Classic Benzodiazepines Modulate the Open-Close Equilibrium in α1β2γ2L γ-Aminobutyric Acid Type A Receptors

Background: Classic benzodiazepine agonists induce their clinical effects by binding to a site on [gamma]-aminobutyric acid type A (GABAA) receptors and enhancing receptor activity. There are conflicting data regarding whether the benzodiazepine site is allosterically coupled to [gamma]-aminobutyric acid binding versus the channel open-close (gating) equilibrium. The authors tested the hypothesis that benzodiazepine site ligands modulate [alpha]1[beta]2[gamma]2L GABAA receptor gating both in the absence of orthosteric agonists and when the orthosteric sites are occupied.

Methods: GABAA receptors were recombinantly expressed in Xenopus oocytes and studied using two-microelectrode voltage clamp electrophysiology. To test gating effects in the absence of orthosteric agonist, the authors used spontaneously active GABAA receptors containing a leucine-to-threonine mutation at residue 264 on the [alpha]1 subunit. To examine effects on gating when orthosteric sites were fully occupied, they activated wild-type receptors with high concentrations of a partial agonist, piperidine-4-sulfonic acid.

Results: In the absence of orthosteric agonists, the channel activity of [alpha]1L264T[beta]2[gamma]2L receptors was increased by diazepam and midazolam and reduced by the inverse benzodiazepine agonist FG7142. Flumazenil displayed very weak agonism and blocked midazolam from further activating mutant channels. In wild-type receptors activated with saturating concentrations of piperidine-4-sulfonic acid, midazolam increased maximal efficacy.     

CNS 7056: a novel ultra-short-acting Benzodiazepine

BACKGROUND: A new benzodiazepine derivative, CNS 7056, has been developed to permit a superior sedative profile to current agents, i.e., more predictable fast onset, short duration of sedative action, and rapid recovery profile. This goal has been achieved by rendering the compound susceptible to metabolism via esterases. The authors now report on the profile of CNS 7056 in vitro and in vivo. METHODS: The affinity of CNS 7056 and its carboxylic acid metabolite, CNS 7054, for benzodiazepine receptors and their selectivity profiles were evaluated using radioligand binding. The activity of CNS 7056 and midazolam at subtypes (alpha1beta2gamma2, alpha2beta2gamma2, alpha3beta2gamma2, alpha5beta2gamma2) of the gamma-aminobutyric acid type A (GABAA) receptor was evaluated using the whole cell patch clamp technique. The activity of CNS 7056 at brain benzodiazepine receptors in vivo was measured in rats using extracellular electrophysiology in the substantia nigra pars reticulata. The sedative profile was measured in rodents using the loss of righting reflex test. RESULTS: CNS 7056 bound to brain benzodiazepine sites with high affinity. The carboxylic acid metabolite, CNS 7054, showed around 300 times lower affinity. CNS 7056 and CNS 7054 (10 mum) showed no affinity for a range of other receptors. CNS 7056 enhanced GABA currents in cells stably transfected with subtypes of the GABAA receptor. CNS 7056, like midazolam and other classic benzodiazepines, did not show clear selectivity between subtypes of the GABAA receptor. CNS 7056 (intravenous) caused a dose-dependent inhibition of substantia nigra pars reticulata neuronal firing and recovery to baseline firing rates was reached rapidly. CNS 7056 (intravenous) induced loss of the righting reflex in rodents. The duration of loss of righting reflex was short (< 10 min) and was inhibited by pretreatment with flumazenil. CONCLUSIONS: CNS 7065 is a high-affinity and selective ligand for the benzodiazepine site on the GABAA receptor. CNS 7056 does not show selectivity between GABAA receptor subtypes. CNS 7056 is a potent sedative in rodents with a short duration of action. Inhibition of substantia nigra pars reticulata firing and the inhibition of the effects of CNS 7056 by flumazenil show that it acts at the brain benzodiazepine receptor.     

Effects of Isoflurane on γ-Aminobutyric Acid Type A Receptors Activated by Full and Partial Agonists

Background: Volatile anesthetics prolong inhibitory postsynaptic potentials in central neurons via an allosteric action on the [gamma]-aminobutyric acid type A (GABAA) receptor, an effect that may underlie the hypnotic actions of these agents. Inhaled anesthetics such as isoflurane act to enhance responses to submaximal concentrations of GABA, but it is not clear whether their effect is mediated by an increase in the binding of the agonist or by changes in receptor gating behavior. To address this question, the authors studied the effects of isoflurane on a mutant GABAA receptor with a gating defect that decreases receptor sensitivity by lowering agonist efficacy. They then compared the effects of clinically relevant concentrations of isoflurane on the actions of GABA and piperidine-4-sulfonic acid (P4S), a partial agonist at the GABAA receptor.

Methods: The authors created a mutant of the GABAA receptor [alpha]1 subunit (L277A) by site-directed mutagenesis. The mutant subunit was coexpressed with [beta]2 and [gamma]2S subunits in HEK293 cells, and responses to GABA and P4S were recorded using the whole-cell patch clamp technique. EC50 values were determined for the full agonist GABA and the partial agonist P4S. The authors also determined the relative efficacy ([epsilon]) of P4S. These measurements were then repeated in the presence of isoflurane.

Results: The concentration-response curve for GABA was shifted to the right (EC50 = 278 [mu]m) in the [alpha]1(L277A)[beta]2[gamma]2S mutant receptor, compared with the corresponding wild-type [alpha]1[beta]2[gamma]2S GABAA receptor (EC50 = 16 [mu]m). P4S is a partial agonist at both receptors, with a dramatically decreased relative efficacy at the mutant receptor ([epsilon] = 0.24). When the mutant receptor was studied in the presence of isoflurane, the concentration-response curves for both GABA and P4S were shifted to the left (EC50 for GABA = 78 [mu]m); the efficacy of P4S also increased significantly ([epsilon] = 0.40).     

Influence of GABAA receptor gamma2 splice variants on receptor kinetics and isoflurane modulation

BACKGROUND: Gamma-aminobutyric acid type A (GABAA) receptors, the major inhibitory receptors in the brain, are important targets of many drugs, including general anesthetics. These compounds exert multiple effects on GABAA receptors, including direct activation, prolongation of deactivation kinetics, and reduction of inhibitory postsynaptic current amplitudes. However, the degree to which these actions occur differs for different agents and synapses, possibly because of subunit-specific effects on postsynaptic receptors. In contrast to benzodiazepines and intravenous anesthetics, there is little information available about the subunit dependency of actions of volatile anesthetics. Therefore, the authors studied in detail the effects of isoflurane on recombinant GABAA receptors composed of several different subunit combinations. METHODS: Human embryonic kidney 293 cells were transiently transfected with rat complementary DNAs of alpha1beta2, alpha1beta2gamma2L, alpha1beta2gamma2S, alpha5beta3, or alpha5beta3gamma2S subunits. Using rapid application and whole cell patch clamp techniques, cells were exposed to 10- and 2,000-ms pulses of gamma-aminobutyric acid (1 mm) in the presence or absence of isoflurane (0.25, 0.5, 1.0 mm). Anesthetic effects on decay kinetics, peak amplitude, net charge transfer and rise time were measured. Statistical significance was assessed using the Student t test or one-way analysis of variance followed by the Tukey post hoc test. RESULTS: Under control conditions, incorporation of a gamma2 subunit conferred faster deactivation kinetics and reduced desensitization. Isoflurane slowed deactivation, enhanced desensitization, and reduced peak current amplitude in alphabeta receptors. Coexpression with a gamma2 subunit caused these effects of isoflurane to be substantially reduced or abolished. Although the two gamma2 splice variants imparted qualitatively similar macroscopic kinetic properties, there were significant quantitative differences between effects of isoflurane on deactivation and peak current amplitude in gamma2S- versus gamma2L-containing receptors. The net charge transfer resulting from brief pulses of gamma-aminobutyric acid was decreased by isoflurane in alphabeta but increased in alphabetagamma receptors. CONCLUSIONS: The results indicate that subunit composition does substantially influence modulation of GABAA receptors by isoflurane. Specifically, the presence of a gamma2 subunit and the identity of its splice variant are important factors in determining physiologic and pharmacologic properties. These results may have functional implications in understanding how anesthetic effects on specific types of GABAA receptors in the brain contribute to changes in brain function and behavior.     

CNS 7056: A Novel Ultra-short-acting Benzodiazepine

Background: A new benzodiazepine derivative, CNS 7056, has been developed to permit a superior sedative profile to current agents, i.e., more predictable fast onset, short duration of sedative action, and rapid recovery profile. This goal has been achieved by rendering the compound susceptible to metabolism via esterases. The authors now report on the profile of CNS 7056 in vitro and in vivo.

Methods: The affinity of CNS 7056 and its carboxylic acid metabolite, CNS 7054, for benzodiazepine receptors and their selectivity profiles were evaluated using radioligand binding. The activity of CNS 7056 and midazolam at subtypes ([alpha]1[beta]2[gamma]2, [alpha]2[beta]2[gamma]2, [alpha]3[beta]2[gamma]2, [alpha]5[beta]2[gamma]2) of the [gamma]-aminobutyric acid type A (GABAA) receptor was evaluated using the whole cell patch clamp technique. The activity of CNS 7056 at brain benzodiazepine receptors in vivo was measured in rats using extracellular electrophysiology in the substantia nigra pars reticulata. The sedative profile was measured in rodents using the loss of righting reflex test.

Results: CNS 7056 bound to brain benzodiazepine sites with high affinity. The carboxylic acid metabolite, CNS 7054, showed around 300 times lower affinity. CNS 7056 and CNS 7054 (10 [mu]m) showed no affinity for a range of other receptors. CNS 7056 enhanced GABA currents in cells stably transfected with subtypes of the GABAA receptor. CNS 7056, like midazolam and other classic benzodiazepines, did not show clear selectivity between subtypes of the GABAA receptor. CNS 7056 (intravenous) caused a dose-dependent inhibition of substantia nigra pars reticulata neuronal firing and recovery to baseline firing rates was reached rapidly. CNS 7056 (intravenous) induced loss of the righting reflex in rodents. The duration of loss of righting reflex was short (< 10 min) and was inhibited by pretreatment with flumazenil.     

Effects of isoflurane on gamma-aminobutyric acid type A receptors activated by full and partial agonists

BACKGROUND: Volatile anesthetics prolong inhibitory postsynaptic potentials in central neurons an allosteric action on the gamma-aminobutyric acid type A (GABA(A)) receptor, an effect that may underlie the hypnotic actions of these agents. Inhaled anesthetics such as isoflurane act to enhance responses to submaximal concentrations of GABA, but it is not clear whether their effect is mediated by an increase in the binding of the agonist or by changes in receptor gating behavior. To address this question, the authors studied the effects of isoflurane on a mutant GABA(A) receptor with a gating defect that decreases receptor sensitivity by lowering agonist efficacy. They then compared the effects of clinically relevant concentrations of isoflurane on the actions of GABA and piperidine-4-sulfonic acid (P4S), a partial agonist at the GABA(A) receptor. METHODS: The authors created a mutant of the GABA receptor alpha subunit (L277A) by site-directed mutagenesis. The mutant subunit was coexpressed with beta(2) and gamma(2S) subunits in HEK293 cells, and responses to GABA and P4S were recorded using the whole-cell patch clamp technique. EC values were determined for the full agonist GABA and the partial agonist P4S. The authors also determined the relative efficacy (epsilon) of P4S. These measurements were then repeated in the presence of isoflurane. RESULTS: The concentration-response curve for GABA was shifted to the right (EC(50) = 278 microm) in the alpha(1)(L277A)beta(2)gamma(2S) mutant receptor, compared with the corresponding wild-type alpha(1)beta(2)gamma(2S) GABA(A) receptor (EC(50) = 16 microm). P4S is a partial agonist at both receptors, with a dramatically decreased relative efficacy at the mutant receptor (epsilon = 0.24). When the mutant receptor was studied in the presence of isoflurane, the concentration-response curves for both GABA and P4S were shifted to the left (EC(50) for GABA = 78 microm); the efficacy of P4S also increased significantly (epsilon = 0.40). CONCLUSION: By studying a mutant GABA receptor with impaired gating, the authors were able to demonstrate clearly that isoflurane can increase the efficacy of a partial agonist, as well as increase agonist potency. These data suggest that the volatile anesthetic isoflurane exerts at least some of its effects on the GABA(A) receptor via alterations in gating rather than simply changing binding or unbinding of the agonist.     

The actions of sevoflurane and desflurane on the gamma-aminobutyric acid receptor type A: effects of TM2 mutations in the alpha and beta subunits

BACKGROUND: Previous studies have shown that specific amino acid residues in the putative second transmembrane segment (TM2) of the gamma-aminobutyric acid receptor type A (GABAA) receptor play a critical role in the enhancement of GABAA receptor function by halothane, enflurane, and isoflurane. However, very little is known about the actions of sevoflurane and desflurane on recombinant GABAA receptors. The aim of this study was to examine the effects of sevoflurane and desflurane on potentiation of GABA-induced responses in the wild-type GABAA receptor and in receptors mutated in TM2 of the alpha1, alpha 2, or beta 2 subunits. METHODS: GABAA receptor alpha 1 or alpha 2, beta 2 or beta 3, and gamma 2s subunit cDNAs were expressed for pharmacologic study by transfection of human embryonic kidney 293 cells and assayed using the whole cell voltage clamp technique. Concentration-response curves and EC50 values for agonist were determined in the wild-type alpha 1 beta 2 gamma 2s and alpha 2 beta 3 gamma 2s receptors, and in receptors harboring mutations in TM2, such as alpha1(S270W)beta 2 gamma 2s, alpha 1 beta 2(N265W)gamma 2s, and alpha2(S270I)beta 3 gamma 2s. The actions of clinically relevant concentration of volatile anesthetics (isoflurane, sevoflurane, and desflurane) on GABA activated Cl- currents were compared in the wild-type and mutant GABAA receptors. RESULTS: Both sevoflurane and desflurane potentiated submaximal GABA currents in the wild-type GABAA alpha 1 beta 2 gamma 2s receptor and alpha 2 beta 3 gamma 2s receptor. Substitution of Ser270 in TM2 of the alpha subunit by a larger amino acid, tryptophan (W) or isoleucine (I), as in alpha1(S270W)beta 2 gamma 2s and alpha 2(S270I)beta 3 gamma 2s, completely abolished the potentiation of GABA-induced currents by these anesthetic agents. In contrast, mutation of Asn265 in TM2 of the beta subunit to tryptophan (W) did not prevent potentiation of GABA-induced responses. The actions of sevoflurane and desflurane in the wild-type receptor and in mutated receptors were qualitatively and quantitatively similar to those observed for isoflurane. CONCLUSIONS: Positions Ser270 of the GABAA alpha1 and alpha2 subunits, but not Asn265 in the TM2 of the beta2 subunit, are critical for regulation of the GABAA receptor by sevoflurane and desflurane, as well as isoflurane, consistent with the idea that these three volatile anesthetics share a common site of actions on the alpha subunit of the GABAA receptor.     

Synergistic interaction between alpha 2-adrenergic agonists and benzodiazepines in rats.

Both alpha 2-adrenergic agonists and benzodiazepines exert anxiolytic and sedative effects when administered as preoperative medications. Clinical effects achieved with a combination of drugs, representative of these classes of compounds, is greater than that which could be expected from a simple additive response. Therefore, we investigated the nature of the interaction between dexmedetomidine, the highly-selective alpha 2-adrenergic agonist, and midazolam in a series of in vivo and in vitro studies in rats. Rats were administered midazolam, dexmedetomidine, or a combination of midazolam and dexmedetomidine intravenously to derive three dose-response curves for loss of righting reflex (LRR). LRR was determined in rats in a rotating cage (4 rotations/min) by observing whether the rat failed to maintain its upright posture for greater than or equal to 15 s exactly 2.5 min after drug administration. The effect of either flumazenil (benzodiazepine receptor antagonist) or atipamezole (the alpha 2-adrenergic antagonist) on the LRR was also determined. A probit analysis was performed and an isobologram for the ED50 was derived to assess the nature of the interaction. Rat brain membranes were prepared for receptor binding assays using [3H]-flumazenil and [3H]-rauwolscine to characterize the benzodiazepine and alpha 2-adrenergic receptors, respectively. The ability of either midazolam or dexmedetomidine to displace the radiolabeled ligand from the alternative receptor was assessed. To detect a possible kinetic interaction between the two drugs, separate cohorts of rats were administered the two drugs individually or in combination at the combination ED50 doses.(ABSTRACT TRUNCATED AT 250 WORDS)     

Influence of GABAA Receptor γ2 Splice Variants on Receptor Kinetics and Isoflurane Modulation

Background: [gamma]-Aminobutyric acid type A (GABAA) receptors, the major inhibitory receptors in the brain, are important targets of many drugs, including general anesthetics. These compounds exert multiple effects on GABAA receptors, including direct activation, prolongation of deactivation kinetics, and reduction of inhibitory postsynaptic current amplitudes. However, the degree to which these actions occur differs for different agents and synapses, possibly because of subunit-specific effects on postsynaptic receptors. In contrast to benzodiazepines and intravenous anesthetics, there is little information available about the subunit dependency of actions of volatile anesthetics. Therefore, the authors studied in detail the effects of isoflurane on recombinant GABAA receptors composed of several different subunit combinations.

Methods: Human embryonic kidney 293 cells were transiently transfected with rat complementary DNAs of [alpha]1[beta]2, [alpha]1[beta]2[gamma]2L, [alpha]1[beta]2[gamma]2S, [alpha]5[beta]3, or [alpha]5[beta]3[gamma]2S subunits. Using rapid application and whole cell patch clamp techniques, cells were exposed to 10- and 2,000-ms pulses of [gamma]-aminobutyric acid (1 mm) in the presence or absence of isoflurane (0.25, 0.5, 1.0 mm). Anesthetic effects on decay kinetics, peak amplitude, net charge transfer and rise time were measured. Statistical significance was assessed using the Student t test or one-way analysis of variance followed by the Tukey post hoc test.

Results: Under control conditions, incorporation of a [gamma]2 subunit conferred faster deactivation kinetics and reduced desensitization. Isoflurane slowed deactivation, enhanced desensitization, and reduced peak current amplitude in [alpha][beta] receptors. Coexpression with a [gamma]2 subunit caused these effects of isoflurane to be substantially reduced or abolished. Although the two [gamma]2 splice variants imparted qualitatively similar macroscopic kinetic properties, there were significant quantitative differences between effects of isoflurane on deactivation and peak current amplitude in [gamma]2S- versus [gamma]2L-containing receptors. The net charge transfer resulting from brief pulses of [gamma]-aminobutyric acid was decreased by isoflurane in [alpha][beta] but increased in [alpha][beta][gamma] receptors.     

Droperidol inhibits GABA(A) and neuronal nicotinic receptor activation

BACKGROUND: Droperidol is used in neuroleptanesthesia and as an antiemetic. Although its antiemetic effect is thought to be caused by dopaminergic inhibition, the mechanism of droperidol's anesthetic action is unknown. Because gamma-aminobutyric acid type A (GABAA) and neuronal nicotinic acetylcholine receptors (nAChRs) have been implicated as putative targets of other general anesthetic drugs, the authors tested the ability of droperidol to modulate these receptors. METHODS: gamma-Aminobutyric acid type A alpha1beta1gamma2 receptor, alpha7 and alpha4beta2 nAChRs were expressed in Xenopus oocytes and studied with two-electrode voltage clamp recording. The authors tested the ability of droperidol at concentrations from 1 nm to 100 microm to modulate activation of these receptors by their native agonists. RESULTS: Droperidol inhibited the GABA response by a maximum of 24.7 +/- 3.0%. The IC50 for inhibition was 12.6 +/- 0.47 nm droperidol. At high concentrations, droperidol (100 microm) activates the GABAA receptor in the absence of GABA. Inhibition of the GABA response is significantly greater at hyperpolarized membrane potentials. The activation of the alpha7 nAChR is also inhibited by droperidol, with an IC50 of 5.8 +/- 0.53 microm. The Hill coefficient is 0.95 +/- 0.1. Inhibition is noncompetitive, and membrane voltage dependence is insignificant. CONCLUSIONS: Droperidol inhibits activation of both the GABAA alpha1beta1gamma2 and alpha7 nAChR. The submaximal GABA inhibition occurs within a concentration range such that it might be responsible for the anxiety, dysphoria, and restlessness that limit the clinical utility of high-dose droperidol anesthesia. Inhibition of the alpha7 nAChR might be responsible for the anesthetic action of droperidol.     

Nonhalogenated Alkane Anesthetics Fail to Potentiate Agonist Actions on Two Ligand-gated Ion Channels

Background: Although ether, alcohol, and halogenated alkane anesthetics potentiate agonist actions or increase the apparent agonist affinity of ligand-gated ion channels at clinically relevant concentrations, the effects of nonhalogenated alkane anesthetics on ligand-gated ion channels have not been studied. The current study assessed the abilities of two representative nonhalogenated alkane anesthetics (cyclopropane and butane) to potentiate agonist actions or increase the apparent agonist affinity of two representative ligand-gated ion channels: the nicotinic acetylcholine receptor and [gamma]-aminobutyric acid type A (GABAA) receptor.

Methods: Nicotinic acetylcholine receptors were obtained from the electroplax organ of Torpedo nobiliana, and human GABAA receptors ([alpha]1[beta]2[gamma]2L) were expressed in human embryonic kidney 293 cells. The Torpedo nicotinic acetylcholine receptors apparent agonist affinity in the presence and absence of anesthetic was assessed by measuring the apparent rates of desensitization induced by a range of acetylcholine concentrations. The GABAA receptor's apparent agonist affinity in the presence and absence of anesthetic was assessed by measuring the peak currents induced by a range of GABA concentrations.

Results: Neither cyclopropane nor butane potentiated agonist actions or increased the apparent agonist affinity (reduced the apparent agonist dissociation constant) of the Torpedo nicotinic acetylcholine receptor or GABAA receptor. At clinically relevant concentrations, cyclopropane and butane reduced the apparent rate of Torpedo nicotinic acetylcholine receptor desensitization induced by low concentrations of agonist.     

The Actions of Sevoflurane and Desflurane on the γ-Aminobutyric Acid Receptor Type A: Effects of TM2 Mutations in the α and β Subunits

Background: Previous studies have shown that specific amino acid residues in the putative second transmembrane segment (TM2) of the [gamma]-aminobutyric acid receptor type A (GABAA) receptor play a critical role in the enhancement of GABAA receptor function by halothane, enflurane, and isoflurane. However, very little is known about the actions of sevoflurane and desflurane on recombinant GABAA receptors. The aim of this study was to examine the effects of sevoflurane and desflurane on potentiation of GABA-induced responses in the wild-type GABAA receptor and in receptors mutated in TM2 of the [alpha]1, [alpha]2, or [beta]2 subunits.

Methods: GABAA receptor [alpha]1 or [alpha]2, [beta]2 or [beta]3, and [gamma]2s subunit cDNAs were expressed for pharmacologic study by transfection of human embryonic kidney 293 cells and assayed using the whole cell voltage clamp technique. Concentration-response curves and EC50 values for agonist were determined in the wild-type [alpha]1[beta]2[gamma]2s and [alpha]2[beta]3[gamma]2s receptors, and in receptors harboring mutations in TM2, such as [alpha]1(S270W)[beta]2[gamma]2s, [alpha]1[beta]2(N265W)[gamma]2s, and [alpha]2(S270I)[beta]3[gamma]2s. The actions of clinically relevant concentration of volatile anesthetics (isoflurane, sevoflurane, and desflurane) on GABA activated Cl- currents were compared in the wild-type and mutant GABAA receptors.

Results: Both sevoflurane and desflurane potentiated submaximal GABA currents in the wild-type GABAA [alpha]1[beta]2[gamma]2s receptor and [alpha]2[beta]3[gamma]2s receptor. Substitution of Ser270 in TM2 of the [alpha] subunit by a larger amino acid, tryptophan (W) or isoleucine (I), as in [alpha]1(S270W)[beta]2[gamma]2s and [alpha]2(S270I)[beta]3[gamma]2s, completely abolished the potentiation of GABA-induced currents by these anesthetic agents. In contrast, mutation of Asn265 in TM2 of the [beta] subunit to tryptophan (W) did not prevent potentiation of GABA-induced responses. The actions of sevoflurane and desflurane in the wild-type receptor and in mutated receptors were qualitatively and quantitatively similar to those observed for isoflurane.     

The effects of benzodiazepines on human opioid receptor binding and function

We performed in vitro studies to investigate the potential interaction of benzodiazepines with cloned human opioid receptor subtypes. Midazolam, chlordiazepoxide, and diazepam directly displaced [(3)H]-diprenorphine binding from kappa and delta receptors, but not mu receptors, whereas flumazenil was inactive. These benzodiazepines also stimulated (35)S-GTPgammaS binding in membranes containing human kappa receptors, and the effect of midazolam was prevented by a selective kappa antagonist. Midazolam was also weakly active at delta-receptor activation, whereas all three were inactive at mu receptors. The results suggest that the analgesic efficacy reported for intrathecal benzodiazepines may be attributed, in part, to direct interaction with kappa-opioid receptors. IMPLICATIONS: Several human and animal studies have shown analgesic effects of benzodiazepines after spinal injection. Our results show that large concentrations of midazolam, chlordiazepoxide, and diazepam displace the binding of [(3)H]-diprenorphine-an opiate radioligand from kappa receptors. In an in vitro functional assay, midazolam is a weak agonist at the delta-opioid receptor, whereas all three benzodiazepines are kappa-opioid agonists. These findings may partially explain the mechanism of benzodiazepine-induced spinal analgesia.     

The synergistic interaction between midazolam and clonidine in spinally-mediated analgesia in two different pain models of rats

Both midazolam, a benzodiazepine gamma-aminobutyric acid type A receptor agonist, and clonidine, an alpha2-adrenergic receptor agonist, induce spinally-mediated analgesia. We investigated the analgesic interaction of spinally-administered midazolam and clonidine in their effects on acute and inflammatory nociception. Rats implanted with lumbar intrathecal catheters were injected intrathecally with saline (control), midazolam (1 to 100 microg), or clonidine (0.1 to 3 microg) to test for their responses to thermal stimulation to the tail (tail-flick test) and subcutaneous formalin injection into the hind paw (formalin test). The effects of the combination of midazolam and clonidine on both stimuli were tested by isobolographic analysis by using the 50% effective doses. The general behavior and motor function were examined as side effects. When combined, the 50% effective doses of midazolam (clonidine) decreased from 1.57 microg (0.26 microg) to 0.29 g (0.05 microg) in the tail-flick test and from 1.34 microg (0.12 microg) and 1.21 microg (0.13 microg) to 0.05 microg (0.005 microg) and 0.13 microg (0.015 microg) in Phase 1 and 2 of the formalin test, respectively. Side effects did not increase by using the combination. These results suggest a favorable combination of intrathecal midazolam and clonidine in the management of acute and inflammatory pain after proper neurotoxicologic studies. IMPLICATIONS: Spinally-administered midazolam, a benzodiazepine, and clonidine, an alpha2-adrenergic receptor agonist, have significant synergistic effects on thermally-induced acute and formalin-induced inflammatory pain.     

Actions of Midazolam on GABAergic Transmission in Substantia Gelatinosa Neurons of Adult Rat Spinal Cord Slices

Background: Although intrathecal administration of midazolam has been found to produce analgesia, how midazolam exerts this effect is not understood fully at the neuronal level in the spinal cord.

Methods: The effects of midazolam on either electrically evoked or spontaneous inhibitory transmission and on a response to exogenous [gamma]-aminobutyric acid (GABA), a GABAA-receptor agonist, muscimol, or glycine were evaluated in substantia gelatinosa neurons of adult rat spinal cord slices by using the whole-cell patch-clamp technique.

Results: Bath-applied midazolam (1 [mu]M) prolonged the decay phase of evoked and miniature inhibitory postsynaptic currents (IPSCs), mediated by GABAA receptors, without a change in amplitudes, while not affecting glycine receptor-mediated miniature inhibitory postsynaptic currents in both the decay phase and the amplitude. Either GABA- or muscimol-induced currents were enhanced in amplitude by midazolam (0.1 [mu]M) in a manner sensitive to a benzodiazepine receptor antagonist, flumazenil (1 [mu]M); glycine currents were, however, unaltered by midazolam.     

Modulation of GABA(A) receptor function by nonhalogenated alkane anesthetics: the effects on agonist enhancement,direct activation,and inhibition

At clinically relevant concentrations, ethers, alcohols, and halogenated alkanes enhance agonist action on the gamma-aminobutyric acid(A) (GABA(A)) receptor, whereas nonhalogenated alkanes do not. Many anesthetics also directly activate and/or inhibit GABA(A) receptors, actions that may produce important behavioral effects; although, the effects of nonhalogenated alkane anesthetics on GABA(A) receptor direct activation and inhibition have not been studied. In this study, we assessed the abilities of two representative nonhalogenated alkanes, cyclopropane and butane, to enhance agonist action, directly activate, and inhibit currents mediated by expressed alpha(1)beta(2)gamma(2L) GABA(A) receptors using electrophysiological techniques. Our studies reveal that cyclopro- pane and butane enhance agonist action on the GABA(A) receptor at concentrations that exceed those required to produce anesthesia. Neither nonhalogenated alkane directly activated nor inhibited GABA(A) receptors, even at concentrations that approach their aqueous saturated solubilities. These results strongly suggest that the behavioral actions of nonhalogenated alkane anesthetics do not result from their abilities to enhance agonist actions, directly activate, or inhibit alpha(1)beta(2)gamma(2L) GABA(A) receptors and are consistent with the hypothesis that electrostatic interactions between anesthetics and their protein binding sites modulate GABA(A) receptor potency. IMPLICATIONS: When normalized to either their in vivo anesthetic potencies or hydrophobicities, cyclopropane and butane are 1-1.5 orders of magnitude less potent enhancers of agonist action on alpha(1beta2gamma2L) GABA(A) receptors than isoflurane. Additionally, cyclopropane and butane fail to directly activate or inhibit receptors, even at near aqueous saturating concentrations. Thus, it is unlikely that either enhancement or inhibition of the most common GABA(A) receptor subtype in the brain accounts for the behavioral activities of cyclopropane and butane.     

Paradoxical reactions to benzodiazepines in intravenous sedation: a report of 2 cases and review of the literature

Paradoxical reactions to benzodiazepines have been thoroughly reported since the introduction of this type of drug. The mechanism of benzodiazepine action is through the gamma-aminobutyric acid receptors. Properties of benzodiazepine include sedation, anxiolysis, amnesia, anticonvulsion, and muscle relaxation. Unfortunately, adverse paradoxical reactions can be stimulated by benzodiazepines and are difficult to predict and diagnose. Two cases of paradoxical reactions associated with the use of intravenous midazolam are presented, and the management of this complication and its different etiologies are reviewed. The relationship of the paradoxical reaction to alteration of the cholinergic homeostasis, serotonin levels, the role of genetics, and gamma-aminobutyric acid receptor configuration is discussed.     

Modulation of Recombination Human gamma-Aminobutyric Acid sub A Receptors by Isoflurane: Influence of the Delta Subunit

Background: The gamma-aminobutyric acid (GABA)A receptor/chloride channel has a broad-spectrum anesthetic sensitivity and is a key regulator of arousal. Each receptor/channel complex is an assembly of five protein subunits. Six subunit classes have been identified, each containing one to six members; many combinations are expressed throughout the brain. Benzodiazepines and intravenous anesthetic agents are clearly subunit dependent, but the literature to date suggests that volatile anesthetics are not. The physiological role of the delta subunit remains enigmatic, and it has not been examined as a determinant of anesthetic sensitivity.

Methods: Combinations of GABAA receptor subunit cDNAs were injected into Xenopus laevis oocytes: alpha1 beta1, alpha1 beta1 gamma2L, alpha1 beta1 delta, and alpha1 beta1 gamma2L delta. Expression of functional ion channels with distinct signalling and pharmacologic properties was demonstrated within 1-4 days by established electrophysiological methods.

Results: Co-expression of the delta subunit produced changes in receptor affinity; current density; and the modulatory efficacy of diazepam, zinc, and lanthanum; it also produced subtle changes in the rate of desensitization in response to GABA. Isoflurane enhanced GABA-induced responses from all combinations: alpha beta delta (> 10-fold) > alpha beta > alpha beta gamma >or= to alpha beta gamma delta ([nearly =] 5-fold). Dose-response plots were bell shaped. Compared with alpha beta gamma receptors (EC50 = 225 micro Meter), both alpha beta delta (EC50 = 372 micro Meter) and alpha beta gamma delta (EC sub 50 = 399 micro Meter) had a reduced affinity for isoflurane. Isoflurane (at a concentration close to the EC50 for each subunit) increased the affinity of GABA for its receptor but depressed the maximal response (alpha beta gamma and alpha beta gamma delta). In contrast, the small currents through alpha beta delta receptors were enhanced, even at saturating agonist concentrations.     

Insertion of alpha7 nicotinic receptors at neocortical layer V GABAergic synapses is induced by a benzodiazepine, midazolam

Benzodiazepines act mainly at postsynaptic gamma-aminobutyric acid type A (GABA(A)) receptors. In rat neocortical layer V pyramidal neurons, we found that midazolam (MDZ), a benzodiazepine, increases the frequency of GABAergic miniature inhibitory postsynaptic currents (mIPSCs) via insertion of alpha7 nicotinic acetylcholine receptors (nAChRs) at presynaptic GABAergic boutons. Although nicotine alone had no effect, MDZ plus nicotine dramatically increased mIPSC frequency. Neostigmine, an acetylcholinesterase inhibitor, mimicked the actions of nicotine. MDZ increased the number of alpha-bungarotoxin-bound boutons that were blocked by protein kinase C (PKC) inhibitors, as revealed by confocal imaging of a neuron-synaptic bouton preparation. Thus, MDZ may induce membrane translocation of alpha7 nAChRs on GABAergic boutons via activation of PKC, enabling endogenous acetylcholine to increase GABA release. The above actions seem unique to MDZ because neither other benzodiazepines (diazepam and flunitrazepam) nor zolpidem had this effect. The findings reveal both a novel cholinergic modulatory mechanism affecting GABAergic transmission and a novel action of some general anesthetics.     

Differential effects of intrathecal midazolam on morphine-induced antinociception in the rat: role of spinal opioid receptors.

The antinociceptive effects of an intrathecally administered benzodiazepine agonist midazolam, alone and in combination with morphine, were examined in the rat by using the tail-flick test. The duration of antinociceptive effect produced by midazolam was significantly less (P less than 0.05) than that produced by morphine. Low doses of midazolam (10 micrograms) and morphine (10 micrograms) produced a synergistic effect in prolonging antinociceptive effect. However, at higher doses (20 or 30 micrograms), these drugs reduced the extent of antinociception produced by each other. Naloxone administration prevented antinociception produced by these drugs, indicating interactions between midazolam and opioid receptors. Midazolam had dual effects on the binding of opioid ligands to the spinal opioid receptors. At low dose, it potentiated the displacement of [3H]naloxone by morphine. At higher doses, midazolam inhibited the binding of opioid ligands to their spinal receptors in the following order: kappa greater than delta greater than mu. These results indicate that differential antinociceptive effects of midazolam on morphine-induced antinociception involve interaction of this benzodiazepine with spinal opioid receptors.