Modulation of recombination human gamma-aminobutyric acidA receptors by isoflurane: influence of the delta subunit

BACKGROUND: The gamma-aminobutyric acid (GABA)A receptor/chloride channel has a broad-spectrum anesthetic sensitivity and is a key regulator of arousal. Each receptor/channel complex is an assembly of five protein subunits. Six subunit classes have been identified, each containing one to six members; many combinations are expressed throughout the brain. Benzodiazepines and intravenous anesthetic agents are clearly subunit dependent, but the literature to date suggests that volatile anesthetics are not. The physiological role of the delta subunit remains enigmatic, and it has not been examined as a determinant of anesthetic sensitivity. METHODS: Combinations of GABA(A) receptor subunit cDNAs were injected into Xenopus laevis oocytes: alpha1beta1, alpha1beta1gamma2L, alpha1beta1delta, and alpha1beta1gamma2Ldelta. Expression of functional ion channels with distinct signalling and pharmacologic properties was demonstrated within 1-4 days by established electrophysiological methods. RESULTS: Co-expression of the delta subunit produced changes in receptor affinity; current density; and the modulatory efficacy of diazepam, zinc, and lanthanum; it also produced subtle changes in the rate of desensitization in response to GABA. Isoflurane enhanced GABA-induced responses from all combinations: alphabeta delta (>10-fold) > alphabeta > alphabeta gamma > or = alphabeta gammadelta (approximately 5-fold). Dose-response plots were bell shaped. Compared with alphabeta gamma receptors (EC50 = 225 microM), both alphabeta delta (EC50 = 372 microM) and alphabeta gammadelta (EC50 = 399 microM) had a reduced affinity for isoflurane. Isoflurane (at a concentration close to the EC50 for each subunit) increased the affinity of GABA for its receptor but depressed the maximal response (alphabeta gamma and alphabeta gammadelta). In contrast, the small currents through alphabeta delta receptors were enhanced, even at saturating agonist concentrations. CONCLUSIONS: Delta subunit expression alters GABA(A) receptor function but is not an absolute determinant of anesthetic sensitivity.     

Changing from isoflurane to desflurane toward the end of anesthesia does not accelerate recovery in humans

BACKGROUND: In an attempt to combine the advantage of the lower solubilities of new inhaled anesthetics with the lesser cost of older anesthetics, some clinicians substitute the former for the latter toward the end of anesthesia. The authors tried to determine whether substituting desflurane for isoflurane in the last 30 min of a 120-min anesthetic would accelerate recovery. METHODS: Five volunteers were anesthetized three times for 2 h using a fresh gas inflow of 2 l/min: 1.25 minimum alveolar concentration (MAC) desflurane, 1.25 MAC isoflurane, and 1.25 MAC isoflurane for 90 min followed by 30 min of desflurane concentrations sufficient to achieve a total of 1.25 MAC equivalent ("crossover"). Recovery from anesthesia was assessed by the time to respond to commands, by orientation, and by tests of cognitive function. RESULTS: Compared with isoflurane, the crossover technique did not accelerate early or late recovery (P > 0.05). Recovery from isoflurane or the crossover anesthetic was significantly longer than after desflurane (P < 0.05). Times to response to commands for isoflurane, the crossover anesthetic, and desflurane were 23 +/- 5 min (mean +/- SD), 21 +/- 5 min, and 11 +/- 1 min, respectively, and to orientation the times were 27 +/- 7 min, 25 +/- 5 min, and 13 +/- 2 min, respectively. Cognitive test performance returned to reference values 15-30 min sooner after desflurane than after isoflurane or the crossover anesthetic. Isoflurane cognitive test performance did not differ from that with the crossover anesthetic at any time. CONCLUSIONS: Substituting desflurane for isoflurane during the latter part of anesthesia does not improve recovery, in part because partial rebreathing through a semiclosed circuit limits elimination of isoflurane during the crossover period. Although higher fresh gas flow during the crossover period would speed isoflurane elimination, the amount of desflurane used and, therefore, the cost would increase.     

Regional deposition and retention of particles in shallow, inhaled boluses: effect of lung volume

We evaluated the effects of volatile anesthetics on T-type calcium current (ICa,T) present in four different cell types using the whole cell version of the patch clamp technique. In dorsal root ganglion neurons and in two neuroendocrine cells--adrenal glomerulosa cells (AG) and thyroid C-cells--ICa,T was reversibly decreased by volatile anesthetics at clinically relevant concentrations, with isoflurane and enflurane being more potent that halothane. In AG cells, the most sensitive cell type tested, ICa,T was reduced 47%+/-4% (n = 6) by isoflurane (0.7 mM) and 56%+/-2% (n = 5) by enflurane (1.2 mM), but by only 24%+/-1% (n = 5; P < 0.05) by halothane (0.7 mM). Isoflurane caused a significant increase in the rate of deactivation of ICa,T in AG cells. In ventricular myocytes, however, ICa,T was much less sensitive to both isoflurane and halothane. The differential sensitivity of ICa,T in various cell types to the anesthetics may reflect differences in the channels expressed in these tissues or differences in the cellular intermediates involved in anesthetic action. Depression of ICa,T in neuronal cells may contribute to anesthetic action through decreases in cellular excitability. IMPLICATIONS: Using the patch clamp technique, we showed that T-type calcium channels, which promote cellular excitability, are inhibited by volatile anesthetics in neuronal and neuroendocrine cells, but not in ventricular myocytes. Inhibition of neuronal T-type channels may contribute to the mechanism of action of volatile anesthetics.     

Positive modulation of human gamma-aminobutyric acid type A and glycine receptors by the inhalation anesthetic isoflurane

The interactions of the inhalation anesthetic agent isoflurane with ligand-gated chloride channels were studied using transient expression of recombinant human receptors in a mammalian cell line. Isoflurane enhanced gamma-aminobutyric acid (GABA)-activated chloride currents in cells that expressed heteromeric GABAA receptors consisting of combinations of alpha 1 or alpha 2, beta 1, and gamma 2 subunits and in cells that expressed receptors consisting of combinations of only alpha and beta subunits. Receptors consisting of alpha 2 and gamma 2 subunits were poorly expressed but were sensitive to isoflurane. Receptors consisting of beta 1 and gamma 2 subunits were not expressed. Isoflurane also enhanced glycine-activated chloride currents through homomeric alpha glycine receptors but did not enhance GABA currents in cells expressing homomeric rho 1 receptors. These results show that not all ligand-gated chloride channel receptors are sensitive to isoflurane and, therefore, that the anesthetic interacts with specific structural determinants of these ion channel proteins.     

Inhibition of sodium/calcium exchange and calcium channels of heart cells by volatile anesthestics

BACKGROUND: Volatile anesthetics exert profound effects on the heart, probably through their effect on Ca2+ movements during the cardiac cycle. Ca2+ movements across the sarcolemma are thought to involve mainly Ca2+ channels and the Na+/Ca2+ exchanger. We have therefore investigated the action of halothane, isoflurane, and enflurane on Na+/Ca2+ exchange and Ca2+ channel activity to assess the contribution of these pathways to the observed effect of the anesthetics on the myocardium. METHODS: Sarcolemmal ion fluxes were investigated using radioisotope uptake by isolated adult rat heart cells in suspension. Na+/Ca2+ exchange activity was measured from 45Ca2+ uptake by Na(+)-loaded cells. Ca2+ channel activity was measured from verapamil-sensitive trace 54Mn2+ uptake during electric stimulation. RESULTS: Halothane, isoflurane, and enflurane inhibited Na+/Ca2+ exchange completely, with similar potency when concentrations were expressed in millimolar units in aqueous medium but not when expressed as minimum alveolar concentration (MAC). The inhibition by enflurane was particularly strong, > 50%, at 2 MAC. In contrast, the three anesthetics inhibited Ca2+ channels with similar potency when concentrations were expressed as MAC but not when expressed in millimolar units in aqueous medium. Hill plots of pooled data with all three anesthetics showed a slope of -3.87 +/- 0.50 for inhibition of Na+/Ca2+ exchange and -1.73 +/- 0.19 for inhibition of Ca2+ channels. CONCLUSIONS: Halothane, isoflurane, and enflurane inhibit both Na+/Ca2+ exchange and Ca2+ channels at concentrations relevant to anesthesia, although they exhibit differences in potency and number of sites of action. At 1.5 MAC, halothane inhibits Ca2+ channels more than Na+/Ca2+ exchange, whereas enflurane inhibits Na+/Ca2+ exchange more than Ca2+ channels. Isoflurane inhibited both systems equally. The inhibition of Ca2+ influx by these agents is likely to contribute to their negative inotropic effect in the heart. The inhibition of Na+/Ca2+ exchange by enflurane may account for its observed action of delaying relaxation in species lacking sarcoplasmic reticulum.     

Patients treated by cardiologists have a lower in-hospital mortality for acute myocardial infarction

Local anesthetics suppress excitability by interfering with ion channel function. Ensheathment of peripheral nerve fibers, however, impedes diffusion of drugs to the ion channels and may influence the evaluation of local anesthetic potencies. Investigating ion channels in excised membrane patches avoids these diffusion barriers. We investigated the effect of local anesthetics with voltage-dependent Na+ and K+ channels in enzymatically dissociated sciatic nerve fibers of Xenopus laevis using the patch clamp method. The outside-out configuration was chosen to apply drugs to the external face of the membrane. Local anesthetics reversibly blocked the transient Na+ inward current, as well as the steady-state K+ outward current. Half-maximal tonic inhibiting concentrations (IC50), as obtained from concentration-effect curves for Na+ current block were: tetracaine 0.7 microM, etidocaine 18 microM, bupivacaine 27 microM, procaine 60 microM, mepivacaine 149 microM, and lidocaine 204 microM. The values for voltage-dependent K+ current block were: bupivacaine 92 microM, etidocaine 176 microM, tetracaine 946 microM, lidocaine 1118 microM, mepivacaine 2305 microM, and procaine 6302 microM. Correlation of potencies with octanol:buffer partition coefficients (logP0) revealed that ester-bound local anesthetics were more potent in blocking Na+ channels than amide drugs. Within these groups, lipophilicity governed local anesthetic potency. We conclude that local anesthetic action on peripheral nerve ion channels is mediated via lipophilic drug-channel interactions. IMPLICATIONS: Half-maximal blocking concentrations of commonly used local anesthetics for Na+ and K+ channel block were determined on small membrane patches of peripheral nerve fibers. Because drugs can directly diffuse to the ion channel in this model, these data result from direct interactions of the drugs with ion channels.     

Selective anesthetic inhibition of brain nitric oxide synthase

BACKGROUND: It has been postulated that nitric oxide (NO) is a neurotransmitter involved in consciousness, analgesia, and anesthesia. Halothane has been shown to attenuate NO-mediated cyclic guanosine monophosphate accumulation in neurons, and a variety of anesthetic agents attenuate endothelium-mediated vasodilation, suggesting an interaction of anesthetic agents and the NO-cyclic guanosine monophosphate pathway. However, the exact site of anesthetic inhibitory action in this multistep pathway is unclear. The current study examines effects of volatile and intravenous anesthetic agents on the enzyme nitric oxide synthase (NOS) in brain. METHODS: NOS activity was determined by in vitro conversion of [14C]arginine to [14C]citrulline. Wistar rats were decapitated and cerebellum quickly harvested and homogenized. Brain extracts were then examined for NOS activity in the absence and presence of the volatile anesthetics halothane and isoflurane, and the intravenous agents fentanyl, midazolam, ketamine, and pentobarbital. Dose-response curves of NOS activity versus anesthetic concentration were constructed. Effects of anesthetics on NOS activity were evaluated by analysis of variance. RESULTS: Control activities were 57.5 +/- 4.5 pmol.mg protein-1.min-1 in the volatile anesthetic experiments and 51.5 +/- 6.5 pmol.mg protein-1.min-1 in the intravenous anesthetic experiments. NOS activity was not affected by ketamine (< or = 1 x 10(-4) M), pentobarbital (< or = 5 x 10(-5) M), fentanyl (< or = 1 x 10(-5) M), and midazolam (< or = 1 x 10(-5) M). Halothane decreased NOS activity to 36.7 +/- 2.5 (64% of control, P < 0.01 from control), 23.8 +/- 4.3 (41%, P < 0.01 from control and < 0.05 from 0.5% halothane), 25.2 +/- 3.8 (44%, P < 0.01 from control and < 0.05 from 0.5% halothane), and 19.7 +/- 2.8 (34%, P < 0.01 from control and < 0.05 from 0.5% halothane) pmol.mg protein-1.min-1 at 0.5, 1.0, 2.0, and 3.0% vapor. Isoflurane decreased NOS activity to 48.9 +/- 6.1 (85% of control), 46.0 +/- 3.2 (80%, P < 0.05 from control), 40.3 +/- 5.1 (70%, P < 0.05 from control), and 34.2 +/- 4.0 (60%, P < 0.05 from control and 0.5% and 1.0% isoflurane) pmol.mg protein-1.min-1 at 0.5, 1.0, 1.5, 2.0% vapor, respectively. CONCLUSIONS: Volatile anesthetics inhibit brain NOS activity in an in vitro system, but the intravenous agents examined have no effect at clinically relevant concentrations. This inhibition suggests a protein-anesthetic interaction between halothane, isoflurane, and NOS. In contrast, intravenous agents appear to have no direct effect on NOS activity. Whether intravenous agents alter signal transduction or regulatory pathways that activate NOS is unknown.     

Bound volatile general anesthetics alter both local protein dynamics and global protein stability

BACKGROUND: Recent studies have demonstrated that volatile general anesthetic agents such as halothane and isoflurane may bind to discrete sites on protein targets. In the case of bovine serum albumin, the sites of halothane and chloroform binding have been identified as being located in the IB and IIA subdomains. This structural information provides a foundation for more detailed studies into the potential mechanisms of anesthetic action. METHODS: The effect of halothane and isoflurane and the nonimmobilizer 1,2-dichlorohexafluorocyclobutane on the mobility of the indole ring in the tryptophan residues of albumin was investigated using measurements of fluorescence anisotropy. Myoglobin served as a negative control. In addition, the effect of bound anesthetic agents on global protein stability was determined by thermal denaturation experiments using near-ultraviolet circular dichroism spectroscopy. RESULTS: The fluorescence anisotropy measurements showed that halothane and isoflurane decreased the mobility of the indole rings in a concentration-dependent manner. The calculated dissociation constants were 1.6+/-0.4 and 1.3+/-0.3 mM for isoflurane and halothane, respectively. In contrast, both agents failed to increase the fluorescence anisotropy of the tryptophan residues in myoglobin, compatible with lack of binding. The nonimmobilizer 1,2-dichlorohexafluorocyclobutane caused no change in the fluorescence anisotropy of albumin. Binding of the anesthetic agents stabilized the native folded form of albumin to thermal denaturation. Analysis of the thermal denaturation data yielded dissociation constant values of 0.98+/-0.10 mM for isoflurane and 1.0+/-0.1 mM for halothane. CONCLUSIONS: Attenuation of local side-chain dynamics and stabilization of folded protein conformations may represent fundamental modes of action of volatile general anesthetic agents. Because protein activity is crucially dependent on inherent flexibility, anesthetic-induced stabilization of certain protein conformations may explain how these important clinical agents change protein function.     

Functional characterization of a mutated chicken alpha7 nicotinic acetylcholine receptor subunit with a leucine residue inserted in transmembrane domain 2

1. Site-directed mutagenesis was used to create an altered form of the chicken alpha7 nicotinic acetylcholine (ACh) receptor subunit (alpha7x61) in which a leucine residue was inserted between residues Leu9' and Ser10' in transmembrane domain 2. The properties of alpha7x61 receptors are distinct from those of the wild-type receptor. 2. Oocytes expressing wild-type alpha7 receptors responded to 10 microM nicotine with rapid inward currents that desensitized with a time-constant of 710+/-409 ms (mean+/-s.e.mean, n=5). However in alpha7x61 receptors 10 microM nicotine resulted in slower onset inward currents that desensitized with a time-constant of 5684+/-3403 ms (mean+/-s.e.mean, n = 4). No significant difference in the apparent affinity of nicotine or acetylcholine between mutant and wild-type receptors was observed. Dihydro-beta-erythroidine (DHbetaE) acted as an antagonist on both receptors. 3. Molecular modelling of the alpha7x61 receptor channel pore formed by a bundle of M2 alpha-helices suggested that three of the channel lining residues would be altered by the leucine insertion i.e.; Ser10 would be replaced by the leucine insertion, Val13' and Phe14' would be replaced, by Thr and Val, respectively. 4 When present in the LEV-1 nicotinic ACh receptor subunit from Caenorhabditis elegans the same alteration conferred resistance to levamisole anthelmintic drug. Levamisole blocked responses to nicotine of wild-type and alpha7x61 receptors. However, block was more dependent on membrane potential for the alpha7x61 receptors. 5. We conclude that the leucine insertion in transmembrane domain 2 has the unusual effect of slowing desensitization without altering apparent agonist affinity.     

Potentiation and inhibition of neuronal nicotinic receptors by atropine: competitive and noncompetitive effects

Atropine, the classic muscarinic receptor antagonist, inhibits ion currents mediated by neuronal nicotinic acetylcholine receptors expressed in Xenopus laevis oocytes. At the holding potential of -80 mV, 1 microM atropine inhibits 1 mM acetylcholine-induced inward currents mediated by rat alpha2beta2, alpha2beta4, alpha3beta2, alpha3beta4, alpha4beta2, alpha4beta4, and alpha7 nicotinic receptors by 12-56%. Inward currents induced with a low agonist concentration are equally inhibited (alpha3beta2, alpha3beta4), less inhibited (alpha2beta4, alpha7), or potentiated (alpha4beta2, alpha4beta4) by 1 microM atropine. Effects on the more sensitive alpha4beta4 nicotinic receptors were investigated in detail by systematic variation of acetylcholine and atropine concentrations and of membrane potential. At high agonist concentration, atropine inhibits alpha4beta4 nicotinic receptor-mediated ion current in a noncompetitive, voltage-dependent way with IC50 values of 655 nM at -80 mV and of 4.5 microM at -40 mV. At low agonist concentration, 1 microM atropine potentiates alpha4beta4 nicotinic receptor-mediated ion current. This potentiating effect is surmounted by high concentrations of acetylcholine, indicating a competitive interaction of atropine with the nicotinic receptor, and potentiation is also reversed at high atropine concentrations. Steady state effects of acetylcholine and atropine are accounted for by a model for combined receptor occupation and channel block, in which atropine acts on two distinct sites. The first site is associated with noncompetitive ion channel block. The second site is associated with competitive potentiation, which appears to occur when the agonist recognition sites of the receptor are occupied by acetylcholine and atropine. The apparent affinity of atropine for the agonist recognition sites of the alpha4beta4 nicotinic acetylcholine receptor is estimated to be 29.9 microM.     

No effects of high-dose omeprazole in patients with severe airway hyperresponsiveness and (a)symptomatic gastro-oesophageal reflux

Uptake of inhaled anesthetics may be measured as the amount of anesthetic infused to maintain a constant alveolar concentration of anesthetic. This method assumes that the patient absorbs all of the infused anesthetic, and that none is lost to circuit components. Using a standard anesthetic circuit with a 3-L rebreathing bag simulating the lungs, and simulating metabolism by input of carbon dioxide, we tested this assumption for halothane, isoflurane, and sevoflurane. Our results suggest that after washin of anesthetic sufficient to eliminate a material difference between inspired and end-tidal anesthetic, washin to other parts of the circuit (probably the ventilator) and absorbent (soda lime) continued to remove anesthetic for up to 15 min. From 30 min to 180 min of anesthetic administration, circuit components absorbed trivial amounts of isoflurane (12 +/- 13 mL vapor at 1.5 minimum alveolar anesthetic concentration, slightly more sevoflurane (39 +/- 15 mL), and still more halothane (64 +/- 9 mL). During this time, absorbent degraded sevoflurane (321 +/- 31 mL absorbed by circuit components and degraded by soda lime). The amount degraded increased with increasing input of carbon dioxide (e.g., the 321 +/- 31 mL increased to 508 +/- 48 mL when carbon dioxide input increased from 250 mL/min to 500 mL/min). Measurement of anesthetic uptake as a function of the amount of anesthetic infused must account for these findings. Implications: Systems that deliver inhaled anesthetics may also remove the anesthetic. Initially, anesthetics may diffuse into delivery components and the interstices of material used to absorb carbon dioxide. Later, absorbents may degrade some anesthetics (e.g., sevoflurane). Such losses may compromise measurements of anesthetic uptake.     

The effects of sevoflurane, isoflurane, halothane, and enflurane on hemodynamic responses during an inhaled induction of anesthesia via a mask in humans

A rapid increase in isoflurane or desflurane concentration induces tachycardia and hypertension and increases-plasma catecholamine concentration. Little information is available as to whether sevoflurane, halothane, and enflurane induce similar responses during anesthesia induction via mask. Fifty ASA physical status I patients, aged 20-40 yr, and scheduled for elective minor surgery, received one of four volatile anesthetics: sevoflurane, isoflurane, halothane, or enflurane. Anesthesia was induced with thiamylal, followed by inhalation of 0.9 minimum alveolar anesthetic concentration (MAC) of the anesthetic in 100% oxygen via mask. The inspired concentration of anesthetic was increased by 0.9 MAC every 5 min to a maximum of 2.7 MAC. Heart rate (HR) and systolic blood pressure (SBP) were measured before and every minute for 15 min during anesthetic inhalation. In the sevoflurane and isoflurane groups, venous blood samples were drawn to determine the concentrations of plasma epinephrine and norepinephrine 3 min after each increase in anesthetic concentration. Sustained increments in HR were observed after increases in inspired isoflurane concentration to 1.8 MAC and 2.7 MAC (peak changes of 15 +/- 3 and 17 +/- 3 bpm, respectively). Isoflurane also increased SBP transiently after the inspired concentration was increased to 2.7 MAC (peak change of 10 +/- 4 mm Hg). Enflurane increased HR after the inspired concentration was increased to 2.7 MAC (peak change of 9 +/- 2 bpm). In contrast, changes in sevoflurane and halothane concentrations did not induce hyperdynamic responses. Plasma norepinephrine concentration in the isoflurane group was significantly higher than that in the sevoflurane group during 2.7 MAC (P = 0.022). We propose that there is a direct relationship between airway irritation of the anesthetic and immediate cardiovascular change during an inhaled induction of anesthesia.     

The interaction of McN-A-343 with muscarine receptors in cardiac and smooth muscle

The interaction of the muscarine receptor partial agonist (4-m-chlorophenylcarbamoyloxy)-2-butynyltrimethylammonium chloride (McN-A-343) was investigated at muscarine receptors in the atria and taenia caeci of the guinea-pig to compare its interaction at the muscarine M2 receptor in the two tissues. In the smooth muscle, the muscarine M3 receptor subtype is responsible for the contractile response but the major subtype detected in binding or antibody experiments is the M2 subtype. In guinea pig atria the dissociation constant of McN-A-343 at muscarine receptors was 15.2 microM determined in functional experiments on left atria in McEwen's solution or 14.8 microM in binding experiments with [3H]-(-)-quinuclidinyl benzilate ([3H]QNB) in the same medium containing 5'-guanylylimododiphosphate (50 microM). In the taenia caeci, the dissociation constant estimated for McN-A-343 at the M3 receptor from functional experiments based on the contractile response to the agonist in McEwen's solution was 4.6 microM. This value was similar to the dissociation constant (6.2 microM) estimated from binding studies versus [3H]QNB conducted in the same medium although studies with 11-[[2-[(diethylamino)methyl]-1-piperidinyl]acetyl]-5,11-dihydro-6H- pyrido[2,3-b][1,4]benzodiazepine 6-one (AF-DX 116) versus [3H]-(-)-N-methylscopolamine suggested that 70% of the receptors were the M2 subtype. The presence of the M2 subtype in the taenia caeci was also confirmed by the ability of oxotremorine to inhibit the increase in cAMP produced by isoprenaline (10 microM) since apparent pKB values for AF-DX 116 and hexahydrosiladiphenidol were 6.95 and 6.75, respectively. McN-A-343 (100 microM) failed to inhibit the response to isoprenaline and did not antagonize the inhibitory response to oxotremorine. It is concluded that the apparent affinity of McN-A-343 for muscarine M2 receptors in the atria and the taenia caeci differs and a number of explanations are discussed.     

Effects of halothane and isoflurane on carbon monoxide-induced relaxations in the rat aorta

BACKGROUND: Halothane and isoflurane previously were reported to attenuate endothelium-derived relaxing factor/nitric oxide-mediated vasodilation and cyclic guanosine monophosphate (cGMP) formation in isolated rat aortic rings. Carbon monoxide has many chemical and physiologic similarities to nitric oxide. This study was designed to investigate the effects of halothane and isoflurane on carbon monoxide-induced relaxations and cGMP formation in the isolated rat aorta. METHODS: Isometric tension was recorded continuously from endothelium denuded rat aortic rings suspended in Krebs-filled organ baths. Rings precontracted with submaximal concentrations of norepinephrine were exposed to cumulative concentrations of carbon monoxide (26-176 microM). This procedure was repeated three times, with anesthetics delivered 10 min before the second procedure. Carbon monoxide responses of rings contracted with the same concentration of norepinephrine (10(-6) M and 2 x 10(-6) M) used in the anesthetic-exposed preparations also were examined. The concentrations of cGMP were determined in denuded rings using radioimmunoassay. The rings were treated with carbon monoxide (176 microM, 30 s) alone, or carbon monoxide after a 10-min incubation with halothane (0.34 mM or 0.72 mM). To determine whether the sequence of anesthetic delivery influenced results, vascular rings pretreated with halothane were compared with nonpretreated rings. RESULTS: Carbon monoxide (26-176 microM) caused a dose-dependent reduction of norepinephrine-induced tension, with a maximal relaxation of 1.51 +/- 0.07 g (85 +/- 7% of norepinephrine-induced contraction). Halothane (0.34 mM and 0.72 mM) significantly attenuated the carbon monoxide-induced relaxations, but only the highest concentration of isoflurane (0.53 mM) significantly attenuated the carbon monoxide-induced relaxations. Carbon monoxide (176 microM) significantly increased cGMP content (+88.1 +/- 7.1%) and preincubation of the aortic rings with halothane (0.34 mM and 0.72 mM) inhibited this increase (-70.7 +/- 6.8% and -108.1 +/- 10.6%, respectively). When aortic rings and carbon monoxide were added simultaneously to Krebs solution equilibrated with halothane (0.72 mM), no inhibition of cGMP formation occurred. CONCLUSION: Carbon monoxide-induced endothelium-independent relaxations of rat aortic rings were decreased by clinically relevant concentrations of halothane and isoflurane. The carbon monoxide-induced elevations of cGMP were attenuated by halothane only when the anesthetic was incubated with aortic rings before carbon monoxide treatment. The possible clinical significance of the actions of the anesthetics on this endogenous vasodilator is yet to be determined.     

The effect of imidazoline receptors and alpha2-adrenoceptors on the anesthetic requirement (MAC) for halothane in rats

BACKGROUND: Recent evidences have documented that several pharmacologic actions of alpha2-adrenoceptor agonists are mediated via activation of not only alpha2-adrenoceptors, but also by imidazoline receptors, which are nonadrenergic receptors in the central nervous system. However, the effect of imidazoline receptors on the anesthesia is not well known, and it is important to clarify the effects of both receptors on anesthesia. METHODS: Seventy-two rats were anesthetized with halothane, and the anesthetic requirement for halothane was evaluated as minimum alveolar concentration (MAC). The MAC for halothane was determined in the presence of dexmedetomidine (0, 10, 20, and 30 microg/kg, intraperitoneally [IP]), a selective alpha2-adrenoceptor agonist with weak affinity for imidazoline receptors. Then, the authors evaluated the inhibitory effect of rauwolscine (20 mg/kg, IP), an alpha2-adrenoceptor antagonist with little affinity for imidazoline receptors, on the MAC-reducing action of dexmedetomidine (30 microg/kg). Further, the effect of rilmenidine (20, 50, 100, 1000 microg/kg, IP), a selective imidazoline receptor agonist, on the MAC for halothane was also investigated. RESULTS: Dexmedetomidine decreased the MAC for halothane dose-dependently, and this MAC-reducing action of dexmedetomidine was completely blocked by rauwolscine. Rilmenidine alone did not change the MAC for halothane. CONCLUSIONS: The present data indicate that the anesthetic sparing action of dexmedetomidine is most likely mediated through alpha2- adrenoceptors, and the stimulation of imidazoline receptors exerts little effect on the anesthetic requirement for halothane.     

Inhibition of nitric oxide synthase decreases anesthetic requirements of intravenous anesthetics in Xenopus laevis

BACKGROUND: Acute inhibition of nitric oxide synthase (NOS) has been demonstrated to reduce the anesthetic requirements of volatile anesthetics. Recent data suggest that not only volatile but also intravenous anesthetic agents interact with nitric oxide (NO) metabolism. The aim of this study was to examine the effect of NOS inhibition by nitroG-L-arginine-methyl-ester (L-NAME) on the anesthetic action of the intravenous anesthetics thiopental, propofol, and ketamine. METHODS: The anesthetic potencies of thiopental, propofol, and ketamine were determined in Xenopus laevis tadpoles in the absence and presence of L-NAME. Anesthesia was defined as loss of righting reflex for 5 s. A nonlinear logistic regression curve was fitted to the data and half-maximal effective concentrations (EC50) were calculated. A second set of experiments was performed with different concentrations of L-NAME in the presence of the previously determined the EC50 of the intravenous anesthetics. RESULTS: The EC50s of the anesthetics thiopental, propofol, and ketamine were determined to be 25.5 +/- 2.0 microM, 1.9 +/- 0.1 microM, and 59.7 +/- 0.7 microM, respectively. The addition of L-NAME shifted the concentration-response curves to the left in a concentration-dependent manner. In the presence of 1 mM L-NAME, the EC50 of thiopental was reduced by 43%, the EC50 of propofol by 26%, and the EC50 of ketamine by 63%. The addition of D-NAME did not change the EC50 values of the three anesthetics. In the presence of L-arginine, the effect of L-NAME on the EC50 of thiopental was reversed. When administered by itself in a concentration range from 0.1 microM to 10 mM, L-NAME did not alter the behavior of the tadpoles. CONCLUSIONS: The results of the present study show that acute inhibition of NOS by L-NAME results in reduced anesthetic requirements of the intravenous anesthetics thiopental, propofol, and ketamine. This interaction of acutely administered L-NAME and intravenous anesthetics indicates that the NO-cyclic guanosine 3',5'-monophosphate system is involved in mediating the anesthetic effect of these compounds.     

Mutation in the M1 domain of the acetylcholine receptor alpha subunit decreases the rate of agonist dissociation

We describe the kinetic consequences of the mutation N217K in the M1 domain of the acetylcholine receptor (AChR) alpha subunit that causes a slow channel congenital myasthenic syndrome (SCCMS). We previously showed that receptors containing alpha N217K expressed in 293 HEK cells open in prolonged activation episodes strikingly similar to those observed at the SCCMS end plates. Here we use single channel kinetic analysis to show that the prolonged activation episodes result primarily from slowing of the rate of acetylcholine (ACh) dissociation from the binding site. Rate constants for channel opening and closing are also slowed but to much smaller extents. The rate constants derived from kinetic analysis also describe the concentration dependence of receptor activation, revealing a 20-fold shift in the EC50 to lower agonist concentrations for alpha N217K. The apparent affinity of ACh binding, measured by competition against the rate of 125I-alpha-bungarotoxin binding, is also enhanced 20-fold by alpha N217K. Both the slowing of ACh dissociation and enhanced apparent affinity are specific to the lysine substitution, as the glutamine and glutamate substitutions have no effect. Substituting lysine for the equivalent asparagine in the beta, epsilon, or delta subunits does not affect the kinetics of receptor activation or apparent agonist affinity. The results show that a mutation in the amino-terminal portion of the M1 domain produces a localized perturbation that stabilizes agonist bound to the resting state of the AChR.     

Volatile anesthetics affect calcium mobilization in bovine endothelial cells

BACKGROUND: The site where volatile anesthetics inhibit endothelium-dependent, nitric oxide-mediated vasodilation is unclear. To determine whether anesthetics could limit endothelium-dependent nitric oxide production by inhibiting receptor-mediated increases in cytosolic Ca2+, experiments were performed to see if the inhalational anesthetics halothane, isoflurane, and enflurane affect intracellular Ca2+ ([Ca2+]i) transients induced by the agonists bradykinin and adenosine triphosphate in cultured bovine aortic endothelial cells. METHODS: Bovine aortic endothelial cells, which had been loaded with the fluorescent Ca2+ indicator Fura-2, were added to medium preequilibrated with volatile anesthetic (1.25% and 2.5% for isoflurane, 1.755 and 3.5% for enflurane, and 0.75% and 1.5% for halothane). In Ca(2+)-containing medium, intracellular Ca2+ transients were elicited in response to bradykinin (10 nM and 1 microM) or adenosine triphosphate (1 microM and 100 microM). RESULTS: Both bradykinin and adenosine triphosphate triggered a rapid rise to peak [Ca2+]i followed by a gradual decline to a plateau above the resting level. Although basal [Ca2+]i was unaltered by the anesthetics, both halothane and enflurane, in a dose-dependent manner, depressed the peak and plateau of the [Ca2+]i transient elicited by 10 nM bradykinin, whereas isoflurane had no effect. When [Ca2+]i transients were elicited by 1 microM bradykinin, halothane (1% and 5%) did not alter peak and plateau levels. Halothane and enflurane also decreased [Ca2+]i transients evoked by 1 microM and 100 microM adenosine triphosphate, whereas isoflurane also had no effect in this setting. CONCLUSIONS: Halothane and enflurane, but not isoflurane, inhibit bradykinin- and adenosine triphosphate-stimulated Ca2+ transients in endothelial cells. Limitations of Ca2+ availability to activate constitutive endothelial nitric oxide synthase could allow for part, but not all, of the inhibition of endothelium-dependent nitric oxide-mediated vasodilation by inhalational anesthetics.     

Critical elements determining diversity in agonist binding and desensitization of neuronal nicotinic acetylcholine receptors

To identify the molecular determinants underlying the pharmacological diversity of neuronal nicotinic acetylcholine receptors, we compared the alpha7 homo-oligomeric and alpha4beta2 hetero-oligomeric receptors. Sets of residues from the regions initially identified within the agonist binding site of the alpha4 subunit were introduced into the alpha7 agonist binding site, carried by the homo-oligomeric alpha7-V201-5HT3 chimera. Introduction of the alpha4 residues 183-191 into alpha7 subunit sequence (chimera C2) selectively increased the apparent affinities for equilibrium binding and for ion channel activation by acetylcholine, resulting in a receptor that no longer displays differences in the responses to acetylcholine and nicotine. Introduction of the alpha4 residues 151-155 (chimera B) produced a approximately 100-fold increase in the apparent affinity for both acetylcholine and nicotine in equilibrium binding measurements. In both cases electrophysiological recordings revealed a much smaller increase (three- to sevenfold) in the apparent affinity for activation, but the concentrations required to desensitize the mutant chimeras parallel the shifts in apparent binding affinity. The data were fitted by a two-state concerted model, and an alteration of the conformational isomerization constant leading to the desensitized state accounts for the chimera B phenotype, whereas alteration of the ligand binding site accounts for the chimera C2 phenotype. Point mutation analysis revealed that several residues in both fragments contribute to the phenotypes, with a critical effect of the G152K and T183N mutations. Transfer of alpha4 amino acids 151-155 and 183-191 into the alpha7-V201-5HT3 chimera thus confers physiological and pharmacological properties typical of the alpha4beta2 receptor.     

Depressant effects of volatile anesthetics on second messenger system in mRNA-expressed Xenopus laevis oocytes

In this study I attempted to elucidate the depressant effects of volatile anesthetics on inositol trisphosphate (IP3)-mediated signal transduction pathway and to identify the site of action. For this purpose, we used Xenopus laevis oocytes which translated and expressed 5-HT receptors after injection of mRNA isolated from the rat brain. In this system, binding of the agonist to G-protein coupled receptors activates phospholipase C that produces IP3. Mobilization of Ca2+ by IP3 from the storage finally opens Ca2+ dependent Cl- channels. Halothane, isoflurane and methoxyflurane depressed Cl- current elicited by 5-HT. For the further quantitative study, methoxyflurane was used because of its better solubility and less vapor pressure that avoided evaporation of the agent. The 5-HT elicited Cl- current was depressed in a non-competitive fashion. Response were 75, 60, 20% of control in the presence of 0.5, 1 and 3 mM methoxyflurane, respectively. Responses elicited by a pressure-injection of Ca2+ or IP3 remained unchanged in the presence of high concentrations of either halothane, isoflurane or methoxyflurane. These results suggest that the depressant mechanism by volatile anesthetics on the signal transduction pathway involves neither Ca2+ dependent Cl- channel dynamics nor intracellular Ca2+ mobilization by IP3. Changes of microdomain characteristics of the membrane in the presence of anesthetic molecules including membrane-bound proteins and enzyme system may be a main mechanism of action of volatile anesthetics.