Takafumi Horishita

n-Alcohols Inhibit Voltage-Gated Na+ Channels Expressed in Xenopus Oocytes

Voltage-gated sodium channels are essential for the initiation and propagation of action potentials in excitable cells and are known as a target of local anesthetics. In addition, inhibition of sodium channels by volatile anesthetics has been proposed as a mechanism of general anesthesia. The n-alcohols produce anesthesia, and their potency increases with carbon number until a “cut-off” is reached. In this study, we examined effects of a range of n-alcohols on Nav1.2 subunits to determine the alcohol cut-off for this channel. We also studied the effect of a short-chain alcohol (ethanol) and a long-chain alcohol (octanol) on Nav1.2, Nav1.4, Nav1.6, and Nav1.8 subunits, and we investigated the effects of alcohol on channel kinetics. Ethanol and octanol inhibited sodium currents of all subunits, and the inhibition of the Nav1.2 channel by n-alcohols indicated a cut-off at nonanol. Ethanol and octanol produced open-channel block, which was more pronounced for Nav1.8 than for the other sodium channels. Inhibition of Nav1.2 was due to decreased activation and increased inactivation. These results suggest that sodium channels may have a hydrophobic binding site for n-alcohols and demonstrate the differences in the kinetic mechanisms of inhibition for n-alcohols and inhaled anesthetics.

Gargling with sodium azulene sulfonate reduces the postoperative sore throat after intubation of the trachea

Postoperative sore throat (POST) is a complication that remains to be resolved in patients undergoing endotracheal intubation. In this study, we investigated whether preoperative gargling with sodium 1,4-dimethyl-7-isopropylazulene-3-sulfonate monohydrate (sodium azulene sulfonate, Azunol) reduces POST after endotracheal intubation. Forty patients scheduled for elective surgery under general anesthesia were randomized into Azunol and control groups. In the Azunol group, patients gargled with 4 mg Azunol diluted with 100 mL tap water (40 &mgr;g/mL). In the control group, patients gargled with 100 mL of tap water. After emergence from general anesthesia, the patients with POST were counted and POST was evaluated using a verbal analog pain scale. There were no significant differences between the two groups by age, height, body weight, gender distribution, or duration of anesthesia and surgery. In the control group, 13 patients (65%) complained of POST, which remained 24 h later in nine patients (45%). In the Azunol group, five patients (25%) also complained of POST, which completely disappeared by 24 h later. The incidence of POST and verbal analog pain scale scores in the Azunol group decreased significantly compared with the control group. We demonstrated that gargling with Azunol effectively attenuated POST with no adverse reactions.

Effects of Acamprosate on Neuronal Receptors and Ion Channels Expressed in Xenopus Oocytes

BACKGROUND Acamprosate (calcium acetylhomotaurinate) has proven to be a moderately effective pharmacological adjunct for the treatment of alcoholism. However, the central nervous system mechanism by which acamprosate reduces alcohol relapse remains unclear. Here we survey a number of metabotropic receptors, ligand-gated ion channels, and voltage-gated ion channels, to determine if acamprosate has actions at these sites in the central nervous system. METHODS Xenopus oocytes were injected with cDNAs or cRNAs encoding metabotropic glutamate receptors 1 and 5, M1 muscarinic receptors, glycine alpha1 homomeric and alpha1beta1 heteromeric receptors, gamma-aminobutyric acid A (GABA(A)alpha4beta3delta, alpha4beta3gamma2s, and alpha1beta2gamma2s) receptors, vanilloid receptor 1, and various combinations of alpha and beta subunits of voltage-gated Na+ channels. Electrophysiological responses were measured using two-electrode voltage clamp parameters after activation with agonists or voltage steps (for the voltage-gated channels). Acamprosate (0.1 to 100 microM) was pre-applied for 1 minute, followed by co-application with agonist. Acamprosate was also applied with ethanol to determine if it altered ethanol responses at some of these receptors and channels. RESULTS None of the receptors or ion channels responded to acamprosate alone. Acamprosate also failed to alter the activation of receptors or channels by agonists or after activation of voltage-gated channels. There was no effect of acamprosate on ethanol responses at GABA(A)alpha1beta2gamma2s receptors or Na+ channels. CONCLUSIONS Acamprosate does not significantly modulate the function of these receptors and ion channels at clinically relevant concentrations. Thus, the clinical effectiveness of acamprosate in the treatment of alcoholism is not likely due to direct effects on these receptors or ion channels.

Gargling with povidone-iodine reduces the transport of bacteria during oral intubation

PurposeNosocomial pneumonia remains a common complication in patients undergoing endotracheal intubation. This study examined the transport of bacteria into the trachea during endotracheal intubation, and evaluated the effects of gargling with povidone-iodine on bacterial contamination of the tip of the intubation tube.MethodsIn the gargling group, patients gargled with 25 mL of povidone-iodine (2.5 mg· mL−1). In the control group, patients gargled with 25 mL of tap water. Before tracheal intubation, microorganisms were obtained from the posterior wall of the patient’s pharynx using sterile cotton swabs. After anesthesia, all patients were extubated and bacteria contaminating the tip of the tracheal tube were sampled and cultured.ResultsBefore orotracheal intubation, all 19 patients who gargled with tap water (control group) had bacterial colonization on the posterior walls of the pharynx. This group included five patients who had methicillin-resistant staphylococcus aureus (MRSA) in their nasal cavity preoperatively and MRSA was also detected in the pharynx of four patients. Bacterial colonization was observed in all 19 patients who gargled with povidone-iodine (gargling group) and four patients carried MRSA in their nasal cavity, although no MRSA was detected in the pharynx. In the control group, all the patients had bacterial colonization at the tip of the tube after extubation. Additionally, MRSA was detected in two of the four patients. In the gargling group, povidone-iodine eradicated general bacteria and MRSA colonies in the pharynx before intubation and at the tip of the tube after extubation.ConclusionGargling with povidone-iodine before oral intubation reduces the transport of bacteria into the trachea.RésuméObjectifLa pneumonie nosocomiale est une complication encore fréquente à la suite d’une intubation endotrachéale. Nous avons vérifié le transport des bactéries à l’intérieur de la trachée pendant l’intubation endotrachéale et évaluons les effets du gargarisme avec povidone iodé sur la contamination bactérienne de la pointe du tube d’intubation.MéthodeLes patients du groupe de gargarisme ont utilisé 25 mL de povidone iodé (2,5 mg· mL−1). Les patients témoins se sont gargarisés avec 25 mL d’eau du robinet. Avant l’intubation trachéale, les microorganismes ont été prélevés sur la paroi postérieure du pharynx au moyen de coton-tiges stériles. Après l’anesthésie, tous les patients ont été extubés et les bactéries de la pointe du tube trachéal ont été prélevées et mises en culture.RésultatsAvant l’intubation orotrachéale, on a détecté des bactéries sur les parois postérieures du pharynx chez les 19 patients témoins. Ce groupe comprenait cinq patients avec staphylocoque aureus résistant à la méthicilline (SARM) dans la cavité nasale avant l’opération. Le SARM a aussi été détecté dans le pharynx de quatre patients. Il y avait une colonisation bactérienne chez les 19 patients qui ont utilisé le mélange povidone iodé. On a retrouvé le SARM dans la cavité nasale de quatre patients, mais non dans le pharynx. Tous les patients témoins présentaient une colonisation bactérienne à la pointe du tube après l’extubation. De plus, le SARM a été détecté chez deux des quatre patients. Par contre, la povidone iodé a éliminé les bactéries en général et les colonies de SARM dans le pharynx avant l’intubation et à la pointe du tube après l’extubation.ConclusionLe gargarisme avec povidone iodé avant l’intubation orale réduit le transport bactérien dans la trachée.

The effects of the tramadol metabolite O-desmethyl tramadol on muscarinic receptor-induced responses in Xenopus oocytes expressing cloned M1 or M3 receptors.

O-desmethyl tramadol is one of the main metabolites of tramadol. It has been widely used clinically and has analgesic activity. Muscarinic receptors are involved in neuronal functions in the brain and autonomic nervous system, and much attention has been paid to these receptors as targets for analgesic drugs in the central nervous system. We have reported that tramadol inhibits the function of type-1 muscarinic (M1) receptors and type-3 muscarinic (M3) receptors, suggesting that muscarinic receptors are sites of action of tramadol. However, the effects of O-desmethyl tramadol on muscarinic receptor functions have not been studied in detail. In this study, we investigated the effects of O-desmethyl tramadol on M1 and M3 receptors, using the Xenopus oocyte expression system. O-desmethyl tramadol (0.1–100 &mgr;M) inhibited acetylcholine (ACh)-induced currents in oocytes expressing the M1 receptors (half-maximal inhibitory concentration [IC50] = 2 ± 0.6 &mgr;M), whereas it did not suppress ACh-induced currents in oocytes expressing the M3 receptor. Although GF109203X, a protein kinase C inhibitor, increased the ACh-induced current, it had little effect on the inhibition of ACh-induced currents by O-desmethyl tramadol in oocytes expressing M1 receptors. The inhibitory effect of O-desmethyl tramadol on M1 receptor was overcome when the concentration of ACh was increased (KD with O-desmethyl tramadol = 0.3 &mgr;M). O-desmethyl tramadol inhibited the specific binding of [3H]quinuclidinyl benzilate ([3H]QNB) to the oocytes expressed M1 receptors (IC50 = 10.1 ± 0.1 &mgr;M), whereas it did not suppress the specific binding of [3H]QNB to the oocytes expressed M3 receptors. Based on these results, O-desmethyl tramadol inhibits functions of M1 receptors but has little effect on those of M3 receptors. This study demonstrates the molecular action of O-desmethyl tramadol on the receptors and may help to explain its neural function.

The effects of volatile aromatic anesthetics on voltage-gated Na+ channels expressed in Xenopus oocytes.

BACKGROUND: Many inhaled anesthetics inhibit voltage-gated sodium channels at clinically relevant concentrations, and suppression of neurotransmitter release by these anesthetics results, at least partly, from decreased presynaptic sodium channel activity. Volatile aromatic anesthetics can inhibit N-methyl-d-aspartate (NMDA) receptor function and enhance &ggr;-amino butyric acid A receptor function, but these effects depend strongly on the chemical properties of the aromatic compounds. In the present study we tested whether diverse aromatic anesthetics consistently inhibit sodium channel function. METHODS: We studied the effect of eight aromatic anesthetics on Nav1.2 sodium channels with β1 subunits, using whole-cell, two-electrode voltage-clamp techniques in Xenopus oocytes. RESULTS: All aromatic anesthetics inhibited INa (sodium currents) at a holding potential which produce half-maximal current (V1/2) (partial depolarization); inhibition was modest with 1,3,5-trifluorobenzene (8% ± 2%), pentafluorobenzene (13% ± 2%), and hexafluorobenzene (13% ± 2%), but greater with benzene (37% ± 2%), fluorobenzene (39% ± 2%), 1,2-difluorobenzene (48% ± 2%), 1,4-difluorobenzene (31 ± 3%), and 1,2,4-trifluorobenzene (33% ± 1%). Such dichotomous effects were noted by others for NMDA and &ggr;-aminobutyric acid A receptors. Parallel, but much smaller inhibition, was found for INa at a holding potential which produced near maximal current (−90 mV) (VH-90), and hexafluorobenzene caused small (6% ± 1%) enhancement of this current. These changes in sodium channel function were correlated with effectiveness for inhibiting NMDA receptors, with lipid solubility of the compounds, with molecular volume, and with cation-&pgr; interactions. CONCLUSION: Aromatic compounds vary in their actions on the kinetics of sodium channel gating and this may underlie their variable inhibition. The range of inhibition produced by minimum alveolar anesthetic concentration concentrations of inhaled anesthetics indicates that sodium channel inhibition may underlie the action of some of these anesthetics, but not others.

The endocannabinoid anandamide inhibits voltage-gated sodium channels Nav1.2, Nav1.6, Nav1.7, and Nav1.8 in Xenopus oocytes.

BACKGROUND:Anandamide is an endocannabinoid that regulates multiple physiological functions by pharmacological actions, in a manner similar to marijuana. Recently, much attention has been paid to the analgesic effect of endocannabinoids in terms of identifying new pharmacotherapies for refractory pain management, but the mechanisms of the analgesic effects of anandamide are still obscure. Voltage-gated sodium channels are believed to play important roles in inflammatory and neuropathic pain. We investigated the effects of anandamide on 4 neuronal sodium channel &agr; subunits, Nav1.2, Nav1.6, Nav1.7, and Nav1.8, to explore the mechanisms underlying the antinociceptive effects of anandamide. METHODS:We studied the effects of anandamide on Nav1.2, Nav1.6, Nav1.7, and Nav1.8 &agr; subunits with &bgr;1 subunits by using whole-cell, 2-electrode, voltage-clamp techniques in Xenopus oocytes. RESULTS:Anandamide inhibited sodium currents of all subunits at a holding potential causing half-maximal current (V1/2) in a concentration-dependent manner. The half-maximal inhibitory concentration values for Nav1.2, Nav1.6, Nav1.7, and Nav1.8 were 17, 12, 27, and 40 &mgr;mol/L, respectively, indicating an inhibitory effect on Nav1.6, which showed the highest potency. Anandamide raised the depolarizing shift of the activation curve as well as the hyperpolarizing shift of the inactivation curve in all &agr; subunits, suggesting that sodium current inhibition was due to decreased activation and increased inactivation. Moreover, anandamide showed a use-dependent block in Nav1.2, Nav1.6, and Nav1.7 but not Nav1.8. CONCLUSION:Anandamide inhibited the function of &agr; subunits in neuronal sodium channels Nav1.2, Nav1.6, Nav1.7, and Nav1.8. These results help clarify the mechanisms of the analgesic effects of anandamide.

The Tramadol Metabolite, O-Desmethyl Tramadol, Inhibits 5-Hydroxytryptamine Type 2C Receptors Expressed in Xenopus Oocytes

Purpose: Tramadol is widely used clinically as an analgesic, yet the mechanism by which it produces antinociception remains unclear. O-Desmethyl tramadol, the main metabolite of tramadol, is a more potent analgesic than tramadol. We reported previously that tramadol inhibits the 5-hydroxytryptamine (5-HT) type 2C receptor (5-HT2CR), a G-protein-coupled receptor that is expressed widely within brain and that mediates several effects of 5-HT, including nociception, feeding, and locomotion. The effects of O-desmethyl tramadol on 5-HT2CR have not been studied. In this study, we investigated the effect of O-desmethyl tramadol on 5-HT2CR expressed in Xenopus oocytes. Methods: We examined the effect of O-desmethyl tramadol on 5-HT2CR using the Xenopus oocyte expression system. Furthermore, we investigated the effects of O-desmethyl tramadol on the binding of [3H]5-HT by 5-HT2CR. Results: O-Desmethyl tramadol, at pharmacologically relevant concentrations, inhibited 5-HT-evoked Ca2+-activated Cl– currents in oocytes that expressed 5-HT2CR. The inhibitory effect of O-desmethyl tramadol on 5-HT2CR was overcome at higher concentrations of 5-HT. Bisindolylmaleimide I (GF109203X), a protein kinase C inhibitor, increased 5-HT-evoked currents but had little effect on the inhibition of 5-HT-evoked currents by O-desmethyl tramadol. O-Desmethyl tramadol inhibited the specific binding of [3H]5-HT by 5-HT2CR expressed in oocytes. O-Desmethyl tramadol altered the apparent dissociation constant for binding of [3H]5-HT by 5-HT2CR without changing maximum binding, which indicated competitive inhibition. Conclusion: These results suggest that O-desmethyl tramadol inhibits 5-HT2CR, which provides further insight into the pharmacological properties of tramadol and O-desmethyl tramadol.

Analysis of the Effects of Halothane on Gi-Coupled Muscarinic M2 Receptor Signaling in Xenopus Oocytes Using a Chimeric Gα Protein

Metabotropic G protein-coupled receptors have recently been recognized as targets for anesthetics and analgesics. In particular, G_q-coupled receptors such as muscarinic M₁ receptors (M₁R) and 5-hydroxytryptamine (5-HT) type 2A receptors have been reported to be targets for anesthetics. Much less is known, however, about the effects of anesthetics on G_i-coupled receptors. Here we report a method to analyze functions of G_i-coupled receptors in Xenopus oocytes expressing a chimeric Gα protein. A chimeric Gα_q protein Gα_qi5, which contains carboxy-terminus five amino acids of Gα_i, enables G_i-coupled receptors to couple to Gq-coupled receptor-mediated downstream pathways such as activation of phospholipase C. We determined acetylcholine (ACh)-induced Ca²⁺-activated Cl^– currents in Xenopus oocytes coexpressing G_i-coupled muscarinic M₂receptors (M₂R) with the chimeric Gα_qi5. Although ACh did not induce any currents in oocytes expressing M₂R alone, it caused robust Cl^– currents in oocytes coexpressing M₂R with Gα_qi5. The EC₅₀ of the ACh-induced Cl^– current mediated through Gα_qi5 was 0.2 µmol/l, which was 2.2 times higher than that of the ACh-induced G protein-activated inwardly rectifying K⁺ currents activated by Gβγ subunits liberated from endogenously expressed Gα_i in Xenopus oocytes. Other G_i-coupled somatostatin type 2, 5-HT_1A and δ-opioid receptors, when coexpressed with Gα_qi5 in oocytes, also caused robust Ca²⁺-activated Cl^– currents. In oocytes coexpressing M₂R and Gα_qi5, a volatile anesthetic halothane inhibited M₂R-induced Cl^– currents in a concentration-dependent manner with the IC₅₀ of 1.1 mmol/l, suggesting that halothane inhibits M₂R-induced cellular responses at clinically relevant concentrations. Treatment with the protein kinase C inhibitor GF109203X produced a 3.5-fold enhancement of the initial Cl^– currents induced by 1 µmol/l ACh in oocytes expressing M₂R and G_qi5. The rate of halothane-induced inhibition of Cl^– currents elicited by ACh, however, was not changed in such oocytes pretreated with GF109203X. These findings suggest that halothane inhibits the M₂R-induced signaling by acting at sites other than PKC activity. Collectively these findings suggest that the use of oocyte expressing Gα_qi5 would be helpful to examine the effects of anesthetics or analgesics on the function of G_i-coupled receptors in the Xenopus oocyte expression system.

Effects of Anesthetics on the Function of Orexin-1 Receptors Expressed in Xenopus Oocytes

Neurons in the hypothalamus containing the neuropeptide orexin have been implicated in the control of sleep and wakefulness and in the pathology of narcolepsy. In this study, we investigated the effects of volatile anesthetics, ethanol and intravenous anesthetics on orexin-A-induced Ca2+-activated Cl– currents using Xenopus oocytes expressing orexin-1 receptors (OX1Rs). The volatile anesthetics isoflurane, enflurane and halothane inhibited Cl– currents elicited by 1-µmol/l orexin-A. Ethanol and the intravenous anesthetics pentobarbital and ketamine also inhibited the action of orexin-A. The inhibitory effects of all of the compounds tested were shown to be caused by the inhibition of OX1R function. These results may, at least in part, explain their hypnotic effects.