LINK: pnas.org/content/96/23/13512.full
Abstract
The neurosteroid 3α-hydroxysteroid-5α-pregnan-20-one (allopregnanolone) acts as a positive allosteric modulator of γ-aminobutyric acid at γ-aminobutyric acid type A receptors and hence is a powerful anxiolytic, anticonvulsant, and anesthetic agent. Allopregnanolone is synthesized from progesterone by reduction to 5α-dihydroprogesterone, mediated by 5α-reductase, and by reduction to allopregnanolone, mediated by 3α-hydroxysteroid dehydrogenase (3α-HSD). Previous reports suggested that some selective serotonin reuptake inhibitors (SSRIs) could alter concentrations of allopregnanolone in human cerebral spinal fluid and in rat brain sections. We determined whether SSRIs directly altered the activities of either 5α-reductase or 3α-HSD, using an in vitro system containing purified recombinant proteins. Although rats appear to express a single 3α-HSD isoform, the human brain contains several isoforms of this enzyme, including a new isoform we cloned from human fetal brains. Our results indicate that the SSRIs fluoxetine, sertraline, and paroxetine decrease the K m of the conversion of 5α-dihydroprogesterone to allopregnanolone by human 3α-HSD type III 10- to 30-fold. Only sertraline(zoloft) inhibited the reverse oxidative reaction. SSRIs also affected conversions of androgens to 3α- and 3α, 17β-reduced or -oxidized androgens mediated by 3α-HSD type IIBrain. Another antidepressant, imipramine, was without any effect on allopregnanolone or androstanediol production. The region-specific expression of 3α-HSD type IIBrain and 3α-HSD type III mRNAs suggest that SSRIs will affect neurosteroid production in a region-specific manner. Our results may thus help explain the rapid alleviation of the anxiety and dysphoria associated with late luteal phase dysphoria disorder and major unipolar depression by these SSRIs.
Enzymatic Activities of Human 3α-HSDs.Human type IIBrain and type III not only differ in sequence but also differ dramatically in their activities. Human 3α-HSD type III and type IIBrain were expressed in bacteria. The K m and V max for the human 3α-HSD type III were determined. The K m for the conversion of DHP to allopregnanolone was 7.2 nM, and the V max was ≈126 nmol/mg/min (Table 2). Fluoxetine decreased the K m to 0.63nM but did not substantially alter the V max. The K m for the conversion of allopregnanolone to DHP was 43 μM, and the V max was 7.1 nmol/mg/min. Fluoxetine decreased the K m slightly but increased the V max 3-fold. Calculation of the enzymatic efficiency for the conversion of DHP to allopregnanolone showed that fluoxetine increased the efficiency 15-fold whereas the effect on the conversion from allopregnanolone to DHP was 4-fold (Table 2). In contrast to the effect seen with the purified rat 3α-HSD, paroxetine appeared to have a greater effect on enzyme kinetics, as it decreased the K m of the conversion of DHP to allopregnanolone from 7.2 to 0.26 nM, resulting in a 18-fold increase in enzyme efficiency. Paroxetine had a slightly lesser effect on the oxidative reaction, increasing the enzyme efficiency only 3-fold. Sertraline also decreased the K m of the conversion of DHP to allopregnanolone from 7.2 to 0.69 nM, a 10-fold increase in enzyme efficiency. Unlike fluoxetine and paroxetine, sertraline increased the K m of the conversion of allopregnanolone to DHP from 43 to 130.4 μM, a 2.5-fold reduction in oxidative enzyme efficiency. Imipramine had no cumulative effect on the enzyme, either in the oxidative or reductive reaction.
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In this windowIn a new windowTable 2 Summary of human type III activity
The effects of fluoxetine, paroxetine, and imipramine on the enzymatic activity of human 3α-HSD type IIBrain also were determined. Unlike human type III, human type IIBrain did not appreciably convert DHP to allopregnanolone or allopregnanolone to DHP. However, the human type IIBrain had considerable 20α-HSD activity and converted progesterone to 20α-dihydroprogesterone (4-pregnen-20α-ol-3, 5-dione). In addition, human type IIBrain possesses 17β-HSD activity and converts androstanediol to androsterone (Fig. 3 A). Fluoxetine affected the K m of the 20α-HSD function of the type II enzyme (Table 3). Fluoxetine, but not paroxetine or imipramine, increased the K m of this reaction from 142 to 238 μM, resulting in a slightly less efficient 20α-HSD activity. Thus, fluoxetine slightly inhibits the side reaction: progesterone to 20α-dihydroprogesterone.
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Download as PowerPoint SlideFigure 3 Schematic representation of the activities of the human type III and type IIBrain 3αHSDs using androgens as substrates. (A) Type IIBrain. (B) Type III. Activities definitively determined by using DHT and androstanediol are shown by thick arrows. Reactions denoted by thin arrows may be catalyzed by the enzymes but were not tested.
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In this windowIn a new windowTable 3 Summary of type IIBrain activity with progesterone
Although the type IIBrain isoform did not use progestins as substrates, it did use androgens as substrates. It did not convert DHP to allopregnanolone but converted androgens such as 5α-dihydrotestosterone (5α-androstan-17β-ol-3-one) to androstanediol (5α-androstan-3α, 17β-diol). By comparison, the rat 3α-HSD is a pure 3α-HSD and has no additional activities. The type IIBrain enzyme did not appreciably oxidize androstanediol to DHT; instead, androstanediol was converted to androsterone (5α-androstan-3α-ol-17-one), through the 17β HSD activity of this 3α-HSD. The type III also has 17β-HSD activity as it converts DHT to androstanedione (5α-androstan-3α, 17β-dione) and androstanediol to androsterone (Fig. 3 B). The 3α activity of type IIBrain was tested to ascertain whether the SSRIs affected the conversion of androgens in a manner similar to the way SSRIs affected the conversion of progestins by human type III (see above).
Fluoxetine and paroxetine affected the reduction of DHT to androstanediol in a similar manner to the way the conversion of DHP to allopregnanolone was affected by the type III enzyme. However, the conversion of DHT to androstanediol required micromolar concentrations of DHT (K m 2.37 μM) whereas the conversion of DHP to allopregnanolone by the type III enzyme or rat 3αHSD required only nanomolar concentrations of substrate. Both fluoxetine and paroxetine decreased the K m of the enzyme (47-fold and 6-fold, respectively) and also increased the V max (3.6-fold and 11-fold) (Table 4). The enzymatic efficiency of the conversion of DHT to androstanediol increased 163-fold when the enzyme was incubated with fluoxetine and 63-fold with paroxetine but did not change substantially with imipramine. These results suggest that both fluoxetine and paroxetine enhance the 3α activity of 3αHSD type IIBrain when androgens are used as a substrate. The 17β-hydroxysteroid dehydrogenase activity of the 3αHSD type IIBrain also was affected by paroxetine. The conversion of androstanediol to androsterone is altered in the presence of paroxetine, with both a 2-fold increase in K m and a 5-fold increase in V max. Paroxetine decreases the K m slightly and increases the V max 5-fold. Imipramine also appeared to have an effect on the conversion of androstanediol to androsterone, the mechanism for which is unknown.