Enhancing the action of serotonin by three different mechanisms prevents spontaneous seizure-induced mortality in Dravet mice.

OBJECTIVE: Sudden unexpected death in epilepsy (SUDEP) is an underestimated complication of epilepsy. Previous studies have demonstrated that enhancement of serotonergic neurotransmission suppresses seizure-induced sudden death in evoked seizure models. However, it is unclear whether elevated serotonin (5-HT) function will prevent spontaneous seizure-induced mortality (SSIM), which is characteristic of human SUDEP. We examined the effects of 5-HT-enhancing agents that act by three different pharmacological mechanisms on SSIM in Dravet mice, which exhibit a high incidence of SUDEP, modeling human Dravet syndrome. METHODS: Dravet mice of both sexes were evaluated for spontaneous seizure characterization and changes in SSIM incidence induced by agents that enhance 5-HT-mediated neurotransmission. Fluoxetine (a selective 5-HT reuptake inhibitor), fenfluramine (a 5-HT releaser and agonist), SR 57227 (a specific 5-HT3 receptor agonist), or saline (vehicle) was intraperitoneally administered over an 8-day period in Dravet mice, and the effect of these treatments on SSIM was examined. RESULTS: Spontaneous seizures in Dravet mice generally progressed from wild running to tonic seizures with or without SSIM. Fluoxetine at 30 mg/kg, but not at 20 or 5 mg/kg, significantly reduced SSIM compared with the vehicle control. Fenfluramine at 1-10 mg/kg, but not .2 mg/kg, fully protected Dravet mice from SSIM, with all mice surviving. Compared with the vehicle control, SR 57227 at 20 mg/kg, but not at 10 or 5 mg/kg, significantly lowered SSIM. The effect of these drugs on SSIM was independent of sex. SIGNIFICANCE: Our data demonstrate that elevating serotonergic function by fluoxetine, fenfluramine, or SR 57227 significantly reduces or eliminates SSIM in Dravet mice in a sex-independent manner. These findings suggest that deficits in serotonergic neurotransmission likely play an important role in the pathogenesis of SSIM, and fluoxetine and fenfluramine, which are US Food and Drug Administration-approved medications, may potentially prevent SUDEP in at-risk patients.

A Comparison of Breathing Stimulants for Reversal of Synthetic Opioid-Induced Respiratory Depression in Conscious Rats.

Potent synthetic opioids are an important cause of death in the United States' opioid epidemic, and a breathing stimulant may have utility in treating opioid overdose. We hypothesized that sufentanil-induced respiratory depression may be reversed by breathing stimulant administration. Using nose-only plethysmography and arterial blood analysis, we compared effects of several breathing stimulants in reversing sufentanil-induced respiratory depression in conscious rats. We studied taltirelin (1 mg/kg i.v.), PKTHPP (5 mg/kg i.v.), CX717 (30 mg/kg i.v.), BIMU8 (1 mg/kg i.v.), A85380 (30 µg/kg i.v.), and 8-hydroxy-2-(di-n-propylamino)tetralin (8-OH-DPAT) (150 µg/kg i.v./i.m.) and used sufentanil (10 µg/kg i.v.). By plethysmography (in % baseline, mean ± S.E.M.), taltirelin restored ventilation in sufentanil-treated rats (from 50 ± 5% to 102 ± 8%) by increased breathing rates (from 80 ± 4% to 160 ± 12%). By arterial blood analysis, however, taltirelin did not correct hypoxia, decreased hypercarbia only after 45 minutes, and worsened metabolic acidosis (base excess from +0 ± 1 to -7 ± 1 mEq/l). Additionally, taltirelin increased exhaled carbon dioxide, an estimate of oxygen consumption, by up to 64%. PKTHPP, CX717, BIMU8, and A85380 failed to significantly change ventilation or arterial blood values in sufentanil-treated rats. 8-OH-DPAT, however, improved ventilation (from 54 ± 8% to 92 ± 10%), reversed hypercarbia (from 64 ± 6 to 47 ± 2 mmHg), and shortened time to righting from 43 ± 4 to 15 ± 1 minutes in sufentanil-treated rats placed supine. Taltirelin has limited therapeutic potential, as its ventilatory effects are offset by metabolic acidosis, possibly from increased oxygen consumption. At the doses studied, PKTHPP, CX717, BIMU8, and A85380 have limited effects in reversing sufentanil-induced respiratory depression; 8-OH-DPAT, however, warrants further study. SIGNIFICANCE STATEMENT: Respiratory depression is an important cause of death after potent synthetic opioid overdose. 8-Hydroxy-2-(di-n-propylamino)tetralin or related compounds may be useful in treating respiratory depression as caused by potent synthetic opioids.

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.

Calabadion 1 selectively reverses respiratory and central nervous system effects of fentanyl in a rat model.

BACKGROUND: We hypothesised that Calabadion 1, an acyclic cucurbit[n]uril molecular container, reverses fentanyl-induced respiratory depression and dysfunction of the CNS. METHODS: Experiments were conducted in male Sprague-Dawley rats. A constant-rate i.v. infusion of fentanyl (12.5 or 25 µg kg-1 over 15 min) was administered followed by an i.v. bolus of Calabadion 1 (0.5-200 mg kg-1) or placebo. The primary outcome was reversal of ventilatory and respiratory depression, assessed by pneumotachography and arterial blood gas analysis, respectively. Key secondary outcomes were effects on fentanyl-induced central nervous dysfunction quantified by righting reflex, balance beam test, and electromyography (EMG). RESULTS: Calabadion 1 reversed fentanyl-induced respiratory depression across the endpoints minute ventilation, pH, and Paco2 (P=0.001). Compared with placebo, Calabadion 1 dose dependently (P for trend <0.001) reversed fentanyl-induced hypoventilation {81.9 [5.1] (mean [standard error of the mean]) vs 45.5 [12.4] ml min-1; P<0.001}, acidosis (pH 7.43 [0.01] vs 7.28 [0.04]; P=0.005), and hypercarbia (Paco2 43.4 [1.6] vs 63.4 [8.1] mm Hg; P=0.018). The effective Calabadion 1 doses required to reverse respiratory depression by 50% and 90% (ED50Res and ED90Res) were 1.7 and 15.6 mg kg-1, respectively. Higher effective doses were needed for recovery of righting reflex (ED50CNS: 9.6 mg kg-1; ED90CNS: 86.1 mg kg-1), which was accelerated by Calabadion 1 (4.6 [0.3] vs 9.0 [0.7] min; P<0.001). Calabadion 1 also significantly accelerated recovery of full functional mobility and reversal of muscle rigidity. CONCLUSIONS: Calabadion 1 selectively and dose dependently reversed the respiratory system and CNS side-effects of fentanyl.

Intravenous and Intratracheal Thyrotropin Releasing Hormone and Its Analog Taltirelin Reverse Opioid-Induced Respiratory Depression in Isoflurane Anesthetized Rats.

Thyrotropin releasing hormone (TRH) is a tripeptide hormone and a neurotransmitter widely expressed in the central nervous system that regulates thyroid function and maintains physiologic homeostasis. Following injection in rodents, TRH has multiple effects including increased blood pressure and breathing. We tested the hypothesis that TRH and its long-acting analog, taltirelin, will reverse morphine-induced respiratory depression in anesthetized rats following intravenous or intratracheal (IT) administration. TRH (1 mg/kg plus 5 mg/kg/h, i.v.) and talitrelin (1 mg/kg, i.v.), when administered to rats pretreated with morphine (5 mg/kg, i.v.), increased ventilation from 50% ± 6% to 131% ± 7% and 45% ± 6% to 168% ± 13%, respectively (percent baseline; n = 4 ± S.E.M.), primarily through increased breathing rates (from 76% ± 9% to 260% ± 14% and 66% ± 8% to 318% ± 37%, respectively). By arterial blood gas analysis, morphine caused a hypoxemic respiratory acidosis with decreased oxygen and increased carbon dioxide pressures. TRH decreased morphine effects on arterial carbon dioxide pressure, but failed to impact oxygenation; taltirelin reversed morphine effects on both arterial carbon dioxide and oxygen. Both TRH and talirelin increased mean arterial blood pressure in morphine-treated rats (from 68% ± 5% to 126% ± 12% and 64% ± 7% to 116% ± 8%, respectively; n = 3 to 4). TRH, when initiated prior to morphine (15 mg/kg, i.v.), prevented morphine-induced changes in ventilation; and TRH (2 mg/kg, i.v.) rescued all four rats treated with a lethal dose of morphine (5 mg/kg/min, until apnea). Similar to intravenous administration, both TRH (5 mg/kg, IT) and taltirelin (2 mg/kg, IT) reversed morphine effects on ventilation. TRH or taltirelin may have clinical utility as an intravenous or inhaled agent to antagonize opioid-induced cardiorespiratory depression.

Halogenated Ether, Alcohol, and Alkane Anesthetics Activate TASK-3 Tandem Pore Potassium Channels Likely through a Common Mechanism.

The TWIK-related acid-sensitive potassium channel 3 (TASK-3; KCNK9) tandem pore potassium channel function is activated by halogenated anesthetics through binding at a putative anesthetic-binding cavity. To understand the pharmacologic requirements for TASK-3 activation, we studied the concentration-response of TASK-3 to several anesthetics (isoflurane, desflurane, sevoflurane, halothane, α-chloralose, 2,2,2-trichloroethanol [TCE], and chloral hydrate), to ethanol, and to a panel of halogenated methanes and alcohols. We used mutagenesis to probe the anesthetic-binding cavity as observed in a TASK-3 homology model. TASK-3 activation was quantified by Ussing chamber voltage clamp analysis. We mutagenized the residue Val-136, which lines the anesthetic-binding cavity, its flanking residues (132 to 140), and Leu-122, a pore-gating residue. The 2-halogenated ethanols activate wild-type TASK-3 with the following rank order efficacy (normalized current [95% confidence interval]): 2,2,2-tribromo-(267% [240-294]) > 2,2,2-trichloro-(215% [196-234]) > chloral hydrate (165% [161-176]) > 2,2-dichloro- > 2-chloro ≈ 2,2,2-trifluoroethanol > ethanol. Similarly, carbon tetrabromide (296% [245-346]), carbon tetrachloride (180% [163-196]), and 1,1,1,3,3,3-hexafluoropropanol (200% [194-206]) activate TASK-3, whereas the larger carbon tetraiodide and α-chloralose inhibit. Clinical agents activate TASK-3 with the following rank order efficacy: halothane (207% [202-212]) > isoflurane (169% [161-176]) > sevoflurane (164% [150-177]) > desflurane (119% [109-129]). Mutations at and near residue-136 modify TCE activation of TASK-3, and interestingly M159W, V136E, and L122D were resistant to both isoflurane and TCE activation. TASK-3 function is activated by a multiple agents and requires a halogenated substituent between ∼30 and 232 cm3/mol volume with potency increased by halogen polarizeability. Val-136 and adjacent residues may mediate anesthetic binding and stabilize an open state regulated by pore residue Leu-122. Isoflurane and TCE likely share commonalities in their mechanism of TASK-3 activation.

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.

A Novel Strategy to Reverse General Anesthesia by Scavenging with the Acyclic Cucurbit[n]uril-type Molecular Container Calabadion 2.

BACKGROUND: Calabadion 2 is a new drug-encapsulating agent. In this study, the authors aim to assess its utility as an agent to reverse general anesthesia with etomidate and ketamine and facilitate recovery. METHODS: To evaluate the effect of calabadion 2 on anesthesia recovery, the authors studied the response of rats to calabadion 2 after continuous and bolus intravenous etomidate or ketamine and bolus intramuscular ketamine administration. The authors measured electroencephalographic predictors of depth of anesthesia (burst suppression ratio and total electroencephalographic power), functional mobility impairment, blood pressure, and toxicity. RESULTS: Calabadion 2 dose-dependently reverses the effects of ketamine and etomidate on electroencephalographic predictors of depth of anesthesia, as well as drug-induced hypotension, and shortens the time to recovery of righting reflex and functional mobility. Calabadion 2 displayed low cytotoxicity in MTS-3-(4,5-dimethylthiazol-2-yl)-5-(3-carboxymethoxyphenyl)-2-(4-sulfophenyl)-2H-tetrazolium-based cell viability and adenylate kinase release cell necrosis assays, did not inhibit the human ether-à-go-go-related channel, and was not mutagenic (Ames test). On the basis of maximum tolerable dose and acceleration of righting reflex recovery, the authors calculated the therapeutic index of calabadion 2 in recovery as 16:1 (95% CI, 10 to 26:1) for the reversal of ketamine and 3:1 (95% CI, 2 to 5:1) for the reversal of etomidate. CONCLUSIONS: Calabadion 2 reverses etomidate and ketamine anesthesia in rats by chemical encapsulation at nontoxic concentrations.

Physostigmine and Methylphenidate Induce Distinct Arousal States During Isoflurane General Anesthesia in Rats.

BACKGROUND: Although emergence from general anesthesia is clinically treated as a passive process driven by the pharmacokinetics of drug clearance, agents that hasten recovery from general anesthesia may be useful for treating delayed emergence, emergence delirium, and postoperative cognitive dysfunction. Activation of central monoaminergic neurotransmission with methylphenidate has been shown to induce reanimation (active emergence) from general anesthesia. Cholinergic neurons in the brainstem and basal forebrain are also known to promote arousal. The objective of this study was to test the hypothesis that physostigmine, a centrally acting cholinesterase inhibitor, induces reanimation from isoflurane anesthesia in adult rats. METHODS: The dose-dependent effects of physostigmine on time to emergence from a standardized isoflurane general anesthetic were tested. It was then determined whether physostigmine restores righting during continuous isoflurane anesthesia. In a separate group of rats with implanted extradural electrodes, physostigmine was administered during continuous inhalation of 1.0% isoflurane, and the electroencephalogram changes were recorded. Finally, 2.0% isoflurane was used to induce burst suppression, and the effects of physostigmine and methylphenidate on burst suppression probability (BSP) were tested. RESULTS: Physostigmine delayed time to emergence from isoflurane anesthesia at doses ≥0.2 mg/kg (n = 9). During continuous isoflurane anesthesia (0.9% ± 0.1%), physostigmine did not restore righting (n = 9). Blocking the peripheral side effects of physostigmine with the coadministration of glycopyrrolate (a muscarinic antagonist that does not cross the blood-brain barrier) produced similar results (n = 9 each). However, during inhalation of 1.0% isoflurane, physostigmine shifted peak electroencephalogram power from δ (<4 Hz) to θ (4-8 Hz) in 6 of 6 rats. During continuous 2.0% isoflurane anesthesia, physostigmine induced large, statistically significant decreases in BSP in 6 of 6 rats, whereas methylphenidate did not. CONCLUSIONS: Unlike methylphenidate, physostigmine does not accelerate time to emergence from isoflurane anesthesia and does not restore righting during continuous isoflurane anesthesia. However, physostigmine consistently decreases BSP during deep isoflurane anesthesia, whereas methylphenidate does not. These findings suggest that activation of cholinergic neurotransmission during isoflurane anesthesia produces arousal states that are distinct from those induced by monoaminergic activation.

Breathing Stimulant Compounds Inhibit TASK-3 Potassium Channel Function Likely by Binding at a Common Site in the Channel Pore.

Compounds PKTHPP (1-{1-[6-(biphenyl-4-ylcarbonyl)-5,6,7,8-tetrahydropyrido[4,3-d]-pyrimidin-4-yl]piperidin-4-yl}propan-1-one), A1899 (2''-[(4-methoxybenzoylamino)methyl]biphenyl-2-carboxylic acid 2,4-difluorobenzylamide), and doxapram inhibit TASK-1 (KCNK3) and TASK-3 (KCNK9) tandem pore (K2P) potassium channel function and stimulate breathing. To better understand the molecular mechanism(s) of action of these drugs, we undertook studies to identify amino acid residues in the TASK-3 protein that mediate this inhibition. Guided by homology modeling and molecular docking, we hypothesized that PKTHPP and A1899 bind in the TASK-3 intracellular pore. To test our hypothesis, we mutated each residue in or near the predicted PKTHPP and A1899 binding site (residues 118-128 and 228-248), individually, to a negatively charged aspartate. We quantified each mutation's effect on TASK-3 potassium channel concentration response to PKTHPP. Studies were conducted on TASK-3 transiently expressed in Fischer rat thyroid epithelial monolayers; channel function was measured in an Ussing chamber. TASK-3 pore mutations at residues 122 (L122D, E, or K) and 236 (G236D) caused the IC50 of PKTHPP to increase more than 1000-fold. TASK-3 mutants L122D, G236D, L239D, and V242D were resistant to block by PKTHPP, A1899, and doxapram. Our data are consistent with a model in which breathing stimulant compounds PKTHPP, A1899, and doxapram inhibit TASK-3 function by binding at a common site within the channel intracellular pore region, although binding outside the channel pore cannot yet be excluded.

Fluoxetine prevents respiratory arrest without enhancing ventilation in DBA/1 mice.

Sudden unexpected death in epilepsy (SUDEP) is a fatal epileptic event. DBA/1 mice are a relevant animal model for the study of SUDEP, as these mice exhibit seizure-induced respiratory arrest (S-IRA) leading to death, which has been observed in patients with witnessed SUDEP. Fluoxetine, a selective serotonin (5-hydroxytryptamine or 5-HT) reuptake inhibitor (SSRI), reduces S-IRA in DBA/1 mice. Given that DBA/1 mice with S-IRA can be resuscitated using a ventilator, we hypothesized that breathing stimulants can prevent S-IRA and that fluoxetine prevents S-IRA by enhancing ventilation in these mice. Spontaneous respiratory function in anesthetized or awake DBA/1 mice was examined using noninvasive plethysmography before and after administering fluoxetine or breathing stimulants, doxapram, and 5,6,7,8-tetrahydropyrido[4,3-d]pyrimidine (PK-THPP). The effects of these drugs on S-IRA in DBA/1 mice were tested. As reported previously, systemic administration of fluoxetine reduced S-IRA in awake DBA/1 mice, but fluoxetine in anesthetized and awake DBA/1 mice did not increase basal ventilation or the ventilatory response to 7% CO2. Both doxapram and PK-THPP increased ventilation in room air and in air+7% CO2 in anesthetized DBA/1 mice. However, neither of the breathing stimulants reduced the incidence of S-IRA. Our studies confirm that fluoxetine reduces S-IRA in DBA/1 mice without enhancing basal ventilation in the absence of seizures. Although breathing stimulants increased ventilation in the absence of seizures, they were ineffective in reducing S-IRA, indicating that drug-induced increases in ventilation are insufficient to compensate for S-IRA in DBA/1 mice.

The pharmacology of cyclopropyl-methoxycarbonyl metomidate: a comparison with propofol.

BACKGROUND: Cyclopropyl-methoxycarbonyl metomidate (CPMM) is a "soft" etomidate analogue currently being developed as a propofol alternative for anesthetic induction and maintenance. METHODS: We compared the potencies of CPMM and propofol by assessing their abilities to directly activate α1(L264T)ß3γ2 gamma-aminobutyric acid type A (GABAA) receptors and induce loss of righting reflexes in tadpoles. We also measured the rates of encephalographic recovery in rats after CPMM and propofol infusions ranging in duration from 5 to 120 minutes. RESULTS: CPMM and propofol activate GABAA receptors and induce loss of righting reflexes in tadpoles with respective 50% effective concentrations (EC50s) of 3.8 ± 0.4 and 3.9 ± 0.2 µM (GABAA receptor) and 2.6 ± 0.19 and 1.3 ± 0.04 µM (tadpole). Encephalographic recovery after prolonged infusion was faster with CPMM and lacked propofol's context sensitivity. CONCLUSION: CPMM and propofol have similar potencies in GABAA receptors and tadpoles; however, CPMM provides more rapid and predictable recovery than propofol, particularly after prolonged infusion.

Adrenocortical suppression and recovery after continuous hypnotic infusion: etomidate versus its soft analogue cyclopropyl-methoxycarbonyl metomidate.

INTRODUCTION: Etomidate is no longer administered as a continuous infusion for anesthetic maintenance or sedation, because it results in profound and persistent suppression of adrenocortical steroid synthesis with potentially lethal consequences in critically ill patients. We hypothesized that rapidly metabolized soft analogues of etomidate could be developed that do not produce persistent adrenocortical dysfunction even after prolonged continuous infusion. We hope that such agents might also provide more rapid and predictable anesthetic emergence. We have developed the soft etomidate analogue cyclopropyl-methoxycarbonyl etomidate (CPMM). Upon termination of 120-minute continuous infusions, hypnotic and encephalographic recoveries occur in four minutes. The aims of this study were to assess adrenocortical function during and following 120-minute continuous infusion of CPMM and to compare the results with those obtained using etomidate. METHODS: Dexamethasone-suppressed rats were randomized into an etomidate group, CPMM group, or control group. Rats in the etomidate and CPMM groups received 120-minute continuous infusions of etomidate and CPMM, respectively. Rats in the control group received neither hypnotic. In the first study, adrenocortical function during hypnotic infusion was assessed by administering adrenocorticotropic hormone (ACTH) 90 minutes after the start of the hypnotic infusion and measuring plasma corticosterone concentrations at the end of the infusion 30 minutes later. In the second study, adrenocortical recovery following hypnotic infusion was assessed by administering ACTH every 30 minutes after infusion termination and measuring plasma corticosterone concentrations 30 minutes after each ACTH dose. RESULTS: During hypnotic infusion, ACTH-stimulated serum corticosterone concentrations were significantly lower in the CPMM and etomidate groups than in the control group (100 ± 64 ng/ml and 33 ± 32 ng/ml versus 615 ± 265 ng/ml, respectively). After hypnotic infusion, ACTH-stimulated serum corticosterone concentrations recovered to control values within 30 minutes in the CPMM group but remained suppressed relative to those in the control group for more than 3 hours in the etomidate group. CONCLUSIONS: Both CPMM and etomidate suppress adrenocortical function during continuous infusion. However, recovery occurs significantly more rapidly following infusion of CPMM.

TASK-1 (KCNK3) and TASK-3 (KCNK9) tandem pore potassium channel antagonists stimulate breathing in isoflurane-anesthetized rats.

BACKGROUND: TASK-1 and TASK-3 tandem pore potassium channel subunits provide a constitutive acidic pH- and hypoxia-inhibited potassium conductance. TASK channels are expressed in a number of tissues involved in regulation of breathing, and the TASK-1/TASK-3 heterodimer provides the predominant hypoxia-sensitive potassium conductance in carotid body type 1 glomus chemosensing cells. The carotid bodies have an important role in regulation of breathing. Doxapram is a potent TASK-1 and TASK-3 potassium channel antagonist and a carotid body and breathing stimulant. PK-THPP and A1899 are potent and selective TASK-1 and TASK-3 antagonists. I hypothesized PK-THPP and A1899 are, like doxapram, breathing stimulants. METHODS: I studied rat TASK-3 potassium channel function by Ussing chamber using Fischer rat thyroid monolayers. To quantify breathing effects, I studied male Sprague-Dawley rats spontaneously breathing 1.5% isoflurane in room air by noninvasive plethysmography and by arterial blood gas analysis. RESULTS: PK-THPP, A1899, and doxapram inhibit rat TASK-3 potassium channel function with IC50s of 42 nM (33-52), 1.6 µM (0.8-3.3), and 22 µM (18-28) (n = 4-6; 95% confidence limits). IV PK-THPP, A1899, and doxapram stimulated breathing by plethysmography with a peak change in minute ventilation relative to baseline of 84% ± 19% and 226% ± 56% (for PK-THPP at 0.5 and 5 mg/kg; mean ± SEM; n = 3-4; P < 0.05 and P < 0.001, respectively, relative to vehicle); 46% ± 2% and 236% ± 48% (for A1899 at 5 and 25 mg/kg; n = 3-4; P > 0.05 and P < 0.001, respectively); 103% ± 20% (for doxapram at 25 mg/kg; n = 4), and 33% ± 9% (for dimethylsulfoxide vehicle at 1 mL/kg; n = 4). PK-THPP and A1899, unlike doxapram, induced a profound and lasting respiratory alkalosis by arterial blood gas analysis. Thirty minutes after IV drug administration, I observed an arterial pH and carbon dioxide partial pressure of 7.62 ± 0.02 and 23 ± 0.8 mm Hg (for PK-THPP after 5 mg/kg; n = 4; P < 0.001 for both relative to vehicle), 7.49 ± 0.02 and 31 ± 2 mm Hg (for A1899 at 25 mg/kg; n = 6; P < 0.05 and 0.001, respectively), 7.43 ± 0.03 and 39 ± 4 mm Hg (for doxapram after 25 mg/kg; n = 4; P > 0.05 for both), and 7.38 ± 0.03 and 48 ± 4 mm Hg (for dimethylsulfoxide vehicle after 1 mL/kg; n = 3). CONCLUSIONS: PK-THPP and A1899 are potent rat TASK-3 antagonists and effective breathing stimulants. PK-THPP and A1899 effects on breathing were of greater magnitude and/or duration relative to that of doxapram. PK-THPP and A1899 or related compounds may have therapeutic potential for treating breathing disorders.

Carboetomidate: an analog of etomidate that interacts weakly with 11ß-hydroxylase.

BACKGROUND: Carboetomidate is a pyrrole etomidate analog that is 3 orders of magnitude less potent an inhibitor of in vitro cortisol synthesis than etomidate (an imidazole) and does not inhibit in vivo steroid production. Although carboetomidate's reduced functional effect on steroid synthesis is thought to reflect lower binding affinity to 11ß-hydroxylase, differential binding to this enzyme has never been experimentally demonstrated. In the current study, we tested the hypothesis that carboetomidate and etomidate bind with differential affinity to 11ß-hydroxylase by comparing their abilities to inhibit photoaffinity labeling of purified enzyme by a photoactivatable etomidate analog and to modify the enzyme's absorption spectrum in a way that is indicative of ligand binding. In addition, we made a preliminary exploration of the manner in which etomidate and carboetomidate might differentially interact with this site using spectroscopic methods as well as molecular modeling techniques to better understand the structural basis for their selectivity. METHODS: The ability of azi-etomidate to inhibit cortisol synthesis was tested by assessing its ability to inhibit cortisol synthesis by H295R cells. The binding affinities of etomidate and carboetomidate to 11ß-hydroxylase were compared by assessing their abilities to (1) inhibit photoincorporation of the photolabile etomidate analog [(3)H]azi-etomidate into the enzyme and (2) modify the absorption spectrum of the enzyme's heme group. In silico docking studies of etomidate, carboetomidate, and azi-etomidate binding to 11ß-hydroxylase were performed using the computer software GOLD. RESULTS: Similar to etomidate, azi-etomidate potently inhibits in vitro cortisol synthesis. Etomidate inhibited [(3)H]azi-etomidate photolabeling of 11ß-hydroxylase in a concentration-dependent manner. At a concentration of 40 µM, etomidate reduced photoincorporation of [(3)H]azi-etomidate by 96% ± 1% whereas carboetomidate had no experimentally detectable effect. On addition of etomidate to 11ß-hydroxylase, a type 2 difference spectrum was produced indicative of etomidate complexation with the enzyme's heme iron; carboetomidate had no effect whereas azi-etomidate produced a reverse type 1 spectrum. Computer modeling studies predicted that etomidate, carboetomidate, and azi-etomidate can fit into the heme-containing pocket that forms 11ß-hydroxylase's active site and pose with their carbonyl oxygens interacting with the heme iron and their phenyl rings stacking with phenylalanine-80. However, additional unique poses were identified for etomidate and azi-etomidate that likely account for their higher affinities. CONCLUSIONS: Carboetomidate's reduced ability to suppress in vitro and in vivo steroid synthesis as compared with etomidate reflects its lower binding affinity to 11ß-hydroxylase and may be attributed to carboetomidate's inability to form a coordination bond with the heme iron located at the enzyme's active site.

Covalent modification of a volatile anesthetic regulatory site activates TASK-3 (KCNK9) tandem-pore potassium channels.

TASK-3 (KCNK9) tandem-pore potassium channels provide a volatile anesthetic-activated and Gα(q) protein- and acidic pH-inhibited potassium conductance important in neuronal excitability. Met-159 of TASK-3 is essential for anesthetic activation and may contribute to the TASK-3 anesthetic binding site(s). We hypothesized that covalent occupancy of an anesthetic binding site would irreversibly activate TASK-3. We introduced a cysteine at residue 159 (M159C) and studied the rate and effect of Cys-159 modification by N-ethylmaleimide (NEM), a cysteine-selective alkylating agent. TASK-3 channels were transiently expressed in Fischer rat thyroid cells, and their function was studied in an Ussing chamber. NEM irreversibly activated M159C TASK-3, with minimal effects on wild-type TASK-3. NEM-modified M159C channels were resistant to inhibition by both acidic pH and active Gα(q) protein. M159C channels that were first inhibited by Gα(q) protein were more-slowly activated by NEM, which suggests protection of Cys-159, and similar results were observed with isoflurane activation of wild-type TASK-3. M159W and M159F TASK-3 mutants behaved like NEM-modified M159C channels, with increased basal currents and resistance to inhibition by active Gα(q) protein or acidic pH. TASK-3 wild-type/M159C dimers expressed as a single polypeptide demonstrated that modification of a single Cys-159 was sufficient for TASK-3 activation, and M159F/M159C and M159W/M159C dimers provided evidence for cross-talk between subunits. The data are consistent with residue 159 contributing to an anesthetic regulatory site or sites, and they suggest that volatile anesthetics, through perturbations at a single site, increase TASK-3 channel activity and disrupt its regulation by active Gα(q) protein, a determinant of central nervous system arousal and consciousness.

Differential effects of etomidate and its pyrrole analogue carboetomidate on the adrenocortical and cytokine responses to endotoxemia.

OBJECTIVE: We developed a novel pyrrole analog of etomidate, (R)-ethyl 1-(1-phenylethyl)-1H-pyrrole-2-carboxylate (carboetomidate), which retains etomidate's desirable anesthetic and hemodynamic properties but lacks its potent inhibitory affect on adrenocorticotropic hormone-stimulated steroid synthesis. The objective of this study was to test the hypothesis that in contrast to etomidate, carboetomidate neither suppresses the adrenocortical response to endotoxemia nor enhances the accompanying production of proinflammatory cytokines. DESIGN: Animal study. SETTING: University research laboratory. SUBJECTS: Male Sprague-Dawley rats. INTERVENTIONS: For both single and multiple anesthetic dose studies, rats were injected with Escherichia coli lipopolysaccharide immediately followed by a hypnotic dose of etomidate, carboetomidate, or vehicle alone (dimethyl sulfoxide) as a control. For single-dose studies, no additional anesthetic (or vehicle) was administered. For multiple anesthetic dose studies, additional doses of anesthetic (or vehicle) were administered every 15 mins for a total of eight anesthetic (or vehicle) doses. MEASUREMENTS AND MAIN RESULTS: Plasma adrenocorticotropic hormone, corticosterone, and cytokine concentrations were measured before lipopolysaccharide administration and intermittently throughout the 5-hr experiment. In single anesthetic dose studies, plasma adrenocorticotropic hormone and cytokine concentrations were not different at any time point among the etomidate, carboetomidate, and vehicle groups, whereas plasma corticosterone concentrations were briefly (60-120 mins) reduced in the etomidate group. In multiple anesthetic dose studies, plasma corticosterone concentrations were persistently lower and peak plasma interleukin-1ß and interleukin-6 concentrations were higher in the etomidate group vs. the carboetomidate and control groups. Peak plasma interleukin-10 concentrations were similarly elevated in the etomidate and carboetomidate groups vs. the control group. CONCLUSIONS: Compared with etomidate, carboetomidate produces less suppression of adrenocortical function and smaller increases in proinflammatory cytokine production in an endotoxemia model of sepsis. These findings suggest that carboetomidate could be a useful alternative to etomidate for maintaining anesthesia for a prolonged period of time in patients with sepsis.

Electroencephalographic and hypnotic recoveries after brief and prolonged infusions of etomidate and optimized soft etomidate analogs.

BACKGROUND: Methoxycarbonyl etomidate is the prototypical soft etomidate analog. Because it has relatively low potency and is extremely rapidly metabolized, large quantities must be infused to maintain hypnosis. Consequently with prolonged infusion, metabolite reaches sufficient concentrations to delay recovery. Dimethyl-methoxycarbonyl metomidate (DMMM) and cyclopropyl-methoxycarbonyl metomidate (CPMM) are methoxycarbonyl etomidate analogs with higher potencies and slower clearance. Because of these properties, we hypothesized that dosing would be lower and electroencephalographic and hypnotic recoveries would be faster - and less context-sensitive - with DMMM or CPMM versus methoxycarbonyl etomidate or etomidate. METHODS: Etomidate, DMMM, and CPMM where infused into rats (n = 6 per group) for either 5 min or 120 min. After infusion termination, electroencephalographic and hypnotic recovery times were measured. The immobilizing ED50 infusion rates were determined using a tail clamp assay. RESULTS: Upon terminating 5-min infusions, electroencephalographic and hypnotic recovery times were not different among hypnotics. However, upon terminating 120-min infusions, recovery times varied significantly with respective values (mean ± SD) 48 ± 13 min and 31 ± 6.5 min (etomidate), 17 ± 7.0 min and 14 ± 3.4 min (DMMM), and 4.5 ± 1.1 min and 4.2 ± 1.6 min (CPMM). The immobilizing ED50 infusion rates were (mean ± SD) 0.19 ± 0.03 mg · kg · min (etomidate), 0.60 ± 0.12 mg · kg · min (DMMM), and 0.89 ± 0.18 mg · kg · min (CPMM). CONCLUSIONS: Electroencephalographic and hypnotic recoveries following prolonged infusions of DMMM and CPMM are faster than those following methoxycarbonyl etomidate or etomidate. In the case of CPMM infusion, recovery times are 4 min and context-insensitive.