Mutations of arginine 222 in pre-transmembrane domain I of mouse 5-HT(3A) receptor abolish 20(R)- but not 20(S)-ginsenoside Rg(3) inhibition of 5-HT-mediated ion currents

Ginsenosides, active ingredients of Panax ginseng, exist as stereoisomers depending on the position of the hydroxyl group on carbon-20; i.e. 20(R)-ginsenoside and 20(S)-ginsenoside are epimers. We previously investigated the structure-activity relationship of the ginsenoside Rg(3) stereoisomers, 20-R-protopanaxatriol-3-[O-beta-D-glucopyranosyl (1-->2)-beta-glucopyranoside], (20(R)-Rg(3)) and 20-S-protopanaxatriol-3-[O-beta-D-glucopyranosyl (1-->2)-beta-glucopyranoside], (20(S)-Rg(3)) in regulating 5-HT(3A) receptor-mediated ion currents (I(5-HT)) expressed in Xenopus oocytes and found that 20(S)-Rg(3) rather than 20(R)-Rg(3) was more stronger inhibitor of I(5-HT). In the present study, we further investigated the effects of 20(R)-Rg(3) and 20(S)-Rg(3) on mouse 5-HT(3A) receptor channel activity after site-directed mutations of 5-HT(3A) receptor facilitation site, which is located at pre-transmembrane domain I (pre-TM1). 5-HT(3A) receptor was expressed in Xenopus oocytes, and I(5-HT) was measured using two-electrode voltage clamp technique. In wild-type, both 20(R)-Rg(3) and 20(S)-Rg(3) inhibited I(5-HT) with concentration-dependent and reversible manner. Induction of 5-HT(3A) receptor facilitation by point mutations of pre-TM1 amino acid residue R222 to R222A, R222D, R222E or R222T not only decreased EC(50) values for I(5-HT) compared to wild-type but also abolished 20(R)-Rg(3)-induced inhibition of I(5-HT). Those mutations also shifted the IC(50) values by 20(S)-Rg(3) into right direction by 2- to 4-folds compared with wild-type. These results indicate that 5-HT(3A) receptor facilitation differentially affects 20(R)-Rg(3)- and 20(S)-Rg(3)-mediated 5-HT(3A) receptor channel regulation.     

Identification of ginsenoside interaction sites in 5-HT3A receptors

We previously demonstrated that 20(S)-ginsenoside Rg(3) (Rg(3)), one of the active components of Panax ginseng, non-competitively inhibits 5-HT(3A) receptor channel activity on extracellular side of the cell. Here, we sought to elucidate the molecular mechanisms underlying Rg(3)-induced 5-HT(3A) receptor regulation. We used the two-microelectrode voltage-clamp technique to investigate the effect of Rg(3) on 5-HT-mediated ion currents (I(5-HT)) in Xenopus oocytes expressing wild-type or 5-HT(3A) receptors harboring mutations in the gating pore region of transmembrane domain 2 (TM2). In oocytes expressing wild-type 5-HT(3A) receptors, Rg(3) dose-dependently inhibited peak I(5-HT) with an IC(50) of 27.6+/-4.3microM. Mutations V291A, F292A, and I295A in TM2 greatly attenuated or abolished the Rg(3)-induced inhibition of peak I(5-HT). Mutation V291A but not F292A and I295A induced constitutively active ion currents with decrease of current decay rate. Rg(3) accelerated the rate of current decay with dose-dependent manner in the presence of 5-HT. Rg(3) and TMB-8, an open channel blocker, dose-dependently inhibited constitutively active ion currents. The IC(50) values of constitutively active ion currents in V291A mutant receptor were 72.4+/-23.1 and 6.5+/-0.7microM for Rg(3) and TMB-8, respectively. Diltiazem did not prevent Rg(3)-induced inhibition of constitutively active ion currents in occlusion experiments. These results indicate that Rg(3) inhibits 5-HT(3A) receptor channel activity through interactions with residues V291, F292, and I295 in the channel gating region of TM2 and further demonstrate that Rg(3) regulates 5-HT(3A) receptor channel activity in the open state at different site(s) from those of TMB-8 and diltiazem.     

Differential regulations of quercetin and its glycosides on ligand-gated ion channels

Quercetin, one of the flavonoids, is a compound of low molecular weight found in various plants and shows a wide range of diverse neuropharmacological actions. In fruits and vegetables, quercetin exists as monomer- (quercetin-3-O-rhamnoside) (Rham1), dimer- (Rutin), or trimer-glycosides [quercetin-3-(2G-rhamnosylrutinoside)] (Rham2) at carbon-3. In the previous studies, we demonstrated that quercetin inhibits both glycine and 5-hydroxytryptamine type 3, (5-HT3A) receptor channel activities expressed in Xenopus oocytes. However, the effects of quercetin glycosides on glycine and 5-HT3A receptor channel activities are not well known. In the present study, we investigated the effects of quercetin glycosides on the human glycine alpha1 receptor and mouse 5-HT3A receptor channel activities expressed in Xenopus oocytes using a two-electrode voltage clamp technique. In oocytes expressing glycine or 5-HT3A receptors, quercetin- or its glycosides-induced inhibitions on glycine- (IGly) and 5-HT-induced current (I5-HT) were dose-dependent and reversible. Applications of quercetin and its glycosides inhibited IGly in order of quercetin>Rutin> or =Rham1>Rham2. Applications of quercetin and its glycosides inhibited I5-HT in order of Rham2> or =quercetin>Rutin=Rham1. The inhibitions of IGly by quercetin glycosides were non-competitive and voltage-sensitive, whereas the inhibitions of I5-HT by quercetin glycosides were competitive and voltage-insensitive manners. These results also indicate that quercetin glycosides might regulate the human glycine alpha1 and mouse 5-HT3A receptors with differential manners.     

Inhibitory effects of dextrorotatory morphinans on the human 5-HT(3A) receptor expressed in Xenopus oocytes: Involvement of the N-terminal domain of the 5-HT(3A) receptor

We previously developed a series of dextromethorphan (DM, 3-methoxy-17-methylmorphinan) analogs modified at positions 3 and 17 of the morphinan ring system. Recent reports have shown that DM attenuates abdominal pain caused by irritable bowel syndrome, and multidrug regimens that include DM prevent nausea/vomiting following cancer surgery. However, little is known regarding the molecular mechanisms underlying the beneficial effects of DM. Here, we investigated the effects of DM, 3 of its analogs (AM, 3-allyloxy-17-methoxymorphian; CM, 3-cyclopropyl-17-methoxymorphinan; and DF, 3-methyl-17-methylmorphinan), and 1 of its metabolites (HM, 3-methoxymorphinan) on the activity of the human 5-HT(3A) receptor channel expressed in Xenopus laevis oocytes, using the 2-microelectrode voltage clamp technique. We found that intra-oocyte injection of human 5-HT(3A) receptor cRNAs elicited an inward current (I(5-HT)) in the presence of 5-HT. Cotreatment with AM, CM, DF, DM, or HM inhibited I(5-HT) in a dose-dependent, voltage-independent, and reversible manner. The IC(50) values for AM, CM, DF, DM, and HM were 24.5±1.4, 21.5±4.2, 132.6±35.8, 181.3±23.5, and 191.3±31.5μM, respectively. The IC(50) values of AM and CM were 7-fold lower than that of DM, and mechanistic analysis revealed that DM, DF, HM, AM, and CM were competitive inhibitors of I(5-HT). Point mutations of Arg241 in the N-terminal, but not amino acids in the pore region, to other amino acid residues attenuated or abolished DM- and DM-analog-induced inhibition of I(5-HT). Together, these results demonstrated that dextrorotatory morphinans might regulate 5-HT(3A) receptor channel activity via interaction with its N-terminal domain.     

Inhibitory Effects of Quercetin on Muscle-type of Nicotinic Acetylcholine Receptor-Mediated Ion Currents Expressed in Xenopus Oocytes

The flavonoid quercetin is a low molecular weight compound generally found in apple, gingko, tomato, onion and other red-colored fruits and vegetables. Like other flavonoids, quercetin has diverse pharmacological actions. However, relatively little is known about the influence of quercetin effects in the regulation of ligand-gated ion channels. Previously, we reported that quercetin regulates subsets of nicotinic acetylcholine receptors such as α3β4, α7 and α9α10. Presently, we investigated the effects of quercetin on muscle-type of nicotinic acetylcholine receptor channel activity expressed in Xenopus oocytes after injection of cRNA encoding human fetal or adult muscle-type of nicotinic acetylcholine receptor subunits. Acetylcholine treatment elicited an inward peak current (I(ACh)) in oocytes expressing both muscle-type of nicotinic acetylcholine receptors and co-treatment of quercetin with acetylcholine inhibited I(ACh). Pre-treatment of quercetin further inhibited I(ACh) in oocytes expressing adult and fetal muscle-type nicotinic acetylcholine receptors. The inhibition of I(ACh) by quercetin was reversible and concentration-dependent. The IC(50) of quercetin was 18.9±1.2 μM in oocytes expressing adult muscle-type nicotinic acetylcholine receptor. The inhibition of I(ACh) by quercetin was voltage-independent and non-competitive. These results indicate that quercetin might regulate human muscle-type nicotinic acetylcholine receptor channel activity and that quercetin-mediated regulation of muscle-type nicotinic acetylcholine receptor might be coupled to regulation of neuromuscular junction activity.     

Automated higher-throughput compound screening on ion channel targets based on the Xenopus laevis oocyte expression system

As numerous diseases have been shown to be related to dysfunction of ion channels and neurotransmitter receptors and to affect regulatory pathways, ion channels have attracted increasing attention as a target class for drug discovery. The concomitant demand of the pharmaceutical industry for adequate electrophysiological methods to investigate drug effects on specific ion channels in secondary and safety screening has resulted in the development of electrophysiological instrumentation that allows automated monitoring of ion channel function with a higher throughput. Here we tested a fully automated screening system based on the Xenopus laevis oocyte expression system. We addressed the questions of data quality and reproducibility obtained by automated oocyte injection and two-electrode voltage-clamp (TEVC) recording using the Roboocyte (Multi Channel Systems GmbH, Reutlingen, Germany) technology compared to conventional oocyte recording. A gamma-aminobutyric acid (GABA)A-receptor subtype (alpha(1)beta(2)) was chosen as an example for a ligand-gated ion channel, and the slowly activating potassium current I(Ks) as a voltage-activated ion channel. Oocytes were injected with cDNA or cRNA via the Roboocyte injection stage. Ion channel currents were successfully recorded after 2-7 days in about 40% of the oocytes injected with GABA(A) receptor cDNA, and after 2-4 days in about 60% of the oocytes injected with KCNE1 cRNA. EC(50) values for the GABA(A) receptor and IC(50) values for blockers of I(Ks) were comparable to values obtained with conventional TEVC recording techniques. In conclusion, our results show that the Roboocyte is a valuable automated tool for oocyte injection and TEVC recording that can be used in drug screening and target validation to enhance the number of compounds and oocytes tested per day.     

Effects of ginsenosides,active components of ginseng,on nicotinic acetylcholine receptors expressed in Xenopus oocytes

We investigated the effects of ginsenosides, the active ingredient of ginseng, on neuronal or muscle-type nicotinic acetylcholine receptor channel activity expressed in Xenopus oocytes after injection of cRNA encoding bovine neuronal alpha3beta4, alpha7 or human muscle alphabetadeltavarepsilon subunits. Treatment with acetylcholine elicited an inward peak current (I(ACh)) in oocytes expressing nicotinic acetylcholine receptor subtypes. Cotreatment with ginsenoside Rg2 and acetylcholine inhibited I(ACh) in oocytes expressing with alpha3beta4 or alphabetadeltavarepsilon but not in oocytes expressing alpha7 nicotinic acetylcholine receptors. The inhibition of I(ACh) by ginsenoside Rg2 was reversible and dose-dependent. The half-inhibitory concentrations (IC50) of ginsenoside Rg2 were 60.2+/-14.1 and 15.7+/-3.5 microM in oocytes expressing alpha3beta4 and alphabetadeltavarepsilon nicotinic acetylcholine receptors, respectively. The inhibition of I(ACh) by ginsenoside Rg2 was voltage-independent and noncompetitive. Other ginsenosides besides ginsenoside Rg2 also inhibited I(ACh) in oocytes expressing alpha3beta4 or alphabetadeltavarepsilon nicotinic acetylcholine receptors. The order of potency for the inhibition of I(ACh) was ginsenoside Rg2>Rf>Re>Rg1>Rc>Rb2>Rb1 in oocytes expressing alpha3beta4 nicotinic acetylcholine receptors and was ginsenoside Rg2>Rf>Rg1>Re>Rb1>Rc>Rb2 in oocytes expressing alphabetadeltavarepsilon nicotinic acetylcholine receptors. These results indicate that ginsenosides might regulate nicotinic acetylcholine receptors in a differential manner and this regulation might be one of the pharmacological actions of Panax ginseng.     

Differences in potency and efficacy of a series of phenylisopropylamine/phenylethylamine pairs at 5-HT(2A) and 5-HT(2C) receptors

The pharmacological profile of a series of (+/-)-2,5-dimethoxy-4-(X)-phenylisopropylamines (X=I, Br, NO(2), CH(3), or H) and corresponding phenylethylamines, was determined in Xenopus laevis oocytes injected with cRNA coding for rat 5-HT(2A) or 5-HT(2C) receptors. The efficacy and relative potency of these drugs were determined and compared to classical 5-HT(2) receptor agonists and antagonists. The rank order of agonist potency at the 5-HT(2A) receptor was: alpha-methyl-5-HT=5-HT>m-CPP>MK-212; at the 5-HT(2C) receptor the order was: 5-HT>alpha-methyl-5-HT>MK-212>m-CPP. All these compounds were full agonists at the 5-HT(2C) receptor, but alpha-methyl-5-HT and m-CPP showed lower efficacy at the 5-HT(2A) receptor. 4-(4-Fluorobenzoyl)-1-(4-phenylbutyl)piperidine (4F 4PP) was 200 times more potent as a 5-HT(2A) antagonist than at 5-HT(2C) receptors. Conversely, RS 102221 was 100 times more potent as a 5-HT(2C) antagonist, confirming their relative receptor selectivities. The phenylisopropylamines were partial agonists at the 5-HT(2A) receptor, with I(max) relative to 5-HT in the 22+/-7 to 58+/-15% range; the corresponding phenylethylamines had lower or undetectable efficacies. All these drugs had higher efficacies at 5-HT(2C) receptors; DOI was a full 5-HT(2C) agonist. 2C-I and the other phenylethylamines examined showed relative efficacies at the 5-HT(2C) receptor ranging from 44+/-10% to 76+/-16%. 2C-N was a 5-HT(2) receptor antagonist; the mechanism was competitive at the 5-HT(2A), but non-competitive at the 5-HT(2C) receptor. The antagonism was time-dependent at the 5-HT(2C) receptor but independent of pre-incubation time at the 5-HT(2A) receptor subtype. The alpha-methyl group determines the efficacy of these phenylalkylamines at the 5-HT(2A) and 5-HT(2C) receptors.     

Differential agonist and inverse agonist profile of antipsychotics at D2L receptors coupled to GIRK potassium channels

The D(2) dopaminergic receptor represents a major target of antipsychotic drugs. Using the coupling of the human D(2long) (hD(2L)) receptor to G protein-coupled inward rectifier potassium (GIRK) channels in Xenopus laevis oocytes, we examined the activity of antipsychotic agents of different classes - typical, atypical, and a "new generation" of compounds, exhibiting a preferential D(2) and 5-HT(1A) receptor profile. When the hD(2L) receptor was coexpressed with GIRK channels, a series of reference compounds exhibited full agonist (dopamine, and quinpirole), partial agonist (apomorphine, (-)3-PPP, and (+)-UH232) or inverse agonist (raclopride, and L741626) properties. Sarizotan exhibited only very weak partial agonist action. At higher levels of receptor cRNA injected per oocyte, both partial agonist activity and inverse agonist properties were generally more pronounced. The inverse agonist action of L741626 was reversed by interaction with sarizotan, thus confirming the constitutive activity of wild-type hD(2L) receptors in the oocyte expression system. When antipsychotic agents were tested for their actions at the hD(2L) receptor, typical (haloperidol) as well as atypical (nemonapride, ziprasidone, and clozapine) compounds acted as inverse agonists. In contrast, among D(2)/5-HT(1A) antipsychotics, only SLV313 and F15063 behaved as inverse agonists, whilst the other members of this group (bifeprunox, SSR181507 and the recently marketed antipsychotic, aripiprazole) exhibited partial agonist properties. Thus, the X. laevis oocyte expression system highlights markedly different activity of antipsychotics at the hD(2L) receptor. These differential properties may translate to distinct therapeutic potential of these compounds.     

Voltage and ionic regulation of human serotonin transporter in Xenopus oocytes

1. The serotoninergic system is known to be involved in the control of multiple behavioural and physiological functions. The serotonin (5-hydroxtryptamine; 5-HT) transporter (SERT), which controls the synaptic 5-HT concentration through re-uptake of this neurotransmitter into presynaptic terminals, has been a primary therapeutic target for various psychiatric and peripheral disorders. The aim of the present study was to identify the regulatory mechanism(s) of the human SERT (hSERT) in heterologously expressed oocytes. 2. The hSERT cRNA was transcribed in vitro and injected into Xenopus oocytes. The 5-HT-induced transporter currents were measured by voltage clamp. The effects of extracellular sodium or chloride were studied by replacement perfusion with tetramethylammonium-chloride (96 mmol/L) or sodium acetate (96 mmol/L). In addition, to alter the internal calcium concentration, CaCl2 (50 micromol/L) and inositol triphosphate (IP3; 50 micromol/L), with or without EGTA (2.5 mmol/L), were injected into oocytes. The specificity of 5-HT-sensitive currents was determined by the use of the SERT antagonist desipramine and niflumic acid to block background chloride currents. 3. The hSERT-expressing oocytes displayed voltage-dependent, 5-HT-induced currents that increased at negative potentials. Replacing extracellular sodium or chloride significantly decreased the hSERT currents by 89 and 45%, respectively (P < 0.05, n = 7 each). Injection of IP3 or CaCl2 increased the hSERT currents by approximately 65% (P < 0.05; n = 10 each) and the effect of IP3 was abolished by preinjection of EGTA. 4. These results demonstrate that hSERT activity is not only voltage dependent, but is also affected by intracellular calcium and extracellular sodium and chloride.     

Hyperpolarization-activated current I(h) in nucleus of solitary tract neurons: regional difference in serotonergic modulation

The nucleus of solitary tract (NTS) contains diverse neural circuits responsible for basic vital functions. We examined the effect of serotonin (5-HT) on hyperpolarization-activated current (I(h)) in neurons acutely isolated from caudal, medial and rostral parts of the NTS. Caudal and medial NTS neurons showed a large amplitude of I(h) compared with rostral neurons. In these neurons, perfusion with 5-HT potentiated Ih amplitude in a concentration-dependent manner. The effect of 5-HT was blocked by NAN-190, a 5-HT1A receptor antagonist. Thus, 5-HT1A receptors may regulate I(h) channel activity in caudal and medial NTS neurons.     

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.     

Evidence for the involvement of 5-HT2A receptors in mild mesenteric ischemia/reperfusion dysfunctions in mice

In this study, the involvement of 5-HT2A receptors on mesenteric ischemia-reperfusion injury was examined in mice. Intestinal ischemia produced by 45 min occlusion of superior mesenteric artery was followed by 24h reperfusion (I/R). The 5-HT2A selective antagonist, ketanserin (0.5 mgkg(-1)) or the 5-HT2A agonist DOI (0.25 mgkg(-1)) was intravenously administered before ischemia and 8h after the beginning of reperfusion. The effects were compared with those obtained in sham operated animals (S). Ketanserin prevented the upper gastrointestinal transit delay induced by I/R (P<0.01), protected intestine from leukocyte recruitment as indicated by jejunal myeloperoxidase activity (P<0.05) and reverted Evans Blue extravasation elicited by I/R in lung, colon and jejunum (P<0.05). On the other hand, 5-HT2A activation by DOI mimicked the effects of I/R in S mice prolonging small intestine transit (P<0.05) and enhancing neutrophil accumulation in jejunal tissues (P<0.05). Furthermore, the reduction of ADP-induced platelet aggregation in plasma of I/R mice was prevented by ketanserin treatment. All together, these findings support the critical involvement of 5-HT2A receptor subtype in mediating the damage induced by mesenteric I/R in mice.     

The L293 residue in transmembrane domain 2 of the 5-HT3A receptor is a molecular determinant of allosteric modulation by 5-hydroxyindole

Allosteric modulation of ligand-gated ion channels can play important roles in shaping synaptic transmission. The function of the 5-hydroxytryptamine (serotonin) type 3 (5-HT(3)) receptor, a member of the Cys-loop ligand-gated ion channel superfamily, is modulated by a variety of compounds such as alcohols, anesthetics and 5-hydroxyindole (5-HI). In this study, the molecular determinants of allosteric modulation by 5-HI were explored in N1E-115 neuroblastoma cells expressing the native 5-HT(3) receptor and HEK 293 cells transfected with the recombinant 5-HT(3A) receptor using molecular biology and whole-cell patch-clamp techniques. 5-HI potentiated 5-HT-activated currents in both N1E-115 cells and HEK 293 cells, and significantly decreased current desensitization and deactivation. Substitution of Leu293 (L293, L15') in the second transmembrane domain (TM2) with cysteine (L293C) or serine (L293S) abolished 5-HI modulation. Other mutations in the TM2 domain, such as D298A and T284F, failed to alter 5-HI modulation. The L293S mutation enhanced dopamine efficacy and converted 5-HI into a partial agonist at the mutant receptor. These data suggest that 5-HI stabilizes the 5-HT(3A) receptor in the open state by decreasing both desensitization and 5-HT unbinding/channel closing; and L293 is a common site for both channel gating and allosteric modulation by 5-HI. Our observations also indicate existence of a second 5-HI recognition site on the 5-HT(3A) receptor, which may overlap with the 5-HT binding site and is not involved in the positive modulation by 5-HI. These findings support the idea that there are two discrete sites for 5-HI allosteric modulation and direct activation in the 5-HT(3A) receptor.     

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-HT(2C)R), 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-HT(2C)R have not been studied. In this study, we investigated the effect of O-desmethyl tramadol on 5-HT(2C)R expressed in Xenopus oocytes. METHODS: We examined the effect of O-desmethyl tramadol on 5-HT(2C)R using the Xenopus oocyte expression system. Furthermore, we investigated the effects of O-desmethyl tramadol on the binding of [(3)H]5-HT by 5-HT(2C)R. RESULTS: O-Desmethyl tramadol, at pharmacologically relevant concentrations, inhibited 5-HT-evoked Ca(2+)-activated Cl(-) currents in oocytes that expressed 5-HT(2C)R. The inhibitory effect of O-desmethyl tramadol on 5-HT(2C)R 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 [(3)H]5-HT by 5-HT(2C)R expressed in oocytes. O-Desmethyl tramadol altered the apparent dissociation constant for binding of [(3)H]5-HT by 5-HT(2C)R without changing maximum binding, which indicated competitive inhibition. CONCLUSION: These results suggest that O-desmethyl tramadol inhibits 5-HT(2C)R, which provides further insight into the pharmacological properties of tramadol and O-desmethyl tramadol.     

Block of human aorta Kir6.1 by the vascular KATP channel inhibitor U37883A.

1. A human aorta cDNA library was screened at low stringency with a rat pancreatic Kir6.1 cDNA probe and a homologue of Kir6.1 (hKir6.1) was isolated and sequenced. 2. Metabolic poisoning of Xenopus laevis oocytes with sodium azide and application of the K+ channel opener drug diazoxide induced K+ channel currents in oocytes co-injected with cRNA for hKir6.1 and hamster sulphonylurea receptor (SUR1), but not in oocytes injected with water or cRNA for hKir6.1 or SUR1 alone. 3. K+ channel currents due to hKir6.1+SUR1 or mouse Kir6.2+SUR1 were strongly inhibited by 1 microM glibenclamide. K+-current carried by hKir6.1+SUR1 was inhibited by the putative vascular-selective KATP channel inhibitor U37883A (IC50 32 microM) whereas current carried by Kir6.2+SUR1 or Shaker K+ channels was unaffected. 4. The data support the hypothesis that hKir6.1 is a component of the vascular KATP channel, although the lower sensitivity of hKir6.1+SUR1 to U37883A compared with native vascular tissues suggests the need for another factor or subunit. Furthermore, the data suggest that pharmacology of KATP channels can be determined by the pore-forming subunit as well as the sulphonylurea receptor and point to a molecular basis for the pharmacological distinction between vascular and pancreatic/cardiac KATP channels.     

Studies on the transmembrane signalling mechanism of the brain using Xenopus oocytes

Xenopus oocytes have been used as an efficient and convenient expression system of messenger ribonucleic acid (mRNA) exogeneously injected. The transmembrane signalling mechanism of acetylcholine (ACh)- and serotonin (5-HT)-evoked inward current and properties of voltage-sensitive Ca channel (VSCC) current were studied using the oocytes injected with rat brain mRNA. M1 ACh or 5-HT1C receptor stimulation causes Cl- current via G-protein activation, inositol phosphate formation, an increase of free Ca2+ and finally gating of Cl channels via Ca2(+)-calmodulin. Protein kinase C seems to modulate the responses negatively. In addition, N type VSCC was also expressed and regulated by both protein kinases A and C. Limitations of the expression system as well as advantages are discussed.     

Functional characterization of human breast cancer resistance protein (BCRP, ABCG2) expressed in the oocytes of Xenopus laevis

To evaluate the function and substrate specificity of human breast cancer resistance protein (BCRP, ABCG2) in the absence of cofactors or heterologous partner proteins, Xenopus laevis oocytes were injected with cRNA of wild-type or mutant (R482T) BCRP. High expression of BCRP was observed on the oocyte surface. Accumulation and efflux assays revealed that oocytes expressing R482T transported daunorubicin (DNR), mitoxantrone (MX), rhodamine 123, and flavopiridol (FLV), whereas wild-type BCRP transported only MX and FLV, in agreement with observations in mammalian and other systems. Transport activity was completely inhibited by fumitremorgin C, a known inhibitor of BCRP. Injection of oocytes with cRNA containing mutations of serine 187 in the ATP-binding cassette signature motif (S187T or S187A) resulted in strong expression of the mutant forms; however, these oocytes were devoid of transporter activity. When oocytes were coinjected with R482T and R482T/S187T, DNR transport was inhibited in a manner dependent on the amount of R482T/S187T cRNA added, consistent with the idea that the active form of BCRP is a homodimer or homomultimer. Substrate interaction studies found that no two substrates reciprocally inhibited the efflux of the other. Although FLV proved to be an effective inhibitor of both MX and DNR transport, and MX inhibited DNR transport, the other substrates tested had only weak or no inhibitory activity, indicating a complex nature of substrate interaction with the BCRP homodimer. We conclude that the X. laevis oocyte heterologous expression system is a valid and effective means of studying BCRP function and substrate specificity.     

Serotonin upregulates the activity of phagocytosis through 5-HT1A receptors

1 In this study, we investigated whether serotonin could regulate the in vitro activity of phagocytosis through 5-hydroxytryptamine or serotonin (5-HT(1A)) receptors. 2 Mouse peritoneal macrophages were cultured with serotonin and the activity of phagocytosis was assessed by the uptake of zymosan and latex particles added to the culture media. Specific binding of [(3)H]8-OH-DPAT and immunohistochemistry using an affinity-purified anti-5-HT(1A)-receptor antibody were assayed in the macrophages. In addition, we took advantage of the availability of pharmacological inhibitors of nuclear factor-kappaB (NF-kappaB) to explore its role in the regulation of the 5-HT(1A) receptor. 3 Serotonin increased the in vitro activity of phagocytosis in a dose-dependent manner. The 5-HT(1A) receptor agonist (+/-)-8-hydroxy-2-(di-n-propyl-amino)-tetralin (R(+)-8-OH-DPAT) reproduced these effects. Serotonin- or R(+)-8-OH-DPAT-induced increases in phagocytosis were blocked by the 5-HT(1A) receptor antagonist WAY100635 and the NF-kappaB inhibitor pyrrolidinedithiocarbamate. Moreover, mouse peritoneal macrophages expressed specific binding sites for [(3)H]8-OH-DPAT when cultivated in the presence of zymosan or latex beads. Immunohistochemistry confirmed the expression of the 5-HT(1A) receptor protein in the macrophages. 4 These results show that serotonin can upregulate the activity of peritoneal macrophages through 5-HT(1A) receptors.     

The acute and chronic effects of MDMA ("ecstasy") on cortical 5-HT2A receptors in rat and human brain.

While the pre-synaptic effects of 3,4-methylenedioxymethamphetamine (MDMA) on serotonin (5-HT) neurons have been studied extensively, little is known about its effects on post-synaptic 5-HT(2) receptors. Therefore, cortical 5-HT(2A) receptor densities and 5-HT concentration were studied in MDMA treated rats (10 mg/kg s.c.). Furthermore, 5-HT(2A) post-synaptic receptor densities in the cerebral cortex of recent as well as ex-MDMA users were studied using [123I]R91150 SPECT. In rats we observed a decrease followed by a time-dependent recovery of cortical 5-HT(2A) receptor densities, which was strongly and positively associated with the degree of 5-HT depletion. In recent MDMA users, post-synaptic 5-HT(2A) receptor densities were significantly lower in all cortical areas studied, while 5-HT(2A) receptor densities were significantly higher in the occipital cortex of ex-MDMA users. The combined results of this study suggest a compensatory upregulation of post-synaptic 5-HT(2A) receptors in the occipital cortex of ex-MDMA users due to low synaptic 5-HT levels.