Halothane inhibition of recombinant cardiac L-type Ca2+ channels expressed in HEK-293 cells

BACKGROUND: Volatile anesthetics depress cardiac contractility, which involves inhibition of cardiac L-type calcium channels. To explore the role of voltage-dependent inactivation, the authors analyzed halothane effects on recombinant cardiac L-type calcium channels (alpha1Cbeta2a and alpha1Cbeta2aalpha2/delta1), which differ by the alpha2/delta1 subunit and consequently voltage-dependent inactivation. METHODS: HEK-293 cells were transiently cotransfected with complementary DNAs encoding alpha1C tagged with green fluorescent protein and beta2a, with and without alpha2/delta1. Halothane effects on macroscopic barium currents were recorded using patch clamp methodology from cells expressing alpha1Cbeta2a and alpha1Cbeta2aalpha2/delta1 as identified by fluorescence microscopy. RESULTS: Halothane inhibited peak current (I(peak)) and enhanced apparent inactivation (reported by end pulse current amplitude of 300-ms depolarizations [I300]) in a concentration-dependent manner in both channel types. alpha2/delta1 coexpression shifted relations leftward as reported by the 50% inhibitory concentration of I(peak) and I300/I(peak)for alpha1Cbeta2a (1.8 and 14.5 mm, respectively) and alpha1Cbeta2aalpha2/delta1 (0.74 and 1.36 mm, respectively). Halothane reduced transmembrane charge transfer primarily through I(peak) depression and not by enhancement of macroscopic inactivation for both channels. CONCLUSIONS: The results indicate that phenotypic features arising from alpha2/delta1 coexpression play a key role in halothane inhibition of cardiac L-type calcium channels. These features included marked effects on I(peak) inhibition, which is the principal determinant of charge transfer reductions. I(peak) depression arises primarily from transitions to nonactivatable states at resting membrane potentials. The findings point to the importance of halothane interactions with states present at resting membrane potential and discount the role of inactivation apparent in current time courses in determining transmembrane charge transfer.     

Halothane Inhibition of Recombinant Cardiac L-type Ca2+ Channels Expressed in HEK-293 Cells

Background: Volatile anesthetics depress cardiac contractility, which involves inhibition of cardiac L-type calcium channels. To explore the role of voltage-dependent inactivation, the authors analyzed halothane effects on recombinant cardiac L-type calcium channels ([alpha]1C[beta]2a and [alpha]1C[beta]2a[alpha]2/[delta]1), which differ by the [alpha]2/[delta]1 subunit and consequently voltage-dependent inactivation.

Methods: HEK-293 cells were transiently cotransfected with complementary DNAs encoding [alpha]1C tagged with green fluorescent protein and [beta]2a, with and without [alpha]2/[delta]1. Halothane effects on macroscopic barium currents were recorded using patch clamp methodology from cells expressing [alpha]1C[beta]2a and [alpha]1C[beta]2a[alpha]2/[delta]1 as identified by fluorescence microscopy.

Results: Halothane inhibited peak current (Ipeak) and enhanced apparent inactivation (reported by end pulse current amplitude of 300-ms depolarizations [I300]) in a concentration-dependent manner in both channel types. [alpha]2/[delta]1 coexpression shifted relations leftward as reported by the 50% inhibitory concentration of Ipeak and I300/Ipeak for [alpha]1C[beta]2a (1.8 and 14.5 mm, respectively) and [alpha]1C[beta]2a[alpha]2/[delta]1 (0.74 and 1.36 mm, respectively). Halothane reduced transmembrane charge transfer primarily through Ipeak depression and not by enhancement of macroscopic inactivation for both channels.     

Interaction of Volatile Anesthetics with Human Kv Channels in Relation to Clinical Concentrations

Background: Recent evidence shows that inhibition of human Kv3 channels by intravenous anesthetics occurs at clinical concentrations. The effects of volatile anesthetics on these human ion channels are unknown. This study was designed to establish whether minimum alveolar concentrations (MAC) of halothane, enflurane, isoflurane, and desflurane exhibit effects on Kv3 channels. To obtain an indication whether these findings may be specific to Kv3 channels, the effects of enflurane and isoflurane on human Kv1.1 channels were also investigated.

Methods: Kv3 channels natively expressed in SH-SY5Y cells and Kv1.1 channels expressed in HEK293 cells were measured with the whole cell patch clamp technique by standard protocols. Concentrations of volatile anesthetics were determined by gas chromatography.

Results: Halothane, enflurane, isoflurane, and desflurane reversibly inhibited Kv3 channels in a concentration-dependent manner. Concentrations at half-maximal effect (IC50 values) ranged between 1,800 and 4,600 [mu]m. Hill coefficients were between 1.7 and 2.5. IC50 values for inhibition of Kv1.1 channels were 2,800 and 5,200 [mu]m, and Hill coefficients were 3.9 and 5.6 for enflurane and isoflurane, respectively.     

Interaction of volatile anesthetics with human Kv channels in relation to clinical concentrations

BACKGROUND: Recent evidence shows that inhibition of human Kv3 channels by intravenous anesthetics occurs at clinical concentrations. The effects of volatile anesthetics on these human ion channels are unknown. This study was designed to establish whether minimum alveolar concentrations (MAC) of halothane, enflurane, isoflurane, and desflurane exhibit effects on Kv3 channeLs. To obtain an indication whether these findings may be specific to Kv3 channels, the effects of enflurane and isoflurane on human Kv1.1 channels were also investigated. METHODS: Kv3 channels natively expressed in SH-SY5Y cells and Kv1.1 channels expressed in HEK293 cells were measured with the whole cell patch clamp technique by standard protocols. Concentrations of volatile anesthetics were determined by gas chromatography. RESULTS: Halothane, enflurane, isoflurane, and desflurane reversibly inhibited Kv3 channels in a concentration-dependent manner. Concentrations at half-maximal effect (IC50 values) ranged between 1,800 and 4,600 microM. Hill coefficients were between 1.7 and 2.5. IC50 values for inhibition of Kv1.1 channels were 2,800 and 5,200 microM, and Hill coefficients were 3.9 and 5.6 for enflurane and isoflurane, respectively. CONCLUSION: Volatile anesthetics inhibit human Kv3 channels at clinical concentrations. At 1-3 MAC, inhibition would account on average for 2-12%. Inhibition would be highest with enflurane (between 3% and 22%) and lowest with isoflurane (between 0.2% and 3%). Kv1.1 channels would only be inhibited by enflurane at clinical concentrations (2% at 2 MAC and 8% at 3 MAC). Whether the degree of K channel inhibition by volatile anesthetics may contribute to their clinical action needs further study.     

Inhibition of Kv4.3/KChIP2.2 Channels by Bupivacaine and Its Modulation by the Pore Mutation Kv4.3V401I

Background: The transient outward current Ito is an important repolarizing K current in human ventricular myocardium mediated by Kv4.3 and KChIP2.2 subunits. Inhibition of Ito by amino-amide local anesthetics may be involved in severe cardiotoxic side effects. This study elucidates the molecular mechanisms of bupivacaine interaction with complexes formed by Kv4.3 and KChIP2.2 as well as the modulatory effect of KChIP2.2. For this purpose, the pharmacologic effects of bupivacaine on Kv4.3wt/KChIP2.2 channels and on the pore mutant Kv4.3V401I were investigated.

Methods: Kv4.3/KChIP2.2 cDNA was transiently expressed in Chinese hamster ovary cells. Site-directed mutagenesis and patch clamp experiments were performed to analyze the effects of bupivacaine on wild-type and mutant channels.

Results: Inhibition of Kv4.3wt/KChIP2.2 channels by bupivacaine was concentration-dependent and reversible. The IC50s for inhibition of the charge conducted by Kv4.3wt/KChIP2.2 channels by bupivacaine and levobupivacaine were 55 +/- 8 and 50 +/- 5 [mu]m, respectively. The local anesthetic accelerated macroscopic current decline of Kv4.3wt/KChIP2.2 and slowed recovery from inactivation without altering steady state inactivation. KChIP2.2 altered the response of Kv4.3wt channels to bupivacaine and bupivacaine modulated KChIP2.2 effects on Kv4.3wt channels. The pore mutation V401I slowed macroscopic current decline of Kv4.3 channels and recovery from inactivation, and it diminished modulation of gating by KChIP2.2. Bupivacaine inhibition of Kv4.3V401I resembled Kv4.3wt and was not changed by coexpression of KChIP2.2.     

Ketamine blockade of voltage-gated sodium channels: evidence for a shared receptor site with local anesthetics.

BACKGROUND: The general anesthetic ketamine is known to be an N-methyl-D-aspartate receptor blocker. Although ketamine also blocks voltage-gated sodium channels in a local anesthetic-like fashion, little information exists on the molecular pharmacology of this interaction. We measured the effects of ketamine on sodium channels. METHODS: Wild-type and mutant (F1579A) recombinant rat skeletal muscle sodium channels were expressed in Xenopus oocytes. The F1579A amino acid substitution site is part of the intrapore local anesthetic receptor. The effect of ketamine was measured in oocytes expressing wild-type or mutant sodium channels using two-electrode voltage clamp. RESULTS: Ketamine blocked sodium channels in a local anesthetic-like fashion, exhibiting tonic blockade (concentration for half-maximal inhibition [IC50] = 0.8 mm), phasic blockade (IC50 = 2.3 mm), and leftward shift of the steady-state inactivation; the parameters of these actions were strongly modified by alteration of the intrapore local anesthetic binding site (IC50 = 2.1 mm and IC50 = 10.3 mm for tonic and phasic blockade, respectively). Compared with lidocaine, ketamine showed greater tonic inhibition but less phasic blockade. CONCLUSIONS: Ketamine interacts with sodium channels in a local anesthetic-like fashion, including sharing a binding site with commonly used clinical local anesthetics.     

Ketamine Blockade of Voltage-gated Sodium Channels: Evidence for a Shared Receptor Site with Local Anesthetics

Background : The general anesthetic ketamine is known to be an N-methyl-d-aspartate receptor blocker. Although ketamine also blocks voltage-gated sodium channels in a local anesthetic-like fashion, little information exists on the molecular pharmacology of this interaction. We measured the effects of ketamine on sodium channels.

Methods : Wild-type and mutant (F1579A) recombinant rat skeletal muscle sodium channels were expressed in Xenopus oocytes. The F1579A amino acid substitution site is part of the intrapore local anesthetic receptor. The effect of ketamine was measured in oocytes expressing wild-type or mutant sodium channels using two-electrode voltage clamp.

Results : Ketamine blocked sodium channels in a local anesthetic-like fashion, exhibiting tonic blockade (concentration for half-maximal inhibition [IC50] = 0.8 mm), phasic blockade (IC50 = 2.3 mm), and leftward shift of the steady-state inactivation; the parameters of these actions were strongly modified by alteration of the intrapore local anesthetic binding site (IC50 = 2.1 mm and IC50 = 10.3 mm for tonic and phasic blockade, respectively). Compared with lidocaine, ketamine showed greater tonic inhibition but less phasic blockade.     

Different Actions of General Anesthetics on the Firing Patterns of Neocortical Neurons Mediated by the GABAA Receptor

Background: In cultured slice preparations of rat neocortical tissue, clinically relevant concentrations of volatile anesthetics mainly decreased action potential firing of neurons by enhancing [Greek small letter gamma]-aminobutyric acid (GABAA) receptor-mediated synaptic inhibition. The author's aim was to determine if other anesthetic agents are similarly effective in this model system and act via the same molecular mechanism.

Methods: The actions of various general anesthetics on the firing patterns of neocortical neurons were investigated by extracellular single-unit recordings.

Results: Pentobarbital, propofol, ketamine, and ethanol inhibited spontaneous action potential firing in a concentration-dependent manner. The estimated median effective concentration (EC50) values were close to or below the EC50 values for general anesthesia. Bath application of the GABAA antagonist bicuculline (100 [micro sign]M) decreased the effectiveness of propofol, ethanol, halothane, isoflurane, enflurane, and diazepam by more than 90%, indicating that these agents acted predominantly via the GABAA receptor. The depressant effects of pentobarbital and ketamine were not significantly reduced by bicuculline treatment. Drugs acting mainly via the GABAA receptor altered the firing patterns of neocortical cells in different manners. Diazepam reduced the discharge rates by decreasing the number of action potentials per burst, leaving the burst rate unaffected. In contrast, muscimol, GABA, propofol, and volatile anesthetics decreased the burst rate.     

Kinetic Modulation of HERG Potassium Channels by the Volatile Anesthetic Halothane

Background: HERG (human ether-a-gogo related gene) encodes the cardiac rapidly activating delayed rectifier potassium currents (Ikr), which play an important role in cardiac action potential repolarization. General anesthetics, like halothane, can prolong Q-T interval, suggesting that they act on myocellular repolarization, possibly involving HERG channels. Evidence for direct modulation of HERG channels by halothane is still lacking. To gain insight on HERG channel modulation by halothane the authors recorded macroscopic currents expressed in Xenopus oocytes and conducted non-stationary noise analysis to evaluate single channel parameters modified by the anesthetic.

Methods: Macroscopic currents were recorded in 120 mm K+ internal-5 mm K+ external solutions with the cut open oocyte technique. Macropatch recordings for non-stationary noise analysis of HERG tail currents were made in symmetrical 120 mm K+ solutions. Pulse protocols designed for HERG current recording were elicited from a holding potential of -80 mV. Halothane was delivered via gravity-fed perfusion.

Results: Halothane (0.7%, 1.5%, and 3%) decreased macroscopic HERG currents in a concentration-dependent manner (average reduction by 14%, 22%, and 35% in the range of -40 mV to 40 mV) irrespective of potential. HERG currents had slower activation and accelerated deactivation and inactivation. Non-stationary noise analysis revealed that halothane, 1.5%, decreased channel Po by 27%, whereas single-channel current amplitudes and number of channels in the patch remained unchanged.     

Effects of Halothane and Isoflurane on Fast and Slow Inactivation of Human Heart hH1a Sodium Channels

Background: Cloning and heterologous expression of ion channels allow biophysical and molecular studies of the mechanisms of volatile anesthetic interactions with human heart sodium channels. Volatile anesthetics may influence the development of arrhythmias arising from cardiac sodium channel dysfunction. For that reason, understanding the mechanisms of interactions between these anesthetics and cardiac sodium channels is important. This study evaluated the mechanisms of volatile anesthetic actions on the cloned human cardiac sodium channel (hH1a) [Greek small letter alpha] subunit.

Methods: Inward sodium currents were recorded from human embryonic kidney (HEK293) cells stably expressing hH1a channels. There effects of halothane and isoflurane on current and channel properties were evaluated using the whole cell voltage-clamp technique.

Results: Halothane at 0.47 and 1.1 mM and isoflurane at 0.54 and 1.13 mM suppressed the sodium current in a dose- and voltage-dependent manner. Steady state activation was not affected, but current decay was accelerated. The voltage dependence of steady state fast and slow inactivations was shifted toward more hyperpolarized potentials. The slope factor of slow but not fast inactivation curves was reduced significantly. Halothane increased the time constant of recovery from fast inactivation. The recovery from slow inactivation was not affected significantly by either anesthetic.     

Differential Effects of General Anesthetics on G Protein-coupled Inwardly Rectifying and Other Potassium Channels

Background: General anesthetics differentially affect various families of potassium channels, and some potassium channels are suggested to be potential targets for anesthetics and alcohols.

Methods: The voltage-gated (ERG1, ELK1, and KCNQ2/3) and inwardly rectifying (GIRK1/2, GIRK1/4, GIRK2, IRK1, and ROMK1) potassium channels were expressed in Xenopus oocytes. Effects of volatile agents [halothane, isoflurane, enflurane, F3 (1-chloro-1,2,2-trifluorocyclobutane), and the structurally related nonimmobilizer F6 (1,2-dichlorohexafluoro-cyclobutane)], as well as intravenous (pentobarbital, propofol, etomidate, alphaxalone, ketamine), and gaseous (nitrous oxide) anesthetics and alcohols (ethanol and hexanol) on channel function were studied using a two-electrode voltage clamp.

Results: ERG1, ELK1, and KCNQ2/3 channels were either inhibited slightly or unaffected by concentrations corresponding to twice the minimum alveolar concentrations or twice the anesthetic EC50 of volatile and intravenous anesthetics and alcohols. In contrast, G protein-coupled inwardly rectifying potassium (GIRK) channels were inhibited by volatile anesthetics but not by intravenous anesthetics. The neuronal-type GIRK1/2 channels were inhibited by 2 minimum alveolar concentrations of halothane or F3 by 45 and 81%, respectively, whereas the cardiac-type GIRK1/4 channels were inhibited only by F3. Conversely, IRK1 and ROMK1 channels were completely resistant to all anesthetics tested. Current responses of GIRK2 channels activated by [mu]-opioid receptors were also inhibited by halothane. Nitrous oxide (~0.6 atmosphere) slightly but selectively potentiated GIRK channels. Results of chimeric and multiple amino acid mutations suggest that the region containing the transmembrane domains, but not the pore-forming domain, may be involved in determining differences in anesthetic sensitivity between GIRK and IRK channels.     

Differential membrane effects of general and local anesthetics.

The authors studied the effects of varying Na+ and Ca++ concentrations and of replacing H2O with D2O in Ringer's solution upon the actions of general and local anesthetics on isolated frog sciatic nerves. This experimental model was used to study whether general anesthetics affect excitable membranes in a manner similar to that of typical membrane stabilizers (local anesthetics). Procaine (2.5-7.5 mM), halothane (9, 18, and 36 mM), enflurane (8 mM), and ketamine (0.15 and 0.73 mM) raised threshold and lowered spike amplitude, and their effects were facilitated by reducing Na+ concentration in the Ringer's solution. The local anesthetic effects of procaine (2.5-7.5 mM) and ketamine (0.73 mM) were antagonized by Ca++, while the axonal depressant effect of halothane was facilitated by increasing Ca++ concentration in the Ringer's solution, indicating a different mode of action. General anesthetics also differed from local anesthetics in their interaction with water: replacement by D2O of H2O in the Ringer's solution selectively increased the axonal depressant effects of halothane and enflurane but not those of ketamine or procaine. Since D2O differs from H2O in its greater ice-likeness, these results are consistent with the view that general anesthetics stabilize excitable membranes via stabilization of the water-biopolymer lattice, as predicted by the hydrate-microcrystal theory of anesthesia. In contrast, local anesthetics may stabilize excitable tissues by binding to the same fixed negative charges of the membrane to which Ca++ is normally bound. (Key words: Theories of anesthesia, hydrate-microcrystal; Nerve, mode of action of anesthetics; Anesthetics, volatile, halothane; Anesthetics, volatile, enflurane; Anesthetics, local, procaine; Anesthetics, intravenous, ketamine.)     

Intravenous Anesthetics Differentially Modulate Ligand-gated Ion Channels

Background: Heteromeric neuronal nicotinic acetylcholine receptors (nAChRs) are potently inhibited by volatile anesthetics, but it is not known whether they are affected by intravenous anesthetics. Ketamine potentiates [gamma]-aminobutyric acid type A (GABAA) receptors at high concentrations, but it is unknown whether there is potentiation at clinically relevant concentrations. Information about the effects of intravenous anesthetics with different behavioral profiles on specific ligand-gated ion channels may lead to hypotheses as to which ion channel effect produces a specific anesthetic behavior.

Methods: A heteromeric nAChR composed of [alpha]4 and [beta]4 subunits was expressed heterologously in Xenopus laevis oocytes. Using the two-electrode voltage clamp technique, peak ACh-gated current was measured before and during application of ketamine, etomidate, or thiopental. The response to GABA of [alpha]1[beta]2[gamma]2s GABAA receptors expressed in human embryonic kidney cells and Xenopus oocytes was compared with and without coapplication of ketamine from 1 [mu]m to 10 mm.

Results: Ketamine caused potent, concentration-dependent inhibition of the [alpha]4[beta]4 nAChR current with an IC50 of 0.24 [mu]m. The inhibition by ketamine was use-dependent; the antagonist was more effective when the channel had been opened by agonist. Ketamine did not modulate the [alpha]1[beta]2[gamma]2s GABAA receptor response in the clinically relevant concentration range. Thiopental caused 27% inhibition of ACh response at its clinical EC50. Etomidate did not modulate the [alpha]4[beta]4 nAChR response in the clinically relevant concentration range, although there was inhibition at very high concentrations.     

Differential Modulation of the Cardiac Adenosine Triphosphate-sensitive Potassium Channel by Isoflurane and Halothane

Background: The cardiac adenosine triphosphate-sensitive potassium (KATP) channel is activated during pathophysiological episodes such as ischemia and hypoxia and may lead to beneficial effects on cardiac function. Studies of volatile anesthetic interactions with the cardiac KATP channel have been limited. The goal of this study was to investigate the ability of volatile anesthetics halothane and isoflurane to modulate the cardiac sarcolemmal KATP channel.

Methods: The KATP channel current (IKATP) was monitored using the whole cell configuration of the patch clamp technique from single ventricular cardiac myocytes enzymatically isolated from guinea pig hearts. IKATP was elicited by extracellular application of the potassium channel openers 2,4-dinitrophenol or pinacidil.

Results: Volatile anesthetics modulated IKATP in an anesthetic-dependent manner. Isoflurane facilitated the opening of the KATP channel. Following initial activation of IKATP by 2,4-dinitrophenol, isoflurane at 0.5 and 1.3 mm further increased current amplitude by 40.4 +/- 11.1% and 58.4 +/- 20.6%, respectively. Similar results of isoflurane were obtained when pinacidil was used to activate IKATP. However, isoflurane alone was unable to elicit KATP channel opening. In contrast, halothane inhibited IKATP elicited by 2,4-dinitrophenol by 50.6 +/- 5.8% and 72.1 +/- 11.6% at 0.4 and 1.0 mm, respectively. When IKATP was activated by pinacidil, halothane had no significant effect on the current.     

Volatile General Anesthetics Produce Hyperpolarization of Aplysia Neurons by Activation of a Discrete Population of Baseline Potassium Channels

Background: The mechanism by which volatile anesthetics act on neuronal tissue to produce reversible depression is unknown. Previous studies have identified a potassium current in invertebrate neurons that is activated by volatile anesthetics. The molecular components generating this current are characterized here in greater detail.

Methods: The cellular and biophysical effects of halothane and isoflurane on neurons of Aplysia californica were studied. Isolated abdominal ganglia were perfused with anesthetic-containing solutions while membrane voltage changes were recorded. These effects were also studied at the single-channel level by patch clamping cultured neurons from the abdominal and pleural ganglia.

Results: Clinically relevant concentrations of halothane and isoflurane produced a slow hyperpolarization in abdominal ganglion neurons that was sufficient to block spontaneous spike firings. Single-channel studies revealed specific activation by volatile anesthetics of a previously described potassium channel. In pleural sensory neurons, halothane and isoflurane increased the open probability of the outwardly rectifying serotonin-sensitive channel (S channel). Halothane also inhibited a smaller noninactivating channel with a linear slope conductance of approximately 40 pS. S channels were activated by halothane with a median effective concentration of approximately 500 micro Meter (0.013 atm), which increased channel activity about four times. The mechanism of channel activation involved shortening the closed-time durations between bursts and apparent recruitment of previously silent channels.     

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.     

Anesthetics Affect the Uptake but Not the Depolarization-evoked Release of GABA in Rat Striatal Synaptosomes

Background: Numerous classes of anesthetic agents have been shown to enhance the effects mediated by the postsynaptic gamma-aminobutyric acid A (GABAA) receptor-coupled chloride channel in the mammalian central nervous system. However, presynaptic actions of anesthetics potentially relevant to clinical anesthesia remain to be clarified. Therefore, in this study, the effects of intravenous and volatile anesthetics on both the uptake and the depolarization-evoked release of GABA in the rat stratum were investigated.

Methods: Assay for specific GABA uptake was performed by measuring the radioactivity incorporated in purified striatal synaptosomes incubated with3 H-GABA (20 nM, 5 min, 37 degrees Celsius) and increasing concentrations of anesthetics in either the presence or the absence of nipecotic acid (1 mM, a specific GABA uptake inhibitor). Assay for GABA release consisted of superfusing3 H-GABA preloaded synaptosomes with artificial cerebrospinal fluid (0.5 ml *symbol* min sup 1, 37 degrees Celsius) and measuring the radioactivity obtained from 0.5 ml fractions over 18 min, first in the absence of any treatment (spontaneous release, 8 min), then in the presence of either KCl alone (9 mM, 15 mM) or with various concentrations of anesthetics (5 min), and finally, with no pharmacologic stimulation (5 min). The following anesthetic agents were tested: propofol, etomidate, thiopental, ketamine, halothane, enflurane, isoflurane, and clonidine.

Results: More than 95% of3 H-GABA uptake was blocked by a 10 sup 3 -M concentration of nipecotic acid. Propofol, etomidate, thiopental, and ketamine induced a dose-related, reversible, noncompetitive, inhibition of3 H-GABA uptake: IC50 = 4.6 plus/minus 0.3 x 105 M, 5.8 plus/minus 0.3 x 10 sup -5 M, 2.1 plus/minus 0.4 x 10 sup -3 M, and 4.9 plus/minus 0.5 x 10 sup -4 M for propofol, etomidate, thiopental, and ketamine, respectively. Volatile agents and clonidine had no significant effect, even when used at concentrations greater than those used clinically. KCl application induced a significant, calcium-dependent, concentration-related, increase from basal3 H-GABA release, +34 + 10% (P < 0.01) and +61 plus/minus 13% (P < 0.001), respectively, for 9 mM and 15 mM KCl. The release of3 H-GABA elicited by KCl was not affected by any of the anesthetic agents tested.     

Temperature-independent Inhibition of L-Type Calcium Currents by Halothane and Sevoflurane in Human Atrial Cardiomyocytes

Background: Cardiac L-type calcium currents (ICa,L) are affected by volatile anesthetics, possibly contributing to their side effects. Actions of anesthetics on ion channels are usually studied in vitro at room temperature. However, the solubility of anesthetic gases as well as ICa,L are markedly sensitive to the study temperature. Therefore, temperature-dependent effects of halothane and sevoflurane on cardiac ICa,L were analyzed.

Methods: ICa,L were studied at 21[degrees]C and 36[degrees]C with the patch clamp technique in isolated human atrial cardiomyocytes. Concentrations of anesthetics brought into solution by gassing at both temperatures were determined with gas chromatography.

Results: The aqueous concentrations of halothane and sevoflurane were linearly related to their concentration in the gas phase (1 to 3 minimum alveolar concentration [MAC]). At 21[degrees]C, the slope of this relation was 0.52 and 0.12 mm/vol % for halothane and sevoflurane, respectively, and decreased at 36[degrees]C to 0.29 and 0.09 mm/vol %, respectively. ICa,L displayed significantly higher current amplitudes at 36[degrees]C than at 21[degrees]C and significantly accelerated time-dependent inactivation. Halothane (1-2 MAC) and sevoflurane (1-3 MAC) evoked stronger inhibitions of ICa,L at 21[degrees]C than at 36[degrees]C. In spite of different temperature-dependent current amplitudes, the fractional (percent) inhibition of ICa,L showed the same linear relationship to the concentrations of halothane and sevoflurane in the bath medium at both temperatures, as revealed from present and previous experiments.     

The effects of general anesthetics on excitatory and inhibitory synaptic transmission in area CA1 of the rat hippocampus in vitro

It is unclear whether general anesthetics induce enhancement of neural inhibition and/or attenuation of neural excitation. We studied the effects of pentobarbital (5 x 10(-4) mol/L), propofol (5 x 10(-4) mol/L), ketamine (10(-3) mol/L), halothane (1.5 vol%), and isoflurane (2.0 vol%) on both excitatory and inhibitory synaptic transmission in rat hippocampal slices. Excitatory or inhibitory synaptic pathways were isolated using pharmacological antagonists. Extracellular microelectrodes were used to record electrically evoked CA1 neural population spikes (PSs). In the presence of the gamma-aminobutyric acid type A (GABA(A)) receptor antagonist (bicuculline), the inhibitory actions of pentobarbital and propofol were completely antagonized, whereas those of ketamine, halothane, and isoflurane were only partially blocked. To induce the N-methyl-D-aspartate (NMDA) receptor-mediated PS (NMDA PS), the non-NMDA and GABA(A) receptors were blocked in the absence of Mg2+. Ketamine, halothane, and isoflurane decreased the NMDA PS, and pentobarbital and propofol had no effect on the NMDA PS. The non-NMDA receptor-mediated PS (non-NMDA PS) was examined using the antagonists for the NMDA and GABA(A) receptors. Volatile, but not i.v., anesthetics reduced the non-NMDA PS. These findings indicate that pentobarbital and propofol produce inhibitory actions due to enhancement in the GABA(A) receptor; that ketamine reduces NMDA receptor-mediated responses and enhances GABA(A) receptor-mediated responses; and that halothane and isoflurane modulate GABA(A), NMDA, and non-NMDA receptor-mediated synaptic transmission. IMPLICATIONS: Volatile anesthetics modulate both excitatory and inhibitory synaptic transmission of in vitro rat hippocampal pathways, whereas i.v. anesthetics produce more specific actions on inhibitory synaptic events. These results provide further support the idea that general anesthetics produce drug-specific and distinctive effects on different pathways in the central nervous system.     

Interaction of Ropivacaine with Cloned Cardiac Kv4.3/KChIP2.2 Complexes

Background: Inhibition of cardiac K channels by local anesthetic may contribute to QTc interval prolongation of the electrocardiogram and induction of ventricular arrhythmia. The transient outward current Ito has been identified as a toxicologically relevant target of bupivacaine. S(-)-ropivacaine has been developed as a safer alternative to bupivacaine. The effects of S(-)-ropivacaine on Ito have not been investigated. In human ventricular myocardium, Ito is formed by Kv4.3 and KChIP2.2 subunits. Therefore, the aim of this study was to establish the effects of S(-)-ropivacaine on human Kv4.3/KChIP2.2 channels.

Methods: Kv4.3/KChIP2.2 complementary DNA cloned from human heart was transiently transfected in Chinese hamster ovary cells. The pharmacologic effects of S(-)-ropivacaine were investigated with the patch clamp method.

Results: Ropivacaine inhibited Kv4.3/KChIP2.2 channels in a concentration-dependent, stereospecific, and reversible manner. The IC50 value of S(-)-ropivacaine for inhibition of the charge conducted by Kv4.3/KChIP2.2 channel was 117 +/- 21 [mu]m (n = 30). The local anesthetic accelerated macroscopic current decline with an IC50 value of 77 +/- 11 [mu]m (n = 30). It shifted the midpoint of channel activation into the depolarizing direction, and it slowed recovery from inactivation without altering steady state inactivation. Kv4.3 channels are more sensitive to the inhibitory effect than Kv4.3/KChIP2.2 channels.