Volatile Anesthetics Depress Spinal Motor Neurons

Background: Depression of spinal alpha-motor neurons apparently plays a role in the surgical immobility induced by isoflurane. Using the noninvasive technique of F-wave analysis, the authors tested the hypothesis that depressed motor neuron excitability is an effect common to other clinically relevant inhaled anesthetics.

Methods: The authors measured F-wave amplitude in rats anesthetized with desflurane, enflurane, halothane, or sevoflurane. Each animal received one anesthetic at five equipotent anesthetic concentrations (0.6, 0.8, 1.2, and 1.6 minimum alveolar concentration [MAC] and 0.8 MAC with 65% N2 O). F waves were detected as late potentials in electromyographic responses evoked in the intrinsic muscles of the hind paw after monopolar stimulation of the ipsilateral posterior tibial nerve.

Results: All tested inhaled anesthetics depressed F-wave amplitude but not M-wave (orthodromic, early muscle activation) amplitude, and increased M-F latency in a dose-dependent manner. At 1.0 MAC, the estimated F/M ratio was 70+/-13% SD of that at baseline (0.6 MAC). Nitrous oxide added to 0.8 MAC of the potent vapors depressed F/M ratio by 63+/-17%.     

Volatile Anesthetics Differentially Affect Immunostimulated Expression of Inducible Nitric Oxide Synthase: Role of Intracellular Calcium

Background: Nitric oxide released by inducible nitric oxide synthase (iNOS) plays an important role in immune responses and systemic vasodilation in septic shock. Volatile anesthetics have been reported to interfere with signal transduction and gene expression. We studied the effect of volatile anesthetics on activity and expression of iNOS and potential mechanisms of action.

Methods: Nitrite release and iNOS expression were determined using the Griess reaction and Western and Northern blot techniques, respectively, in J774 murine macrophages stimulated with lipopolysaccharide and [gamma]-interferon in the absence and presence of various concentrations (0.25-2.0 minimum alveolar concentration [MAC]) of volatile anesthetics (i.e., halothane, enflurane, isoflurane, desflurane). Furthermore, potential interference of volatile anesthetics with specific signal transduction pathways was investigated.

Results: All volatile anesthetics, studied in a time- and dose-dependent manner, suppressed nitrite production and iNOS expression in J774 macrophages stimulated by lipopolysaccharide or [gamma]-interferon at clinically relevant concentrations. The inhibition was completely antagonized by ionomycin but unaffected by diacylglycerol, phorbol myristate acetate, and C2-ceramide. In contrast, in cells costimulated by lipopolysaccharide plus [gamma]-interferon, volatile anesthetics significantly increased nitrite production and iNOS expression independent of ionomycin and other mediators studied.     

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 Calcium2+, experiments were performed to see if the inhalational anesthetics halothane, isoflurane, and enflurane affect intracellular Calcium2+ ([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 Calcium2+ 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 Calcium2+ -containing medium, intracellular Calcium sup 2+ transients were elicited in response to bradykinin (10 nM and 1 micro Meter) or adenosine triphosphate (1 micro Meter and 100 micro Meter).

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+] sub i transient elicited by 10 nM bradykinin, whereas isoflurane had no effect. When [Ca2+]i transients were elicited by 1 micro Meter bradykinin, halothane (1% and 5%) did not alter peak and plateau levels. Halothane and enflurane also decreased [Ca2+]i transients evoked by 1 micro Meter and 100 micro Meter adenosine triphosphate, whereas isoflurane also had no effect in this setting.     

Volatile Anesthetics Reduce Low-voltage-activated Calcium Currents in a Thyroid C-Cell Line

Background: Volatile anesthetics may act in part by inhibiting voltage-dependent calcium channels. The effects of several volatile agents on three types of calcium channels in a thyroid C-cell line were examined.

Methods: Whole-cell calcium currents were recorded using standard patch clamp techniques. Current-voltage relationships were derived before, during, and after application of isoflurane, enflurane, or halothane. Low-voltage-activated (LVA; T type) calcium currents were isolated based on the voltage range of activation. High-voltage-activated (HVA) calcium currents were separated into L and N types using omega-conotoxin GVIA (omega-CTX) and nicardipine.

Results: All three agents reversibly decreased both LVA and HVA currents at clinically relevant concentrations. Isoflurane and enflurane both reduced peak LVA current more than peak HVA current: -33 +/- 6% (mean +/- SE) versus -22 +/- 4% for 0.71 mM isoflurane (n = 6), and -46 +/- 6% versus -35 +/- 5% for 1.21 mM enflurane (n = 6). In contrast, halothane depressed LVA and HVA currents to a similar extent: -22 +/- 4% versus -29 +/- 3% for 0.65 mM halothane (n = 6). Isoflurane had no effect on LVA whole-cell current kinetics. Pretreatment with either omega-CTX (400 nM) or nicardipine (1 micro Meter) did not change the sensitivity of HVA current to isoflurane.     

Lidocaine Suppresses the Anoxic Depolarization and Reduces the Increase in the Intracellular Ca sup 2+ Concentration in Gerbil Hippocampal Neurons

Background: The movement of ions, particularly Ca2+, across the plasma membrane of neurons is regarded as an initial element of the development of ischemic neuronal damage. Because the mechanism by which lidocaine protects neurons against ischemia is unclear, the effects of lidocaine on the ischemia-induced membrane depolarization, histologic outcome, and the change in the intracellular Ca2+ concentration in the gerbil hippocampus were studied.

Methods: The changes in the direct-current potential shift in the hippocampal CA1 area produced by transient forebrain ischemia for 4 min were compared in animals given lidocaine (0.8 micro mol administered intracerebroventricularly) 10 min before ischemia and those given saline. The histologic outcome was evaluated 7 days after ischemia by assessing delayed neuronal death in hippocampal CA1 pyramidal cells in these animals. In a second study, hypoxia-induced intracellular Ca2+ increases were evaluated by in vitro microfluorometry in gerbil hippocampal slices, and the effects of lidocaine (10, 50, and 100 micro Meter) on the Ca2+ accumulation were examined. In addition, the effect of lidocaine (100 micro Meter) drug perfusion with a Ca2+ -free ischemia-like medium was investigated.

Results: The preischemic administration of lidocaine delayed the onset of the ischemia-induced membrane depolarization (anoxic depolarization) and reduced its maximal amplitude. The histologic outcome was improved by the preischemic treatment with lidocaine. The in vitro hypoxia-induced increase in the intracellular concentration of Ca2+ was suppressed by the perfusion with lidocaine-containing mediums (50 and 100 micro Meter), regarding the initiation and the extent of the increase. The hypoxia-induced intracellular Ca2+ elevation in the Ca2+ -free condition was similar to that in the Ca2+ -containing condition. Perfusion with lidocaine (100 micro Meter) inhibited this elevation in the Ca2+ -free condition.     

Effects of Volatile Anesthetics on Sarcolemmal Calcium Transport and Sarcoplasmic Reticulum Calcium Content in Isolated Myocytes

Background: The surface membrane Ca2+-adenosine triphosphatase and Na+-Ca2+ exchanger transport Ca2+ out of the ventricular myocyte, competing for cytosolic Ca2+ with the Ca2+-adenosine triphosphatase located in the sarcoplasmic reticulum. In this study the authors examined the effects of halothane, isoflurane, and sevoflurane on Ca2+ extrusion from the cell and sarcoplasmic reticulum Ca2+ content.

Methods: Single myocytes from the right ventricular free wall of adult male ferret hearts were isolated, loaded with the acetoxymethyl ester of the fluorescent Ca2+ indicator fluo-3, and electrically stimulated at 0.25 Hz to reach a steady state level of intracellular Ca2+ stores. The effects of halothane, isoflurane, and sevoflurane (1 minimum alveolar concentration) on the peak and rate of decline of the Ca2+ transient induced by 10 mm caffeine were examined. The peak was used as an index of sarcoplasmic reticulum Ca2+ content, and the rate of decline was used to monitor Ca2+ extrusion from the cell.

Results: During control conditions, halothane reduced the Ca2+ content of the sarcoplasmic reticulum, isoflurane maintained it, and sevoflurane caused it to increase. Halothane did not affect Ca2+ extrusion from the cell, but both isoflurane and sevoflurane inhibited it. When Na+-Ca2+ exchange was inhibited by ionic substitution, isoflurane and sevoflurane still reduced the rate of Ca2+ efflux from the cell. However, when the sarcolemmal Ca2+-adenosine triphosphatase was inhibited by carboxyeosin, isoflurane and sevoflurane had no effect on Ca2+ efflux.     

Inhibition of Sodium/Calcium Exchange and Calcium Channels of Heart Cells by Volatile Anesthetics

Background: Volatile anesthetics exert profound effects on the heart, probably through their effect on Calcium2+ movements during the cardiac cycle. Calcium2+ movements across the sarcolemma are thought to involve mainly Calcium2+ channels and the Sodium sup +/Calcium2+ exchanger. We have therefore investigated the action of halothane, isoflurane, and enflurane on Sodium sup +/Calcium2+ exchange and Calcium2+ 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. Sodium sup +/Calcium2+ exchange activity was measured from45 Calcium2+ uptake by Sodium sup + -loaded cells. Calcium2+ channel activity was measured from verapamil-sensitive trace54 Manganese2+ uptake during electric stimulation.

Results: Halothane, isoflurane, and enflurane inhibited Sodium sup +/Calcium2+ 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 Calcium2+ 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 plus/minus 0.50 for inhibition of Sodium sup +/Calcium2+ exchange and 1.73 plus/minus 0.19 for inhibition of Calcium2+ channels.     

Effect of Volatile Anesthetics with and without Verapamil on Intracellular Activity in Vascular Smooth Muscle

Background: Although halothane and isoflurane inhibit receptor agonist-induced smooth muscle contraction by inhibiting Calcium2+ influx via the L-type voltage-dependent Calcium2+ channels, their effects on pharmacomechanical coupling remained to be clarified. The intracellular action of both anesthetics was studied during agonist-induced contractions using the Calcium2+ channel blocker verapamil.

Methods: Isolated spiral strips of rat thoracic aorta with endothelium removed were suspended for isometric tension recordings in physiologic salt solution. Cytosolic concentration of Calcium2+ ([Ca sup 2+]i) was measured concomitantly using fura-2-Calcium2+ fluorescence. Muscle contraction was evoked by the receptor agonists with 30 nM norepinephrine or 10 micro Meter prostaglandin F2 alpha (PGF2 alpha), followed by exposure to halothane, at 0%, 1%, 2%, and 3% or isoflurane, at 2% and 4%. The effects of the anesthetics were compared with those of 0.1-1 micro Meter verapamil (n = 8 for each condition). To clarify the intracellular action of the volatile anesthetics on agonist-induced contractions, this procedure was repeated for the anesthetics only in the presence of 1 micro Meter verapamil (n = 8 for each condition). The effects of both anesthetics were also examined in nonreceptor-mediated contractions evoked with a 1-micro Meter dose of the protein kinase C activator, 12-deoxyphorbol 13-isobutylate, which increases the Calcium2+ sensitivity of the contractile elements (n = 8 for each).

Results: Halothane, isoflurane, and verapamil suppressed norepinephrine- and PGF2 alpha-induced increases in muscle tension and [Ca sup 2+]i in a concentration-dependent manner. The Calcium2+ -tension regression lines suggested that the volatile anesthetics reduced Calcium2+ sensitivity of the contractile elements during PGF2 alpha-induced contraction. Pretreatment of the muscle strip with verapamil revealed that halothane and isoflurane released Calcium2+ during norepinephrine-induced contraction and that [Ca2+]i -tension relationship was modulated during PGF2 alpha-induced contractions. Halothane at 2% and 3% and isoflurane at 4% suppressed 12-deoxyphorbol 13-isobutylate-induced increases in muscle tension, whereas they enhanced increases in [Ca2+]i, indicating that both anesthetics suppressed Calcium2+ sensitivity during 12-deoxyphorbol 13-isobutylate-induced contraction.     

Local Anesthetics Inhibit Substance P Binding and Evoked Increases in Intracellular Calcium sup 2+

Background: During spinal and epidural anesthesia, local anesthetics reach concentrations in cerebrospinal fluid and spinal cord tissues at which their actions may extend beyond the classic blockade of sodium channels. This study examines the effects of several clinical and experimental local anesthetics on the binding and actions of a peptide neurotransmitter, substance P, known to be important in nociceptive transmission in the dorsal horn.

Methods: The binding of radiolabeled (Bolton-Hunter modified) substance P was studied in chick brain membranes in the presence of local anesthetics. The increase in intracellular calcium [Ca2+]in evoked by substance P was measured by the fluorescent indicator fura-2 loaded in a murine cell line expressing substance P (NK1) receptors. Cells were preincubated with bupivacaine before and during the transient addition of substance P.

Results: Both substance P binding and Calcium2+ increase were inhibited half-maximally by approximately 1 mM bupivacaine at pH 7.5, whereas tetracaine, lidocaine, and benzocaine were slightly less potent at inhibiting binding. Concentration-dependent substance P-binding studies showed that bupivacaine's inhibition was not competitive. Inhibition of substance P binding by bupivacaine increased with increasing pH, but the protonated species appears to have some inhibitory activity, and quaternary lidocaine also inhibited binding. There was no stereoselectivity to the binding inhibition.     

Increase of Glutamate Uptake in Astrocytes: A Possible Mechanism of Action of Volatile Anesthetics

Background: Glutamate is the most ubiquitous excitatory neurotransmitter in the vertebrate central nervous system. Astrocytes play an important role in terminating glutamatergic neurotransmission by removing released glutamate from the synaptic cleft. The authors examined the effects of several anesthetics on the glutamate uptake activity of astrocytes.

Methods: Cultured astrocytes from hippocampi of rat embryos were incubated with solution containing [sup 3 H]glutamate, which was pre-equilibrated with 0-4% halothane at 37 degrees Celsius. The uptake activity was evaluated as the amount of radioactivity per cell of protein.

Results: When the reaction solution was equilibrated with 4% halothane, glutamate uptake increased to about 165% of the control. The effect of halothane was dose-dependent, and a significant augmentation (30-50%) of glutamate uptake was observed at a range in clinical use concentrations (1-2%). On the other hand, the uptake of gamma-aminobutyric acid, an inhibitory transmitter, was hardly affected by 1-4% halothane. The effect of halothane on glutamate uptake was also examined in neuron-rich culture, and similar augmentation was observed, although the extent was less than that in astrocyte culture. Biochemical subcellular fractions (i.e., glial plasmalemmal vesicles and synaptosomes) were also examined, however, only slight (not significant) increase was detected in the glutamate uptake activity. Other volatile anesthetics, such as enflurane, isoflurane, and sevoflurane, also enhanced glutamate uptake, whereas the intravenous anesthetics ketamine and pentobarbital showed no effect on glutamate uptake.     

Volatile Anesthetics Selectively Inhibit the Calcium sup 2+ -transporting ATPase in Neuronal and Erythrocyte Plasma Membranes

Background: The activity of the plasma membrane Calcium2+ - transporting adenosine triphosphatase (PMCA) is inhibited by volatile anesthetics at clinical concentrations. The goal of the current study was to determine whether the inhibition is selective as compared to other adenosine triphosphatases (ATPases) and another group of general anesthetics, barbiturates. In addition, the authors determined whether the response to anesthetics of the enzymes in neuronal membranes is similar to that in erythrocyte membranes.

Methods: The effects of halothane, isoflurane, and sodium pentobarbital on four different ATPase activities were studied at 37 degrees C in two distinct plasma membrane preparations, human red blood cells and synaptosomal membranes from rat cerebellum.

Results: Inhibition patterns of the PMCA by halothane and isoflurane at anesthetic concentrations were very similar in red blood cells and synaptosomal membranes. The half-maximal inhibition (I50) occurred at 0.25-0.30 mM halothane and 0.30-0.32 mM isoflurane. The PMCA in both membranes was significantly more sensitive to the inhibitory action of volatile anesthetics (I50 = 0.75-1.15 minimum alveolar concentration) than were other ATPases, such as the Sodium sup +, Potassium sup + -ATPase (I50 [nearly equal] 3 minimum alveolar concentration) or Magnesium sup 2+ -ATPase (I50 greater or equal to 5 minimum alveolar concentration). In contrast, sodium pentobarbital inhibited the PMCA in both membranes only at [nearly equal] 100-200-fold above its anesthetic concentrations. The other ATPases were inhibited at similar pentobarbital concentrations (I50 = 11-22 mM).     

Effects of Small Concentrations of Volatile Anesthetics on Action Potential Firing of Neocortical Neurons In Vitro

Background: Volatile general anesthetics depress neuronal activity in the mammalian central nervous system and enhance inhibitory Cl- currents flowing across the [Greek small letter gamma]-aminobutyric acidA (GABA (A)) receptor - ion channel complex. The extent to which an increase in GABAA-mediated synaptic inhibition contributes to the decrease in neuronal firing must be determined, because many further effects of these agents have been reported on the molecular level.

Methods: The actions of halothane, isoflurane, and enflurane on the firing patterns of single neurons were investigated by extracellular recordings in organotypic slice cultures derived from the rat neocortex.

Results: Volatile anesthetics depressed spontaneous action potential firing of neocortical neurons in a concentration-dependent manner. The estimated median effective concentration (EC50) values were about one half the EC50 values for general anesthesia. In the presence of the GABA (A) antagonist bicuculline (20 [micro sign]M), the effectiveness of halothane, isoflurane, and enflurane in reducing the discharge rates were diminished by 48 - 65%, indicating that these drugs act via the GABAA receptor.     

Use of Volatile Anesthetics in Extracorporeal Circulation

Background and objectivesThe use of volatile anesthetics in cardiac surgery is not recent. Since the introduction of halothane in clinical practice, several cardiac surgery centers started to use these anesthetics constantly.ContentIn the last years a great number of studies have shown that the volatile anesthetics have a protecting effect against myocardial ischemic dysfunction. Experimental evidences have shown that the halogenated anesthetics have cardioprotective effects that cannot be only explained by coronary flow alterations or by the balance between myocardium available and consumed oxygen. In addition to that, the use of volatile anesthetics during extracorporeal circulation (ECC) in cardiac surgery plays an important role. Recent studies have proven that these agents have cardioprotective properties and produce better results when the volatile anesthetic is used during the whole surgery procedure, including ECC. The use of halogenated anesthetics through calibrated vaporizers adapted to the ECC circuit via oxygenator membranes has become popular. Therefore, the professionals involved such as anesthesiologists and perfusionists should learn specifcs in order to solve possible doubts.     

Contribution of Calcium Channel Subtypes to the Intracellular Calcium Signal in Sensory Neurons: The Effect of Injury

Background: Although the activation-induced intracellular Ca2+ signal is disrupted by sensory neuron injury, the contribution of specific Ca2+ channel subtypes is unknown.

Methods: Transients in dissociated rat dorsal root ganglion neurons were recorded using fura-2 microfluorometry. Neurons from control rats and from neuropathic animals after spinal nerve ligation were activated either by elevated bath K+ or by field stimulation. Transients were compared before and after application of selective blockers of voltage-activated Ca2+ channel subtypes.

Results: Transient amplitude and area were decreased by blockade of the L-type channel, particularly during sustained K+ stimulation. Significant contributions to the Ca2+ transient are attributable to the N-, P/Q-, and R-type channels, especially in small neurons. Results for T-type blockade varied widely between cells. After injury, transients lost sensitivity to N-type and R-type blockers in axotomized small neurons, whereas adjacent small neurons showed decreased responses to blockers of R-type channels. Axotomized large neurons were less sensitive to blockade of N- and P/Q-type channels. After injury, neurons adjacent to axotomy show decreased sensitivity of K+-induced transients to L-type blockade but increased sensitivity during field stimulation.     

Behavioral Effects of Volatile Anesthetics in Caenorhabditis elegans

Background: The nematode Caenorhabditis elegans offers many advantages as a model organism for studying volatile anesthetic action: It has a simple, well-understood nervous system; it allows the researcher to do forward genetics; and its genome will soon be completely sequenced. C. elegans is immobilized by volatile anesthetics only at high concentrations and with an unusually slow time course. Here other behavioral dysfunctions are considered as anesthetic endpoints in C. elegans.

Methods: The potency of halothane for disrupting eight different behaviors was determined by logistic regression of concentration and response data. Other volatile anesthetics were also tested for some behaviors. Established protocols were used for behavioral endpoints that, except for pharyngeal pumping, were set as complete disruption of the behavior. Time courses were measured for rapid behaviors. Recovery from exposure to 1 or 4 vol% halothane was determined for mating, chemotaxis, and gross movement. All experiments were performed at 20 to 22 degrees Celsius.

Results: The median effective concentration values for halothane inhibition of mating (0.30 vol% - 0.21 mM), chemotaxis (0.34 vol% - 0.24 mM), and coordinated movement (0.32 vol% - 0.23 mM) were similar to the human minimum alveolar concentration (MAC; 0.21 mM). In contrast, halothane produced immobility with a median effective concentration of 3.65 vol% (2.6 mM). Other behaviors had intermediate sensitivities. Halothane's effects reached steady-state in 10 min for all behaviors tested except immobility, which required 2 h. Recovery was complete after exposure to 1 vol% halothane but was significantly reduced after exposure to immobilizing concentrations.     

注射挥发性麻醉药对动物的效应

研究了注射挥发性麻醉药对动物的效应，在小鼠，乙醚的催眠ＥＤ５０是５．３５（ｓｃ）和１．６２（ｉｐ）ｍｌ／ｋｇ；ＬＤ５０是１３．７（ｓｃ）和２．３２（ｉｐ）ｍｌ／ｋｇ。甲氧氟烷的ＥＤ５０为０．７５（ｉｐ）和４．１２（ｉｍ）ｍｌ／ｋｇ；ＬＤ５０为１．２３（ｉｐ）５．８１（ｉｍ）ｍｌ／ｋｇ，ｉｐ氟烷的ＥＤ５０和ＬＤ５０分别是１．２０和１．９４ｍｌ／ｋｇ，ｉｐ安氟醚的ＥＤ５０和ＬＤ５０分别是３．０１和７．