Selective serotonin reuptake inhibitors directly alter activity of neurosteroidogenic enzymes

Abstract

The neurosteroid 3alpha-hydroxysteroid-5alpha-pregnan-20-one (allopregnanolone) acts as a positive allosteric modulator of gamma-aminobutyric acid at gamma-aminobutyric acid type A receptors and hence is a powerful anxiolytic, anticonvulsant, and anesthetic agent. Allopregnanolone is synthesized from progesterone by reduction to 5alpha-dihydroprogesterone, mediated by 5alpha-reductase, and by reduction to allopregnanolone, mediated by 3alpha-hydroxysteroid dehydrogenase (3alpha-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 5alpha-reductase or 3alpha-HSD, using an in vitro system containing purified recombinant proteins. Although rats appear to express a single 3alpha-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 5alpha-dihydroprogesterone to allopregnanolone by human 3alpha-HSD type III 10- to 30-fold. Only sertraline inhibited the reverse oxidative reaction. SSRIs also affected conversions of androgens to 3alpha- and 3alpha, 17beta-reduced or -oxidized androgens mediated by 3alpha-HSD type II(Brain). Another antidepressant, imipramine, was without any effect on allopregnanolone or androstanediol production. The region-specific expression of 3alpha-HSD type II(Brain) and 3alpha-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.

Figure 1

Effect of fluoxetine on 5α-reductase activity. Rat type I 5α-reductase was expressed in COS-1 cells after transfection. Cells were incubated with ¹⁴C-progesterone for 1 hour at 37°C, 72 hours after transfection in the presence (+) or absence (−) of 50 μM fluoxetine. Steroid product was extracted and analyzed by thin layer chromatography. Conversion of progesterone (PROG) to DHP was determined by phosphoimager analysis of the thin layer chromatography and was determined to be 43.5 and 42.5% in the absence and the presence of fluoxetine, respectively.

Figure 2

(A) Nucleotide sequence and the predicted amino acid sequence of the human brain 3α-HSD clone. The ORF contains 969 nucleotides and encodes a protein of 323 amino acids. (B) Comparison of the amino acid sequences of mammalian 3α-HSDs. Only amino acids differing from the human brain type II_Brain 3α(20α, 17β)-HSD sequence are shown. Human type 1 is also chlordecone reductase and DD4 (21, 33); human type 3 is also bile acid binding protein (31); human 20αHSD is also DD1 (34, 35); x, no amino acid (rat 3αHSD is one amino acid shorter than the human forms).

Figure 3

Schematic representation of the activities of the human type III and type II_Brain 3αHSDs using androgens as substrates. (A) Type II_Brain. (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.

Figure 4

Regional distribution of type II_Brain and type III in adult human brain. Northern blots containing 2 μg poly-A⁺ RNA per lane from human brain were hybridized with a PCR-generated probe corresponding to the less conserved 3′ ends of type II_Brain and type III. (A) Lanes: 1, cerebellum; 2, cortex; 3, medulla; 4, spinal cord; 5, occipital lobe; 6, frontal lobe; 7, temporal lobe; 8, putamen. (B) Lanes: 1, amygdala; 2, caudate nucleus; 3, corpus callosum; 4, hippocampus; 5, whole brain; 6, substantia nigra; 7, subthalamic nucleus; 8, thalamus.