Harry J. Karavolas

Regional distribution of cytosolic and particulate 5α-dihydroprogesterone 3α-hydroxysteroid oxidoreductases in female rat brain

Numerous studies have indicated that progesterone metabolites, particularly 3alpha,5alpha-tetrahydroprogesterone, can potently influence multiple brain functions, e.g. they have the capacity to mediate gonadotropin regulation and various anticonvulsive, anesthetic and anxiolytic effects. These circulating progesterone metabolites are likely to represent only a fraction of the bioavailable pool of these steroids in that the central nervous system (CNS) also possesses enzymes that can synthesize these metabolites in situ. Therefore, because the ability of the CNS to produce these neuroactive progestins is an important consideration when assessing overall progestin function and metabolism, we measured the major progesterone metabolizing enzyme activities, namely the cytosolic NADPH and particulate NADH 5alpha-dihydroprogesterone 3alpha-hydroxysteroid oxidoreductase (3alpha-HSOR) and progesterone 5alpha-reductase activities in nine brain regions from random cycling and ovariectomized rats. These assays entailed the use of reverse isotopic dilution analysis and revealed that all three enzymic activities were present in each of the brain regions examined, but that these regions displayed differential patterns with regard to their levels of cytosolic and particulate 3alpha-HSOR activity. The cytosolic 3alpha-HSOR activity was highest in the olfactory bulb/tubercle and colliculi regions which were greater than levels in the hypothalamus/preoptic area and cerebellum which were greater than levels in the amygdala/striatum and hippocampus/dentate gyrus. Midbrain/thalamus, cerebral cortex and pons/medulla were different only from the olfactory bulb/tubercle and colliculi regions. The particulate 3alpha-HSOR activity was highest in the olfactory bulb/tubercle region followed by colliculi, hippocampus/dentate gyrus and pons/medulla which were greater than levels in the hypothalamus/preoptic area, cerebellum and amygdala/striatum. Cerebral cortex and midbrain/thalamus were different only from the olfactory bulb/tubercle area. The highest levels of 5alpha-reductase activity were found in the pons/medulla region followed by the colliculi, midbrain/thalamus, cerebellum and olfactory bulb/tubercle which were greater than levels in the amygdala/striatum, hippocampus/dentate gyrus, hypothalamus/preoptic area and cerebral cortex. It is interesting to note that although 5alpha-reductase may control, at least in part, substrate levels for the 3alpha-HSORs, the distribution of 5alpha-reductase activity in these nine brain regions did not correlate with 3alpha-HSOR levels. The differences in the levels of activity of these three enzymes in various brain regions suggests a role in maintaining a differential balance of the neuroactive steroid, 3alpha,5alpha-tetrahydroprogesterone, and its precursor, 5alpha-dihydroprogesterone, in various regions of the CNS.

Binding of 5α-dihydroprogesterone and other progestins to female rat anterior pituitary nuclear extracts

The specific binding of 5 alpha-dihydroprogesterone (5 alpha-DHP), progesterone and R5020 to anterior pituitary nuclear extracts was studied using ovariectomized rats treated with estradiol benzoate and progesterone. The binding equilibrium association constant for 5 alpha-dihydroprogesterone with different preparations of nuclear extract ranged from 4.0 +/- 0.54 microM-1 to 59 +/- 10 microM-1. The association constants for progesterone and R5020 were 0.39 +/- 0.81 nM-1 and 1.5 +/- 0.15 nM-1, respectively. The binding of 5 alpha-DHP was specific in that it could be competed only by R5020, progesterone and 5 alpha-DHP and not by other progesterone metabolites and other hormonal steroids tested. With [3H]-progesterone and [3H]R5020 as ligands the most efficient competitors also were R5020, progesterone and 5 alpha-DHP. Estrogen priming of ovariectomized rats consistently and significantly increased the number of binding sites for all three progestins and subsequent progesterone treatment enabled their detection at higher levels in the nuclei.

Comparative facilitation and inhibition of lordosis in the guinea pig with progesterone, 5α-pregnane-3,20-dione, or 3α-hydroxy-5α-pregnan-20-one

Abstract The major 5α-reduced metabolites of progesterone tentatively identified in neural tissue of the guinea pig were evaluated in this species for their ability to facilitate and inhibit lordosis responses of spayed females after estradiol benzoate (EB) pretreatment. 5α-Dihydroprogesterone was found to be an effective facilitative agent, but at doses of 0.05-0.3 mg administered at time intervals from 12–60 hr after estradiol, it was not as potent as progesterone. The steroids 3α-hydroxy-5α-pregnan-20-one and 5β-pregnane-3,20-dione, evaluated at only one dose level (0.18 mg) and at one time interval after estradiol (36 hr), were found to have moderate facilitative effects, but they were not as effective as 5α-dihydroprogesterone. The inhibitory influences of the metabolites studied were found to be weak relative to progesterone when given at doses of 0.6 mg 1 hr after EB. However, when 5α-dihydroprogesterone was given at a higher dose (3.6 mg) it was then found to be an effective inhibitor of the lordosis response. The results indicate that this metabolite has behavioral influences similar to those of progesterone for both facilitation and inhibition of estrus. It was suggested that the superior potency of injected progesterone may be due to mechanisms of bioavailability, including relative solubility differences of the two steroids when administered subcutaneously.

Progesterone metabolism in the pineal, brain stem, thalamus and corpus callosum of the female rat

Specific brain regions, namely, thalamus, tectum, tegmentum, cerebellum, medulla and pineal, from five proestrous rats were incubated for 30 min with [3H]progesterone. After reverse isotopic dilution analysis, the following metabolites were identified in all incubations by purification to constant specific activity, derivative formation and/or gas liquid chromatography trapping: [3H]5alpha-pregnane-3, 20-dione (10-20% of the starting substrate except pineal -- 0.7%), [3H]3alpha-hydroxy-5alpha-pregnan-20-one (1.6-3.8% except for pineal -- 0.5%) and [3H]20alpha-hydroxy-4-pregnen-3-one (0.05-0.11%). Preliminary results from the corpus collosum incubation indicated the presence of the same metabolites. Although some apparent constant specific activities were obtained for 20alpha-hydroxy-5alpha-pregnan-3-one and 5beta-pregnane-3, 20-dione, the low levels of 3H associated with these steroids did not permit a definitive identification. The results indicate the presence of at least delta1-steroid 5alpha-reductase, 3alpha-hydroxysteroid dehydrogenase and 20alpha-hydroxysteroid dehydrogenase activities with progesterone as substrate in the brain regions examined.

Subcellular location of hypothalamic progesterone metabolizing enzymes and evidence for distinct NADH- and NADHP-linked 3α-hydroxysteroid oxidoreductase activities

Abstract The subcellular location of adult female rat hypothalamic steroid 5α-reductase and 3α-hydroxysteroid oxidoreductase (3α-HSD) activities, which catalyze the conversion of progesterone to 3α-hydroxy-5α-pregnan-20-one via 5α-pregnane-3,20-dione, have been investigated using 3 H-labeled substrates and an isotopic dilution assay system. In crude homogenates, appreciable conversion of 5α-pregnane-3,20-dione to 3α-hydroxy-5α-pregnan-20-one is observed with either NADH or NADPH as cofactor. The NADH-and NADPH-linked 3α-HSD activities can be partially separated and enriched using subcellular fractionation by differential centrifugation. The subcellular location of these 3α-HSD activities as well as progesterone 5α-reductase activity was determined with the aid of subcellular markers and electron microscopic techniques. When NADPH is the added cofactor, 3α-HSD activity is present primarily in the cytosol and cofractionates with lactate dehydrogenase activity. When NADH is the added cofactor, 3α-HSD activity is enriched in the 1000 g pellet and is further localized to a cell debris-membranes fraction derived from this pellet. Using the muscarinic cholinergic receptor as a plasma membrane marker, the NADH-linked 3α-HSD activity appears to cofractionate with plasma membranes in subcellular fractions. Progesterone 5α-reductase activity is enriched in the 105,000 g pellet and cofractionates with NADPH cytochrome c reductase activity, suggesting a microsomal location. On the basis of these results, there appear to be at least two hypothalamic 3α-HSDs, a cytosolic NADPH-linked activity and a plasma membrane NADH-linked 3α-HSD activity.

Distribution and ovarian control of progestin-metabolizing enzymes in various rat hypothalamic regions☆

The three principal hypothalamic progesterone metabolizing enzyme activities, namely the progesterone 5 alpha-reductase and 5 alpha-dihydroprogesterone NADH- and NADPH-linked 3 alpha-hydroxysteroid oxidoreductase (3 alpha-HSOR) activities, were examined in discrete rat hypothalamic subsections throughout the estrous cycle and from ovariectomized rats treated with estradiol benzoate or vehicle. The regions studied included the median eminence, the medial preoptic area and the ventromedial and arcuate nuclei. The enzyme assays were performed using radiolabeled steroid substrates and reverse isotopic dilution analysis. While all four hypothalamic regions obtained from intact cycling animals possessed substantial amounts of these three enzyme activities, the median eminence generally had the highest activity levels (2- to 4-fold greater) except during estrus. The other three regions usually had comparable levels. No significant fluctuations were observed in any enzyme activity over the estrous cycle. After ovariectomy, there was a significant decrease (approximately 35%) in the level of the NADPH-linked 3 alpha-HSOR activity in the median eminence compared to the level observed in intact cycling animals, suggesting ovarian control. Estrogen treatment for 3 days did not restore this enzyme level to that observed in intact animals. The NADPH-linked 3 alpha-HSOR activity from the other three hypothalamic regions, as well as the NADH-linked 3 alpha-HSOR and the 5 alpha-reductase activities from all four brain regions, did not change significantly after ovariectomy. These results indicate that the median eminence possesses an increased capacity for progesterone metabolism relative to the other hypothalamic regions tested, and that the NADPH-linked 3 alpha-HSOR activity in this region may be under ovarian control.

The kinetic mechanism of the hypothalamic progesterone 5α-reductase

Abstract The kinetic mechanism of the hypothalamic NADPH-linked progesterone 5α-reductase from female rats was determined to be equilibrium ordered sequential by initial velocity, product inhibition and dead-end inhibition studies. Analysis of the initial velocity data resulted in intersecting double reciprocal plots indicating a sequential mechanism (apparent K m (progesterone) = 95.4 ± 4.5 nM; apparent K ia (NADPH) = 9.9 ± 0.7±M). The plot of l/v vs l/progesterone intersected on the ordinale which is consistent with an equilibrium ordered mechanism. Ordered addition of the substrates was also supported by product inhibition studies with NADP versus NADPH and NADP versus progesterone. NADP is a competitive inhibitor versus NADPH (apparent K is = 4.3 ± 1.3μM) and a noncompetitive inhibitor versus progesterone (apparent K is = 31.9 ± 1.4μM and apparent K ii = 145.4 ± 15.5μM). These inhibition patterns show that NADPH binds prior to progesterone. Taken together, these analyses indicate that the cofactor, NADPH, binds to the enzyme in rapid equilibrium and preferentially precedes the binding of progesterone.

Characterization of the purified pituitary cytosolic NADPH: 5α-dihydroprogesterone 3α-hydroxysteroid oxidoreductase

The purified cytosolic 3 alpha-hydroxysteroid oxidoreductase (3 alpha-HSOR) from female rat pituitary which catalyzes the reversible conversion of 5 alpha-dihydroprogesterone (5 alpha-DHP) to 3 alpha, 5 alpha-tetrahydroprogesterone (3 alpha, 5 alpha-THP) has been characterized in terms of its steroid substrate specificity, dihydrodiol dehydrogenase activity and inhibition by drugs such as medroxyprogesterone and indomethacin. The purified enzyme has a strong preference for the C21 progestin steroid substrates, 5 alpha-DHP and 3 alpha, 5 alpha-THP, over the corresponding C19 androgenic steroid substrates, 5 alpha-dihydrotesterone (5 alpha-DHT) and 3 alpha, 5 alpha-tetrahydrotestosterone (3 alpha, 5 alpha-THT). The apparent Km for 5 alpha-DHP (80 nM) is about 250 times lower than the Km for the androgenic steroid, 5 alpha-DHT (21 microM). In the oxidative direction, the apparent Km for 3 alpha, 5 alpha-TP (1.4 microM) is about 3-fold lower than the Km for the androgenic steroid, 3 alpha, 5 alpha-THT (4.2 microM). A number of other naturally occurring 3-keto- and 3 alpha(beta)-hydroxy-steroids were assessed for their ability to act as inhibitors (alternate substrates) of the 3 alpha-reduction of 5 alpha-DHP catalyzed by the purified 3 alpha-HSOR. None of the 3 beta- or 5 beta-isomers had any effect. Of the other 3-keto and 3 alpha- steroids tested, only deoxycorticosterone and the ovarian progestins showed any significant inhibition. These may be acting as inhibitors since there was little, if any, direct 3 alpha-reduction of progesterone to 3 alpha-hydroxy-4-pregnen-20-one. Unlike the liver cytosolic 3 alpha-HSOR, the pituitary enzyme has no associated dihydrodiol (quinone) dehydrogenase activity. This enzyme is similar to other cytosolic 3 alpha-HSORs from liver and brain in that it is potentially inhibited by indomethacin and by medroxyprogesterone.

Partial characterization of the microsomal and solubilized hypothalamic progesterone 5α-reductase

Microsomal progesterone 5 alpha-reductase activity from female rat hypothalamus has been solubilized and partially characterized in terms of kinetic and physical properties. The solubilization of progesterone 5 alpha-reductase has been accomplished through the use of a digitonin/KCL-extraction. Both the microsomal and solubilized enzyme activities exhibit similar kinetic and physical characteristics. These include their apparent Km for progesterone (Microsomal Km = 113 +/- 11 nM; solubilized Km = 144 +/- 20 nM) and their affinity (approximately 7 nM) for the 5 alpha-dihydroprogesterone analog, 4-aza-4-methyl-5 alpha-pregnane-3,20-dione, which is a potent inhibitor of progesterone 5 alpha-reduction. Both activities are inhibited by divalent cations (Zn2+ and Cu2+) and the sulfhydryl-blocking agent p-chloromercuribenzoic acid. Studies aimed at optimizing isolation and assay conditions for the hypothalamic progesterone 5 alpha-reductase indicate that the microsomal activity is enhanced in the presence of monovalent cations (particularly K+ and Li+) and the metal chelator EDTA, but is unaffected by the sulfhydryl reducing agent dithiothreitol. The activity of the solubilized enzyme is also enhanced by EDTA but slightly stimulated by dithiothreitol. Analysis of hypothalamic progesterone 5 alpha-reduction for possible flavin involvement (as a hydride carrier between NADPH and the steroid) indicates that the enzyme activity is decreased by high levels of flavins, flavin analogs and riboflavin deficiency.

Properties of the hypothalamic 5α-dihydroprogesterone NADH- and NADPH-linked 3α-hydroxysteroid oxidoreductase activities

Abstract Rat hypothalamic 5α-dihydroprogesterone NADH-linked 3α-hydroxysteroid oxidoreductase, (3α-HSD) activity, which is associated with plasma membranes, has a relatively sharp pH optimum of 5.5 and a temperature optimum range of 45–52°C. This enzyme exhibited apparent K m s for the reductive reaction of 0.40 ± 0.09 μm and 29 ± 12 μ M for 5α-dihydroprogesterone and NADH, respectively. For the oxidative reaction, apparent K m s of 0.11 ± 0.01 μ M and 84.3 ± 22.8 μ M were observed for 3α-hydroxy-5α-pregnan-20-one and NAD + respectively. The NADPH-linked 3α-HSD activity, which is present in the cytosol, proceeded optimally between pH 6–10 and at a temperature of 45°C. This enzyme exhibited apparent K m s for the reductive reaction of 0.083 ± 0.009 μ M and 0.71 ± 0.10 μ M for 5α-dihydroprogesterone and NADPH, respectively. For the oxidative reaction, apparent K m s of 2.33 ± 0.15 μ M and 21.0 ± 3.1 μ M were observed for 3α-hydroxy-5α-pregnan-20-one and NADP + , respectively. The results suggest that the NADH-linked and NADPH-linked 5α-dihydroprogesterone 3α-HSD activities of female rat hypothalamus differ in their intra-cellular interconversion of 5α-dihydroprogesterone and 3α-hydroxy-5α-pregnan-20-one.