Kazuyoshi Ueta

Nitrous oxide and xenon inhibit the human (α7)5 nicotinic acetylcholine receptor expressed in Xenopus oocyte

The neuronal nicotinic acetylcholine (nACh) receptor is one of the ligand-gated ion channels that regulate the synaptic release of neurotransmitters in the central nervous system. Recently, neuronal nACh receptors have received attention as a potential target for general anesthetics because many general anesthetics inhibit their functions at clinical concentrations. Several general anesthetics are known to inhibit the homomeric (&agr;7)5 nACh receptor, a subtype of neuronal nACh receptors, but the effects of two gaseous anesthetics, nitrous oxide (N2O) and xenon (Xe), remain unknown. Using the two-electrode voltage-clamping technique, we investigated the effects of N2O and Xe at the human (&agr;7)5 nACh receptor expressed in Xenopus oocytes. At clinically relevant concentrations, N2O and Xe reversibly inhibited the ACh-induced currents of the (&agr;7)5 nACh receptor in a concen-tration-dependent manner. The inhibitory actions of both anesthetics at the (&agr;7)5 nACh receptor were noncompetitive and voltage-independent. Our results suggest that inhibition of the (&agr;7)5 nACh receptor by N2O and Xe may play a role in their anesthetic effects.

Antiallodynic and antihyperalgesic effect of milnacipran in mice with spinal nerve ligation.

BACKGROUND: The antidepressant, milnacipran, has been reported to have antinociceptive, antiallodynic, and antihyperalgesic effects. In this study, we examined the mechanisms of the antiallodynic and antihyperalgesic effects of milnacipran in a model of neuropathic pain induced by spinal nerve ligation in mice. METHODS: The fifth left lumbar nerve of male C57BL6 mice was tightly ligated. Withdrawal threshold to tactile stimulation and withdrawal latency to heat stimulation in the injured or contralateral paw was tested by using von Frey filaments and radiant heat, respectively. RESULTS: Milnacipran was administered either orally (7.5–120 mg/kg), intrathecally, intracerebroventricularly, or locally (210 ng–21 &mgr;g). Both systemic, intrathecal and intracerebroventricular milnacipran increased withdrawal threshold and withdrawal latency in nerve-ligated mice whereas local injection had no effect. Depletion of spinal serotonergic or noradrenergic neurons was achieved by use of the specific neurotoxins, 6-hydroxydopamine or 5,7-dihydroxytryptamine, applied intrathecally 3 days before evaluation of the analgesic effect of milnacipran. Spinal serotonergic and noradrenergic denervation attenuated the effect of milnacipran in sham-operated mice. In nerve-ligated mice, however, the effect of milnacipran was lost after noradrenergic denervation but not after serotonergic denervation. CONCLUSIONS: We concluded that the antiallodynic and antihyperalgesic effects of milnacipran on neuropathic pain induced by spinal nerve ligation are principally mediated through action at supraspinal and spinal sites via activation of the spinal noradrenergic system.

Local Anesthetics Have Different Mechanisms and Sites of Action at Recombinant 5-HT3 Receptors

Background and Objectives: In addition to their blockade of voltage-dependent sodium channels, the action of local anesthetics at 5-hydroxytryptamine-3 (5-HT3) receptors may be clinically relevant. Because local anesthetics have different clinical properties, we have tested the hypothesis that differences in interactions at the 5-HT3 receptor may be clinically relevant by investigating the effects of 4 local anesthetics on recombinant wild-type and 4 mutant 5-HT3A receptors. Methods: The cRNAs from human wild-type and 4 mutant 5-HT3A subunit clones were synthesized in vitro and expressed in Xenopus oocytes. Four mutant receptors were obtained by site-directed mutagenesis in the N-terminal extracellular region, which contains the agonist binding domain. Tryptophan (W) at positions 62 and 155 were replaced by tyrosine (Y) and glutamate (E) at position 101 by aspartate (D) or asparagine (N). The 2-electrode voltage clamp technique was used to measure peak currents induced by 5-HT in these receptors in the presence and absence of local anesthetics. Results: All local anesthetics inhibited 5-HT-induced currents in a dose-dependent manner in the wild-type receptor. Inhibition by procaine and tetracaine were competitive whereas those of bupivacaine and lidocaine were both noncompetitive and competitive. The 4 mutants (W62Y, W155Y, E101D, E101N) could all form functional receptors. All mutant receptors exhibited a major increase (> 10-fold) in the half-maximum inhibitory concentration for procaine. The half-maximum inhibitory concentrations of tetracaine, bupivacaine, and lidocaine in mutant receptors were increased 2- to 3-fold except that of tetracaine in W62Y receptor (6-fold). Conclusions: The ester type local anesthetics, procaine and tetracaine, may act at a different site on the 5-HT3A receptor and with a different mechanism than the amide-type local anesthetics. Clinical differences between local anesthetics may be at least partially due to differences in interactions at the 5-HT3A receptor.

In vitro inhibition of recombinant ligand-gated ion channels by high concentrations of milnacipran

AbstractRationaleTricyclic antidepressants are known to inhibit various ligand-gated ion-channel (LGIC) receptors, and some of their clinical features may be associated with this activity. The effects of milnacipran, a selective inhibitor of the reuptake of serotonin and noradrenaline, on LGIC receptors have not yet been investigated on such ion-channel receptors.ObjectivesTo determine the in vitro effect of milnacipran on four recombinant LGIC receptors, nicotinic acetylcholine, N-methyl-d-aspartate, γ-amino butyric acid (GABA) and 5-hydroxytryptamine3A receptors.MethodsReceptors were expressed in Xenopus oocytes and LGIC activity measured using a two-voltage clamp technique.ResultsThere was no interaction at the GABA receptor. The results at the other LGIC receptors showed that they could be inhibited by high concentrations of milnacipran.ConclusionsAt high concentrations, milnacipran can inhibit certain LGICs. It is, however, unlikely that these interactions have any clinical consequence under normal therapeutic conditions, since the concentrations required are considerably higher than those achieved in plasma of treated patients.

In vitro antagonism of recombinant ligand-gated ion-channel receptors by stereospecific enantiomers of bupivacaine.

Bupivacaine is a racemic mixture of S(−)- and R(+)-enantiomers. Both isomers have similar potency as local anesthetics, but the S(−)-enantiomer produces less central nervous system and cardiovascular toxicity. Local anesthetic-induced convulsion is likely to be associated with not only sodium channel but also ligand-gated ion channel. The present study investigates the direct effects of the stereoenantiomers of bupivacaine on 4 recombinant ligand-gated ion-channel receptors. Methods: The antagonist activities of the S(−)- and R(+)-enantiomers of bupivacaine were tested at the nicotinic acetylcholine, N-methyl-D-aspartate (NMDA), γ-aminobutyric acidA (GABAA), and 5-hydroxytryptamine3A (5-HT3A) receptors expressed in Xenopus oocytes using a 2-voltage clamp technique. Results: Racemic bupivacaine and its 2 enantiomers all antagonized the 4 receptors in a concentration-dependent manner. Potencies at nicotinic acetylcholine, NMDA, and 5-HT3A receptors were similar. At GABAA receptors, the potency of R(+)-bupivacaine was less than racemic bupivacaine or levobupivacaine. Conclusions: Comparison of the antagonist potencies with local concentrations obtained in clinical use suggests that bupivacaine and its enantiomers are likely to produce extensive inhibition at the nicotinic acetylcholine, NMDA, and 5-HT3A receptors but a much weaker and probably not clinically relevant effect at the GABAA receptor. It is possible that direct effects at these receptors may contribute, at least in part, to the spinal and epidural anesthesia induced by these compounds. It is unlikely, however, that the difference of the toxicity in bupivacaine enantiomers is because of the stereoselectivities of bupivacaine at ligand-gated ion-channel receptors studied.

Influence of Humidification on Comfort During Noninvasive Ventilation With a Helmet

OBJECTIVE: To evaluate optimal humidifier water temperature when using a helmet for noninvasive ventilation. METHODS: Twenty-eight healthy individuals underwent 8 cm H2O CPAP ventilation with FIO2 of 0.21 and 0.5. Each was sequentially tested in the following order: using the helmet without humidification at ambient temperature; with humidification with unheated chamber water; and with humidification with the chamber water at 31°C, 34°C, and 37°C. At each setting, after a 20 min stabilization period, measurements were taken. Comfort level at each setting was evaluated using a visual analog scale rated zero (least comfortable) to 10 (most comfortable). RESULTS: Temperature and relative and absolute humidity inside the helmet increased; however, the comfort scores significantly decreased as the humidification chamber water temperature increased. Regardless of the FIO2, statistically significantly highest comfort scores were obtained when humidification water, with and without active humidification, was at ambient temperature. Unacceptable absolute humidity was obtained only without humidification at room temperature when FIO2 was 0.5. CONCLUSIONS: With the clinical use of a helmet, for patient comfort and mucosal humidification during CPAP, the most desirable conditions are likely to be obtained by humidifying without heating, that is by leaving the water in the humidifier chamber at room temperature.