Positive Modulator Of GABAA Receptors Research Articles

GABAA positive modulator and NMDA antagonist-like discriminative stimulus effects of isoflurane vapor in mice

Several neurotransmitter systems have been hypothesized to be involved in the in vivo effects of volatile anesthetics. Drug discrimination may represent a novel procedure to explore the neurochemical systems underlying the sub-anesthetic behavioral effects of these compounds. The purpose of the present study was to examine the contribution of GABA(A) and NMDA receptors to the discriminative stimulus effects of a behaviorally active sub-anesthetic concentration of isoflurane vapor. Sixteen B6SJLF1/J mice were trained to discriminate 10 min of exposure to 6,000 ppm isoflurane vapor from air. Substitution tests were conducted with volatile anesthetics, abused vapors, GABA(A) positive modulators, NMDA antagonists, and nitrous oxide. The volatile anesthetics, enflurane and halothane as well as the abused vapors toluene and 1,1,1-trichloroethane fully substituted for isoflurane. The GABA(A) positive modulators, pentobarbital, midazolam, and zaleplon but not the direct GABA(A) agonist, muscimol, produced high levels of partial substitution for isoflurane. The anticonvulsant, valproic acid fully substituted for isoflurane but a second, tiagabine, did not substitute. The competitive NMDA antagonist, CGS-19755, fully and the non-competitive NMDA antagonist, dizocilpine, partially substituted for isoflurane. The glycine-site NMDA antagonist, L-701,324 did not substitute for isoflurane. Gamma-hydroxybutric acid and nitrous oxide gas also failed to substitute for isoflurane. The discriminative stimulus effects of sub-anesthetic concentrations of isoflurane vapor are shared by other vapor anesthetics and abused inhalants. The discriminative stimulus effects of isoflurane vapor appear to be mediated by both positive allosteric modulation of GABA(A) receptors as well as antagonism of NMDA receptors.

Prenatal ethanol exposure attenuates GABAergic inhibition in basolateral amygdala leading to neuronal hyperexcitability and anxiety-like behavior of adult rat offspring

Prenatal exposure to a relatively high-dose ethanol (EtOH) caused anxiety-like behavior of adult male rat offspring. Previous studies have demonstrated that GABA system in the basolateral amygdala complex (BLA) is involved in the pathogensis of anxiety-related disorders. The role of GABAergic system in the BLA was investigated in anxiety-like behavior evoked by prenatal EtOH exposure. The infusion of midazolam (MDZ), a positive modulator of GABAA receptor, into the BLA prevented anxiety-like behavior in EtOH-offspring without affecting the corresponding behavior of control offspring. The data suggest that anxiety-like behavior could be causally related to increased neuronal excitability attributable to depressed GABAergic inhibition in the BLA. To test this hypothesis, evoked potential was studied using brain slices from EtOH-offspring. Potential evoked in the BLA by single stimuli applied to external capsule showed multispike responses, indicative of GABAergic disinhibition. These multiple responses were no longer evident after the perfusion with MDZ. In the slices from EtOH-offspring, paired-pulse inhibition (GABAA-dependent) was suppressed. Also, in EtOH-offspring, long-term potentiation (LTP) was induced by a single train of high frequency stimulation, which did not induce LTP in control rats. Moreover, MDZ pretreatment prevented the facilitating effect of EtOH on LTP induction. The data provide the functional evidence that prenatal EtOH exposure attenuates GABAergic inhibition in the BLA resulting in neuronal hyperexcitability and anxiety-like behavior of adult rat offspring.

P.1.c.044 SH-I-048A, a novel positive modulator of GABA-A receptor: in vitro and behavioral profile

P.1.c.044 SH-I-048A, a novel positive modulator of GABA-A receptor: in vitro and behavioral profile

Novel GABAB receptor positive modulators: a patent survey

Importance of the field: Positive allosteric modulators of GABAB receptors may have a similar potential as positive modulators of GABAA receptors, the benzodiazepines discovered in 1957. The discovery of positive allosteric modulators of GABAB receptors at Novartis in Basel in 2000 opened the way to search for compounds, which activate GABAB receptors without the drawbacks of full agonists, such as desensitization, tolerance, muscle-relaxant effects, hypothermia, and central and gastrointestinal side effects.Areas covered in this review: Numerous animal experiments point out that several indications can be addressed with positive modulators of GABAB receptors, such as depression, anxiety, schizophrenia, neuropathic and chronic pain and treatment of craving for drugs of abuse, such as alcohol, cocaine and nicotine. Peripherally acting compounds may be valuable drugs for the treatment of gastroesophageal reflux disease and irritable bowel syndrome.What the reader will gain: An overview on 19 patents in this field, of the different scaffolds for positive modulators of GABAB receptors and of the major players in the field.Take home message: The search for subtype selective benzodiazepine receptor ligands has proved to be extremely difficult. Positive modulators of GABAB receptors may provide novel anxiolytic drugs faster.

GABAA receptor signaling in caudal periaqueductal gray regulates maternal aggression and maternal care in mice

Maternal aggression (maternal defense) is exhibited by lactating females towards intruders and contributes to the protection of offspring. Enhancement of Gamma-Aminobutyric acid (GABA)A receptor signaling by benzodiazepines elevates maternal aggression, and we previously found indirect evidence (via c-Fos immunohistochemistry) that caudal periaqueductal gray (cPAG) and lateral septum (LS) could be sites where benzodiazepines increase aggression. We recently found that GABAA receptor signaling in LS modulates maternal aggression, and in this study, we tested the hypothesis that GABAA receptor signaling in cPAG also regulates this behavior. Site-directed injections to cPAG were made in lactating mice using the GABAA receptor antagonist, bicuculline (3–9ng) or the GABAA receptor positive modulator, chlordiazepoxide (CDP), a benzodiazepine (2.5–20μg). Maternal aggression, other maternal behaviors, and anxiety-like measures (using the light–dark box) were then examined. GABAA receptor positive modulator did not increase aggression, which could have resulted from a ceiling effect. However, 8ng and 9ng of bicuculline in cPAG significantly decreased maternal aggression without altering other maternal behaviors or light–dark box performance, suggesting some GABAA receptor signaling in cPAG is required for full maternal aggression expression. Additionally, 7ng of bicuculline significantly increased licking/grooming of pups, and decreased the number of transitions between the light and dark compartments of the light–dark box without affecting aggression. Given these results indicating that antagonizing GABAA receptor in cPAG dose-dependently promotes offspring grooming behavior while impairing aggression, it is possible that the cPAG represents a key site for decision making (aggression versus other behaviors) in the lactating female.

Ganaxolone suppression of behavioral and electrographic seizures in the mouse amygdala kindling model

Ganaxolone (3alpha-hydroxy-3beta-methyl-5alpha-pregnan-20-one), a synthetic analog of the endogenous neurosteroid allopregnanolone and a positive allosteric modulator of GABAA receptors, may represent a new treatment approach for epilepsy. Here we demonstrate that pretreatment with ganaxolone (1.25-20 mg/kg, s.c.) causes a dose-dependent suppression of behavioral and electrographic seizures in fully amygdala-kindled female mice, with nearly complete seizure protection at the highest dose tested. The ED50 for suppression of behavioral seizures was 6.6 mg/kg. The seizure suppression produced by ganaxolone was comparable to that of clonazepam (ED50, 0.1 mg/kg, s.c.). To the extent that amygdala kindling represents a model of mesial temporal lobe epilepsy, this study supports the utility of ganaxolone in the treatment of patients with temporal lobe seizures.

Novel positive allosteric modulators of GABAA receptors: Do subtle differences in activity at α1 plus α5 versus α2 plus α3 subunits account for dissimilarities in behavioral effects in rats?

Over the last years, genetic studies have greatly improved our knowledge on the receptor subtypes mediating various pharmacological effects of positive allosteric modulators at GABA A receptors. This stimulated the development of new benzodiazepine (BZ)-like ligands, especially those inactive/low-active at GABA A receptors containing the α 1 subunit, with the aim of generating more selective drugs. Hereby, the affinity and efficacy of four recently synthesized BZ site ligands: SH-053-2′N, SH-053-S-CH3-2′F, SH-053-R-CH3-2′F and JY-XHe-053 were assessed. They were also studied in behavioral tests of spontaneous locomotor activity, elevated plus maze, and water maze in rats, which are considered predictive of, respectively, the sedative, anxiolytic, and amnesic influence of BZs. The novel ligands had moderately low to low affinity and mild to partial agonistic efficacy at GABA A receptors containing the α 1 subunit, with variable, but more pronounced efficacy at other BZ-sensitive binding sites. While presumably α 1 receptor-mediated sedative effects of GABA A modulation were not fully eliminated with any of the ligands tested, only SH-053-2′N and SH-053-S-CH3-2′F, both dosed at 30 mg/kg, exerted anxiolytic effects. The lack of clear anxiolytic-like activity of JY-XHe-053, despite its efficacy at α 2- and α 3-GABA A receptors, may have been partly connected with its preferential affinity at α 5-GABA A receptors coupled with weak agonist activity at α 1-containing subtypes. The memory impairment in water-maze experiments, generally reported with BZ site agonists, was completely circumvented with all four ligands. The results suggest that a substantial amount of activity at α 1 GABA A receptors is needed for affecting spatial learning and memory impairments, while much weaker activity at α 1- and α 5-GABA A receptors is sufficient for eliciting sedation.

Novel positive allosteric modulators of GABAA receptors: Do subtle differences in activity at α1 plus α5 versus α2 plus α3 subunits account for dissimilarities in behavioral effects in rats?

Novel positive allosteric modulators of GABAA receptors: Do subtle differences in activity at α1 plus α5 versus α2 plus α3 subunits account for dissimilarities in behavioral effects in rats?

Anticonvulsant doses of ganaxolone do not compromise motor performance in immature rats

Neuroactive steroids that function as positive modulators of GABA-A receptors are potential anticonvulsant drugs. We previously demonstrated that ganaxolone is effective against pentetrazol-induced motor seizures in immature rats. In the present study, we examined the effects of ganaxolone in another model, cortical epileptic afterdischarges (ADs). The possible side effects of ganaxolone were studied in rats 12, 18, and 25 days of age following the implantation of epidural electrodes. Low-frequency stimulation of the sensorimotor cortical area elicited ADs characterized by a spike-and-wave rhythm and clonic seizures. Ganaxolone (5, 10, 20, or 40 mg/kg) was administered intraperitoneally after the first AD and stimulation was repeated five more times. The highest dose of ganaxolone (40 mg/kg) suppressed progressive prolongation of ADs in 25-day-old rats and postponed it in 12-day-old rats. No significant effect was observed in 18-day-old animals. Movements during stimulation and clonic seizures accompanying ADs were not affected by ganaxolone. Ganaxolone at doses of 20 and 40 mg/kg had no significant effect on motor function, such as surface righting, negative geotaxis, wire mesh ascending, and bar holding. After administration of 40 mg/kg ganaxolone to 18- and 25-day-old rats, spontaneous locomotion in the open field tended to decrease. Doses of ganaxolone with a moderate anticonvulsant effect in the present model did not seriously compromise motor performance.

5α‐reductase mRNA is increased in the rostral ventrolateral medulla (RVLM) of term pregnant rats

In term pregnant rats, baseline GABAergic inhibition of the RVLM is increased, while baseline GABAergic inhibition of the hypothalamic paraventricular nucleus (PVN) is decreased. The major metabolite of progesterone, 3α‐hydroxy‐dihydroprogesterone (3α‐OH‐DHP), is a potent endogenous positive modulator of GABAA receptors and CNS levels of this neurosteroid are increased in pregnancy. Local regulation of the enzymes responsible for formation of 3α‐OH‐DHP, 5α‐reductase (5α‐RDA) and 3α‐hydroxysteroid dehydrogenase (3α‐HSD), is one factor which determines brain region specific actions of 3α‐OH‐DHP. Expression of mRNA for 5α‐RDA and 3α‐HSD (reference gene = MAPK) was determined in tissue punches of the RVLM and PVN from nonpregnant (NP) and term pregnant (P) rats. In the PVN, pregnancy did not change relative expression of 5α‐RDA [NP(7)= 1.0 ±0.23; P(7)= 0.64± 0.15] or 3α‐HSD [NP(5)= 1.0 ±0.38; P(6)= 0.84± 0.23]. In the RVLM, expression of 3α‐HSD [NP(5)= 1.0 ±0.54; P(6)= 0.67± 0.44] was not different between NP and P rats. However, there was a strong trend for increased expression of 5α‐RDA in P (5) [2.44± 0.89] compared to NP(5) [1.0 ±0.28] rats. Although preliminary, these data suggest that increased expression of 5α‐RDA in the RVLM of pregnant rats, could result in increased formation of the major metabolite of progesterone and potential positive modulation of GABAA receptors. (NIH HL 36245)

GABA Regulates the Rat Hypothalamic‐Pituitary‐Adrenocortical Axis via Different GABA‐A Receptor α‐Subtypes

The control of the corticotropin-releasing hormone (CRH) neurons in the hypothalamic paraventricular nucleus (PVN) is balanced by excitatory and inhibitory inputs. The GABA-A receptor, which is a major target for the inhibitory control, is composed of five subunits. The presence of an alpha(1)-, alpha(2)-, alpha(3)-, or alpha(5)-subunit in the GABA-A receptor protein complex is necessary for benzodiazepines to exert their potentiating effect on the receptor. We postulate that the effect of nonselective benzodiazepines on the hypothalamo-pituitary adrenocortical (HPA) axis is critically dependent on the composition of the GABA-A receptor subunits through which they act. We show here that positive modulators of alpha(1)-subtype containing GABA-A receptors with zolpidem (10 mg/kg) increase HPA activity in terms of increase in plasma corticosterone and induction of Fos in the PVN, whereas activation of non-alpha(1)-subtype GABA-A receptors using L-818,417 (10 mg/kg) likely inhibits the activity. We also show that the alpha(2)-subunit gene is highly expressed in the PVN, but its expression is not affected by chronic mild stress. These results show that the stimulatory effect on HPA activity after positive modulation of GABA-A receptors composed of alpha(1)-subunit(s) affects a selective afferent system than the PVN, which is distinct from another afferent system(s) activated by non alpha(1)-containing GABA-A receptors.

P.4.a.012 Adjunctive levetiracetam for treatment refractory post-traumatic stress disorder (PTSD)

There is a remarkable array of new chemical entities in the current antiepileptic drug (AED) development pipeline. In some cases, the compounds were synthesized in an attempt improve upon the activity of marketed AEDs. In other cases, the discovery of antiepileptic potential was largely serendipitous. Entry into the pipeline begins with the demonstration of activity in one or more animal screening models. Results from testing in a panel of such models provide a basis to differentiate agents and may offer clues as to the mechanism. Target activity may then be defined through cell-based studies, often years after the initial identification of activity. Some pipeline compounds are believed to act through conventional targets, whereas others are structurally novel and may act by novel mechanisms. Follow-on agents include the levetiracetam analogs brivaracetam and seletracetam that act as SV2A-ligands; the valproate-like agents valrocemide, valnoctamide, propylisopropyl acetamide, and isovaleramide; the felbamate analog flurofelbamate, a dicarbamate, and the unrelated carbamate RWJ-333369; the oxcarbazepine analog licarbazepine, which probably acts as a use-dependent sodium channel blockers, and its prodrug acetate BIA 2-093; various selective partial benzodiazepine receptor agonists, including ELB139, which is a positive allosteric modulator of α3-containing GABAA receptors. A variety of AEDs that may act through novel targets are also in clinical development: lacosamide, a functionalized amino acid; talampanel, a 2,3-benzodiazepine selective noncompetitive AMPA receptor antagonist; NS1209, a competitive AMPA receptor antagonist; ganaxolone, a neuroactive steroid that acts as a positive modulator of GABAA receptors; retigabine, a KCNQ potassium channel opener with activity as a GABAA receptor positive modulator; the benzanilide KCNQ potassium channel opener ICA-27243 that is more selective than retigabine; and rufinamide, a triazole of unknown mechanism.

GABA homeostasis contributes to the developmental programming of anxiety-related behavior

During development, when inhibitory and excitatory synapses are formed and refined, homeostatic mechanisms act to adjust inhibitory input in order to maintain neural activity within a normal range. As the brain matures, synaptogenesis slows and a relatively stable level of inhibition is achieved. Deficits in inhibitory neurotransmission are associated with increased anxiety-related behavior and drugs that potentiate GABA function, the major inhibitory neurotransmitter in the brain, are effective anxiolytics. These observations raise the possibility that transient perturbations in the activity of neural circuits during development might induce compensatory changes in inhibition that could persist into adulthood and contribute to changes in anxiety-related behavior. To test this hypothesis, we treated mice continuously during the major period of forebrain synaptogenesis (P14–28) with the GABA-A receptor positive modulator diazepam and assessed anxiety-related behavior in adulthood. Control experiments confirmed anxiolytic effects of the drug following one day of treatment and the development of tolerance following two weeks of treatment. When tested in adulthood, one month after the end of treatment, diazepam-treated mice exhibited significantly increased behavioral inhibition in the open-field, elevated-plus maze, and novel object behavioral paradigms. Levels of benzodiazepine binding sites in amygdala and frontal cortex were specifically decreased in diazepam-treated mice demonstrating that homeostatic adjustments in GABA function persist into adulthood. Our results show that increased GABAergic activity can affect the developmental programming of anxiety-related behavior.

Reduced GABA neurotransmission underlies hyperalgesia induced by repeated forced swimming stress

We determined if cutaneous hyperalgesia and pain-induced c-Fos overexpression in the spinal cord produced by repeated forced swimming (FS) stress in the rat were related to changes in GABA neurotransmission by studying spinal release of GABA and the effect of positive modulation of GABA-A receptors with diazepam. Male rats were daily submitted to 10–20 min of either forced swimming or sham swimming (SS) for 3 consecutive days. Two days later, spinal GABA release was estimated by in vivo microdialysis. In other set of rats, either diazepam (2 mg/kg, i.p.) or saline was administered 1 h before either SS or FS and inflammatory nociception was assessed with the formalin test; it was followed by removal of lumbar spinal cords for c-Fos immunocytochemistry. Basal and pain-evoked release of GABA in the spinal cord was lower in FS rats than in SS rats. In contrast, pain scores during formalin test late phase and pain-induced c-Fos expression in laminae I–VI of ipsilateral dorsal horn were significantly higher in FS rats than in SS rats. In FS rats, diazepam did not have effect on GABA release but reduced pain scores and overexpression of c-Fos whereas flumazenil (0.1 mg/kg, i.p.), an antagonist of the benzodiazepine binding site, reversed these effects. When diazepam was given only 1 h before the formalin test, it slightly but significantly reduced pain scores during late phase in FS rats but not in SS rats. In conclusion, stress-induced reduction in GABA-A receptor activation is involved in the development of FS stress-induced hyperalgesia.

Étifoxine et récepteurs GAB A

The targets of a number of anxiolytic compounds are the GABA A receptors, complexes composed of 5 protein subunits surrounding a central channel permeable to Cl − ions. These subunits are produced from a set of 16 genes (α 1–6, β 1–3, γ 1–3, δ, ɛ, π and θ) and assembled to produce various receptors composed of two α subunits, two β subunits and one other subunit, usually of the γ type. Benzodiazepines, the most widely prescribed anxiolytics at present, bind to an allosteric site localised at the α-γ 2 interface that potentiates the inhibitory effects of GABA. Despite their efficacy as anxiolytics, classic benzodiazepines exhibit adverse effects, which has prompted the search for alternative compounds. One of these, etifoxine, is chemically unrelated to the benzodiazepines but also potentiates GABAergic transmission without causing drug dependence or impaired cognitive function. The existence of interactions between etifoxine and the GABA A receptors is demonstrated by both binding and electrophysiological studies. Etifoxine inhibits the binding of [ 35S] TBPS to GABA A receptors without inhibiting the binding of [ 3H]-muscimol or [ 3H]-flunitrazepam, indicating that the etifoxine binding site differs from that of GABA and of the benzodiazepines, respectively. Conversely, etifoxine slightly increases the binding of these two radioligands and other positive modulators of GABA Areceptors, but by an original mechanism (increase in the number of binding sites without any increased affinity for these sites). The currents induced by GABA in neuronal cultures are potentiated by etifoxine, even in the presence of a benzodiazepine site antagonist, thus confirming that etifoxine does not bind to this site. Other studies of recombinant receptors show that the potentiation persists in the absence of α or γ subunits, which suggests that the β subunit plays a major role in the effects of etifoxine. This is confirmed by the fact that the degree of potentiation depends on the nature of the β subunit co-expressed with α and β ; the potentiation obtained is in fact markedly more pronounced for receptors containing a β 2 or β 3 than for those containing a β 1 subunit. This preference for β 2 and β 3 distinguishes etifoxine from other positive GABA A receptor modulators such as neurosteroids or benzodiazepines, the modulatory effects of which are relatively unaffected by the nature of the β subunit. The specificity of the mechanism of action of etifoxine on GABA A receptors might contribute to its original therapeutic properties.

The neurosteroid system: an emerging therapeutic target for hepatic encephalopathy

Both acute and chronic liver failure induce cerebral complications known as hepatic encephalopathy (HE) and thought to selectively involve brain astrocytes. Alterations of astrocytic-neuronal cross talk occurs affecting brain function. In acute liver failure, astrocyte undergo swelling, which results in increased intracranial pressure and may lead to brain herniation. In chronic liver failure, Alzheimer-type II astrocytosis is a characteristic change. Neurosteroids (NS) synthesized in the brain mainly by astrocytes independent of peripheral steroidal sources (adrenals and gonads) are suggested to play a role in HE. NS bind and modulate different types of membrane receptors. Effects on the gamma amino butyric acid (GABA)-A receptor complex are the most extensively studied. For example, the NS tetrahydroprogesterone (allopregnanolone), and tetrahydrodeoxycorticosterone (THDOC) are potent positive allosteric modulators of GABA-A receptors. As a consequence of modulation of these receptors, NS are well-known to modulate inhibitory neurotransmission in the central nervous system. Some NS bind to intracellular receptors, and in this way may also regulate gene expression. In HE, it has been well documented that neurotransmission and gene expression alterations occur during the progression of the disease. This review summarizes findings of relevance for the involvement of NS in human and experimental HE.

Changes in relative potency among positive GABAA receptor modulators upon discontinuation of chronic benzodiazepine treatment in rhesus monkeys

Benzodiazepine treatment can result in dependence as evidenced by signs of withdrawal upon discontinuation of use. Positive GABA(A) receptor modulators were examined for their capacity to attenuate flumazenil-like discriminative stimulus effects (i.e., withdrawal) that emerge upon discontinuation of chronic benzodiazepine treatment. Rhesus monkeys receiving chronic diazepam (5.6 mg(-1) kg(-1) 24 h(-1) p.o.) discriminated flumazenil (0.1 mg/kg s.c.) from vehicle. Upon discontinuation of diazepam treatment, responding switched from the vehicle to the flumazenil lever, although at different times among monkeys. The shorter-acting benzodiazepine lorazepam (3.2 mg(-1) kg(-1) 8 h(-1)) was substituted for diazepam and, 11 h after lorazepam, monkeys consistently responded on the flumazenil lever. Flumazenil-lever responding after acute lorazepam deprivation was attenuated not only by benzodiazepines (lorazepam and midazolam) but also by positive GABA(A) receptor modulators acting at neuroactive steroid (pregnanolone and alfaxalone) and barbiturate sites (pentobarbital). Deprivation-induced responding on the flumazenil lever was not attenuated by low efficacy positive GABA(A) modulators (bretazenil and L-838,417) or non-GABA(A) receptor ligands (ketamine and cocaine). Neuroactive steroids were relatively more potent than other positive GABA(A) receptor modulators in attenuating deprivation-induced flumazenil-lever responding, as compared to their relative potency in monkeys discriminating midazolam and otherwise not receiving benzodiazepine treatment. These results suggest that positive GABA(A) receptor modulators acting at different sites attenuate withdrawal induced by discontinuation of benzodiazepine treatment, consistent with previous studies suggesting that the same compounds attenuate flumazenil-precipitated withdrawal. Differences in the relative potency of positive modulators as a function of acute versus chronic benzodiazepine treatment suggest that neuroactive steroids, in particular, are especially potent in benzodiazepine-dependent animals.

Serum Profiles of Free and Conjugated Neuroactive Pregnanolone Isomers in Nonpregnant Women of Fertile Age

Pregnanolone isomers (PI) with a hydroxy group in the 3alpha-position are neuroinhibitors operating via positive modulation of GABA(A) receptors. The 3beta-PI and sulfates of PI and pregnenolone exert the opposite effect. In addition to the brain's in situ synthesis, some circulating steroids can penetrate the blood-brain barrier. To assess the physiological impact of peripheral endogenous neuroactive pregnanolone isomers and their polar conjugates in women, serum allopregnanolone (P3alpha5alpha), isopregnanolone (P3beta5alpha), pregnanolone (P3alpha5beta), epipregnanolone (P3beta5beta), pregnenolone, estradiol (including their polar conjugates), and additional steroids were measured in 16 women in the follicular and luteal phases of the menstrual cycle using gas chromatography/mass spectrometry and RIA for the analysis. Linear models and Spearman's correlations were used for data evaluation. The levels of conjugated PI were from one to almost three orders of magnitude higher in comparison with the free steroids. The results indicate that a substantial proportion of the progesterone is metabolized in the sequence progesterone-->5beta-dihydroprogesterone-->P3alpha5beta-->conjugated P3alpha5beta. The sulfation of PI and particularly of P3alpha5beta moderates the levels of free PI and restrains estradiol biosynthesis via progesterone degradation. PI including the conjugates reflected changing progesterone formation during the menstrual cycle. In the follicular phase, the positive correlation with conjugated pregnenolone, the independence of progesterone, and the negative age relationships of PI indicate their adrenal origin. The dependence on progesterone and the independence of conjugated pregnenolone suggest a gonadal source of PI in the luteal phase. The neuroactivating PI prevailed over neuroinhibiting PI.

Impact of ε and θ subunits on pharmacological properties of α3β1 GABAA receptors expressed in Xenopus oocytes

Backgroundγ-Aminobutyric acid type A (GABAA) receptors provide the main inhibitory control in the brain. Their heterogeneity may make it possible to precisely target drug effects to selected neuronal populations. In situ hybridization using rat brain sections has revealed a unique expression of GABAA receptor ε and θ subunit transcripts in the locus coeruleus, where they are accompanied at least by α3, α2, β1 and β3 subunits. Here, we studied the pharmacology of the human α3β1, α3β1ε, α3β1θ and α3β1εθ receptor subtypes expressed in Xenopus oocytes and compared them with the γ2 subunit-containing receptors.ResultsThe GABA sensitivites and effects of several positive modulators of GABAA receptors were studied in the absence and the presence of EC25 GABA using the two-electrode voltage-clamp method. We found 100-fold differences in GABA sensitivity between the receptors, α3β1ε subtype being the most sensitive and α3β1γ2 the least sensitive. Also gaboxadol dose-response curves followed the same sensitivity rank order, with EC50 values being 72 and 411 μM for α3β1ε and α3β1γ2 subtypes, respectively. In the presence of EC25 GABA, introduction of the ε subunit to the receptor complex resulted in diminished modulatory effects by etomidate, propofol, pregnanolone and flurazepam, but not by pentobarbital. Furthermore, the α3β1ε subtype displayed picrotoxin-sensitive spontaneous activity. The θ subunit-containing receptors were efficiently potentiated by the anesthetic etomidate, suggesting that θ subunit could bring the properties of β2 or β3 subunits to the receptor complex.ConclusionThe ε and θ subunits bring additional features to α3β1 GABAA receptors. These receptor subtypes may constitute as novel drug targets in selected brain regions, e.g., in the brainstem locus coeruleus nuclei.

Glutamate- and GABA-based CNS therapeutics

Glutamate- and GABA-releasing neurons form the basis for neurotransmission in the mammalian central nervous system (CNS). The co-ordination of these excitatory and inhibitory systems, together with intrinsic voltage-gated ion channels and G-protein-coupled receptor modulation, provides the diverse neuronal firing patterns, network activity and synaptic plasticity that are required for the complexity of CNS function. Virtually all of the known molecular components of the gamma-aminobutyric acid (GABA) and glutamate neurotransmitter systems have been considered as potential therapeutic targets. Positive allosteric modulators of GABAA receptors, such as the benzodiazepines, have found wide clinical use, and the N-methyl-D-aspartate receptor antagonists ketamine and memantine have therapeutic utility. In these fundamental neurotransmitter systems, drugs that provide allosteric modulation of ligand-gated ion channels or G-protein-coupled receptors, or seek to selectively target receptor subtypes, appear to hold the greatest promise for the desired balance of efficacy and tolerability. This might also be achieved through targeting transporter subtypes. A large number of compounds based on these strategies are currently in clinical trials for diseases that span a wide range of CNS disorders.