Enzyme-assisted synthesis and characterization of glucuronide conjugates of neuroactive steroids

Synthesis of reference standards is needed to determine the presence and function of steroid glucuronides in the brain or other tissues, because commercial sources of steroid glucuronide standards are limited or unavailable. In the present study porcine, rat, and bovine liver microsomes were tested to evaluate their ability to glucuronidate eight neurosteroids and neuroactive steroids of various types: dehydroepiandrosterone, pregnenolone, isopregnanolone, 5alpha-tetrahydrodeoxycorticosterone, corticosterone, cortisol, beta-estradiol, and testosterone. In general, the glucuronidation efficiency of rat liver was rather poor compared with that of bovine and porcine liver microsomes. Since porcine liver apparently has a relatively large amount of dehydrogenase, its microsomes also produced dehydrogenated steroids and their glucuronides, as well as various regioisomers in which the site of glucuronidation varied. In contrast, bovine liver microsomes produced mainly a single major glucuronidation product and few dehydrogenation products and gave the best overall yield for two-third of the steroids tested. The enzymatic synthesis of five glucuronides of four steroids was carried out and the conditions, purification, and analytical methods for the glucuronidation products were optimized. The steroid glucuronides synthesized were characterized by nuclear magnetic resonance spectroscopy (NMR) and liquid chromatography-mass spectrometry (LC-MS). The stereochemically pure steroid glucuronide conjugates were recovered in milligram amounts (yield 10-78%) and good purity (>85-90%), which is sufficient for LC-MS/MS method development and analyses of steroid glucuronides in biological matrices such as brain, urine, or plasma.     

Effects of neuroactive steroids on myelin of peripheral nervous system

Peripheral nervous system (PNS) possess both classical (e.g. progesterone receptor, PR, androgen receptor, AR) and non-classical (e.g. GABA_A receptor) steroid receptors and consequently may represent a target for the action of neuroactive steroids. Our data have indicated that neuroactive steroids, like for instance, progesterone, dihydroprogesterone, tetrahydroprogesterone, dihydrotestosterone and 3-diol, stimulate both in vivo and in vitro (Schwann cell cultures), the expression of two important proteins of the myelin of peripheral nerves, the glycoprotein Po (Po) and the peripheral myelin protein 22 (PMP22). It is important to highlight that the mechanisms by which neuroactive steroids exert their effects on the expression of Po and PMP22 involve different kind of receptors depending on the steroid and on the myelin protein considered. In particular, at least in culture of Schwann cells, the expression of Po seems to be under the control of PR, while that of PMP22 needs the GABA_A receptor.

Because Po and PMP22 play an important physiological role for the maintenance of the multilamellar structure of the myelin of the PNS, the present observations might suggest the utilization of neuroactive steroids as new therapeutically approaches for the rebuilding of the peripheral myelin.     

Neurosteroids and neuroactive drugs in mental disorders

Clinical and preclinical studies have suggested that fluctuations in the peripheral and brain concentrations of progesterone and deoxycorticosterone and its metabolites 3alpha,5alpha-tetrahydroprogesterone and 3alpha,5alpha-tetrahydrodeoxycorticosterone, respectively, might play an important role in certain pathological conditions characterized by emotional or affective disturbances, including major depression, anxiety disorders, and schizophrenia. Moreover, it has been shown that administration of drugs having clinical relevance in the treatment of these pathologies influence the secretion of these steroids. It remains to be determined, however, whether such changes in the concentrations of neuroactive steroids are a cause of, a risk factor for, or a consequence of mental disorders. The observation that effective pharmacological treatment of some of these pathologies influences the concentrations of neuroactive steroids suggests that these endogenous compounds might themselves prove to be efficacious in the treatment of mental illness.     

Hormones, nicotine, and cocaine: Clinical studies

Nicotine and cocaine each stimulate hypothalamic-pituitary-adrenal and -gonadal axis hormones, and there is increasing evidence that the hormonal milieu may modulate the abuse-related effects of these drugs. This review summarizes some clinical studies of the acute effects of cigarette smoking or IV cocaine on plasma drug and hormone levels and subjective effects ratings. The temporal covariance between these dependent measures was assessed with a rapid (2 min) sampling procedure in nicotine-dependent volunteers or current cocaine users. Cigarette smoking and IV cocaine each stimulated a rapid increase in LH and ACTH, followed by gradual increases in cortisol and DHEA. Positive subjective effects ratings increased immediately after initiation of cigarette smoking or IV cocaine administration. However, in contrast to cocaine's sustained positive effects (< 20 min), ratings of “high” and “rush” began to decrease within one or two puffs of a high-nicotine cigarette while nicotine levels were increasing. Peak nicotine levels increased progressively after each of three successive cigarettes smoked at 60 min intervals, but the magnitude of the subjective effects ratings and peak ACTH and cortisol levels diminished. Only DHEA increased consistently after successive cigarettes. The possible influence of neuroactive hormones on nicotine dependence and cocaine abuse and the implications for treatment of these addictive disorders are discussed.     

The steroid metabolome in lamotrigine-treated women with epilepsy

Background

Epilepsy in women may be associated with reproductive disorders and alterations in serum steroid levels. Some steroids can be induced by epilepsy and/or treatment with antiepileptic drugs; however, there are still limited data available concerning this effect on the levels of other neuroactive steroid metabolites such as 3a-hydroxy-5a/b-reduced androstanes.

Aim

To evaluate steroid alterations in women with epilepsy (WWE) on lamotrigine monotherapy.

Subjects and methods

Eleven WWE and 11 age-matched healthy women underwent blood sampling in both phases of their menstrual cycles (MCs). The steroid metabolome, which included 30 unconjugated steroids, 17 steroid polar conjugates, gonadotropins, and sex hormone-binding globulin (SHBG), was measured using gas chromatography–mass spectrometry (GC–MS) and radioimmunoassay (RIA).

Results

WWE had lower cortisol levels (status p < 0.001), but elevated levels of unconjugated 17-hydroxypregnenolone (status p < 0.001). Progesterone was higher in the follicular menstrual phase (FP) in WWE than in the controls (status × menstrual phase p < 0.05, Bonferroni multiple comparisons p < 0.05), whereas 17-hydroxyprogesterone was higher in WWE in both menstrual phases (status p < 0.001). The steroid conjugates were mostly elevated in WWE. The levels of 5α/β-reduced androstanes in WWE that were significantly higher than the controls were etiocholanolone (status p < 0.001), 5α-androstane-3α,17β-diol (status p < 0.001), and the 5α/β-reduced androstane polar conjugates (status p < 0.001).

Conclusions

WWE showed a trend toward higher circulating 3α-hydroxy-5α/β-reduced androstanes, increased activity of 17α-hydroxylase/17,20 lyase in the Δ⁵-steroid metabolic pathway, and increased levels of the steroid polar conjugates.     

Steroid modulation of the GABA/benzodiazepine receptor-linked chloride lonophore

Recent findings suggest that steroids with sedative-hypnotic properties interact specifically with the gamma-aminobutyric acidA/benzodiazepine receptor-chloride ionophore complex (GBRC). They show positive heterotropic cooperativity by allosterically enhancing the binding of GABA agonists and the clinically useful benzodiazepines (BZs) to their respective recognition sites. These steroids have stringent structural requirements for activity at the GBRC, with the essential requirements for high potency being a 3 alpha-hydroxyl group and a 5 alpha-reduced A-ring. Some of these steroids are naturally occurring metabolites of progesterone and deoxycorticosterone and have nanomolar potencies as potentiators of chloride channel conductance. These 3 alpha-hydroxylated, 5 alpha-reduced steroids do not act through any known sites on the GBRC. Thus, the exact site and mechanism of action remain to be determined. Together with the observation that physiological levels of these metabolites are sufficient to influence the function of the GBRC, the evidence clearly suggests a role for these steroids in the normal regulation of brain excitability by potentiating the postsynaptic effects of gamma-aminobutyric acid (GABA). Pharmacological studies of the GBRC-active steroids show that they possess anxiolytic and anticonvulsant activities. The potential therapeutic application of these steroids in the treatment of mood disorders and catamenial exacerbation of seizures associated with the menstrual cycle is discussed. Collectively, the evidence from the studies of these steroids imply that another mechanism by which the endocrine system influences brain function has been identified. Its characterization will provide important insight into how steroids modulate brain excitability under normal and pathophysiological states.     

Transmitter-Like Release of Endogenous 3,4-Dihydroxyphenylalanine from Rat Striatal Slices

Biphasic electrical field stimulation (0.5-5 Hz, 2 ms, 25 V, 3 min) and high K+ (10-30 mM, 5 min) released endogenous 3,4-dihydroxyphenylalanine (DOPA) from superfused rat striatal slices. Characteristics of the DOPA release were compared with those of 3,4-dihydroxyphenylethylamine (dopamine, DA). Electrical stimulation at 2 Hz evoked DOPA and DA over similar time courses. alpha-Methyl-p-tyrosine (0.2 mM) markedly reduced release of DOPA but not of DA. Maximal release (0.3 pmol) of DOPA was obtained at 2 Hz and at 15 mM K+. The impulse-evoked release of DOPA and DA was completely tetrodotoxin (0.3 microM) sensitive and Ca2+ dependent and the 15 mM K+-evoked release was also Ca2+ dependent. On L-[3,5-3H]tyrosine (1 microM) superfusion, high K+ (15 and 60 mM) released DOPA and DA together with concentration-dependent decreases in tyrosine 3-monooxygenase (EC 1.14.16.2) activity as indicated by [3H]H2O formation, followed by concentration-dependent increases after DOPA and DA release ended. These findings suggest that striatal DOPA is released by a Ca2+-dependent excitation-secretion coupling process similar to that involved in transmitter release.     

Effects of neuroactive steroids on cochlear hair cell death induced by gentamicin

As neuroactive steroids, sex steroid hormones have non-reproductive effects. We previously reported that 17β-estradiol (βE2) had protective effects against gentamicin (GM) ototoxicity in the cochlea. In the present study, we examined whether the protective action of βE2 on GM ototoxicity is mediated by the estrogen receptor (ER) and whether other estrogens (17α-estradiol (αE2), estrone (E1), and estriol (E3)) and other neuroactive steroids, dehydroepiandrosterone (DHEA) and progesterone (P), have similar protective effects. The basal turn of the organ of Corti was dissected from Sprague-Dawley rats and cultured in a medium containing 100 μM GM for 48 h. The effects of βE2 and ICI 182,780, a selective ER antagonist, were examined. In addition, the effects of other estrogens, DHEA and P were tested using this culture system. Loss of outer hair cells induced by GM exposure was compared among groups. βE2 exhibited a protective effect against GM ototoxicity, but its protective effect was antagonized by ICI 182,780. αE2, E1, and E3 also protected hair cells against gentamicin ototoxicity. DHEA showed a protective effect; however, the addition of ICI 182,780 did not affect hair cell loss. P did not have any effect on GM-induced outer hair cell death. The present findings suggest that estrogens and DHEA are protective agents against GM ototoxicity. The results of the ER antagonist study also suggest that the protective action of βE2 is mediated via ER but that of DHEA is not related to its conversion to estrogen and binding to ER. Further studies on neuroactive steroids may lead to new insights regarding cochlear protection.     

Transmitter-Like Basal and K+-Evoked Release of 3,4-Dihydroxyphenylalanine from the Striatum in Conscious Rats Studied by Microdialysis

Using microdialysis and HPLC, characteristics of the release of endogenous 3,4-dihydroxyphenylalanine (DOPA) from striatum in conscious rats were studied in comparison with those of 3,4-dihydroxyphenylethylamine (dopamine; DA). Purified L-aromatic amino acid decarboxylase (AADC) converted a putative peak of DOPA to DA. The retention time of DOPA differed from that of DA and major metabolites of DA and norepinephrine. The DOPA peak of dialysates comigrated with that of authentic DOPA when the pH of the HPLC buffer was modified. The ratio of the basal release of DOPA:DA was 1:2. 3-Hydroxybenzylhydrazine (NSD-1015; 100 mg/kg, i.p.), an AADC inhibitor, markedly increased the basal release of DOPA but produced no effect on DA. The basal release of DOPA was markedly decreased by alpha-methyl-p-tyrosine (200 mg/kg, i.p.), substantially tetrodotoxin (1 microM) sensitive, and Ca2+ (removal plus 12.5 mM Mg2+ addition) dependent. Fifty millimolar K+ released DOPA and this release was also Ca2+ dependent. These characteristics of the basal and evoked release of DOPA were similar to those of DA. The ratio of the evoked release of DOPA:DA was 1:3. These results indicate that DOPA is released under physiological conditions and by K(+)-induced depolarization in a manner similar to that for transmitter DA from striatum in freely moving rats.     

Neuroactive and other free amino acids in seed and young plants of Panax ginseng

The seeds and one to three years old plants of Asian ginseng (Panax ginseng C.A. Meyer) were analyzed for their free amino acid contents. The neuro-excitatory beta-ODAP (beta-N-oxalyl-L-alpha,beta-diaminopropionic acid), suggested to be the cause of the crippling neurolathyrism, was the major component in the seed extract (70% of the total free amino acids detected) and showed the highest concentration (0.43% by wt) compared to that in the different parts of young plants. beta-ODAP concentration was higher in the shoots as compared to roots and declined in older plants. The amount of beta-ODAP in the roots may be considered as an indirect measure of age and quality. Another neuro-active non-protein amino acid, GABA (gamma-aminobutyric acid), increased dramatically after germination and reached highest concentration in different parts of 3 year-old plants. Glutamine and arginine were the two major free proteinogenic amino acids in the ginseng plants and together they constituted over 50% of all the free amino acids detected in the root.