Muscarinic receptors are involved in neuronal function and are targets of analgesic drugs. We here report that tramadol inhibits type-3 muscarinic receptors function via quinuclidinyl benzilate-binding sites at clinically relevant concentrations. These findings may explain the modulation of neuronal function and the anticholinergic effects of tramadol.

.-We studied the functions of ␤␥-subunits of Gi/o protein using the Xenopus oocyte expression system. Isoproterenol (ISO) elicited cAMP production and slowly activating Cl Ϫ currents in oocytes expressing ␤2-adrenoceptor and the protein kinase A-dependent Cl , and baclofen enhanced ISO-induced cAMP levels and CFTR currents in oocytes expressing ␤2-adrenoceptor-CFTR and 5-HT1A receptor (5-HT1AR), ␦-opioid receptor, or GABAB receptor, respectively. 5-HT also enhanced pituitary adenylate cyclase activating peptide (PACAP) 38-induced cAMP levels and CFTR currents in oocytes expressing PACAP receptor, CFTR and 5-HT1AR. The 5-HT-induced enhancement of Gs-coupled receptormediated currents was abrogated by pretreatment with pertussis toxin (PTX) and coexpression of G transducin ␣ (Gt␣). The 5-HT-induced enhancement was further augmented by coexpression of the G␤␥-activated form of adenylate cyclase (AC) type II but not AC type III. Thus ␤␥-subunits of Gi/o protein contribute to the enhancement of Gs-coupled receptor-mediated responses. 5-HT and DADLE did not elicit any currents in oocytes expressing 5-HT1AR or ␦-opioid receptor alone. They elicited Ca 2ϩ -activated Cl Ϫ currents in oocytes coexpressing these receptors with the G␤␥-activated form of phospholipase C (PLC)-␤2 but not with PLC-␤1. These currents were inhibited by pretreatment with PTX and coexpression of Gt␣, suggesting that ␤␥-subunits of Gi/o protein activate PLC-␤2 and then cause intracellular Ca 2ϩ mobilization. Our results indicate that ␤␥-subunits of Gi/o protein participate in diverse intracellular signals, enhancement of Gs-coupled receptor-mediated responses, and intracellular Ca 2ϩ mobilization.

No abstract

Although a potential target site of general anesthetics is primarily the GABA A receptor, a chloride ion channel, a previous study suggested that the intravenous general anesthetic propofol attenuates the M1 muscarinic acetylcholine receptor (M1 receptor)-mediated signal transduction. In the present study, we examined the target site of propofol in M1 receptor-mediated signal transduction. Two-electrode voltage-clamp method was used in Xenopus oocytes expressing both M1 receptors and associated G protein ␣ subunits (Gq␣). Propofol inhibited M1 receptor-mediated signal transduction in a dose-dependent manner (IC 50 ϭ 50 nM). Injection of guanosine 5Ј-3-O-(thio)triphosphate (GTP␥S) into oocytes overexpressing Gq␣ was used to investigate direct effects of propofol on G protein coupled with the M1 receptor. Propofol did not affect activation of Gq␣-mediated signal transduction with the intracellular injection of GTP␥S. We also studied effects of propofol on l-[N- Effects of propofol on Gs-and Gi/o-coupled signal transduction were investigated, using oocytes expressing the ␤2 adrenoceptor (␤2 receptor)/cystic fibrosis transmembrane conductance regulator or oocytes expressing the M2 muscarinic acetylcholine receptor (M2 receptor)/Kir3.1 (a member of G protein-gated inwardly rectifying K ϩ channels). Neither ␤2 receptor-mediated nor M2 receptor-mediated signal transduction was inhibited by a relatively high concentration of propofol (50 M). These results indicate that propofol inhibits M1 receptor-mediated signal transduction by selectively disrupting interaction between the receptor and associated G protein.

Alphaxalone, a neurosteroid anesthetic, inhibited the function of muscarinic M(1) and M(3) receptors and the specific binding of [(3)H]quinuclidinyl benzilate ([(3)H]QNB) to oocytes expressing these receptors. These findings suggest that alphaxalone inhibits these receptors by interfering with the QNB binding sites.