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.     

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.     

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.     

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.     

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.     

Systemically Administered α2-Agonist-induced Peripheral Vasoconstriction in Humans

Background: [alpha]2-Adrenoceptors mediate both sympatholytic and vasoconstrictive hemodynamic effects. The goal of this study was to profile the peripheral vasoconstrictive effects of a selective [alpha]2-adrenoceptor agonist in isolation from the sympatholytic effects it also induces.

Methods: The authors administered increasing plasma target concentrations of dexmedetomidine (0.075, 0.15, 0.3, and 0.6 ng/mL) or saline placebo to healthy young volunteers in whom the sympatholytic effects of the drug were attenuated in one of two ways: general anesthesia (propofol-alfentanil-nitrous oxide) or axillary brachial plexus block. Measurements were made of finger blood volume (an indicator of vasoconstriction) by photoplethysmographic determination of light transmitted through a finger (LTF) and hemodynamic variables. Measurements made before and during the four steps of infusion were compared by repeated-measures ANOVA.

Results: In anesthetized volunteers, all concentrations of dexmedetomidine increased LTF (vasoconstriction) and systolic blood pressure (P < 0.001 for both), whereas placebo did not. In awake volunteers, all concentrations decreased systolic blood pressure (P < 0.001). Concentrations of 0.15, 0.3, and 0.6 ng/mL decreased LTF (vasodilation) in the neurally intact hand; in contrast, the same concentrations increased LTF (vasoconstriction) in the sympathectomized hand (P < 0.001 for both).     

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 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.     

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.     

β2-Adrenoceptor Genotype Affects Vasopressor Requirements during Spinal Anesthesia for Cesarean Delivery

Background: Maternal hypotension is common after spinal anesthesia for cesarean delivery. There is wide variability in the incidence and severity of hypotension and in the response to treatment. The [beta]2 adrenoceptor ([beta]2AR) possesses several polymorphic sites. Codons 16 (Arg16Gly) and 27 (Glu27Gln) have been shown to affect desensitization of the receptor. The goal of this study was to determine whether genetic variants of the [beta]2AR alter incidence of hypotension or the amount of vasopressor treatment required during spinal anesthesia for cesarean delivery.

Methods: One hundred seventy healthy women undergoing elective cesarean delivery were studied. Spinal anesthesia was performed with 12 mg hyperbaric bupivacaine, 25 [mu]g fentanyl, and 200 [mu]g morphine. Hypotension was treated with ephedrine and/or phenylephrine intravenously, and [beta]2AR genotype at codons 16 and 27 was determined. Analysis of variance was used to compare variables between genotypes, with data expressed as mean +/- SD.

Results: Ephedrine or phenylephrine was used in more than 90% of patients, with no difference in the incidence of hypotension between [beta]2AR genotypes. However, there was a significant effect of genotype on the amount of vasopressor required. Gly16 homozygotes received significantly less ephedrine (18 +/- 14 mg) than Arg16 homozygotes (28 +/- 13 mg) and Arg16Gly heterozygotes (30 +/- 20 mg; P = 0.0005). Glu27 homozygotes required significantly less ephedrine than Gln 27 homozygotes (14 +/- 13 vs. 30 +/- 19 mg; P = 0.002). Gln27Glu heterozygotes received less ephedrine than Gln27 homozygotes (23 +/- 16 vs. 30 +/- 19 mg; P = 0.03).     

α2-Adrenoceptor Activation by Clonidine Enhances Stimulation-evoked Acetylcholine Release from Spinal Cord Tissue after Nerve Ligation in Rats

Background: Spinally administered clonidine produces analgesia via [alpha]2-adrenergic receptors. The analgesic potency of clonidine and its dependency on muscarinic acetylcholine receptors increase in rats after nerve injury. The authors hypothesized that these changes reflect greater acetylcholine release from the spinal cord by clonidine, either through direct or indirect effects.

Methods: Male Sprague-Dawley rats were divided into two groups: no surgery or left L5 and L6 spinal nerve ligation (SNL). All experiments were performed 3 weeks after SNL. Crude synaptosomes were prepared from the spinal enlargement and loaded with [3H]choline. Samples were incubated with clonidine in the absence or presence of KCl depolarization. The authors also examined the effect of clonidine on KCl evoked acetylcholine release using perfusion of spinal cord slices, in which some spinal circuitry is maintained.

Results: In synaptosomes, clonidine alone induced minimal acetylcholine release, which was actually greater in tissue from normal rats than in tissue from SNL rats. In the presence of KCl depolarization, however, clonidine enhanced acetylcholine release in tissue from SNL rats but inhibited release in tissue from normal rats. Similarly, in spinal cord slices, clonidine enhanced KCl evoked acetylcholine release in tissue from SNL animals but inhibited such release in tissue from normal animals. The [alpha]2-adrenoceptor antagonist idazoxan inhibited the effects of clonidine in slices from SNL rats.     

Moxonidine, a Selective Imidazoline-α2-Adrenergic Receptor Agonist, Produces Spinal Synergistic Antihyperalgesia with Morphine in Nerve-injured Mice

Background: Moxonidine, a novel imidazoline-[alpha]2-adrenergic receptor-selective analgesic, was recently identified as antinociceptive but has yet to be evaluated in neuropathic pain models. [alpha]2-adrenergic receptor-selective analgesics, and high-efficacy opioids, effectively inhibit neuropathic pain behaviors in rodents. In contrast, morphine potency and efficacy decreases in states of neuropathic pain, both in rodents and in humans, but may be restored or enhanced by coadministration of morphine with [alpha]2-adrenergic receptor-selective analgesics. The current experiments extend the evaluation of opioid-coadjuvant interactions in neuropathic subjects by testing the respective antihyperalgesic interactions of moxonidine and clonidine with morphine in a test of mechanical hyperalgesia.

Methods: Nerve-injured mice (Chung model) were spinally administered moxonidine, clonidine, morphine, and the combinations moxonidine-morphine and clonidine-morphine. Hyperalgesia was detected by von Frey monofilament stimulation (3.3 mN) to the hind paws (plantar surface). The ED50 values were calculated and the interactions tested by isobolographic analysis.

Results: In nerve-injured mice, moxonidine, clonidine, and morphine all dose-dependently inhibited mechanical hyperalgesia. Furthermore, the combinations of moxonidine-morphine and clonidine-morphine resulted in substantial leftward shifts in the dose-response curves compared with those of each agonist administered separately. The calculated ED50 values of the dose-response curves of these combinations were significantly lower than their corresponding theoretical additive ED50 values. These results confirmed that both interactions were synergistic.     

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.     

Effects of the α2-Adrenoceptor Agonist Dexmedetomidine on Bronchoconstriction in Dogs

Background: Tracheal intubation can elicit reflex bronchoconstriction in patients with asthma or chronic obstructive pulmonary disease, complicating mechanical ventilation and weaning from mechanical support. In vitro studies of human and animal bronchial tissue indicate that [alpha]2-adrenoceptor stimulation can lead to smooth muscle relaxation and prevention of bronchoconstriction. Dexmedetomidine is a selective [alpha]2-adrenoceptor agonist approved for sedation in the intensive care unit. Whether dexmedetomidine can affect reflex bronchoconstriction is unknown.

Methods: After the approval of the institutional animal care and use committee, five mongrel dogs were anesthetized with thiopental, endotracheally intubated, and ventilated, and their airways were challenged with histamine. High-resolution computed tomography was used to measure airway luminal areas at baseline and after nebulized histamine. After recovery to baseline, on separate days, dexmedetomidine (0.5 [mu]g/kg) was administered either intravenously or as an aerosol, and the histamine challenge was repeated.

Results: At baseline, histamine constricted the airways to 66 +/- 27% (mean +/- SD) (P < 0.0001) and 59 +/- 30% (P < 0.0001) of maximum on the days dexmedetomidine was administered by intravenous and inhalational means, respectively. After recovery, intravenous administration of dexmedetomidine blocked the histamine-induced bronchoconstriction (87 +/- 30.4% of maximum, compared with histamine alone (P < 0.0001), whereas dexmedetomidine administered by inhalation showed no protective effect (45 +/- 30% of maximum; P < 0.0001 compared with histamine alone).     

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.     

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.     

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.