Receptor mediation and nociceptin inhibition of bradykinin-induced plasma extravasation in the knee joint of the rat.

OBJECTIVE AND DESIGN: The aim was to investigate the signaling mechanisms and regulation of bradykinin (BK)-induced inflammation in rat knee joint. MATERIALS AND METHODS: Knee joints of anesthetized rats were perfused with BK (0.1-1.0 microM), and synovial plasma extravasation (PE) was evaluated by spectrophotometrical measurement of Evans Blue leakage. To examine the signaling pathway, B1 antagonist [des-Arg10]-HOE140 (0.1-1.0 microM) and B2 antagonist HOE140 (0.05-1.0 microM), calcitonin gene-related peptide (CGRP) antagonist CGRP8-37 (0.5-1.0 microM), prostaglandin E2 antagonist AH-6809 (0.1-1.0 microM), and histamine H1 antagonist mepyramine (0.1-1.0 microM) were used. Nociceptin (0.0001-1.0 microM) and antagonist J-113397 were tested for modulation of BK-induced PE. The analyses were compared side-by-side with 5-hydroxytryptamine-induced PE. RESULTS: BK perfusion dose-dependently induced PE, which was blocked by HOE140, CGRP8-37, AH-6809, and mepyramine. It was also inhibited by nociceptin, which could be reversed by antagonist J-113397. In contrast, 5-hydroxytryptamine-induced PE was biphasically regulated by nociceptin and was not antagonized by CGRP8-37. CONCLUSIONS: BK-induced PE is mediated by B2 receptors and may involve CGRP, prostaglandin, and histamine pathways. BK-induced PE is inhibited by nociceptin through the activation of ORL1 receptors. There are differences between BK- and 5-hydroxytryptamine-induced inflammation in signaling and modulation.

Mouse chromosome 7 harbors a quantitative trait locus for isoflurane minimum alveolar concentration.

BACKGROUND: The minimum alveolar concentration (MAC) of isoflurane is a quantitative trait because it varies continuously in a population. The location on the genome of genes or other genetic elements controlling quantiative traits is called quantitative trait loci (QTLs). In this study we sought to detect a quantitative trait locus underlying isoflurane MAC in mice. METHODS: To accomplish this, two inbred mouse strains differing in isoflurane MAC, the C57BL/6J and LP/J mouse strains, were bred through two generations to produce genetic recombination. These animals were genotyped for microsatellite markers. We also applied an independent, computational method for identifying QTL-regulating differences in isoflurane MAC. In this approach, the isoflurane MAC was measured in a panel of 19 inbred strains, and computationally searched for genomic intervals where the pattern of genetic variation, based on single nucleotide polymorphisms, correlated with the differences in isoflurane MAC among inbred strains. RESULTS AND CONCLUSIONS: Both methods of genetic analysis identified a QTL for isoflurane MAC that was located on the proximal part of mouse chromosome 7.

Do N-methyl-D-aspartate receptors mediate the capacity of inhaled anesthetics to suppress the temporal summation that contributes to minimum alveolar concentration?

Antagonism of N-methyl-d-aspartate (NMDA) receptors markedly decreases the minimum alveolar concentration (MAC) of inhaled anesthetics. To assess the importance of suppression of the temporal summation NMDA receptor component of MAC, we stimulated the tail of rats with trains of electrical pulses of varying interstimulus intervals (ISIs) and determined the inhaled anesthetic concentrations (crossover concentrations) that suppressed movement at different ISIs. The slopes of crossover concentrations versus ISIs provided a measure of temporal summation for each anesthetic. We studied five anesthetics that differ widely in their in vitro capacity to block NMDA receptors. To block NMDA receptor transmission and reveal the NMDA receptor component, the NMDA receptor antagonist, MK801, was separately added during each anesthetic. Halothane, isoflurane, and hexafluorobenzene did not appreciably suppress the NMDA receptor components of temporal summation, which contributed to 21% to 29% of MAC (P < 0.05 for each). Xenon and o-difluorobenzene suppressed these components to 8% to 0%, respectively, of MAC (neither significant), consistent with their greater NMDA receptor blocking action in vitro. NMDA receptor blockade may contribute to the MAC produced by inhaled anesthetics that potently inhibit NMDA receptors in vitro but not those that have a limited in vitro effect.

Effect of isoflurane and other potent inhaled anesthetics on minimum alveolar concentration, learning, and the righting reflex in mice engineered to express alpha1 gamma-aminobutyric acid type A receptors unresponsive to isoflurane.

BACKGROUND: Enhancement of the function of gamma-aminobutyric acid type A receptors containing the alpha1 subunit may underlie a portion of inhaled anesthetic action. To test this, the authors created gene knock-in mice harboring mutations that render the receptors insensitive to isoflurane while preserving sensitivity to halothane. METHODS: The authors recorded miniature inhibitory synaptic currents in hippocampal neurons from hippocampal slices from knock-in and wild-type mice. They also determined the minimum alveolar concentration (MAC), and the concentration at which 50% of animals lost their righting reflexes and which suppressed pavlovian fear conditioning to tone and context in both genotypes. RESULTS: Miniature inhibitory postsynaptic currents decayed more rapidly in interneurons and CA1 pyramidal cells from the knock-in mice compared with wild-type animals. Isoflurane (0.5-1 MAC) prolonged the decay phase of miniature inhibitory postsynaptic currents in neurons of the wild-type mice, but this effect was significantly reduced in neurons from knock-in mice. Halothane (1 MAC) slowed the decay of miniature inhibitory postsynaptic current in both genotypes. The homozygous knock-in mice were more resistant than wild-type controls to loss of righting reflexes induced by isoflurane and enflurane, but not to halothane. The MAC for isoflurane, desflurane, and halothane did not differ between knock-in and wild-type mice. The knock-in mice and wild-type mice did not differ in their sensitivity to isoflurane for fear conditioning. CONCLUSIONS: gamma-Aminobutyric acid type A receptors containing the alpha1 subunit participate in the inhibition of the righting reflexes by isoflurane and enflurane. They are not, however, involved in the amnestic effect of isoflurane or immobilizing actions of inhaled agents.

Administration of epinephrine does not increase learning of fear to tone in rats anesthetized with isoflurane or desflurane.

Previous reports suggest that the administration of epinephrine increases learning during deep barbiturate-chloral hydrate anesthesia in rats but not during anesthesia with 0.4% isoflurane in rabbits. We revisited this issue, using fear conditioning to a tone in rats as our experimental model for learning and memory and isoflurane and desflurane as our anesthetics. Expressed as a fraction of the minimum alveolar anesthetic concentration (MAC) preventing movement in 50% of rats, the amnestic 50% effective dose (ED(50)) for fear to tone in control rats inhaling isoflurane and injected with saline intraperitoneally (i.p.) was 0.32 +/- 0.03 MAC (mean +/- se) compared with 0.37 +/- 0.06 MAC in rats injected with 0.01 mg/kg of epinephrine i.p. and 0.38 +/- 0.03 MAC in rats injected with 0.1 mg/kg of epinephrine i.p. For desflurane, the amnestic ED(50) were 0.32 +/- 0.05 MAC in control rats receiving a saline injection i.p. versus 0.36 +/- 0.04 MAC in rats injected with 0.1 mg/kg of epinephrine i.p. We conclude that exogenous epinephrine does not decrease amnesia produced by inhaled isoflurane or desflurane, as assessed by fear conditioning to a tone in rats.

Alpha 1 subunit-containing GABA type A receptors in forebrain contribute to the effect of inhaled anesthetics on conditioned fear.

Inhaled anesthetics are believed to produce anesthesia by their actions on ion channels. Because inhaled anesthetics robustly enhance GABA A receptor (GABA(A)-R) responses to GABA, these receptors are considered prime targets of anesthetic action. However, the importance of GABA(A)-Rs and individual GABA(A)-R subunits to specific anesthetic-induced behavioral effects in the intact animal is unknown. We hypothesized that inhaled anesthetics produce amnesia, as assessed by loss of fear conditioning, by acting on the forebrain GABA(A)-Rs that harbor the alpha1 subunit. To test this, we used global knockout mice that completely lack the alpha1 subunit and forebrain-specific, conditional knockout mice that lack the alpha1 subunit only in the hippocampus, cortex, and amygdala. Both knockout mice were 75 to 145% less sensitive to the amnestic effects of the inhaled anesthetic isoflurane. These results indicate that alpha1-containing GABA(A)-Rs in the hippocampus, amygdala, and/or cortex influence the amnestic effects of inhaled anesthetics and may be an important molecular target of the drug isoflurane.

Acetylcholine receptors and thresholds for convulsions from flurothyl and 1,2-dichlorohexafluorocyclobutane.

UNLABELLED: There are acetylcholine receptors throughout the central nervous system, and they may mediate some forms and aspects of convulsive activity. Most high-affinity binding sites on nicotinic acetylcholine receptors for nicotine, cytisine, and epibatidine in the brain contain the beta2 subunit of the receptor. Transitional inhaled compounds (compounds less potent than predicted from their lipophilicity and the Meyer-Overton hypothesis) and nonimmobilizers (compounds that do not produce immobility despite a lipophilicity that suggests anesthetic qualities as predicted from the Meyer-Overton hypothesis) can produce convulsions. The nonimmobilizer flurothyl [di-(2,2,2,-trifluoroethyl)ether] blocks the action of gamma-aminobutyric acid on gamma-aminobutyric acid(A) receptors, whereas the nonimmobilizer 1,2-dichlorohexafluorocyclobutane (2N, also called F6) does not. 2N can block the action of acetylcholine on nicotinic acetylcholine receptors. We examined the relative capacities of these compounds to cause convulsions in mice having and lacking the beta2 subunit of the acetylcholine receptor. The partial pressure causing convulsions in half the mice (the 50% effective concentration [EC(50)]) was the same as in control mice. For the knockout mice, the EC(50) for flurothyl was 0.00170 +/- 0.00030 atm (mean +/- SD), and for 2N, it was 0.0345 +/- 0.0041 atm. For the control mice, the respective values were 0.00172 +/- 0.00057 atm and 0.0341 +/- 0.0048 atm. The ratio of the 2N to flurothyl EC(50) values was 20.8 +/- 3.5 for the knockout mice and 21.7 +/- 7.0 for the control mice. These results do not support the notion that acetylcholine receptors are important mediators of the capacity of 2N or flurothyl to cause convulsions. However, we also found that both nonimmobilizers inhibit rat alpha4beta2 neuronal nicotinic acetylcholine receptors at EC(50) partial pressures (0.00091 atm and 0.062 atm for flurothyl and 2N, respectively) that approximate those that produce convulsions (0.0015 atm and 0.04 atm). IMPLICATIONS: The results from the present study provide conflicting data concerning the notion that acetylcholine receptors mediate the capacity of nonimmobilizers to produce convulsions.

Halothane and isoflurane have additive minimum alveolar concentration (MAC) effects in rats.

UNLABELLED: Studies suggest that at concentrations surrounding MAC (the minimum alveolar concentration suppressing movement in 50% of subjects in response to noxious stimulation), halothane depresses dorsal horn neurons more than does isoflurane. Similarly, these anesthetics may differ in their effects on various receptors and ion channels that might be anesthetic targets. Both findings suggest that these anesthetics may have effects on movement in response to noxious stimulation that would differ from additivity, possibly producing synergism or even antagonism. We tested this possibility in 20 rats. MAC values for halothane and (separately) for isoflurane were determined in duplicate before and after testing the combination (also in duplicate; six determinations of MAC for each rat). The sum of the isoflurane and halothane MAC fractions for individual rats that produced immobility equaled 1.037 +/- 0.082 and did not differ significantly from a value of 1.00. That is, the combination of halothane and isoflurane produced immobility in response to tail clamp at concentrations consistent with simple additivity of the effects of the anesthetics. These results suggest that the immobility produced by inhaled anesthetics need not result from their capacity to suppress transmission through dorsal horn neurons. IMPLICATIONS: Despite differences in their capacities to inhibit spinal dorsal horn cells, isoflurane and halothane are additive in their ability to suppress movement in response to a noxious stimulus.

Alpha-2 adrenoreceptors probably do not mediate the immobility produced by inhaled anesthetics.

UNLABELLED: Agonism of alpha-adrenoreceptors has a powerful anesthetic result mediated, in part, by effects on the spinal cord. Alpha-adrenoreceptor agonists (e.g., dexmedetomidine) can decrease the minimum alveolar anesthetic concentration (MAC) of inhaled anesthetics (e.g., halothane) to zero, with an apparently additive interaction between halothane and dexmedetomidine. We tested whether the capacity of the inhaled anesthetic isoflurane to produce immobility in the face of noxious stimulation resulted from agonism of alpha-adrenoreceptors. MAC (the concentration required to eliminate movement in response to a noxious stimulus in 50% of subjects) of isoflurane was determined before and after intraperitoneal administration of the alpha-adrenoreceptor antagonists yohimbine and atipamezole. The doses of yohimbine and atipamezole equaled or exceeded those that reverse the ability of agonism of alpha-adrenoreceptors to decrease MAC. Smaller doses of yohimbine or atipamezole slightly increased (by 10%) the MAC of isoflurane, an increase we interpret as the result of blockade of a small amount of tonically active alpha-adrenoreceptor activity. Doses five-fold larger did not change MAC. Doses 10-fold larger decreased MAC. We conclude that alpha-adrenoreceptors do not or minimally mediate the capacity of inhaled anesthetics to produce immobility. IMPLICATIONS: Although stimulation (agonism) of alpha-2 adrenoreceptors can decrease the inhaled anesthetic concentration required to produce immobility in the face of noxious stimulation, blockade of alpha-2 adrenoreceptors minimally affects the concentration. Thus, augmentation of the effect of alpha-2 adrenoreceptors is not an appreciable part of the mechanism whereby inhaled anesthetics produce immobility.

The use of the potassium channel activator riluzole to test whether potassium channels mediate the capacity of isoflurane to produce immobility.

UNLABELLED: Inhaled anesthetics produce immobility during noxious stimulation, primarily by actions on the spinal cord. In this study, we examined whether activation of potassium channels of the KCNK subfamily alters volatile anesthetic potency. We measured the change in isoflurane minimum alveolar anesthetic concentration (MAC) during 4-h intrathecal or IV infusions of the nonspecific KCNK activator riluzole in 54 Sprague-Dawley rats. IV or intrathecal infusions of riluzole doses that did not result in permanent injury or death equally decreased isoflurane MAC. We conclude that although riluzole exhibited anesthetic effects, the similar dose response from IV or intrathecal infusion suggests systemic absorption and actions in the brain rather than the spinal cord. IMPLICATIONS: Riluzole, a drug that activates potassium channels and decreases glutamatergic neurotransmission, primarily acts on supraspinal sites to produce immobility in response to noxious stimuli. This finding does not support the hypothesis that potassium channels mediate the capacity of inhaled anesthetics to produce immobility in the face of noxious stimulation.

Spinal N-methyl-d-aspartate receptors may contribute to the immobilizing action of isoflurane.

UNLABELLED: We examined whether N-methyl-D-aspartate (NMDA) receptors influence the immobilizing effect of isoflurane by a spinal or supraspinal action. We antagonized NMDA receptors by intrathecal (IT), intracerebroventricular (ICV), and IV administration of MK 801 (a noncompetitive NMDA antagonist) and measured the decrease in isoflurane minimum alveolar anesthetic concentration (MAC). We also measured MK 801 tissue concentrations in homogenates of upper and lower spinal cord, a slice of cerebral cortex, and the whole brain. IT infusion of MK 801 decreased isoflurane MAC more potently than ICV or IV infusions. The change in MAC correlated with the MK 801 concentration in the lower part of the spinal cord (P < 0.01) but not with concentrations in supraspinal tissue. The maximal effect of IT MK 801 reached a plateau without achieving anesthesia. IV doses 270-fold larger than the largest IT dose also did not produce anesthesia in the absence of isoflurane. These results suggest that the capacity of MK 801 to decrease the MAC of isoflurane results from an effect on the spinal cord but that spinal NMDA receptors provide only partial mediation of the immobility produced by isoflurane. Because neither IT nor IV MK 801 provide complete anesthesia, these findings also call into question the notion that NMDA blockade alone suffices to produce anesthesia as defined by immobility in the face of noxious stimulation. IMPLICATIONS: Spinal cord NMDA receptors may mediate a portion of the immobilizing effect of isoflurane. Blockade of NMDA receptors in the cord by MK 801 has a MAC-sparing effect, but MK 801 does not, by itself, produce complete anesthesia.

GABA(A) receptor blockade antagonizes the immobilizing action of propofol but not ketamine or isoflurane in a dose-related manner.

UNLABELLED: The enhancing action of propofol on gamma-amino-n-butyric acid subtype A (GABA(A)) receptors purportedly underlies its anesthetic effects. However, a recent study found that a GABA(A) antagonist did not alter the capacity of propofol to depress the righting reflex. We examined whether the noncompetitive GABA(A) antagonist picrotoxin and the competitive GABA(A) antagonist gabazine affected a different anesthetic response, immobility in response to a noxious stimulus (a tail clamp in rats), produced by propofol. This effect was compared with that seen with ketamine and isoflurane. Picrotoxin increased the 50% effective dose (ED(50)) for propofol by approximately 379%; gabazine increased it by 362%, and both antagonists acted in a dose-related manner with no apparent ceiling effect (i.e., no limit). Picrotoxin maximally increased the ED(50) for ketamine by approximately 40%-50%, whereas gabazine increased it by 50%-60%. The isoflurane minimum alveolar anesthetic concentration increased by approximately 60% with the picrotoxin and 70% with the gabazine infusion. The ED(50) for propofol was also antagonized by strychnine, a non-GABAergic glycine receptor antagonist and convulsant, to determine whether excitation of the central nervous system by a non-GABAergic mechanism could account for the increases in propofol ED(50) observed. Because strychnine only increased the immobilizing ED(50) of propofol by approximately 50%, GABA(A) receptor antagonism accounted for the results seen with picrotoxin and gabazine. We conclude that GABA(A) antagonism can influence the ED(50) for immobility of propofol and the non-GABAergic anesthetic ketamine, although to a different degree, reflecting physiologic antagonism for ketamine (i.e., an indirect effect via a modulatory effect on the neural circuitry underlying immobility) versus physiologic and pharmacologic antagonism for propofol (i.e., a direct effect by antagonism of propofol's mechanism of action). This study also suggests that the immobilizing action of isoflurane probably does not involve the GABA(A) receptor because antagonism of GABA(A) receptors for animals anesthetized with isoflurane produces results quantitatively and qualitatively similar to ketamine and markedly different from propofol. IMPLICATIONS: IV picrotoxin and gabazine antagonized the immobilizing action of propofol in a dose-related manner, whereas antagonism of the immobilizing action of ketamine and isoflurane was similar, smaller than for propofol, and not dose-related. These results are consistent with a role for gamma-amino-n-butyric acid subtype A receptors in mediating propofol anesthesia but not ketamine or isoflurane anesthesia.

Insulin decreases isoflurane minimum alveolar anesthetic concentration in rats independently of an effect on the spinal cord.

UNLABELLED: The observation that insulin supplies an element of analgesia suggests that insulin administration might decrease the concentration of inhaled anesthetic required to produce MAC (the minimum alveolar anesthetic concentration required to eliminate movement in response to noxious stimulation in 50% of subjects). We hypothesized that insulin decreases MAC by directly affecting the nervous system, by decreasing blood glucose, or both. To test these hypotheses, we infused increasing doses of insulin either intrathecally or IV in rats anesthetized with isoflurane and determined the resulting MAC change (assessing forelimb and hindlimb movement separately). Infusion of insulin produced a dose-related decrease in MAC that did not differ among groups. That is, the IV and intrathecal infusions caused similar decreases in MAC at a given infusion rate. Blood glucose concentrations were larger in the rats given insulin with 5% dextrose. However, the percentage change in MAC determined from forelimb versus hindlimb movement did not differ. For a given insulin infusion rate, MAC changes and glucose levels did not correlate with each other, except, possibly, for the most rapid infusion rate, for which smaller glucose concentrations were associated with a marginally larger decrease in MAC. Intrathecal infusions of insulin did not produce spinal cord injury. In summary, we found that insulin decreases isoflurane MAC in a dose-related manner independently of its effects on the blood concentration of glucose. The sites at which insulin acts to decrease MAC appear to be supraspinal rather than spinal. The effect may be due to a capacity of insulin to produce analgesia through an action on one or more neurotransmitter receptors. IMPLICATIONS: Intrathecal and IV insulin administration equally decrease isoflurane MAC in rats, regardless of the concentration of blood sugar. These findings indicate that although insulin decreases MAC, the decrease is not mediated by actions on the spinal cord.

Isoflurane antagonizes the capacity of flurothyl or 1,2-dichlorohexafluorocyclobutane to impair fear conditioning to context and tone.

UNLABELLED: In animals, the conventional inhaled anesthetic, isoflurane, impairs learning fear to context and fear to tone, doing so at concentrations that produce amnesia in humans. Nonimmobilizers are inhaled compounds that do not produce immobility in response to noxious stimulation, nor do they decrease the requirement for conventional inhaled anesthetics. Like isoflurane, the nonimmobilizer 1,2-dichlorohexafluorocyclobutane (2N) impairs learning at concentrations less than those predicted from its lipophilicity to produce anesthesia. The capacity of the nonimmobilizer di-(2,2,2,-trifluoroethyl) ether (flurothyl) to affect learning and memory has not been studied. Both nonimmobilizers can cause convulsions. We hypothesized that if isoflurane, 2N, and flurothyl act by the same mechanism to impair learning and memory, their effects should be additive. We found that isoflurane, 2N, and flurothyl (each, alone) impaired learning fear to context and fear to tone in rats, with the nonimmobilizers doing so at concentrations less than those that cause convulsions. (Fear was defined by freezing [volitional immobility] in the presence of the conditioned stimulus [context or tone].) However, the combination of isoflurane and 2N or flurothyl produced an antagonistic rather than an additive effect on learning, a finding in conflict with our hypothesis. And flurothyl was no less potent than 2N (at least no less potent relative to the concentration of each that produced convulsions) in its capacity to impair learning. We conclude that conventional inhaled anesthetics and nonimmobilizers impair learning and memory by different mechanisms. The basis for this impairment remains unknown. IMPLICATIONS: Conventional inhaled anesthetics and nonimmobilizers are antagonistic in their effects on learning and memory, and this finding suggests that they impair learning and memory, at least in part, by different mechanisms.