Effects of nonimmobilizers and halothane on Caenorhabditis elegans

We studied the effects of two nonimmobilizers, a transitional compound, and halothane on the nematode, Caenorhabditis elegans, by using reversible immobility as an end point. By themselves, the nonimmobilizers did not immobilize any of the four strains of animals tested. Toluene appears to be a transitional compound for all strains tested. The additive effects of the nonimmobilizers with halothane were also studied. Similar to results seen in studies of mice, the nonimmobilizers were antagonistic to halothane in the wild type nematode. However, the nonimmobilizers did not affect the 50% effective concentrations of halothane for two other mutant strains. For halothane, the slopes of the dose response curves were smaller in more sensitive strains compared with the wild type. As in mammals, nonimmobilizers antagonize the effects of halothane on the nematode, C. elegans. The variation in slopes in the response to halothane in different strains is consistent with multiple sites of action. These results support the use of C. elegans as a model for the study of anesthetics.     

An evolutionarily conserved presynaptic protein is required for isoflurane sensitivity in Caenorhabditis elegans.

Background: Volatile general anesthetics inhibit neurotransmitter release by an unknown mechanism. A mutation in the presynaptic soluble NSF attachment protein receptor (SNARE) protein syntaxin 1A was previously shown to antagonize the anesthetic isoflurane in Caenorhabditis elegans. The mechanism underlying this antagonism may identify presynaptic anesthetic targets relevant to human anesthesia.

Methods: Sensitivity to isoflurane concentrations in the human clinical range was measured in locomotion assays on adult C. elegans. Sensitivity to the acetylcholinesterase inhibitor aldicarb was used as an assay for the global level of C. elegans neurotransmitter release. Comparisons of isoflurane sensitivity (measured by the EC50) were made by simultaneous curve fitting and F test as described by Waud.

Results: Expression of a truncated syntaxin fragment (residues 1-106) antagonized isoflurane sensitivity in C. elegans. This portion of syntaxin interacts with the presynaptic protein UNC-13, suggesting the hypothesis that truncated syntaxin binds to UNC-13 and antagonizes an inhibitory effect of isoflurane on UNC-13 function. Consistent with this hypothesis, overexpression of UNC-13 suppressed the isoflurane resistance of the truncated syntaxins, and unc-13 loss-of-function mutants were highly isoflurane resistant. Normal anesthetic sensitivity was restored by full-length UNC-13, by a shortened form of UNC-13 lacking a C2 domain, but not by a membrane-targeted UNC-13 that might bypass isoflurane inhibition of membrane translocation of UNC-13. Isoflurane was found to inhibit synaptic localization of UNC-13.     

GAS-1: A Mitochondrial Protein Controls Sensitivity to Volatile Anesthetics in the Nematode Caenorhabditis elegans

Background: Mutations in several genes of Caenorhabditis elegans confer altered sensitivities to volatile anesthetics. A mutation in one gene, gas-1(fc21), causes animals to be immobilized at lower concentrations of all volatile anesthetics than in the wild-type, and it does not depend on mutations in other genes to control anesthetic sensitivity. gas-1 confers different sensitivities to stereoisomers of isoflurane, and thus may be a direct target for volatile anesthetics. The authors have cloned and characterized the gas-1 gene and the mutant allele fc21.

Methods: Genetic techniques for nematodes were as previously described. Polymerase chain reaction, sequencing, and other molecular biology techniques were performed by standard methods. Mutant rescue was done by injecting DNA fragments into the gonad of mutant animals and scoring the offspring for loss of the mutant phenotype.

Results: The gas-1 gene was cloned and identified. The protein GAS-1 is a homologue of the 49-kDa (IP) subunit of the mitochondrial NADH:ubiquinone-oxidoreductase (complex I of the respiratory chain). gas-1(fc21) is a missense mutation replacing a strictly conserved arginine with lysine.     

Behavioral Effects of Volatile Anesthetics in Caenorhabditis elegans

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

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

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

Tail clamp responses in stomatin knockout mice compared with mobility assays in Caenorhabditis elegans during exposure to diethyl ether, halothane, and isoflurane

BACKGROUND: The gene unc-1 plays a central role in determining volatile anesthetic sensitivity in Caenorhabditis elegans. Because different unc-1 alleles cause strikingly different phenotypes in different volatile anesthetics, the UNC-1 protein is a candidate to directly interact with volatile anesthetics. UNC-1 is a close homologue of the mammalian protein stomatin, for which a mouse knockout was recently constructed. Because the stomatin gene is expressed in dorsal root ganglion cells, the authors hypothesized that the knockout would have an effect on anesthetic sensitivity in mice similar to that seen in nematodes. METHODS: Mice were placed in semiclosed chambers and exposed to continuous flows of diethyl ether, halothane, or isoflurane in air. Using lack of response to tail clamp as an endpoint, the authors determined the EC50s for the knockout strain compared with the nonmutated parental strain. They compared the differences seen in the mouse strains with the differences seen in the nematode strains. RESULTS: Stomatin-deficient mice had a 12% increase in sensitivity to diethyl ether but no significant change in sensitivity to halothane or isoflurane compared with wild type. No defect in locomotion was noted in the mutant mouse. CONCLUSIONS: Nematodes and mice with deletions of the stomatin gene both have increased sensitivity to diethyl ether. Neither nematodes nor mice with stomatin deficiencies have significantly altered sensitivity to isoflurane or halothane. The effects of stomatin deficiency cross phylogenetic boundaries and support the importance of this protein in anesthetic response and the use of C. elegans as a model for anesthetic action in mammals.     

Volatile Anesthetic Preconditioning Present in the Invertebrate Caenorhabditis elegans

Background: Volatile anesthetics (VAs) have been found to induce a delayed protective response called preconditioning to subsequent hypoxic/ischemic injury. VA preconditioning has been primarily studied in canine and rodent heart. A more genetically tractable model of VA preconditioning would be extremely useful. Here, the authors report the development of the nematode Caenorhabditis elegans as a model of VA preconditioning.

Methods: Wild-type and mutant C. elegans were exposed to isoflurane, halothane, or air under otherwise identical conditions. After varying recovery periods, the animals were challenged with hypoxic, azide, or hyperthermic incubations. After recovery from these incubations, mortality was scored.

Results: Isoflurane- and halothane-preconditioned animals had significantly reduced mortality to all three types of injuries compared with air controls. Concentrations as low as 1 vol% isoflurane (0.64 mm) and halothane (0.71 mm) induced significant protection. The onset and duration of protection after anesthetic were 6 and 9 h, respectively. A mutation that blocks inhibition of neurotransmitter release by isoflurane did not attenuate the preconditioning effect. A loss-of-function mutation of the Apaf-1 homolog CED-4 blocked the preconditioning effect of isoflurane, but mutation of the downstream caspase CED-3 did not.     

Common Genetic Determinants of Halothane and Isoflurane Potencies in Caenorhabditis elegans

Background: Genetics provides a way to evaluate anesthetic action simultaneously at the molecular and behavioral levels. Results from strains that differ in anesthetic sensitivity have been mixed in their support of unitary theories of anesthesia. Here the authors use the previously demonstrated large variation of halothane sensitivities in Caenorhabditis elegans recombinant inbred strains to assess the similarities of the determinants of halothane action with those of another volatile anesthetic, isoflurane.

Methods: The recombinant inbred strains, constructed from two evolutionarily distinct C. elegans lineages, were phenotyped. A coordination assay on agar quantified the sensitivity to the volatile anesthetics; median effective concentrations (EC50 s) were calculated by nonlinear regression of concentration-response data and were correlated between the drugs for those strains tested in common. Genetic loci were identified by statistical association between EC50 s and chromosomal markers.

Results: The recombinant inbred strains varied dramatically in sensitivity to halothane and isoflurane, with a 10-fold range in EC50 s. Heritability estimates for each drug were imprecise but altogether high (49-80%). Halothane and isoflurane EC50 s were significantly correlated (r = 0.71, P < 10-9). Genetic loci controlling sensitivity were found for both volatile anesthetics; the most significant determinant colocalized on chromosome V. A smaller recombinant inbred strain study of ethanol-induced immobility segregated different genetic effects that did not correlate with sensitivity to either halothane or isoflurane.     

Xenon acts by inhibition of non-N-methyl-D-aspartate receptor-mediated glutamatergic neurotransmission in Caenorhabditis elegans

BACKGROUND: Electrophysiologic experiments in rodents have found that nitrous oxide and xenon inhibit N-methyl-D-aspartate (NMDA)-type glutamate receptors. These findings led to the hypothesis that xenon and nitrous oxide along with ketamine form a class of anesthetics with the identical mechanism, NMDA receptor antagonism. Here, the authors ask in Caenorhabditis elegans whether xenon, like nitrous oxide, acts by a NMDA receptor-mediated mechanism. METHODS: Xenon:oxygen mixtures were delivered into sealed chambers until the desired concentration was achieved. The effects of xenon on various behaviors were measured on wild-type and mutant C. elegans strains. RESULTS: With an EC50 of 15-20 vol% depending on behavioral endpoint, xenon altered C. elegans locomotion in a manner indistinguishable from that of mutants in glutamatergic transmission. Xenon reduced the frequency and duration of backward locomotion without altering its speed or other behaviors tested. Mutation of glr-1, encoding a non-NMDA glutamate receptor subunit, abolished the behavioral effects of xenon; however, mutation of nmr-1, which encodes the pore-forming subunit of an NMDA glutamate receptor previously shown to be required for nitrous oxide action, did not significantly alter xenon response. Transformation of the glr-1 mutant with the wild-type glr-1 gene partially restored xenon sensitivity, confirming that glr-1 was necessary for the full action of xenon. CONCLUSIONS: Xenon acts in C. elegans to alter locomotion through a mechanism requiring the non-NMDA glutamate receptor encoded by glr-1. Unlike for the action of nitrous oxide in C. elegans, the NMDA receptor encoded by nmr-1 is not essential for sensitivity to xenon.     

The effect of two genes on anesthetic response in the nematode Caenorhabditis elegans

The authors studied the wild type strain, N2, and three mutant strains of the nematode, Caenorhabditis elegans, in order to measure genetically produced changes in responses to nine volatile anesthetics. They determined the anesthetic ED50s of N2 for thiomethoxyflurane, methoxyflurane, chloroform, halothane, enflurane, isoflurane, fluroxene, flurothyl, and diethylether. The log-log relationship of the oil-gas partition coefficients (O/G) and the ED50s of these agents for N2 yields a straight line with a slope of -.997 with a R2 of .98 over a range of O/G (at 37 degrees C) from 48 to 7230. When the O/Gs are corrected to 22 degrees C, the slope is -.964 with an R2 of .98. This relationship is similar to that found in other animals. Two mutant strains, unc-79 and unc-80, show altered responses to these anesthetics. These strains are two to three times more sensitive than N2 to anesthetics with an O/G greater than that of halothane (220 at 37 degrees C), yet they differ little from N2 in response to anesthetics with lower O/Gs. unc-79 and unc-80 are about 30% more sensitive than N2 to diethylether. The double mutant unc-79; unc-80 is more sensitive to halothane, isoflurane, and fluroxene than is either mutant alone. The authors believe these data indicate an alteration at the site of action of volatile anesthetics in unc-79 and unc-80. They also postulate that the interaction of unc-79 and unc-80 indicate these genes code for enzymes in a common pathway, and that unc-79 precedes unc-80 in this pathway.     

Desflurane inhibits platelet function in vitro similar to halothane

BACKGROUND AND OBJECTIVE: The effects of the volatile anaesthetic desflurane on platelet activation in vitro were studied and compared to those of halothane. METHODS: Platelet-rich plasma was exposed to 2 MAC of desflurane or halothane, or air only and stimulated by platelet agonists ADP (2.5, 5 and 10 micromol L(-10) and collagen (10 microg m(L-1)). Platelet response was measured by Born aggregometry (maximum aggregation response, area under the curve) and flow cytometry (mean channel fluorescence, percentage of CD62P-positive cells, index of platelet activation for positive platelets). RESULTS: Aggregation response was significantly reduced in platelets exposed to desflurane or halothane; the inhibitory effect was more pronounced when the areas under the curve were analysed: values ranged from 37.5% to 73.3% of control samples for ADP stimulation and 77.1% to 79.8% for collagen stimulation. CD62P expression before and after stimulation with receptor agonists was not statistically different in platelets exposed to desflurane, halothane or air. CONCLUSIONS: By impairing platelet aggregation while not affecting alpha-degranulation desflurane has a differential effect on various aspects of platelet activation similar to halothane. Our results are compatible with the hypothesis of an impairment of platelet thromboxane receptor signalling by halothane. We suggest a similar mechanism for desflurane.     

Mitochondrial function in partially nephrectomized rats

Summary: Previous demonstrations of increased oxygen consumption and renal iron accumulation in partially nephrectomized (RK) rats on the one hand, and acute mitochondrial toxicity of iron in normal rats on the other suggest a complex relationship between mitochondrial function and iron. Renal cortical mitochondrial respiratory function was therefore investigated in RK and iron-loaded rats. State 4 (resting) respiration was increased in RK rats (49±1 vs 43±1 natom 0/min, P. <0.001) in comparison to control. Mitochondrial enzyme activity was lower in RK than control rats (e.g. succinate dehydrogenase 158.2 ± 13.6 vs 237.2±17.4 Δlog[cyt C]/min/mg/prot, P <0.01). Acute iron-loading impaired creatinine clearance (1.39 ± 0.07 vs 0.40 ± 0.29 mL/min) and mitochondrial enzyme activity (e.g. cytochrome oxidase 362.0 ± 32.8 vs 185.0 ± 46.6 Δlog[cyt C]/min/mg/prot, P <0.05) in control rats, but RK rats were more resistant to injury and there was no change in mitochondrial enzyme activity. Mitochondrial fragility was similar in RK and control rats, and in both was unaffected by iron-loading. In conclusion, rates of resting mitochondrial respiration are increased in partially nephrectomized rats, despite reduced intrinsic activity of two iron-dependent mictchondrial enzymes. Mitochondrial function is more resistant to acute injury with iron in partially nephrectomized rats.     

Effect of halothane on diaphragmatic muscle function in pentobarbital-anesthetized dogs

The mechanism underlying the decrease in minute ventilation (VE) observed under halothane anesthesia was investigated in nine spontaneously breathing dogs. Anesthesia was induced with pentobarbital sodium and was maintained with halothane. Inspired fraction of halothane (FIhal) was increased every 30 min, from 0.005 to 0.02. VE decreased from 8.1 +/- 0.9 to 4.8 +/- 0.4 l . min-1 (P less than 0.001), as FIhal increased from 0 to 0.02. This resulted from a decrease in both mean inspiratory flow (VT/TI) and the duty ratio (TI/TTOT). Transdiaphragmatic pressure (Pdi) and the integrated electrical activity of both hemidiaphragms (Edi) were measured during normal breathing, and during breathing against closed airways (P0di, E0di), in order to obtain an index of the inspiratory neuromuscular output of the diaphragm. With increasing FIhal, there was a significant decrease in Pdi, P0di, Edi, and E0di. The authors measured Pdi and Edi generated during supramaximal stimulation of the two phrenic nerves (PSdi, Esdi) at frequencies of 10, 20, 50, and 100 Hz, in order to eliminate in this decrease the role played by a decrease in the neural drive to breathing. PSdi and ESdi decreased significantly with increasing FIhal, and had not returned to the control values 30 min after discontinuation of halothane administration. The authors conclude that, in pentobarbital-anesthetized dogs, halothane is responsible for a diaphragmatic dysfunction, which may be located either at the neuromuscular junction, on the contractile processes of the muscle, or on both, and for a decrease in the activation time of the inspiratory muscles. Both of these effects contribute to the decrease in VE observed under halothane anesthesia.     

Changes in kidney function in abdominoperineal rectum excision under halothane anesthesia

In 17 cases of abdomino-perineal rectum extirpation performed under combined intratracheal halothane anaesthesia, the volume of urine and the osmolality of serum and urine were determined. Though urinary volume decreased significantly, its osmolality did not rise. According to the operational stress this type of operation can be divided into an: 1) intra-abdominal and a 2) perineal phase. In the secretory and concentrating capacity of the kidney, again two phases can be distinguished. Owing to the great operational stress patients with deficient renal function often develop renal failure. It is stressed that such patients close observation, careful hydration prior to operation and the administration of diuretics are of particular importance.