Benzodiazepines affect channel opening of GABA A receptors induced by either agonist binding site

Benzodiazepines are widely used as anxiolytics, sedatives, muscle relaxants, and anticonvulsants. They allosterically modulate GABA type A (GABA(A)) receptors by increasing the apparent affinity of the agonist GABA to elicit chloride currents. Such an increase in apparent affinity of channel gating could either be caused by an increase in affinity for GABA or by a facilitation of channel opening. In the first case, conformation of the affected sites would have to be altered. In the second case, the affected sites are not necessarily altered, because diazepam could facilitate conformational changes leading to the open channel. It is controversial as to whether benzodiazepines affect only channel opening induced by the occupation of one of the two agonist binding sites or by both. We used receptors formed by concatenated subunits to selectively destroy one of the two agonist sites by point mutation. Both of the receptors harboring only one active agonist site could be stimulated by diazepam. We therefore present evidence that binding of diazepam can affect channel opening induced by either agonist binding site.     

Ethanol modulates the interaction of the endogenous neurosteroid allopregnanolone with the alpha1beta2gamma2L GABAA receptor

We have examined alpha1beta2gamma2L GABAA receptor modulation by the endogenous steroids allopregnanolone (3alpha5alphaP), pregnenolone sulfate, and beta-estradiol in the absence and presence of ethanol. Coapplication of 0.1 to 1% (17-170 mM) ethanol influenced receptor modulation by 3alpha5alphaP but not that by pregnenolone sulfate or beta-estradiol. One of the three kinetic effects evident in channel potentiation by 3alpha5alphaP, prolongation of the longest-lived open time component (OT3), was affected by ethanol with the midpoint of its dose-response curve moved to lower steroid concentrations by 2 orders of magnitude without significantly affecting the maximal effect. Manipulations designed to affect the ability of 3alpha5alphaP to prolong OT3 also affected OT3 prolongation in the presence of ethanol. A mutation to the gamma2 subunit, which reduces the ability of 3alpha5alphaP to prolong OT3, also reduces the interaction between ethanol and 3alpha5alphaP. And the presence of the competitive steroid antagonist (3alpha,5alpha)-17-phenylandrost-16-en-3-ol (17-PA) diminishes the positive interaction between ethanol and 3alpha5alphaP on the GABAA receptor. Together, the findings suggest that steroid interactions with the classic steroid binding site underlie the effect seen in the presence of ethanol, and that ethanol acts by increasing the affinity of 3alpha5alphaP for the site. Tadpole behavioral assays showed that the presence of 3alpha5alphaP at a concentration ineffective at causing changes in tadpole behavior shifted the ethanol dose-response curve for loss of righting reflex to lower concentrations and that this effect was neutralized by coapplication of 17-PA with 3alpha5alphaP.     

Benzodiazepine binding site heterogeneity in the purified GABAA receptor

The displacement of [3H]flunitrazepam binding activity by ethyl-beta-carboline-3-carboxylate (beta CCE) was studied in both membrane-bound and purified GABAA receptors from adult bovine cerebral cortex, hippocampus and cerebellum. It was found that the best fit for the displacement of benzodiazepine binding in the cerebellar membranes was a single site with IC50 = 0.55 +/- 0.21 nM, whereas the best fit for cortical and hippocampal membranes was a two-site model with respective values of IC50 = 0.2 +/- 0.09 nM (high affinity), IC50 = 21 +/- 6 nM (low affinity) (cortex) and IC50 = 0.25 +/- 0.05 nM, IC50 = 20 +/- 2 nM (hippocampus). These same properties were retained in the purified GABAA receptor from the three brain regions. Thus, we have demonstrated that binding site heterogeneity as defined by the displacement of beta CCE is preserved in purified GABAA receptors and we suggest that this provides evidence for the existence of GABAA receptor isoforms.     

Defining the propofol binding site location on the GABAA receptor

The GABAA receptor is a target of many general anesthetics. The low affinity of general anesthetics has complicated the search for the location of anesthetic binding sites. Attention has focused on two pairs of residues near the extracellular ends of the M2 and M3 membrane-spanning segments, alpha1Ser270/beta2Asn265 (15' M2) and alpha1Ala291/beta2Met286 (M3). In the 4-A resolution acetylcholine receptor structure, the aligned positions are separated by approximately 10 A. To determine whether these residues are part of a binding site for propofol, an intravenous anesthetic, we probed propofol's ability to protect cysteines substituted for these residues from modification by the sulfhydryl-specific reagent p-chloromercuribenzenesulfonate (pCMBS-). pCMBS- reacted with cysteines substituted at the four positions in the absence and presence of GABA. Because propofol binding induces conformational change in the GABAAreceptor, we needed to establish a reference state of the receptor to compare reaction rates in the absence and presence of propofol. We compared reaction rates in the presence of GABA with those in the presence of propofol +GABA. The GABA concentration was reduced to give a similar fraction of the maximal GABA current in both conditions. Propofol protected, in a concentration-dependent manner, the cysteine substituted for beta2Met286 from reaction with pCMBS-. Propofol did not protect the cysteine substituted for the aligned alpha1 subunit position or the 15' M2 segment Cys mutants in either subunit. We infer that propofol may bind near the extracellular end of the betasubunit M3 segment.     

Modulation of GABAA receptors and neuropeptide secretion by the neurosteroid allopregnanolone in posterior and intermediate pituitary

A number of neurosteroids bind to GABAA receptors and alter their responsiveness to neurotransmitters. Considerable effort has been devoted to understanding how this form of receptor modulation alters inhibitory synaptic function. Neurosteroid-sensitive GABAA receptors have also been demonstrated in many endocrine cells, but little is known about how neurosteroids modulate the release of hormones. Here, the action of allopregnanolone, a neurosteroid that enhances GABAA receptor-mediated responses, was investigated in posterior pituitary nerve terminals and intermediate pituitary endocrine cells. Patch clamp recordings showed that GABA-evoked currents were enhanced to similar degrees and with similar concentration dependences in both locations. An organ bath preparation of the neurointermediate lobe was used to investigate drug effects on secretion of vasopressin and alpha-melanocyte stimulating hormone. GABA increased the basal release of vasopressin and alpha-melanocyte stimulating hormone from the posterior and intermediate pituitary lobe, respectively, an effect that could be blocked by picrotoxinin. Vasopressin release evoked by electrical stimulation was also examined, and a small statistically significant inhibition by 5 microM GABA was observed. Allopregnanolone increased the basal release of vasopressin, and this effect was blocked by the GABAA receptor antagonist picrotoxinin. Allopregnanolone had no effect in conjunction with GABA. In contrast to the posterior lobe, allopregnanolone had no effect on release from the intermediate lobe. Thus, allopregnanolone in physiological relevant concentrations modulates GABAA receptors in both the posterior and intermediate lobes, but only affects hormone release in the posterior lobe.     

Reinforcing effects of the neurosteroid allopregnanolone in rats

The GABA(A) receptor positive modulator allopregnanolone (3alpha-hydroxy-5alpha-pregnan-20-one) is a potent neurosteroid with behavioral and biochemical characteristics similar to ethanol, barbiturates, and benzodiazepines. This suggests that neurosteroids may provide an alternative class of sedative/hypnotic, anticonvulsant, and anxiolytic pharmacotherapies. However, there is evidence from animal models that neurosteroids may be susceptible to abuse by humans. Thus, the present study evaluated the reinforcing effects of orally administered allopregnanolone in rats. In the first experiment, male Long-Evans rats (n=9) were allowed to voluntarily consume a 50-microg/ml allopregnanolone (50A) solution or water in an unlimited-access two-bottle choice procedure for 10 days. Subsequently, the same animals were trained to lever-press to receive a 50A solution in daily 30-min operant sessions using a sucrose substitution procedure. In the two-bottle choice procedure, rats consumed significantly more allopregnanolone than water, suggesting that allopregnanolone was serving as a reinforcer. In the operant self-administration procedure, allopregnanolone did not maintain levels of responding that were different from water, suggesting that allopregnanolone did not function as a reinforcer in this procedure. These results suggest that orally administered allopregnanolone possesses reinforcing properties; however, additional studies are necessary to determine whether operant oral self-administration will be a viable index of allopregnanolone's reinforcing effects.     

Phosphorylation of GABAA receptors influences receptor trafficking and neurosteroid actions

Rationale

Gamma-aminobutyric acid type A receptors (GABA_ARs) are the principal mediators of inhibitory transmission in the mammalian central nervous system. GABA_ARs can be localized at post-synaptic inhibitory specializations or at extrasynaptic sites. While synaptic GABA_ARs are activated transiently following the release of GABA from presynaptic vesicles, extrasynaptic GABA_ARs are typically activated continuously by ambient GABA concentrations and thus mediate tonic inhibition. The tonic inhibitory currents mediated by extrasynaptic GABA_ARs control neuronal excitability and the strength of synaptic transmission. However, the mechanisms by which neurons control the functional properties of extrasynaptic GABA_ARs had not yet been explored.

Objectives

We review GABA_ARs, how they are assembled and trafficked, and the role phosphorylation has on receptor insertion and membrane stabilization. Finally, we review the modulation of GABA_ARs by neurosteroids and how GABA_AR phosphorylation can influence the actions of neurosteroids.

Conclusions

Trafficking and stability of functional channels to the membrane surface are critical for inhibitory efficacy. Phosphorylation of residues within GABA_AR subunits plays an essential role in the assembly, trafficking, and cell surface stability of GABA_ARs. Neurosteroids are produced in the brain and are highly efficacious allosteric modulators of GABA_AR-mediated current. This allosteric modulation by neurosteroids is influenced by the phosphorylated state of the GABA_AR which is subunit dependent, adding temporal and regional variability to the neurosteroid response. Possible links between neurosteroid actions, phosphorylation, and GABA_AR trafficking remain to be explored, but potential novel therapeutic targets may exist for numerous neurological and psychological disorders which are linked to fluctuations in neurosteroid levels and GABA_A subunit expression.     

Pharmacology of structural changes at the GABA(A) receptor transmitter binding site

BACKGROUND AND PURPOSE

The binding of transmitter to specialized binding pockets leads to rearrangements in the structure of the receptor eventually resulting in channel opening. We used voltage-clamp fluorometry to investigate the pharmacological basis and biophysical processes that underlie structural changes at the transmitter binding site of the rat α1β2γ2L GABA_A receptor.

EXPERIMENTAL APPROACH

Simultaneous electrophysiological and site-specific fluorescence measurements were conducted on receptors expressed in Xenopus oocytes and labelled with an environmentally-sensitive fluorophore, Alexa 546 maleimide, at the α1L127C site.

KEY RESULTS

Receptors activated by GABA demonstrate a concentration-dependent increase in fluorescence intensity, indicating that the environment surrounding the fluorophore becomes less polar upon activation. Qualitatively similar responses were observed with other GABA site ligands such as piperidine-4-sulphonic acid, muscimol, β-alanine and 4,5,6,7-tetrahydroisoxazolo[5,4-c]pyridin-3-ol. Fluorescence changes were not affected by the direction of current flow. During long applications of GABA significant desensitization developed, which was not accompanied by additional changes in fluorescence. Pentobarbital was an efficacious agonist of the labelled mutant receptor but did not cause changes in fluorescence. Direct activation by etomidate or the steroid allopregnanolone also did not result in fluorescence changes. Functional potentiation of GABA-activated receptors by allopregnanolone or etomidate enhanced both the GABA-elicited functional response and the fluorescence change. In contrast, potentiation by pentobarbital was not accompanied by an enhanced fluorescence response.

CONCLUSIONS AND IMPLICATIONS

The data indicate that there is no direct correlation between current flow or position of the activation gate and the structural changes as detected by Alexa 546-labelled α1L127Cβ2γ2L GABA_A receptors. Channel potentiation by pentobarbital qualitatively differs from potentiation by etomidate or allopregnanolone.     

Effects of subunit types of the recombinant GABAA receptor on the response to a neurosteroid

When vertebrate brain poly(A)⁺ RNA is expressed in Xenopus oocytes the response of the GABA receptors formed is found to be inhibited allosterically by a neurosteroid, pregnenolone sulphate (PS). This negative modulation was reproduced after expressing RNAs enconding bovine GABA_A receptor subunits in the combinations α1 + ß1, or αi + ß1 + γ2 (where i = 1. 2 or 3). The characteristics of this inhibition vary significantly with the type of the α subunit (α1, α2, or α3) used. When the bovine γ2L alternate form of the γ2 subunit was replaced by the human γ2S subunit, the behaviour was unchanged: the human γ2S subunit used is a newly-cloned form, which encodes a polypeptide with two amino acid differences from the human γ2 subunit previously described. The results of co-application of PS and 3α-hydroxy-5α-pregnan-ol-20-one, a neurosteroid which is a positive modulator of the GABA_A receptor, indicate that these act at different sites on the receptor. PS also increases the desensitisation of the receptor by GABA. This effect, also, is α-subunit-type dependent and occurs by an acceleration of the fast phase of desensitisation.     

Phosphatidylinositol 4,5-bisphosphate depletion fails to affect neurosteroid modulation of GABAA receptor function

Rationale

Neurosteroids and likely other lipid modulators access transmembrane sites on the GABA_A receptor (GABA_AR) by partitioning into and diffusing through the plasma membrane. Therefore, specific components of the plasma membrane may affect the potency or efficacy of neurosteroid-like modulators. Here, we tested a possible role for phosphatidylinositol 4,5-bisphosphate (PIP2), a phospholipid that governs activity of many channels and transporters, in modulation or function of GABA_ARs.

Objectives

In these studies, we sought to deplete plasma-membrane PIP2 and probe for a change in the strength of potentiation by submaximal concentrations of the neurosteroid allopregnanolone (3α5αP) and other anesthetics, including propofol, pentobarbital, and ethanol. We also tested for a change in the behavior of negative allosteric modulators pregnenolone sulfate and dipicrylamine.

Methods

We used Xenopus oocytes expressing the ascidian voltage-sensitive phosphatase (Ci-VSP) to deplete PIP2. Voltage pulses to positive membrane potentials were used to deplete PIP2 in Ci-VSP-expressing cells. GABA_ARs composed of α1β2γ2L and α4β2δ subunits were challenged with GABA and 3α5αP or other modulators before and after PIP2 depletion. KV7.1 channels and NMDA receptors (NMDARs) were used as positive controls to verify PIP2 depletion.

Results

We found no evidence that PIP2 depletion affected modulation of GABA_ARs by positive or negative allosteric modulators. By contrast, Ci-VSP-induced PIP2 depletion depressed KV7.1 activation and NMDAR activity.

Conclusions

We conclude that despite a role for PIP2 in modulation of a wide variety of ion channels, PIP2 does not affect modulation of GABA_ARs by neurosteroids or related compounds.     

Two flavones from Scutellaria baicalensis Georgi and their binding affinities to the benzodiazepine site of the GABAA receptor complex

A new flavone 6,2'-dihydroxy-5,7,8,6'-tetramethoxyflavone (1) together with one known flavone 5,7,2'-trihydroxy-6,8-dimethoxyflavone (2) were isolated from the roots of Scutellaria baicalensis Georgi. Their structures were elucidated on the basis of spectral evidence and their affinities for the benzodiazepine (BDZ) site of the GABAA receptor complex were evaluated with a radioligand receptor binding assay.     

Effects of subunit types of the recombinant GABAA receptor on the response to a neurosteroid.

When vertebrate brain poly(A)+ RNA is expressed in Xenopus oocytes the response of the GABA receptors formed is found to be inhibited allosterically by a neurosteroid, pregnenolone sulphate (PS). This negative modulation was reproduced after expressing RNAs encoding bovine GABAA receptor subunits in the combinations alpha i + beta 1, or alpha i + beta 1 + gamma 2 (where i = 1, 2 or 3). The characteristics of this inhibition vary significantly with the type of the alpha subunit (alpha 1, alpha 2, or alpha 3) used. When the bovine gamma 2L alternate form of the gamma 2 subunit was replaced by the human gamma 2S subunit, the behaviour was unchanged: the human gamma 2S subunit used is a newly-cloned form, which encodes a polypeptide with two amino acid differences from the human gamma 2 subunit previously described. The results of co-application of PS and 3 alpha-hydroxy-5 alpha-pregnan-ol-20-one, a neurosteroid which is a positive modulator of the GABAA receptor, indicate that these act at different sites on the receptor. PS also increases the desensitisation of the receptor by GABA. This effect, also, is alpha-subunit-type dependent and occurs by an acceleration of the fast phase of desensitisation.     

Effects of subunit types of the cloned GABAA receptor on the response to a neurosteroid.

Combinations of cloned GABAA receptor subtypes, having the subunit combinations alpha i + beta 1 or alpha i + beta 1 + gamma 2 (i = 1, 2, 3), were expressed in Xenopus oocytes. The endogenous steroid 3 alpha-hydroxy-5 alpha-pregnan-20-one potentiates GABA currents induced therein by GABA. This potentiation was greater in the alpha 1 + beta and alpha 3 + beta 1 than in the alpha 2 + beta 1 combinations. The presence of the gamma 2-subunit increased the steroid potency in alpha 1 + beta 1 and alpha 2 + beta 1, but the combination alpha 3 + beta 1 + gamma 2 became much less steroid-sensitive. It is concluded that the steroid modification of the GABAA receptor is strongly influenced by the alpha- and the gamma 2-subunit types.     

Differential anesthetic activity of ketamine and the GABAergic neurosteroid allopregnanolone in mice lacking progesterone receptor A and B subtypes

Progesterone affects the function of the brain by multiple mechanisms. The physiological effects of progesterone are mediated by the interaction of the hormone with progesterone receptors (PRs), which are widely expressed in the hypothalamus, hippocampus and limbic areas. The PR is composed of two protein isoforms, PR-A and PR-B, which are expressed from a single PR gene. In addition, progesterone influences neuronal activity through its conversion to allopregnanolone, a neurosteroid that acts as a positive allosteric modulator of GABA(A) receptors. However, the role of PRs in the sedative-hypnotic action of neurosteroids is unclear. In this study, PR knockout (PRKO) mice were used as model to study the sedative-anesthetic actions of the progesterone-derived neurosteroid allopregnanolone and the noncompetitive NMDA receptor antagonist ketamine. Mice were confirmed to be PR deficient by genotyping and immunohistochemistry of PR expression in the brain. Anesthetic potency was evaluated by the loss of the righting reflex paradigm. Allopregnanolone-induced anesthetic activity was similar in PRKO mice and their wild-type littermates, suggesting that PRs are not involved in the anesthetic response to allopregnanolone. However, the noncompetitive NMDA receptor antagonist ketamine has significantly reduced anesthetic potency in PRKO mice, suggesting a possible developmental plasticity of glutamate receptors. There was no marked gender-related difference to ketamine response in both genotypes. In conclusion, these results suggest that the neurosteroid allopregnanolone and ketamine produce differential anesthetic response in mice lacking PRs.     

Alteration of voluntary ethanol and saccharin consumption by the neurosteroid allopregnanolone in mice

RATIONALE: The neurosteroid 3 alpha-hydroxy-5 alpha-pregnan-20-one (allopregnanolone, ALLOP) is a positive modulator of gamma-aminobutyric acid type A (GABA(A)) receptors. Recent findings indicate that ethanol (EtOH) and ALLOP share common mechanisms of action and that ALLOP may modulate some of EtOH's abuse-related effects. OBJECTIVES: The present studies investigated whether ALLOP pretreatment altered voluntary EtOH consumption in male and female C57BL/6J mice, and voluntary saccharin and quinine consumption in male C57BL/6J mice. METHODS: Mice had access to two drinking tubes containing water versus 5% or 10% (v/v) EtOH or a tastant for 2 h each day at the beginning of the dark cycle. Following establishment of stable consumption, animals received 2 days of vehicle followed by 3 days of ALLOP injections (0, 3.2, 10, or 17 mg/kg, IP), immediately prior to EtOH or tastant access. RESULTS: Prior to injection, the 2-h baseline dose of the 10% EtOH solution consumed was 1.31 g/kg (expt 1) or 2.46 g/kg (expt 3) for male and 2.21 g/kg (expt 2) for female mice. Baseline intake of the 5% EtOH solution was 0.60 g/kg for males and 0.75 g/kg for females (expt 5). In males, ALLOP administration significantly and dose-dependently increased consumption of both EtOH solutions during the first hour of availability without affecting water intake. In females, ALLOP did not significantly alter EtOH consumption. Lastly, ALLOP significantly increased saccharin, but not quinine, consumption in males (females were not tested). CONCLUSIONS: ALLOP may increase voluntary EtOH consumption in male mice by altering its reinforcing effects. The lack of significant effect on quinine and water consumption suggests that ALLOP does not simply increase consumption of all fluids.     

Individually monitoring ligand-induced changes in the structure of the GABAA receptor at benzodiazepine binding site and non-binding-site interfaces

The mechanisms by which the GABA and benzodiazepine (BZD) binding sites of the GABA-A receptor are allosterically coupled remain elusive. In this study, we separately monitored ligand-induced structural changes in the BZD binding site (alpha/gamma interface) and at aligned positions in the alpha/beta interface. alpha(1)His101 and surrounding residues were individually mutated to cysteine and expressed with wild-type beta2 and gamma2 subunits in Xenopus laevis oocytes. The accessibilities of introduced cysteines to modification by methanethiosulfonate ethylammonium (MTSEA)-Biotin were measured in the presence and absence of GABA-site agonists, antagonists, BZDs, and pentobarbital. The presence of flurazepam or the BZD-site antagonist flumazenil (Ro15-1788) decreased the rate of modification of alpha(1)H101C at the BZD binding site. GABA and muscimol each increased MTSEA-Biotin modification of alpha(1)H101C located at the BZD-site, gabazine (SR-95531, a GABA binding site antagonist) decreased the rate, whereas pentobarbital had no effect. Modification of alpha(1)H101C at the alpha/beta interface was significantly slower than modification of alpha(1)H101C at the BZD site, and the presence of GABA or flurazepam had no effect on its accessibility, indicating the physicochemical environments of the alpha/gamma and alpha/beta interfaces are different. The data are consistent with the idea that GABA-binding site occupation by agonists causes a GABA binding cavity closure that is directly coupled to BZD binding cavity opening, and GABA-site antagonist binding causes a movement linked to BZD binding cavity closure. Pentobarbital binding/gating resulted in no observable movements in the BZD binding site near alpha(1)H101C, indicating that structural mechanisms underlying allosteric coupling between the GABA and BZD binding sites are distinct.     

Synthesis of GABAA receptor agonists and evaluation of their alpha-subunit selectivity and orientation in the GABA binding site

Drugs used to treat various disorders target GABA A receptors. To develop alpha subunit selective compounds, we synthesized 5-(4-piperidyl)-3-isoxazolol (4-PIOL) derivatives. The 3-isoxazolol moiety was substituted by 1,3,5-oxadiazol-2-one, 1,3,5-oxadiazol-2-thione, and substituted 1,2,4-triazol-3-ol heterocycles with modifications to the basic piperidine substituent as well as substituents without basic nitrogen. Compounds were screened by [(3)H]muscimol binding and in patch-clamp experiments with heterologously expressed GABA A alpha ibeta 3gamma 2 receptors (i = 1-6). The effects of 5-aminomethyl-3 H-[1,3,4]oxadiazol-2-one 5d were comparable to GABA for all alpha subunit isoforms. 5-piperidin-4-yl-3 H-[1,3,4]oxadiazol-2-one 5a and 5-piperidin-4-yl-3 H-[1,3,4]oxadiazol-2-thione 6a were weak agonists at alpha 2-, alpha 3-, and alpha 5-containing receptors. When coapplied with GABA, they were antagonistic in alpha 2-, alpha 4-, and alpha 6-containing receptors and potentiated alpha 3-containing receptors. 6a protected GABA binding site cysteine-substitution mutants alpha 1F64C and alpha 1S68C from reacting with methanethiosulfonate-ethylsulfonate. 6a specifically covalently modified the alpha 1R66C thiol, in the GABA binding site, through its oxadiazolethione sulfur. These results demonstrate the feasibility of synthesizing alpha subtype selective GABA mimetic drugs.     

黄芩茎叶黄酮作为GABAA受体BZD结合部位配体

目的从黄芩地上部分中寻找活性成分。方法利用各种柱色谱 (硅胶、SephadexLH 2 0、D10 1大孔吸附树脂和聚酰胺 )分离黄芩地上部分的活性成分。结果从黄芩地上部分的水提取物中分离得到 7种黄酮类化合物。其中白杨素对GABAA 受体的BZD结合部位有较高的亲和力 ,其IC50 值为 0 6 7μmol/L。 7种黄酮类化合物亲和力的排列顺序为 :白杨素 >汉黄芩苷元 >红花素 >黄芩素 >5 ,6 ,7 三羟基 4′ 甲氧基黄酮 >异红花素 >白杨素 7 葡萄糖醛酸苷。     

The influence of subchronic administration of the neurosteroid allopregnanolone on sleep in the rat.

The endogenous neurosteroid allopregnanolone has recently been demonstrated to have somnogenic properties that are very similar to those of other agonistic modulators of GABA(A) receptors, especially of short-acting benzodiazepines. Short-acting benzodiazepines are established to rapidly lose their hypnotic effect upon repeated administration. To investigate the tolerance potential of allopregnanolone, we assessed sleep-wake behavior in rats during subchronic treatment (once daily for five days) with placebo or 15 mg/kg allopregnanolone (n = 8 each). The sleep patterns of the placebo and allopregnanolone group did not differ significantly before and after treatment. Throughout the entire treatment period the allopregnanolone group exhibited shorter non-rapid eye movement sleep (non-REMS) latencies, prolonged REMS latencies, longer non-REMS episodes, more pre-REMS and less low-frequency, but higher spindle activity in the electroencephalogram (EEG) within non-REMS than the placebo group. The lack of tolerance effects suggests that allopregnanolone may be an efficacious modulator of sleep-wake behavior over longer time periods than most drugs targeting the benzodiazepine binding site of the GABA(A) receptor.     

On the receptor binding site of relaxins

Relaxin plays a critical role in viviparity and has recently been implicated as a hormone of oviparity as well. In most mammals relaxin causes the widening of the birth canal during parturition and suppresses uterine motility during pregnancy. Relaxins isolated from several species have shown a great deal of sequence variability, and speculations regarding a putative receptor interaction site have, as a consequence, varied considerably. The isolation of skate relaxin in combination with our chemical modification data enable us to suggest a unique site for the interaction of relaxin with its uterine and symphyseal receptors.