Kouichiro Minami

The Effects of Tramadol and Its Metabolite on Glycine, ??-Aminobutyric AcidA, and N-Methyl-d-Aspartate Receptors Expressed in Xenopus Oocytes

We assessed the effects of tramadol, a centrally acting analgesic, and its major metabolite, on neurotransmitter-gated ion channels. Tramadol binds to &mgr;-opioid receptors with low affinity and inhibits reuptake of monoamines in the central nervous system. These actions are believed to primarily contribute to its antinociceptive effects. However, little is known about other sites of tramadols action. We tested the effects of tramadol and its M1 metabolite (0.1–100 &mgr;M) on human recombinant neurotransmitter-gated ion channels, including glycine, γ-aminobutyric acidA (GABAA), and N-methyl-d-aspartate (NMDA) receptors, expressed in Xenopus oocytes. Tramadol and M1 metabolite did not have any effects on glycine receptors. GABAA receptors were significantly inhibited only at large concentrations (100 &mgr;M). NMDA receptors were inhibited in a concentration-dependent manner. Tramadol and M1 metabolite inhibited the glutamate-concentration response curve without changing the half-maximal effective concentration or the Hill coefficient, indicating a noncompetitive inhibition. This study suggests that glycine receptors do not provide the antinociceptive effect of tramadol and that the inhibition of GABAA receptors at large concentration might correlate with convulsions. The inhibitory effect on NMDA receptors may contribute to the antinociceptive effect of tramadol at relatively large concentrations.

Inhibitory effects of anesthetics and ethanol on muscarinic receptors expressed in Xenopus oocytes

Anesthetics (and ethanol) are known to produce amnesia as well as immobilization. Recent identification of a nonimmobilizing (nonanesthetic) agent (F6 or 1,2-dichlorohexafluorocyclobutane) that impairs learning and memory suggests that distinct mechanisms may be responsible for these two actions of anesthetic agents. Muscarinic receptors are believed to play a role in memory and learning, and we asked if a specific subtype of these receptors is affected by anesthetics as well as the new nonanesthetic. We investigated the effects of halothane, a novel halogenated anesthetic compound F3 (1-chloro-1,2,2-trifluorocyclobutane) and ethanol on acetylcholine-induced current mediated by a muscarinic m1 receptor expressed in Xenopus oocytes. We also studied the effects of halogenated nonanesthetic compounds, F6 and F8 (2,3-chlorooctafluorobutane) on muscarinic m1 receptors. Halothane, F3, F6 and ethanol inhibited muscarinic m1 receptor-induced Ca2+-dependent Cl- currents at pharmacologically relevant concentrations. F8 had no effect on acetylcholine-induced muscarinic m1 receptor function. The protein kinase C inhibitor, bisindolylmaleimide I (GF109203X), enhanced the acetylcholine-induced current and the protein kinase C activator, phorbol 12-myristate 13-acetate (PMA), inhibited this current. GF109203X abolished the inhibitory effects of halothane, F3 and ethanol on muscarinic m1 receptors but had no effect on actions of F6. These results demonstrate that anesthetics and a nonanesthetic inhibit the function of muscarinic m1 receptors and suggest activation of protein kinase C as the mechanism of action of anesthetics and ethanol on these receptors.

Potentiation of ghrelin signaling attenuates cancer anorexia – cachexia and prolongs survival

Cancer anorexia–cachexia syndrome is characterized by decreased food intake, weight loss, muscle tissue wasting and psychological distress, and this syndrome is a major source of increased morbidity and mortality in cancer patients. This study aimed to clarify the gut–brain peptides involved in the pathogenesis of the syndrome and determine effective treatment for cancer anorexia–cachexia. We show that both ghrelin insufficiency and resistance were observed in tumor-bearing rats. Corticotropin-releasing factor (CRF) decreased the plasma level of acyl ghrelin, and its receptor antagonist, α-helical CRF, increased food intake of these rats. The serotonin 2c receptor (5-HT2cR) antagonist SB242084 decreased hypothalamic CRF level and improved anorexia, gastrointestinal (GI) dysmotility and body weight loss. The ghrelin receptor antagonist (D-Lys3)-GHRP-6 worsened anorexia and hastened death in tumor-bearing rats. Ghrelin attenuated anorexia–cachexia in the short term, but failed to prolong survival, as did SB242084 administration. In addition, the herbal medicine rikkunshito improved anorexia, GI dysmotility, muscle wasting, and anxiety-related behavior and prolonged survival in animals and patients with cancer. The appetite-stimulating effect of rikkunshito was blocked by (D-Lys3)-GHRP-6. Active components of rikkunshito, hesperidin and atractylodin, potentiated ghrelin secretion and receptor signaling, respectively, and atractylodin prolonged survival in tumor-bearing rats. Our study demonstrates that the integrated mechanism underlying cancer anorexia–cachexia involves lowered ghrelin signaling due to excessive hypothalamic interactions of 5-HT with CRF through the 5-HT2cR. Potentiation of ghrelin receptor signaling may be an attractive treatment for anorexia, muscle wasting and prolong survival in patients with cancer anorexia–cachexia.

Inhibitory Effect of Adrenomedullin on Rat Mesangial Cell Mitogenesis

We investigated the effects of adrenomedullin on rat mesangial cell proliferation. Adrenomedullin (10(-10)-10(-7) M inhibited both [3H]thymidine incorporation into cultured rat mesangial cells and cell proliferation in a concentration-dependent manner. Adrenomedullin (10(-10)-10(-6) M) stimulated cyclic adenosine monophosphate accumulation in rat mesangial cells in a concentration-dependent manner. These findings suggest that adrenomedullin inhibits proliferation of rat mesangial cells probably through a cyclic adenosine monophosphate-dependent mechanism.

The inhibitory effects of tramadol on muscarinic receptor-induced responses in Xenopus oocytes expressing cloned M(3) receptors.

Tramadol is a widely used analgesic, but its mechanism of action is not completely understood. Muscarinic receptors are involved in neuronal function in the brain and autonomic nervous system, and much attention has been paid to these receptors as targets of analgesic drugs in the central nervous system. In this study, we investigated the effects of tramadol on type-3 muscarinic (M3) receptors using the Xenopus oocyte expression system. Tramadol (10 nM–100 &mgr;M) inhibited acetylcholine-induced currents in oocytes expressing M3 receptor. Although GF109203X, a protein kinase C inhibitor, increased the basal current, it had little effect on the inhibition of acetylcholine-induced currents by tramadol. Moreover, tramadol inhibited the specific binding sites of [3H]quinuclidinyl benzilate. These findings suggest that tramadol at clinically relevant concentrations inhibits M3 function via quinuclidinyl benzilate-binding sites. This may explain the modulation of neuronal function and the anticholinergic effects of tramadol.

KETAMINE INTERACTS WITH THE NORADRENALINE TRANSPORTER AT A SITE PARTLY OVERLAPPING THE DESIPRAMINE BINDING SITE

Effects of the intravenous anaesthetic ketamine on the desipramine-sensitive noradrenaline transporter (NAT) were examined in cultured bovine adrenal medullary cells and in transfected Xenopus laevis oocytes expressing the bovine NAT (bNAT). Incubation (1–3 h) of adrenal medullary cells with ketamine (10–300 µM) caused an increase in appearance of catecholamines in culture medium. Ketamine (10–1000 µM) inhibited desipramine-sensitive uptake of [3H] noradrenaline (NA) (IC50=97 µM). Saturation analysis showed that ketamine reduced Vmax of [3H]NA uptake without changing Km, indicating a non-competitive inhibition. Other inhibitors of NAT, namely cocaine and desipramine, showed a competitive inhibition of [3H]NA uptake while a derivative of ketamine, phencyclidine, showed a mixed type of inhibition. Ketamine (10–1000 µM) also inhibited the specific binding of [3H]desipramine to plasma membranes isolated from bovine adrenal medulla. Scatchard analysis of [3H]desipramine binding revealed that ketamine increased Kd without altering Bmax, indicating a competitive inhibition. In transfected Xenopus oocytes expressing the bNAT, ketamine attenuated [3H]NA uptake with a kinetic characteristic similar to that of cultured adrenal medullary cells. These findings are compatible with the idea that ketamine non-competitively inhibits the transport of NA by interacting with a site which partly overlaps the desipramine binding site on the NAT.

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.

Sites of positive allosteric modulation by neurosteroids on ionotropic γ-aminobutyric acid receptor subunits

Neurosteroids are known as allosteric modulators of ionotropic γ‐aminobutyric acid (GABA) receptors. Here, we investigated sites of positive allosteric modulation by allotetrahydrodeoxycorticosterone (5α‐THDOC) at GABA receptors using the technique of chimeragenesis and the Xenopus oocyte expression system. Our findings have demonstrated that the region from transmembrane segment (TM) 4 to the C‐terminus of the GABAA receptor α1 subunit is crucial for the action of 5α‐THDOC, but insufficient for the action of another neurosteroid allopregnanolone, suggesting that a specific region critical for neurosteroid action at GABA receptors exists in the domain between TM4 and the C‐terminus of GABA receptor subunits.

Tramadol inhibits norepinephrine transporter function at desipramine-binding sites in cultured bovine adrenal medullary cells.

Tramadol is a widely used analgesic, but its mode of action is not well understood. To study the effects of tramadol on norepinephrine transporter (NET) function, we assayed the effect of tramadol on [3H]-norepinephrine ([3H]-NE) uptake and [3H]-desipramine binding to plasma membranes isolated from bovine adrenal medulla. We then characterized [14C]-tramadol binding in cultured bovine adrenal medullary cells. Tramadol inhibited the desipramine-sensitive uptake of [3H]-NE by the cells in a concentration-dependent manner (50% inhibitory concentration = 21.5 ± 6.0 &mgr;M). Saturation analysis revealed that tramadol increased the apparent Michaelis constant of [3H]-NE uptake without changing the maximal velocity, indicating that inhibition occurred via competition for the NET (inhibition constant, Ki = 13.7 &mgr;M). Tramadol inhibited the specific binding of [3H]-desipramine to plasma membranes. Scatchard analysis of [3H]-desipramine binding revealed that tramadol increased the apparent dissociation constant (Kd) for binding without altering maximal binding, indicating competitive inhibition (Ki = 11.2 &mgr;M). The binding of [14C]-tramadol to the cells was specific and saturable, with a Kd of 18.1 ± 2.4 &mgr;M. These findings indicate that tramadol competitively inhibits NET function at desipramine-binding sites.

Inhibition by carbamazepine of various ion channels-mediated catecholamine secretion in cultured bovine adrenal medullary cells

The effects of carbamazepine (CBZ) on 22Na+ influx, 45Ca2+ influx, catecholamine secretion and cyclic GMP production were examined in cultured bovine adrenal medullary cells. 1 CBZ (40–120 μmol/l) inhibited 22Na+ influx evoked by carbachol in a concentration-dependent manner. CBZ inhibited carbachol-evoked 45Ca2+ influx and catecholamine secretion at concentrations similar to those which suppressed 22Na+ influx. 2 CBZ (4–120 μmol/l) inhibited veratridine-induced 22Na+ influx, 45Ca2+ influx and catecholamine secretion. 3 CBZ (12 or 40–120 μmol/l) suppressed 56 mmol/1 K+-evoked 45Ca2+ influx and catecholamine secretion, respectively. 4 Combination of CBZ with nitrendipine or ω-agatoxin-IVA produced further inhibition of 56 mmol/l K+ - evoked 45Ca2+ influx and catecholamine secretion, compared to the effect of CBZ alone, whereas CBZ plus ω-conotoxin-GVIA did not produce any further inhibition. 5 CBZ (40 μmol/1) attenuated the production of cyclic GMP caused by muscarine. These results suggest that CBZ at therapeutic concentrations (16–48 μmol/l: 4–12 μg/ml) inhibits catecholamine secretion by interfering with nicotinic acetylcholine receptor-associated ion channels, voltage-dependent Na+ channels and N-type voltage-dependent Ca2+ channels, and may have an antimuscarinic effect in adrenal medullary cells.