5alpha-R Isozymes Research Articles

Effects of metoclopramide on mRNA levels of 5α-reductase isozymes in rat brain

Prolactin and 3alpha, 5alpha-reduced neurosteroids are implicated in maternal behavior. Sulpiride, an inductor of hyperprolactinemia, increased mRNA levels of 5alpha-reductase (5alpha-R), the key enzyme in the biosynthesis of these neurosteroids, indicating a possible interrelationship between prolactin levels and 5alpha-R, although the effects of sulpiride per se cannot be ruled out. In this study, we quantified mRNA levels of both 5alpha-R isozymes in prefrontal cortex of rats after the administration of metoclopramide, another inductor of prolactin secretion. The mRNA levels of both 5alpha-R isozymes were significantly increased in male and female rats by metoclopramide, although the effect was not identical to that of sulpiride. Therefore, other factors induced by these drugs, besides prolactin, may intervene in the regulation of both 5alpha-R isozymes.

Effects of Sulpiride on Prolactin and mRNA Levels of Steroid 5α-reductase Isozymes in Adult Rat Brain

Prolactin (PRL) promotes maternal behavior (MB), a complex pattern of behavior aimed at maximizing offspring survival. 3alpha,5alpha-reduced neurosteroids may also regulate MB. Indeed, PRL, 3alpha,5alpha-reduced neurosteroids, and 5alpha-reductase (5alpha-R), the key enzyme in the biosynthesis of these neuroactive steroids, are all increased in stress situations These facts led us to hypothesize a possible interrelation between PRL levels and 5alpha-R. In the present study we quantified mRNA levels of both 5alpha-R isozymes in prefrontal cortex of male and female rats after administration of sulpiride, an inductor of PRL secretion. Our results demonstrated that mRNA levels of both 5alpha-R isozymes were significantly increased in male and female rats by sulpiride, directly or via sulpiride-induced hyperprolactinemia. Since 3alpha,5alpha-reduced neurosteroids and PRL exert anxiolytic effects in response to stress, these molecules and 5alpha-R may possibly participate in a common pathway of significant adaptation to stress situations.

Effects of Dihydrotestosterone on Brain mRNA Levels of Steroid 5α-Reductase Isozymes in Early Postnatal Life of Rat

We studied the expression of type 1 (5alpha-R1) and type 2 (5alpha-R2) 5alpha-reductase isozymes (5alpha-R) and their regulation by dihydrotestosterone (DHT) in the prefrontal cortex of male and female rats during postnatal sexual differentiation of the central nervous system (CNS), using one-step quantitative RT-PCR coupled with laser-induced fluorescence capillary electrophoresis. We found a higher expression of 5alpha-R2, which is considered a masculinizing enzyme, in the female versus male CNS, and observed sexual dimorphism in the regulation of both 5alpha-R isozymes by DHT. These results open up a new research line that could improve understanding of the role of 5alpha-R isozymes in the physiology of the CNS.

Differential regulation of steroid 5α‐reductase isozymes expression by androgens in the adult rat brain

The enzyme 5alpha-reductase (5alpha-R) is present in many mammalian tissues, including the brain. The physiological importance of 5alpha-R in the brain derives from its capability to convert testosterone (T) to a more potent androgen, dihydrotestosterone (DHT), and to convert progesterone and deoxycorticosterone (DOC) to their respective 5alpha-reduced derivatives, precursors of allopregnanolone and tetrahydroDOC, potent allosteric modulators of the gamma-aminobutyric acid receptor (GABA(A)-R). 5alpha-R occurs as two isoforms, 5alpha-R type 1 (5alpha-R1) and 5alpha-R type 2 (5alpha-R2). We studied the effects of T and DHT on the mRNA levels of both 5alpha-R isozymes in the prefrontal cortex of the adult rat, using an accurate and precise method that combines the high specificity of one-step quantitative RT-PCR with the sensitivity of capillary electrophoresis. Our results demonstrate that both isozymes of 5alpha-R are expressed in the cerebral cortex of adult rats. The gene expression of 5alpha-R type 2 is under the positive control of T and DHT. The gene that codes for 5alpha-R type 1 is not constitutive, because its expression is negatively regulated by T and DHT. These results open up a new research line that may lead to a better understanding of the role of 5alpha-R isozymes in the physiology of the central nervous system.

Development of a quantitative RT-PCR method to study 5alpha-reductase mRNA isozymes in rat prostate in different androgen status.

The incidence of prostate cancer (PCa) and benign prostatic hypertrophy (BPH) continues to rise in the Western world. The development and growth of the prostate gland depends on androgen stimulation. Dihydrotestosterone (DHT) is the primary androgen responsible for prostate development and also for the pathogenesis of benign prostatic hyperplasia (BPH). DHT is synthesized in prostate from circulating testosterone (T) through the action of 5alpha-Reductase (5alpha-R) (EC 1.3.99.5), which occurs as two isozymes, type-1 and type-2. Both types are expressed in the prostate: type-2 isozyme is predominantly expressed in prostate and is implicated in BPH and PCa; type-1 isozyme is also increased in some prostatic adenocarcinomas. In recent years, various inhibitors of type-2 isozyme or of both type-1 and type-2 isozyme have been used in prostatic diseases. We present the first published measurements of mRNA levels of steroid 5alpha-R isozymes in the ventral prostate of rats of different androgen status. We used a novel method that combines the high specificity of competitive PCR with the sensitivity of laser-induced capillary electrophoresis (LIF-CE). We demonstrated that T and DHT androgens control the expression of both 5alpha-R isozymes in rat prostrate. This approach could be of great value for the study of prostate diseases in humans and would allow study at the transcriptional level of the effects of drugs that inhibit either or both of these isozymes.

Semiquantitative RT-PCR method coupled to capillary electrophoresis to study 5alpha-reductase mRNA isozymes in rat ventral prostate in different androgen status.

The development and growth of the prostate gland depends on androgen stimulation. Dihydrotestosterone (DHT) is the primary androgen responsible for prostate development and also for the pathogenesis of benign prostatic hyperplasia (BPH). The incidence of prostate cancer (PCa) and benign prostatic hypertrophy (BPH) continues to rise in the Western world. DHT is synthesized in prostate from circulating testosterone (T) through the action of 5alpha-Reductase (5alpha-R) (EC 1.3.99.5), which occurs as two isozymes, type 1 and type 2. Type-1 5alpha-R is widely distributed in the body, and type-2 5alpha-R is confined to androgen-dependent structures. Both types are expressed in the prostate: type-2 isozyme is implicated in BPH and PCa; type-1 isozyme is also increased in some prostatic adenocarcinomas. In recent years, various inhibitors of type-2 isozyme or of both type-1 and type-2 isozyme have been used in prostatic diseases. In this work we present measurements of mRNA levels of steroid 5alpha-R isozymes in the ventral prostate of rats of different androgen status. We used a novel method that combines the high specificity of semiquantitive PCR with the sensitivity of laser-induced fluorescence capillary electrophoresis (LIF-CE). We demonstrated that T control the expression of 5alpha-R2 isozyme in rat prostrate. This approach could be of great value for the study of prostate diseases in humans and would allow study at the transcriptional level the effects of drugs that inhibit either or both of these isozymes.

Human osteoblast-like cells express predominantly steroid 5alpha-reductase type 1.

In previous studies we established that human bone and human osteoblast-like cells (hOB cells) cultured from bone express 5alpha-reductase (5alpha-R) activity, as demonstrated by the conversion of testosterone and androstenedione to their corresponding 5alpha-reduced metabolites, 5alpha-dihydrotestosterone (DHT) and 5alpha-androstanedione. Two 5alpha-R isozymes (types 1 and 2) have been identified in various tissues. As their nature in bone is unknown, we investigated which isozymes were expressed in first passage hOB cells cultured from bone specimens obtained from six donors (five women and one man). For comparison, 5alpha-reductase isozyme expression in genital skin fibroblasts cultured from foreskin of three males was determined. Pharmacological and biochemical studies using selective inhibitors of the 5alpha-R isozymes were performed, and gene expression was assessed by RT-PCR. In hOB cells, LY191704, a potent nonsteroidal selective inhibitor of 5alpha-R type 1, and the 4-azasteroid 17beta-(N,N,-diethyl-carbamoyl)-4-methyl-4-aza-5alpha-androstan-3-one (a dual inhibitor of 5alpha-R types 1 and 2) inhibited 5alpha-R activity with a 50% inhibitory concentration (IC(50)) of approximately 4 nM. Finasteride, a selective inhibitor of 5alpha-R type 2, blocked 5alpha-R activity with an IC(50) of approximately 60 nM. The IC(50) of progesterone, a physiological substrate for 5alpha-R, was approximately 200 nM. In genital skin fibroblasts, LY191704 inhibited 5alpha-R with an IC(50) of more than 5000 nM, whereas finasteride and 17beta-(N,N,-diethyl-carbamoyl)-4-methyl-4-aza-5alpha-androstan-3-one effectively inhibited 5alpha-R with IC(50) of approximately 4 nM. Experiments to determine 5alpha-reductase activity in homogenates of hOB cells as a function of pH showed very low activity at pH 5.5, but a broad shoulder of activity from pH 6.0-9.0, which was not inhibited by finasteride, but was nearly completely blocked by LY191704. RT-PCR revealed that 5alpha-R type 1 and 2 mRNAs were expressed in both bone and genital skin fibroblasts. Based on our pharmacological and biochemical studies, it appears that 5alpha-R activity in hOB cells is catalyzed predominantly by the type 1 rather than the type 2 isozyme. This expression pattern is in contrast to that in genital skin fibroblasts, where the activity of the type 2 isozyme prevails. As in most androgen target tissues DHT is biologically more active as an androgen than testosterone, DHT is formed in bone by 5alpha-R type 1 action from circulating testosterone, and bone cells also express the androgen receptor, local DHT production may play a physiological role in human bone homeostasis.

Morphological and hormonal changes in the ventral and dorsolateral prostatic lobes of rats treated with finasteride, a 5-alpha reductase inhibitor.

In rats, the prostate is divided into three distinct lobes, and the lobes are dependent on androgens [testosterone (T) and dihydrotestosterone (DHT)] as trophic hormones. However, the reasons for the difference in the incidence of proliferative changes reported are not well-understood. Administration of finasteride, a 5-alpha reductase (5alphaR) inhibitor which selectively inhibits the conversion of T to DHT, results in elevated intraprostatic T levels. However, long-term (2 years) administration of finasteride results in no increase in proliferative changes in the ventral lobes of the rat prostate. Therefore, studies were designed to determine the differences in intraprostatic hormonal levels, morphology, and 5alphaR activity in different lobes of the rat prostate. Sexually mature male Sprague-Dawley rats were used in all studies. Finasteride was administered orally to rats. The methodology included determination of intraprostatic T and DHT levels by radioimmunoassay, qualitative and quantitative evaluation of prostatic morphology, and in vitro determination of 5alphaR activities in rat prostatic lobes. A significant amount of 5alphaR activity was observed in the dorsal, ventral, and lateral lobes of the rat prostate. Both 5alphaR isozymes (types 1 and 2) were present in all lobes, based on 5alphaR activities observed at both acidic and neutral pH. Oral administration of finasteride (160 mg/kg/day) for 15 days resulted in significant (P < or = 0.001) decreases in intraprostatic DHT levels and increases in T levels; when compared to controls, the mean decrease in DHT levels in the ventral and the dorsolateral lobes was 86% and 94%, respectively, and the mean increase in T levels in the ventral and the dorsolateral lobes was approximately 3 times and 20 times, respectively, higher than in controls. Chronic administration of finasteride (80 mg/kg/day) for 6 months resulted in significant (P < or = 0.001) decreases in the weights of the prostatic lobes, which correlated with significant (P < or = 0.001) decreases in the total number of epithelial and stromal cells per gland in both the ventral and dorsolateral lobes of the prostate. There were no qualitative differences in prostatic morphology between the control and finasteride-treated groups. A short-term study in control rats exposed to bromodeoxyuridine (Brdu) showed that the number of Brdu-labeled cells in the dorsolateral lobe was significantly (P < or = 0.05) greater than in the ventral lobe. This first comparative study has highlighted some of the similarities and differences among the prostatic lobes of the rat. Inhibition of conversion of T to DHT with finasteride resulted in a significant increase in intraprostatic T levels and a significant decrease in DHT levels in rats; despite a significant increase in intraprostatic T levels, the prostate remained atrophic, indicating that DHT alone has a trophic effect on the prostate.