynthesis.

The synthesis of new progesterone derivatives substituted at the 18 methyl group is described. These compounds are designed as 18-monooxygenase, cytochrome P-450-dependent potential kcat inhibitors. Preliminary results on the in vitro biological investigation of these modified progesterones are presented.

Use of tandem mass spectrometry (MS-MS) for aldosterone assay at the nanogram level in complex biological mixtures.

Aldosterone in biological samples was measured comparatively by two methods: radioimmunoassay (RIA) and tandem mass spectrometry. The last method is described in this paper. Aldosterone was derivatized as butane boronate cyclic ester. This derivative gives an intense molecular ion under 70-eV electron impact conditions. This ion exhibits an abundant metastable decomposition in the second field free region by loss of formic acid, which can be used for the assay. A linear relationship was observed between the ionic current and the amount of aldosterone from 10 to 200 ng. A good correlation between this new method and the radioimmunological assay is observed. Aldosterone can be readily detected at the nanogram level.

Synthesis and activity of new inhibitors of aldosterone biosynthesis.

A new family of aldosterone biosynthesis inhibitors, designed as 18-mono-oxygenase, cytochrome-P450-dependent, potential Kcat inhibitors, is described. These compounds are progesterone derivatives substituted at the 18-methyl group. Preliminary results on the in vitro biological evaluation of these modified progesterones are presented. Aldosterone biosynthesis is completely inhibited by 18-vinyl progesterone 5 at a concentration of 0.8 microM and by 18-ethynyl progesterone 6 at 8 microM. It appears that products designed as alkylating agents for the prosthetic heme group are the most potent inhibitors in that series.

A new type of tachykinin binding site in the rat brain characterized by specific binding of a labeled eledoisin derivative.

A new ligand for investigating tachykinin-binding site subtypes was synthesized by coupling the 125I-Bolton and Hunter reagent to eledoisin (125I-BHE). Using a synaptosomal preparation (P2 fraction) of rat cerebral cortex, 125I-BHE was shown to bind with apparent high affinity (apparent Kd = 15.3 nM). When concentrations of up to 30 nM 125I-BHE were used, 125I-BHE binding was specific, saturable, reversible, and temperature-dependent. In contrast to [3H]dopamine, 125I-BHE was not taken up within synaptosomes by an ouabain-sensitive process. Eledoisin, kassinin, and substance P were examined for their ability to inhibit specific 125I-BHE binding to cortical synaptosomes. Eledoisin and kassinin were considerably more potent than substance P, in contrast to the order of potency observed for specific 125I-Bolton-Hunter substance P (125I-BHSP) binding. Specific 125I-BHE binding was highest in the cerebral cortex and hypothalamus; intermediate in the hippocampus, striatum, and thalamus; low in the mesencephalon, septum, and substantia nigra; and absent in the cerebellum. Comparison of these data with those previously obtained for 125I-BHSP binding to synaptosomes indicated that 125I-BHE-labeled binding sites differ markedly from those of 125I-BHSP-labeled binding sites. Therefore, tachykinin receptors other than substance P receptors seem to be present in the central nervous system.

Properties of a 125I-substance P derivative binding to synaptosomes from various brain structures and the spinal cord of the rat.

Using crude synaptosomal fractions (P2 fractions) and 125I-Bolton and Hunter substance P (125I-BHSP) as a ligand, the characteristics of specific binding sites were examined in various brain structures and in the spinal cord (dorsal and ventral parts) of the rat. Scatchard plots revealed the occurrence of a single class of binding sites in the various structures studied with comparable Kd values (from 0.46 to 1.10 nmol/l in the brain and 0.51, 0.56 nmol/l in the spinal cord dorsal and ventral parts respectively) and of marked differences in the number of binding sites (Bmax) (septum greater than striatum greater than hippocampus, hypothalamus greater than mesencephalon greater than cerebral cortex and dorsal part of the spinal cord greater than ventral part). In the brain no correlation was found between the number of 125I-BHSP binding sites and the amount of substance P levels (substance P-like immunoreactivity) in synaptosomes, particularly in the hippocampus and the substantia nigra since the former structure was characterized by its low substance P content and its high number of binding sites and the reverse was observed in the substantia nigra. The ability of several C- and N-terminal fragments of substance P and of tachykinins to compete with 125I-BHSP binding to synaptosomes from the hippocampus, the hypothalamus and the dorsal part of the spinal cord was then determined. Results obtained were closely similar from one structure to another and comparable to those previously reported using whole brain synaptosomes.(ABSTRACT TRUNCATED AT 250 WORDS)

A reinvestigation of the mechanism of delta 5-3-ketosteroid isomerase from bovine adrenal glands.

The mechanism of the isomerization of androst-5-ene 3,17-dione by the isomerase of bovine adrenals has been reinvestigated using the methodology previously developed for the study of the bacterial enzyme of Pseudomonas testosteroni. However, owing to the lower activity of the mammalian enzyme, competitive non-enzymic reaction cannot be neglected. It has been shown that even in the absence of spontaneous isomerization, epimerization and exchange of the label on C4 takes place in the buffer. This prevents any quantitative discussion of the course of the reaction. It is however possible to conclude that the mechanism we have proposed for the bacterial enzyme that is, besides the classical 4 beta leads to 6 beta transfer and exchange with the medium, a competitive abstraction of the 4 alpha proton, accounts for the data obtained with the mammalian microsomes.

A reinvestigation of the mechanism of Pseudomonas testosteroni delta 5-3-ketosteroid isomerase.

The mechanism of the isomerisation of delta 5-3,17-androstenedione by the isomerase (3-oxosteroid delta 4-delta 5-isomerase, EC 5.3.3.1) of Pseudomonas testosteroni has been reinvestigated with delta 5-[4-beta-2H]androstenedione as substrate in H2O and delta 5-androstenedione in 2H2O. A precise localisation of the label in delta 4-androstenendione has revealed that the previously reported 4 beta leads to 6 beta deuterium transfer accounts for only a part of the reaction. Along with this process, removal of the 4 alpha proton is also occurring. This has already been observed with mammalian isomerases. Hence the assumed difference in mechanism between the bacterial and mammalian enzymes is very unlikely.