Robert H. Purdy

Neurosteroids act on recombinant human GABAA receptors

The endogenous steroid metabolites 3 alpha,21dihydroxy-5 alpha-pregnan-20-one and 3 alpha-hydroxy-5 alpha-pregnan-20-one potentiate GABA-activated Cl- currents recorded from a human cell line transfected with the beta 1, alpha 1 beta 1, and alpha 1 beta 1 gamma 2 combinations of human GABAA receptor subunits. These steroids are active at nanomolar concentrations in potentiating GABA-activated Cl- currents and directly elicit bicuculline-sensitive Cl- currents when applied at micromolar concentrations. The potentiating and direct actions of both steroids were expressed with every combination of subunits tested. However, an examination of single-channel currents recorded from outside-out patches excised from these transfected cells suggests that despite the common minimal structural requirements for expressing steroid and barbiturate actions, the mechanism of GABAA receptor modulation by these pregnane steroids may differ from that of barbiturates.

Social Isolation‐Induced Decreases in Both the Abundance of Neuroactive Steroids and GABAA Receptor Function in Rat Brain

The effects of social isolation on behavior, neuroactive steroid concentrations, and GABAA receptor function were investigated in rats. Animals isolated for 30 days immediately after weaning exhibited an anxiety‐like behavioral profile in the elevated plus‐maze and Vogel conflict tests. This behavior was associated with marked decreases in the cerebrocortical, hippocampal, and plasma concentrations of pregnenolone, progesterone, allopregnanolone, and allotetrahydrodeoxycorticosterone compared with those apparent for group‐housed rats ; in contrast, the plasma concentration of corticosterone was increased in the isolated animals. Acute footshock stress induced greater percentage increases in the cortical concentrations of neuroactive steroids in isolated rats than in group‐housed rats. Social isolation also reduced brain GABAA receptor function, as evaluated by measuring both GABA‐evoked Cl‐ currents in Xenopus oocytes expressing the rat receptors and tert‐[35S]butylbicyclophosphorothionate ([35S]TBPS) binding to rat brain membranes. Whereas the amplitude of GABA‐induced Cl‐ currents did not differ significantly between group‐housed and isolated animals, the potentiation of these currents by diazepam was reduced at cortical or hippocampal GABAA receptors from isolated rats compared with that apparent at receptors from group‐housed animals. Moreover, the inhibitory effect of ethyl‐β‐carboline‐3‐carboxylate, a negative allosteric modulator of GABAA receptors, on these currents was greater at cortical GABAA receptors from socially isolated animals than at those from group‐housed rats. Finally, social isolation increased the extent of [35S]TBPS binding to both cortical and hippocampal membranes. The results further suggest a psychological role for neurosteroids and GABAA receptors in the modulation of emotional behavior and mood.

Brain steroidogenesis mediates ethanol modulation of GABAA receptor activity in rat hippocampus.

An interaction with the GABA type A (GABAA) receptor has long been recognized as one of the main neurochemical mechanisms underlying many of the pharmacological actions of ethanol. However, more recent data have suggested that certain behavioral and electrophysiological actions of ethanol are mediated by an increase in brain concentration of neuroactive steroids that results from stimulation of the hypothalamic-pituitary-adrenal (HPA) axis. Neuroactive steroids such as 3α-hydroxy-5α-pregnan-20-one (3α,5α-THP) are, in fact, potent and efficacious endogenous positive modulators of GABAA receptor function. Because neurosteroids can be synthesized de novo in the brain, we have investigated whether ethanol might affect both neurosteroid synthesis and GABAA receptor function in isolated rat hippocampal tissue. Here, we show that ethanol increases the concentration of 3α,5α-THP as well as the amplitude of GABAA receptor-mediated IPSCs recorded from CA1 pyramidal neurons in isolated hippocampal slices. These effects are shared by the neurosteroid precursor progesterone, the peripheral benzodiazepine receptor-selective agonist CB34, and γ-hydroxybutyrate, all of which are known to increase the formation of neuroactive steroids in plasma and in the brain. The action of ethanol on GABAA receptor-mediated IPSC amplitude is biphasic, consisting of a rapid, direct effect on GABAA receptor activity and an indirect effect that appears to be mediated by neurosteroid synthesis. Furthermore, ethanol affects GABAA receptor activity through a presynaptic action, an effect that is not dependent on neurosteroid formation. These observations suggest that ethanol may modulate GABAA receptor function through an increase in de novo neurosteroid synthesis in the brain that is independent of the HPA axis. This novel mechanism may have a crucial role in mediating specific central effects of ethanol.

Radioimmunoassay of 3α-hydroxy-5α-pregnan-20-one in rat and human plasma

Abstract A radioimmunoassay for measuring 3α-hydroxy-5α-pregnan-20-one in plasma has been developed. Polyclonal antibodies were raised in rabbits against 3α-hydroxy-20-oxo-5α-pregnan-11α-yl carboxymethyl ether coupled to bovine serum albumin. 3α-Hydroxy-5α-pregnan-20-one was purified from ether extracts of plasma by high-performance liquid chromatography. These antibodies were then used for the radioimmunoassay of this centrally active progesterone metabolite in rat and human plasma. 3α-Hydroxy-5α-pregnan-20-one was detected in plasma from female rats on the day ofestrus (2.0 to 9.3 ng/ml) and in the plasma of women during the luteal phase of the menstrual cycle at levels ranging from 0.25 to 2.5 ng/ml. The latter was highly correlated with plasma progesterone levels.

Pregnenolone sulfate augments NMDA receptor mediated increases in intracellular Ca2+ in cultured rat hippocampal neurons

The ability of the neuroactive steroid pregnenolone sulfate to alter N-methyl-D-aspartate (NMDA) receptor-mediated elevations in intracellular Ca2+ ([Ca2+]i) was studied in cultured fetal rat hippocampal neurons using microspectrofluorimetry and the Ca2+ sensitive indicator fura-2. Pregnenolone sulfate (5-250 microM) caused a concentration-dependent and reversible potentiation of the rise (up to approximately 800%) in [Ca2+]i induced by NMDA. In contrast, the steroid failed to alter basal (unstimulated) [Ca2+]i or to modify the rise in [Ca2+]i that occurs when hippocampal neurons are depolarized by high K+ in the presence of the NMDA receptor antagonist CPP. These data suggest that the previously reported excitatory properties of pregnenolone sulfate may be due, in part, to an augmentation of the action of glutamic acid at the NMDA receptor.

Binding of 2-hydroxyestradiol and 4-hydroxyestradiol to estrogen receptors from human breast cancers

The binding of catechol estrogens, epoxyenones and methoxyestrogens was evaluated using estrogen receptors in cytosol prepared from human breast cancers. The relative affinity of 2-hydroxyestradiol, a metabolite formed in vitro from estradiol-17 beta by breast cancer cells, was indistinguishable from that of estradiol-17 beta. 4-Hydroxyestradiol, which is also a metabolite of estradiol-17 beta, associated with the estrogen receptor with a relative affinity approximately 1.5-fold greater than that of estradiol-17 beta. Epoxyenones and methoxyestrogens were weak competitors compared to the binding of estradiol-17 beta, exhibiting relative affinities 3% or less than the affinity of estradiol-17 beta. Sucrose density gradient centrifugation revealed that both 2- and 4-hydroxyestradiol inhibited the binding of estradiol-17 beta to both the 4S and 8S isoforms of the estrogen receptor in a competitive manner, with a Ki = 0.94 nM for 2-hydroxyestradiol and a Ki = 0.48 nM for 4-hydroxyestradiol. It can be concluded that these data demonstrate a specific receptor-mediated estrogenic action for both of these catechol estrogens.

Relative rates of 2- and 4-hydroxyestrogen synthesis are dependent on both substrate and tissue

1. introduction 2. Materials and methods The variety of physiological effects attributed to estrogens, as well as the pathological characteristics of estrogen-sensitive human breast tumors, have prompted numerous studies on the possible metabolic conver- sion of the parent estrogens to activated metabolites. Most of these efforts have focused on the formation and properties of the catecholestrogens of El and E?. The enzyme system catalyzing their synthesis is a cytochrome WSOdependent monooxygenase localized primarily in liver, but also found in numerous estrogen-sensitive mammalian tissues [ 1,2] including human mammary neoplasma [3]. Little information is available on the substrate specificity of this microsomal enzyme system. It is usually described as an estrogen 2-hydroxylase, since derivatives of 2-OHE, and 2-OHEa are the principal products identified from incubations of El and Ez, respectively, with microsomes from rat tissues [4,7]. The objective of this study was to determine if differ- ences in the structure of estrogens of major therapeutic interest would significantly affect the relative rates of formation of their 2- and 4-hydroxy metabolites. Evi- dence was also sought for the biosynthesis of cate- cholestrogens by microsomes from a cultured line of human mammary tumor cells whose growth was known to be stimulated by these hormones [2]. Moxestrol, the 2- and 4-hydroxy derivatives of moxestrol [8] and ethynylestradiol were generously provided by Drs J. P. Raynaud and G. Deltour (Roussel-UCLAF, Romainville). The method in [9] was used for the synthesis of 4.hydroxyequilenin. Other catecholestrogens and their monomethyl ethers were obtained from Steraloids (Wilton NH) or pre- pared by established procedures [ 11. Hepes, NADPH and Lascorbic acid were purchased from Sigma (St Louis MD). [ 3H] SAM (13.5 Ci/mmol) was obtained from New England Nuclear (Boston MA). Spectro- analyzed grade dimethylsulfoxide and HPLC grade n-heptane were from Fisher Scientific (Fair Lawn NJ) and USPgrade absolute ethanol was from US Industrial Chemicals (New York NY).

Analysis of metabolites of progesterone in bovine liver, kidney, kidney fat, and milk by high performance liquid chromatography

An investigation by high-performance liquid chromatography (HPLC) of the metabolites from the livers of cows and steers treated with [4-14C]-progesterone revealed them to consist principally of 3α-hydroxy-5β-pregnan-20-one and 5β-pregnane-3α,20β-diol. In bovine kidney tissue the principal metabolites were found to be 20β-hydroxypregn-4-en-3-one, 3α and 3β-hydroxy-5α-pregnan-20-one and 5α-pregnane-3β,20β-diol in about equal amount, plus about 15% of the total as unconverted progesterone. In milk the major product was 5α-pregnane-3,20-dione in addition to about 80% of the total as unconverted progesterone. Only traces of compounds other than progesterone were found in kidney fat. These metabolites were further characterized by crystallization to constant specific acitivity, including derivative formation. This study demonstrates for the first time the utility of HPLC for the rapid analysis of a complete mixture of metabolites of progesterone from crude extracts of mammalian tissues.

Changes in GABAA Receptor Gene Expression Induced by Withdrawal of, but Not by Long-Term Exposure to, Ganaxolone in Cultured Rat Cerebellar Granule Cells

The effects of ganaxolone, a synthetic analog of the endogenous neuroactive steroid allopregnanolone, on the function and expression of GABAA receptors were determined. Electrophysiological recordings demonstrated that ganaxolone potentiated with a potency and efficacy similar to those of allopregnanolone the Cl−currents evoked by GABA at recombinant human GABAAreceptors (comprising α1β2γ2L or α2β2γ2L subunit assemblies) expressed in Xenopus oocytes. Exposure of cultured rat cerebellar granule cells to 1 μM ganaxolone for 5 days had no effect on the abundance of mRNAs encoding the α1, α2, α3, α4, α5, γ2L, or γ2S subunits of the GABAA receptor. Withdrawal of ganaxolone after such long-term treatment, however, induced an increase in the abundance of α2, α4, and α5 subunit mRNAs and a decrease in the amounts of α1, γ2L, and γ2S subunit mRNAs. These changes were maximal 3 to 6 h after drug withdrawal and were reversible, being no longer apparent after 24 h. These results suggest that long-term exposure of cerebellar granule cells to ganaxolone does not affect the sensitivity of the GABAAreceptor to several positive modulators. Nevertheless, the reduction in the amounts of the α1 and γ2 subunit mRNAs together with the increase in the abundance of the α4 subunit mRNA induced by abrupt discontinuation of long-term treatment with ganaxolone suggest that withdrawal of this drug might result in a reduced response to classic benzodiazepines.

Potentiation of GABAergic Neurotransmission by Steroids

The endogenous 3α-hydroxy ring A-reduced steroids allopregnanolone and pregnanolone are among the most active ligands of gamma amino butric acid A (GABAA) receptors, with affinities equal to or greater than those of other known ligands such as barbiturates, and even benzodiazepines (1,2). It is, therefore, not surprising that numerous 3α-hydroxy steroids have anxiolytic, hypnotic, anticonvulsant, and anesthetic effects when administered to laboratory animals (2). These pharmacological actions, together with their low intrinsic toxicity and lack of hormonal activity, have led an expanding number of laboratories to investigate their physiological and pharmacological properties in both laboratory animals and humans. Efforts are also underway to develop novel synthetic anxiolytic, hypnotic, and anticonvulsant steroids as therapeutic agents. In this chapter, we discuss a few selected areas related to the pharmacology, physiology, and chemistry of GABAergic neuroactive steroids, including their potential roles in alcohol dependence and withdrawal, as well as in affective disorders.