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The molecular site of action for volatile anesthetics remains unknown despite many years of study. Members of the K(2P) potassium channel family, whose currents are potentiated by volatile anesthetics have emerged as possible anesthetic targets. In fact, a mouse model in which the gene for TREK-1 (KCNK2) has been inactivated shows resistance to volatile anesthetics. In this study we tested whether inactivation of another member of this ion channel family, KCNK7, in a knockout mouse displayed altered sensitivity to the anesthetizing effect of volatile anesthetics. KCNK7 knockout mice were produced by standard gene inactivation methods. Heterozygous breeding pairs produced animals that were homozygous, heterozygous or wild-type for the inactivated gene. Knockout animals were tested for movement in response to noxious stimulus (tail clamp) under varying concentrations of isoflurane, halothane, and desflurane to define the minimum alveolar concentration (MAC) preventing movement. Mice homozygous for inactivated KCNK7 were viable and indistinguishable in weight, general development and behavior from heterozygotes or wild-type littermates. Knockout mice (KCNK7-/-) displayed no difference in MAC for the three volatile anesthetics compared to heterozygous (+/-) or wild-type (+/+) littermates. Because inactivation of KCNK7 does not alter MAC, KCNK7 may play only a minor role in normal CNS function or may have had its function compensated for by other inhibitory mechanisms. Additional studies with transgenic animals will help define the overall role of the K(2P) channels in normal neurophysiology and in volatile anesthetic mechanisms.

Background: Previous studies demonstrated that MAC (the minimum alveolar concentration of an inhaled anesthetic that produces immobility in 50% of subjects exposed to noxious stimulation) for halothane directly correlates with the central nervous system concentration of Na+. However, those studies globally altered Na+ concentrations, and thus did not distinguish effects on the spinal cord from cerebral effects. This is an important distinction because the cord appears to be the primary site for mediation of the immobility produced by inhaled anesthetics. Accordingly, in the present study, we examined the effect of altering intrathecal versus intracerebroventricular concentrations of Na+ on MAC., Methods: In rats prepared with chronic indwelling catheters or stylets, we infused solutions deficient in Na+ and with an excess of Na+ into the lumbar subarachnoid and intracerebroventricular spaces and measured MAC for isoflurane before, during, and after infusion., Results: MAC of isoflurane correlated directly with concentrations of Na+ infused intrathecally but did not correlate with concentrations infused intracerebroventricularly., Conclusion: The results are consistent with a mediation or modulation of MAC by Na+ channels. These might include voltage-gated or ligand-gated channels or other Na-sensitive targets (e.g., pumps, transporters, exchangers).

Background: The minimum alveolar concentration (MAC) of isoflurane is a quantitative trait because it varies continuously in a population. The location on the genome of genes or other genetic elements controlling quantiative traits is called quantitative trait loci (QTLs). In this study we sought to detect a quantitative trait locus underlying isoflurane MAC in mice., Methods: To accomplish this, two inbred mouse strains differing in isoflurane MAC, the C57BL/6J and LP/J mouse strains, were bred through two generations to produce genetic recombination. These animals were genotyped for microsatellite markers. We also applied an independent, computational method for identifying QTL-regulating differences in isoflurane MAC. In this approach, the isoflurane MAC was measured in a panel of 19 inbred strains, and computationally searched for genomic intervals where the pattern of genetic variation, based on single nucleotide polymorphisms, correlated with the differences in isoflurane MAC among inbred strains., Results and Conclusions: Both methods of genetic analysis identified a QTL for isoflurane MAC that was located on the proximal part of mouse chromosome 7.

Background: Enhancement of the function of gamma-aminobutyric acid type A receptors containing the alpha1 subunit may underlie a portion of inhaled anesthetic action. To test this, the authors created gene knock-in mice harboring mutations that render the receptors insensitive to isoflurane while preserving sensitivity to halothane., Methods: The authors recorded miniature inhibitory synaptic currents in hippocampal neurons from hippocampal slices from knock-in and wild-type mice. They also determined the minimum alveolar concentration (MAC), and the concentration at which 50% of animals lost their righting reflexes and which suppressed pavlovian fear conditioning to tone and context in both genotypes., Results: Miniature inhibitory postsynaptic currents decayed more rapidly in interneurons and CA1 pyramidal cells from the knock-in mice compared with wild-type animals. Isoflurane (0.5-1 MAC) prolonged the decay phase of miniature inhibitory postsynaptic currents in neurons of the wild-type mice, but this effect was significantly reduced in neurons from knock-in mice. Halothane (1 MAC) slowed the decay of miniature inhibitory postsynaptic current in both genotypes. The homozygous knock-in mice were more resistant than wild-type controls to loss of righting reflexes induced by isoflurane and enflurane, but not to halothane. The MAC for isoflurane, desflurane, and halothane did not differ between knock-in and wild-type mice. The knock-in mice and wild-type mice did not differ in their sensitivity to isoflurane for fear conditioning., Conclusions: gamma-Aminobutyric acid type A receptors containing the alpha1 subunit participate in the inhibition of the righting reflexes by isoflurane and enflurane. They are not, however, involved in the amnestic effect of isoflurane or immobilizing actions of inhaled agents.

Background: Depletion of central nervous system catecholamines, including dopamine, can decrease MAC (the minimum alveolar concentration of an inhaled anesthetic required to suppress movement in response to a noxious stimulus in 50% of test subjects); release of central nervous system catecholamines, including dopamine, can increase MAC; and increased free dopamine concentrations in the striatum can decrease MAC. Such findings suggest that dopamine receptors might mediate part of the capacity of inhaled anesthetics to provide immobility in the face of noxious stimulation., Methods: We measured the effect of blockade of D2 dopamine-mediated transmission with 0.3 mg/kg or 3.0 mg/kg droperidol on the MAC of cyclopropane, desflurane, halothane, isoflurane, or sevoflurane in rats, and the effect of 3.0 mg/kg droperidol on the dose or concentration of etomidate (an anesthetic known to act principally by enhancing the response of gamma-aminobutyric acid(A) receptors to gamma-aminobutyric acid) required to suppress movement in response to noxious stimulation., Results: Blockade of D2 dopamine-mediated transmission with droperidol does not decrease the MAC of cyclopropane, desflurane, halothane, isoflurane, or sevoflurane or its equivalent for etomidate in rats., Conclusions: These data, plus data from studies by others about D1 dopamine receptors, indicate that dopamine receptors do not mediate the immobility produced by inhaled anesthetics.

Most studies of chirality in inhaled anesthetic action have used the enantiomers of isoflurane. These enantiomers are expensive and scarce, which limits studies, such as the preliminary identification of molecular targets of anesthetic action, that can be performed with these isomers. We hypothesized that secondary alcohols (i.e., compounds having a -CH2-CHOH-CH3 group) that are experimental anesthetics would show enantioselectivity. To test this hypothesis, we determined the minimum alveolar anesthetic concentration (MAC) of the enantiomers of the homologous series of 2-alcohols from 2-butanol to 2-heptanol in rats. Because these alcohols are partially metabolized to 2-ketones during the course of study (i.e., having a -CH2-CO-CH3 group), we independently measured the MAC of the 2-ketones. Assuming additivity of MAC of the ketones with the alcohols, we corrected for the anesthetic effect of the ketones in rats to determine the MAC of the alcohols. We found that the 2-butanol and 2-pentanol isomers were enantioselective. S-(+)-2-butanol had a MAC that was 17% larger than for the R-(-)-enantiomer, whereas S-(+)-2-pentanol had a MAC that was 38% larger than the R-(-)- enantiomer. No stereoselectivity was observed for 2-hexanol and 2-heptanol. These findings may permit studies of chirality in anesthesia, particularly in in vitro systems where metabolism does not occur, using inexpensive volatile compounds.

Antagonism of N-methyl-d-aspartate (NMDA) receptors markedly decreases the minimum alveolar concentration (MAC) of inhaled anesthetics. To assess the importance of suppression of the temporal summation NMDA receptor component of MAC, we stimulated the tail of rats with trains of electrical pulses of varying interstimulus intervals (ISIs) and determined the inhaled anesthetic concentrations (crossover concentrations) that suppressed movement at different ISIs. The slopes of crossover concentrations versus ISIs provided a measure of temporal summation for each anesthetic. We studied five anesthetics that differ widely in their in vitro capacity to block NMDA receptors. To block NMDA receptor transmission and reveal the NMDA receptor component, the NMDA receptor antagonist, MK801, was separately added during each anesthetic. Halothane, isoflurane, and hexafluorobenzene did not appreciably suppress the NMDA receptor components of temporal summation, which contributed to 21% to 29% of MAC (P < 0.05 for each). Xenon and o-difluorobenzene suppressed these components to 8% to 0%, respectively, of MAC (neither significant), consistent with their greater NMDA receptor blocking action in vitro. NMDA receptor blockade may contribute to the MAC produced by inhaled anesthetics that potently inhibit NMDA receptors in vitro but not those that have a limited in vitro effect.

The Meyer-Overton hypothesis predicts that anesthetic potency correlates inversely with lipophilicity; e.g., MAC times the olive oil/gas partition coefficient equals a constant of approximately 1.82 +/- 0.56 atm (mean +/- sd) for conventional inhaled anesthetics. MAC is the minimum alveolar concentration of anesthetic required to eliminate movement in response to a noxious stimulus in 50% of subjects. In contrast to conventional inhaled anesthetics, MAC times the olive oil/gas partition coefficient for normal alcohols from methanol through octanol equals a constant one tenth as large as that for conventional inhaled anesthetics. The alcohol (C-OH) group causes a great affinity of alcohols to water, and the C-OH may tether the alcohol at the hydrophobic-hydrophilic interface where anesthetics are thought to act. We hypothesized that the position of the C-OH group determined potency, perhaps by governing the maximum extent to which the acyl portion of the molecule might extend into a hydrophobic phase. Using the same reasoning, we added studies of ketones with similar numbers of carbon atoms between the C=O group and the terminal methyl group. The results for both alcohols and ketones showed the predicted correlation, but the correlation was no better than that with carbon chain length regardless of the placement of the oxygen. The oil/gas partition coefficient predicted potency as well as, or better than, either chain length or oxygen placement. Hydrophilicity, as indicated by the saline/gas partition coefficient, also seemed to influence potency.

Several previous studies concluded that opioid receptors do not mediate the capacity of inhaled anesthetics to produce immobility in the face of noxious stimulation because administration of naloxone (a nonspecific opioid receptor antagonist) does not increase the minimum alveolar anesthetic concentration (MAC) of inhaled anesthetic that produces immobility in 50% of subjects given a noxious stimulation. In contrast, a recent study found that 0.1 mg/kg naloxone given intraperitoneally increased sevoflurane MAC in mice by 18% (P < 0.01). We repeated the recent study with sevoflurane in the same strain of mice, administering nothing (control), 0.1 mg/kg, and 1.0 mg/kg of naloxone. Our study differed in that we also tested a parallel group given saline rather than naloxone. We were blinded to drug administration. MAC decreased 4.8% +/- 11.0% (mean+/- sd) and 2.4% +/- 12.5% with the first and second administrations of saline. Similarly, MAC decreased 4.7% +/- 7.1% and 5.5% +/- 10.0% with the administration of 0.1 mg/kg and 1.0 mg/kg of naloxone. We do not find that naloxone increases MAC. Opioid receptors do not underlie a portion of the capacity of inhaled anesthetics to produce immobility.

Despite the known capacity of hypothermia to increase anesthetic potency (decrease the partial pressure required to produce anesthesia), many in vitro studies examine the effects of ethanol and other anesthetics in oocytes or isolated neurons at room temperature. We tested whether, as predicted for potent inhaled anesthetics, a proportionate increase in solubility with hypothermia matched a decrease in ethanol minimum alveolar concentration (MAC), and thereby made the use of a single anesthetic concentration appropriate regardless of temperature. We determined ethanol MAC in normothermic (37.3 degrees C) and hypothermic (28.5 degrees C) rats, and, at the two temperatures, also determined ethanol solubilities in olive oil and saline. Ethanol MAC decreased, while olive oil/gas and saline/gas partition coefficients increased. However, the increase in the saline/gas partition coefficient did not match the decrease in MAC, and thus the aqueous-phase partial pressure producing absence of movement in 50% of rats (EC50) values for ethanol decreased by 17%. Although this decrease is not large, it may be important for comparative estimates of the in vitro effects of ethanol at different temperatures.

Background: A recent report finds that elderly Japanese women given xenon have a significantly smaller (26% less) MAC (minimum alveolar concentration required to eliminate movement in response to surgical incision in 50% of patients) than Japanese men of the same age. The authors assessed whether this finding applied to other/all anesthetics., Methods: The authors reviewed data obtained previously for 258 patients (127 women and 131 men) anesthetized with desflurane, diethyl ether, halothane, methoxyflurane, sevoflurane, or xenon. Data were normalized to the MAC for the anesthetic as determined by logistic regression (i.e., MAC would equal a value of 1.000)., Results: The MAC for the normalized combined (all) data for women (1.013 +/- 0.017; mean +/- SEM) did not differ significantly from the normalized combined data for men (1.005 +/- 0.009), and neither differed significantly from 1.000. However, a significantly smaller MAC value was found for women in two studies of sevoflurane (subsets of the above studies) given to Japanese patients: 12% in one study and 16% in the other., Conclusions: Overall, no difference in MAC was found for women versus men. Whether women (particularly older Japanese women) have a smaller MAC than men remains to be confirmed by prospective studies.

Studies using molecular modeling, genetic engineering, neurophysiology/pharmacology, and whole animals have advanced our understanding of where and how inhaled anesthetics act to produce immobility (minimum alveolar anesthetic concentration; MAC) by actions on the spinal cord. Numerous ligand- and voltage-gated channels might plausibly mediate MAC, and specific amino acid sites in certain receptors present likely candidates for mediation. However, in vivo studies to date suggest that several channels or receptors may not be mediators (e.g., gamma-aminobutyric acid A, acetylcholine, potassium, 5-hydroxytryptamine-3, opioids, and alpha(2)-adrenergic), whereas other receptors/channels (e.g., glycine, N-methyl-D-aspartate, and sodium) remain credible candidates.

Background: General anesthesia may delay the onset of movement in response to noxious stimulation. The authors hypothesized that the production of immobility could involve depression of time-related processes involved in the generation of movement., Methods: The delays (latencies) between onset of tail clamp (n = 16) or 50-Hz continuous electrical stimulation (n = 8) and movement were measured in rats equilibrated at 0.1-0.2% increasing steps of isoflurane. In other rats (n = 8), the isoflurane concentrations just permitting and preventing movement (crossover concentrations) in response to trains of 0.5-ms 50-V square-wave pulses of interstimulus intervals of 10, 3, 1, 0.3, or 0.1 s during the step increases were measured. These measures were again made during administration of intravenous MK801, an N-methyl-D-aspartate receptor antagonist that can block temporal summation (n = 6). Temporal summation refers to the cumulative effect of repeated stimuli. Crossover concentrations to 10- and 0.1-s interstimulus interval pulses ranging in voltage from 0.25-50 V were also measured (n = 4)., Results: The increase in concentrations from 0.6 to nearly 1.0 minimum alveolar concentration progressively increased latency from less than 1 s to 58 s. Shortening the interstimulus interval (50 V) pulses from 10 to 0.1 s progressively increased crossover concentrations from 0.6 to 1.0 minimum alveolar concentration. In contrast, during MK801 administration shortening interstimulus intervals did not change crossover concentrations, producing a flat response to change in the interstimulus interval. Increasing the voltage of interstimulus interval pulses increased the crossover concentrations but did not change the response to change in interstimulus intervals for pulses greater than 1 V., Conclusions: Increasing the duration or frequency (interstimulus interval) of stimulation increases the concentration of isoflurane required to suppress movement by a 0.4 minimum alveolar concentration MK801 blocks this effect, a finding consistent with temporal summation (which requires intact N-methyl-D-aspartate receptor activity) at concentrations of up to 1 minimum alveolar concentration isoflurane.

Background: Neuronal nicotinic acetylcholine receptors (nAChRs) have been implicated in the mechanism of action of isoflurane as they are inhibited at subanesthetic concentrations. Despite clear evidence for nicotinic inhibition at relevant isoflurane concentrations, it is unclear what behavioral result ensues, if any., Methods: The authors have modeled two behaviors common to all general anesthetics, immobility and hypnosis, as minimum alveolar concentration that prevents movement in response to a supramaximal stimulus (MAC) and loss of righting reflex (LORR). They have tested the ability of nicotinic pharmacologic modulators and congenital absence of most heteromeric nAChRs to affect concentration of isoflurane required for these behaviors., Results: Neither mecamylamine, 5 mg/kg, nor chlorisondamine, 10 mg/kg, affected isoflurane MAC. Nicotine caused a small decrease in MAC. None of the above agents had any effect on the concentration of isoflurane required for LORR. Mice genetically engineered to lack the beta 2 nicotinic gene product were not different in MAC or LORR from controls., Conclusions: Nicotinic antagonists do not cause MAC or LORR. Inhibition of nicotinic acetylcholine receptors by isoflurane is not likely related to its ability to provide immobility and hypnosis in a surgical setting. This is perhaps not surprising as the inhibition of nAChRs in vitro is complete at an isoflurane concentration equal to one half of MAC. Nicotinic inhibition may, however, be involved in anesthetic behaviors such as amnesia and analgesia, which occur at lower anesthetic concentrations.

Implications: The two experimental designs (quantal and bracketing) used for population minimum alveolar anesthetic concentration studies give equivalent results. An expression relating variability in terms of Hill coefficients and SD is presented. Evolutionary implications of low population variability in anesthetic phenotypes is discussed.