Anesthetic pretreatment confers thermotolerance on Saccharomyces cerevisiae yeast.

Saccharomyces cerevisiae yeast, when pretreated with elevated temperatures, undergo adaptive changes that promote survival after an otherwise lethal heat stress. The heat shock response, a cellular stress response variant, mediates these adaptive changes. Ethanol, a low-potency anesthetic, promotes thermotolerance possibly through heat shock response activation. Therefore, we hypothesized other anesthetic compounds, like ethanol, may invoke the heat shock response to promote thermotolerance. To test this hypothesis, we pretreated yeast with a series of non-volatile anesthetic and anesthetic-related compounds and quantified survival following lethal heat shock (52 °C for 5 min). Most compounds invoked thermoprotection and promoted survival with a potency proportional to hydrophobicity: tribromoethanol (5.6 mM, peak survival response), trichloroethanol (17.8 mM), dichloroethanol (100 mM), monochloroethanol (316 mM), trifluoroethanol (177.8 mM), ethanol (1 M), isopropanol (1 M), propofol (316 µM), and carbon tetrabromide (32 µM). Thermoprotection conferred by pretreatment with elevated temperatures was "left shifted" by anesthetic co-treatment from (in °C) 35.3 ± 0.1 to 32.2 ± 0.1 with trifluoroethanol (177.8 mM), to 31.2 ± 0.1 with trichloroethanol (17.8 mM), and to 29.1 ± 0.3 with tribromoethanol (5.6 mM). Yeast in postdiauxic shift growth phase, relative to mid-log, responded with greater heat shock survival; and media supplementation with tryptophan and leucine blocked thermoprotection, perhaps by reversing the amino acid starvation response. Our results suggest S. cerevisase may serve as a model organism for understanding anesthetic toxicity and anesthetic preconditioning, a process by which anesthetics promote tissue survival after hypoxic insult.

The effect of atomoxetine, a selective norepinephrine reuptake inhibitor, on respiratory arrest and cardiorespiratory function in the DBA/1 mouse model of SUDEP.

Sudden unexpected death in epilepsy (SUDEP) is a significant public health burden. The mechanisms of SUDEP are elusive, although cardiorespiratory dysfunction is a likely contributor. Clinical and animal studies indicate that seizure-induced respiratory arrest (S-IRA) is the primary event leading to death in many SUDEP cases. Our prior studies demonstrated that intraperitoneal (IP) injection of atomoxetine, a norepinephrine reuptake inhibitor (NRI) widely used to treat attention deficit hyperactivity disorder, suppresses S-IRA in DBA/1 mice. In the current study, we injected atomoxetine intracerebroventricularly (ICV) and measured its effect on S-IRA in DBA/1 mice to determine its central effects. Additionally, to test our hypothesis that atomoxetine reduces S-IRA via altering cardiorespiratory function, we examined the effect of atomoxetine on respiratory and cardiac function using non-invasive plethysmography and ECG in anesthetized DBA/1 mice, and on blood pressure and heart rate using a tail-cuff system in conscious DBA/1 mice. ICV administration of atomoxetine at 200-250nmol significantly reduced S-IRA evoked by acoustic stimulation in DBA/1 mice, consistent with a central atomoxetine effect on S-IRA. Peripheral atomoxetine administration at a dosage that reduces S-IRA (15mg/kg, IP) slightly increased basal ventilation and the ventilatory response to 7% CO2, but exerted no effect on heart rate in anesthetized DBA/1 mice. IP injection of atomoxetine produced no effect on the heart rate and blood pressures in conscious mice. These data suggest that atomoxetine suppresses S-IRA through direct effects on the CNS and potentially through enhanced lung ventilation in DBA/1 mice.

5-Hydroxytryptophan, a precursor for serotonin synthesis, reduces seizure-induced respiratory arrest.

OBJECTIVE: The DBA/1 mouse is a relevant animal model of sudden unexpected death in epilepsy (SUDEP), as it exhibits seizure-induced respiratory arrest (S-IRA) evoked by acoustic stimulation, followed by cardiac arrhythmia and death. Defects in serotonergic neurotransmission may contribute to S-IRA. The tryptophan hydroxylase-2 (TPH2) enzyme converts L-tryptophan to 5-hydroxytryptophan (5-HTP), a precursor for central nervous system (CNS) serotonin (5-HT) synthesis; and DBA/1 mice have a polymorphism that decreases TPH2 activity. We, therefore, hypothesized that supplementation with 5-HTP may bypass TPH2 and suppress S-IRA in DBA/1 mice. METHODS: TPH2 expression was examined by Western blot in the brainstem of DBA/1 and C57BL/6J mice both with and without acoustic stimulation. Changes in breathing and cardiac electrical activity in DBA/1 and C57BL/6J mice that incurred sudden death during generalized seizures evoked by pentylenetetrazole (PTZ) were studied by plethysmography and electrocardiography. The effect of 5-HTP administration on seizure-induced mortality evoked by acoustic stimulation or by PTZ was investigated in DBA/1 mice. RESULTS: Repetitive acoustic stimulation resulted in reduced TPH2 protein in the brainstem of DBA/1 mice as compared with C57BL/6J mice. S-IRA evoked by acoustic stimulation in DBA/1 mice was significantly reduced by 5-HTP. Following S-IRA, cardiac electrical activity could be detected for minutes before terminal asystole and death in both DBA/1 and C57BL/6J mice after PTZ treatment. The incidence of S-IRA by PTZ administration was greater in DBA/1 than in C57BL/6J mice, and administration of 5-HTP also significantly reduced S-IRA by PTZ in DBA/1 mice. SIGNIFICANCE: Our data suggest that S-IRA is the primary event leading to death incurred in most DBA/1 and some C57BL/6J mice during PTZ-evoked seizures. Suppression of S-IRA by 5-HTP suggests that 5-HT transmission contributes to the pathophysiology of S-IRA, and that 5-HTP, an over-the-counter supplement available for human consumption, may be clinically useful in preventing SUDEP.

Assessing the effects of pharmacological agents on respiratory dynamics using time-series modeling.

Developing quantitative descriptions of how stimulant and depressant drugs affect the respiratory system is an important focus in medical research. Respiratory variables-respiratory rate, tidal volume, and end tidal carbon dioxide-have prominent temporal dynamics that make it inappropriate to use standard hypothesis-testing methods that assume independent observations to assess the effects of these pharmacological agents. We present a polynomial signal plus autoregressive noise model for analysis of continuously recorded respiratory variables. We use a cyclic descent algorithm to maximize the conditional log likelihood of the parameters and the corrected Akaike's information criterion to choose simultaneously the orders of the polynomial and the autoregressive models. In an analysis of respiratory rates recorded from anesthetized rats before and after administration of the respiratory stimulant methylphenidate, we use the model to construct within-animal z-tests of the drug effect that take account of the time-varying nature of the mean respiratory rate and the serial dependence in rate measurements. We correct for the effect of model lack-of-fit on our inferences by also computing bootstrap confidence intervals for the average difference in respiratory rate pre- and postmethylphenidate treatment. Our time-series modeling quantifies within each animal the substantial increase in mean respiratory rate and respiratory dynamics following methylphenidate administration. This paradigm can be readily adapted to analyze the dynamics of other respiratory variables before and after pharmacologic treatments.

Potent activation of the human tandem pore domain K channel TRESK with clinical concentrations of volatile anesthetics.

The tandem pore domain K channel family mediates background K currents present in excitable cells. Currents passed by certain members of the family are enhanced by volatile anesthetics, thus suggesting a novel mechanism of anesthesia. The newest member of the family, termed TRESK (TWIK [tandem pore domain weak inward rectifying channel]-related spinal cord K channel), has not been studied for anesthetic sensitivity. We isolated the coding sequence for TRESK from human spinal cord RNA and functionally expressed it in Xenopus oocytes and transfected COS-7 cells. With both whole-cell voltage-clamp and patch-clamp recording, TRESK currents increased up to three-fold by clinical concentrations of isoflurane, halothane, sevoflurane, and desflurane. Nonanesthetics (nonimmobilizers) had no effect on TRESK. Various IV anesthetics, including etomidate, thiopental, and propofol, have a minimal effect on TRESK currents. Amide and ester local anesthetics inhibit TRESK in a concentration-dependent manner but at concentrations generally larger than those that inhibit other tandem pore domain K channels. We also determined that TRESK is found not only in spinal cord, but also in human brain RNA. These results identify TRESK as a target of volatile anesthetics and suggest a role for this background K channel in mediating the effects of inhaled anesthetics in the central nervous system.