The tramadol metabolite O-desmethyl tramadol inhibits substance P-receptor functions expressed in Xenopus oocytes

Tramadol has been widely used as analgesic. O-Desmethyl tramadol (ODT) is one of the main metabolites of tramadol, having much greater analgesic potency than tramadol itself. Substance P receptors (SPR) are well known to modulate nociceptive transmission within the spinal cord. In this study, we investigated the effects of ODT on SPR expressed in Xenopus oocytes by examining SP-induced Ca(2+)-activated Cl(-) currents. ODT inhibited the SPR-induced Cl(-) currents at pharmacologically relevant concentrations. The protein kinase C (PKC) inhibitor bisindolylmaleimide I did not abolish the inhibitory effects of ODT on SP-induced Ca(2+)-activated Cl(-) currents. The results suggest that the tramadol metabolite ODT inhibits the SPR functions, which may be independent of activation of PKC-mediated pathways.     

Interaction of the central analgesic, tramadol, with the uptake and release of 5-hydroxytryptamine in the rat brain in vitro.

1. Tramadol is a centrally acting analgesic with low opioid receptor affinity and therefore presumably other mechanisms of analgesic action. Tramadol inhibits noradrenaline uptake but since 5-hydroxytryptamine (5-HT) is also involved in the modulation of pain perception, we tested the effects of tramadol on 5-HT uptake and release in vitro. 2. Tramadol inhibited the uptake of [3H]-5-HT into purified rat frontal cortex synaptosomes with an IC50 of 3.1 microM. The (+)-enantiomer was about four times more potent than the (-)-enantiomer; the main metabolite of tramadol, O-desmethyltramadol, was about ten times less potent. 3. Rat frontal cortex slices were preincubated with [3H]-5-HT, then superfused and stimulated electrically. Tramadol facilitated the basal outflow of [3H]-5-HT, at concentrations greater than 1 microM, while the uptake inhibitor 5-nitroquipazine enhanced both basal and stimulation-evoked overflow. Effects of the (+)-enantiomer were more potent than either the racemate, the (-)-enantiomer or the principal metabolite. 4. The effects of tramadol on the basal outflow of [3H]-5-HT were almost completely abolished when the superfusion medium contained a high concentration of the selective 5-HT uptake blocker, 6-nitroquipazine. 5. The results provide evidence for an interaction of tramadol with the neuronal 5-HT transporter. An intact uptake system is necessary for the enhancement of extraneuronal 5-HT concentrations by tramadol indicating an intraneuronal site of action.     

Effects of chronic tramadol on pre- and post-synaptic measures of monoamine function

The atypical analgesic tramadol has strong structural similarities to the antidepressant venlafaxine and is a mixed noradrenaline (NA) and serotonin (5-HT) uptake inhibitor. Because tramadol has been found active in the forced swim test, a common predictor of antidepressant efficacy, we therefore examined the effects of chronic tramadol on various pre- and post-synaptic monoamine measures. Male Wistar rats (150-200 g) received tramadol (20 mg/kg i.p.) or vehicle for 21 days and were sacrificed 24 h after the last dose. Quantitative autoradiography revealed that specific frontocortical [3H]dihydroalprenolol and [3H]ketanserin binding was lower in the chronic tramadol group than controls (beta: 37+/-8 and 217+/-56 fmol/mg; 5-HT2A: 23+/-3 and 44+/-7 fmol/mg, respectively, p < 0.05). Chronic tramadol had no effect on the magnitude of electrically stimulated noradrenaline (NA) efflux or uptake in locus coeruleus (LC) slices. Although dexmedetomidine (10 nM) decreased LC NA efflux equally (by approximately 60%) in chronic tramadol and vehicle groups, desipramine (50 nM) increased LC NA efflux more in vehicle (to 164+/-7%) than tramadol-treated rats (144+/-6%; p < 0.05). Chronic tramadol had no effect on dorsal raphé (DRN) or median raphé (MRN) 5-HT efflux. However, 5-HT uptake in tramadol-treated rats was slower (p < 0.05) in MRN and nearly so (p = 0.055) in DRN. The selective 5-HT1A agonist 8-OH-DPAT reduced 5-HT efflux in both DRN and MRN. Its effect in DRN was greater in rats given chronic tramadol than in vehicle controls (54+/-2 versus 32+/-6% reduction in 5-HT efflux, respectively). In conclusion, we suggest that tramadol has many of the pre- and postsynaptic neurochemical features of a conventional antidepressant, as might be predicted from its pharmacology.     

Inhibitory effects of tramadol on nicotinic acetylcholine receptors in adrenal chromaffin cells and in Xenopus oocytes expressing alpha 7 receptors

1. Tramadol has been used clinically as an analgesic; however, the mechanism of its analgesic effects is still unknown. 2. We used bovine adrenal chromaffin cells to investigate effects of tramadol on catecholamine secretion, nicotine-induced cytosolic Ca(2+) concentration ([Ca(2+)](i)) increases and membrane current changes. We also investigated effects of tramadol on alpha7 nicotinic acetylcholine receptors (AChRs) expressed in Xenopus oocytes. 3. Tramadol concentration-dependently suppressed carbachol-induced catecholamine secretion to 60% and 27% of the control at the concentration of 10 and 100 microM, respectively, whereas it had little effect on veratridine- or high K(+)-induced catecholamine secretion. 4. Tramadol also suppressed nicotine-induced ([Ca(2+)](i)) increases in a concentration-dependent manner. Tramadol inhibited nicotine-induced inward currents, and the inhibition was unaffected by the opioid receptor antagonist naloxone. 5. Tramadol inhibited nicotinic currents carried by alpha7 receptors expressed in Xenopus oocytes. 6. Tramadol inhibited both alpha-bungarotoxin-sensitive and -insensitive nicotinic currents in bovine adrenal chromaffin cells. 7. In conclusion, tramadol inhibits catecholamine secretion partly by inhibiting nicotinic AChR functions in a naloxone-insensitive manner and alpha7 receptors are one of those inhibited by tramadol.     

Release studies with rat brain cortical synaptosomes indicate that tramadol is a 5-hydroxytryptamine uptake blocker and not a 5-hydroxytryptamine releaser.

Tramadol is a centrally acting opioid analgesic whose mechanism of action could also involve an increase in central serotoninergic transmission. Thus, tramadol inhibits synaptosomal serotonin (5-hydroxytryptamine, 5-HT) reuptake and induces tritium release from [3H]5-HT-preloaded slices. We investigated the effect of (+/-)-tramadol in release studies with superfused rat brain cortex synaptosomes preloaded with [3H]5-HT. Tramadol had no releasing effect up to 30 microM, whereas at 10 microM tramadol significantly inhibited by 45% D-fenfluramine-induced [3H]5-HT release. At 100 microM, tramadol showed a slight releasing effect in the absence or in the presence of pargyline, which was not augmented in synaptosomes pre-exposed to Ro 04-1284 (2-ethyl-1,3,4,6,7,11b-hexahydro-3-isobutyl-9,10-dimethoxy-2H-benzo [a]quinolizin-2-ol hydrochloride), a reserpine-like compound that enhances cytoplasmic 5-HT levels. In summary, (+/-)-tramadol behaved as a classical 5-HT uptake blocker (like citalopram) and not as a substrate of the 5-HT carrier with indirect 5-HT mimetic properties (like D-fenfluramine).     

Suppressive effects of isorhynchophylline on 5-HT2A receptor function in the brain: behavioural and electrophysiological studies

Isorhynchophylline is a major oxindole alkaloid found in Uncaria species which have long been used in traditional Chinese medicine. Here, we investigated the effects of isorhynchophylline and isorhynchophylline-related alkaloids on 5-hydroxytryptamine (5-HT) receptor-mediated behavioural responses in mice and 5-HT-evoked current responses in Xenopus oocytes expressing 5-HT2A or 5-HT2C receptors. Isorhynchophylline dose-dependently inhibited 5-HT2A receptor-mediated head-twitch but not 5-HT1A receptor-mediated head-weaving responses evoked by 5-methoxy-N,N-dimethyltryptamine. Pretreatment with reserpine, a monoamine-depleting agent, enhanced the head-twitching, but did not influence the effect of isorhynchophylline on the behavioural response. Isocorynoxeine, an isorhynchophylline-related alkaloid in which the configuration of the oxindole moiety is the same as in isorhynchophylline, also reduced the head-twitch response in reserpinized mice over the same dose range as isorhynchophylline, while both rhynchophylline and corynoxeine, stereoisomers of isorhynchophylline and isocorynoxeine, did not. None of the alkaloids tested had an effect on meta-chlorophenylpiperazine-induced hypolocomotion, a 5-HT2C receptor-mediated behavioural response. In experiments in vitro, isorhynchophylline and isocorynoxeine dose-dependently and competitively inhibited 5-HT-evoked currents in Xenopus oocytes expressing 5-HT2A receptors, but had less of a suppressive effect on those in oocytes expressing 5-HT2C receptors. These results indicate that isorhynchophylline and isocorynoxeine preferentially suppress 5-HT2A receptor function in the brain probably via a competitive antagonism at 5-HT2A receptor sites and that the configuration of the oxindole moiety of isorhynchophylline is essential for their antagonistic activity at the 5-HT2A receptor.     

Role of 5-HT1A and 5-HT1B receptors in the antinociceptive effect of tramadol

Tramadol, (1RS,2RS)-2-[(dimethylamine)-methyl]-1-(3-methoxyphenyl)-cyclohexanol hydrochloride, is an atypical centrally acting analgesic agent with relatively weak opioid receptor affinity and which, like some antidepressants, is able to inhibit the reuptake of serotonin (5-hydroxytryptamine, 5-HT) in the raphe nucleus. We have previously demonstrated that pindolol, a beta-adrenoceptor blocker/5-hydroxytryptamine(1A/1B) receptor antagonist, enhanced tramadol antinociception and that the selective 5-HT1A agonist 8-Hydroxy-2-(di-n-propylamino)tetralin (8-OH-DPAT) reduced it. These effects were related to the negative feedback control that regulates raphe region neurones. The current study examines the ability of the selective antagonist at somatodendritic 5-HT1A receptors, N-[2-[4-(2-methoxyphenyl)-1-piperazinyl] ethyl]-N-(2-pyridinyl) cyclohexane carboxamide (WAY100635, 0.8 mg/kg), the selective antagonist at terminal 5-HT1B receptors, N-[3-(2-dimethylamino) ethoxy-4-methoxyphenyl]-2'-methyl-4'-(5-methyl-1,2,4-oxadiazol-3-yl)-(1,1'-biphenyl)-4-carboxamide (SB216641, 0.1-0.8 mg/kg) and the selective agonist at 5-HT1B receptors, 1,4-tDihydro-3-(1,2,3,6-tetrahydro-4-pyridinyl)-5H-pyrrolo[3,2-b] pyridin-5-one (CP93129, 0.2-0.4 mg/kg), to modify the antinociceptive effect of 4-64 mg/kg of tramadol in the hot plate test in mice. The results show that 0.8 mg/kg of WAY100635 enhanced antinociceptive effect of tramadol while neither agonism nor antagonism at the 5-HT1B receptor modifies it significantly at the doses tested. These results account for involvement of the somatodendritic 5-HT1A receptors in the analgesic effect of tramadol and support the supraspinal interaction of serotonin and the opioid system in the regulation of pain.     

Competitive and non-competitive effects of 5-hydroxyindole on 5-HT3 receptors in N1E-115 neuroblastoma cells.

1. Effects of 5-hydroxyindole (5-OH-indole) (the aromatic moiety of 5-hydroxytryptamine (5-HT)) on 5-HT-evoked ion current and the nature of these effects on 5-HT3 receptors have been investigated in whole-cell voltage clamp and radioligand binding experiments on cultured N1E-115 mouse neuroblastoma cells. 2. The amplitude of 10 microM 5-HT-evoked ion currents was enhanced up to 150% of the control value by increasing concentrations up to 10 mM 5-OH-indole with half maximum effect of 0.8 mM. At concentrations between 10 mM and 50 mM, 5-OH-indole blocked the 5-HT-evoked ion current. Both the enhancement and the block by 5-OH-indole were accompanied by a marked slowing of the kinetics of decay of the 5-HT-evoked inward currents. 3. The blocking effect was surmounted when the 5-HT concentration was raised from 10 microM to 100 microM. Conversely, the increase in amplitude and the slowing of the decay of the 5-HT-evoked ion current induced by 1 mM 5-OH-indole were not reversed by the same increase of 5-HT concentration. 4. The binding of the selective antagonist [3H]-GR65630 to 5-HT3 receptors was displaced by 5-OH-indole in a concentration-dependent manner with a pKi of 1.96. In saturation binding experiments 10 mM 5-OH-indole reduced the affinity of [3H]-GR65630, whereas the total number of binding sites remained unaffected.(ABSTRACT TRUNCATED AT 250 WORDS)     

Analysis of the effects of halothane on Gi-coupled muscarinic M2 receptor signaling in Xenopus oocytes using a chimeric G alpha 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 M(1) receptors (M(1)R) 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 G alpha protein. A chimeric G alpha(q) protein G alpha(qi5), which contains carboxy-terminus five amino acids of G alpha(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 Ca(2+)-activated Cl(-) currents in Xenopus oocytes coexpressing G(i)-coupled muscarinic M(2)receptors (M(2)R) with the chimeric G alpha(qi5). Although ACh did not induce any currents in oocytes expressing M(2)R alone, it caused robust Cl(-) currents in oocytes coexpressing M(2)R with G alpha(qi5). The EC(50) of the ACh-induced Cl(-) current mediated through G alpha(qi5) was 0.2 micromol/l, which was 2.2 times higher than that of the ACh-induced G protein-activated inwardly rectifying K(+) currents activated by G beta gamma subunits liberated from endogenously expressed G alpha(i) in Xenopus oocytes. Other G(i)-coupled somatostatin type 2, 5-HT(1A) and delta-opioid receptors, when coexpressed with G alpha(qi5) in oocytes, also caused robust Ca(2+)-activated Cl(-) currents. In oocytes coexpressing M(2)R and G alpha(qi5), a volatile anesthetic halothane inhibited M(2)R-induced Cl(-) currents in a concentration-dependent manner with the IC(50) of 1.1 mmol/l, suggesting that halothane inhibits M(2)R-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 micromol/l ACh in oocytes expressing M(2)R 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 M(2)R-induced signaling by acting at sites other than PKC activity. Collectively these findings suggest that the use of oocyte expressing G alpha(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.     

The interaction of trichloroethanol with murine recombinant 5-HT3 receptors.

1. The effects of ethanol, chloral hydrate and trichloroethanol upon the 5-HT3 receptor have been investigated by use of electrophysiological techniques applied to recombinant 5-HT3 receptor subunits (5-HT3R-A or 5-HT3R-As) expressed in Xenopus laevis oocytes. Additionally, the influence of trichloroethanol upon the specific binding of [3H]-granisetron to membrane preparations of HEK 293 cells stably transfected with the murine 5-HT3R-As subunit and 5-HT3 receptors endogenous to NG 108-15 cell membranes was assessed. 2. Ethanol (30-300 mM), chloral hydrate (1-30 mM) and trichloroethanol (0.3-10 mM), produced a reversible, concentration-dependent, enhancement of 5-HT-mediated currents recorded from oocytes expressing either the 5-HT3R-A, or the 5-HT3R-As subunit. 3. Trichloroethanol (5 mM) produced a parallel leftward shift of the 5-HT concentration-response curve, reducing the EC50 for 5-HT from 1 +/- 0.04 microM (n = 4) to 0.5 +/- 0.01 microM (n = 4) for oocytes expressing the 5-HT3R-A. A similar shift, from 2.1 +/- 0.05 microM (n = 11) to 1.3 +/- 0.1 microM (n = 4), was observed in oocytes expressing the 5-HT3R-As subunit. Trichloroethanol (5 mM) had little or no effect upon the maximum current produced by 5-HT for either recombinant receptor. 4. Trichloroethanol (5 mM) similarly reduced the EC50 for 2-methyl-5-HT from 13 +/- 0.4 microM (n = 4) to 4.6 +/- 0.2 microM (n = 4) and from 15 +/- 2 microM (n = 4) to 5 +/- 0.4 microM (n = 4) for oocytes expressing the 5-HT3R-A and 5-HT3R-As subunit respectively. Additionally, trichloroethanol (5 mM) produced a clear enhancement of the maximal current to 2-methyl-5-HT (expressed as a percentage of the maximal current to 5-HT) from 63 +/- 0.7% (n = 4) to 101 +/- 1.6% (n = 4) and from 9 +/- 0.2% (n = 4) to 74 +/- 2% (n = 4) for oocytes expressing the 5-HT3R-A and 5-HT3R-As subunit respectively. 5. Trichloroethanol (2.5 mM) had no effect upon the Kd, or Bmax, of specific [3H]-granisetron binding to membrane homogenates of NG 108-15 cells or HEK 293 cells. Similarly, competition for [3H]-granisetron binding by the 5-HT3 receptor antagonists ondansetron and tropisetron was unaffected. However, competition for [3H]-granisetron binding by the 5-HT3 receptor agonists, 5-HT, 2-methyl-5-HT and phenylbiguanide was enhanced by trichloroethanol (2.5 mM).(ABSTRACT TRUNCATED AT 400 WORDS)     

In vivo effect of tramadol on locus coeruleus neurons is mediated by alpha2-adrenoceptors and modulated by serotonin

Tramadol is a centrally-acting analgesic endowed with opioid, noradrenergic and serotonergic properties. Various data suggest that, in addition to its analgesic effect, tramadol may have antidepressant and anxiolytic-like effects. This study investigates, through single-unit extracellular recording techniques, the in vivo effects of tramadol on locus coeruleus (LC) neurons and its possible effects on alpha(2)-adrenoceptors, opioid receptors and the 5-HT system. Tramadol produced a dose-dependent and complete inhibition of LC activity (ED(50)=2.1mg/kg). This inhibitory effect was prevented and reversed by the selective alpha(2)-adrenoceptor antagonist, idazoxan, but not by the opioid receptor antagonist, naloxone. The inhibition of the synthesis of 5-HT by p-chlorophenylalanine and the pre-administration of the 5-HT(1A) receptor agonist, 8-OH-DPAT at 40microg/kg, caused a significant potentiation of the tramadol effect decreasing the ED(50) by 53% and 67% respectively. Lower doses of 8-OH-DPAT, of 1 and 4microg/kg, did not significantly modify the tramadol effect. In summary, the results indicate that tramadol elicits an inhibitory effect on LC neurons in vivo through alpha(2)-adrenoceptors. Moreover, this effect is modulated by the 5-HT system and particularly by 5-HT(1A) receptors.     

Multiple conformations of native and recombinant human 5-hydroxytryptamine(2a) receptors are labeled by agonists and discriminated by antagonists

We have expanded previous studies with the 5-hydroxytryptamine (5-HT)(2) receptor agonist (+/-)-1-(2,5-dimethoxy-4-[(125)I]iodophenyl)-2-aminopropane [(+/-)-[(125)I]DOI] in human brain that had shown biphasic competition curves for several 5-HT(2A) receptor antagonists by using new selective antagonists of 5-HT(2A) (MDL100,907) and 5-HT(2C) (SB242084) receptors together with ketanserin and mesulergine. Autoradiographic competition experiments were performed with these antagonists in human brain regions where (+/-)-[(125)I]DOI labels almost exclusively 5-HT(2A) receptors (frontal cortex and striosomes). Furthermore, the effect of uncoupling receptor/G protein complexes on antagonist competition was studied with guanosine-5'-(beta,gamma-imido)triphosphate [Gpp(NH)p]. Competition experiments with (+/-)-[(3)H]1-(4-bromo-2,5-dimethoxyphenil)-2-aminopropane [(+/-)-[(3)H]DOB] were also performed in membranes from Chinese hamster ovary cells (CHOFA4) expressing cloned human 5-HT(2A) receptors. In both systems, ketanserin and MDL100,907 displayed biphasic competition profiles, whereas SB242084 and mesulergine competed monophasically. In absence of antagonist, 100 microM Gpp(NH)p decreased brain (+/-)-[(125)I]DOI specific binding by 40 to 50% and (+/-)-[(3)H]DOB specific binding to CHOFA4 cells by 30%. The remaining agonist-labeled uncoupled sites were still displaced biphasically by ketanserin and MDL100,907, with unaltered affinities. Saturation experiments were performed in CHOFA4 cells. (+/-)-[(3)H]DOB labeled two sites (K(d(h))= 0.8 nM, K(d(l)) = 31.22 nM). Addition of 100 microM Gpp(NH)p resulted in a single low-affinity (K(d) = 24.44 nM) site with unchanged B(max). [(3)H]5-HT showed no specific binding to 5-HT(2A) receptors. These results conform with the extended ternary complex model of receptor action that postulates the existence of partly activated receptor conformation(s) (R*) in equilibrium with the ground (R) and the activated G protein-coupled (R*G) conformations. Thus, both in human brain and CHOFA4 cells, the agonists possibly label all three conformations and ketanserin and MDL100,907 recognize with different affinities at least two of these conformations.     

Resveratrol enhances 5-hydroxytryptamine type 3A receptor-mediated ion currents: the role of arginine 222 residue in pre-transmembrane domain I

Resveratrol, which is found in grapes, red wine, and berries, has many beneficial health effects, such as anti-cancer, neuro-protective, anti-inflammatory, and life-prolonging effects. However, the cellular mechanisms by which resveratrol acts are relatively unknown, especially in terms of possible regulation of receptors involved in synaptic transmission. 5-Hydroxytryptamine type 3A (5-HT(3A)) receptor is one of several ligand-gated ion channels involved in fast synaptic transmission. In the present study, we investigated the effect of resveratrol on mouse 5-HT(3A) receptor channel activity. 5-HT(3A) receptor was expressed in Xenopus oocytes, and the current was measured using a two-electrode voltage clamp technique. Treatment of resveratrol itself had no effect on the oocytes injected with H(2)O as well as on the oocytes injected with 5-HT(3A) receptor cRNA. In the oocytes injected with 5-HT(3A) receptor cRNA, co- or pre-treatment of resveratrol with 5-HT potentiated 5-HT-induced inward peak current (I(5-HT)) with concentration-, reversible, and voltage-independent manners. The EC(50) of resveratrol was 28.0±2.4 μM. The presence of resveratrol caused a leftward shift of 5-HT concentration-response curve. Protein kinase C (PKC) activator or inhibitor had no effect on resveratrol action on I(5-HT). Site-directed mutations of pre-transmembrane domain 1 (pre-TM1) such as R222A, R222D, R222E, R222K, and R222T abolished or attenuated resveratrol-induced enhancement of I(5-HT), indicating that resveratrol might interact with pre-TM1 of 5-HT(3A) receptor. These results indicate that resveratrol might regulate 5-HT(3A) receptor channel activity via interaction with the N-terminal domain and these results further show that resveratrol-mediated regulation of 5-HT(3A) receptor channel activity might be one of cellular mechanisms of resveratrol action.     

Effects of tramadol and O-demethyl-tramadol on human 5-HT reuptake carriers and human 5-HT3A receptors: a possible mechanism for tramadol-induced early emesis

([3H]5-HT)-uptake and patch-clamp techniques were used to study the actions of (+) and (-) tramadol and the active metabolites of tramadol, (+) and (-) O-demethyl-tramadol on the human serotonin (5-HT) transporter and the human 5-HT3A receptor, stably expressed in HEK-293 cells. The (+) and (-) enantiomers of tramadol suppressed the human 5-HT transporter concentration-dependently (IC50=1.0 and 0.8 microM, respectively), resulting in 97% and 87% transport inhibition at their respective initial plasma concentrations (9.5 microM). The (+) and (-) enantiomers of the active tramadol metabolite were less potent than tramadol in inhibiting the human 5-HT transporter (IC50=15 and 44 microM, respectively), resulting in 19.2% and 4.8% transport inhibition at their highest plasma concentrations (2.5 microM). In contrast to their potent suppression of the 5-HT transporter, both, (+) and (-) tramadol inhibited 5-HT (30 microM)-induced currents only at substantially higher concentrations (IC50=199 and 251 microM, respectively), resulting in only 6% and 4% inhibition at the initial maximum plasma concentration. A similar low potent inhibition of human 5-HT(3A) receptors was found for (+) and (-) O-demethyl-tramadol (IC50=158 and 63 microM, respectively). In conclusion, at clinical plasma concentrations tramadol potently suppresses the human 5-HT transporter, whereas it has only a slight effect on the human 5-HT3A receptor. The results are compatible with a possible mechanism for tramadol-induced early emesis involving the serotonergic system.     

Contribution of active and inactive states of the human 5-HT4d receptor to the functional activities of 5-HT4-receptor agonists

In the present study, binding affinities of 5-hydroxytryptamine-4 (5-HT(4)) ligands for the human 5-HT(4d) receptor were determined using the agonist [(3)H]5-HT and the selective 5-HT(4) antagonist [(3)H]GR113,808. We also compared the affinity differences between [(3)H]5-HT binding (K(H)) and [(3)H]GR113,808 binding (K(L)) with their activities as 5-HT(4) ligands. Binding studies using [(3)H]5-HT revealed that the human 5-HT(4d) receptor has two binding sites, whereas [(3)H]GR113,808 yielded a single binding site. Additionally, the number of [(3)H]5-HT binding sites decreased in the presence of guanosine-5'-O-(3-thiotriphosphate) (GTPgammaS), but the number of [(3)H]GR113,808 sites did not change. In competitive binding assays, full agonists such as 5-methoxytryptamine and tegaserod showed 2- to 8-fold higher affinities for [(3)H]5-HT binding (K(H)) than for [(3)H]GR113,808 binding (K(L)) (K(H)K(L)). Finally, partial agonists displayed similar binding affinities for both radioligands (K(H) = K(L)). These findings suggest that the equilibrium between active and inactive states of the human 5-HT(4d) receptor relies on the functional activities of 5-HT(4) ligands, and these states affect the affinities of 5-HT(4) ligands in the competitive binding assay.     

Functional analysis of human sodium-phosphate transporter 4 (NPT4/SLC17A3) polymorphisms

We analyzed the functional properties of five nonsynonymous single nucleotide polymorphisms (SNPs) in the sodium-phosphate transporter NPT4 gene (SLC17A3) using the Xenopus oocyte expression system. NPT4 variants carrying SNP V257F, G279R, or P378L exhibited reduced transport of [(14)C]para-aminohippurate, [(3)H]bumetanide, [(3)H]estrone sulfate, and [(14)C]urate, when each variant clone was expressed in the plasma membrane of oocytes. This study suggests the possibility that the genetic variation of NPT4 contributes to inter-individual differences in disposition of anionic drugs such as diuretics as well as certain endogenous organic anions such as urate.     

PSAB-OFP,a selective alpha 7 nicotinic receptor agonist,is also a potent agonist of the 5-HT3 receptor

5-Hydroxytryptamine 3 (5-HT(3)) and alpha 7 nicotinic receptors share high sequence homology and pharmacological cross-reactivity. An assessment of the potential role of alpha 7 receptors in many neurophysiological processes, and hence their therapeutic value, requires the development of selective alpha 7 receptor agonists. We used a recently reported selective alpha 7 receptor agonist, (R)-(-)-5'Phenylspiro[1-azabicyclo[2.2.2] octane-3,2'-(3'H)furo[2,3-b]pyridine (PSAB-OFP) and confirmed its activity on human recombinant alpha 7 receptors. However, PSAB-OFP also displayed high affinity binding to 5-HT(3) receptors. To assess the functional activity of PSAB-OFP on 5-HT(3) receptors we studied recombinant human 5-HT(3) receptors expressed in Xenopus oocytes, as well as native mouse 5-HT(3) receptors expressed in N1E-115 neuroblastoma cells, using whole-cell patch clamp and Ca(2+) imaging. Our results show that PSAB-OFP is an equipotent, partial agonist of both alpha 7 and 5-HT(3) receptors. We conclude that it will be necessary to identify the determinant of this overlapping pharmacology in order to develop more selective alpha 7 receptor ligands.     

Voltage-dependent inhibition of recombinant NMDA receptor-mediated currents by 5-hydroxytryptamine

The effect of 5-HT and related indolealkylamines on heteromeric recombinant NMDA receptors expressed in Xenopus oocytes was investigated using the two-electrode voltage-clamp recording technique. In the absence of external Mg(2+) ions, 5-HT inhibited NMDA receptor-mediated currents in a concentration-dependent manner. The inhibitory effect of 5-HT was independent of the NR1a and NR2 subunit combination.The inhibition of glutamate-evoked currents by 5-HT was use- and voltage-dependent. The voltage sensitivity of inhibition for NR1a+NR2 subunit combinations by 5-HT was similar, exhibiting an e-fold change per approximately 20 mV, indicating that 5-HT binds to a site deep within the membrane electric field. The inhibition of the open NMDA receptor by external Mg(2+) and 5-HT was not additive, suggesting competition between Mg(2+) and 5-HT for a binding site in the NMDA receptor channel. The concentration-dependence curves for 5-HT and 5-methoxytryptamine (5-MeOT) inhibition of NMDA receptor-mediated currents are shifted to the right in the presence of external Mg(2+). The related indolealkylamines inhibited glutamate-evoked currents with the following order of inhibitory potency: 5-MeOT=5-methyltryptamine>tryptamine>7-methyltryptamine>5-HT>tryptophan=melatonin.Taken together, these data suggest that 5-HT and related compounds can attenuate glutamate-mediated excitatory synaptic responses and may provide a basis for drug treatment of excitoxic neurodegeneration.     

The "selective" dopamine D1 receptor antagonist, SCH23390, is a potent and high efficacy agonist at cloned human serotonin2C receptors

RATIONALE: The benzazepine and "selective" dopamine D1 receptor antagonist, SCH23390 [(R)-(+)-8-chloro-2,3,4,5-tetrahydro-3-methyl-5-phenyl-1H-benzazepine-7-ol], shows significant affinity at native serotonin (5-HT)2C receptors. OBJECTIVES: We examined its functional actions at cloned human (h)5-HT2C receptors (VSV isoform) stably expressed in CHO cells. METHODS: Since 5-HT2C receptors are positively coupled to phospholipase C (PLC), their activation was determined by depletion of membrane-bound pools of pre-labelled [3H]phosphotidylinositol ([3H]PI). RESULTS: SCH23390 showed high affinity (Ki, 9.3 nM) at h5-HT2C sites and depleted [3H]PI with an EC50 of 2.6 nM. Its efficacy was equivalent to that of 5-HT. [3H]PI depletion elicited by SCH23390 was concentration-dependently abolished by the selective 5-HT2C antagonist, SB242,084, with a K(B) of 0.55 nM. Further, in the presence of a fixed concentration of SB242,084 (10 nM), the concentration-response curve for SCH23390 was shifted to the right without loss of maximal effect, yielding a K(B) of 0.57 nM. CONCLUSIONS: SCH23390 is a potent and high efficacy agonist at h5-HT2C receptors. Activation of 5-HT2C receptors by SCH23390 may contribute to its functional properties both in animals and in humans.     

Meperidine, remifentanil and tramadol but not sufentanil interact with alpha(2)-adrenoceptors in alpha(2A)-, alpha(2B)- and alpha(2C)-adrenoceptor knock out mice brain

alpha(2)-adrenoceptor agonists like clonidine or dexmedetomidine increase the sedative and analgesic actions of opioids. Furthermore opioids like meperidine show potent anti-shivering effects like alpha(2)-adrenoceptor agonists. The underlying molecular mechanisms of these effects are still poorly defined. The authors therefore studied the ability of four different opioids (meperidine, remifentanil, sufentanil and tramadol) to interact with different alpha(2)-adrenoceptor subtypes in mice lacking individual alpha(2A)-, alpha(2B)- or alpha(2C)-adrenoceptors (alpha(2)-adrenoceptor knock out (alpha(2)-AR KO) mice)). The interaction of opioids with alpha(2)-adrenoceptors was investigated by quantitative receptor autoradiography in brain slices of alpha(2A)-, alpha(2B)- or alpha(2C)-adrenoceptor deficient mice. Displacement of the radiolabelled alpha(2)-adrenoceptor agonist [(125)I]-paraiodoclonidine ([(125)I]-PIC) from alpha(2)-adrenoceptors in different brain regions by increasing opioid concentrations was measured, and binding affinity of the analysed opioids to alpha(2)-adrenoceptor subtypes in different brain regions was quantified. Meperidine, remifentanil and tramadol but not sufentanil provoked dose dependent displacement of specifically bound [(125)I]-PIC from all alpha(2)-adrenoceptor subtypes in cortex, cerebellum, medulla oblongata, thalamus, hippocampus and pons. Required concentrations of meperidine and remifentanil for [(125)I]-PIC displacement from alpha(2B)- and alpha(2C)-adrenoceptors were lower than from alpha(2A)-adrenoceptors, indicating higher binding affinity for alpha(2B)- and alpha(2C)-adrenoceptors. In contrast, [(125)I]-PIC displacement by tramadol indicated higher binding affinity to alpha(2A)-adrenoceptors than to alpha(2B)- and alpha(2C)-adrenoceptors. Our results indicate that meperidine, remifentanil and tramadol interact with alpha(2)-adrenoceptors in mouse brain showing different affinity for alpha(2A)-, alpha(2B)- and alpha(2C)-adrenoceptors. In contrast, the micro-agonist sufentanil did not show any alpha(2)-adrenoceptor interaction. These effects may have an impact on the pharmacologic actions of these opioids.