Conserved role of unc-79 in ethanol responses in lightweight mutant mice.

The mechanisms by which ethanol and inhaled anesthetics influence the nervous system are poorly understood. Here we describe the positional cloning and characterization of a new mouse mutation isolated in an N-ethyl-N-nitrosourea (ENU) forward mutagenesis screen for animals with enhanced locomotor activity. This allele, Lightweight (Lwt), disrupts the homolog of the Caenorhabditis elegans (C. elegans) unc-79 gene. While Lwt/Lwt homozygotes are perinatal lethal, Lightweight heterozygotes are dramatically hypersensitive to acute ethanol exposure. Experiments in C. elegans demonstrate a conserved hypersensitivity to ethanol in unc-79 mutants and extend this observation to the related unc-80 mutant and nca-1;nca-2 double mutants. Lightweight heterozygotes also exhibit an altered response to the anesthetic isoflurane, reminiscent of unc-79 invertebrate mutant phenotypes. Consistent with our initial mapping results, Lightweight heterozygotes are mildly hyperactive when exposed to a novel environment and are smaller than wild-type animals. In addition, Lightweight heterozygotes exhibit increased food consumption yet have a leaner body composition. Interestingly, Lightweight heterozygotes voluntarily consume more ethanol than wild-type littermates. The acute hypersensitivity to and increased voluntary consumption of ethanol observed in Lightweight heterozygous mice in combination with the observed hypersensitivity to ethanol in C. elegans unc-79, unc-80, and nca-1;nca-2 double mutants suggests a novel conserved pathway that might influence alcohol-related behaviors in humans.

Mutations M287L and Q266I in the glycine receptor α1 subunit change sensitivity to volatile anesthetics in oocytes and neurons, but not the minimal alveolar concentration in knockin mice.

BACKGROUND: Volatile anesthetics (VAs) alter the function of key central nervous system proteins but it is not clear which, if any, of these targets mediates the immobility produced by VAs in the face of noxious stimulation. A leading candidate is the glycine receptor, a ligand-gated ion channel important for spinal physiology. VAs variously enhance such function, and blockade of spinal glycine receptors with strychnine affects the minimal alveolar concentration (an anesthetic EC50) in proportion to the degree of enhancement. METHODS: We produced single amino acid mutations into the glycine receptor α1 subunit that increased (M287L, third transmembrane region) or decreased (Q266I, second transmembrane region) sensitivity to isoflurane in recombinant receptors, and introduced such receptors into mice. The resulting knockin mice presented impaired glycinergic transmission, but heterozygous animals survived to adulthood, and we determined the effect of isoflurane on glycine-evoked responses of brainstem neurons from the knockin mice, and the minimal alveolar concentration for isoflurane and other VAs in the immature and mature knockin mice. RESULTS: Studies of glycine-evoked currents in brainstem neurons from knockin mice confirmed the changes seen with recombinant receptors. No increases in the minimal alveolar concentration were found in knockin mice, but the minimal alveolar concentration for isoflurane and enflurane (but not halothane) decreased in 2-week-old Q266I mice. This change is opposite to the one expected for a mutation that decreases the sensitivity to volatile anesthetics. CONCLUSION: Taken together, these results indicate that glycine receptors containing the α1 subunit are not likely to be crucial for the action of isoflurane and other VAs.

Ross C. Terrell, PhD, an anesthetic pioneer.

On December 30, 2010, Ross C. Terrell, PhD, died. With his passing at age 85, we lost one of the pioneers of modern anesthesia. Terrell synthesized most of the inhalation anesthetics used today, including desflurane, enflurane, isoflurane, and sevoflurane.

Gamma-aminobutyric acid type A receptor ß3 subunit forebrain-specific knockout mice are resistant to the amnestic effect of isoflurane.

BACKGROUND: ß3 containing γ-aminobutyric acid type A receptors (GABA(A)-Rs) mediate behavioral end points of IV anesthetics such as immobility and hypnosis. A knockout mouse with targeted forebrain deletion of the ß3 subunit of the GABA(A)-R shows reduced sensitivity to the hypnotic effect of etomidate, as measured by the loss of righting reflex. The end points of amnesia and immobility produced by an inhaled anesthetic have yet to be evaluated in this conditional knockout. METHODS: We assessed forebrain selective ß3 conditional knockout mice and their littermate controls for conditional fear to evaluate amnesia and MAC, the minimum alveolar concentration of inhaled anesthetic necessary to produce immobility in response to noxious stimulation, to assess immobility. Suppression of conditional fear was assessed for etomidate and isoflurane, and MAC was assessed for isoflurane. RESULTS: Etomidate equally suppressed conditional fear for both genotypes. The knockout showed resistance to the suppression of conditional fear produced by isoflurane in comparison with control littermates. Controls and knockouts did not differ in isoflurane MAC values. CONCLUSIONS: These results suggest that ß3 containing GABA(A)-Rs in the forebrain contribute to hippocampal-dependent memory suppressed by isoflurane, but not etomidate.

Slowing of the hippocampal θ rhythm correlates with anesthetic-induced amnesia.

BACKGROUND: Temporary, antegrade amnesia is one of the core desirable endpoints of general anesthesia. Multiple lines of evidence support a role for the hippocampal θ rhythm, a synchronized rhythmic oscillation of field potentials at 4-12 Hz, in memory formation. Previous studies have revealed a disruption of the θ rhythm at surgical levels of anesthesia. We hypothesized that θ-rhythm modulation would also occur at subhypnotic but amnestic concentrations. Therefore, we examined the effect of three inhaled agents on properties of the θ rhythm considered critical for the formation of hippocampus-dependent memories. METHODS: We studied the effects of halothane and nitrous oxide, two agents known to modulate different molecular targets (GABAergic [γ-aminobutyric acid] vs. non-GABAergic, respectively) and isoflurane (GABAergic and non-GABAergic targets) on fear-conditioned learning and θ oscillations in freely behaving rats. RESULTS: All three anesthetics slowed θ peak frequency in proportion to their inhibition of fear conditioning (by 1, 0.7, and 0.5 Hz for 0.32% isoflurane, 60% N2O, and 0.24% halothane, respectively). Anesthetics inconsistently affected other characteristics of θ oscillations. CONCLUSIONS: At subhypnotic amnestic concentrations, θ-oscillation frequency was the parameter most consistently affected by these three anesthetics. These results are consistent with the hypothesis that modulation of the θ rhythm contributes to anesthetic-induced amnesia.

Isoflurane suppresses stress-enhanced fear learning in a rodent model of post-traumatic stress disorder.

BACKGROUND: A minority of patients who experience awareness and/or pain during surgery subsequently develop post-traumatic stress disorder. In a rodent model of post-traumatic stress disorder, stress-enhanced fear learning (SEFL), rats are preexposed to a stressor of 15 foot shocks. Subsequent exposure to a single foot shock produces an enhanced fear response. This effect is akin to sensitized reactions shown by some post-traumatic stress disorder patients to cues previously associated with the traumatic event. METHODS: The authors studied the effect of isoflurane and nitrous oxide on SEFL. Rats were exposed to the inhaled anesthetic during or after a 15-foot shock stressor. Then, rats were given a single foot shock in a different environment. Their fear response was quantified in response to the 15-foot shock and single-foot shock environments. SEFL longevity was tested by placing a 90-day period between the 15 foot shocks and the single foot shock. In addition, the intensity of the foot shock was increased to evaluate treatment effectiveness. RESULTS: Increasing isoflurane concentrations decreased SEFL when given during, but not after, the stressor. At 0.40 minimum alveolar concentration (MAC), isoflurane given during the stressor blocked SEFL 90 days later. A threefold increase in the stressor intensity increased the isoflurane concentration required to block SEFL to no more than 0.67 MAC. As with isoflurane, nitrous oxide suppressed SEFL at a similar MAC fraction. CONCLUSIONS: These results suggest that sufficient concentrations (perhaps 0.67 MAC or less) of an inhaled anesthetic may prevent SEFL.

A comparison of the molecular bases for N-methyl-D-aspartate-receptor inhibition versus immobilizing activities of volatile aromatic anesthetics.

BACKGROUND: Aromatic anesthetics exhibit a wide range of N-methyl-d-aspartate (NMDA) receptor inhibitory potencies and immobilizing activities. We sought to characterize the molecular basis of NMDA receptor inhibition using comparative molecular field analysis (CoMFA), and compare the results to those from an equivalent model for immobilizing activity. METHODS: Published potency data for 14 compounds were supplemented with new values for 2 additional agents. The anesthetics were divided into a training set (n = 12) used to formulate the activity models and a test set (n = 4) used to independently assess the models' predictive capability. The anesthetic structures were geometry optimized using ab initio quantum mechanics and aligned by field-fit minimization to provide the best correlation between the steric and electrostatic fields of the molecules and one or more lead structures. Orientations that yielded CoMFA models with the greatest predictive capability (assessed by leave-one-out cross-validation) were retained. RESULTS: The final CoMFA model for the inhibition of NR1/NR2B NMDA receptors explained 99.3% of the variance in the observed activities of the 12 training set agents (F(2,)(9) = 661.5, P < 0.0001). The model effectively predicted inhibitory potency for the training set (cross-validated r(2)(CV) = 0.944) and 4 excluded test set compounds (predictive r(2)(Pred) = 0.966). The equivalent model for immobility in response to noxious stimuli explained 98.0% of the variance in the observed activities for the training set (F(2,)(9) = 219.2, P < 0.0001) and exhibited adequate predictive capability for both the training set (r(2)(CV) = 0.872) and test set (r(2)(Pred) = 0.926) agents. Comparison of pharmacophoric maps showed that several key steric and electrostatic regions were common to both activity models, but differences were observed in the relative importance of these key regions with respect to the two aspects of anesthetic activity. CONCLUSIONS: The similarities in the pharmacophoric maps are consistent with NMDA receptors contributing part of the immobilizing activity of volatile aromatic anesthetics.

Increasing the duration of isoflurane anesthesia decreases the minimum alveolar anesthetic concentration in 7-day-old but not in 60-day-old rats.

BACKGROUND: While studying neurotoxicity in rats, we observed that the anesthetic minimum alveolar anesthetic concentration (MAC) of isoflurane decreases with increasing duration of anesthesia in 7-day-old but not in 60-day-old rats. After 15 min of anesthesia in 7-day-old rats, MAC was 3.5% compared with 1.3% at 4 h. We investigated whether kinetic or dynamic factors mediated this decrease. METHODS: In 7-day-old rats, we measured inspired and cerebral partial pressures of isoflurane at MAC as a function of duration of anesthesia. In 60-day-old rats, we measured inspired partial pressures of isoflurane at MAC as a function of duration of anesthesia. Finally, we determined the effect of administering 1 mg/kg naloxone and of delaying the initiation of the MAC determination (pinching the tail) on MAC in 7-day-old rats. RESULTS: In 7-day-old rats, both inspired and cerebral measures of MAC decreased from 1 to 4 h. The inspired MAC decreased 56%, whereas the cerebral MAC decreased 33%. At 4 h, the inspired MAC approximated the cerebral MAC (i.e., the partial pressures did not differ appreciably). Neither administration of 1 mg/kg naloxone nor delaying tail clamping until 3 h reversed the decrease in MAC. In 60-day-old rats, inspired MAC of isoflurane was stable from 1 to 4 h of anesthesia. CONCLUSIONS: MAC of isoflurane decreases over 1-4 h of anesthesia in 7-day-old but not in 60-day-old rats. Both pharmacodynamic and a pharmacokinetic components contribute to the decrease in MAC in 7-day-old rats. Neither endorphins nor sensory desensitization mediate the pharmacodynamic component.

Gamma-aminobutyric acid type A receptor alpha 4 subunit knockout mice are resistant to the amnestic effect of isoflurane.

BACKGROUND: General anesthesia produces multiple end points including immobility, hypnosis, sedation, and amnesia. Tonic inhibition via gamma-aminobutyric acid type A receptors (GABA(A)-Rs) may play a role in mediating behavioral end points that are suppressed by low concentrations of anesthetics (e.g., hypnosis and amnesia). GABA(A)-Rs containing the alpha4 subunit are highly concentrated in the hippocampus and thalamus, and when combined with delta subunits they mediate tonic inhibition, which is sensitive to low concentrations of isoflurane. METHODS: In this study, we used a GABA(A) alpha4 receptor knockout mouse line to evaluate the contribution of alpha4-containing GABA(A)-Rs to the effects of immobility, hypnosis, and amnesia produced by isoflurane. Knockout mice and their wild-type counterparts were assessed on 3 behavioral tests: conditional fear (to assess amnesia), loss of righting reflex (to assess hypnosis), and the minimum alveolar concentration of inhaled anesthetic necessary to produce immobility in response to noxious stimulation in 50% of subjects (to assess immobility). RESULTS: Genetic inactivation of the alpha4 subunit reduced the amnestic effect of isoflurane, minimally affected loss of righting reflex, and had no effect on immobility. CONCLUSIONS: These results lend support to the hypothesis that different sites of action mediate different anesthetic end points and suggest that alpha4-containing GABA(A)-Rs are important mediators of the amnestic effect of isoflurane on hippocampal-dependent declarative memory.

Knockout of the gene encoding the K(2P) channel KCNK7 does not alter volatile anesthetic sensitivity.

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.

The effects of volatile aromatic anesthetics on voltage-gated Na+ channels expressed in Xenopus oocytes.

BACKGROUND: Many inhaled anesthetics inhibit voltage-gated sodium channels at clinically relevant concentrations, and suppression of neurotransmitter release by these anesthetics results, at least partly, from decreased presynaptic sodium channel activity. Volatile aromatic anesthetics can inhibit N-methyl-D-aspartate (NMDA) receptor function and enhance gamma-amino butyric acid A receptor function, but these effects depend strongly on the chemical properties of the aromatic compounds. In the present study we tested whether diverse aromatic anesthetics consistently inhibit sodium channel function. METHODS: We studied the effect of eight aromatic anesthetics on Na(v)1.2 sodium channels with beta(1) subunits, using whole-cell, two-electrode voltage-clamp techniques in Xenopus oocytes. RESULTS: All aromatic anesthetics inhibited I(Na) (sodium currents) at a holding potential which produce half-maximal current (V(1/2)) (partial depolarization); inhibition was modest with 1,3,5-trifluorobenzene (8% +/- 2%), pentafluorobenzene (13% +/- 2%), and hexafluorobenzene (13% +/- 2%), but greater with benzene (37% +/- 2%), fluorobenzene (39% +/- 2%), 1,2-difluorobenzene (48% +/- 2%), 1,4-difluorobenzene (31 +/- 3%), and 1,2,4-trifluorobenzene (33% +/- 1%). Such dichotomous effects were noted by others for NMDA and gamma-aminobutyric acid A receptors. Parallel, but much smaller inhibition, was found for I(Na) at a holding potential which produced near maximal current (-90 mV) (V(H-90)), and hexafluorobenzene caused small (6% +/- 1%) enhancement of this current. These changes in sodium channel function were correlated with effectiveness for inhibiting NMDA receptors, with lipid solubility of the compounds, with molecular volume, and with cation-pi interactions. CONCLUSION: Aromatic compounds vary in their actions on the kinetics of sodium channel gating and this may underlie their variable inhibition. The range of inhibition produced by minimum alveolar anesthetic concentration concentrations of inhaled anesthetics indicates that sodium channel inhibition may underlie the action of some of these anesthetics, but not others.

Obesity modestly affects inhaled anesthetic kinetics in humans.

BACKGROUND: Few studies have determined the effect of obesity on inhaled anesthetic pharmacokinetics. We hypothesized that the solubility of potent inhaled anesthetics in fat and increased body mass index (BMI) in obese patients interact to increase anesthetic uptake and decrease the rate at which the delivered (FD) and inspired (FI) concentrations of an inhaled anesthetic approach a constantly maintained alveolar concentration (end-tidal or FA). This hypothesis implies that the effect of obesity would be greater with a more soluble anesthetic such as isoflurane versus desflurane. METHODS: In 107 ASA physical status I-III patients, anesthesia was induced with propofol, tracheal intubation facilitated with neuromuscular blockade, and ventilation controlled with 50% nitrous oxide in oxygen to maintain end-tidal carbon dioxide concentrations between 35 and 45 mm Hg. Isoflurane or desflurane was administered in a 1 L/min inflow rate at FD concentrations sufficient to maintain FA at 0.6 minimum alveolar anesthetic concentration (0.7% or 3.7%, respectively). FD, FI, and FA were measured 5, 10, 20, 40, 60, 90, 120,150, and 180 min after starting potent inhaled anesthetic delivery. RESULTS: Fifty-nine patients received isoflurane and 48 received desflurane. BMI ranged between 18 and 63 kg/m(2) and demographic variables did not differ between anesthetic groups. For isoflurane, FD/FA or FI/FA weakly (but significantly) correlated with BMI at 9/18 time points whereas for desflurane FD/FA or FI/FA correlated significantly with BMI at only one time point (P < 0.01). After dividing each group into nonobese (BMI < 30) and obese (BMI > or = 30) patients, with isoflurane, FD/FA or FI/FA was higher in obese patients at four time points whereas there was no difference between nonobese and obese patients for desflurane. Patients receiving isoflurane took longer to respond to command after discontinuing anesthesia but obesity did not increase or decrease awakening time for either isoflurane or desflurane. When BMI was used to normalize FI/FA and FD/FA the median values for isoflurane consistently exceeded the median value for desflurane by factors ranging from 3 to 5, values comparable to the ratios of their blood/gas (3.1), muscle/gas (4.6), and fat/gas (5.4) partition coefficients. CONCLUSION: BMI modestly affects FD/FA and FI/FA, and this effect is most apparent for an anesthetic having a greater solubility in all tissues. An increased BMI increases anesthetic uptake and, thus, the need for delivered anesthetic to sustain a constant alveolar anesthetic concentration, particularly with a more soluble anesthetic. However, the increase with an increased body mass is small.

Is synergy the rule? A review of anesthetic interactions producing hypnosis and immobility.

BACKGROUND: Drug interactions may reveal mechanisms of drug action: additive interactions suggest a common site of action, and synergistic interactions suggest different sites of action. We applied this reasoning in a review of published data on anesthetic drug interactions for the end-points of hypnosis and immobility. METHODS: We searched Medline for all manuscripts listing propofol, etomidate, methohexital, thiopental, midazolam, diazepam, ketamine, dexmedetomidine, clonidine, morphine, fentanyl, sufentanil, alfentanil, remifentanil, droperidol, metoclopramide, lidocaine, halothane, enflurane, isoflurane, sevoflurane, desflurane, N2O, and Xe that contained terms suggesting interaction: interaction, additive, additivity, synergy, synergism, synergistic, antagonism, antagonistic, isobologram, or isobolographic. When available, data were reanalyzed using fraction analysis or response surface analysis. RESULTS: Between drug classes, most interactions were synergistic. The major exception was ketamine, which typically interacted in either an additive or infra-additive (antagonistic) manner. Inhaled anesthetics typically showed synergy with IV anesthetics, but were additive or, in the case of nitrous oxide and isoflurane, possibly infra-additive, with each other. CONCLUSIONS: Except for ketamine, IV anesthetics acting at different sites usually demonstrated synergy. Inhaled anesthetics usually demonstrated synergy with IV anesthetics, but no pair of inhaled anesthetics interacted synergistically.

Is a new paradigm needed to explain how inhaled anesthetics produce immobility?

A paradox arises from present information concerning the mechanism(s) by which inhaled anesthetics produce immobility in the face of noxious stimulation. Several findings, such as additivity, suggest a common site at which inhaled anesthetics act to produce immobility. However, two decades of focused investigation have not identified a ligand- or voltage-gated channel that alone is sufficient to mediate immobility. Indeed, most putative targets provide minimal or no mediation. For example, opioid, 5-HT3, gamma-aminobutyric acid type A and glutamate receptors, and potassium and calcium channels appear to be irrelevant or play only minor roles. Furthermore, no combination of actions on ligand- or voltage-gated channels seems sufficient. A few plausible targets (e.g., sodium channels) merit further study, but there remains the possibility that immobilization results from a nonspecific mechanism.

Intrathecal veratridine administration increases minimum alveolar concentration in rats.

BACKGROUND: Results from several studies point to sodium channels as potential mediators of the immobility produced by inhaled anesthetics. We hypothesized that the intrathecal administration of veratridine, a drug that enhances the activity or effect of sodium channels, should increase MAC. METHODS: We measured the change in isoflurane MAC caused by intrathecal infusion of various concentrations of veratridine into the lumbothoracic subarachnoid space of rats. We compared these result with those obtained from intracerebroventricular infusion. RESULTS: As predicted, intrathecal infusion of veratridine increased MAC. The greatest infused concentration (25 microM) also produced neuronal injury in the hindlimbs of two rats and decreased the peak effect on MAC. A concentration of 1.6 microM produced the largest (21%) increase in MAC. Intraventricular infusion of 1.6 and 6.4 microM veratridine did not alter MAC. Rats given 25 microM died. CONCLUSIONS: Intrathecal administration of veratradine increases MAC of isoflurane, a finding consistent with a role for sodium channels as potential mediators of the immobility produced by inhaled anesthetics.

General anesthetics have additive actions on three ligand gated ion channels.

BACKGROUND: The purpose of this study was to determine whether pairs of compounds, including general anesthetics, could simultaneously modulate receptor function in a synergistic manner, thus demonstrating the existence of multiple intraprotein anesthetic binding sites. METHODS: Using standard electrophysiologic methods, we measured the effects of at least one combination of benzene, isoflurane (ISO), halothane (HAL), chloroform, flunitrazepam, zinc, and pentobarbital on at least one of the following ligand gated ion channels: N-methyl-D-aspartate receptors, glycine receptors and gamma-aminobutyric acid type A receptors. RESULTS: All drug-drug-receptor combinations were found to exhibit additive, not synergistic modulation. ISO with benzene additively depressed N-methyl-D-aspartate receptors function. ISO with HAL additively enhanced glycine receptors function, as did ISO with zinc. ISO with HAL additively enhanced gamma-aminobutyric acid type A receptors function as did all of the following: HAL with chloroform, pentobarbital with ISO, and flunitrazepam with ISO. CONCLUSION: The simultaneous allosteric modulation of ligand gated ion channels by general anesthetics is entirely additive. Where pairs of general anesthetic drugs interact synergistically to produce general anesthesia, they must do so on systems more complex than a single receptor.

Additivity versus synergy: a theoretical analysis of implications for anesthetic mechanisms.

BACKGROUND: Inhaled anesthetics have been postulated to act at multiple receptors, with modest action at each site summing to produce immobility to noxious stimulation. Recent experimental results affirm prior findings that inhaled anesthetics interact additively. Synergy implies multiple sites of action by definition. In this essay, we explore the converse: does additivity imply a single site of action? METHODS: The interaction of one versus two ligands competing for the same binding site at a receptor was explored using the law of mass action. Circuits were then constructed to investigate how the potency of drugs and the steepness of the concentration versus response relationship is amplified by the arrangement of suppressors into serial circuits, and enhancers into parallel circuits. Assemblies of suppressor and enhancer circuits into signal processing units were then explored to investigate the constraints signal processing units impose on additive interactions. Lastly, the relationship between synergy, additivity, and fractional receptor occupancy was explored to understand the constraints imposed by additivity. RESULTS: Drugs that compete for a single receptor, and that similarly affect the receptor, must be additive in their effects. Receptors that bind suppressors in serial circuits, or enhancers in parallel circuits, increase the apparent potency of the drugs and the steepness of the concentration versus response relationship. When assemblies of suppressor and enhancer circuits are arranged into signal processing units, the interactions may be additive or synergistic. The primary determinant is the relationship between the concentration of drug associated with the effect of interest and the concentration associated with 50% receptor occupancy, k(d). Effects mediated by very low concentrations are more likely to be additive. Similarly, inhaled anesthetics that act at separate sites are unlikely to exhibit additive interactions if anesthetic drug effect occurs at concentrations at or above 50% receptor occupancy. However, if anesthetic drug effect occurs at very low levels of receptor occupancy, then additivity is expected even among anesthetics acting on different receptors. CONCLUSIONS: Additivity among drugs acting on different receptors is only likely if the concentrations responsible for the drug effect of interest are well below the concentration associated with 50% receptor occupancy.