Inhibition of Presynaptic Sodium Channels by Halothane

Background: Recent electrophysiologic studies indicate that clinical concentrations of volatile general anesthetic agents inhibit central nervous system sodium (Na sup +) channels. In this study, the biochemical effects of halothane on Na sup + channel function were determined using rat brain synaptosomes (pinched-off nerve terminals) to assess the role of presynaptic Na sup + channels in anesthetic effects.

Methods: Synaptosomes from adult rat cerebral cortex were used to determine the effects of halothane on veratridine-evoked Na sup + channel-dependent Na sup + influx (using22 Na sup +), changes in intrasynaptosomal [Na sup +] (using ion-specific spectrofluorometry), and neurotoxin interactions with specific receptor sites of the Na sup + channel (by radioligand binding). The potential physiologic and functional significance of these effects was determined by measuring the effects of halothane on veratridine-evoked Na sup + channel-dependent glutamate release (using enzyme-coupled spectrofluorometry).

Results: Halothane inhibited veratridine-evoked22 Na sup + influx (IC50 = 1.1 mM) and changes in intrasynaptosomal [Na sup +] (concentration for 50% inhibition [IC50] = 0.97 mM), and it specifically antagonized [sup 3 H]batrachotoxinin-A 20-alpha-benzoate binding to receptor site two of the Na sup + channel (IC50 = 0.53 mM). Scatchard and kinetic analysis revealed an allosteric competitive mechanism for inhibition of toxin binding. Halothane inhibited veratridine-evoked glutamate release from synaptosomes with comparable potency (IC50 = 0.67 mM).     

Conformational State-dependent Effects of Halothane on Cardiac Na sup + Current

Background: The Na sup + channel is voltage gated and characterized by three distinct states: closed, open, and inactivated. To identify the effects of halothane on the cardiac Na sup + current (INa) at various membrane potentials, the effects of 1.2 mm halothane at different holding potentials (VH) on INa were examined in single, enzymatically isolated guinea pig ventricular myocytes.

Methods: The INa was recorded using the whole-cell configuration of the patch-clamp technique. Currents were generated from resting VH s of -110, -80, or -65 mV. State-dependent block was characterized by monitoring frequency dependence, tonic block, and removal of inactivation by veratridine.

Results: Halothane produced significant (P < 0.05) VH -dependent depressions of peak INa (mean +/- SEM): 24.4 +/- 4.1% (VH = -110 mV), 42.1 +/- 3.4% (VH = -80 mV), and 75.2 +/- 1.5% (VH = -65 mV). Recovery from inactivation was significantly increased when cells were held at -80 mV (control, tau = 6.0 +/- 0.3 ms; halothane, tau = 7.1 +/- 0.4 ms), but not at -110 mV. When using a VH of -80 mV, halothane exhibited a use-dependent block, with block of INa increasing from 8.6 +/- 1.4% to 30.7 +/- 3.5% at test pulse rates of 2 and 11 Hz, respectively. Use-dependent inhibition was not apparent at VH of -110 mV. When inactivation of INa was removed by exposure to 100 micro Meter veratridine, no significant difference was observed in the depressant effect of halothane at both VH s: 26.6 +/- 4.5% (VH = -80 mV) and 26.4 +/- 5.6% (VH = -110 mV).     

A comparison of the effects of halothane and tetrodotoxin on the regional repolarization characteristics of canine Purkinje fibers

The effects of halothane, tetrodotoxin (TTX), veratridine (VTD), and alterations of extracellular calcium ion concentration [Ca++]0) on regional differences of canine Purkinje fiber action potential duration (APD50 and APD90) were investigated in vitro at a paced rate of 75 beats per min. Under control conditions (n = 15 hearts) APD90 of proximal (false tendon) fibers (289 +/- 6 ms) always exceeded (P less than or equal to 0.01) that of distal (apical) fibers (213 +/- 4 ms). Halothane (0.35-1.07 mM) reduced regional differences of APD90 by producing dose-dependent decreases of proximal APD90 without decreases of distal APD90. The regional actions of halothane were similar to those of low (0.33-1.0 microM) concentrations of the Na+ channel antagonist TTX, which also decreased proximal APD90 more than distal APD90. The actions of halothane in combination with TTX further decreased proximal APD90, whereas the Na+ channel agonist VTD, which increased proximal APD90 more than distal APD90, reversed the regional actions of halothane. Decreasing Ca++ influx by reducing [Ca++]0 from 1.8 to 0.6 mM increased proximal APD90 more than distal APD90 in a manner opposite to the regional actions of halothane. Although there was no difference between the values of APD90 obtained for each region in the presence of halothane at 0.6, 1.8, and 3.6 mM [Ca++]0, the action of halothane decreasing APD90 of proximal fibers was more prominent at 0.6 mM [Ca++]0 because of the increased APD90 of fibers under this condition. The findings are consistent with, but do not definitively prove, the hypothesis that halothane may decrease APD90 of proximal Purkinje fibers by a mechanism similar to that of TTX involving inhibition of plateau-phase inward Na+ current.     

Differential use-dependent (frequency-dependent) effects in single mammalian axons: data and clinical considerations

The potential clinical scope of use-dependent block of conduction (UDB) was assessed by studying characteristics of UDB in vitro in individual mammalian axons. Single and repetitive stimulation was applied to rabbit cervical sympathetic and vagus nerves exposed to solutions containing lidocaine 0, 0.3, or 0.6 mmol/l (9.1 or 18.2 mg/dl) at 37 degrees C. Unit responses were recorded in dissected filaments or extracellularly in the vagus nodose ganglion. With lidocaine 0.3 mM, equilibrium conduction block, tested by single shocks, was rare. 40-Hz trains produced a significantly greater increase in latency (slowing of conduction) and a much greater incidence of UDB in the sympathetic units than in myelinated vagus axons of equivalent control conduction velocities or in unmyelinated axons. 10-Hz stimulation did not produce UDB. With lidocaine 0.6 mM, the incidence of equilibrium conduction block was too high among sympathetic axons to assess UDB, and significantly higher than among nonsympathetic myelinated and unmyelinated units. The observations support the hypothesis that the differential block of sympathetics observed clinically with spinal anesthesia may be, at least in part, a use-dependent (frequency-dependent) effect. UDB seems unlikely to contribute to local anesthetic block of pain impulses.     

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 [approximately 0.8 minimum alveolar concentration (MAC)] or veratridine (IC50 = 0.42 mM [approximately 0.4 MAC]), and veratridine-evoked increases in [Ca2+]i (IC50 = 0.5 mM [approximately 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 [approximately 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. CONCLUSIONS: The anesthetic cyclobutane F3 significantly inhibited Na+ channel-mediated glutamate release and increases in [Ca2+]i. In contrast, the nonanesthetic cyclobutane F6 had no significant effects at predicted anesthetic concentrations. F3 inhibited dorsal root ganglion neuron Na+ channels with a potency and by mechanisms similar to those of conventional volatile anesthetics; F6 was less effective and did not produce voltage-dependent block. This concordance between anesthetic activity and Na+ channel inhibition supports a role for presynaptic Na+ channels as targets for general anesthetic effects and suggests that shifting the voltage-dependence of Na+ channel inactivation is an important property of volatile anesthetic compounds.     

Modulation of the Cardiac Sodium Current by Inhalational Anesthetics in the Absence and Presence of beta-Stimulation

Background: Cardiac dysrhythmias during inhalational anesthesia in association with catecholamines are well known, and halothane is more "sensitizing" than isoflurane. However, the underlying mechanisms of action of volatile anesthetics with or without catecholamines on cardiac Na channels are poorly understood. In this study, the authors investigated the effects of halothane and isoflurane in the absence and presence of beta-stimulation (isoproterenol) on the cardiac Na sup + current (INa) in ventricular myocytes enzymatically isolated from adult guinea pig hearts.

Methods: A standard whole-cell patch-clamp technique was used. The INa was elicited by depolarizing test pulses from a holding potential of -80 mV in reduced Na sup + solution (10 mM).

Results: Isoproterenol alone depressed peak INa significantly by 14.6 +/- 1.7% (means +/- SEM). Halothane (1.2 mM) and isoflurane (1.0 mM) also depressed peak INa significantly by 42.1 +/- 3.4% and 21.3 +/- 1.9%, respectively. In the presence of halothane, the effect of isoproterenol (1 micro Meter) was potentiated, further decreasing peak I sub Na by 34.7 +/- 4.1%. The halothane effect was less, although significant, in the presence of a G-protein inhibitor (GDP beta S) or a specific protein kinase A inhibitor [PKI-(6-22)-amide], reducing peak I sub Na by 24.2 +/- 3.3% and 24 +/- 2.4%, respectively. In combination with isoflurane, the effect of isoproterenol on INa inhibition was less pronounced, but significant, decreasing current by 12.6 +/- 3.9%. GDP beta S also reduced the inhibitory effect of isoflurane. In contrast, PKI-(6-22)-amide had no effect on isoflurane INa inhibition.     

Halothane Blocks Synaptic Excitation of Inhibitory Interneurons

Background: Activation of principal hippocampal neurons is controlled by feedforward and feedback inhibition mediated by gamma-aminobutyric acidergic interneurons. The effects of halothane on glutamate receptor-mediated synaptic excitation of inhibitory interneurons have not been reported yet.

Methods: The effects of halothane on glutamatergic excitatory postsynaptic currents and on spike threshold in visually identified interneurons were studied with tight-seal, whole-cell voltage- and current-clamp recordings in thin slices from adult mouse hippocampus. The excitatory postsynaptic currents were pharmacologically isolated into their N-methyl-D-aspartate and non-N-methyl-D-aspartate receptor-mediated components using selective antagonists.

Results: Halothane (0.37-2.78 mM) reversibly blocked non-N-methyl-D-aspartate and N-methyl-D-aspartate excitatory postsynaptic currents in hippocampal oriens-alveus interneurons. Half-maximal inhibition was observed at similar concentrations (0.59 mM and 0.50 mM, respectively). Halothane inhibited synaptically generated action potentials at concentrations that did not elevate the spike threshold.     

Effects of halothane and isoflurane on calcium and potassium channel currents in canine coronary arterial cells.

The effects of halothane (0.75% and 1.5%) and isoflurane (2.6%) on macroscopic Ca2+ and K+ channel currents (ICa and IK, respectively) were investigated in voltage-clamped vascular muscle cells from the canine coronary artery. Single coronary arterial cells were dialyzed with K+ glutamate solution and superfused with Tyrode's solution for measurement of IK (n = 45). Stepwise depolarization from a holding potential of -60 mV to beyond -30 mV elicited an outward, slowly inactivating IK that had a macroscopic slope conductance of 18 nS. IK was reduced 75% by 10 mM 4-aminopyridine, a K+ channel antagonist. Compared to 4-aminopyridine, halothane at 0.75% and 1.5% reduced peak IK amplitude only by 14 +/- 2% and 36 +/- 3%, respectively. At approximately equianesthetic concentrations, 2.6% isoflurane suppressed IK less than did 1.5% halothane, reducing peak amplitude by 15 +/- 3%. In other sets of experiments, cells were dialyzed with 120 Cs(+)-glutamate solution and superfused with 10 mM BaCl2 or CaCl2 solutions to isolate ICa (n = 39) pharmacologically. Under these conditions, progressive depolarizing steps from -60 mV elicited a small inward current, which was potentiated 3.4-fold by equimolar substitution of Ba2+ for Ca2+ in the external solution and was blocked by 1 microM nifedipine. This inward current, which resembled L-type ICa, was blocked 37 +/- 4% and 70 +/- 4% in the presence of 0.75% and 1.5% halothane, respectively. Isoflurane (2.6%) also decreased ICa by 55 +/- 5%. It appears that while halothane and isoflurane suppress both IK and ICa, these anesthetics preferentially reduce ICa.(ABSTRACT TRUNCATED AT 250 WORDS)     

Effects of halothane, thiopental, and lidocaine on fluidity of synaptic plasma membranes and artificial phospholipid membranes.

The effects of halothane, thiopental, and lidocaine were studied with spin-labeling methods in synaptic plasma membranes (order parameter) and artificial phospholipid membranes (lateral diffusion). Halothane had a biphasic action, low concentrations (0.64 mM) ordering and high concentrations (2.9 mM) fluidizing both types of membranes. A biphasic effect in phospholipid membranes was also seen with thiopental, 0.1 mM ordering and 10 mM fluidizing, whereas in synaptic plasma membranes both low and high concentrations caused an increased order in the lipid bilayer region. At high thiopental concentrations, a considerable number of molecules may have reacted with membrane proteins or accumulated in the highly fluidic hydrophobic interior region of the membrane without affecting the rotational movement of the labeled fatty acid. Lidocaine alone, or together with calcium chloride, at various concentrations to 10 mM had no significant effect, and a fluidizing effect of 1 mM calcium chloride was possibly a result of interaction of calcium chloride with the label. The results indicate that the three lipid-soluble anesthetics interact differently with the lipid part of membranes. Lidocaine did not seem to affect bilyer lipids, while thiopental and halothane in phospholipid vesicles and halothane alone in synaptic membranes caused a dose-dependent biphasic effect.     

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.     

Halothane and Isoflurane Decrease the Open State Probability of Potassium sup + Channels in Dog Cerebral Arterial Muscle Cells

Background: Both halothane and isoflurane evoke cerebral vasodilation. One of the potential mechanisms for arterial vasodilation is enhanced Potassium sup + efflux resulting from an increased opening frequency of membrane Potassium sup + channels. The current study was designed to determine the effects of volatile anesthetics on Potassium sup + channel current in single vascular smooth muscle cells isolated from dog cerebral arteries.

Methods: Patch clamp recording techniques were used to investigate the effects of volatile anesthetics on macroscopic and microscopic Potassium sup + channel currents.

Results: In the whole-cell patch-clamp mode, in cells dialyzed with pipette solution containing 2.5 mM EGTA and 1.8 mM CaCl2, depolarizing pulses from 60 to +60 mV elicited an outward Potassium sup + current that was blocked 65 plus/minus 5% by 3 mM tetraethylammonium (TEA). Halothane (0.4 and 0.9 mM) depressed the amplitude of this current by 18 plus/minus 4% and 34 plus/minus 6%, respectively. When 10 mM EGTA was used in the pipette solution to strongly buffer intracellular free Calcium2+, an outward Potassium sup + current insensitive to 3 mM TEA was elicited. This Potassium sup + current, which was reduced 51 plus/minus 4% by 1 mM 4-aminopyridine, was also depressed by 17 plus/minus 5 and 29 plus/minus 7% with application of 0.4 and 0.9 mM halothane, respectively. In cell-attached patches using 145 mM KCl in the pipette solution and 5.2 mM KCl in the bath, the unitary conductance of the predominant channel type detected was 99 pS. External application of TEA (0.1 to 3 mM) reduced the unitary current amplitude of the 99 pS Potassium sup + channel in a concentration-dependent manner. The open state probability of this 99 pS Potassium sup + channel was increased by 1 micro Meter Calcium2+ ionophore (A23187). These findings indicate that the 99 pS channel measured in cell-attached patches was a TEA-sensitive, Calcium2+ -activated Potassium sup + channel. Halothane and isoflurane reversibly decreased the open state probability (NPo), mean open time, and frequency of opening of this 99 pS Potassium sup + channel without affecting single channel amplitude or the slope of the current-voltage relationship.     

Halothane presynaptically depresses synaptic transmission in wild-type Drosophila larvae but not in halothane-resistant (har) mutants.

BACKGROUND: General anesthetics produce important changes in neural function, but the relation between the many individual changes produced by anesthetics in neural components and the responsiveness of the whole organism is uncertain. An analysis of genetically altered animals that have modified responses to volatile anesthetics may help to allay this uncertainty. METHODS: The authors evaluated the effect of halothane on synaptic transmission at the larval neuromuscular junction in wild-type (Ore-R) and halothane-resistant (har) mutants of Drosophila melanogaster. The body wall muscles, which are innervated by glutamatergic nerves, were voltage clamped at -60 mV using the patch-clamp technique in the whole cell configuration. Nerve-evoked excitatory junctional currents and miniature excitatory junctional currents were recorded. The effects of halothane on the amplitude of these currents were compared in Ore-R and two bar mutants derived from the Ore-R strain. The time course and frequency of miniature excitatory junctional currents also were analyzed in the presence of halothane. RESULTS: In Ore-R, halothane (1.8%; 1.01 mM) significantly reduced the amplitude of nerve-evoked excitatory junctional currents (61.9+/-17% of control, mean +/- SD; n = 7), but not that of miniature excitatory junctional currents. Conversely, in two har mutants, halothane had no effect on the amplitude of either nerve-evoked excitatory junctional currents or miniature excitatory junctional currents. In Ore-R, the frequency of miniature excitatory junctional currents was decreased significantly in the presence of halothane (0.9-2.7%; 0.52-1.46 mM), whereas halothane did not change the frequency in two har mutants. The miniature excitatory junctional current decay time constant, thought to reflect the kinetic properties of junctional glutamate receptor channels, was not changed by halothane in either the Ore-R strain or the har mutants. CONCLUSIONS: Halothane depresses synaptic transmission at the wild-type Drosophila neuromuscular junction, most likely by affecting presynaptic properties. The absence of an effect by halothane in the har mutants provides evidence that the depression of presynaptic function at the glutamate-mediated synapses is an important contributor to the way halothane alters the responsiveness of the whole animal.     

Kinetic modulation of HERG potassium channels by the volatile anesthetic halothane

BACKGROUND: (human ether-a-gogo related gene) encodes the cardiac rapidly activating delayed rectifier potassium currents (I(kr)), 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 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 P(o) by 27%, whereas single-channel current amplitudes and number of channels in the patch remained unchanged. CONCLUSIONS: Halothane inhibits HERG currents expressed in oocytes in a concentration-dependent manner. It slowed down activation and accelerated deactivation and inactivation of HERG channels. The authors' results demonstrate that halothane decreased HERG currents by modulating kinetic properties of HERG channels, decreasing their open probability. Partial block of I(kr) currents could contribute to delayed myocellular repolarization and altered cardiac electrophysiology.     

Halothane relaxes previously constricted isolated porcine coronary artery segments more than isoflurane

Coronary vasodilation by halothane and isoflurane were compared using in vitro tension recording. Porcine left anterior descending coronary arterial segments (1.5-2.0 mm o.d.) were constricted with either K+ (30 mM) or prostanoid U44069 (6 X 10(-7) M) in the absence of other drugs or anesthetics. Following stabilization of constriction, arteries were exposed to halothane or isoflurane at 0.5, 1.0, 1.5, 2.0, and 3.0% concentrations. K+ (30 mM) induced constriction was reduced by halothane at 1.5, 2.0, and 3.0% and U44069 (6 X 10(-7) M) induced constriction was reduced at 0.5, 1.0, 1.5, 2.0, and 3.0%. K+ (30 mM) induced constriction was reduced by isoflurane only at 3.0% and U44069 (6 X 10(-7) M) induced constriction was reduced by isoflurane only at 2.0 and 3.0%. U44069 induced constriction was more susceptible than K+ induced constriction to relaxation by halothane or isoflurane. Halothane was more potent than isoflurane as a direct relaxant of porcine epicardial left anterior descending arterial segments previously constricted with K+ (30 mM) or U44069 (6 X 10(-7) M).     

AMPA receptor competitive antagonism reduces halothane MAC in rats.

Various subtypes of receptors have been identified for glutamate, an excitatory neurotransmitter. Previous studies have shown that antagonism of glutamate at the NMDA receptors reduces minimum alveolar concentration (MAC) for volatile anesthetics. NBQX (2,3-dihydroxy-6-nitro-7-sulfamoyl-benzo(f)quinoxaline) is a selective antagonist at the glutamatergic AMPA receptor. The purpose of this experiment was to determine whether AMPA receptor antagonism influences halothane MAC in the rat. Sprague-Dawley rats were anesthetized with halothane in 50% O2/balance N2, tracheally intubated and the lungs were mechanically ventilated. Increasing doses of NBQX were intravenously infused in three groups while the control group was infused with vehicle (D5W). Halothane MAC was then determined by the tail-clamp method. Halothane MAC was log-linearly related to plasma NBQX concentrations (MAC = 0.125 (In plasma concentration NBQX) + 1.035, r2 = 0.77). A maximal 58% reduction of halothane MAC was achieved with an NBQX loading dose of 42 mg/kg followed by a continuous infusion rate of 36 mg x kg-1 x h-1 (control = 1.02 +/- 0.07%; NBQX = 0.43 +/- 0.12%; P < .01). Larger doses of NBQX were not possible because of the poor aqueous solubility of this compound. In a separate experiment, awake rats were randomly assigned to groups based on the dose of NBQX infused. Pa(CO2) and mean arterial pressure were measured at time 0 and at 5 and 30 min after start of NBQX infusion. The infusion was then stopped. Time until recovery of the righting reflex was recorded.(ABSTRACT TRUNCATED AT 250 WORDS)     

Effects of halothane on delayed afterdepolarization and calcium transients in dog ventricular myocytes exposed to isoproterenol

The effects of halothane on isoproterenol-induced delayed after-depolarizations (DADs) were investigated in canine ventricular myocytes. In addition, the effects of halothane on the intracellular free calcium transient were determined in fura-2-loaded myocytes exposed to isoproterenol to explore the mechanisms underlying halothane effects on DADs. Isoproterenol (100 nM) induced DADs and/or undriven action potentials in myocytes stimulated electrically with the use of trains of 10 stimuli delivered at basic drive cycle lengths of 200-1,000 ms. Isoproterenol (100 nM) increased the peak ratio (350/380 nm excitation) of stimulated myocyte calcium transients; furthermore, isoproterenol induced a second spontaneous component in the calcium transients of 62% of treated myocytes (n = 72). Halothane (1.5%, 0.53 mM) significantly decreased the amplitude of isoproterenol-induced DADs (P less than 0.01). Halothane not only reduced the peak ratio of the stimulated calcium transient, but also eliminated the second spontaneous component in myocytes previously exposed to isoproterenol (n = 14). Elevated extracellular calcium concentrations (5 mM) restored the amplitudes of DADs and the second components of the calcium transients in myocytes exposed to isoproterenol and halothane. These data suggest that halothane opposes isoproterenol-induced DADs by altering intracellular calcium stores. The authors' findings do not support a role for DAD-induced triggered activity in the genesis of anesthetic-catecholamine dysrhythmias.     

Halothane does not decrease amiloride-sensitive alveolar fluid clearance in rabbits

Halothane decreases alveolar fluid clearance (AFC), a function required for efficient gas exchange in the rat. Further, halothane decreases amiloride-sensitive Na(+) transport in rat alveolar type II cells, a process responsible for a significant portion of AFC. We tested the hypothesis that halothane would decrease amiloride-sensitive AFC in rabbits. Rabbits anesthetized with 1.8% halothane had 5% albumin in 0.9% NaCl instilled into the right lung with (n = 11) or without (n = 11) 1 mM amiloride present in the instillate. Similarly, animals anesthetized with IV fentanyl and droperidol were administered 5% albumin solution with (n = 11) or without (n = 11) amiloride. At 90 min after instillation, alveolar fluid samples were obtained, and AFC was determined by changes in fluid protein concentration. Rabbits anesthetized with halothane or fentanyl and droperidol in the absence of amiloride had similar AFC values (35% +/- 12% and 35% +/- 7%, respectively, mean +/- SD). Rabbits anesthetized with halothane or fentanyl and droperidol in the presence of amiloride had similar AFC values (20% +/- 10% and 16% +/- 12%, respectively) that were significantly less than the groups not administered amiloride (P < 0.01). Unlike the rat, the ability of the rabbit to clear fluid from the alveolar space through amiloride-sensitive pathways is not decreased by halothane anesthesia. Implications: Unlike the rat, the ability of the rabbit to clear fluid from the alveolar space through amiloride-sensitive pathways is not decreased by halothane anesthesia.     

N-Phenylethyl Amitriptyline in Rat Sciatic Nerve Blockade

Background: The antidepressant amitriptyline is commonly used orally for the treatment of chronic pain, particularly neuropathic pain, which is thought to be caused by high-frequency ectopic discharge. Among its many properties, amitriptyline is a potent Na+ channel blocker in vitro, has local anesthetic properties in vivo, and confers additional blockade at high stimulus-discharge rates (use-dependent blockade). As with other drug modifications, adding a phenylethyl group to obtain a permanently charged quaternary ammonium derivative may improve these advantageous properties.

Methods: The electrophysiologic properties of N-phenylethyl amitriptyline were assessed in cultured neuronal GH3 cells with the whole cell mode of the patch clamp technique, and the therapeutic range and toxicity were evaluated in the rat sciatic nerve model.

Results: In vitro, N-phenylethyl amitriptyline at 10 [mu]m elicits a greater block of Na+ channels than amitriptyline (resting block of approximately 90%vs. approximately 15%). This derivative also retains the attribute of amitriptyline in evoking high-degree use-dependent blockade during repetitive pulses. In vivo, duration to full recovery of nociception in the sciatic nerve model was 1,932 +/- 72 min for N-phenylethyl amitriptyline at 2.5 mm (n = 7) versus 72 +/- 3 min for lidocaine at 37 mm (n = 4; mean +/- SEM). However, there was evidence of neurotoxicity at 5 mm.     

Effects of halothane and diltiazem on L-type calcium currents in single smooth muscle cells from rabbit portal veins

We have studied the effects of halothane and diltiazem on L-typevoltage-dependent calcium currents (I_Ca) in single smooth musclecells from rabbit portal veins using a whole cell voltage clamptechnique. The threshold of I_Ca was –30 mV and the peakcurrent was reached at 0 mV. Halothane (0.25, 0.5, 1.0, 1.5and 2.07%) decreased I_Ca in a concentration-dependent mannerand shifted the I_Ca activation threshold to the depolarizingside. Halothane 2.0% abolished Diltiazem 10^–8–10^–6mol litre^–1, a calcium channel antagonist, also depressedI_Ca in a concentration-dependent manner. Administration of both0.5% halothane and diltiazem 10 mol litre^–1 (concentrationslower than the clinical therapeutic range) abolished I_Ca however,halothane did not exhibit use-dependent inhibition of I_Ca whereasdiltiazem showed partial use-dependency. We conclude that thedecrease in I_Ca produced by halothane is associated with a directvasodilator effect of this anaesthetic, but is not explainedby block of Ca²⁺ channels similar to the action of diltiazem.Furthermore, administration of low concentrations of both halothaneand diltiazem decreased I_Ca and may reduce the contractilityof the vascular smooth muscle cells.     

Lamotrigine Inhibits Extracellular Glutamate Accumulation during Transient Global Cerebral Ischemia in Rabbits

Background: During cerebral ischemia, an influx of Na+ may be partially responsible for the release of the excitatory amino acid glutamate. When glutamate is released in excessive concentrations during ischemia, it may become neurotoxic. The ability of the Na+ channel blocker lamotrigine to inhibit glutamate release during episodes of transient global cerebral ischemia was investigated.

Methods: After approval was given by the animal care and use committee, 24 New Zealand white rabbits were randomly assigned to one of four groups each containing six animals (control, L20, L50, and a hypothermic group). After anesthesia (1% halothane) was induced, the tracheas were intubated and the lungs mechanically ventilated before microdialysis probes were placed in the hippocampus. Ninety minutes before the onset of ischemia, either 20 or 50 mg/kg lamotrigine was administered intravenously (in the L20 and L50 groups). Esophageal temperature was maintained at 38 degrees C in the control, L20, and L50 groups, whereas the animals in the hypothermic group were cooled to 30 degrees Celsius. Two 10-min periods of cerebral ischemia, separated by a 90-min interval, were generated by inflating a neck tourniquet. High-performance liquid chromatography was used to determine the glutamate concentration in the microdialysate. Analysis of variance and Dunnett's test were used for statistical analysis. Data are presented as means +/- SD.

Results: During the first ischemic period, glutamate concentration increased only slightly from baseline. A significant increase was observed during the second ischemic period for the control (sixfold) and the L20 (threefold) groups. Glutamate concentrations in the L50 and the hypothermic groups were significantly lower than in the other two groups and remained at the baseline level during the entire experiment.