Junichi Ogata

Ketamine suppresses proinflammatory cytokine production in human whole blood in vitro.

UNLABELLED The production of proinflammatory cytokines, such as tumor necrosis factor (TNF) a, interleukin (IL)-6, and IL-8, increases in patients with sepsis; marked production causes organ failure and septic shock. We previously reported that ketamine suppressed lipopolysaccharide (LPS)-induced TNF-alpha production in mice. However, there are no reports on the effect of ketamine on cytokine production in human whole blood. Therefore, in this study, we investigated the efficacy of ketamine on LPS-induced TNF-alpha, IL-6, and IL-8 production and recombinant human (rh) TNF-a-induced IL-6 and IL-8 production in human whole blood. After adding different doses of ketamine to whole blood, the blood was stimulated with LPS or rhTNF. After incubation, the plasma TNF-alpha activity and IL-6 and IL-8 concentrations were measured using the L929 cell cytotoxic assay or an enzyme-linked immunoassay. Ketamine significantly suppressed LPS-induced TNF-alpha production at concentrations >20 microg/mL. At concentrations >100 microg/mL, ketamine also significantly suppressed both LPS-induced and rhTNF-induced IL-6 and IL-8 production. In this study, we demonstrated that ketamine directly inhibits the production of proinflammatory cytokines such as TNF-alpha, IL-6, and IL-8 in human whole blood. IMPLICATIONS We found that ketamine suppressed lipopolysaccharide-induced tumor necrosis factor alpha, interleukin (IL)-6, and IL-8 production and recombinant human tumor necrosis factor-induced IL-6 and IL-8 production in human whole blood. Ketamine directly suppresses proinflammatory cytokine production.

Effects of Anesthetics on Mutant N-Methyl-d-Aspartate Receptors Expressed in Xenopus Oocytes

Alcohols, inhaled anesthetics, and some injectable anesthetics inhibit the function of N-methyl-d-aspartate (NMDA) receptors, but the mechanisms responsible for this inhibition are not fully understood. Recently, it was shown that ethanol inhibition of NMDA receptors was reduced by mutation of residues in the transmembrane (TM) segment 3 of the NR1 subunit (F639A) or in TM4 of the NR2A subunit (A825W), suggesting putative ethanol binding sites. We hypothesized that the actions of other anesthetics might also require these amino acids and evaluated the effects of anesthetics on the NMDA receptors expressed in Xenopus oocytes with two-electrode voltage-clamp recording. Effects of hexanol, octanol, isoflurane, halothane, chloroform, cyclopropane, 1-chloro-1,2,2-trifluorocyclobutane, and xenon were reduced or eliminated in the mutant NMDA receptors, whereas the inhibitory effects of nitrous oxide, ketamine, and benzene were not affected by these mutations. Rapid applications of glutamate and glycine by a T-tube device provided activation time constants, which suggested different properties of ketamine and isoflurane inhibition. Thus, amino acids in TM3 and TM4 are important for the actions of many anesthetics, but nitrous oxide, benzene, and ketamine seem to have distinct mechanisms for inhibition of the NMDA receptors.

The inhibitory effects of tramadol on 5-hydroxytryptamine type 2C receptors expressed in Xenopus oocytes

Although tramadol is widely available as an analgesic, its mechanism of antinociception remains unresolved. Serotonin (5-hydroxytryptamine, 5-HT) is a monoaminergic neurotransmitter that modulates numerous sensory, motor, and behavioral processes. The 5-HT type 2C receptor (5-HT2CR) is one of the major 5-HT receptor subtypes and is implicated in many important effects of 5-HT, including pain, feeding, and locomotion. In this study, we used a whole-cell voltage clamp to examine the effects of tramadol on 5-HT-induced Ca2+-activated Cl− currents mediated by 5-HT2CR expressed in Xenopus oocytes. Tramadol inhibited 5-HT-induced Cl− currents at pharmacologically relevant concentrations. The protein kinase C (PKC) inhibitor, bisindolylmaleimide I (GF109203×), did not abolish the inhibitory effects of tramadol on the 5-HT2CR-mediated events. We also studied the effects of tramadol on [3H]5-HT binding to 5-HT2CR expressed in Xenopus oocytes, and found that it inhibited the specific binding of [3H]5-HT to 5-HT2CR. Scatchard analysis of [3H]5-HT binding revealed that tramadol altered the apparent dissociation constant for binding without changing maximal binding, indicating competitive inhibition. The results suggest that tramadol inhibits 5-HT2CR function, and the mechanism of this inhibitory effect seems to involve competitive displacement of the 5-HT binding to the 5-HT2CR, rather than via activation of the PKC pathway.

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.

What is the main mechanism of tramadol

Tramadol is an analgesic that is used worldwide for pain, but its mechanisms of action have not been fully elucidated. The majority of studies to date have focused on activation of the μ-opioid receptor (μOR) and inhibition of monoamine reuptake as mechanisms of tramadol. Although it has been speculated that tramadol acts primarily through activation of the μOR, no evidence has revealed whether tramadol directly activates the μOR. During the past decade, major advances have been made in our understanding of the physiology and pharmacology of ion channels and G protein-coupled receptor (GPCR) signaling. Several studies have shown that GPCRs and ion channels are targets for tramadol. In particular, tramadol has been shown to affect GPCRs. Here, the effects of tramadol on GPCRs, monoamine transporters, and ion channels are presented with a discussion of recent research on the mechanisms of tramadol.

Landiolol for the treatment of tachyarrhythmia associated with atrial fibrillation

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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.

Kisspeptin-10 potentiates miniature excitatory postsynaptic currents in the rat supraoptic nucleus.

Kisspeptin is the natural ligand of the G protein-coupled receptor -54 and plays a major role in gonadotropin-releasing hormone secretion in the hypothalamus. Kisspeptin-10 is an endogenous derivative of kisspeptin and has 10 -amino acids. Previous studies have demonstrated that central administration of kisspeptin-10 stimulates the secretion of arginine vasopressin (AVP) in male rats. We examined the effects of kisspeptin-10 on- excitatory synaptic inputs to magnocellular neurosecretory cells (MNCs) including AVP neurons in the supraoptic nucleus (SON) by obtaining in vitro whole-cell patch-clamp recordings from slice preparations of the rat brain. The application of kisspeptin-10 (100 nM-1 μM) significantly increased the frequency of miniature excitatory postsynaptic currents (mEPSCs) in a dose-related manner without affecting the amplitude. The kisspeptin-10-induced potentiation of the mEPSCs was significantly attenuated by previous exposure to the kisspeptin receptor antagonist kisspeptin-234 (100 nM) and to the protein kinase C inhibitor bisindolylmaleimide I (20 nM). These results suggest that kisspeptin-10 participates in the regulation of synaptic inputs to the MNCs in the SON by interacting with the kisspeptin receptor.