Neurosteroid analogues. 10. The effect of methyl group substitution at the C-6 and C-7 positions on the GABA modulatory and anesthetic actions of (3alpha,5alpha)- and (3alpha,5beta)-3-hydroxypregnan-20-one.

The planar 5alpha-reduced steroid (3alpha,5alpha)-3-hydroxypregnan-20-one and the nonplanar 5beta-reduced steroid (3alpha,5beta)-3-hydroxypregnan-20-one act at GABA(A) receptors to induce general anesthesia. The structural features of the binding sites for these anesthetic steroids on GABA(A) receptors have not been determined. To determine how structural modifications at the steroid C-6 and C-7 positions effect the actions of these anesthetic steroids, an axial or equatorial methyl group was introduced at these positions. The analogues were evaluated (1) in [(35)S]-tert-butylbicyclophosphorothionate binding experiments, (2) in electrophysiological experiments using rat alpha(1)beta(2)gamma(2L) GABA(A) receptors expressed in Xenopus laevis oocytes, and (3) as tadpole anesthetics. The effects of methyl group substitution in the 5alpha- and 5beta-reduced series of compounds were strikingly similar. In both series, a 6beta-Me group gave compounds with actions similar to or greater than those of the parent steroids. A 6alpha-, 7beta- or 7alpha-Me substituent resulted in reduced potency for inhibition of radioligand binding, GABA(A) receptor modulation and tadpole anesthesia. Because of the similar effects of methyl group substitution in the two series of compounds and previous results from other studies showing that structural modifications in the steroid D ring/side chain region produce similar effects regardless of the stereochemistry of the A,B-ring fusion, we propose that either the 3alpha-hydroxyl groups of planar and nonplanar anesthetic steroids hydrogen bond to different amino acids on GABA(A) receptors or that this critical hydrogen bonding group interacts with membrane lipids instead of the receptor.

Photoaffinity labeling with a neuroactive steroid analogue. 6-azi-pregnanolone labels voltage-dependent anion channel-1 in rat brain.

Neuroactive steroids modulate the function of gamma-aminobutyric acid, type A (GABA(A)) receptors in the central nervous system by an unknown mechanism. In this study we have used a novel neuroactive steroid analogue, 3 alpha,5 beta-6-azi-3-hydroxypregnan-20-one (6-AziP), as a photoaffinity labeling reagent to identify neuroactive steroid binding sites in rat brain. 6-AziP is an effective modulator of GABA(A) receptors as evidenced by its ability to inhibit binding of [(35)S]t-butylbicyclophosphorothionate to rat brain membranes and to potentiate GABA-elicited currents in Xenopus oocytes and human endothelial kidney 293 cells expressing GABA(A) receptor subunits (alpha(1)beta(2)gamma(2)). [(3)H]6-AziP produced time- and concentration-dependent photolabeling of protein bands of approximately 35 and 60 kDa in rat brain membranes. The 35-kDa band was half-maximally labeled at a [(3)H]6-AziP concentration of 1.9 microM, whereas the 60-kDa band was labeled at higher concentrations. The photolabeled 35-kDa protein was isolated from rat brain by two-dimensional PAGE and identified as voltage-dependent anion channel-1 (VDAC-1) by both matrix-assisted laser desorption ionization time-of-flight and ESI-tandem mass spectrometry. Monoclonal antibody directed against the N terminus of VDAC-1 immunoprecipitated labeled 35-kDa protein from a lysate of rat brain membranes, confirming that VDAC-1 is the species labeled by [(3)H]6-AziP. The beta(2) and beta(3) subunits of the GABA(A) receptor were co-immunoprecipitated by the VDAC-1 antibody suggesting a physical association between VDAC-1 and GABA(A) receptors in rat brain membranes. These data suggest that neuroactive steroid effects on the GABA(A) receptor may be mediated by binding to an accessory protein, VDAC-1.

3beta -hydroxypregnane steroids are pregnenolone sulfate-like GABA(A) receptor antagonists.

Endogenous neurosteroids have rapid actions on ion channels, particularly GABA(A) receptors, which are potentiated by nanomolar concentrations of 3alpha-hydroxypregnane neurosteroids. Previous evidence suggests that 3beta-hydroxypregnane steroids may competitively antagonize potentiation induced by their 3alpha diastereomers. Because of the potential importance of antagonists as experimental and clinical tools, we characterized the functional effect of 3beta-hydroxysteroids. Although 3beta-hydroxysteroids reduced the potentiation induced by 3alpha-hydroxysteroids, 3beta-hydroxysteroids acted noncompetitively with respect to potentiating steroids and inhibited the largest degrees of potentiation most effectively. Potentiation by high concentrations of barbiturates was also reduced by 3beta-hydroxysteroids. 3beta-Hydroxysteroids are also direct, noncompetitive GABA(A) receptor antagonists. 3beta-Hydroxysteroids coapplied with GABA significantly inhibited responses to > or =15 microm GABA. The profile of block was similar to that exhibited by sulfated steroids, known blockers of GABA(A) receptors. This direct, noncompetitive effect of 3beta-hydroxysteroids was sufficient to account for the apparent antagonism of potentiating steroids. Mutated receptors exhibiting decreased sensitivity to sulfated steroid block were insensitive to both the direct effects of 3beta-hydroxysteroids on GABA(A) responses and the reduction of potentiating steroid effects. At concentrations that had little effect on GABAergic synaptic currents, 3beta-hydroxysteroids and low concentrations of sulfated steroids significantly reversed the potentiation of synaptic currents induced by 3alpha-hydroxysteroids. We conclude that 3beta-hydroxypregnane steroids are not direct antagonists of potentiating steroids but rather are noncompetitive, likely state-dependent, blockers of GABA(A) receptors. Nevertheless, these steroids may be useful functional blockers of potentiating steroids when used at concentrations that do not affect baseline neurotransmission.

Gastrointestinal absorption and plasma kinetics of soy Delta(5)-phytosterols and phytostanols in humans.

Our objective was to measure the systemic absorption of lecithin-emulsified Delta(5)-phytosterols and phytostanols during test meals by use of dual stable isotopic tracers. Ten healthy subjects underwent two single-meal absorption tests in random order 2 wk apart, one with intravenous dideuterated Delta(5)-phytosterols and oral pentadeuterated Delta(5)-phytosterols and the other with the corresponding labeled stanols. The oral-to-intravenous tracer ratio in plasma, a reflection of absorption, was measured by a sensitive negative ion mass spectroscopic technique and became constant after 2 days. Absorption from 600 mg of Delta(5)-soy sterols given with a standard test breakfast was 0.512 +/- 0.038% for sitosterol and 1.89 +/- 0.27% for campesterol. The absorption from 600 mg of soy stanols was 0.0441 +/- 0.004% for sitostanol and 0.155 +/- 0.017% for campestanol. Reduction of the double bond at position 5 decreased absorption by 90%. Plasma t(1/2) for stanols was significantly shorter than that for Delta(5)-sterols. We conclude that the efficiency of phytosterol absorption is lower than what was reported previously and is critically dependent on the structure of both sterol nucleus and side chain.