General Anesthetics Modify the Kinetics of Nicotinic Acetylcholine Receptor Desensitization at Clinically Relevant Concentrations

Background: General anesthetics are thought to induce anesthesia through their actions on ligand-gated ion channels. One such channel, the nicotinic acetylcholine receptor (nAcChoR), can be found in different subtypes in the central nervous system and at the periphery in the neuromuscular junction. The latter subtype of the nAcChoR is a useful model for examining interactions between general anesthetics and ligand-gated ion channels, because it can be isolated and purified in sufficient quantities to allow for biophysical and biochemical studies. This study examines the actions of general anesthetics on agonist-induced conversion of the nAcChoR to inactive desensitized conformational states.

Methods: Nicotinic acetylcholine receptor membranes were purified from the electric organ of Torpedo nobiliana. Agonist-induced desensitization was characterized from the time-dependent increase in fluorescence intensity that results from the binding of the fluorescent acetylcholine analog, Dns-C6 -Cho, to the nAcChoR.

Results: Mixing Dns-C6 -Cho with nAcChoR-rich membranes results in an increase in fluorescence that is characterized by four rate processes. Concentrations of isoflurane and butanol, which range from subclinical to toxic increase the rates of the third and fourth components of fluorescence, corresponding to fast and slow desensitization, respectively. At concentrations that are twice their EC sub 50 s for anesthesia, isoflurane, butanol, chloroform, methanol, and cyclopentane-methanol increase the apparent rates of fast and slow desensitization by an average of 92 plus/minus 22% and 108 plus/minus 22%, respectively.     

The Alkyl Chain Dependence of the Effect of Normal Alcohols on Agonist-induced Nicotinic Acetylcholine Receptor Desensitization Kinetics

Background: The nAcChoR is the prototypical member of a superfamily of ligand-gated ion channels that are all relevant targets of anesthetics and undergo desensitization upon prolonged exposure to agonist. This study was designed to investigate the effects of representative normal alcohols on the apparent rate of acetylcholine-induced nAcChoR desensitization.

Methods: Nicotinic acetylcholine receptors were obtained from the electroplax organ of Torpedo nobiliana. The apparent rate of acetylcholine-induced desensitization in the presence and absence of normal alcohols was measured using stopped-flow fluorescence.

Results: Normal alcohols as long as octanol (the longest studied) increased the apparent rate of desensitization induced by low concentrations of acetylcholine, shifting the agonist concentration-response curve for desensitization to the left. Ethanol, butanol, and, to a lesser extent, hexanol increased the maximal rate of desensitization induced by high, saturating concentrations of agonist. Beyond hexanol, heptanol and octanol had no effect on this maximal apparent rate of desensitization, even at concentrations that approach those that directly induce desensitization in the absence of agonist.     

Inhibitory Effects of Isoflurane and Nonimmobilizing Halogenated Compounds on Neuronal Nicotinic Acetylcholine Receptors

Background: Neuronal nicotinic acetylcholine receptors (nAchRs) are inhibited by low concentrations of volatile anesthetics. However, it is not clear whether this phenomenon contributes to the anesthetic effects of volatile anesthetics. Effects of a volatile anesthetic (isoflurane) and structurally related nonimmobilizers (F6: 1,2-dichlorohexafluorocyclobutane, F8: 2,3-dichlorooctafluorobutane) on the current mediated through neuronal nAchRs were studied.

Method: This study investigated neuronal nAchRs in PC12 cells and acutely dissociated rat medial habenula (MHb) neurons. Whole cell currents elicited by 30 [mu]m nicotine were recorded in the absence and presence of the halogenated agents. The minimum alveolar concentrations (MACs) for F6 and F8 were predicted from Meyer-Overton correlation.

Results: All halogenated compounds inhibited the nicotine-induced current in a concentration-dependent manner in PC12 cells. In MHb neurons, while isoflurane and F6 significantly inhibited the nicotine-induced peak current, F8 failed to inhibit it. The peak currents in the presence of isoflurane at 1.7 MAC, of F6 at 2.4 MAC, and of F8 at 2.2 MAC were 12, 31, and 97% of control, respectively.     

Isoflurane and Sevoflurane Interact with the Nicotinic Acetylcholine Receptor Channels in Micromolar Cconcentrations

Background: This study was performed to elucidate and compare the effects of sevoflurane and of isoflurane on the nicotinic acetylcholine receptor of mouse myotubes. The experiments were done with special reference to anesthetic concentrations considerably less than those used for clinical anesthesia.

Methods: The patch-clamp technique was used to record acetylcholine-activated currents from the embryonic type of the nicotinic acetylcholine receptor in the outside-out mode. A piezo-driven liquid filament switch was used for the ultrafast application of acetylcholine alone or in combination with isoflurane or sevoflurane. In addition, the patches were preexposed to either anesthetic, preceding the activation with acetylcholine.

Results: The current elicited by acetylcholine was reduced reversibly and in a concentration-dependent manner by both anesthetics, which were equally effective. Preexposure of the patches to isoflurane or sevoflurane showed an additional inhibition that was present at micromolar concentrations. The time courses of current decay could be fitted by single exponentials for isoflurane. At higher concentrations of sevoflurane, the current decay became biexponential. In contrast to isoflurane, sevoflurane increased the time constants of desensitization when applied in low concentrations.     

Differential Effects of Anesthetic and Nonanesthetic Cyclobutanes on Neuronal Voltage-gated Sodium Channels

Background: Despite their key role in the generation and propagation of action potentials in excitable cells, voltage-gated sodium (Na+) channels have been considered to be insensitive to general anesthetics. The authors tested the sensitivity of neuronal Na+ channels to structurally similar anesthetic (1-chloro-1,2,2-trifluorocyclobutane; F3) and nonanesthetic (1,2-dichlorohexafluorocyclobutane; F6) polyhalogenated cyclobutanes by neurochemical and electrophysiologic methods.

Methods: Synaptosomes (pinched-off nerve terminals) from adult rat cerebral cortex were used to determine the effects of F3 and F6 on 4-aminopyridine- or veratridine-evoked (Na+ channel-dependent) glutamate release (using an enzyme-coupled spectrofluorimetric assay) and increases in intracellular Ca2+ ([Ca2+]i) (using ion-specific spectrofluorimetry). Effects of F3 and F6 on Na+ currents were evaluated directly in rat lumbar dorsal root ganglion neurons by whole-cell patch-clamp recording.

Results: F3 inhibited glutamate release evoked by 4-aminopyridine (inhibitory concentration of 50% [IC50] = 0.77 mM [~ 0.8 minimum alveolar concentration (MAC)] or veratridine (IC50 = 0.42 mM [~ 0.4 MAC]), and veratridine-evoked increases in [Ca2+]i (IC50= 0.5 mM [~ 0.5 MAC]) in synaptosomes; F6 had no significant effects up to 0.05 mM (approximately twice the predicted MAC). F3 caused reversible membrane potential-independent inhibition of peak Na+ currents (70 +/- 9% block at 0.6 mM [~ 0.6 MAC]), and a hyperpolarizing shift in the voltage-dependence of steady state inactivation in dorsal root ganglion neurons (-21 +/- 9.3 mV at 0.6 mM). F6 inhibited peak Na+ currents to a lesser extent (16 +/- 2% block at 0.018 mM [predicted MAC]) and had minimal effects on steady state inactivation.     

Human Neuronal Nicotinic Acetylcholine Receptors Expressed in Xenopus Oocytes Predict Efficacy of Halogenated Compounds That Disobey the Meyer-Overton Rule

Background: According to the Meyer-Overton rule, anesthetic potency of a substance can be predicted by its lipid solubility, but a group of halogenated volatile compounds predicted to induce anesthesia does not obey this rule. Thus, these compounds are useful tools for studies of molecular targets of anesthetics. Human neuronal nicotinic acetylcholine receptor (hnAChR) subunits have been recently cloned, which allowed the authors to assess whether these receptors could differentiate among volatile anesthetic and nonimmobilizer compounds. This study provides the first data regarding anesthetic sensitivity of hnAChRs.

Methods: [alpha]2[beta]4, [alpha]3[beta]4, and [alpha]4[beta]2 hnAChRs were expressed in Xenopus oocytes, and effects of volatile anesthetics isoflurane and F3 (1-chloro-1,2,2-triflurocyclobutane, 1A) and nonimmobilizers F6 (1,2-dichlorohexafluorocyclobutane, 2N) and F8 (2,3-dichlorooctafluorobutane) on the peak acetylcholine-gated currents were studied using the two-electrode voltage-clamp technique.

Results: Isoflurane and F3 inhibited all the hnAChRs tested in a concentration-dependent manner. Isoflurane at a concentration corresponding to 1 minimum alveolar concentration (MAC) inhibited 83, 69, and 71% of ACh-induced currents in [alpha]2[beta]4, [alpha]3[beta]4, and [alpha]4[beta]2 hnAChRs, respectively, and 1 MAC of F3 inhibited 64, 44, and 61% of currents gated in those receptors. F6 (8-34[mu]M) did not cause any changes in currents gated by any of the receptors tested. F8 (4-18[mu]M) did not alter the currents gated in either [alpha]3[beta]4 or [alpha]4[beta]2 receptors, but caused a small potentiation of [alpha]2[beta]4 hnAChRs without a concentration-response relation.     

Halothane Interactions with Nicotinic Acetylcholine Receptor Membranes: Steady-state and Kinetic Studies of Intrinsic Fluorescence Quenching

Background: Although it has been suggested that anesthetics alter protein conformational states by binding to nonpolar sites within the interior regions of proteins, the rate and extent to which anesthetics penetrate membrane proteins has not been characterized. The authors report the use of steady-state and stopped-flow spectroscopy to characterize the interactions of halothane with receptor membranes.

Methods: Steady-state and stopped-flow fluorescence spectroscopy was used to characterize halothane quenching of nicotinic acetylcholine receptor (nAcChoR)-rich membrane intrinsic fluorescence and the rate of isoflurane-induced nAcChoR desensitization.

Results: At equilibrium, halothane quenched only 54 +/- 1.4% of all tryptophan fluorescence. Diethyl ether failed to reduce fluorescence quenching by halothane, suggesting that it does not bind to the same protein sites as halothane. Stopped-flow fluorescence traces defined two kinetic components of quenching: a fast component that occurred in less than 1 ms followed by a slower biphasic fluorescence decay. Protein unfolding with sodium dodecyl sulfate reduced halothane's Stern-Volmer quenching constant, eliminated the biphasic decay, and rendered fluorescence accessible to quenching by halothane within 1 ms. Functional studies indicate that anesthetic-induced desensitization of nAcChoR occurs in less than 2 ms.     

Effects of Thiopental and Its Optical Isomers on Nicotinic Acetylcholine Receptors

Background: With the exception of [gamma]-aminobutyric acidA (GABAA) receptors, the major molecular targets underlying the anesthetizing actions of thiopental have yet to be established. Neuronal nicotinic acetylcholine receptors (nAChRs) are closely related to GABAA receptors and hence might also be major targets. If so, they might be expected to be substantially inhibited by surgical concentrations (EC50 = 25 [mu]m) of thiopental and to display the same stereoselectivity as does general anesthesia.

Methods: Neuronal [alpha]4[beta]2, neuronal [alpha]7 and muscle [alpha][beta][gamma][delta] nAChRs were expressed in Xenopus oocytes. Peak acetylcholine-activated currents were measured at -70 mV using the two-electrode voltage clamp technique. Racemic thiopental and its two optical isomers were applied with and without preincubation and at high and low concentrations of acetylcholine.

Results: Inhibition of all three nAChRs was enhanced by preincubation with thiopental, a protocol that mimics the pharmacologic situation in vivo. Using this protocol, inhibition was further enhanced by high concentrations of acetylcholine, with IC50 = 18 +/- 2, 34 +/- 4, and 20 +/- 2 [mu]m (mean +/- SEM) thiopental for the neuronal [alpha]4[beta]2, neuronal [alpha]7 and muscle [alpha][beta][gamma][delta] nAChRs, respectively, with Hill coefficients near unity. Neither the neuronal [alpha]7 nor the muscle [alpha][beta][gamma][delta] nAChR differentiated between the optical isomers of thiopental. However, R (+)-thiopental was significantly more effective than the S (-) isomer at inhibiting the neuronal [alpha]4[beta]2 nAChR; interestingly, this is diametrically opposite to their stereoselectivity for general anesthesia.     

Differential Effects of Thiopental on Neuronal Nicotinic Acetylcholine Receptors and P sub 2X Purinergic Receptors in PC12 Cells

Background: PC12 cells, derived from rat pheochromocytoma, express neuronal nicotinic acetylcholine receptors (nAchRs) and P2X purinergic receptors, both of which resemble the receptors in postganglionic sympathetic neurons. The former is the established and the latter is the putative receptor to mediate fast synaptic transmission. The authors investigated effects of thiopental on these two ligand-gated ion channels.

Methods: Whole cell currents were recorded in PC12 cells without treatment of nerve growth factor, using conventional whole cell patch clamp technique. Nicotine or adenosine tri-phosphate (ATP) 30 micro Meter was applied for 4-5 s in the absence or presence of thiopental 3-300 micro Meter.

Results: Nicotine induced the rapidly decaying inward current at -60 mV, which exhibited the characteristics of the neuronal nAchR-mediated current. Thiopental inhibited the nicotine-induced inward current and accelerated the current decay in a dose-dependent manner, resulting in the greater effects on the steady current than the peak current. IC50s for the peak and steady current were 56.7 and 7.4 micro Meter, when the anesthetic was coapplied with nicotine. Thiopental's inhibition was not associated with a change in the reversal potential and was voltage-independent at membrane potential of -30 to -70 mV. Most of thiopental's effects seemed to require channel opening. In contrast to the nicotine-induced current, thiopental had little effect on the current elicited by ATP.     

Effects of Volatile Anesthetic Agents on In Situ Vascular Smooth Muscle Transmembrane Potential in Resistance- and Capacitance-regulating Blood Vessels

Introduction: This study was designed to compare the inhibitory effect of inhaled volatile anesthetic agents on in situ sympathetic neural versus nonneural regulation of vascular smooth muscle transmembrane potentials as correlates of vascular smooth muscle tone in resistance- and capacitance-regulating blood vessels.

Methods: Vascular smooth muscle transmembrane potentials were measured in situ with glass microelectrodes in neurally intact, small (200-300 m OD) mesenteric arteries and veins of rats before, during, and after inhaled halothane, isoflurane, or sevoflurane (0.5 or 1.0 minimum alveolar concentration [MAC]). Such transmembrane potentials and their anesthetically induced changes were compared, respectively, with those measured in similar vessel preparations after local sympathetic neural denervation with 6-hydroxydopamine.

Results: In neurally intact vessels, transmembrane potentials (in millivolts, mean +/- SD) before inhalation of the anesthetic agent were -39 +/- 2.8 (artery) and -43 +/- 4.6 (vein). At 1.0 MAC, halothane, isoflurane, and sevoflurane induced respective hyperpolarizations (in millivolts, mean +/- SD) of 9 +/- 3.1, 6 +/- 2.7, and 9 +/- 4.0 in arteries and 6 +/- 4.4, 2.8 +/- 3.0, and 8.7 +/- 5.6 in veins. Sympathetic denervation significantly attenuated these hyperpolarizations (except for venous response to isoflurane). At 0.5 MAC, transmembrane potential responses to all three volatile anesthetic agents were small and not consistently significant in either the intact or denervated vessels.     

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.     

竹柏果皮和果壳中挥发油成分及其抗肿瘤活性研究

摘要目的 比较瑶药竹柏果皮和果壳中挥发油成分,并对两部位的挥发油进行抗肿瘤活性研究。方法应用水蒸气蒸馏法分别从竹柏果皮和果壳中提取挥发油,并结合气相色谱 质谱法分析其化学成分;采用噻唑蓝（MTT）法分别对果皮和果壳挥发油进行抗肿瘤活性研究。结果竹柏果皮挥发油收率为0.23%,分离出21种成分,鉴定18种,主要含有石竹烯,占挥发油总量的72.44%;竹柏果壳挥发油收率为0.15%,分离出15种成分,鉴定其中13种,与果皮共有4种成分;竹柏果皮和果壳挥发油对鼻咽癌细胞均具有抑制增殖活性,半数抑制浓度分别为54.58和114.83 μg·mL－1,果皮挥发油抑制活性较强。结论从竹柏果皮和果壳中提取挥发油主要成分为烯类、醇类、萜类,其中果皮挥发油具有较强的抗肿瘤活性。     

油茶树嫩枝挥发油GC-MS分析及其体内外抗菌作用

分析了油茶树嫩枝挥发油的化学成分,研究了其体内外抗菌活性,比较其与民间所用"刀烟"和油茶籽油之间的抗菌效果.采用水蒸气蒸馏法提取,运用毛细管气相色谱-质谱联用法结合计算机检索对其化学成分进行分析和鉴定,用气相色谱面积归一化法测定各组分的相对百分含量,用NCCLS肉汤稀释法测定MIC值,并依照中药药理研究方法建立了小鼠皮肤感染模型,进行体表抗感染试验.结果检出并鉴定了35个峰的相对分子质量、分子式及相对含量,表明该挥发油和"刀烟"均具有一定的体外抗菌活性,且对体表感染金黄色葡萄球菌的小鼠具有显著的保护作用.     

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.