Chronic exposure to inhaled anesthetics increases cholesterol content in Acholeplasma laidlawii.

Acholeplasma laidlawii cells were grown in cholesterol-enriched medium and exposed continuously to either air (control), 4.0 vol.% halothane in air at 1 atm pressure (4% atm halothane), or 80% cyclopropane in oxygen for 24 h at 37 degrees C. Cells grown in the presence of 4% atm halothane or 80% cyclopropane had approximately twice as much membrane cholesterol content/mg protein as the control cells. Cells grown in an anesthetic environment also tended to have a higher membrane cholesterol/phospholipid molar ratio compared to control cells. Membranes isolated from halothane-exposed cells grown in a cholesterol-enriched medium were more ordered at 37 degrees C (measurements were made with no anesthetic present) than membranes from control cells grown in an identically enriched medium. This difference in membrane physical state between control and anesthetic-exposed cells decreased as the temperature decreased, and disappeared at approx. 23 degrees C. Continuous exposure of A. laidlawii to 4% atm halothane or 80% cyclopropane for 24 h did not markedly affect membrane fatty acid composition, either in cells grown on an unsupplemented medium or in cells grown in a medium enriched in myristic, palmitic or stearic acids. These results further support the hypothesis that an increased membrane cholesterol content may play a role in the tolerance or dependence that develops after chronic exposure to anesthetic agents.

Aging, chronic administration of ethanol, and acute exposure to nitrous oxide: effects on vitamin B12 and folate status in rats.

Elderly patients with alcoholism often require surgery and receive nitrous oxide (N2O) as a component of their anesthetic. Since aging, ethanol, and N2O may all perturb folate and/or vitamin B12 metabolism, we examined the combined influence of these parameters on vitamin B12/folate status in a rodent model. Aged male Fischer 344 rats (24 months old) were given a liquid ethanol diet (35% of calories as ethanol) and control rats were pair-fed a liquid diet with carbohydrate substituting for the caloric content of ethanol. After receiving liquid diets for 7 weeks, rats were exposed to 60% N2O/40% 0(2) for 6 h. Urinary excretion of formic acid, formiminoglutamic acid (FIGLU), and methylmalonic acid (MMA) were used as indirect markers of folate/vitamin B12 status. In both the aged ethanol-fed and control groups, excretion of formic acid and FIGLU markedly increased the first day after N2O exposure and returned towards background values by the second day. No changes occurred in MMA excretion. Exposure to N2O decreased methionine synthase activities in liver, kidney and brain, and recovery of methionine synthase activities occurred over a period of 4 days in both the aged ethanol-fed and control groups. Ethanol treatment for 7 weeks combined with acute exposure to N2O did not deplete the aged rats of folate or vitamin B12 in blood, liver, kidney or brain. Thus, in this animal model, aging, chronic ethanol administration, and acute N2O exposure did not act synergistically to produce prolonged and severe disturbances in folate and vitamin B12 metabolism.

Methionine synthase activities in mice following acute exposures to ethanol and nitrous oxide.

Acute or chronic exposure to nitrous oxide or chronic exposure to ethanol decreases the activity of the vitamin B12-dependent enzyme methionine synthase. To assess the combined effect of acute exposure to nitrous oxide and ethanol, mice were given an intraperitoneal injection of ethanol (3 g/kg) and exposed to an inspired mixture of 66% nitrous oxide and 34% oxygen for 4 hr. Methionine synthase activities in liver, kidney, and brain were measured immediately after exposure to nitrous oxide and at various times over a 4-day recovery period. Methionine synthase activities in liver and kidney returned to control levels 2-4 days following inactivation. In brain, a significant 16% decrease in methionine synthase activity remained after a 4-day recovery period. The acute administration of ethanol did not alter the magnitude of the inactivation induced by nitrous oxide nor the time course of recovery of methionine synthase activity following inactivation. Moreover, in mice that were not exposed to nitrous oxide, methionine synthase activity was not altered by the acute administration of ethanol alone or in combination with 0.4% atm isoflurane. Thus, in this animal model, an acute dose of ethanol does not alter methionine synthase activity nor does it enhance the inactivation produced by nitrous oxide.

Effects of halothane on the nicotinic acetylcholine receptor from Torpedo californica.

To determine whether the binding of anesthetics to key membrane receptors is a plausible mode of action, we modeled the effect of the general anesthetic halothane in the nicotinic acetylcholine receptor membrane system isolated from Torpedo californica. Our results demonstrated that halothane inhibits the binding of [3H]phencyclidine ([3H]PCP) to the acetylcholine receptor. The inhibition was reversible, concentration dependent, and had an equilibrium dissociation constant (Kd) of 2.2% atm halothane at 25 degrees. Double-reciprocal plots of the halothane effects at various phencyclidine (PCP) concentrations imply that, under equilibrium conditions, halothane inhibits [3H]PCP binding competitively. In contrast, results from kinetic studies showed that the rate of PCP dissociation is highly sensitive to halothane with EC50 = 0.8% atm halothane in nitrogen. Several possible interpretations are discussed; however, the basic observation was that the kinetics of [3H]PCP binding to the nicotinic acetylcholine receptor was affected by halothane at low concentrations in this model system.

Saturable binding of anesthetics to nicotinic acetylcholine receptors. A possible mechanism of anesthetic action.

Recent controversies in the existence of saturable binding of general anesthetics in brain tissues prompted a careful examination of specific binding of anesthetics to neural receptors. We examined the binding of both local and general anesthetics using electron spin resonance and radioligand criteria. Our results suggested that the hydrophobic path, most probably through the lipid bilayer, figures importantly in the binding of the uncharged moieties of anesthetics. Competitive interactions by hydrophobic compounds for the high-affinity site in the nicotinic acetylcholine receptor led us to propose a hypothesis that includes a hydrophobic crevice of limited volume as part of the high-affinity site. Association of anesthetic at this crevice is in turn dependent on the anesthetic concentration in the lipid phase of the membrane. The hypothesis provides a mechanism for the saturable interaction of anesthetics with their protein target site in the membrane without violating the correlations expressed by the Meyer-Overton rule of anesthetic action.

Nitrous oxide: a cause of cancer or chemotherapeutic adjuvant?

The administration of nitrous oxide rapidly inactivates the vitamin B12-dependent enzyme methionine synthase. This inactivation disrupts the normal interrelationships between vitamin B12 and folic acid, and results in altered levels of folic acid derivatives and certain amino acids and their metabolites. Attempts have been made to use the antifolate properties of nitrous oxide to treat patients with leukemia. Although transient improvements may be observed in patients with leukemia who are given nitrous oxide, prolonged administration of nitrous oxide is highly toxic and causes marked hematological and neurological abnormalities. Animal and in vitro studies suggest that the action of nitrous oxide may be tumor selective, and that nitrous oxide may interact with and enhance the therapeutic effect of other antitumor agents. However, there is a delicate balance between the possible beneficial and harmful effects of nitrous oxide, and the conditions for which nitrous oxide may prove useful as a chemotherapeutic adjuvant remain to be defined. Concern has also been raised that nitrous oxide may have carcinogenic potential, especially in operating room and dental personnel who are chronically exposed to trace concentrations of this gas. However, there is no convincing evidence to date that nitrous oxide causes cancer in either animals or humans.

Characteristics and implications of desflurane metabolism and toxicity.

The metabolism of desflurane has been assessed both in animals and humans by measuring the appearance of fluoride metabolites (fluoride ion, nonvolatile organic fluoride, trifluoroacetic acid) in blood and urine. Desflurane administered to rats (either pretreated or not pretreated with phenobarbital or ethanol) for 3.2 MAC-hours and to swine for 5.5 MAC-hours produced fluoride ion levels in blood that were almost indistinguishable from values measured in control animals. In contrast, a significant 17% increase in plasma fluoride ion concentration in swine was detected 4 h after exposure to desflurane. In human studies, desflurane administered to patients (3.1 MAC-hours) and volunteers (7.35 MAC-hours) resulted in postanesthesia serum fluoride in concentrations that did not differ from background fluoride ion concentrations. Similarly, postanesthetic urinary excretion of fluoride ion and organic fluoride in volunteers was comparable to preanesthetic excretion rates. Small but statistically significant levels of trifluoroacetic acid were found in both serum and urine from volunteers after exposure to desflurane. Peak serum concentrations averaging 0.38 +/- 0.17 microM trifluoroacetic acid (mean +/- SD) and peak urinary excretion rates averaging 0.169 +/- 0.107 mumol/h were detected in volunteers 24 h after desflurane exposure. Although these increases in trifluoroacetic acid after exposure to desflurane were statistically significant, they are approximately 10-fold less than levels seen after exposure to isoflurane. Desflurane strongly resists biodegradation, and only a small amount is metabolized in animals and humans.

Anesthetic requirement in the quaking mouse.

Anesthetic requirements for nitrous oxide, cyclopropane, enflurane, and isoflurane were determined in quaking mice (autosomal recessive mutants with a deficiency in central nervous system myelin) and their littermate (non-quaking) controls. Although quaking mice had significantly (15 to 23 per cent) lower nitrous oxide and cyclopropane rolling-response ED50S than did littermate controls, enflurane and isoflurane rolling-response ED50S were not significantly different. Tail-clamp ED50S for cyclopropane, enflurane, and isoflurane in quaking and control mice were not significantly different. Myelin from quaking mice showed higher levels of palmitic, stearic, and docosahexaenoic acids, lower levels of oleic, eicosenoic, and docosatetraenoic (and/or nervonic) acids, and a lower cholesterol/phospholipid ratio. In contrast, the phospholipid, fatty acid, and cholesterol compositions of synaptic plasma membranes isolated from quaking and control mice were essentially identical. It is concluded that gross alterations in the lipid composition of central nervous system myelin have little or no influence on anesthetic requirement.

Anaesthetic requirement in mice selectively bred for differences in ethanol sensitivity.

Anaesthetic requirements for nitrous oxide, enflurane and isoflurane were determined in mice selectively bred for their susceptibility ("long-sleep" mice) or resistance ("short-sleep" mice) to alcohol. Nitrous oxide and enflurane requirements, measured by the rolling-response test, were 34 and 20% greater, respectively, in short-sleep mice than in long-sleep mice. Although isoflurane requirement was 39% greater when measured by the tail-clamp test, it was not significantly different when measured by the rolling-response test. The greater anaesthetic requirement for short-sleep mice was not associated with a different synaptic membrane phospholipid, fatty acid or cholesterol composition.

Cross-mating of mice selectively bred for resistance or susceptibility to nitrous oxide anesthesia: potencies of nitrous oxide in offspring.

To investigate the genetic bases of differences in nitrous oxide requirement in mice selectively bred for resistance (HI mice) or susceptibility (LO mice) to nitrous oxide anesthesia, the potency of nitrous oxide in offspring having one HI and one LO parent was measured. Nitrous oxide ED50S (+/- SD) in offspring having HI male and LO female parents or LO male and HI female parents were 1.53 +/- 0.10 and 1.63 +/- 0.14 atm, respectively. In contrast, the nitrous oxide ED50 for offspring having two LO parents was 1.26 +/- 0.09 atm, and for offspring having two HI parents, 1.96 +/- 0.10 atm. Thus, the nitrous oxide ED50S for offspring produced by cross-mating HI and LO animals approximated the average values for their parents. We conclude that no single one gene determines dominance or recessiveness for nitrous oxide requirement in mice, and that the genetic control of resistance or susceptibility to nitrous oxide anesthesia probably involves many genes.

Vitamin B12 and folate status in rats after chronic administration of ethanol and acute exposure to nitrous oxide.

The chronic administration of ethanol or brief exposure to nitrous oxide (N2O) decreases the activity of hepatic methionine synthase and disrupts normal metabolic processes that require folate and vitamin B12. This combination of drugs has clinical relevance since alcoholic patients often require surgery and receive N2O as a component of their anesthetic. To assess this clinical problem using a rodent model, rats were given a liquid ethanol diet (35% of calories as ethanol) and control rats were pair-fed a liquid diet with carbohydrate substituting for the caloric content of ethanol. After receiving liquid diets for 6 weeks, rats were exposed to 60% N2O/40% O2 for 6 hr. Urinary excretions of formic acid and formiminoglutamic acid (FIGLU) were used as indirect markers of folate status. In both the ethanol-fed and control groups, excretion of formic acid and FIGLU markedly increased the first day after N2O and returned towards background values by the second day after N2O exposure. Ethanol treatment alone decreased methionine synthase activities in liver, but not kidney or brain. Exposure to N2O further decreased methionine synthase activities, and recovery of methionine synthase activity after N2O occurred over a period of 4 days at the same rate in both the ethanol-fed and control groups. Ethanol treatment for 6 weeks combined with acute exposure to N2O did not deplete the rats of vitamin B12 in blood, liver, kidney, or brain. We conclude that in this animal model, chronic treatment with ethanol does not markedly exacerbate the disturbances in folate/vitamin B12 metabolism caused by brief exposure to N2O.

Dimethylthiourea, a hydroxyl radical scavenger, impedes the inactivation of methionine synthase by nitrous oxide in mice.

Dimethylthiourea (DMTU), a potent scavenger of hydroxyl radicals, was studied to see if it attenuated the inactivation of methionine synthase produced by nitrous oxide in mice. Mice were given i.p. injections of DMTU 0.5-4.0 mg g-1 or saline and, 1 h after injection, were exposed to 66% nitrous oxide in oxygen for periods of 0.5-8 h. At given times after nitrous oxide exposure, higher methionine synthase activities were found in the livers, kidneys and brains of mice injected with DMTU than in the saline-injected animals. These higher methionine synthase activities in the DMTU-treated animals represented a delay in the enzyme inactivation produced by nitrous oxide, as the difference in activities between the DMTU-injected and saline-injected mice decreased with increasing duration of exposure to nitrous oxide. Greater differences in methionine synthase activities between the DMTU- and saline-injected animals were observed with increasing doses of DMTU. The rate of enzyme inactivation following exposure to nitrous oxide was greater in liver and least in brain, and the difference in activities between the two groups varied with the organ examined. DMTU exhibited its greatest effect in the kidney, where methionine synthase activities were nearly doubled in the DMTU 2.0 mg g-1-injected compared with the saline-injected mice after 1-h exposure to 66% nitrous oxide. Following a marked inactivation of methionine synthase by exposing mice to 66% nitrous oxide for 4 h, injection of DMTU 2.0 mg g-1 at the end of exposure to nitrous oxide did not enhance, but impaired, the recovery of enzyme activity. The findings are consistent with the hypothesis that nitrous oxide combines with the vitamin B12 molecule of methionine synthase to form a hydroxyl radical that reacts with an inactivates the enzyme, and that DMTU slows this inactivation by scavenging hydroxyl radicals.

Tolerance of mice to nitrous oxide.

Mice continuously exposed to a subanesthetic dose of nitrous oxide (N2O) become tolerant to nitrous oxide, as measured by an increase in their ED50 (dose required to abolish the righting reflex in 50% of animals) for N2O. The maximum increase in ED50 is approximately 0.25 atm for mice exposed to 40, 50 or 70% N2O for 2 to 3 weeks. Mice exposed to 25% N2O for 3 weeks do not develop tolerance. Mice exhibit a withdrawal syndrome when removed from the subanesthetic environment after exposure to 40 to 70% but not 25% N2O for 3 weeks. Tolerance to 50 or 70% N2O develops within 1 week and is maximal at 2 weeks. Tolerance to nitrous oxide is lost within 6 days following removal of the animals from the subanesthetic environment. Synaptic membrane fatty acid, phospholipid, and cholesterol compositions of tolerant animals are not significantly altered.

Role of voltage-sensitive receptors in nicotinic transmission.

This paper compares the conductance induced by bath-applied acetyl-choline (ACh) and by the same transmitter released from nerve terminals at Electrophorus electroplaques. For the former case, dose-response relations are characterized by the maximal agonist-induced conductance, rgamma (130 mmho/cm2), and by the concentration which induces half this conductance; this concentration is termed Kapp and equals 50 micron at -85 mV. For the latter case, neurally evoked postsynaptic currents (PSCs) are characterized by the peak conductance during strongly facilitated release, gPSC, and by the rate constant for decay, alpha. Since gPSC roughly equals rgamma, it is concluded that the PSC activates nearly all available receptor channels. These and other data agree with recent estimates that during the growth phase of the quantal response, (a) the ACh concentration is at least several hundred micromolar; and (b) most nearby channels are activated. However both alpha and Kapp increase during depolarization, at a rate of about e-fold per 86 mV. These observations on voltage sensitivity suggest that a suprathreshold synaptic event is rapidly terminated because the action potential abruptly releases ACh molecules from receptors.

Dioxychlorane: a challenge to the correlation of anesthetic potency and lipid solubility.

Dioxychlorane is a five-membered cyclic halogenated diether which has been reported to be anesthetic at an inspired concentration of 0.3% atm and to have an oil/gas partition coefficient to 260 to 275. These values suggested that dioxychlorane might represent a significant deviation from the correlation of anesthetic potency with lipid solubility. However, the results of this study did not confirm the above data: MAC in four dogs was 0.11 +/- 0.02 (SE) % atm, and the oil/gas partition coefficient was 1,286 +/- 44. Dioxychlorane appears not to be an exception to the correlation of anesthetic potency with lipid solubility.

Disruption of folate and vitamin B12 metabolism in aged rats following exposure to nitrous oxide.

The ability of nitrous oxide (N2O) to disrupt folate and vitamin B12 metabolism was examined in young (2-month), middle-aged (12-month), and elderly (24-month) Fischer 344 rats. Abnormalities in folate metabolism were assessed in a noninvasive manner by measuring the urinary excretion of formic acid and formiminoglutamic acid (FIGLU), compounds that are elevated in the urine of mammals with a deficiency in folate. After a 6-h exposure to 60% N2O/40% O2, urinary formic acid excretion increased 3- to 25-fold the first day following N2O exposure and returned to background levels by the second day after exposure in all age groups. Urinary FIGLU excretion increased 100- to 300-fold in the first day following N2O exposure, with the highest FIGLU excretion rates found in the elderly rats and the lowest in the young rats. By the second day after N2O exposure, FIGLU excretion rates returned to baseline levels in all age groups. Plasma folate progressively decreased with increasing age, whereas no age-dependent changes were observed in red cell folate, liver folate, or plasma vitamin B12 levels. The elderly rats demonstrated the highest vitamin B12 content in the liver and the lowest vitamin B12 content in the kidney compared to the other age groups. Hepatic methionine synthase activities (measured 16-21 days after N2O exposure) were elevated in the elderly compared to the middle-aged or young rats, but methionine synthase activities in kidney and brain were not different among the three different age groups. It was concluded that in rats, aging per se only slightly influences the disruption of folate metabolism produced by exposure to N2O.