A neurosteroid analogue with T-type calcium channel blocking properties is an effective hypnotic, but is not harmful to neonatal rat brain.

BACKGROUND: More than 4 million children are exposed annually to sedatives and general anaesthetics (GAs) in the USA alone. Recent data suggest that common GAs can be detrimental to brain development causing neurodegeneration and long-term cognitive impairments. Challenged by a recent US Food and Drug Administration (FDA) warning about potentially neurotoxic effects of GAs in children, there is an urgent need to develop safer GAs. METHODS: Postnatal Day 7 (P7) rat pups of both sexes were exposed to six (repeated every 2 h) injections of equipotent hypnotic doses of ketamine or the neuroactive steroid (3ß,5ß,17ß)-3-hydroxyandrostane-17-carbonitrile (3ß-OH) for 12 h. Loss of righting reflex was used to assess hypnotic properties and therapeutic index; quantitative caspase-3 immunohistochemistry was used to assess developmental neuroapoptosis; patch-clamp recordings in acute brain slices were used to assess the effects of 3ß-OH on neuronal excitability and synaptic transmission. Cognitive abilities of rats exposed to ketamine, 3ß-OH, or vehicle at P7 were assessed in young adulthood using the radial arm maze. RESULTS: The neuroactive steroid 3ß-OH has a therapeutic index similar to ketamine, a commonly used clinical GA. We report that 3ß-OH is safe and, unlike ketamine, does not cause neuroapoptosis or impair cognitive development when administered to P7 rat pups. Interestingly, 3ß-OH blocks T-type calcium channels and presynaptically dampens synaptic transmission at hypnotically-relevant brain concentrations, but it lacks a direct effect on γ-aminobutyric acid A or glutamate-gated ion channels. CONCLUSIONS: The neurosteroid 3ß-OH is a relatively safe hypnotic that warrants further consideration for paediatric anaesthesia.

Structural requirements of steroidal agonists of transient receptor potential melastatin 3 (TRPM3) cation channels.

BACKGROUND AND PURPOSE: Transient receptor potential melastatin 3 (TRPM3) proteins form non-selective but calcium-permeable membrane channels, rapidly activated by extracellular application of the steroid pregnenolone sulphate and the dihydropyridine nifedipine. Our aim was to characterize the steroid binding site by analysing the structural chemical requirements for TRPM3 activation. EXPERIMENTAL APPROACH: Whole-cell patch-clamp recordings and measurements of intracellular calcium concentrations were performed on HEK293 cells transfected with TRPM3 (or untransfected controls) during superfusion with pharmacological substances. KEY RESULTS: Pregnenolone sulphate and nifedipine activated TRPM3 channels supra-additively over a wide concentration range. Other dihydropyridines inhibited TRPM3 channels. The natural enantiomer of pregnenolone sulphate was more efficient in activating TRPM3 channels than its synthetic mirror image. However, both enantiomers exerted very similar inhibitory effects on proton-activated outwardly rectifying anion channels. Epiallopregnanolone sulphate activated TRPM3 almost equally as well as pregnenolone sulphate. Exchanging the sulphate for other chemical moieties showed that a negative charge at this position is required for activating TRPM3 channels. CONCLUSIONS AND IMPLICATIONS: Our data demonstrate that nifedipine and pregnenolone sulphate act at different binding sites when activating TRPM3. The latter activates TRPM3 by binding to a chiral and thus proteinaceous binding site, as inferred from the differential effects of the enantiomers. The double bond between position C5 and C6 of pregnenolone sulphate is not strictly necessary for the activation of TRPM3 channels, but a negative charge at position C3 of the steroid is highly important. These results provide a solid basis for understanding mechanistically the rapid chemical activation of TRPM3 channels.

Cyclodextrins sequester neuroactive steroids and differentiate mechanisms that rate limit steroid actions.

BACKGROUND AND PURPOSE: Neuroactive steroids are potent modulators of GABA(A) receptors and are thus of interest for their sedative, anxiolytic, anticonvulsant and anaesthetic properties. Cyclodextrins may be useful tools to manipulate neuroactive effects of steroids on GABA(A) receptors because cyclodextrins form inclusion complexes with at least some steroids that are active at the GABA(A) receptor, such as (3alpha,5alpha)-3-hydroxypregnan-20-one (3alpha5alphaP, allopregnanolone). EXPERIMENTAL APPROACH: To assess the versatility of cyclodextrins as steroid modulators, we investigated interactions between gamma-cyclodextrin and neuroactive steroids of different structural classes. KEY RESULTS: Both a bioassay based on electrophysiological assessment of GABA(A) receptor function and optical measurements of cellular accumulation of a fluorescent steroid analogue suggest that gamma-cyclodextrin sequesters steroids rather than directly influencing GABA(A) receptor function. Neither a 5beta-reduced A/B ring fusion nor a sulphate group at carbon 3 affected the presumed inclusion complex formation between steroid and gamma-cyclodextrin. Apparent dissociation constants for interactions between natural steroids and gamma-cyclodexrin ranged from 10-60 microM. Although gamma-cyclodextrin accommodates a range of natural and synthetic steroids, C(11) substitutions reduced inclusion complex formation. Using gamma-cyclodextrin to remove steroid not directly bound to GABA(A) receptors, we found that cellular retention of receptor-unbound steroid rate limits potentiation by 3alpha- hydroxysteroids but not inhibition by sulphated steroids. CONCLUSIONS AND IMPLICATIONS: We conclude that gamma-cyclodextrins can be useful, albeit non-specific, tools for terminating the actions of multiple classes of naturally occurring neuroactive steroids.

5alpha-reduced neuroactive steroids alleviate thermal and mechanical hyperalgesia in rats with neuropathic pain.

5alpha-reduced neuroactive steroids with selective modulatory action in vitro on T or combined modulatory action on T and GABA(A) currents present in peripheral sensory neurons have been shown to induce potent peripheral analgesia in vivo in intact animals. Although the role of T and GABA(A) currents in pathophysiology of neuropathic pain (NPP) is not established, it appears that blockade of T currents and/or potentiation of GABA(A) currents could be beneficial in the management of NPP. To study the potential usefulness of 5alpha-reduced neuroactive steroids in alleviating NPP, we selected two newly synthesized steroids-ECN and CDNC24-with a selective blocking effect on T currents and a selective potentiating effect on GABA(A) currents, respectively, and commercial analogs-alphaxalone and 3alpha5alphaP-with the effects on both ion channels. We used a sciatic nerve ligation model to induce thermal and mechanical hyperalgesia in adult rats and tested peripheral thermal and mechanical nociception following local injection of neuroactive steroids into the peripheral receptive fields of a ligated hind paw. We found that 5alpha-reduced neuroactive steroids alleviate thermal and mechanical hyperalgesia in NPP rats. ECN and CDNC24 were more selective in alleviating thermal nociception in NPP than in sham animals when compared to 3alpha5alphaP and alphaxalone although the anti-nociceptive effect induced by 3alpha5alphaP and alphaxalone was more profound. CDNC24 was most selective since it had very minimal anti-nociceptive effect in sham animals but a very profound anti-nociceptive effect in NPP animals suggesting that, under pathological conditions, peripheral GABA(A) receptors might be an attractive therapeutic target.

Epipregnanolone and a novel synthetic neuroactive steroid reduce alcohol self-administration in rats.

This study was designed to compare the effects of several neuroactive steroids with varying patterns of modulation of gamma-aminobutyric acid (GABA)(A) and NMDA receptors on operant self-administration of ethanol or water. Once stable responding for 10% (w/v) ethanol was achieved, separate test sessions were conducted in which male Wistar rats were allowed to self-administer ethanol or water following pre-treatment with vehicle or one of the following neuroactive steroids: (3beta,5beta)-3-hydroxypregnan-20-one (epipregnanolone; 5, 10, 20 mg/kg; n=12), (3alpha,5beta)-20-oxo-pregnane-3-carboxylic acid (PCA; 10, 20, 30 mg/kg n=10), (3alpha,5beta)-3-hydroxypregnan-20-one hemisuccinate (pregnanolone hemisuccinate; 5, 10, 20 mg/kg; n=12) and (3alpha,5alpha)-3-hydroxyandrostan-17-one hemisuccinate (androsterone hemisuccinate; 5, 10, 20 mg/kg; n=11). The effect of the 3beta-epimer of PCA, (3beta,5beta)-20-oxo-pregnane-3-carboxylic acid (10, 20, 30 mg/kg; n=9), on ethanol self-administration was also examined. The compounds were administered using a Latin-square design 45 min prior to the weekly test sessions. The effects of the 30 mg/kg dose of the steroidal hemisuccinates on ethanol intake were also examined 5 min after administration of these drugs. Both epipregnanolone and PCA attenuated ethanol self-administration. However, neither of the hemisuccinate compounds significantly altered this behavior. The steroidal hemisuccinates (30 mg/kg; n=7) were also tested 5 min before behavior testing and had no effect on ethanol intake 5 min after administration. The 3beta-epimer of PCA also failed to alter ethanol intake. None of the test compounds altered water intake. In electrophysiological studies, the effects of PCA and androsterone hemisuccinate on evoked GABA(A) receptor-mediated inhibitory postsynaptic currents (GABA(A)-IPSCs) was examined in brain slices of the amygdala. PCA had a stimulatory effect at concentrations of 5 and 25 muM. Androsterone hemisuccinate had no agonist activity. The ability of epipregnanolone and PCA to alter ethanol intake appears to be related to different inhibitory actions of these compounds on either GABA(A) or NMDA receptors, respectively. Thus, dual modulation of these systems by selected neuroactive steroids may offer potential for modifying the reinforcing effects of alcohol.

Inhibition of type A GABA receptors by L-type calcium channel blockers.

Modulation of type A GABA receptors (GABAA) by L-type Ca++ channel blockers was investigated. The dihydropyridines nifedipine and nitrendipine, and the phenylalkylamine verapamil inhibited recombinant rat alpha1beta2gamma2 receptors recorded from human embryonic kidney (HEK) 293 cells; nifedipine at low concentrations also elicited modest stimulatory effects on GABA-gated current. The IC50 for GABA current inhibition was lowest for nitrendipine (17.3 +/- 1.3 microM), so subsequent studies were focused on further exploring its mechanism and possible site of action. When co-applied with GABA, nitrendipine had minimal effects on initial current amplitude, but significantly enhanced current decay rate. Nitrendipine-mediated inhibition was subunit-selective, as its IC50 was 10-fold lower in alpha1beta2 receptors. Nitrendipine's effect in recombinant human alpha1beta2gamma2 receptors was similar (IC50=23.0 +/- 1.3 microM) to that observed in rat receptors of the same configuration, indicating the site of action is conserved in the two species. The inhibitory effects were dependent on channel gating, were independent of transmembrane voltage, and were also observed in GABAA receptors recorded from hypothalamic brain slices. The pharmacologic mechanism of inhibition by nitrendipine was non-competitive, indicating it does not act at the GABA binding site. Nitrendipine block was retained in the presence of the benzodiazepine antagonist flumazenil, indicating it does not interact at the benzodiazepine site. The actions of nitrendipine were not affected by a mutation (beta2T246F) that confers resistance to the channel blocker picrotoxin, and they were not altered in the presence of the picrotoxin site antagonist alpha-isopropyl-alpha-methyl-gamma-butyrolactone, demonstrating nitrendipine does not act at the picrotoxin site of the GABAA receptor. Possible interaction of nitrendipine with the Zn++ site was also eliminated, as mutation of beta2 H267 to A, which confers resistance to Zn++, had no effect on nitrendipine-mediated inhibition. Our data suggest some of the central effects of dihydropyridines may be due to actions at GABAA receptors. Moreover, the effects may be mediated through interaction with a novel modulatory site on the GABAA receptor.

The enantiomer of cholesterol.

Cholesterol plays a variety of significant roles in biological systems. However, the mechanisms by which cholesterol functions remain largely unclear. The enantiomer of cholesterol (ent-cholesterol)--which has identical physical properties, but opposite three-dimensional configuration compared to cholesterol--is a unique tool that can be used to better understand the mechanisms of cholesterol function. We review the literature pertaining to ent-cholesterol, focusing in particular on its use in biological studies.

Neuroprotective agent riluzole potentiates postsynaptic GABA(A) receptor function.

The antiepileptic drug riluzole is a use-dependent blocker of voltage-gated Na(+) channels and selectively depresses action potential-driven glutamate over gamma-aminobutyric acid (GABA) release. Here we report that in addition to its presynaptic effect, riluzole at higher concentrations also strongly potentiates postsynaptic GABA(A) responses both in cultured hippocampal neurons and in Xenopus oocytes expressing recombinant receptors. Although peak inhibitory postsynaptic currents (IPSCs) of autaptic hippocampal neurons were inhibited, 20-100 microM riluzole significantly prolonged the decay of IPSCs, resulting in little change in total charge transfer. The effect was dose-dependent and reversible. Riluzole selectively increased miniature IPSC fast and slow decay time constants, without affecting their relative proportions. Miniature IPSC peak amplitude, rise time and frequency were unaffected, indicating a postsynaptic mechanism. In the Xenopus oocyte expression system, riluzole potentiated GABA responses by lowering the EC(50) for GABA activation. Riluzole directly gated a GABA(A) current that was partially blocked by bicuculline and gabazine. Pharmacological experiments suggest that the action of riluzole did not involve a benzodiazepine, barbiturate, or neurosteroid site. Instead, riluzole-induced potentiation was inhibited by the lactone antagonist alpha-isopropyl-alpha-methyl-gamma-butyrolatone (alpha-IMGBL). While most anticonvulsants either block voltage-gated Na(+) channels or potentiate GABA(A) receptors, our results suggest that riluzole may define an advantageous class of anticonvulsants with both effects.

Recent developments in structure-activity relationships for steroid modulators of GABA(A) receptors.

GABAergic neurotransmission can be both positively and negatively modulated by steroids. The steroid effects are thought to be mediated by binding of steroids to specific sites on GABA(A) receptors. It appears that the receptor sites for positive and negative modulatory steroids are different. Thus far, the location and number of binding sites for steroids on these receptors have not been established. In this brief review, we concentrate largely on results from our own structure-activity studies. Novel analogues have been studied to further delineate the structural features required for compounds to modulate receptor function via steroid binding sites. Non-naturally occurring enantiomers of both positive and negative modulators have been studied to provide further evidence for the existence of specific steroid binding sites on the receptors.

The synthetic enantiomer of pregnenolone sulfate is very active on memory in rats and mice, even more so than its physiological neurosteroid counterpart: distinct mechanisms?

The demonstration that the neurosteroid pregnenolone sulfate (PREGS) is active on memory function at both the physiological and pharmacological levels led to us examining in detail the effects of the steroid on spatial working memory by using a two-trial recognition task in a Y-maze, a paradigm based on the natural drive in rodents to explore a novel environment. Dose-response studies in young male adult Sprague-Dawley rats and Swiss mice, after the postacquisition intracerebroventricular injection of steroid, showed an U-inverted curve for memory performance and indicated a greater responsiveness in rats compared with mice. Remarkably, the synthetic (-) enantiomer of PREGS not only also displayed promnesiant activity, but its potency was 10 times higher than that of the natural steroid. Intracerebroventricular coadministration experiments with DL-2-amino-5-phosphonovaleric acid, a competitive selective antagonist of the N-methyl-D-aspartate receptor, abolished the memory-enhancing effect of PREGS, but not that of the PREGS enantiomer, evoking enantiomeric selectivity at the N-methyl-d-aspartate receptor and/or different mechanisms for the promnestic function of the two enantiomers.

Enantiospecificity of cholesterol function in vivo.

The importance of the absolute configuration of cholesterol for its function in vivo is unknown. To directly test this question in vivo, we synthesized the enantiomer of cholesterol (ent-cholesterol) and tested its ability to substitute for natural cholesterol (nat-cholesterol) in the growth, viability, and behavior of Caenorhabditis elegans, a cholesterol auxotroph. First-generation animals grown on ent-cholesterol were viable with only mild behavioral defects. However, ent-cholesterol produced 100% lethality/arrest of their second generation progeny. Isotopically labeled ent-cholesterol incorporated into animals, indicating that its lethality was not secondary to cholesterol starvation. When mixed with nat-cholesterol, ent-cholesterol was not inert; rather, it antagonized the activity of nat-cholesterol. These results demonstrate for the first time that the absolute configuration of cholesterol, not just its physical properties, is essential for its functions in vivo.

Effects on gamma-aminobutyric acid (GABA)(A) receptors of a neuroactive steroid that negatively modulates glutamate neurotransmission and augments GABA neurotransmission.

Neurosteroids positively and negatively modulate gamma-aminobutyric acid (GABA)(A) receptors and glutamate receptors, which underlie most fast inhibition and excitation in the central nervous system. We report the identification of a neuroactive steroid, (3 alpha,5 beta)-20-oxo-pregnane-3-carboxylic acid (3 alpha 5 beta PC), with unique cellular actions. 3 alpha 5 beta PC positively modulates GABA(A) receptor function and negatively modulates N-methyl-D-aspartate (NMDA) receptor function, a combination that may be of particular clinical benefit. 3 alpha 5 beta PC promotes net GABA(A) potentiation at low steroid concentrations (<10 microM) and at negative membrane potentials. At higher concentrations, the steroid also blocks GABA receptors. Because this block would presumably counteract the NMDA receptor blocking actions of 3 alpha 5 beta PC, we characterize the GABA receptor block in some detail. Agonist concentration, depolarization, and high extracellular pH increase the block. The apparent pK for both potentiation and block was 6.4 to 6.9, substantially higher than expected from carboxylated steroid in an aqueous environment. Block is not dependent on the stereochemistry of the carboxylic acid at carbon 3 and is relatively insensitive to placement of the carboxylic acid at the opposite end of the steroid (carbon 24). Potentiation is critically dependent on the stereochemistry of the carboxylic acid group at carbon 3. Consistent with the pH dependence of potentiation, effects of the amide derivative (3 alpha,5 beta)-20-oxo-pregnane-3-carboxamide, suggest that the un-ionized form of 3 alpha 5 beta PC is important for potentiation, whereas the ionized form is probably responsible for block. Further refinement of the neuroactive steroid to promote GABA potentiation and NMDA receptor block and diminish GABA receptor block may lead to a clinically useful neuroactive steroid.

Pentylenetetrazole-induced inhibition of recombinant gamma-aminobutyric acid type A (GABA(A)) receptors: mechanism and site of action.

Pentylenetetrazole (PTZ) is a central nervous system convulsant that is thought, based on binding studies, to act at the picrotoxin (PTX) site of the gamma-aminobutyric acid type A (GABA(A)) receptor. In the present study, we have investigated the mechanism and site of action of PTZ in recombinant GABA(A) receptors. In rat alpha 1 beta 2 gamma 2 receptors, PTZ inhibited GABA-activated Cl(-) current in a concentration-dependent, voltage-independent manner, with an IC(50) of 0.62 +/- 0.13 mM. The mechanism of inhibition appeared competitive with respect to GABA in both rat and human alpha 1 beta 2 gamma 2 receptors. Varying subunit configuration (change or lack of alpha subunit isoform or lack of gamma 2 subunit) had modest effects on PTZ-induced inhibition, as evidenced by comparable IC(50) values (0.6-2.2 mM) in all receptor configurations tested. This contrasts with PTX and other PTX-site ligands, which have greater affinity in receptors lacking an alpha subunit. Using a one-site model for PTZ interaction with alpha 1 beta 2 gamma 2 receptors, the association rate (k(+1)) was found to be 1.14 x 10(3) M(-1) s(-1) and the dissociation rate (k(-1)) was 0.476 s(-1), producing a functional k(d) of 0.418 mM. PTZ could only gain access to its binding site extracellularly. Single-channel recordings demonstrated that PTZ decreased open probability by increasing the duration of closed states but had no effect on single-channel conductance or open state duration. alpha-Isopropyl-alpha-methyl-gamma-butyrolactone, a compound known to antagonize effects of PTX, also diminished the effects of PTZ. Taken together, our results indicate that pentylenetetrazole and picrotoxin interact with overlapping but distinct domains of the GABA(A) receptor.

Synthesis of (5alpha)-17-azaandrostan-3-ols and (5alpha)-17-aza-D-homoandrostan-3-ols and their N-acylated derivatives.

Two groups of N-acylated D-azasteroids (4 and 5) were synthesized to explore structure-activity relationships for steroid modulation of GABA(A) receptor function. The target compounds were prepared conveniently from (5alpha)-3-hydroxyandrostan-17-ones (6 and 7) via the intermediate (5alpha)-17-aza-D-homoandrostan-3-ols (14 and 15) or (5alpha)-17-azaandrostan-3-ols (18 and 19) precursors in high overall yields. A Beckmann rearrangement and a Hofmann rearrangement were employed as two key steps in the synthetic sequences.

The nonfeminizing enantiomer of 17beta-estradiol exerts protective effects in neuronal cultures and a rat model of cerebral ischemia.

Estrogens are potent neuroprotective compounds in a variety of animal and cell culture models, and data indicate that estrogen receptor (ER)-mediated gene transcription is not required for some of these effects. To further address the requirement for an ER in estrogen enhancement of neuronal survival, we assessed the enantiomer of 17beta-estradiol (ENT-E(2)), which has identical chemical properties but interacts only weakly with known ERs, for neuroprotective efficacy. ENT-E(2) was both as potent and efficacious as 17beta-estradiol in attenuating oxidative stress-induced death in HT-22 cells, a murine hippocampal cell line. Further, ENT-E(2) completely attenuated H(2)O(2) toxicity in human SK-N-SH neuroblastoma cells at a 10 nM concentration. In a rodent model of focal ischemia, 17beta-estradiol (100 microgram/kg) or ENT-E(2) (100 microgram/kg), injected 2 h before middle cerebral artery occlusion, resulted in a 60 and 61% reduction in lesion volume, respectively. ENT-E(2), at the doses effective in this study, did not stimulate uterine growth or vaginal opening in juvenile female rats when administered daily for 3 days. These data indicate that the neuroprotective effects of estrogens, both in vitro and in vivo, can be disassociated from the peripheral estrogenic actions.

Steroid structure and pharmacological properties determine the anti-amnesic effects of pregnenolone sulphate in the passive avoidance task in rats.

Pregnenolone sulphate (PREGS) has generated interest as one of the most potent memory-enhancing neurosteroids to be examined in rodent learning studies, with particular importance in the ageing process. The mechanism by which this endogenous steroid enhances memory formation is hypothesized to involve actions on glutamatergic and GABAergic systems. This hypothesis stems from findings that PREGS is a potent positive modulator of N-methyl-d-aspartate receptors (NMDARs) and a negative modulator of gamma-aminobutyric acid(A) receptors (GABA(A)Rs). Moreover, PREGS is able to reverse the amnesic-like effects of NMDAR and GABA(A)R ligands. To investigate this hypothesis, the present study in rats examined the memory-altering abilities of structural analogs of PREGS, which differ in their modulation of NMDAR and/or GABA(A)R function. The analogs tested were: 11-ketopregnenolone sulphate (an agent that is inactive at GABA(A)Rs and NMDARs), epipregnanolone ([3beta-hydroxy-5beta-pregnan-20-one] sulphate, an inhibitor of both GABA(A)Rs and NMDARs), and a newly synthesized (-) PREGS enantiomer (which is identical to PREGS in effects on GABA(A)Rs and NMDARs). The memory-enhancing effects of PREGS and its analogs were tested in the passive avoidance task using the model of scopolamine-induced amnesia. Both PREGS and its (-) enantiomer blocked the effects of scopolamine. The results show that, unlike PREGS, 11-ketopregnenolone sulphate and epipregnanolone sulphate failed to block the effect of scopolamine, suggesting that altering the modulation of NMDA receptors diminishes the memory-enhancing effects of PREGS. Moreover, enantioselectivity was demonstrated by the ability of natural PREGS to be an order of magnitude more effective than its synthetic enantiomer in reversing scopolamine-induced amnesia. These results identify a novel neuropharmacological site for the modulation of memory processes by neuroactive steroids.

Correlation of the apparent affinities and efficacies of gamma-aminobutyric acid(C) receptor agonists.

gamma-Aminobutyric acid (GABA), trans-4-aminocrotonic acid (TACA), muscimol, imidazole-4-acetic acid (I4AA), cis-4-aminocrotonic acid (CACA), and isoguvacine are all GABA(C) receptor agonists. These compounds have different apparent sensitivities (EC(50)) and efficacies (I(max)) on exogenously expressed human rho1 homomeric GABA(C) receptors. It is not clear if these differences are due to distinct binding affinities and/or distinct gating kinetics. In this study, using a recently developed single oocyte binding technique, we determined the apparent dissociation constants (K(i) values) of these compounds from their IC(50) values for [(3)H]GABA displacement. The apparent K(i) values fell into two distinct groups. The high affinity group was comprised of agonists with longer distances between the nitrogen atom of the amino or imidazole group and the carbon atom of the carboxyl or isoxazole group. The single oocyte binding technique, in conjunction with two-electrode voltage clamp, has allowed a direct correlation of the apparent affinity, efficacy, and potency of agonists on intact functional GABA(C) receptors. The correlation and coupling of these parameters are discussed in terms of a simple proposed activation mechanism.

Formation of stable vesicles from N- or 3-alkylindoles: possible evidence for tryptophan as a membrane anchor in proteins.

Twelve indole derivatives have been prepared and studied. Five were 1-substituted: 1, methyl; 2, n-hexyl; 3, n-octyl; 4, n-octadecyl; and 5, cholestanyloxycarbonylmethyl. Four were 3-substituted: 6, methyl; 7, n-hexyl; 8, n-octyl; and 9, n-octadecyl. Three were disubstituted as follows: 10, 1-n-decyl-3- n-decyl; 11, 1-methyl-3-n-decyl; and 12, 1,3-bis(n-octadecyl)indole. Sonication of aqueous suspensions afforded stable aggregates from 3-5 and 8-12. Laser light scattering, dye entrapment, and electron microscopy were used to characterize the aggregates. Aggregates formed from N-substituted indoles proved to be more robust than those formed from 3-alkylindoles. A stable monolayer formed from 3-n-octadecylindole but not from N- or 1,3-disubstituted analogues by using a Langmuir-Blodgett trough. The formation of aggregates was explained in terms of stacking by the relatively polar indole headgroup. In the monolayer experiment, this force was apparently overwhelmed by H-bonding interactions with the aqueous phase.

Steroid inhibition of rat neuronal nicotinic alpha4beta2 receptors expressed in HEK 293 cells.

Steroids, in addition to regulating gene expression, directly affect a variety of ion channels. We examined the action of steroids on human embryonic kidney 293 cells stably transfected to express rat alpha4beta2 neuronal nicotinic receptors. Each steroid that was tested inhibited acetylcholine responses from these receptors, with slow kinetics requiring seconds for block to develop and recover. The action of one steroid [3alpha,5alpha, 17beta-3-hydroxyandrostane-17-carbonitrile (ACN)] was studied in detail. Block showed enantioselectivity, with an IC(50) value of 1.5 microM for ACN and 4.5 microM for the enantiomer. Inhibition curves had Hill slopes larger than 1, indicating more than one binding site per receptor. Block did not require intracellular compounds containing high-energy phosphate bonds and was not affected by analogs of GTP, suggesting that the mechanism does not require the activation of second messengers. Block did not appear to be strongly selective between open and closed channel states or to involve changes in desensitization. A comparison of different steroids showed that a beta-orientation of groups at the 17 position produced more block than alpha-orientated diastereomers. The stereochemistry at the 3 and 5 positions was less influential for block of alpha4beta2 nicotinic receptors, despite its importance for potentiation of gamma-aminobutyric acid(A) receptors. The ability of steroids to block neuronal nicotinic receptors correlated with their ability to produce anesthesia in Xenopus tadpoles, but the concentrations required for inhibition are generally greater. Similarly, the concentrations of endogenous neurosteroids required to inhibit receptors are larger than estimates of brain concentrations.