Classic benzodiazepines modulate the open-close equilibrium in alpha1beta2gamma2L gamma-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 alpha1beta2gamma2L 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 alpha1 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 alpha1L264Tbeta2gamma2L 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. CONCLUSIONS: Independent of orthosteric site occupancy, classic benzodiazepines modulate the gating equilibrium in alpha1beta2gamma2L GABAA receptors and are therefore allosteric coagonists. A Monod-Wyman-Changeux coagonist gating model quantitatively predicts these effects, suggesting that benzodiazepines minimally alter orthosteric ligand binding.     

The γ Subunit Determines Whether Anesthetic-induced Leftward Shift Is Altered by a Mutation at α1S270 in α1β2γ2L GABAA Receptors

Background: A major action of volatile anesthetics is enhancement of [gamma]-aminobutyric acid receptor type A (GABAAR) currents. In recombinant GABAARs consisting of several subunit mixtures, mutating the [alpha]1 subunit serine at position 270 to isoleucine [[alpha]1(S270I)] was reported to eliminate anesthetic-induced enhancement at low GABA concentrations. In the absence of studies at high GABA concentrations, it remains unclear whether [alpha]1(S270I) affects enhancement versus inhibition by volatile anesthetics. Furthermore, the majority of GABAARs in mammalian brain are thought to consist of [alpha]1, [beta]2, and [gamma]2 subunits, and the [alpha]1(S270I) mutation has not been studied in the context of this combination.

Methods: Recombinant GABAARs composed of [alpha]1[beta]2 or [alpha]1[beta]2[gamma]2L subunit mixtures were studied electrophysiologically in whole Xenopus oocytes in the voltage clamp configuration. Currents elicited by GABA (0.03 [mu]m to 1 mm) were measured in the absence and presence of isoflurane or halothane. Anesthetic effects on GABA concentration responses were evaluated for individual oocytes.

Results: In wild-type [alpha]1[beta]2[gamma]2L GABAA, anesthetics at approximately 2 minimum alveolar concentration (MAC) shifted GABA concentration response curves to the left approximately threefold, decreased the Hill coefficient, and enhanced currents at all GABA concentrations. The [alpha]1(S270I) mutation itself rendered the GABAAR more sensitive to GABA and reduced the Hill coefficient. At low GABA concentrations (EC5), anesthetic enhancement of peak current was much smaller in [alpha]1(S270I)[beta]2[gamma]2Lversus wild-type channels. Paradoxically, the leftward shift of the whole GABA concentration-response relation by anesthetics was the same in both mutant and wild-type channels. At high GABA concentrations, volatile anesthetics reduced currents in [alpha]1(S270I)[beta]2[gamma]2L GABAARs. In parallel studies on [alpha]1[beta]2 ([gamma]-less) GABAARs, anesthetic-induced leftward shifts in wild-type receptors were more than eightfold at 2 MAC, and the [alpha]1(S270I) mutation nearly eliminated anesthetic-induced leftward shift.     

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.     

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.     

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).     

Isoflurane and Nociception: Spinal α2A Adrenoceptors Mediate Antinociception while Supraspinal α1 Adrenoceptors Mediate Pronociception

Background: The authors recently established that the analgesic actions of the inhalation anesthetic nitrous oxide were mediated by noradrenergic bulbospinal neurons and spinal [alpha]2B adrenoceptors. They now determined whether noradrenergic brainstem nuclei and descending spinal pathways are responsible for the antinociceptive actions of the inhalation anesthetic isoflurane, and which [alpha] adrenoceptors mediate this effect.

Methods: After selective lesioning of noradrenergic nuclei by intracerebroventricular application of the mitochondrial toxin saporin coupled to the antibody directed against dopamine [beta] hydroxylase (D[beta]H-saporin), the antinociceptive action of isoflurane was determined. Antagonists for the [alpha]1 and [alpha]2 adrenoceptors were injected at spinal and supraspinal sites in intact and spinally transected rats to identify the noradrenergic pathways mediating isoflurane antinociception. Null mice for each of the three [alpha]2-adrenoceptor subtypes ([alpha]2A, [alpha]2B, and [alpha]2C) and their wild-type cohorts were tested for their antinociceptive response to isoflurane.

Results: Both D[beta]H-saporin treatment and chronic spinal transection enhanced the antinociceptive effects of isoflurane. The [alpha]1-adrenoceptor antagonist prazosin also enhanced isoflurane antinociception at a supraspinal site of action. The [alpha]2-adrenoceptor antagonist yohimbine inhibited isoflurane antinociception, and this effect was mediated by spinal [alpha]2 adrenoceptors. Null mice for the [alpha]2A-adrenoceptor subtype showed a reduced antinociceptive response to isoflurane.     

Halothane Does Not Inhibit the Functional Coupling between the β2-Adrenergic Receptor and the Gαs Heterotrimeric G Protein

Background: This study investigated whether halothane affects the functional coupling between the [beta]2 adrenergic receptor and the [alpha] subunit of its cognate stimulatory heterotrimeric guanosine-5'-triphosphate (GTP)-binding protein (G[alpha]s). The authors hypothesized that halothane does not affect isoproterenol-promoted guanosine nucleotide exchange at G[alpha]s and hence would not affect isoproterenol-induced relaxation of airway smooth muscle.

Methods: Halothane effects on isoproterenol-induced inhibition of calcium sensitivity were measured in permeabilized porcine airway smooth muscle. G[alpha]s nucleotide exchange was measured in crude membranes prepared from COS-7 cells transfected to transiently coexpress the human [beta]1 or [beta]2 receptor each with human short G[alpha]s. A radioactive, nonhydrolyzable analog of GTP, [35S]GTP[gamma]S, was used as the reporter for nucleotide exchange at G[alpha]s.

Results: Halothane (0.75 mm, approximately 2.8 minimum alveolar concentration [MAC] in pigs) did not affect isoproterenol-induced inhibition of calcium sensitivity. Isoproterenol caused a time- and concentration-dependent increase in G[alpha]s nucleotide exchange. Halothane, even at concentrations of 1.5 mm (approximately 5.6 MAC), had no effect on basal G[alpha]s nucleotide exchange in the absence of isoproterenol, whereas halothane inhibited isoproterenol-promoted G[alpha]s nucleotide exchange in both the [beta]1-G[alpha]s and [beta]2-G[alpha]s expressing membranes. However, the effect was significantly greater on [beta]1-G[alpha]s coupling compared with [beta]2-G[alpha]s coupling, with no effect on [beta]2-G[alpha]s coupling at 2.8 MAC halothane.     

The α2-Adrenoceptor Agonist Dexmedetomidine Converges on an Endogenous Sleep-promoting Pathway to Exert Its Sedative Effects

Background: The authors investigated whether the sedative, or hypnotic, action of the general anesthetic dexmedetomidine (a selective [alpha]2-adrenoceptor agonist) activates endogenous nonrapid eye movement (NREM) sleep-promoting pathways.

Methods: c-Fos expression in sleep-promoting brain nuclei was assessed in rats using immunohistochemistry and in situ hybridization. Next, the authors perturbed these pathways using (1) discrete lesions induced by ibotenic acid, (2) local and systemic administration of [gamma]-aminobutyric acid receptor type A (GABAA) receptor antagonist gabazine, or (3) [alpha]2-adrenoceptor antagonist atipamezole in rats, and (4) genetic mutation of the [alpha]2A-adrenoceptor in mice.

Results: Dexmedetomidine induced a qualitatively similar pattern of c-Fos expression in rats as seen during normal NREM sleep, i.e., a decrease in the locus ceruleus (LC) and tuberomammillary nucleus (TMN) and an increase in the ventrolateral preoptic nucleus (VLPO). These changes were attenuated by atipamezole and were not seen in mice lacking functional [alpha]2A-adrenoceptors, which do not show a sedative response to dexmedetomidine. Bilateral VLPO lesions attenuated the sedative response to dexmedetomidine, and the dose-response curve to dexmedetomidine was shifted right by gabazine administered systemically or directly into the TMN. VLPO lesions and gabazine pretreatment altered c-Fos expression in the TMN but in not the LC after dexmedetomidine administration, indicating a hierarchical sequence of changes.     

α2A Adrenoceptors Contribute to Feedback Inhibition of Capsaicin-induced Hyperalgesia

Background: Studies on receptor knockout mice have so far shown that of the three [alpha]2-adrenoceptor subtypes, the [alpha]2A adrenoceptor has a major role in mediating the powerful central analgesia induced by synthetic [alpha]2-adrenoceptor agonists. However, because a knockout of the gene for the [alpha]2A adrenoceptor has produced only little if any change in the pain sensitivity of control, nerve-injured, or inflamed animals, it has not been clear whether activation of [alpha]2A-adrenoceptors by endogenous ligands has a significant pain regulatory role.

Methods: The authors assessed spontaneous pain behavior and mechanical hypersensitivity induced by administration of capsaicin in the colon or paw of [alpha]2A-adrenoceptor knockout mice versus their wild-type controls.

Results: Enhanced pain hypersensitivity was observed in [alpha]2A-adrenoceptor knockout mice 20 min or more after administration of capsaicin, but before, hypersensitivity and spontaneous pain were of equal magnitude in [alpha]2A-adrenoceptor knockout and wild-type mice. When wild-type mice were pretreated with an [alpha]2-adrenoceptor antagonist, capsaicin-induced pain hypersensitivity increased to a level equal to that in [alpha]2A-adrenoceptor knockout mice. Capsaicin-induced hypersensitivity was suppressed in wild-type but not [alpha]2A-adrenoceptor knockout mice by a centrally acting [alpha]2-adrenoceptor agonist, whereas a peripherally acting [alpha]2-adrenoceptor agonist was without effect on hypersensitivity, although it attenuated capsaicin-induced spontaneous pain behavior in wild-type mice.     

Effect of Halothane on Gαi-3 and Its Coupling to the M2 Muscarinic Receptor

Background: Halothane is an effective bronchodilator and inhibits airway smooth muscle contraction in part by inhibiting intracellular signaling pathways activated by the M2 muscarinic receptor and its cognate inhibitory heterotrimeric guanosine-5'-triphosphate (GTP)-binding protein (G protein), Gi. This study hypothesized that halothane inhibits nucleotide exchange at the [alpha] isoform-3 subunit of Gi (G[alpha]i-3), but only when regulated by the M2 muscarinic receptor.

Methods: GTP hydrolysis by G[alpha]i-3 and the G[alpha]i-3[beta]1[gamma]2HF heterotrimer expressed in Spodoptera frugiperda cells was measured using a phosphohydrolase assay with [[gamma]32Pi]-labeled GTP. Anesthetic binding to G[alpha]i-3 was measured by saturation transfer difference nuclear magnetic resonance spectroscopy. G[alpha]i-3 nucleotide exchange was measured in crude membranes prepared from COS-7 cells transiently coexpressing the M2 muscarinic receptor and G[alpha]i-3. A radioactive analog of GTP, [35S]GTP[gamma]S, was used as a reporter for G[alpha]i-3 nucleotide exchange.

Results: Although spectroscopy demonstrated halothane binding to G[alpha]i-3, this binding had no effect on [[gamma]32Pi]-labeled GTP hydrolysis by the G[alpha]i-3[beta]1[gamma]2HF heterotrimer expressed in Spodoptera frugiperda cells, nor basal G[alpha]i-3 nucleotide exchange measured in crude membranes when the muscarinic receptor agonist acetylcholine was omitted from the assay. Conversely, halothane caused a concentration-dependent inhibition of G[alpha]i-3 nucleotide exchange with acetylcholine included in the assay.     

Droperidol Inhibits GABAA 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 [alpha]1[beta]1[gamma]2 receptor, [alpha]7 and [alpha]4[beta]2 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 [mu]m 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 [mu]m) 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 [alpha]7 nAChR is also inhibited by droperidol, with an IC50 of 5.8 +/- 0.53 [mu]m. The Hill coefficient is 0.95 +/- 0.1. Inhibition is noncompetitive, and membrane voltage dependence is insignificant.     

Dual Actions of Enflurane on Postsynaptic Currents Abolished by the γ-Aminobutyric Acid Type A Receptor β3(N265M) Point Mutation

Background: At concentrations close to 1 minimum alveolar concentration (MAC)-immobility, volatile anesthetics display blocking and prolonging effects on [gamma]-aminobutyric acid type A receptor-mediated postsynaptic currents. It has been proposed that distinct molecular mechanisms underlie these dual actions. The authors investigated whether the blocking or the prolonging effect of enflurane is altered by a point mutation (N265M) in the [beta]3 subunit of the [gamma]-aminobutyric acid type A receptor. Furthermore, the role of the [beta]3 subunit in producing the depressant actions of enflurane on neocortical neurons was elucidated.

Methods: Spontaneous inhibitory postsynaptic currents were sampled from neocortical neurons in cultured slices derived from wild-type and [beta]3(N265M) mutant mice. The effects of 0.3 and 0.6 mm enflurane on decay kinetics, peak amplitude, and charge transfer were quantified. Furthermore, the impact of enflurane-induced changes in spontaneous action potential firing was evaluated by extracellular recordings in slices from wild-type and mutant mice.

Results: In slices derived from wild-type mice, enflurane prolonged inhibitory postsynaptic current decays and decreased peak amplitudes. Both effects were almost absent in slices from [beta]3(N265M) mutant mice. At clinically relevant concentrations between MAC-awake and MAC-immobility, the anesthetic was less effective in depressing spontaneous action potential firing in slices from [beta]3(N265M) mutant mice compared with wild-type mice.     

Enflurane Actions on Spinal Cords from Mice That Lack the β3 Subunit of the GABAA Receptor

Background: [gamma]-Aminobutyric acid type A (GABAA) receptors are considered important in mediating anesthetic actions. Mice lacking the [beta]3 subunit of this receptor ([beta]3-/-) have a higher enflurane minimum alveolar concentration (MAC) than wild types (+/+). MAC is predominantly determined in spinal cord.

Methods: The authors measured three population-evoked responses in whole spinal cords, namely, the excitatory postsynaptic potential (pEPSP), the slow ventral root potential (sVRP), and the dorsal root potential. Synaptic and glutamate-evoked currents from motor neurons in spinal cord slices were also measured.

Results: Sensitivity of evoked responses to enflurane did not differ between +/+ and -/- cords. The GABAA receptor antagonist bicuculline significantly (P < 0.05) attenuated the depressant effects of enflurane on pEPSP, sVRP and glutamate-evoked currents in +/+ but not -/- cords. The glycine antagonist strychnine elevated the pEPSP to a significantly greater extent in -/- than in +/+ cords, but the interactions between strychnine and enflurane did not differ between -/- and +/+ cords.     

Effect of N-methyl-d-aspartate Receptor ε1 Subunit Gene Disruption of the Action of General Anesthetic Drugs in Mice

Background: Recent molecular strategies demonstrated that the N-methyl-d-aspartate (NMDA) receptor is a major target site of anesthetic agents. In a previous article, the authors showed that knocking out the NMDA receptor [epsilon]1 subunit gene markedly reduced the hypnotic effect of ketamine in mice. In the current study, the authors examined the in vivo contribution of the NMDA receptor [epsilon]1 subunit to the action of other anesthetic drugs.

Methods: The authors determined the anesthetic effects of nitrous oxide on sevoflurane potency in NMDA receptor [epsilon]1 subunit knockout mice compared with those in wild-type mice. They then tested the hypnotic effect of [gamma]-aminobutyric acid-mediated agents, such as propofol, pentobarbital, diazepam, and midazolam, in knockout mice and wild-type mice.

Results: The anesthetic action of sevoflurane itself was unaffected by the abrogation of the NMDA receptor [epsilon]1 subunit. Adding nitrous oxide reduced the required concentration of sevoflurane to induce anesthesia in wild-type mice, whereas this sparing effect was diminished in knockout mice. Furthermore, propofol, pentobarbital, diazepam, and midazolam also had markedly attenuated effects in knockout mice.     

Meperidine Exerts Agonist Activity at the α2B-Adrenoceptor Subtype

Background: The opioid agonist meperidine has actions, such as antishivering, that are more pronounced than those of other opioid agonists and that are not blocked with nonselective opioid antagonists. Agonists at the [alpha]2 adrenoceptors, such as clonidine, are very effective antishivering drugs. Preliminary evidence also indicates that meperidine interacts with [alpha]2 adrenoceptors. The authors therefore studied the ability of meperidine to bind and activate each of the [alpha]2-adrenoceptor subtypes in a transfected cell system.

Methods: The ability of meperidine to bind to and inhibit forskolin-stimulated cyclic adenosine monophosphate formation as mediated by the three [alpha]2-adrenoceptor subtypes transiently transfected into COS-7 cells has been tested. The ability of the opioid antagonist naloxone and the [alpha]2-adrenoceptor antagonists yohimbine and RX821002 to block the analgesic action of meperidine in the hot-plate test was also assessed. The ability of meperidine to fit into the [alpha]2B adrenoceptor was assessed using molecular modeling techniques.

Results: Meperidine bound to all [alpha]2-adrenoceptor subtypes, with [alpha]2B having the highest affinity ([alpha]2B, 8.6 +/- 0.3 [mu]m; [alpha]2C, 13.6 +/- 1.5 [mu]m, P < 0.05; [alpha]2A, 38.6 +/- 0.7 [mu]m). Morphine was ineffective at binding to any of the receptor subtypes. Meperidine inhibited the production of forskolin-stimulated cyclic adenosine monophosphate mediated by all receptor subtypes but was most effective at the [alpha]2B adrenoceptor ([alpha]2B, 0.6 [mu]m; [alpha]2A, 1.3 mm; [alpha]2C, 0.3 mm), reaching the same level of inhibition (approximately 70%) as achieved with the [alpha]2-adrenoceptor agonist dexmedetomidine. The analgesic action of meperidine was blocked by naloxone but not by the [alpha]2-adrenoceptor antagonists yohimbine and RX821002. The modeling studies demonstrated that meperidine can fit into the [alpha]2B-adrenoceptor subtype.     

Activation of α2B-Adrenoceptors Mediates the Cardiovascular Effects of Etomidate

Background: The intravenous anesthetic etomidate exhibits structural similarities to specific [alpha]2-adrenoceptor agonists of the type such as dexmedetomidine. The current study was performed to elucidate the possible interaction of etomidate with [alpha]2-adrenoceptors in mice lacking individual [alpha]2-adrenoceptor subtypes ([alpha]2-KO).

Methods: Sedative and cardiovascular responses to etomidate and the [alpha]2-agonist, dexmedetomidine, were determined in mice deficient in [alpha]2-receptor subtypes. Inhibition of binding of the [alpha]2-receptor antagonist [3H]RX821002 to recombinant [alpha]2-receptors by etomidate was tested in human embryonic kidney (HEK293) cells in vitro.

Results: In vivo, loss and recovery of the righting reflex required similar times after intraperitoneal injection of etomidate in wild-type and in [alpha]2A-receptor-deficient mice, indicating that the hypnotic effect of etomidate in mice does not require the [alpha]2A-receptor subtype. Intravenous injection of etomidate resulted in a transient increase (duration 2.4 +/- 0.2 min) in arterial blood pressure in wild-type mice (17 +/- 3 mmHg). Etomidate did not affect blood pressure in [alpha]2B-KO or [alpha]2AB-KO mice. In membranes from HEK293 cells transfected with [alpha]2-receptors, etomidate inhibited binding of the [alpha]2-antagonist, [3H]RX821002, with higher potency from [alpha]2B- and [alpha]2C-receptors than from [alpha]2A-receptors (Ki [alpha]2A 208 [mu]m , [alpha]2B 26 [mu]m, [alpha]2C 56 [mu]m). In [alpha]2B-receptor-expressing HEK293 cells, etomidate rapidly increased phosphorylation of the extracellular signal-related kinases ERK1/2.     

Lidocaine Enhances Gαi Protein Function

Background: Local anesthetics inhibit several G protein-coupled receptors by interaction with the G[alpha]q protein subunit. It is not known whether this effect on G protein function can be extrapolated to other classes of G proteins. The authors investigated interactions of lidocaine with the human adenosine 1 receptor (hA1R)-coupled signaling pathway. Activated A1Rs couple to adenylate cyclase via the pertussis toxin sensitive G[alpha]i protein, thereby decreasing cyclic adenosine monophosphate formation. A1Rs are widely expressed and abundant in the spinal cord, brain, and heart. Interactions of LAs with the hA1R-coupled transduction cascade therefore might produce a broad range of clinically relevant effects.

Methods: The function of hA1Rs stably expressed in Chinese hamster ovary cells was determined with assays of cyclic adenosine monophosphate, receptor binding, and guanosine diphosphate/guanosine triphosphate [gamma]35S exchange by using reconstituted defined G protein subunits. Involvement of phosphodiesterase and G[alpha]i was characterized by using the phosphodiesterase inhibitor rolipram and pertussis toxin, respectively.

Results: Lidocaine (10-9-10-1 M) had no significant effects on agonist or antagonist binding to the hA1R or on receptor-G protein interactions. However, cyclic adenosine monophosphate levels were reduced significantly to 50% by the LAs, even in the absence of an A1R agonist or presence of an A1R antagonist. This effect was unaffected by rolipram (10 [mu]m), but abolished completely by pretreatment with pertussis toxin, which inactivates the G[alpha]i protein. Therefore, the main target site for LAs in this pathway is located upstream from adenylate cyclase.     

Differential Effects of Volatile Anesthetics on M3 Muscarinic Receptor Coupling to the Gαq Heterotrimeric G Protein

Background: Halothane inhibits airway smooth muscle contraction in part by inhibiting the functional coupling between muscarinic receptors and one of its cognate heterotrimeric G proteins, G[alpha]q. Based on previous studies indicating a more potent effect of halothane and sevoflurane on airway smooth muscle contraction compared with isoflurane, the current study hypothesized that at anesthetic concentrations of 2 minimum alveolar concentration (MAC) or less, halothane and sevoflurane but not isoflurane inhibit acetylcholine-promoted G[alpha]q guanosine nucleotide exchange.

Methods: G[alpha]q guanosine nucleotide exchange was measured in crude membranes prepared from COS-7 cells transiently coexpressing the human M3 muscarinic receptor and human G[alpha]q. A radioactive, nonhydrolyzable analog of guanosine-5'-triphosphate, [35S]GTP[gamma]S, was used as a reporter for nucleotide exchange at G[alpha]q.

Results: Acetylcholine caused a concentration-dependent increase in G[alpha]q [35S]GTP[gamma]S-GDP exchange. Neither anesthetic affected constitutive G[alpha]q [35S]GTP[gamma]S-GDP exchange in the absence of acetylcholine. Conversely, each anesthetic caused a concentration-dependent and reversible inhibition of G[alpha]q [35S]GTP[gamma]S-GDP exchange when promoted by acetylcholine. At concentrations of 3 MAC or less, the effect of halothane and sevoflurane were significantly greater than that of isoflurane, with only a minimal inhibition by isoflurane observed at 2 MAC.     

Fentanyl Attenuates α1B-Adrenoceptor-Mediated Pulmonary Artery Contraction

Background: The authors tested the hypothesis that the intravenous anesthetic fentanyl would attenuate the pulmonary vasoconstrictor response to [alpha]1-adrenoceptor activation. They also investigated the [alpha]1-adrenoceptor subtypes that could potentially mediate this effect of fentanyl.

Methods: Endothelium-denuded canine pulmonary arterial rings were suspended for isometric tension recording. Dose-response curves for the [alpha]1-adrenoceptor agonist phenylephrine were generated in the absence and presence of fentanyl. The effects of inhibiting [alpha]2 (rauwolscine), [alpha]1 (prazosin), [alpha]1A (5-methylurapidil), [alpha]1B (chloroethylclonidine), and [alpha]1D (BMY 7378) adrenoceptors on phenylephrine contraction were also investigated. Receptor "protection" studies were performed to investigate the specific role of [alpha]1B adrenoceptors in mediating fentanyl-induced changes in phenylephrine contraction. Finally, competition binding studies were performed in rat-1 fibroblasts stably transfected with human [alpha]1-adrenoceptor complementary DNAs corresponding to the [alpha]1A-, [alpha]1B-, or [alpha]1D-adrenoceptor subtypes to directly assess whether fentanyl can compete for the [alpha]1-adrenoceptor activation pocket.

Results: Fentanyl attenuated phenylephrine contraction in a dose-dependent fashion. Rauwolscine had no effect on phenylephrine contraction. Phenylephrine contraction was inhibited by prazosin and abolished by chloroethylclonidine but was relatively resistant to inhibition by 5-methylurapidil and BMY 7378. Pretreatment with fentanyl before exposure to chloroethylclonidine increased the maximal contractile response to phenylephrine compared to chloroethylclonidine pretreatment alone. Competition binding studies revealed that fentanyl binds to all three [alpha]1-adrenoceptor subtypes, with a fivefold greater affinity for the [alpha]1B-adrenoceptor compared with the [alpha]1D-adrenoceptor subtype.     

Production of Paradoxical Sensory Hypersensitivity by α2-Adrenoreceptor Agonists

Background: Administration of opioid receptor agonists is followed by paradoxical sensory hypersensitivity. This hypersensitivity has been suggested to contribute to the antinociceptive tolerance observed with opioids. The authors hypothesized that [alpha]2-adrenoreceptor agonists, which also produce antinociceptive tolerance, would produce sensory hypersensitivity.

Methods: [alpha]2-Adrenoreceptor agonists were administered to male Sprague-Dawley rats as a single subcutaneous injection, a continuous subcutaneous infusion, a single intrathecal injection, or a continuous intrathecal infusion. Thermal sensitivity was determined using latency to withdrawal of the hind paw from radiant heat. Tactile sensitivity was determined using withdrawal threshold to von Frey filaments. Spinal dynorphin content was measured by enzyme immunoassay.

Results: Single systemic or intrathecal injections of clonidine or dexmedetomidine produced antinociception followed by delayed thermal and tactile hypersensitivity. Six-day systemic or intrathecal infusion of clonidine produced tactile and thermal hypersensitivity observed even during clonidine infusion. Sensory hypersensitivity was prevented by coadministration of the [alpha]2-adrenoreceptor-selective antagonist idazoxan or the N-methyl-d-aspartate receptor-selective antagonist MK-801. Six-day infusion of intrathecal clonidine increased dynorphin content in dorsal lumbar spinal cord. MK-801 and dynorphin antiserum reversed clonidine-induced sensory hypersensitivity.