General Consideration on the 3aHOR

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The enzyme(s) 3a-HOR may be considered the second element of the 5a-R/3a-HOR system; as previously mentioned, the enzyme may transform 5a-DHT into 3a,5a-diol, 5a-DHPROG into 3a,5a-THPROG, and the gluco- and mineralo-corticoster-oids into their corresponding tetrahydroderivatives (Fig. 2). The formation of these 3a-hydroxylated derivatives is a process that may subserve different physiological roles; this reaction may be of importance: 1. for decreasing the amounts of intracellular 5 a-reduced compounds available for binding to the corresponding intracellular steroid receptors; or 2. for producing steroid derivatives that may act through nongenomic mechanisms (e.g., via the GABAa receptor). At variance with the two isoforms of the 5 a-R, the enzymes involved, appear to be able to catalyze the reaction both in the oxidative and in the reductive direction. The amounts of 5a-reduced/3a-hydroxylated compounds present at any given moment in a cell are therefore dependent on the equilibrium of this reaction.

The first 3a-HOR enzyme to be purified and then cloned has been that present in the rat liver cytosol (146-148). The cDNA of this enzyme codes for a protein of 322 amino acids with an estimated molecular weight of 37 kDa and catalyzes the oxidoreduction of steroids using either NADP+/NADPH or NAD+/NADH as cofactors.

The analysis of the kinetic mechanisms of the reaction supports the obligatory binding of the cofactor before that of the substrate. The enzyme can work both in the oxidative and in the reductive directions, but oxidation appears to be the preferential function.

Fig. 2. The 5a-reductase/3a-hydroxysteroid oxidoreductase (3a-HOR) pathways.

The three-dimensional structure and relevant features of the rat liver cytosolic enzyme have been recently reported (149-151). This enzyme shares 84% sequence similarity with the human liver dihydrodiol dehydrogenase (DD2), which is considered the human counterpart of the rat 3a-HOR (152,153). The human enzyme has been defined dihydrodiol dehydrogenase because it is able to oxidize benzo(a)pyrene 7,8 epoxide to benzo(a)pyrene dihydrodiol. Also the rat enzyme possesses a similar metabolic activity on aromatic hydrocarbons. Multiple DDZ isoforms exist in the human liver, and at least three of them—DD1, DD2, and DD4—exhibit 3a-HOR activity (153). The human gene is more than 47 kb in length, and contains, in the 5'-flanking region, consensus sequences for AP-1, and Oct -1 as well as multiple copies of perfect and imperfect steroid responsive elements (SRE) for the ER, and the GR/PR (150,153). The rat liver enzyme has a low substrate specificity, acting both on C19 and C21 steroids and as previously mentioned, on nonsteroidal substrates.

Several metabolic studies suggest that the rat CNS possesses at least two 3a-HOR. It has indeed been shown that there are one cytosolic and one microsomal enzyme. Both types of enzymes appear to be endowed with oxidoreductase capabilities, but they might act in opposite directions (148,154,155). The cytosolic form has been purified from the rat brain to apparent homogeneity (156) and preliminary attempts to clone it produced clones very similar to that of the liver enzyme (153). The purified brain enzyme is a monomer, with an apparent molecular weight of about 31 kDa which shows a preference for NADPH, and high affinity for 5a-DHT; the enzyme shows a specific activity about 100-fold higher than that of the 5a-R type 1, indicating that in vivo it could rapidly transform the 5a-reduced compounds formed by the 5a-R (156). Karavolas et al. (155) have isolated a cytosolic enzyme from the hypothalamus which shows an affinity for 3 a,5 a-THPROG much lower than that for 5a-DHPROG, indicating that the reductive activity might be its prevailing function. The brain cytosolic enzyme(s), like that of liver, is/are inhibited by all the major classes of nonsteroidal anti-inflammatory drugs (153,156). Much less information is available on the microsomal 3a-HOR. This enzyme has not been purified so far; it appears to be NADH-linked with 300-fold lower affinity for 5a-DHPROG than for 3a,5a-THPROG, suggesting that the reaction may proceed in the oxidative direction (155).

The distribution of the 3a-HOR(s) in the rat brain has been studied evaluating either the enzymatic activity or by immunocytochemistry (using a monoclonal antibody raised against the liver enzyme) in different brain regions (148,153,154). All the data have consistently shown that the highest activities are in the olfactory bulb and in the olfactory tubercle. It has been shown that 3a-HOR is localized in astrocytes, particularly of the type 1 subfamily (128). The formation of 3a,5a-diol from 5a-DHT is usually considered a mechanism of steroid catabolism, because 3a,5a-diol does not bind to the androgen receptor.

As already mentioned, some physiological metabolites formed through the 5a-R/3a-HOR pathway may interact with the GABAa receptor, and consequently may exert anxiolytic and anesthetic properties. This is particularly true for the 5a-reduced-3a-hydroxylated derivatives of PROG and DOC (157-160), 3a,5a-THPROG and 3a,5a-THDOC, respectively. These two steroids are the most potent natural ligands of the GABAA receptor (159), and do have potent sleep-inducing properties in rats (161). Also some synthetic steroids (e.g., alphaxalone) possessing a 5a-reduced/3a-hydroxy-lated structure have been shown to be anxiolytic and/or hypnotic.

The role of the 5a-R in providing the 3a-HOR system with possible precursors for the final transformation into hypnotic compounds has been recently investigated using PROG as the test compound. It has been found that PROG induces a deep anesthetic effect both in male and female rats, which can be counteracted by 5a-R inhibitors (e.g., 4-MA and finasteride). These were totally ineffective when alphaxalone, nembutal, or diazepam, (agents known to interact with the GABAA receptor) were used. These results support the concept that PROG induces its anxyolytic/anesthetic effect through the formation of 5a-reduced/3a-hydroxylated compounds. These results are also supported by the recent observation that the anxiolytic effect of PROG is highly correlated with increased levels of 3a,5a-THPROG in the blood and in the brain (162). It is possible that the process of 5a-reduction/3a-hydroxylation may acquire a particular physiological relevance in conditions in which the secretion of PROG and/or corticosteroids is particularly elevated (e.g., during pregnancy, in the second phase of the menstrual cycle, during stress, and so forth).

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