Isoflurane disrupts excitatory neurotransmitter dynamics via inhibition of mitochondrial complex I.

BACKGROUND: The mechanisms of action of volatile anaesthetics are unclear. Volatile anaesthetics selectively inhibit complex I in the mitochondrial respiratory chain. Mice in which the mitochondrial complex I subunit NDUFS4 is knocked out [Ndufs4(KO)] either globally or in glutamatergic neurons are hypersensitive to volatile anaesthetics. The volatile anaesthetic isoflurane selectively decreases the frequency of spontaneous excitatory events in hippocampal slices from Ndufs4(KO) mice. METHODS: Complex I inhibition by isoflurane was assessed with a Clark electrode. Synaptic function was measured by stimulating Schaffer collateral fibres and recording field potentials in the hippocampus CA1 region. RESULTS: Isoflurane specifically inhibits complex I dependent respiration at lower concentrations in mitochondria from Ndufs4(KO) than from wild-type mice. In hippocampal slices, after high frequency stimulation to increase energetic demand, short-term synaptic potentiation is less in KO compared with wild-type mice. After high frequency stimulation, both Ndufs4(KO) and wild-type hippocampal slices exhibit striking synaptic depression in isoflurane at twice the 50% effective concentrations (EC50). The pattern of synaptic depression by isoflurane indicates a failure in synaptic vesicle recycling. Application of a selective A1 adenosine receptor antagonist partially eliminates isoflurane-induced short-term depression in both wild-type and Ndufs4(KO) slices, implicating an additional mitochondria-dependent effect on exocytosis. When mitochondria are the sole energy source, isoflurane completely eliminates synaptic output in both mutant and wild-type mice at twice the (EC50) for anaesthesia. CONCLUSIONS: Volatile anaesthetics directly inhibit mitochondrial complex I as a primary target, limiting synaptic ATP production, and excitatory vesicle endocytosis and exocytosis.

Comparison of proteomic and metabolomic profiles of mutants of the mitochondrial respiratory chain in Caenorhabditis elegans.

Single-gene mutations that disrupt mitochondrial respiratory chain function in Caenorhabditis elegans change patterns of protein expression and metabolites. Our goal was to develop useful molecular fingerprints employing adaptable techniques to recognize mitochondrial defects in the electron transport chain. We analyzed mutations affecting complex I, complex II, or ubiquinone synthesis and discovered overarching patterns in the response of C. elegans to mitochondrial dysfunction across all of the mutations studied. These patterns are in KEGG pathways conserved from C. elegans to mammals, verifying that the nematode can serve as a model for mammalian disease. In addition, specific differences exist between mutants that may be useful in diagnosing specific mitochondrial diseases in patients.

Metabolic pathway profiling of mitochondrial respiratory chain mutants in C. elegans.

Caenorhabditis elegans affords a model of primary mitochondrial dysfunction that provides insight into cellular adaptations which accompany mutations in nuclear genes that encode mitochondrial proteins. To this end, we characterized genome-wide expression profiles of C. elegans strains with mutations in nuclear-encoded subunits of respiratory chain complexes. Our goal was to detect concordant changes among clusters of genes that comprise defined metabolic pathways. Results indicate that respiratory chain mutants significantly upregulate a variety of basic cellular metabolic pathways involved in carbohydrate, amino acid, and fatty acid metabolism, as well as cellular defense pathways such as the metabolism of P450 and glutathione. To further confirm and extend expression analysis findings, quantitation of whole worm free amino acid levels was performed in C. elegans mitochondrial mutants for subunits of complexes I, II, and III. Significant differences were seen for 13 of 16 amino acid levels in complex I mutants compared with controls, as well as overarching similarities among profiles of complex I, II, and III mutants compared with controls. The specific pattern of amino acid alterations observed provides novel evidence to suggest that an increase in glutamate-linked transamination reactions caused by the failure of NAD(+)-dependent ketoacid oxidation occurs in primary mitochondrial respiratory chain mutants. Recognition of consistent alterations both among patterns of nuclear gene expression for multiple biochemical pathways and in quantitative amino acid profiles in a translational genetic model of mitochondrial dysfunction allows insight into the complex pathogenesis underlying primary mitochondrial disease. Such knowledge may enable the development of a metabolomic profiling diagnostic tool applicable to human mitochondrial disease.

C. elegans and volatile anesthetics.

The mechanism of action of volatile anesthetics remains an enigma, despite their worldwide use. The nematode C. elegans has served as an excellent model to unravel this mystery. Genes and gene sets that control the behavior of the animal in volatile anesthetics have been identified, using multiple endpoints to mimic the phenomenon of anesthesia in man. Some of these studies have clear translational implications in more complicated organisms.

Mitochondrial respiration and reactive oxygen species in C. elegans.

A powerful approach to understanding complex processes such as aging is to study longevity in organisms that are amenable to genetic dissection. The nematode Caenorhabditis elegans represents a superb model system in which to study the effects of mitochondrial function on longevity. Several mutant strains have been identified that indicate that mitochondrial function is a major factor affecting the organism's lifespan. Taken as a group, these mutant strains indicate that metabolic rate, per se, only affects longevity indirectly. Mutations causing lowered metabolic rate potential are capable of decreasing or increasing longevity.

A stomatin and a degenerin interact in lipid rafts of the nervous system of Caenorhabditis elegans.

In Caenorhabditis elegans, the gene unc-1 controls anesthetic sensitivity and normal locomotion. The protein UNC-1 is a close homolog of the mammalian protein stomatin and is expressed primarily in the nervous system. Genetic studies in C. elegans have shown that the UNC-1 protein interacts with a sodium channel subunit, UNC-8. In humans, absence of stomatin is associated with abnormal sodium and potassium levels in red blood cells. Stomatin also has been postulated to participate in the formation of lipid rafts, which are membrane microdomains associated with protein complexes, cholesterol, and sphingolipids. In this study, we isolated a low-density, detergent-resistant fraction from cell membranes of C. elegans. This fraction contains cholesterol, sphingolipids, and protein consistent with their identification as lipid rafts. We then probed Western blots of protein from the rafts and found that the UNC-1 protein is almost totally restricted to this fraction. The UNC-8 protein is also found in rafts and coimmunoprecipitates UNC-1. A second stomatin-like protein, UNC-24, also affects anesthetic sensitivity, is found in lipid rafts, and regulates UNC-1 distribution. Mutations in the unc-24 gene alter the distribution of UNC-1 in lipid rafts. Each of these mutations alters anesthetic sensitivity in C. elegans. Because lipid rafts contain many of the putative targets of volatile anesthetics, they may represent a novel class of targets for volatile anesthetics.

Mitochondrial mutations differentially affect aging, mutability and anesthetic sensitivity in Caenorhabditis elegans.

In the nematode Caenorhabditis elegans, mutations have been previously isolated that affect the activities of Complex I (gas-1) and Complex II (mev-1), two of the five membrane-bound complexes that control electron flow in mitochondrial respiration. We compared the effects of gas-1 and mev-1 mutations on different traits influenced by mitochondrial function. Mutations in Complex I and II both increased sensitivity to free radicals as measured during development and in aging animals. However, gas-1 and mev-1 mutations differentially affected mutability and anesthetic sensitivity. Specifically, gas-1 was anesthetic hypersensitive but not hypermutable while mev-1 was hypermutable but displayed normal responses to anesthetics. These results indicate that Complexes I and II may differ in their effects on behavior and development, and are consistent with the wide variation in phenotypes that result from mitochondrial changes in other organisms.

Model organisms: new insights into ion channel and transporter function. Stomatin homologues interact in Caenorhabditis elegans.

In C. elegans the protein UNC-1 is a major determinant of anesthetic sensitivity and is a close homologue of the mammalian protein stomatin. In humans stomatin is missing from erythrocyte membranes in the hemolytic disease overhydrated hereditary stomatocytosis, despite an apparently normal stomatin gene. Overhydrated hereditary stomatocytosis is characterized by alteration of the normal transmembrane gradients of sodium and potassium. Stomatin has been shown to interact genetically with sodium channels. It is also postulated that stomatin is important in the organization of lipid rafts. We demonstrate here that antibodies against UNC-1 stain the major nerve tracts of Caenorhabditis elegans, with very intense staining of the nerve ring. We also found that a gene encoding a stomatin-like protein, UNC-24, affects anesthetic sensitivity and is genetically epistatic to unc-1. In the absence of UNC-24, the staining of the nerve ring by anti-UNC-1 is abolished, despite normal transcriptional levels of the unc-1 mRNA. Western blots indicate that UNC-24 probably affects the stability of the UNC-1 protein. UNC-24 may therefore be necessary for the correct placement of UNC-1 in the cell membrane and organization of lipid rafts.

Mitochondrial expression and function of GAS-1 in Caenorhabditis elegans.

A mutation in the gene gas-1 alters sensitivity to volatile anesthetics, fecundity, and life span in the nematode Caenorhabditis elegans. gas-1 encodes a close homologue of the 49-kDa iron protein subunit of Complex I of the mitochondrial electron transport chain from bovine heart. gas-1 is widely expressed in the nematode neuromuscular system and in a subcellular pattern consistent with that of a mitochondrial protein. Pharmacological studies indicate that gas-1 functions partially via presynaptic effects. In addition, a mutation in the gas-1 gene profoundly decreases Complex I-dependent metabolism in mitochondria as measured by rates of both oxidative phosphorylation and electron transport. An increase in Complex II-dependent metabolism also is seen in mitochondria from gas-1 animals. There is no apparent alteration in physical structure in mitochondria from gas-1 nematodes compared with those from wild type. These data indicate that gas-1 is the major 49-kDa protein of complex I and that the GAS-1 protein is critical to mitochondrial function in C. elegans. They also reveal the importance of mitochondrial function in determining not only aging and life span, but also anesthetic sensitivity, in this model organism.

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.

The sequence and associated null phenotype of a C. elegans neurocalcin-like gene.

The neuronal calcium sensor (NCS) proteins belong to a subfamily of the EF-hand calcium binding proteins. These proteins are primarily expressed in the nervous system and currently include more than 20 members across species [Nakayama et al., J Mol Evol 34:416-448, 1992]. Two homologues of the ncs genes, Ce-ncs-1 and Ce-ncs-2, have recently been identified in the nematode C. elegans. Here we report the cDNA sequence of a third C. elegans ncs homologue, Ce-ncs-3. We demonstrate that a null mutation in this gene caused by a large deletion in the locus does not confer a visible phenotype in C. elegans. This, in addition to the strong homology between Ce-NCS-3 and the other C. elegans NCS proteins, may indicate functional redundancy between the three genes.

A stomatin and a degenerin interact to control anesthetic sensitivity in Caenorhabditis elegans.

The mechanism of action of volatile anesthetics is unknown. In Caenorhabditis elegans, mutations in the gene unc-1 alter anesthetic sensitivity. The protein UNC-1 is a close homologue of the mammalian protein stomatin. Mammalian stomatin is thought to interact with an as-yet-unknown ion channel to control sodium flux. Using both reporter constructs and translational fusion constructs for UNC-1 and green fluorescent protein (GFP), we have shown that UNC-1 is expressed primarily within the nervous system. The expression pattern of UNC-1 is similar to that of UNC-8, a sodium channel homologue. We examined the interaction of multiple alleles of unc-1 and unc-8 with each other and with other genes affecting anesthetic sensitivity. The data indicate that the protein products of these genes interact, and that an UNC-1/UNC-8 complex is a possible anesthetic target. We propose that membrane-associated protein complexes may represent a general target for volatile anesthetics.

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. CONCLUSIONS: The function of the 49-kDa (IP) subunit of complex I is unknown. The finding that mutations in complex I increase sensitivity of C elegans to volatile anesthetics may implicate this physiologic process in the determination of anesthetic sensitivity. The hypersensitivity of animals with a mutation in the gas-1 gene may be caused by a direct anesthetic effect on a mitochondrial protein or secondary effects at other sites caused by mitochondrial dysfunction.

Unc-1: a stomatin homologue controls sensitivity to volatile anesthetics in Caenorhabditis elegans.

To identify sites of action of volatile anesthetics, we are studying genes in a functional pathway that controls sensitivity to volatile anesthetics in the nematode Caenorhabditis elegans. The unc-1 gene occupies a central position in this pathway. Different alleles of unc-1 have unique effects on sensitivity to the different volatile anesthetics. UNC-1 shows extensive homology to human stomatin, an integral membrane protein thought to regulate an associated ion channel. We postulate that UNC-1 has a direct effect on anesthetic sensitivity in C. elegans and may represent a molecular target for volatile anesthetics.

Control of anesthetic response in C. elegans.

We describe the use of the animal model C. elegans to understand how the volatile anesthetics work at the molecular level. Mutations in several different genes can profoundly change the behavior of this animal under volatile anesthetics. Protein products of two of these genes are discussed. One gene is an integral membrane protein thought to regulate ion channels. The other is a subunit of the first protein complex of the electron transport chain.

Genetic differences affecting the potency of stereoisomers of isoflurane.

BACKGROUND: In previous studies, researchers demonstrated the ability of a variety of organisms and in vitro sites of anesthetic action to distinguish between stereoisomers of isoflurane or halothane. However, it was not shown whether organisms with differing sensitivities to stereoisomers of one volatile anesthetic are able to distinguish between stereoisomers of another. In this study, the responses of mutants of Caenorbabditis elegans to stereoisomers of isoflurane were determined for comparison to previous results in halothane. METHODS: Mutant strains of C. elegans were isolated and grown by standard techniques. The EC50s (the effective concentrations of anesthetia at which 50% of the animals are immobilized for 10 s) of stereoisomers of isoflurane and the racemate were determined in wild type and mutant strains of C. elegans. RESULTS: Wild type C. elegans and strains with high EC50S of the racemate were more sensitive to the (+) isomer of isoflurane by approximately 30%. The racemate showed a EC50s similar to the less potent isomer, the (-) form. In the strains with low EC50s, one strain showed no ability to differentiate between the stereoisomers, whereas two showed a 60% difference between the (+) and (-) forms. CONCLUSIONS: The ability to distinguish between stereoisomers of isoflurane is associated with genetic loci separate from those that distinguish between stereoisomers of halothane. These results are consistent with multiple sites of action for these anesthetics.

Mutations affecting sensitivity to ethanol in the nematode, Caenorhabditis elegans.

Mutations in nine genes have been identified in the nematode, Caenorhabditis elegans, which control sensitivity to ethanol. The interaction of these genes has been examined and used to determine a genetic pathway controlling sensitivity to ethanol. The nature of this pathway indicates that ethanol exerts its anesthetic actions at more than one site of action. These results also indicate that ethanol is similar in its effects to the volatile anesthetics, enflurane and isoflurane.

Mutations conferring new patterns of sensitivity to volatile anesthetics in Caenorhabditis elegans.

BACKGROUND: We previously described the use of the nematode Caenorabditis elegans as a genetic model for studying the mechanism of action of volatile anesthetics. All previous strains of C. elegans with altered responses to anesthetics have been identified by screening the response to halothane. The current study was designed to identify classes of mutations by screening for alterations in sensitivity to enflurane, isoflurane, and diethylether. METHODS: Nematodes were mutated and the resulting mutant strains were screened for immobility in low doses of enflurane, isoflurane, or diethylether. Concentrations of halothane, enflurane, isoflurane, and diethylether that anesthetized 50% of the animals were determined in all mutations. Interactions of some new mutations with previously identified mutations were determined by construction of double mutants. RESULTS: Mutations in six genes were identified and were divided into two classes. One class primarily affected sensitivity to enflurane and isoflurane; a second class affected sensitivity to all of the volatile anesthetics studied. The effects of the latter group dominated the effects of previously identified mutations. CONCLUSIONS: The interaction of these mutations indicates that multiple sites of anesthetic action exist and that there are at least three such sites. A pathway for control of sensitivity to volatile anesthetics is proposed.

Genetic differences affecting the potency of stereoisomers of halothane.

The mechanism of action of volatile anesthetics is the subject of some debate. Much of the controversy has centered on whether the site of such actions is purely lipid in nature or may contain a protein target. This report studies the interaction of stereoisomers of halothane on the wild type and on a variety of genetic mutants of Caenorhabditis elegans. The mutants studied have previously been shown to have altered sensitivities to volatile anesthetics. In one mutant, fc34, (R)-halothane [the (+) isomer] was 3 times more potent than its S (-) isomer. Other mutants and wild-type animals displayed more modest differences in sensitivity to the enantiomers. The results indicate that a genetic pathway exists in C. elegans controlling sensitivity to halothane and that both lipid and protein targets may mediate halothane's effects.