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.

Treatment of cocaine overdose with lipid emulsion.

We describe the management and recovery of a 28-year-old man following a history of overdose by nasal inhalation of cocaine. The patient was presented in a comatose state suffering from seizures and marked cardiovascularly instability. Intravenous lipid emulsion was administered following initial resuscitation and tracheal intubation, as a means of treating persistent cardiac arrhythmias and profound hypotension. Following lipid emulsion therapy, the patient's life-threatening cardiovascular parameters rapidly improved and he recovered well without any side effects, thus being discharged within 2 days.

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.

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 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.

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.

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.

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.

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.

Small cell carcinoma of the lung.

Over a 5-year period 230 cases of small cell carcinoma presented to Brompton Hospital, London. The male to female ratio was 2.5:1 and 39% had evidence of extrathoracic disease. Forty-five patients were untreated and had a median survival of 70 days. The 5-year survival for surgery and radiotherapy was 6.6% and 4.2% respectively. Of 13 patients treated with chemotherapy the median survival was 285 days. The factors which influence survival are described.

Effect of anesthetics and a convulsant on normal and mutant Caenorhabditis elegans.

The authors have developed a method for studying the action of volatile anesthetics in Caenorhabditis elegans (C.e.), a free living nematode. C.e. appears to be a useful model for the study of the influence of genetics on susceptibility to anesthetics. This worm has a small, completely defined nervous system, easily manipulated genetics, and a large number of nervous system mutants. Under normal conditions C.e. moves almost constantly. When exposed to anesthetics there is an initial phase of increased locomotion, followed by uncoordinated motion that progresses to immobility. Motion returns quickly when the nematodes are removed from the anesthetic. The authors called loss of locomotion "anesthesia." The ED50S of various anesthetics with C.e. are as follows: methoxyflurane 0.45%, chloroform 1.25%, halothane 2.7%, enflurane 4.2%, isoflurane 5.6%, fluroxene 9.9%. The authors also studied the action of a convulsant, flurothyl, on C.e. Flurothyl has anesthetizing properties in these animals with an ED50 of 8.1%. No convulsant activity was noted. However, mixtures of halothane and flurothyl were antagonistic in their effects, while halothane and enflurane were additive. Furthermore, the authors isolated a mutant strain (HS1) of C.e. that shows altered responses to several anesthetics and a convulsant. HS1 is uncoordinated when not exposed to anesthetics. Like the normal strain (N2) HS1 loses mobility when exposed to anesthetics. The ED50S for various anesthetics in HS1 were as follows: methoxyflurane 0.04%, chloroform 0.52%, halothane 0.85%, isoflurane 4.9%, enflurane 6.0%, fluroxene 10.9%.(ABSTRACT TRUNCATED AT 250 WORDS)

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.

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.

Pulmonary leiomyosarcomas.

Four cases of pulmonary leiomyosarcoma are presented. The characteristic histological features are described with specific reference to the criteria of malignancy. It is suggested that diagnosis by bronchoscopy provides insufficient material for histological grading and open lung biopsy is the diagnostic tool of choice. The available methods of treatment are discussed.

First report of byssinosis in Hong Kong.

There has been no report of byssinosis in Hong Kong although the textile industry has been one of the leading industries for many years. Three workers with a long history of exposure to cotton dust had chronic obstructive airways disease precipitated by their work environment. One had irreversible airways obstruction but none had chronic bronchitis, emphysema, or asthma. Only one gave a history of "Monday morning tightness," and this was attributed to the fact that most of the textile workers in Hong Kong work seven days a week. It was suggested that a survey be carried out to ascertain the importance of byssinosis in the textile workers of Hong Kong and tha byssinosis should there be added to the list of notifiable occupational diseases.

Prognostic features of large cell anaplastic carcinoma of the bronchus.

The clinical features of 208 patients with large cell anaplastic carcinoma of the bronchus are reviewed. Of the sample 47.6% had disseminated disease at presentation, and 95% were cigarette smokers. The median survival was 6.0 months and five-year survival was 5.9%. Twenty-seven per cent of the patients had surgical resection of tumour. The median survival of resected patients was 13.0 months and five-year-survival was 21%. Radiotherapy was ineffective in controlling the disease in this series.

Multiple sites of action of volatile anesthetics in Caenorhabditis elegans.

The mechanism and site(s) of action of volatile anesthetics are unknown. In all organisms studied, volatile anesthetics adhere to the Meyer-Overton relationship--that is, a ln-ln plot of the oil-gas partition coefficients versus the potencies yields a straight line with a slope of -1. This relationship has led to two conclusions about the site of action of volatile anesthetics. (i) It has properties similar to the lipid used to determine the oil-gas partition coefficients. (ii) All volatile anesthetics cause anesthesia by affecting a single site. In Caenorhabditis elegans, we have identified two mutants with altered sensitivities to only some volatile anesthetics. These two mutants, unc-79 and unc-80, confer large increases in sensitivity to very lipid soluble agents but have little or no increases to other agents. In addition, a class of extragenic suppressor mutations exists that suppresses some altered sensitivities but specifically does not suppress the altered sensitivity to diethyl ether. There is much debate concerning the molecular nature of the site(s) of anesthetic action. One point of discussion is whether the site(s) consists of a purely lipid binding site or if protein is involved. The simplest explanation of our observations is that volatile anesthetics cause immobility in C. elegans by specifically interacting with multiple sites. This model is in turn more consistent with involvement of protein at the site(s) of action.

Interaction of GABA and volatile anesthetics in the nematode Caenorhabditis elegans.

The authors tested whether mutant strains of Caenorhabditis elegans with altered sensitivity to volatile anesthetics have altered responses to GABA or GABA-agonists. They determined the ED50s of the wild-type strain N2 and two mutant strains of C. elegans to a GABA-mimetic ivermectin (IVM) and to GABA. unc-79, a strain with increased sensitivity to halothane, was more sensitive than N2 to IVM and GABA. unc-9, a strain that suppresses the increased sensitivity of unc-79 to halothane, was less sensitive than N2 to IVM and GABA. The authors also tested whether doses of GABA or IVM and volatile anesthetics were additive in their effects on C. elegans. Halothane (2.1%) did not shift the ED50 of IVM, but was antagonistic to GABA. Enflurane (4%) was antagonistic to both IVM and GABA. However, ED50s of halothane and enflurane were unchanged in the presence of IVM (35 nM) or GABA (150 mM). The authors conclude that GABA by itself does not appear to mediate halothane or enflurane sensitivity in C. elegans.

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.