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.     

Sevoflurane Enhances γ-Aminobutyric Acid Type A Receptor Function and Overall Inhibition of Inspiratory Premotor Neurons in a Decerebrate Dog Model

Background: Inspiratory premotor neurons in the caudal ventral medulla relay excitatory drive to phrenic and inspiratory intercostal motoneurons in the spinal cord. These neurons are subject to tonic [gamma]-aminobutyric acid type A (GABAA)ergic inhibition. In a previous study, 1 minimum alveolar concentration (MAC) sevoflurane depressed overall glutamatergic excitatory drive and enhanced overall GABAAergic inhibitory drive to the neurons. This study investigated in further detail the effects of sevoflurane on GABAAergic inhibition by examining postsynaptic GABAA receptor activity in these neurons.

Methods: Studies were performed in decerebrate, vagotomized, paralyzed, and mechanically ventilated dogs during hypercapnic hyperoxia. The effect of 1 MAC sevoflurane on extracellularly recorded neuronal activity was measured during localized picoejection of the GABAA receptor antagonist bicuculline and the GABAA agonist muscimol. Complete blockade of GABAAergic inhibition by bicuculline allowed estimation of the prevailing overall inhibition of the neuron. The neuronal response to muscimol was used to assess the anesthetic effect on the postsynaptic GABAA receptor function.

Results: One MAC sevoflurane depressed the spontaneous activity of 21 inspiratory premotor neurons by (mean +/- SD) 32.6 +/- 20.5% (P < 0.001). Overall excitatory drive was depressed 17.9 +/- 19.8% (P < 0.01). Overall GABAAergic inhibition was enhanced by 18.5 +/- 18.2% (P < 0.001), and the postsynaptic GABAA receptor function was increased by 184.4 +/- 121.8% (n = 20; P < 0.001).     

Selective γ-Aminobutyric Acid Type A Receptor Antagonism Reverses Isoflurane Ischemic Neuroprotection

Background: Isoflurane provides protection against severe forebrain ischemia in the rat. The authors hypothesized that this is attributable to interaction with the [gamma]-aminobutyric acid type A (GABAA) receptor resulting in altered time to onset of ischemic hippocampal depolarization.

Methods: Organotypic hippocampal slices were subjected to oxygen-glucose deprivation in the presence of isoflurane and combinations of GABAA (bicuculline) and GABAB (phaclofen) receptor antagonists. Cell death was measured. Rats were subjected to severe forebrain ischemia while anesthetized with fentanyl-nitrous oxide or 1.4% isoflurane. In the isoflurane group, rats also received intravenous bicuculline (0, 1, or 2 mg/kg). Neurologic and histologic outcomes and time to depolarization were assessed.

Results: In slices, 2% isoflurane caused near-complete protection against oxygen-glucose deprivation. This was unaffected by coadministration of phaclofen but largely reversed by bicuculline. The GABAA agonist muscimol was also protective, having an effect equivalent to 1% isoflurane. In rats, isoflurane (0 mg bicuculline) improved neurologic and histologic outcome versus fentanyl-nitrous oxide (CA1 percentage of alive neurons: fentanyl-nitrous oxide, 15 +/- 7; isoflurane, 61 +/- 24). The isoflurane effect was reversed in a dose-dependent manner by bicuculline (CA1 percentage alive: 1 mg/kg, 44 +/- 22; 2 mg/kg, 21 +/- 15). Time to depolarization was delayed with isoflurane versus fentanyl-nitrous oxide (137 vs. 80 s) but was not affected by bicuculline (149 s). In contrast, postischemic time to repolarization was more rapid with fentanyl-nitrous oxide or isoflurane plus bicuculline versus isoflurane alone.     

γ-Aminobutyric Acid Type A Receptor β2 Subunit Mediates the Hypothermic Effect of Etomidate in Mice

Background: The authors have previously described that the [gamma]-aminobutyric acid type A (GABAA) receptor [beta]2N265S mutation results in a knock-in mouse with reduced sensitivity to etomidate. After recovery from etomidate anesthesia, these mice have improved motor performance and less slow wave sleep. Because most clinically used anesthetics produce hypothermia, the effect of this mutation on core body temperature was investigated.

Methods: The effect of etomidate and propofol on core body temperature were measured using radiotelemetry in freely moving GABAA receptor [beta]2N265S mutant mice and wild-type controls.

Results: [beta]2N265S mutant mice have a reduced hypothermic response to anesthetic doses of etomidate compared with wild-type controls and after a transient loss of righting reflex regain normothermia more rapidly compared with wild-type controls. Subanesthetic doses of etomidate produce hypothermia, which was not observed in the mutant mice. Vehicle administration resulted in a stress-induced hyperthermic response in both genotypes. Propofol produced a hypothermic response that was similar in both genotypes.     

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).     

Opposing Actions of Etomidate on Cortical Theta Oscillations Are Mediated by Different γ-Aminobutyric Acid Type A Receptor Subtypes

Background: Cortical networks generate diverse patterns of rhythmic activity. Theta oscillations (4-12 Hz) are commonly observed during spatial learning and working memory tasks. The authors ask how etomidate, acting predominantly via [gamma]-aminobutyric acid type A (GABAA) receptors containing [beta]2 or [beta]3 subunits, affects theta activity in vitro.

Methods: To characterize the effects of etomidate, the authors recorded action potential firing together with local field potentials in slice cultures prepared from the neocortex of the [beta]3(N265M) knock-in mutant and wild type mice. Actions of etomidate were studied at 0.2 [mu]m, which is approximately 15% of the concentration causing immobility (~1.5 [mu]m).

Results: In preparations derived from wild type and [beta]3(N265M) mutant mice, episodes of ongoing activity spontaneously occurred at a frequency of approximately 0.1 Hz and persisted for several seconds. Towards the end of these periods, synchronized oscillations in the theta band developed. These oscillations were significantly depressed in slices from [beta]3(N265M) mutant mice (P < 0.05). In this preparation etomidate acts almost exclusively via [beta]2 subunit containing GABAA receptors. In contrast, no depression was observed in slices from wild type mice, where etomidate potentiates both [beta]2- and [beta]3-containing GABAA receptors.     

A Conserved Tyrosine in the β2 Subunit M4 Segment Is a Determinant of γ-Aminobutyric Acid Type A Receptor Sensitivity to Propofol

Background: The [gamma]-aminobutyric acid type A receptor (GABAA-R) [beta] subunits are critical targets for the actions for several intravenous general anesthetics, but the precise nature of the anesthetic binding sites are unknown. In addition, little is known about the role the fourth transmembrane (M4) segment of the receptor plays in receptor function. The aim of this study was to better define the propofol binding site on the GABAA-R by conducting a tryptophan scan in the M4 segment of the [beta]2 subunit.

Methods: Seven tryptophan mutations were introduced into the C-terminal end of the M4 segment of the GABAA-R [beta]2 subunit. GABAA-R subunit complementary DNAs were transfected into human embryonic kidney 293 cells grown on glass coverslips. After transfection (36-72 h), coverslips were transferred to a perfusion chamber to assay receptor function. Cells were whole cell patch clamped and exposed to GABA, propofol, etomidate, and pregnenolone. Chemicals were delivered to the cells using two 10-channel infusion pumps and a rapid solution exchanger.

Results: All tryptophan mutations were well tolerated, and with one exception, all resulted in minimal changes in receptor activation by GABA. One mutation, [beta]2(Y444W), selectively suppressed the ability of propofol to enhance receptor function while retaining normal sensitivity to etomidate and pregnenolone.     

Propofol Modulates γ-Aminobutyric Acid-mediated Inhibitory Neurotransmission to Cardiac Vagal Neurons in the Nucleus Ambiguus

Background: Although it is well recognized that anesthetics modulate the central control of cardiorespiratory homeostasis, the cellular mechanisms by which anesthetics alter cardiac parasympathetic activity are poorly understood. One common site of action of anesthetics is inhibitory neurotransmission. This study investigates the effect of propofol on [gamma]-aminobutyric acid-mediated (GABAergic) and glycinergic neurotransmission to cardiac parasympathetic neurons.

Methods: Cardiac parasympathetic neurons were identified in vitro by the presence of a retrograde fluorescent tracer, and spontaneous GABAergic and glycinergic synaptic currents were examined using whole cell patch clamp techniques.

Results: Propofol at concentrations of 1.0 [mu]m and greater significantly (P < 0.05) increased the duration and decay time of spontaneous GABAergic inhibitory postsynaptic currents. To determine whether the action of propofol was at presynaptic or postsynaptic sites, tetrodotoxin was applied to isolate miniature inhibitory postsynaptic currents. Propofol at concentrations of 1.0 [mu]m and greater significantly (P < 0.05) prolonged the decay time and duration of miniature inhibitory postsynaptic currents, indicating that propofol directly alters GABAergic neurotransmission at a postsynaptic site. Propofol at high concentrations (>=50 [mu]m) also inhibited the frequency of both GABAergic inhibitory postsynaptic currents and miniature inhibitory postsynaptic currents. Propofol at concentrations up to 50 [mu]m had no effect on glycinergic neurotransmission.     

Halothane Enhances γ-Aminobutyric Acid Receptor Type A Function but Does Not Change Overall Inhibition in Inspiratory Premotor Neurons in a Decerebrate Dog Model

Background: Inspiratory premotor neurons in the caudal ventral medulla relay excitatory drive to phrenic and inspiratory intercostal motoneurons in the spinal cord. These neurons are subject to tonic [gamma]-aminobutyric acid type A (GABAA)-mediated (GABAAergic) inhibition. In a previous study, 1 minimum alveolar concentration (MAC) halothane depressed overall glutamatergic excitatory drive but did not change overall inhibitory drive to the neurons. This study investigated in further detail the effects of halothane on GABAAergic inhibition by examining postsynaptic GABAA receptor activity in these neurons.

Methods: Studies were performed in decerebrate, vagotomized, paralyzed, and mechanically ventilated dogs during hypercapnic hyperoxia. The effect of 1 MAC halothane on extracellularly recorded neuronal activity was measured during localized picoejection of the GABAA receptor antagonist bicuculline and the GABAA agonist muscimol. Complete blockade of GABAergic inhibition by bicuculline allowed estimation of the prevailing overall inhibition of the neuron. The neuronal response to muscimol was used to assess the anesthetic effect on the postsynaptic GABAA receptor function.

Results: One minimum alveolar concentration halothane depressed the spontaneous activity of 19 inspiratory premotor neurons by 22.9 +/- 29.1% (mean +/- SD; P < 0.01). Overall excitatory drive was depressed 23.6 +/- 16.9% (P < 0.001). Overall GABAergic inhibition was not changed (+8.7 +/- 27.5%; P = 0.295), but the postsynaptic GABAA receptor function was increased by 110.3 +/- 97.5% (P < 0.001).     

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.     

Ketamine Inhibits Inspiratory-evoked γ-Aminobutyric Acid and Glycine Neurotransmission to Cardiac Vagal Neurons in the Nucleus Ambiguus

Background: Ketamine can be used for perioperative pain management as well as a dissociative anesthetic agent in emergency situations. However, ketamine can induce both cardiovascular and respiratory depression, especially in pediatric patients. Although ketamine has usually been regarded as sympathoexcitatory, recent work has demonstrated that ketamine has important actions on parasympathetic cardiac vagal efferent activity. The current study tests the hypothesis that ketamine, at clinical relevant concentrations, alters central cardiorespiratory interactions in the brainstem and, in particular, the inspiration-evoked increase in [gamma]-aminobutyric acid-mediated and glycinergic neurotransmission to parasympathetic cardiac efferent neurons.

Methods: Cardiac vagal neurons were identified by the presence of a retrograde fluorescent tracer. Respiratory evoked [gamma]-aminobutyric acid-mediated and glycinergic synaptic currents were recorded in cardiac vagal neurons using whole cell patch clamp techniques while spontaneous rhythmic respiratory activity was recorded simultaneously.

Results: Ketamine, at concentrations from 0.1 to 10 [mu]m, evoked a concentration-dependent inhibition of inspiratory burst frequency. Inspiration-evoked [gamma]-aminobutyric acid-mediated neurotransmission to cardiac vagal neurons was inhibited at ketamine concentrations of 0.5 and 1 [mu]m. The increase in glycinergic activity to cardiac vagal neurons during inspiration was also inhibited at ketamine concentrations of 0.5 and 1 [mu]m.     

Modulation of γ-Aminobutyric Acid Type A Receptor-mediated Spontaneous Inhibitory Postsynaptic Currents in Auditory Cortex by Midazolam and Isoflurane

Background: Anesthetic agents that target [gamma]-aminobutyric acid type A (GABAA) receptors modulate cortical auditory evoked responses in vivo, but the cellular targets involved are unidentified. Also, for agents with multiple protein targets, the relative contribution of modulation of GABAA receptors to effects on cortical physiology is unclear. The authors compared effects of the GABAA receptor-specific drug midazolam with the volatile anesthetic isoflurane on spontaneous inhibitory postsynaptic currents (sIPSCs) in pyramidal cells of auditory cortex.

Methods: Whole cell recordings were obtained in murine brain slices at 34[degrees]C. GABAA sIPSCs were isolated by blocking ionotropic glutamate receptors. Effects of midazolam and isoflurane on time course, amplitude, and frequency of sIPSCs were measured.

Results: The authors detected no effect of midazolam at 0.01 [mu]m on sIPSCs, whereas midazolam at 0.1 and 1 [mu]m prolonged the decay of sIPSCs by approximately 25 and 70%, respectively. Isoflurane at 0.1, 0.25, and 0.5 mm prolonged sIPSCs by approximately 45, 150, and 240%, respectively. No drug-specific effects were observed on rise time or frequency of sIPSCs. Isoflurane at 0.5 mm caused a significant decrease in sIPSC amplitude.     

Intracerebroventricular Morphine Produces Antinociception by Evoking γ-Aminobutyric Acid Release through Activation of 5-Hydroxytryptamine 3 Receptors in the Spinal Cord

Background: It has been generally considered that supraspinal morphine activates the serotonergic descending inhibitory system and releases serotonin (5-hydroxytryptamine [5-HT]) in the spinal cord, producing antinociception through activation of 5-HT receptors. The involvement of a spinal [gamma]-aminobutyric acid-mediated (GABAergic) system is also suggested in supraspinal morphine antinociception. It has been reported that spinal GABAergic system contributes to 5-HT3 receptor-mediated antinociception. In this study, the authors investigated the contribution of spinal 5-HT3 receptor and the GABAergic system in the intracerebroventricular morphine-induced antinociception.

Methods: Male Sprague-Dawley rats were used. Using the spinal microdialysis method, concentrations of 5-HT and GABA were measured after intracerebroventricular morphine administration. The effect of intracerebroventricular naloxone or spinal perfusion of a selective 5-HT3 receptor antagonist 3-tropanyl-indole-3-carboxylate methiodide on the spinal release of GABA after intracerebroventricular morphine administration was also examined. In the behavioral study, involvement of 5-HT3 receptors or GABAA receptors in the intracerebroventricular morphine-induced antinociceptive effect was investigated using the tail-flick test.

Results: Intracerebroventricular morphine (40 nmol) significantly increased spinal GABA and 5-HT release. Evoked spinal GABA release was reversed by intracerebroventricular naloxone (40 nmol) or spinal perfusion of 3-tropanyl-indole-3-carboxylate methiodide (1 mm). In the behavioral study, intracerebroventricular morphine produced significant antinociception. Intrathecal administration of either GABAA receptor antagonist bicuculine or 3-tropanyl-indole-3-carboxylate methiodide but not vehicle reversed the morphine-induced antinociceptive effect.     

Anesthetic Potency and Influence of Morphine and Sevoflurane on Respiration in μ-Opioid Receptor Knockout Mice

Background: The involvement of the [mu]-opioid receptor ([mu]OR) system in the control of breathing, anesthetic potency, and morphine- and anesthesia-induced respiratory depression was investigated in mice lacking the [mu]OR.

Methods: Experiments were performed in mice lacking exon 2 of the [mu]OR gene ([mu]OR-/-) and their wild-type littermates ([mu]OR+/+). The influence of saline, morphine, naloxone, and sevoflurane on respiration was measured using a whole body plethysmographic method during air breathing and elevations in inspired carbon dioxide concentration. The influence of morphine and naloxone on anesthetic potency of sevoflurane was determined by tail clamp test.

Results: Relative to wild-type mice, [mu]OR-deficient mice displayed approximately 15% higher resting breathing frequencies resulting in greater resting ventilation levels. The slope of the ventilation-carbon dioxide response did not differ between genotypes. In [mu]OR+/+ but not [mu]OR-/- mice, a reduction in resting ventilation and slope, relative to placebo, was observed after 100 mg/kg morphine. Naloxone increased resting ventilation and slope in both genotypes. Sevoflurane at 1% inspired concentration induced similar reductions in resting ventilation and slope in the two genotypes. Anesthetic potency was 20% lower in mutant relevant to wild-type mice. Naloxone and morphine caused an increase and decrease, respectively, in anesthetic potency in [mu]OR+/+ mice only.     

High Spinal Anesthesia for Cardiac Surgery: Effects on β-Adrenergic Receptor Function, Stress Response, and Hemodynamics

Background: This double-blind, randomized, controlled trial examined the effect of high-dose intrathecal bupivacaine in combination with general anesthesia on atrial [beta]-adrenergic receptor function, the stress response, and hemodynamics during coronary artery bypass graft surgery.

Methods: Thirty-eight patients were randomized to either control (n = 19) or intrathecal bupivacaine (ITB) groups (n = 19). Patients in the ITB group received 37.5 mg intrathecal hyperbaric bupivacaine before induction of general anesthesia. Control patients received an injection of local anesthetic into the skin and subcutaneous tissues (sham spinal). Comparisons were made between groups with respect to atrial receptor desensitization and down-regulation, in addition to circulating catecholamines and hemodynamics.

Results: In patients with cardiopulmonary bypass (CPB) times in excess of 1 h, the ITB group had significantly less atrial [beta]-receptor dysfunction, as measured by maximal isproteronol, 50% maximal isoproterenol, sodium fluoride-stimulated activity, and zinterol stimulation assays of adenylyl cyclase activity (P <= 0.02) and [beta]-adrenergic receptor density (P = 0.02). Serum epinephrine, norepinephrine, and cortisol concentrations were significantly lower in the ITB group, independent of CPB times (P < 0.0001, P < 0.001, and P < 0.05, respectively). ITB patients had a higher cardiac index and a lower pulmonary vascular resistance index in the post-CPB time period (P < 0.01 and P < 0.05, respectively). In the pre-CPB period, mean arterial pressure and systemic vascular resistance index were significantly lower in the ITB group.     

Pulmonary Vascular Effects of Propofol at Baseline, during Elevated Vasomotor Tone, and in Response to Sympathetic α- and β-Adrenoreceptor Activation

Background: This in vivo study had two primary objectives. The first goal was to determine whether the pulmonary vascular effects of propofol depend on the preexisting level of vasomotor tone, and the second was to investigate the effects of propofol on the pulmonary vascular responses to sympathetic [alpha]- and [beta]-adrenoreceptor activation.

Methods: Thirty-one mongrel dogs were chronically instrumented to measure the left pulmonary vascular pressure-flow (LPQ) relation. Left lung autotransplantation (LLA) was also performed in eight additional dogs to induce a long-term increase in pulmonary vascular resistance. LPQ plots were measured on separate days in the conscious state and during propofol anesthesia. LPQ plots were measured at baseline and when vasomotor tone was acutely increased with the [alpha] agonist, phenylephrine, or the thromboxane mimetic, U46619. In separate experiments, cumulative dose-response curves to [alpha]- (phenylephrine) and [beta]- (isoproterenol) adrenoreceptor agonists were generated in conscious and propofol-anesthetized dogs.

Results: Compared with the conscious state, propofol had no effect on the baseline LPQ relation in normal or post-LLA dogs. However, propofol caused pulmonary vasoconstriction (P < 0.05) when vasomotor tone was acutely increased with either phenylephrine or U46619 in normal or post-LLA dogs. The pulmonary vasoconstrictor response to [alpha]-adrenoreceptor activation was potentiated (P < 0.05) during propofol anesthesia, whereas the pulmonary vasodilator response to [beta]-adrenoreceptor activation was not altered.     

Neonatal Exposure to a Combination of N-Methyl-d-aspartate and γ-Aminobutyric Acid Type A Receptor Anesthetic Agents Potentiates Apoptotic Neurodegeneration and Persistent Behavioral Deficits

Background: During the brain growth spurt, the brain develops and modifies rapidly. In rodents this period is neonatal, spanning the first weeks of life, whereas in humans it begins during the third trimester and continues 2 yr. This study examined whether different anesthetic agents, alone and in combination, administered to neonate mice, can trigger apoptosis and whether behavioral deficits occur later in adulthood.

Methods: Ten-day-old mice were injected subcutaneously with ketamine (25 mg/kg), thiopental (5 mg/kg or 25 mg/kg), propofol (10 mg/kg or 60 mg/kg), a combination of ketamine (25 mg/kg) and thiopental (5 mg/kg), a combination of ketamine (25 mg/kg) and propofol (10 mg/kg), or control (saline). Fluoro-Jade staining revealed neurodegeneration 24 h after treatment. The behavioral tests-spontaneous behavior, radial arm maze, and elevated plus maze (before and after anxiolytic)-were conducted on mice aged 55-70 days.

Results: Coadministration of ketamine plus propofol or ketamine plus thiopental or a high dose of propofol alone significantly triggered apoptosis. Mice exposed to a combination of anesthetic agents or ketamine alone displayed disrupted spontaneous activity and learning. The anxiolytic action of diazepam was less effective when given to adult mice that were neonatally exposed to propofol.     

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.     

Clinically Relevant Concentrations of Propofol but Not Midazolam Alter In Vitro Dendritic Development of Isolated γ-Aminobutyric Acid-positive Interneurons

Background: Recent laboratory studies showed that exposure to supraclinical concentrations of propofol can induce cell death of immature neurons. However, no data are available regarding the effects of clinically relevant concentrations of this agent on neuronal development. The authors addressed this issue by evaluating the effect of propofol on dendritic growth and arbor expansion of developing [gamma]-aminobutyric acid-positive (GABAergic) interneurons.

Methods: Immature neuroblasts were isolated from the newborn rat subventricular zone and differentiated into GABAergic interneurons in culture. In addition to cell death, the effects of increasing concentrations and durations of propofol exposure on neuronal dendritic development were evaluated using the following morphologic parameters: total dendritic length, primary dendrites, branching point, and Scholl analysis.

Results: The authors demonstrate that propofol induced cell death of GABAergic neurons at concentrations of 50 [mu]g/ml or greater. As little as 1 [mu]g/ml propofol significantly altered several aspects of dendritic development, and as little as 4 h of exposure to this agent resulted in a persistent decrease in dendritic growth. In contrast, application of midazolam did not affect neuronal development.