Regional distribution of cytosolic and particulate 5alpha-dihydroprogesterone 3alpha-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.

Barbiturates modulate the activity of the pituitary 3 alpha-hydroxysteroid oxidoreductases and inhibit their production of 3 alpha,5 alpha-tetrahydroprogesterone.

The effects of sodium phenobarbital and sodium barbital on the activity of the particulate and cytosolic 5 alpha-dihydroprogesterone 3 alpha-hydroxysteroid oxidoreductases (3 alpha-HSORs) of female rat anterior pituitary were investigated. By altering the 3 alpha-HSOR catalyzed conversion of 5 alpha-dihydroprogesterone (5 alpha-DHP) to 3 alpha,5 alpha-tetrahydroprogesterone (3 alpha,5 alpha-THP), these barbiturates could influence the in situ production of 3 alpha,5 alpha-THP. 3 alpha,5 alpha-THP has potent barbiturate-like effects on brain GABAA receptors. Both phenobarbital and 3 alpha,5 alpha-THP can affect gonadotropin release in female rats. In vitro incubations of each 3 alpha-HSOR activity were assayed in the presence of sodium phenobarbital (0.1 to 10.0 mM) or sodium barbital (1.0 to 10.0 mM). Since both 3 alpha-HSOR activities catalyze the reversible oxidoreduction of 5 alpha-DHP and 3 alpha,5 alpha-THP, we examined the effect of these barbiturates not only on the conversion of 5 alpha-DHP to 3 alpha,5 alpha-THP (reductive reaction) but also on the "back conversion" of 3 alpha,5 alpha-THP to 5 alpha-DHP (oxidative reaction). The results indicate that both phenobarbital and, to a lesser extent barbital, significantly affected the activities of the two 3 alpha-HSORs in both reductive and oxidative directions. In the reductive direction, phenobarbital inhibited the activity (33%) of both cytosolic and particulate enzymes which would presumably decrease the levels of 3 alpha,5 alpha-THP. In the oxidative direction, a pattern of stimulation was observed (20 to 100%). Thus, this stimulatory effect on the oxidative conversion of 3 alpha,5 alpha-THP to 5 alpha-DHP, which would presumably also decrease 3 alpha,5 alpha-THP levels, appears correlated with the inhibitory effect of these barbiturates on the reductive conversion of 5 alpha-DHP to 3 alpha,5 alpha-THP. Sodium barbital exhibited somewhat similar effects. These changes suggest that barbiturates can lower 3 alpha,5 alpha-THP levels in the anterior pituitary. The results also suggest the possibility that lowered 3 alpha,5 alpha-THP levels may be involved, at least in part, in the reduction of gonadotropin release by barbiturates.

Changes in pituitary, hypothalamic and brain progestin-metabolizing enzyme activities during lactation.

Progesterone 5 alpha-reductase activity and 5 alpha-dihydroprogesterone 3 alpha-hydroxysteroid oxidoreductase (3 alpha-HSOR) activities (NADH- and NADPH-linked) were measured in anterior pituitaries, hypothalami and brains from lactating rats (8 and 21 days postpartum) and non-lactating (60-day-old cycling) rats. Tissue levels of these three enzyme activities varied significantly among the three groups examined. In terms of pituitary, mean levels of both of its 3 alpha-HSOR activities were 40-140% higher in actively lactating rats (8 days postpartum) relative to mean levels in lactating rats at weaning (21 days postpartum) or in non-lactating rats. There were no differences in pituitary progesterone 5 alpha-reductase activity among the three experimental groups. In the hypothalamus, the NADPH-linked 3 alpha-HSOR was elevated (50%) at 8 days of lactation compared to the group at 21 days. Hypothalamic NADH-linked 3 alpha-HSOR levels did not vary among the 3 groups. Hypothalamic progesterone 5 alpha-reductase levels in the actively lactating and weaning groups were 30% lower than those of the non-lactating group. Brain levels of progesterone 5 alpha-reductase were also lower in these two lactating groups (35-55%) as compared to the non-lactating control group. In brain, NADPH 3 alpha-HSOR activity did not vary among the three groups, but levels of NADH 3 alpha-HSOR activity were lower (40-50%) in the weaning group as compared to the actively lactating and control groups. These findings suggest the possibility that tissue changes in these progesterone-metabolizing enzyme activities during lactation and at weaning are influencing the in situ supply of 3 alpha,5 alpha-tetrahydroprogesterone and 5 alpha-dihydroprogesterone and their derivative effects on GABAA receptor activity and prolactin and gonadotropin release. The decreased activity of progesterone 5 alpha-reductase in hypothalamus and brain would presumably reduce in situ 5 alpha-dihydroprogesterone formation while increases in 3 alpha-HSOR activity would suggest higher in situ 3 alpha,5 alpha-tetrahydroprogesterone formation, especially in the pituitary.

Pituitary progestin-metabolizing enzyme activities in the aged female rat.

Progesterone 5 alpha-reductase activity and 5 alpha-dihydroprogesterone 3 alpha-hydroxysteroid oxidoreductase (3 alpha-HSOR) enzymic activities (NADH-linked and NADPH-linked) were measured in anterior pituitaries (AP) from aged female rats during three stages of reproductive senescence (constant estrus: CE; repeated pseudopregnancies: PSP; and anestrus: AN). To assess ovarian influence on these enzymes during these stages of reproductive aging, we also determined enzyme levels from ovariectomized rats from each stage treated with estrogen or vehicle. Progesterone 5 alpha-reductase and NADH-linked 3 alpha-HSOR activities were 2-fold higher in pituitaries of CE rats as compared to those of PSP and AN rats. NADPH-linked 3 alpha-HSOR levels did not differ among the three stages. All three enzyme levels were elevated 2- to 5-fold as compared to the corresponding enzyme levels from young cycling rats. After ovariectomy (10 days), 5 alpha-reductase activity in PSP and AN rats was elevated 3- to 4-fold relative to mean levels in intact PSP and AN rats. Ovariectomy had no effect on 5 alpha-reductase levels in CE rats. Under similar conditions, young cycling rats exhibit a 10-12-fold increase. Treatment of ovariectomized PSP and AN rats for 3 days with estradiol benzoate (10 micrograms/day) restored 5 alpha-reductase levels. Ovariectomy had no effect on the NADPH-linked 3 alpha-HSOR levels in CE, PSP or AN animals which is similar to that observed with young rats. Ovariectomy also had no effect on the NADH-linked 3 alpha-HSOR levels except for the CE group. The ovariectomized CE rats exhibited reduced pituitary NADH-linked 3 alpha-HSOR levels (30%). In contrast, young rats exhibit elevated pituitary NADH-linked 3 alpha-HSOR levels after ovariectomy (4- to 5-fold). These changes suggest the possibility that altered processing of progesterone and its 5 alpha- and 3 alpha-reduced products may be one means by which the effectiveness of progesterone is reduced during aging. The results also suggest an altered ovarian role in the regulation of these enzymes.

Characterization of the purified pituitary cytosolic NADPH:5 alpha-dihydroprogesterone 3 alpha-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.

Purification of the NADPH:5 alpha-dihydroprogesterone 3 alpha-hydroxysteroid oxidoreductase from female rat pituitary cytosol.

The NADPH:5 alpha-dihydroprogesterone 3 alpha-hydroxysteroid oxidoreductase (3 alpha-HSOR) [EC 1.1.1.50] which catalyzes the reversible conversion of 5 alpha-pregnane-3,20-dione (5 alpha-dihydroprogesterone; 5 alpha-DHP) to 3 alpha-hydroxy-5 alpha-pregnan- 20-one (3 alpha-,5 alpha-tetrahydroprogesterone; 3 alpha,5 alpha-THP) was purified to apparent homogeneity from female rat anterior pituitary cytosol by a three step micro-purification procedure. Specific activity of purified 3 alpha-HSOR was enriched 438-fold from that in pituitary cytosol using successive ion exchange, chromatofocusing and affinity column chromatography purification steps. 3 alpha-HSOR appears to be a monomer with an approximate molecular weight of 36 kDa and an isoelectric point of about 5.75. The purified enzyme appears as a single protein staining band (36 kDa) when examined by polyacrylamide gel electrophoresis and with both silver or Coomassie blue staining. Under non-dissociating electrophoretic conditions, all of the 3 alpha-HSOR activity co-migrated with the 36 kDa protein staining band. The purified enzyme in the presence of the preferred cofactor, NADPH, has an apparent Km for 5 alpha-DHP of 82 nM and a Vmax of 1.2 mumol of 3 alpha,5 alpha-THP formed per mg protein/30 min. The Km for NADPH was 0.71 microM. In the oxidative direction, the enzyme in the presence of NADP+ has a Km for 3 alpha,5 alpha-THP of 1.4 microM and a Vmax of 9.7 mumol of 5 alpha-DHP formed per mg protein/30 min. The Km for NADP+ was 1.6 microM.

Neuroendocrine metabolism of progesterone and related progestins.

In mammalian neuroendocrine structures the metabolic processing of progesterone and related natural progestins is primarily a reductive process involving the C-4,5 double bond and the C-3 and C-20 ketones. The principal products of the neuroendocrine metabolism of progesterone in female rats are the two 5 alpha- and 3 alpha-reduced metabolites, 5 alpha-dihydroprogesterone and 3 alpha,5 alpha-tetrahydroprogesterone, with lesser amounts of the corresponding 20 alpha-reduced products. Certain of these metabolites produce some, but not all, of progesterone's biological effects. 5 alpha-Dihydroprogesterone and 3 alpha,5 alpha-tetrahydroprogesterone, in particular, have potent progesterone-like effects on neuroendocrine functions, such as gonadotropin regulation. The two other principal ovarian progestins, 20 alpha-dihydroprogesterone and 17 alpha-hydroxyprogesterone, are metabolized in an analogous manner. The major neuroendocrine progestin conversions therefore appear to be 5 alpha-reduction and 3 alpha-hydroxysteroid oxidoreduction. In the hypothalamus and anterior pituitary, the enzymic activities that catalyse these conversions appear to be under ovarian control and appear to vary with changing reproductive states. These quantitative changes in processing, together with the potent progesterone-like effects of certain metabolites, suggest that these neuroendocrine conversions may provide an important mechanism for mediating some of the effects of progesterone. Alternatively, some metabolites, by duplicating selected effects of progesterone, may provide a means of prolonging certain of its effects while others are terminated.

The kinetic mechanism of the hypothalamic progesterone 5 alpha-reductase.

The kinetic mechanism of the hypothalamic NADPH-linked progesterone 5 alpha-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 Km (progesterone) = 95.4 +/- 4.5 nM; apparent Kia(NADPH) = 9.9 +/- 0.7 microM). The plot of 1/v vs 1/progesterone intersected on the ordinate 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 Kis = 4.3 +/- 1.3 microM) and a noncompetitive inhibitor versus progesterone (apparent Kis = 31.9 +/- 1.4 microM and apparent Kii = 145.4 +/- 15.5 microM). 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.

Conversion of 17 alpha-hydroxyprogesterone to 5 alpha, 3 alpha, and 20 alpha-reduced metabolites by female rat anterior pituitary and hypothalamus.

The metabolism of 17 alpha-[3H]hydroxyprogesterone was examined in female rat anterior pituitary and hypothalamic tissues. After reverse isotopic dilution analysis and purification to constant specific activity, the following 5 alpha-, 3 alpha- and 20 alpha-reduced products were detected in both tissues: 17 alpha-hydroxy-5 alpha-pregnane-3,20-dione; 3 alpha,17 alpha-dihydroxy-5 alpha-pregnan-20-one; 17 alpha,20 alpha-dihydroxy-4-pregnen-3-one and 5 alpha-pregnane-3 alpha,17 alpha,20 alpha-triol. While the metabolites formed were qualitatively the same, there were quantitative differences between the two tissues. The 3 alpha,5 alpha-reduced metabolite, 3 alpha,17 alpha-dihydroxy-5 alpha-pregnan-20-one, was the principal product in the anterior pituitary while the 5 alpha-reduced metabolite, 17 alpha-hydroxy-5 alpha-pregnane-3,20-dione, was produced in largest amount by the hypothalamus. With both tissues, the aforementioned four products plus starting substrate accounted for nearly all of the starting radioactivity. There was no evidence for the formation of C19 steroids (androgens) despite the presence of the 17 alpha-hydroxy group.

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 anterior pituitary progesterone 5 alpha-reductase.

An analysis of the kinetic mechanism of the microsomal NADPH-linked progesterone 5 alpha-reductase obtained from female rat anterior pituitaries was performed. Initial velocity, product inhibition and dead-end inhibition studies indicate that the kinetic mechanism for the progesterone 5 alpha-reductase is equilibrium ordered sequential. Analysis of the initial velocity data resulted in intersecting double reciprocal plots suggesting a sequential mechanism [apparent Km(progesterone) = 88.2 +/- 8.2 nM; apparent Kia(NADPH) = 7.7 +/- 1.1 microM]. Furthermore, the plot of 1/v vs 1/progesterone intersected on the ordinate which is indicative of an equilibrium ordered mechanism. Additional support for ordered substrate binding was provided by the product inhibition studies with NADPH versus NADP and progesterone versus NADP. NADP is a competitive inhibitor versus NADPH (apparent Kis = 7.8 +/- 1.0 microM) and a noncompetitive inhibitor versus progesterone (apparent Kis = 9.85 +/- 2.1 microM and apparent Kii = 63.2 +/- 12.5 microM). These inhibition patterns suggest that NADPH binds prior to progesterone. In sum, these kinetic studies indicate that NADPH binds to the microsomal enzyme in rapid equilibrium and preferentially precedes the binding of progesterone.

Binding of 5 alpha-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.

Pituitary progesterone 5 alpha-reductase: solubilization and partial characterization.

The microsomal progesterone 5 alpha-reductase activity from female rat anterior pituitary has been solubilized and partially characterized with regard to some of its kinetic and physical properties. The solubilization of progesterone 5 alpha-reductase has been achieved through the use of either an n-octyl glucoside (OG)-KCl- or a digitonin-KCl-extraction. The total yield and specific activity of solubilized enzyme activity is greater using the OG-KCl method. Kinetic analyses of microsomal and OG-KCl-solubilized progesterone 5 alpha-reductase have indicated that both of these preparations exhibit a similar apparent Km for progesterone (microsomal Km = 117 +/- 12 nM; solubilized Km = 123 +/- 11 nM), suggesting that the solubilization procedure does not appreciably alter the kinetic behavior of this enzyme activity. The OG-KCl-extracted progesterone 5 alpha-reductase activity also appears quite stable, since essentially no enzyme activity is lost following dialysis at 4 degrees C for 22 h. In addition, the activity of the solubilized-dialyzed enzyme preparation can be slightly stimulated via the addition of phospholipids. Studies on the properties of the microsomal enzyme activity have indicated that this preparation is unaffected by metal chelators (EDTA or EGTA) but can be completely inhibited by the powerful sulfhydryl blocking agent p-chloromercuribenzoic acid. An evaluation of the possible role of flavins (as a hydride carrier between NADPH and the steroid) has shown that progesterone 5 alpha-reduction is inhibited by high levels of flavins and flavin analogs.

Partial characterization of the microsomal and solubilized hypothalamic progesterone 5 alpha-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.

Progestin receptors in dispersed rat anterior pituitary cells: enriched binding in lactotrope fractions.

Cytosolic progesterone and R5020 binding activities were demonstrated in Pronase-dispersed anterior pituitary cells from estrogen-primed ovariectomized and adrenalectomized rats. Pronase-dispersed pituitary cells were also separated into six cellular fractions on the basis of size and density by sedimentation velocity at unit gravity in a BSA gradient. Fractions enriched in lactotropes or gonadotropes were identified by the cellular contents of radioimmunoassayable prolactin and LH, respectively. Cytosolic progestin receptors appeared to be predominantly associated with lactotrope-rich fractions. Since there was some cross-over between the LH and prolactin enriched fractions, progestin receptors may also be associated with a subpopulation of gonadotropes, as well.

Potent inhibition of the hypothalamic progesterone 5 alpha-reductase by a 5 alpha-dihydroprogesterone analog.

The ability of the 5 alpha-dihydroprogesterone analog, 4-aza-4-methyl-5 alpha-pregnane-3,20-dione (AMPD), to inhibit the progesterone 5 alpha-reductase and the two 5 alpha-dihydroprogesterone 3 alpha-hydroxysteroid oxidoreductase activities (NADH- and NADPH-linked) from female rat hypothalamus has been studied. Dose response experiments indicate that AMPD is a potent antagonist of hypothalamic progesterone 5 alpha-reduction but is an ineffective inhibitor of the NADPH- and NADH-linked 3 alpha-hydroxysteroid oxidoreductase activities, even at concentrations up to 10 microM. Kinetic analyses of the interaction of AMPD with the progesterone 5 alpha-reductase show that it is a competitive inhibitor versus progesterone (Ki(slope) = 6.2 +/- 0.5 nM; apparent Km (progesterone) = 130 +/- 12 nM) and an uncompetitive inhibitor versus NADPH (Ki(intercept) = 11.8 +/- 0.8 nM). These inhibition patterns are consistent with the concept that NADPH binding precedes that of either AMPD or progesterone. The inhibition of the progesterone 5 alpha-reductase by AMPD does not appear irreversible since preincubation of the enzymatic activity (at 37 degrees C) with inhibitor and NADPH, for periods of time up to 60 min, does not lead to a time-dependent loss of activity. Furthermore, this inhibition can be easily removed via dilution, even following a 60-min preincubation with AMPD and NADPH. It is postulated that the specific and powerful inhibition of the progesterone 5 alpha-reductase by AMPD may be due to this compound functioning as a transition state analog. This inhibitor should prove valuable in studying the characteristics of the progesterone 5 alpha-reductase and the function of hypothalamic progestin metabolism.

A high affinity inhibitor of pituitary progesterone 5 alpha-reductase.

The capacity of the 5 alpha-dihydroprogesterone analog, 4-aza-4-methyl-5 alpha-pregnane-3,20-dione (AMPD), to inhibit progesterone 5 alpha-reductase and both 5 alpha-dihydroprogesterone 3 alpha-hydroxysteroid oxidoreductase activities (NADPH- and NADH-linked) from the female rat anterior pituitary has been investigated. Dose response studies demonstrate that AMPD is a powerful inhibitor of pituitary progesterone 5 alpha-reduction but is ineffective at inhibiting either of the 3 alpha-hydroxysteroid oxidoreductase activities, even at concentrations up to 10 microM. A kinetic analysis of the interaction of AMPD with progesterone 5 alpha-reductase indicates that it is a competitive inhibitor vs. progesterone [Kislope = 7.2 +/- 0.6 nM; apparent Michaelis-Menten constant (Km) (progesterone) = 193 +/- 18 nM] and an uncompetitive inhibitor vs. NADPH (Kiintercept = 17.9 +/- 1.4 nM). These inhibition patterns are consistent with the view that NADPH binding precedes that of either AMPD or progesterone. Furthermore, AMPD does not appear to be an irreversible inhibitor since preincubation of the enzyme (at 37 C) with AMPD and NADPH, for periods of time up to 60 min, does not lead to a time-dependent loss of activity. The inhibition can also be readily removed by dilution, even after a 60-min preincubation with the inhibitor and NADPH. It is postulated that the selective and potent inhibition of the 5 alpha-reduction of progesterone by AMPD may be due to the steroid functioning as a transition state analog. This inhibitor should prove useful in studying the properties of progesterone 5 alpha-reductase and the function of anterior pituitary progestin metabolism.

Progesterone metabolism by the hypothalamus, pituitary, and uterus of the aged rat.

Progesterone metabolism was examined in tissues of rats in three stages of reproductive senescence (constant estrus, repeated pseudopregnancies, and anestrus) and in young rats. Metabolites were quantitated by reverse isotopic dilution analysis after incubation of the hypothalamus, pituitary, and uterus with [3H]progesterone. The metabolism of progesterone to 5 alpha-dihydroprogesterone and to 20 alpha-hydroxy-5 alpha-pregnan-3-one and the formation of total 5 alpha-reduced products was significantly reduced (by half) in pituitaries of constant estrous rats compared to rats in all other stages. The formation of 3 alpha-hydroxy-5 alpha-pregnan-20-one and total 3 alpha-reduced products was about 2-fold higher in pituitaries and hypothalami of pseudopregnant and anestrous rats than in constant estrous and young rats, but these differences were statistically significant only in the pituitary samples. In the uterus, progesterone metabolism to 20 alpha-dihydroprogesterone was significantly increased in anestrous rats compared to that in constant estrous and pseudopregnant rats. The results indicate that progesterone metabolism by target tissues, particularly the pituitary, is altered during reproductive senescence. They suggest the possibility that changes in the tissue metabolism of progesterone may be one means by which the effectiveness of progesterone is decreased during aging.

Progesterone metabolism by the hypothalamus, pituitary, and uterus of the rat during pregnancy.

Metabolites of [3H]progesterone were quantitated from incubations of hypothalamus, pituitary, and uterus of rats during different stages of pregnancy. The hypothalamus, anterior pituitary, and a section of uterus from five rats on Days 1, 8, 15, and 21 of pregnancy were incubated individually with [3H]progesterone and analyzed for metabolite formation by reverse isotopic dilution analysis. The radioactive metabolites present were 5 alpha-pregnane-3,20-dione (5 alpha-DHP), 3 alpha-hydroxy-5 alpha-pregnan-20-one, 20 alpha-hydroxy-4-pregnen-3-one, 20 alpha-hydroxy-5 alpha-pregnan-3-one, and 5 alpha-pregnane-3 alpha, 20 alpha-diol. The major metabolite formed by the hypothalamus and pituitary was 5 alpha-DHP. In the pituitary samples, formation of 5 alpha-DHP was decreased on Days 15 and 21 of pregnancy compared to Day 1, and formation of 20 alpha-hydroxy-5 alpha-pregnan-3-one was decreased on Day 21 compared to Day 1. In the uterine samples, 3 alpha-hydroxy-5 alpha-pregnan-20-one was the major metabolite formed at all stages of pregnancy. The formation of all metabolic products of progesterone by the uterus was increased on Day 21 compared to Days 1, 8, and 15 of pregnancy. No changes in the formation of progesterone metabolites were observed in the hypothalamic samples during pregnancy. It is concluded that there are different profiles in the in vitro metabolism of [3H]progesterone by the hypothalamus, pituitary, and uterus of the rat during the course of pregnancy.