Search

Background and purpose In addition to binding to the classical high-affinity extracellular benzodiazepine binding site of the GABAA receptor, some benzodiazepines occupy transmembrane inter-subunit anesthetic sites that bind etomidate (β+ /α- sites) or the barbiturate derivative R-mTFD-MPAB (α+ /β- and γ+ /β- sites). We aimed to define the functional effects of these interactions on GABAA receptor activity and animal behavior. Experimental approach With flumazenil blocking classical high-affinity extracellular benzodiazepine site effects, modulation of GABA-activated currents by diazepam, midazolam, and flurazepam was measured electrophysiologically in wildtype and M2-15' mutant α1 β3 γ2L GABAA receptors. Zebrafish locomotive activity was also assessed in the presence of each benzodiazepine plus flumazenil. Key results In the presence of flumazenil, micromolar concentrations of diazepam and midazolam both potentiated and inhibited wildtype GABAA receptor currents. β3 N265M (M2-15´ in the β+ /α- sites) and α1 S270I (M2-15´ in the α+ /β- site) mutations reduced or abolished potentiation by these drugs. In contrast, the γ2 S280W mutation (M2-15´ in the γ+ /β- site) abolished inhibition. Flurazepam plus flumazenil only inhibited wildtype receptor currents, an effect unaltered by M2-15' mutations. In the presence of flumazenil, zebrafish locomotion was (1) enhanced by diazepam at concentrations up to 30 μM and suppressed at 100 μM; (2) suppressed by midazolam; and (3) enhanced by flurazepam. Conclusions and implications Benzodiazepine binding to transmembrane anesthetic binding sites of the GABAA receptor can produce positive or negative modulation manifesting as decreases or increases in locomotion, respectively. Selectivity for these sites may contribute to the distinct GABAA receptor and behavioral actions of different benzodiazepines, particularly at high (i.e. anesthetic) concentrations.

BACKGROUND AND PURPOSE Enhancement of GABAergic function is the primary mechanism of important therapeutic agents such as benzodiazepines, barbiturates, neurosteroids, general anaesthetics and some anticonvulsants. Despite their chemical diversity, many studies have postulated that these agents may bind at a common or overlapping binding site, or share an activation domain. Similarly, we found that flavan-3-ol esters act as positive modulators of GABAA receptors, and noted that this action resembled the in vitro profile of general anaesthetics. In this study we further investigated the interactions between these agents. EXPERIMENTAL APPROACH Using two-electrode voltage clamp electrophysiological recordings on receptors of known subunit composition expressed in Xenopus oocytes, we evaluated positive modulation by etomidate, loreclezole, diazepam, thiopentone, 5α-pregnan-3α-ol-20-one (THP) and the flavan-3-ol ester 2S,3R-trans 3-acetoxy-4′-methoxyflavan (Fa131) on wild-type and mutated GABAA receptors. KEY RESULTS The newly identified flavan, 2S,3S-cis 3-acetoxy-3′,4′-dimethoxyflavan (Fa173), antagonized the potentiating actions of Fa131, etomidate and loreclezole at α1β2 and α1β2γ2L GABAA receptors. Furthermore, Fa173 blocked the potentiation of GABA responses by high, but not low, concentrations of diazepam, but did not block the potentiation induced by propofol, the neurosteroid THP or the barbiturate thiopental. Mutational studies on ‘anaesthetic-influencing’ residues showed that, compared with wild-type GABAA receptors, α1M236Wβ2γ2L and α1β2N265Sγ2L receptors are resistant to potentiation by etomidate, loreclezole and Fa131. CONCLUSIONS AND IMPLICATIONS Fa173 is a selective antagonist that can be used for allosteric modulation of GABAA receptors. Flavan-3-ol derivatives are potential ligands for etomidate/loreclezole-related binding sites at GABAA receptors and the low-affinity effects of diazepam are mediated via the same site.

BACKGROUND AND PURPOSE Pre-synaptic neurotransmitter release is largely dependent on Ca2+ entry through P/Q-type (CaV2.1) voltage-gated Ca2+ channels (PQCCs) at most mammalian, central, fast synapses. Barbiturates are clinical depressants and inhibit pre-synaptic Ca2+ entry. PQCC barbiturate pharmacology is generally unclear, specifically in man. The pharmacology of the barbiturate pentobarbital (PB) in human recombinant α1A PQCCs has been characterized.

We studied amobarbital's effects on membrane properties and currents, and electrically evoked inhibitory postsynaptic currents (IPSCs) mediated by γ-aminobutyric acid (GABA) in rat thalamic slices. Using concentration–response relationships, we compared amobarbital's effects in nociceptive nuclei and non-nociceptive nucleus reticularis thalami (nRT). Amobarbital decreased input resistance by activating GABAA receptors. Amobarbital produced a larger decrease in ventrobasal than nRT neurons. Amobarbital depressed burst and tonic firing. Depression of burst firing was more effective, particularly in ventrobasal and intralaminar neurons. Depression was reversed by GABAA antagonists, and surmountable by increasing current injection, implicating a receptor-mediated shunt mechanism. Amobarbital did not affect the tetrodotoxin-isolated low threshold Ca2+ spike during GABAA blockade. Amobarbital reduced excitability without altering outward leak, or hyperpolarisation-activated inward currents. Amobarbital increased mean conductance and burst duration of single GABAA channels. Consistent with this, amobarbital increased amplitude and decay time of IPSCs with distinct EC50s, implicating actions at two GABAA receptor sites. Activation of GABAA receptors by low concentrations, fast IPSC amplitude modulation, and failure to affect intrinsic currents distinguished amobarbital's mechanism of action from previously characterised barbiturates. The selective actions of amobarbital on GABAA receptor may have relevance in explaining anaesthetic and analgesic uses. British Journal of Pharmacology (2004) 143, 485–494. doi:10.1038/sj.bjp.0705974

Investigation into the modulatory effects of chlormethiazole at human recombinant γ-aminobutyric acid A receptor (GABAA) and N-methyl-D-aspartate (NMDA) receptors was undertaken to gain insight into its mechanism of action and determine if the drug exhibited any subtype-selective activity. Despite a structural similarity to the β-subunit-selective compound loreclezole, chlormethiazole did not show any difference in maximum efficacy and only a slight difference in EC50 in its potentiating action at α1β1γ2 and α1β2γ2 GABAA receptor subtypes with preference for α1β1γ2. Similar to the previously reported subtype-dependent activity of pentobarbital, chlormethiazole elicited a significantly greater degree of maximum potentiation on receptors lacking a γ2 subunit, and also those receptors containing an α4 or α6 subunit. This also demonstrates that chlormethiazole does not act via the benzodiazepine binding site. Unlike pentobarbital and propofol, chlormethiazole elicited only a slight direct GABAA receptor activation at concentrations up to 1 mM. In addition, the drug did not potentiate anaesthetic-mediated currents elicited by pentobarbital or propofol, suggesting that chlormethiazole may be acting via an anaesthetic binding site. Chlormethiazole produced weak nonselective inhibition of human NMDA NR1a+NR2A and NR1a+NR2B receptors. IC50's were approximately 500 μM that likely exceed the therapeutic dose range for chlormethiazole, indicating that the primary mechanism of the compounds in vivo activity is via GABAA receptors. British Journal of Pharmacology (2003) 140, 1045–1050. doi:10.1038/sj.bjp.0705540

As pharmacokinetic differences between the thiopentone enantiomers seem insufficient to explain the ∼2 fold greater potency for CNS effects of (−)-S- over (+)-R-thiopentone, this study was performed to determine any enantioselectivity of thiopentone at the GABAA receptor, the primary receptor for barbiturate hypnotic effects. Two electrode voltage clamp recording was performed on Xenopus laevis oocytes expressing human GABAA receptor subtype α1β2γ2 to determine relative differences in potentiation of the GABA response by rac-, (+)-R- and (−)-S-thiopentone, and rac-pentobarbitone. Changes in the cellular environment pH and in GABA concentrations were also evaluated. With 3 μM GABA, the EC50 values were (−)-S-thiopentone (mean 26.0±s.e.mean 3.2 μM, n=9 cells) >rac-thiopentone (35.9±4.2 μM, n=6, P=0.1) >(+)-R-thiopentone (52.5±5.0 μM, n=8, P rac-pentobarbitone (97.0±11.2 μM, n=11, P100 μM (−)-S- and R-thiopentone. This direct response was abolished by intracellular oocyte injection of 1,2-bis(2-aminophenoxy)ethane-N,N,N1,N1-tetraacetic acid (BAPTA), a Ca2+ chelating agent. With BAPTA, the EC50 values were (−)-S-thiopentone (20.6±3.2 μM, n=8)

Many structurally diverse general anaesthetics enhance inhibitory neurotransmission in the central nervous system by interacting with the GABAA receptor. By contrast, GABA receptors composed of the ρ1 subunit are anaesthetic-insensitive. Here, we demonstrate that both δ-hexachlorocyclohexane (δ-HCH; 1–100 μM), a positive allosteric modulator of the GABAA receptor, and the anaesthetic pentobarbitone (10–600 μM) have no effect on GABA-evoked currents mediated by wild-type ρ1 recombinant receptors (expressed in Xenopus laevis oocytes). By contrast, these agents produce up to a 10 fold enhancement of GABA responses transduced by a ρ1 receptor in which a transmembrane located isoleucine residue is replaced by serine. However, not all general anaesthetics were similarly influenced by this mutation, because propofol and 5β-pregnan-3α-ol-20-one (5β3α) remained ineffective. These data are discussed in relation to the specificity of general anaesthetic action. British Journal of Pharmacology (1999) 127, 601–604; doi:10.1038/sj.bjp.0702611

1. Patch-clamp recording techniques were used to examine the effects of barbiturates upon the ATP-K+ channel, and voltage-activated channels present in the plasma membrane of CRI-G1 insulin-secreting cells. 2. Thiopentone inhibited ATP-K+ channel activity when applied to cell-attached patches or the intracellular or extracellular surface of cell-free patches. Secobarbitone and pentobarbitone were also effective inhibitors of ATP-K+ channels in cell-free patches, whereas phenobarbitone was ineffective. 3. The diabetogenic agent, alloxan, which is structurally related to the barbiturates also produced an inhibition of ATP-K+ channel activity in outside-out patches. 4. Whole-cell ATP-K+ currents were used to quantify the effects of the barbiturates: concentration-inhibition curves for thiopentone, secobarbitone and pentobarbitone resulted in IC50 values of 62, 250 and 360 microM respectively. Phenobarbitone at a concentration of 1 mM was virtually ineffective. 5. Calculation of the apparent membrane concentrations for these drugs indicate that for a given degree of ATP-K+ channel inhibition a similar concentration of each barbiturate is present in the membrane. This suggests that hydrophobicity plays a primary role in their mechanism of action. The pH-dependence and additive nature of barbiturate block also indicates a membrane site of action. 6. Thiopentone, (100 microM) was also found to inhibit differentially voltage-activated whole-cell currents. The relative potency of thiopentone at this concentration was 0.64, 0.38 and 0.12 for inhibiting Ca2+, K+ and Na+ currents respectively when compared with its ability to inhibit the ATP-K+ channel.

1. The structural requirements of an allosteric barbiturate binding site on acetylcholine receptor-rich membranes isolated from Torpedo electroplaques have been characterized by the ability of fourteen barbiturates to displace [14C]-amobarbitone binding. 2. The barbiturates could be grouped into two classes with ten barbiturates producing a strong inhibition of [14C]-amobarbitone binding (class one) and with four exerting minimal effects (class two). 3. Eight of the ten class one barbiturates displaced essentially all of the [14C]-amobarbitone from its binding site, while, at their respective aqueous solubility limits, two of these barbiturates (thiopentone and dimethylbutylbarbitone (DMBB) inhibited [14C]-amobarbitone binding by nearly 80%. The apparent inhibition constants (KI) for the class one barbiturates ranged from 13 microM for amobarbitone to 2.8 mM for barbitone with the other eight agents lying in the range 100-600 microM, and having the rank order pentobarbitone approximately secobarbitone greater than thiopentone greater than DMBB greater than butabarbitone approximately phenobarbitone greater than aprobarbitone greater than allylbarbitone. 4. By contrast, the class two barbiturates had minimal effects even at close to saturating concentrations. [14C]-amobarbitone binding was reduced slightly (less than 30%) by hexobarbitone, mephobarbitone and methohexitone and was enhanced slightly (less than 20%) by metharbitone. 5. All of the class two, but none of the class one barbiturates, were N-methylated.

1 Injection of pentobarbitone sodium into a lateral cerebral ventricle of rats produced a loss of righting reflex. The duration of anaesthesia was dose-dependent. 2 The optimum dose of pentobarbitone to allow study of the factors affecting the sleeping time was considered to be 650 μg injected in 25 μl water. 3 In a study of the effect of age and sex on the sleeping time, the youngest rats used (88 g body weight) were found to be the most sensitive to barbiturate. Female rats were more sensitive than male animals. 4 The duration of anaesthesia was not affected by induction or inhibition of hepatic drug-metabolizing enzyme activity. 5 Prior administration (acute) of central nervous system depressant drugs shortened the latent period and prolonged the duration of sleep. Prior administration of stimulant drugs antagonized the effect of pentobarbitone. 6 Animals withdrawn following chronic administration of a number of drugs, barbitone, barbitone/bemegride mixture, Mandrax (methaqualone: diphenhydramine; 10: 1), chlordiazepoxide, nitrazepam, chlorpromazine or ethanol, exhibited a significant tolerance to intracerebroventricularly administered pentobarbitone. 7 Withdrawal of amphetamine, morphine, methyprylon or diazepam did not result in tolerance to intracerebroventricularly administered pentobarbitone. 8 Chronic administration of all drugs except amphetamine and morphine induced a tolerance to intraperitoneally administered hexobarbitone (100 mg/kg). 9 The usefulness of sleeping time determination following intracerebroventricular administration of pentobarbitone as an assessment of central nervous system excitability is discussed. It is concluded that this method gives a valid indication of the sensitivity of the central nervous system to barbiturate and of the level of excitability in general. The method is particularly applicable in situations where the activity of hepatic drug-metabolizing enzyme activity may be altered.

1 A study was made in cats anaesthetized with barbiturate or alpha-chloralose, of the excitation of dorsal horn neurones by impulses in unmyelinated (C) primary afferent fibres of the tibial nerve. 2 Block of conduction in the first lumbar segment by cooling produced large increases in the number of action potentials evoked by C fibre afferents in neurones of more caudal segments. 3 Morphine (0.3 to 1.0 mg/kg) reduced the excitation of neurones by C fibre afferents and also reduced the increase produced by blocking conduction in the spinal cord. Naloxone (0.1 to 0.3 mg/kg) reversed these effects of morphine. 4 This decrease in descending inhibition supports findings in the decerebrate cat but gives no support to the hypothesis that an important component of morphine analgesia is an activation of descending inhibitory pathways.

1. The sensitivity of the central nervous system to barbiturate was determined in rats during the chronic administration of barbitone sodium and after its withdrawal.2. The brain barbiturate concentration determined on awakening from a hypnotic dose administered intraperitoneally was found to increase throughout the period of barbitone administration.3. A similar gradual development of central nervous system tolerance was indicated by measuring the duration of anaesthesia following an intraventricular injection of pentobarbitone.4. The change in sensitivity of the brain which occurred during the period of barbitone administration was not demonstrable from the measurement of sleeping time following intraperitoneal injection of barbitone or pentobarbitone.5. After withdrawal, the sensitivity of the brain to barbiturate gradually returned to normal.6. It was concluded that the hypersensitivity to pentobarbitone, but not to barbitone, which develops after withdrawal of barbitone sodium is due to a decreased drug-metabolizing capacity.

Summary 1 Female rats, 3 weeks after pretreatment with 200 or 400 (mg/kg)/day barbitone for 2 or 30 days, exhibited a prolonged sleeping time and a reduced awakening barbiturate brain level when challenged with either barbitone or pentobarbitone. After 3 additional weeks, the latter responses had returned, or were returning to, control values. 2 Barbitone pretreatment schedules had no residual effect upon in vitro hepatic pentobarbitone-metabolizing activity measured 3 or 6 weeks later, except in one instance, when hepatic enzyme activity was significantly enhanced 3 weeks after 30 daily doses of 200 mg/kg barbitone. In this case, however, an enhanced barbiturate sleeping time, together with a reduced awakening barbiturate brain level, were observed. 3 It is concluded that barbitone administered intraperitoneally in doses of 200 to 400 (mg/kg)/day for 2 or 30 days induces a non-hepatogenic intolerance to barbiturates, related to an increased sensitivity of the central nervous system to these drugs. This central intolerance is seen 3 weeks, but not 6 weeks, after pretreatment. Furthermore, this central intolerance has been observed to co-exist with an hepatic tolerance, a situation which could result in a reduced LD50 coupled with an increase in ED50.

1 In rats and hamsters under barbiturate anaesthesia, laser radiation to venules about 50 micrometer in diameter in mesoappendix and cheek pouch respectively caused the formation of platelet thrombi which occluded the vessels in about 9 min. 2 This occlusion time was significantly prolonged by the intravenous injection of N-acetyl neuraminic acid (NANA) but not by D-glucuronic acid or beta-methoxyneuraminic acid, in doses which had no effect on blood pH or on the condition of the animals. 3 The results confirm the anti-thrombotic effect of NANA previously demonstrated with another technique.

1 The effects of pentobarbitone (PB) and other sedative/hypnotic drugs have been examined in relation to γ-aminobutyric acid (GABA) in vitro on the superfused isolated superior cervical ganglion of the rat and in vivo on single units in the brain stem of the anaesthetized rat. 2 PB, and other barbiturates, depolarized the ganglion in a dose-dependent manner (threshold concentration 100–300 μm, cf. GABA depolarization threshold 1 μm). The depolarization was reduced in the presence of the selective GABA antagonist (+)-bicuculline methochloride (Bic). Other non-barbiturate sedatives e.g. chlordiazepoxide, amitriptyline, promethazine at concentrations up to 2mM produced no depolarization. 3 PB, tested at concentrations up to 80 μm, produced variable effects on the dose-response curve to GABA. On most occasions a slight potentiation occurred in responses to low concentrations of GABA (below 10 μm) coupled with a depression in the responses to concentrations of GABA greater than 10 μm. 4 Superfusion with PB in the presence of Bic reversed the depression in the response to GABA produced by Bic. This reversal phenomenon occurred at concentrations of PB too low to depolarize the ganglion and was dependent not only on the concentration of PB but also on that of Bic. 5 The reversal potency within an homologous series of barbiturates increased with the size of the alkyl substituent (R2) at C5 on the barbiturate ring. The most potent occurred when the substi-tuent contained 5 carbon atoms (pentobarbitone and amylobarbitone); above this, activity decreased. 6 PB reversed the effects of the other GABA antagonists, tetramethylenedisulphotetramine and isopropyl bicyclophosphate and also the non-selective antagonism produced by strychnine. A concomitant reduction by strychnine of responses to the cholinomimetic, carbachol, was not reversed by PB. 7 Non-barbiturate sedative/hypnotics also reversed the GABA antagonism produced by Bic. The benzodiazepines were effective at lower concentrations than PB (chlordiazepoxide threshold concentration 0.5 μm, cf. PB 5 μm), however, they only produced a partial reversal even at concentrations much higher than the maximally effective concentration of PB. 8 The Bic reversal effect of chlordiazepoxide (and other benzodiazepines) lasted many hours after removal from the superfusion solution. By contrast the effect of PB lasted only 15–30min after its removal. 9 Chlordiazepoxide (30 μm) applied in the absence of Bic did not affect the response to GABA but did reduce the depression produced by the subsequent application of Bic even though the chlordiazepoxide had been removed 40min earlier. 10 In the rat brain stem in vivo PB, applied iontophoretically in amounts which neither decreased the spontaneous neuronal firing rate nor affected the response to GABA or glycine, reversed the GABA antagonism induced by iontophoretic application of Bic (in all 23 neurones tested). PB also reversed the antagonism produced by strychnine of responses to glycine although this was less readily observed (5 out of 14 neurones tested). 11 Iontophoretic application of other barbiturates and chlordiazepoxide also reversed the effect of Bic. Chlordiazepoxide only produced a partial reversal, as in the isolated ganglion, and no reversal could be demonstrated with flurazepam. 12 Intravenous administration of thiopentone (1.3mg/kg) pentobarbitone (0.4–5.5 mg/kg) hexobar-bitone (0.4-0.8 mg/kg) and clonazepam (0.1-0.2 mg/kg) also reversed the effect of iontophoretically applied Bic. The reversal by clonazepam was of much longer duration than that produced by the barbiturates. 13 It is suggested that the reversal exhibited by PB and the other hypnotics may be explained by assuming that the amino acids and their antagonists bind to the membrane at separate sites. If the reversal agent has particular affinity only for the antagonist binding site then it may displace the antagonist without affecting the receptor.

1. The interactions of chlormethiazole with gamma-aminobutyric acid (GABA) synthesis and release, and with ligand binding to sites associated with the GABAA-receptor complex and the GABAB-receptor have been studied in the rat. The GABAA-receptor was studied using [3H]-muscimol, [3H]-flunitrazepam was used to label the benzodiazepine modulatory site, and [35S]-butyl-bicyclophosphorothionate ([35S]-TBPS) to label the chloride channel. 2. Chlormethiazole had no effect on GABA synthesis in the cortex, hippocampus and striatum or on GABA release from cortical slices in vitro. Chlormethiazole did not displace [3H]-baclofen binding to the GABAB-receptor. 3. Chlormethiazole (IC50 = 140 microM) and pentobarbitone (IC50 = 95 microM) both inhibited [35S]-TBPS binding by increasing the rate of [35S]-TBPS dissociation. In addition, chlormethiazole caused an apparent decrease in the affinity of [35S]-TBPS binding. 4. Chlormethiazole enhanced the binding of [3H]-muscimol but had no effect on [3H]-flunitrazepam binding. In contrast, the sedative barbiturate pentobarbitone enhanced both [3H]-muscimol and [3H]-flunitrazepam binding. 5. It is concluded that the sedative and anticonvulsant effects of chlormethiazole are probably mediated through an action at the GABAA-receptor. However, chlormethiazole does not interact with the GABAA-receptor complex in an identical manner to the sedative barbiturate pentobarbitone.

Summary 1 Drug-metabolizing capacity was assessed in 8 barbiturate-dependent and in 3 Mandrax-dependent patients using, as indices, plasma antipyrine half-life and in some cases urinary output of 6β-hydroxycortisol. For comparison, antipyrine half-life was also measured in volunteers before and after a period of taking hypnotic doses of these agents. 2 Both indices indicated a very high drug-metabolizing capacity in the dependent subjects on admission, the antipyrine half-life value in the barbiturate patients being the shortest reported to date for any drug-exposed group. The urinary output of 6β-hydroxycortisol was approximately three times that in a control population. 3 This induction of drug-metabolizing capacity presumably contributed to the marked drug tolerance observed in the dependent patients. 4 It appeared that drug-metabolizing capacity eventually returned to normal levels after withdrawal of the barbiturate. 5 It is concluded that the abnormal drug-metabolizing capacity of dependent patients must be taken into account in assessing dose requirements for other drugs.