gamma-aminobutyric acid(B) receptors: first of the functional metabotropic heterodimers

Activation of the metabotropic gamma-aminobutyric acid(B) (GABA(B)) receptor increases K(+) conductance and decreases Ca(2+) channel activity in neuronal membranes. Studies with a number of new GABA(B) receptor agonists and antagonists reveal that in addition to their muscle relaxant effects, agonists display analgesic activity and reduce the craving for cocaine. With regard to GABA(B) receptor antagonists, preclinical data suggest they improve cognitive performance and possess antidepressant and antiepileptic potential. With a high-affinity GABA(B) antagonist, the structural properties of the receptor were characterized through expression cloning. Moreover, it has been found that expression of a fully functional GABA(B) receptor requires coupling between two separate and distinct gene products: GABA(B) R1 and GABA(B) R2. Besides being the first example of a functional heterodiameric metabotropic receptor, the components and molecular configuration of the GABA(B) receptor suggest novel mechanisms for producing pharmacologically distinct subtypes of G protein-coupled receptors.     

SL651498: an anxioselective compound with functional selectivity for alpha2- and alpha3-containing gamma-aminobutyric acid(A) (GABA(A)) receptors.

SL651498 [6-fluoro-9-methyl-2-phenyl-4-(pyrrolidin-1-yl-carbonyl)-2,9-dihydro-1H-pyrido[3,4-b]indol-1-one] is a novel pyridoindole derivative that displays high affinity for rat native GABA(A) receptors containing alpha(1) (K(i) = 6.8 nM) and alpha2 (K(i) = 12.3 nM) subunits, and weaker affinity for alpha5-containing GABA(A) receptors (K(i) = 117 nM). Studies on recombinant rat GABA(A) receptors confirm these data (K(i), alpha1beta2gamma2 = 17, alpha2beta2gamma2 = 73, alpha5beta3gamma2 = 215 nM) and indicate intermediate affinity for the alpha3beta2gamma2 subtype (K(i) = 80 nM). SL651498 behaves as a full agonist at recombinant rat GABA(A) receptors containing alpha2 and alpha3 subunits and as a partial agonist at recombinant GABA(A) receptors expressing alpha1 and alpha5 subunits. SL651498 elicited anxiolytic-like activity similar to that of diazepam [minimal effective dose (MED): 1-10 mg/kg, i.p.] in three conflict models, in the elevated plus-maze, the light/dark test, and the defense test battery in rats and mice. Results from activity tests and electroencephalogram analysis indicated that SL651498 induced muscle weakness, ataxia, or sedation at doses much higher than those producing anxiolytic-like activity (MED > or = 30 mg/kg, i.p.). Repeated treatment for 10 days with SL651498 (30 mg/kg, i.p., b.i.d.) in mice was not associated with the development of tolerance to its anticonvulsant effects or physical dependence. Furthermore, SL651498 was much less active than diazepam in potentiating the depressant effects of ethanol in mice. The "anxioselective" profile of SL651498 points to a major role for GABA(A) alpha2 subtype in regulating anxiety and suggests that selectively targeting GABA(A) receptor subtypes can lead to drugs with increased clinical specificity.     

Contribution of metabotropic GABA(B) receptors to neuronal network construction

In the 1980s, Bowery and colleagues discovered the presence of a novel, bicuculline-resistant and baclofen-sensitive type of GABA receptor on peripheral nerve terminals, the GABA_B receptor. Since this pioneering work, GABA_B receptors have been identified in the Central Nervous System (CNS), where they provide an important inhibitory control of postsynaptic excitability and presynaptic transmitter release. GABA_B receptors have been implicated in a number of important processes in the adult brain such as the regulation of synaptic plasticity and modulation of rhythmic activity. As a result of these studies, several potential therapeutic applications of GABA_B receptor ligands have been identified. Recent advances have further shown that GABA_B receptors play more than a classical inhibitory role in adult neurotransmission, and can in fact function as an important developmental signal early in life. Here we summarize current knowledge on the contribution of GABA_B receptors to the construction and function of developing neuronal networks.     

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.     

Interleukin-1beta inhibits gamma-aminobutyric acid type A (GABA(A)) receptor current in cultured hippocampal neurons

Interleukin-1beta (IL-1beta), a polypeptide immune mediator, is induced within the central nervous system in response to a variety of pathological stimuli, including systemic infection, hypoxia, brain trauma, and seizure. IL-1beta action on the gamma-aminobutyric acid type A (GABA(A)) inhibitory neurotransmitter receptor was investigated in whole cell patch-clamped cultured hippocampal neurons. Application of IL-1beta at concentrations encountered in pathophysiological conditions (1-10 ng/ml; 59-590 pM) irreversibly decreased the peak magnitude of current elicited by 30 microM GABA. Current inhibition was IL-1beta concentration- and time-dependent and was prevented by a specific IL-1beta type I receptor antagonist. No significant changes in current kinetics or reversal potential were observed. The IL-1beta depression of GABA current was inhibited by high concentrations of nonspecific kinase inhibitors staurosporine (500 nM) and 1-(5-isoquinolinyl-sulfonyl)-2-methylpiperazine (H-7; 50 microM), but not by a protein kinase C selective inhibitor calphostin C (5 microM). We conclude that IL-1beta inhibits GABA(A) receptor function in hippocampal neurons by the involvement of an unidentified kinase. This blockade of the GABA(A) inhibitory neurotransmitter receptor may underlie the central nervous system hyperexcitability seen in many pathophysiological conditions.     

Ethanol enhances gamma-aminobutyric acid responses in a subpopulation of nucleus accumbens neurons: role of metabotropic glutamate receptors

The nucleus accumbens (NAcc) may be a key area in the rewarding effects of abused drugs. We previously showed that low ethanol concentrations decreased both N-methyl-D-aspartate (NMDA)-induced and kainate-induced currents in NAcc core neurons. To explore the effects of ethanol on gamma-aminobutyric acid (GABA) responses in NAcc, we used intracellular voltage-clamp recordings and locally applied GABA in a slice preparation containing the NAcc. Ethanol (11-200 mM) had no effect on resting membrane properties, but 11, 22, 44, 100, and 200 mM ethanol increased GABA currents in 17, 33, 45, 50, and 22% of cells, respectively. Superfusion of low glutamate concentrations that had no direct effect on membrane properties enhanced ethanol potentiation of GABA currents in more than half the NAcc cells. Neither alpha-amino-3-hydroxy-5-methylisoxazole-4-propionic acid/kainate receptor nor NMDA receptor antagonists affected the percentage of cells showing ethanol enhancement of GABA responses or the degree of ethanol enhancement of GABA currents in NAcc neurons. However, in ethanol-sensitive cells, the metabotropic receptor antagonist alpha-methyl-4-carboxyphenylglycine (MCPG) blocked the ethanol enhancement of GABA currents. In addition, the metabotropic receptor agonist trans-1-aminocyclopentane-1,3-dicarboxylic acid enhanced GABA responses in 50% of cells tested, an effect blocked by MCPG. These data suggest that NAcc core neurons possess both ethanol-sensitive and -insensitive GABA receptors and that glutamate can mimic and enhance the ethanol potentiation of GABA currents in many of these neurons. Furthermore, the ethanol potentiation of GABA currents may involve metabotropic glutamate receptors, perhaps via a phosphorylation mechanism that regulates ethanol sensitivity of GABA receptors in some NAcc neurons.     

Kindled seizures result in decreased responsiveness of benzodiazepine receptors to gamma-aminobutyric acid (GABA)

Repeated kindled seizures result in increased numbers of benzodiazepine receptors in fascia dentata membranes. Our previous work localized these receptors to a discrete neuronal population, the dentate granule cells. The present investigation characterizes the regulation of this binding by NaCl, pentobarbital and gamma-aminobutyric acid (GABA). [3H]Flunitrazepam binding in membranes from kindled rats exceeded that from controls by 24%. NaCl increased this binding in membranes prepared from control and kindled animals by roughly equivalent amounts (18 and 14%, respectively). No significant differences were found in the effects of pentobarbital on benzodiazepine binding between kindled and control groups. In contrast, addition of GABA increased benzodiazepine binding by a significantly smaller percentage in the kindled (116 +/- 5%) compared to the control (138 +/- 11%) group. We propose that this altered molecular response to GABA is intimately related to the enhanced GABA-mediated inhibition of granule cells present after kindled seizures.     

Ethanol inhibition of N-methyl-D-aspartate responses involves presynaptic gamma-aminobutyric acid(B) receptors

Ethanol alters N-methyl-D-aspartate (NMDA) and gamma-aminobutyric acid subtype A (GABA(A)) receptor-mediated neurotransmission. We have previously demonstrated that GABA(B) receptor blockade uncovers ethanol enhancement of GABA(A) responses in the hippocampus. Therefore, we evaluated in vivo and in vitro the role of GABA(B) receptors in ethanol-induced inhibition of neuronal activity as well as NMDA responses in the hippocampus, ventral tegmental area (VTA), and nucleus accumbens (NAcc), three brain areas with known sensitivity to low doses of ethanol. In vivo, in situ microelectrophoretic application of ethanol enhanced inhibition of VTA GABA neuron firing rate by the GABA(B) agonist baclofen and reduced inhibition of VTA GABA firing rate by the GABA(A) agonist muscimol. The GABA(B) antagonist CGP35348 blocked baclofen- and ethanol-induced, but not muscimol-induced, reduction of NMDA-activated firing of hippocampal hilar mossy cells, hilar interneurons, and VTA GABA neurons, as well as ethanol inhibition of NMDA receptor-sensitive, amygdala-driven NAcc neurons. We performed in vitro studies in NAcc slices to evaluate the mechanism of GABA(B) receptor-mediated ethanol inhibition of NMDA neurotransmission. In the presence of the non-NMDA receptor antagonist 6-cyano-7-nitroquinoxaline-2,3-dione and the GABA(A) receptor antagonist bicuculline, superfusion of the GABA(B) antagonist CGP55845 blocked ethanol (66 mM) inhibition of evoked NMDA receptor-mediated excitatory postsynaptic potentials. However, CGP55845 did not significantly affect ethanol inhibition of NMDA currents produced by pressure application of NMDA or non-NMDA glutamatergic excitatory postsynaptic potentials evoked in the presence of the bicuculline and the NMDA antagonist DL-2-amino-5-phosphonovalerate. Taken together, these findings suggest that the sensitivity of NMDA receptor-mediated neurotransmission to ethanol is regulated by GABA(B) receptors, possibly at presynaptic sites.     

Blockade of forebrain gamma-aminobutyric acid (GABA) receptors and reflex activation of the cardiac vagus in anesthetized cats

gamma-Aminobutyric acid (GABA) antagonists have been shown to produce sympathetically mediated increases in blood pressure and heart rate when restricted to the forebrain cerebral ventricles of anesthetized cats. This study explored the possibility that similar administration of these agents might produce reciprocal effects on reflex cardiac vagal excitability. Drugs were infused into and restricted to the forebrain ventricles of cats anesthetized with chloralose and urethane. Arterial pressure and heart rate were continuously monitored and reproducible reflex vagal bradycardia was periodically elicited by bolus i.v. injections of phenylephrine. In early experiments in intact cats and in later studies in spinal transected animals, i.c.v. administration of the GABA antagonist bicuculline methiodide (1-32 micrograms) suppressed phenylephrine-induced reflex bradycardia in a dose-related fashion. When tested in spinal transected cats, i.c.v. picrotoxin, another GABA antagonist, mimicked this effect of bicuculline methiodide. Intraventricular muscimol (10 micrograms), a GABA agonist, had no effect in untreated cats but reversed the effects of bicuculline methiodide and picrotoxin. These data point to tonic GABAergic inhibition in the periventricular forebrain which suppresses the activity of a descending vagal inhibitory mechanism.     

Effects of gamma-aminobutyric acid (GABA) receptor agonists on the neurotoxicity and anticonvulsant activity of barbiturates in mice

The effects of gamma-aminobutyric acid (GABA) receptor agonists [4,5,6,7-tetrahydroisoxazolo(5,4-c)pyridin-3-ol THIP]; [progabide and baclofen] on the minimal neurotoxicity and anticonvulsant activity of pentobarbital and phenobarbital in mice were investigated. When either progabide, THIP or baclofen were administered with pentobarbital, the components of this combination interacted additively by the rotorod test. Combinations of pentobarbital and progabide or phenobarbital and progabide interacted additively when subjected to the pentylenetetrazol (PTZ) minimal threshold seizure (clonic) test. THIP, even at toxic doses, did not alter the anti-PTZ activity of either pentobarbital or phenobarbital. In contrast, baclofen at toxic doses potentiated the anti-PTZ activity of pentobarbital and phenobarbital. Combinations of progabide and pentobarbital or progabide and phenobarbital interacted additively by the maximal electroshock seizure (MES) test. THIP, even when given in toxic doses, had no effect on the anti-MES activity of pentobarbital and phenobarbital. However, baclofen at nontoxic doses potentiated the anti-MES activity of phenobarbital but not that of pentobarbital. These results suggest that 1) in vitro interactions between barbiturates and GABAa receptor agonists may not be the same in vivo, 2) GABAa receptors may play a minor role in the minimal neurotoxicity and anticonvulsant activity of barbiturates and 3) inhibition of glutamate-induced excitation by baclofen may be an important action in potentiating the anti-MES activity of phenobarbital.     

Modeling the binding and function of metabotropic glutamate receptors

A mathematical model for the binding and function of metabotropic glutamate receptors was developed, with the aim to gain new insights into the functioning of these complex receptors. These receptors are homodimers, and each subunit is composed of a ligand binding [Venus flytrap (VFT)] domain and a heptahelical domain (HD) responsible for G-protein activation. Our mechanistic model integrates all structural information available so far: the various states of the VFT dimer (open-open, closed-open, and closed-closed), as well as the fact that a single HD is active at a time. To provide the model with parameters with biological meaning, two published experimental studies were reanalyzed. The first one reports a negative cooperativity in agonist binding (J Biol Chem 279:35526-35534, 2004), whereas the other indicates a positive cooperativity in agonist-mediated response (Nat Struct Mol Biol 11:706-713, 2004). The former study allowed us to explain the mechanistic features associated with VFT recognition by agonists and antagonists integrating a negative allosteric interaction for agonist binding. The second study helped us to quantitatively describe the functional dynamics of transduction of the VFT occupation into functional response, confirming a putative positive cooperativity at the level of receptor coupling efficacy. This model will help both to better understand the functioning of these receptors and to characterize the mechanism of action of various types of allosteric modulators. Moreover, this model may be of general utility for oligomeric systems in which the ligand binding and effector domains correspond to distinct structural domains.     

Benzodiazepine receptors are coupled to a subpopulation of gamma-aminobutyric acid (GABA) receptors: evidence from a quantitative autoradiographic study

The colocalization and interaction of gamma-aminobutyric acid (GABA) and benzodiazepine (BZ) receptors in the rat brain were characterized using standardized, quantitative, light microscopic autoradiographic methods. In serial sections, striking differences were observed in the distribution of high affinity GABA and BZ receptors in areas such as the cerebral cortex, globus pallidus, thalamus, hypothalamus and cerebellar cortex. However, in a semiquantitative visual examination of more than 200 brain regions, added exogenous GABA increased BZ binding in all regions. In a quantitative analysis of 19 regions, exogenous GABA uniformly stimulated [3H]flunitrazepam binding, the effect being proportional to the regional density of BZ receptors. No relationship was seen between the magnitude of the stimulation and the distribution of high affinity GABA receptors. In the mounted tissue sections, BZ binding appeared influenced by endogenous GABA since it was reduced by preincubation or by the addition of bicuculline. Taken together, these data suggest that most or all BZ receptors can be influenced by GABA and are coupled to a type of GABA receptor. However, the BZ-linked GABA receptor could represent either a subpopulation of GABA binding sites or a distinct receptor not labeled under the conditions used in these and other experiments.     

General anesthetic potencies of a series of propofol analogs correlate with potency for potentiation of gamma-aminobutyric acid (GABA) current at the GABA(A) receptor but not with lipid solubility

A series of 27 analogs of the general anesthetic propofol (2,6-diisopropylphenol) were examined for general anesthetic activity in Xenopus laevis tadpoles and for the ability to produce enhancement of submaximal GABA responses and/or direct activation at recombinant GABA(A) receptors. Fourteen of the propofol analogs produced loss of righting reflex in the tadpoles, whereas 13 were inactive as anesthetics. The same pattern of activity was noted with the actions of the compounds at the GABA(A) alpha(1)beta(2)gamma(2s) receptor. The potencies of the analogs as general anesthetics in tadpoles correlated better with potentiation of GABA responses than direct activation at the GABA(A) alpha(1)beta(2)gamma(2s) receptor. The calculated octanol/water partition coefficients for the analogs did not explain the lack of activity exhibited by the 13 nonanesthetic analogs, although this physicochemical parameter did correlate modestly with in vivo anesthetic potency. The actions of one nonanesthetic analog, 2,6-di-tert-butylphenol, were examined in detail. 2,6-Di-tert-butylphenol was inactive at GABA(A) receptors, did not function as an anesthetic in the tadpoles, and did not antagonize any of the actions of propofol at GABA(A) receptors or in tadpoles. A key influence on the potency of propofol analogs appears to be the size and shape of the alkyl groups at positions 2 and 6 of the aromatic ring relative to the substituent at position 1. These data suggest steric constraints for the binding site for propofol on the GABA(A) receptor.     

Biphasic effects of baclofen on phrenic motoneurons: possible involvement of two types of gamma-aminobutyric acid (GABA) receptors

Intravenous injections of baclofen have two general dose-dependent effects on phrenic motoneurons in anesthetized cats. Small doses (0.5-1.5 mg/kg) increase the frequency of action potentials recorded from single motoneurons and from the phrenic nerve, whereas large doses (2-10 mg/kg) reduce or abolish action potentials. The increase in frequency produced by small doses is accompanied by membrane depolarization and, in most experiments, by increased input resistance. Large doses hyperpolarize phrenic motoneurons and produce greater increases in input resistance. Extracellular recording during microelectrophoretic application of baclofen reveals only one effect, depression of cell firing, at all effective current strengths. The low dose stimulatory effect of i.v. baclofen is attributed to disinhibition, whereas the depression by large doses is attributed to disfacilitation. During incomplete inhibition by baclofen, CO2 administration further depresses phrenic nerve activity. Bicuculline (100-600 micrograms/kg i.v.) and picrotoxin (900 micrograms/kg i.v.) restore firing depressed by baclofen, whereas strychnine (80-1280 micrograms/kg) does not. 3-Aminopropanesulfonic acid (5-75 mg/kg i.v.) an agonist at gamma-aminobutyric acid-A receptor sites, depresses phrenic nerve activity. It is suggested that the low dose stimulatory effects are related to actions at gamma-aminobutyric acid-B receptors, whereas the high dose depressant effects are related, at least in part, to activation of gamma-aminobutyric acid-A receptors.     

Role of gamma-aminobutyric acid (GABA)A and GABAB receptors in paraventricular nucleus in control of sympathetic vasomotor tone in hypertension

The paraventricular nucleus (PVN) of the hypothalamus is involved in tonic regulation of sympathetic outflow. Impaired GABAergic control of PVN neurons may contribute to the elevated sympathetic drive in hypertension. In this study, we examined the function of GABA(A) and GABA(B) receptors in the PVN in control of sympathetic nerve activity and arterial blood pressure (ABP) in normotensive and hypertensive rats. Lumbar sympathetic activity (LSNA) and ABP were recorded from anesthetized spontaneously hypertensive rats (SHRs), Sprague-Dawley (SD) rats, and Wistar-Kyoto (WKY) rats. Bilateral microinjection of bicuculline (0.01-0.15 nmol), a GABA(A) receptor antagonist, into the PVN increased LSNA and ABP in normotensive WKY and SD rats in a dose-dependent manner. This response was significantly attenuated in SHRs. Furthermore, the decrease in LSNA and ABP induced by a GABA(A) receptor agonist, muscimol (0.05-1.5 nmol), in the PVN was significantly less in SHRs than in normotensive controls. In contrast, microinjection of the GABA(B) receptor agonist baclofen (0.3-4.5 nmol) into the PVN decreased LSNA and ABP in SHRs. However, in WKY and SD rats, baclofen only decreased LSNA and ABP at the highest dose tested. In addition, blockade of GABA(B) receptors in the PVN with CGP52432 (3-[[(3,4-dichlorophenyl)methyl]amino]propyl]diethoxymethyl)phosphinic acid) (0.15-3.0 nmol) dose-dependently increased LSNA and ABP in SHRs but not in normotensive controls. Collectively, this study provides new evidence that GABA(A) receptor function is attenuated, whereas the function of GABA(B) receptors is enhanced, in the PVN of SHRs.     

Reductions in blood pressure, heart rate and renal sympathetic nervous discharge after imidazole-4-acetic acid: mediation through central gamma-aminobutyric acid (GABA) receptor stimulation

Intracerebroventricular administration of imidazole-4-acetic acid (IAA) (0.3--10 microgram/kg) significantly reduced mean arterial pressure, heart rate and renal sympathetic nerve discharge in chloralose-anesthetized cats. In contrast, i.v. administration of IAA did not lower arterial pressure or heart rate. This would suggest that the hypotensive and bradycardic effects of IAA were centrally mediated. The cardiovascular effects of IAA were attenuated by the central administration of bicuculline methiodide (15 microgram/kg), a gamma-aminobutyric acid receptor antagonist. Reflex bradycardia evoked during the pressor response to norepinephrine (0.03--1.0 microgram/kg i.v.) was reversed to tachycardia after only the central administration of IAA. The vasoconstrictor response evoked during a 20-sec period of bilateral occlusion of carotid arteries was not altered by IAA. These results suggest that the cardiovascular effects of IAA are mediated through the activation of central gamma-aminobutyric acid receptors, to inhibit sympathetic vasomotor outflow.     

Reduction of gamma-aminobutyric acid (GABA)-mediated transmission by a convulsant benzodiazepine.

In contrast to other benzodiazepine, Ro 5-3663 produces convulsions in mice. The CD50 of 7.0 mg/kg i.v. falls between that of picrotoxin and pentylenetetrazol. An electrophysiological study was made of the effects of this convulsant benzodiazepine on spinal reflexes and on ganglionic depolarization evoked by gamma-aminobutyric acid (GABA). In the unanesthetized spinal cat, Ro 5-3663 (15 mg/kg i.v.)depressed the dorsal root potentials and abolished the dorsal root reflexes evoked by muscle and cutaneous afferent inputs. The monosynaptic reflex was typically depressed, whereas polysynaptic potentials were enhanced. Diazepam reversed the depression of the dorsal root reflex and dorsal root potential produced by the convulsant benzodiazepine and reduced the enhancement of the polysynaptic potential. Presynaptic inhibition was attenuated by the convulsant, whereas strychnine-sensitive postsynaptic inhibition was slightly potentiated. Ro 5-3663 reduced the amplitude and duration of the GABA-evoked negative surface potential recorded from the superior cervical ganglion. The results indicate that the convulsant benzodiazepine acts in an opposite manner to the depressant benzodiazepines and support the hypothesis that these two types of compounds act through a modulation of GABAergic mechanisms.     

Enantioselectivity of pregnanolone-induced gamma-aminobutyric acid(A) receptor modulation and anesthesia

This study reports the actions of enantiomer pairs of anesthetic steroids 3alpha5alphaP/ent-3alpha5alphaP and 3alpha5betaP/ent-3alpha5betaP as modulators of gamma-aminobutyric acid (GABA)(A) receptors and as anesthetics. The enantiomers of structurally related 17-carbonitrile analogs also are examined. These studies were aimed at 1) determining whether the steroid recognition site could distinguish between molecules differing in shape, but not other physical properties (enantioselectivity); 2) providing further insight into the structure-activity relationships of anesthetic steroids; and 3) determining whether modulation of GABA(A) receptor function correlates with anesthetic potency for anesthetic steroid enantiomers. Stereoselective actions of the compounds were evaluated in four different bioassays: 1) noncompetitive displacement of [(35)S]t-butylbicyclophosphorothionate from the picrotoxin site of GABA(A) receptors present in rat brain membrane preparations; 2) modulation of GABA currents in cultured rat hippocampal neurons; 3) loss of righting reflex in tadpoles; and 4) loss of righting reflex in mice. The data indicate that 5alpha-reduced steroids, but not 5beta-reduced steroids, show a high degree of enantioselectivity/enantiospecificity in their actions as modulators of GABA(A) receptors and as anesthetics. For all compounds studied, the effects on GABA(A) receptor function closely tracked with anesthetic effects. These data show that the anesthetic steroid recognition site is capable of distinguishing enantiomers, suggesting a protein-binding site of specific dimensions and shape. The results are compatible either with a structural model of the binding site that can accommodate 3alpha5alphaP, 3alpha5betaP, and ent-3alpha5betaP, but not ent-3alpha5alphaP, or with two different binding sites for steroid anesthetics.     

Comparison of norepinephrine- and benzodiazepine-induced augmentation of Purkinje cell responses to gamma-aminobutyric acid (GABA)

The hypothesis tested in the present study was that the benzodiazepines (i.e., flurazepam) and norepinephrine (NE) share a common mechanism to facilitate cerebellar Purkinje neuron responsiveness to iontophoretically applied gamma-aminobutyric acid (GABA). Extracellular activity was recorded from Purkinje neurons in halothane-anesthetized rats from each of the following groups: 1) naive, 2) acute or chronic flurazepam treated, 3) chronic desmethylimipramine treated and 4) injected with 6-hydroxydopamine. Single unit responses to pulsatile (10 sec duration at 45-sec intervals) iontophoretic administration of GABA were examined before, during and after NE or flurazepam microiontophoresis in all treatment groups. Drug response histograms were generated and used to quantitate NE and flurazepam effects on spontaneous activity and GABA-induced inhibitory responses. Doses of GABA sufficient to produce depression of Purkinje cell activity in naive rats (4-40 nA) suppressed firing rate in all Purkinje cells tested in drug-treated animals. In contrast to its consistent GABA facilitating action in naive controls, iontophoretically applied flurazepam was ineffective in augmenting GABA-induced suppression of Purkinje cell discharge in acute and chronic flurazepam-treated animals. Although GABA facilitation by NE was unaffected by acute systemic administration of a benzodiazepine, chronic treatment with flurazepam produced a subsensitivity to the noradrenergic GABA facilitating effects. Within 48 hr of withdrawal from chronic benzodiazepine treatment, both NE and flurazepam again enhanced GABA-induced suppression of Purkinje cell discharge routinely. Chronic desmethylimipramine treatment as well as iontophoresis of the blocking agents sotalol and fluphenazine which have been shown previously to block or reduce NE-mediated enhancement of GABA actions were ineffective in altering the facilitating effect of flurazepam on GABA. Likewise, 6-hydroxydopamine pretreatment had no effect on GABA augmentation by flurazepam. Thus, although flurazepam appears to act independently from the noradrenergic receptor system in augmenting GABA-induced depression of Purkinje cell discharge, a reversible subsensitivity to the GABA facilitating effects of both flurazepam and NE can be produced by chronic treatment with this benzodiazepine. On the basis of this "cross-subsensitivity" to NE and flurazepam actions, it seems reasonable to suggest that these two agents might enhance GABA inhibitory actions by a common biophysical mechanism subsequent to noradrenergic receptor activation.