Effects of dextrorotatory morphinans on alpha3beta4 nicotinic acetylcholine receptors expressed in Xenopus oocytes

We previously demonstrated that dextromethorphan (DM; 3-methoxy-17-methylmorphinan) analogs have neuroprotective effects, and a recent report showed that DM reduces the adverse effects of morphine and blocks alpha3beta4 nicotinic acetylcholine receptors, a major target of anti-addictive agents. Here, we investigated the effects of DM, three of its analogs (DF, 3-methyl-17-methylmorphinan; AM, 3-allyloxy-17-methoxymorphian; and CM, 3-cyclopropyl-17-methoxymorphinan) and one of its metabolites (HM; 3-methoxymorphinan), on neuronal alpha3beta4 nicotinic acetylcholine receptor channel activity expressed in Xenopus laevis oocytes, using the two-microelectrode voltage clamp technique. We found that intraoocyte injection of neuronal alpha3 and beta4 nicotinic acetylcholine receptor subunit cRNAs elicited an inward current (IACh) in the presence of acetylcholine. Co-treatment with DM, DF, AM, CM or HM inhibited IACh in a dose-dependent, voltage-independent and reversible manner. The IC50 values for DM, DF, AM, CM and HM were 19.5+/-5.2, 15.8+/-4.5, 16.3+/-1.7, 10.1+/-2.8, and 13.5+/-4.0 microM, respectively. The order of potency for the inhibition of IACh was CM>HM>DF=AM>DM in oocytes expressing alpha3beta4 nicotinic acetylcholine receptors. The inhibitions of (IACh) by DM, DF and HM, AM and CM were non-competitive. These results indicate that AM, CM and HM could be novel non-competitive agents regulating alpha3beta4 nicotinic acetylcholine receptor channel activity.     

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.     

Characteristics of ginsenoside Rg3-mediated brain Na+ current inhibition

We demonstrated previously that ginsenoside Rg(3) (Rg(3)), an active ingredient of Panax ginseng, inhibits brain-type Na(+) channel activity. In this study, we sought to elucidate the molecular mechanisms underlying Rg(3)-induced Na(+) channel inhibition. We used the two-microelectrode voltage-clamp technique to investigate the effect of Rg(3) on Na(+) currents (I(Na)) in Xenopus laevis oocytes expressing wild-type rat brain Na(V)1.2 alpha and beta1 subunits, or mutants in the channel entrance, the pore region, the lidocaine/tetrodotoxin (TTX) binding sites, the S4 voltage sensor segments of domains I to IV, and the Ile-Phe-Met inactivation cluster. In oocytes expressing wild-type Na(+) channels, Rg(3) induced tonic and use-dependent inhibitions of peak I(Na). The Rg(3)-induced tonic inhibition of I(Na) was voltage-dependent, dose-dependent, and reversible, with an IC(50) value of 32 +/- 6 microM. Rg(3) treatment produced a 11.2 +/- 3.5 mV depolarizing shift in the activation voltage but did not alter the steady-state inactivation voltage. Mutations in the channel entrance, pore region, lidocaine/TTX binding sites, or voltage sensor segments did not affect Rg(3)-induced tonic blockade of peak I(Na). However, Rg(3) treatment inhibited the peak and plateau I(Na) in the IFMQ3 mutant, indicating that Rg(3) inhibits both the resting and open states of Na(+) channel. Neutralization of the positive charge at position 859 of voltage sensor segment domain II abolished the Rg(3)-induced activation voltage shift and use-dependent inhibition. These results reveal that Rg(3) is a novel Na(+) channel inhibitor capable of acting on the resting and open states of Na(+) channel via interactions with the S4 voltage-sensor segment of domain II.     

Synthesis and evaluation of 3-substituted 17-methylmorphinan analogs as potential anticonvulsant agents.

Dextromethorphan (1,(+)-3-methoxy-17-methylmorphinan) demonstrates anticonvulsant activity in a variety of in vitro and in vivo models of convulsive action. It is well known that 1 is metabolized to its phenolic derivative dextrorphan (2) and this metabolite is also a potent anticonvulsant. A series of (+)-3-substituted-17-methylmorphinans, which are structurally similar to 1 but are either not expected to be metabolized to 2 or might do so at a reduced rate, as compared to 1, were prepared. Three analogs, 5 ((+)-3-amino-17-methylmorphinan), 14 ((+)-3-ethoxy-17-methylmorphinan), and 15 ((+)-3-(2-propoxy)-17-methylmorphinan) were found to possess potent anticonvulsant activity with full efficacy (ED50 25, 5.6, and 3.9 mg/kg, sc, respectively) in the rat supramaximal electroshock (MES) test. Binding potencies of these compounds to receptor sites labeled with [3H]dextromethorphan ([3H]1), in rat brain and guinea pig brain subcellular fractions, and [3H]thienylcyclohexylpiperidine (TCP) and [3H]glycine in rat brain, were determined. Most of the analogs displaced [3H]1 from its binding sites, with compounds 14 (IC50 0.42 microM) and 15 (IC50 0.88 microM) having equivalent potencies to 1 (IC50 0.59 microM), in rat brain, and no appreciable activity at the [3H]TCP or [3H]glycine-labeled sites. Compound 5 did not bind with appreciable activity to the [3H]1 site, in rat brain, but did bind to the [3H]TCP site with lower potency than the parent 1 (IC50 7.8 and 2.0 microM, respectively). The mechanism of anticonvulsant action of these agents is not clear although it appears that interaction at the [3H]1 sites may be involved.     

Preferential block of late sodium current in the LQT3 DeltaKPQ mutant by the class I(C) antiarrhythmic flecainide

Flecainide block of Na(+) current (I(Na)) was investigated in wild-type (WT) or the long QT syndrome 3 (LQT3) sodium channel alpha subunit mutation with three amino acids deleted (DeltaKPQ) stably transfected into human embryonic kidney 293 cells using whole-cell, patch-clamp recordings. Flecainide (1-300 mM) caused tonic and use-dependent block (UDB) of I(Na) in a concentration-dependent manner. Compared with WT, DeltaKPQ I(Na) was more sensitive to flecainide, and flecainide preferentially inhibited late I(Na) (mean current between 20 and 23.5 ms after depolarization) compared with peak I(Na). The IC(50) value of peak and late I(Na) for WT was 127 +/- 6 and 44 +/- 2 microM (n = 20) and for DeltaKPQ was 80 +/- 9 and 19 +/- 2 microM (n = 31) respectively. UDB of peak I(Na) was greater and developed more slowly during pulse trains for DeltaKPQ than for WT. The IC(50) value for UDB of peak I(Na) for WT was 29 +/- 4 microM (n = 20) and for DeltaKPQ was 11 +/- 1 microM (n = 26). For DeltaKPQ, UDB of late I(Na) was greater than for peak I(Na). Recovery from block was slower for DeltaKPQ than for WT. We conclude that DeltaKPQ interacts differently with flecainide than with WT, leading to increased block and slowed recovery, especially for late I(Na). These data provide insights into mechanisms for flecainide block and provide a rationale at the cellular and molecular level that open channel block may be a useful pharmacological property for treatment of LQT3.     

An analysis of the variations in potency of grayanotoxin analogs in modifying frog sodium channels of differing subtypes

Responses of tetrodotoxin-sensitive (TTX-s) and insensitive (TTX-i) Na(+) channels, in frog dorsal root ganglion (DRG) cells and frog heart Na(+) channels, to two grayanotoxin (GTX) analogs, GTX-I and alpha-dihydro-GTX-II, were examined using the patch clamp method. GTX-evoked modification occurred only when repetitive depolarizing pulses preceded a single test depolarization; modification, during the test pulse, was manifested by a decrease in peak Na(+) current accompanied by a sustained Na(+) current. GTX-evoked modification of whole-cell Na(+) currents was quantified by normalizing the conductance for sustained currents through GTX-modified Na(+) channels to that for the peak current through unmodified Na(+) channels. The dose-response relation for GTX-modified Na(+) channels was constructed by plotting the normalized slope conductance against GTX concentration. With respect to DRG TTX-i Na(+) channels, the EC(50) and maximal normalized slope conductance were estimated to be 31 microM and 0.23, respectively, for GTX-I, and 54 microM and 0.37, respectively, for alpha-dihydro-GTX-II. By contrast, TTX-s Na(+) channels in DRG cells and Na(+) channels in ventricular myocytes were found to have a much lower sensitivity to both GTX analogs. In single-channel recording on DRG cells and ventricular myocytes, Na(+) channels modified by the two GTX analogs (both at 100 microM), had similar relative conductances (range, 0.25-0.42) and open channel probabilities (range, 0.5-0.71). From these observations, we conclude that the differences in responsiveness of DRG TTX-i, and ventricular whole cell Na(+) currents to the GTX analogs studied are related to the number of Na(+) channels modified.     

OATP1B1, OATP1B3, and mrp2 are involved in hepatobiliary transport of olmesartan, a novel angiotensin II blocker.

Hepatic uptake and biliary excretion of olmesartan, a new angiotensin II blocker, were investigated in vitro using human hepatocytes, cells expressing uptake transporters and canalicular membrane vesicles, and in vivo using Eisai hyperbilirubinemic rats (EHBR), inherited multidrug resistance-associated protein (mrp2)-deficient rats. The uptake by human hepatocytes reached saturation with a Michaelis constant (K(m)) of 29.3 +/- 9.9 microM. Both Na(+)-dependent and Na(+)-independent uptake of olmesartan by human hepatocytes were observed. The uptake by Na(+)-independent human liver-specific organic anion transporters OATP1B1 and OATP1B3 expressed in Xenopus laevis oocytes was also saturable, with K(m) values of 42.6 +/- 28.6 and 71.8 +/- 21.6 microM, respectively. The Na(+)-dependent taurocholate-cotransporting polypeptide expressed in HEK 293 cells did not transport olmesartan. The cumulative biliary excretion in EHBR was one-sixth compared with that in Sprague-Dawley rats. ATP-dependent uptake of olmesartan was observed in both human canalicular membrane vesicles (hCMVs) and MRP2-expressing vesicles. An MRP inhibitor, MK-571 ([[[3-[2-(7-chloro-2-quinolinyl)ethenyl]phenyl][3-(dimethylamino)-3-oxopropyl]thio]methyl]thio]-propanoic acid) completely inhibited the uptake of olmesartan by hCMVs. In conclusion, the hepatic uptake and biliary excretion of olmesartan are mediated by transporters in humans. OATP1B1 and OATP1B3 are involved in hepatic uptake, at least in part, and MRP2 plays a dominant role in the biliary excretion.     

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.     

Ribavirin uptake by cultured human choriocarcinoma (BeWo) cells and Xenopus laevis oocytes expressing recombinant plasma membrane human nucleoside transporters

We investigated the mechanism of the transport of ribavirin (1-beta-D-ribofuranosyl-1,2,4-trizole-3-carboxamide) into placental epithelial cells using human choriocarcinoma (BeWo) cells and Xenopus oocytes expressing human nucleoside transporters. In BeWo cells, when a relatively low concentration (123 nM) of ribavirin was used, both Na(+)-dependent uptake and -independent uptake of ribavirin were observed. On the other hand, when a higher concentration (100 microM) of ribavirin was used, Na(+)-independent uptake was observed, but there was only a slight Na(+)-dependent uptake. In Xenopus oocytes, influxes of ribavirin mediated by hCNT2 (concentrative nucleoside transporter 2), hCNT3 (concentrative nucleoside transporter 3), hENT1 (equilibrative nucleoside transporter 1) and hENT2 (equilibrative nucleoside transporter 2) were saturable, and apparent K(m) values were 18.0 microM, 14.2 microM, 3.46 mM and 3.71 mM, respectively. These data indicate that hCNT2 and hCNT3 have higher affinity for ribavirin than do hENT1 and hENT2. Moreover, analysis by RT-PCR showed that BeWo cells express mRNA of hCNT3, hENT1 and hENT2. These results suggest that ribavirin is taken up by BeWo cells via both the high-affinity Na(+)-dependent transporter hCNT3 and the low-affinity Na(+)-independent transporters hENT1 and hENT2.     

Neuropsychotoxic and neuroprotective potentials of dextromethorphan and its analogs

Dextromethorphan (3-methoxy-17-methylmorphinan) has complex pharmacologic effects on the central nervous system. Although some of these effects are neuropsychotoxic, this review focuses on the neuroprotective effects of dextromethorphan and its analogs. Some of these analogs, particularly dimemorfan (3-methyl-17-methylmorphinan) and 3-hydroxymorphinan, have promising neuroprotective properties with negligible neuropsychotoxic effects. Their neuroprotective effects, the mechanisms underlying these effects, and their therapeutic potential for the treatment of diverse neurodegenerative disorders are discussed.     

Molecular basis of ranolazine block of LQT-3 mutant sodium channels: evidence for site of action

1 We studied the effects of ranolazine, an antianginal agent with promise as an antiarrhythmic drug, on wild-type (WT) and long QT syndrome variant 3 (LQT-3) mutant Na(+) channels expressed in human embryonic kidney (HEK) 293 cells and knock-in mouse cardiomyocytes and used site-directed mutagenesis to probe the site of action of the drug. 2 We find preferential ranolazine block of sustained vs peak Na(+) channel current for LQT-3 mutant (DeltaKPQ and Y1795C) channels (IC(50)=15 vs 135 microM) with similar results obtained in HEK 293 cells and knock-in myocytes. 3 Ranolazine block of both peak and sustained Na(+) channel current is significantly reduced by mutation (F1760A) of a single residue previously shown to contribute critically to the binding site for local anesthetic (LA) molecules in the Na(+) channel. 4 Ranolazine significantly decreases action potential duration (APD) at 50 and 90% repolarization by 23+/-5 and 27+/-3%, respectively, in DeltaKPQ mouse ventricular myocytes but has little effect on APD of WT myocytes. 5 Computational modeling of human cardiac myocyte electrical activity that incorporates our voltage-clamp data predicts marked ranolazine-induced APD shortening in cells expressing LQT-3 mutant channels. 6 Our results demonstrate for the first time the utility of ranolazine as a blocker of sustained Na(+) channel activity induced by inherited mutations that cause human disease and further, that these effects are very likely due to interactions of ranolazine with the receptor site for LA molecules in the sodium channel.     

The novel Na(+)/Ca(2+) exchange inhibitor KB-R7943 also blocks native and expressed neuronal nicotinic receptors

We studied the effects of the novel Na(+)/Ca(2+) exchange inhibitor KB-R7943, 2-[2-[4-(4-nitrobenzyloxy)phenyl]ethyl]isothiourea methanesulphonate, on the native nicotinic receptors present at the bovine adrenal chromaffin cells, as well as on rat brain alpha(3)beta(4) and alpha(7) nicotinic acetylcholine receptors (AChRs) expressed in XENOPUS: oocytes. As expected, KB-R7943 blocked the Na(+)-gradient dependent (45)Ca(2+) uptake into chromaffin cells (IC(50) of 5.5 microM); but in addition, the compound also inhibited the (45)Ca(2+) entry and the increase of cytosolic Ca(2+) concentration, [Ca(2+)](c), stimulated by 5 s pulses of ACh (IC(50) of 6.5 and 1.7 microM, respectively). In oocytes expressing alpha(3)beta(4) and alpha(7) nicotinic AChRs, voltage-clamped at -60 mV, inward currents elicited by 1 s pulses of 100 microM ACh (I(ACh)) were blocked by KB-R7943 with an IC(50) of 0.4 microM and a Hill coefficient of 0.9. Blockade of alpha(3)beta(4) currents by KB-R7943 was noncompetitive; moreover, the blocker (0.3 microM) became more active as the ACh concentration increased (34 versus 66% blockade at 30 microM and 1 mM ACh, respectively). Inhibition of alpha(3)beta(4) currents by 0.3 microM KB-R7943 was more pronounced at hyperpolarized potentials. If given within the ACh pulse (10 microM), the inhibition amounted to 33, 64 and 80% in oocytes voltage-clamped at -40, -60 and -100 mV, respectively. The onset of blockade was faster and the recovery slower at -100 mV; the reverse was true at -40 mV. In conclusion, KB-R7943 is a potent blocker of nicotinic AChRs; moreover, it displays many features of an open-channel blocker at the rat brain alpha(3)beta(4) AChR. These results should be considered when KB-R7943 is to be used to study Ca(2+) homeostasis in cells expressing nicotinic AChRs and the Na(+)/Ca(2+) exchanger.     

Stereoselective interaction of thiopentone enantiomers with the GABA(A) receptor.

1. As pharmacokinetic differences between the thiopentone enantiomers seem insufficient to explain the approximately 2 fold greater potency for CNS effects of (-)-S- over (+)-R-thiopentone, this study was performed to determine any enantioselectivity of thiopentone at the GABA(A) receptor, the primary receptor for barbiturate hypnotic effects. 2. Two electrode voltage clamp recording was performed on Xenopus laevis oocytes expressing human GABA(A) receptor subtype alpha1beta2gamma2 to determine relative differences in potentiation of the GABA response by rac-, (+)-R- and (-)-S-thiopentone, and rac-pentobarbitone. Changes in the cellular environment pH and in GABA concentrations were also evaluated. 3. With 3 microM GABA, the EC50 values were (-)-S-thiopentone (mean 26.0+/-s.e.mean 3.2 microM, n=9 cells) >rac-thiopentone (35.9+/-4.2 microM, n=6, P=0.1) >(+)-R-thiopentone (52.5+/-5.0 microM, n=8, P<0.02) >rac-pentobarbitone (97.0+/-11.2 microM, n=11, P<0.01). Adjustment of environment pH to 7.0 or 8.0 did not alter the EC50 values for (+)-R- or (-)-S-thiopentone. 4 Uninjected oocytes responded to >100 microM (-)-S- and R-thiopentone. This direct response was abolished by intracellular oocyte injection of 1,2-bis(2-aminophenoxy)ethane-N, N,N1,N1-tetraacetic acid (BAPTA), a Ca2+ chelating agent. With BAPTA, the EC50 values were (-)-S-thiopentone (20.6+/-3.2 microM, n=8) <(+)-R-thiopentone (36.2+/-3.2 microM, n=9, P<0.005). 5 (-)-S-thiopentone was found to be approximately 2 fold more potent than (+)-R-thiopentone in the potentiation of GABA at GABA(A) receptors expressed on Xenopus oocytes. This is consistent with the differences in potency for CNS depressant effects found in vivo.     

A novel Na+ channel agonist, dimethyl lithospermate B, slows Na+ current inactivation and increases action potential duration in isolated rat ventricular myocytes

Voltage-gated Na(+) channel blockers have been widely used as local anaesthetics and antiarrhythmic agents. It has recently been proposed that Na(+) channel agonists can be used as inotropic agents. Here, we report the identification of a natural substance that acts as a Na(+) channel agonist. Using the patch-clamp technique in isolated rat ventricular myocytes, we investigated the electrophysiological effects of the substances isolated from the root extract of Salvia miltiorrhiza, which is known as 'Danshen' in Asian traditional medicine. By the intensive activity-guided fractionation, we identified dimethyl lithospermate B (dmLSB) as the most active component, while LSB, which is the major component of the extract, showed negligible electrophysiological effect. Action potential duration (APD(90)) was increased by 20 microM dmLSB from 58.8 +/- 12.1 to 202.3 +/- 9.5 ms. In spite of the prolonged APD, no early after-depolarization (EAD) was observed. dmLSB had no noticeable effect on K(+) or Ca(2+) currents, but selectively affected Na(+) currents (I(Na)). dmLSB slowed the inactivation kinetics of I(Na) by increasing the proportion of slowly inactivating component without inducing any persistent I(Na). The relative amplitude of slow component compared to the peak fast I(Na) was increased dose dependently by dmLSB (EC(50) = 20 microM). Voltage dependence of inactivation was not affected by dmLSB, while voltage dependence of activation shifted by 5 mV to the depolarised direction. Since the APD prolongation by dmLSB did not provoke EAD, which is thought as a possible mechanism for the proarrhythmia seen in other Na(+) channel agonists, dmLSB might be an excellent candidate for a Na(+) channel agonist.     

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)     

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.     

NA+- and K+-channels as molecular targets of the alkaloid ajmaline in skeletal muscle fibres

BACKGROUND AND PURPOSE: Ajmaline is a widely used antiarrhythmic drug. Its action on voltage-gated ion channels in skeletal muscle is not well documented and we have here elucidated its effects on Na(+) and K(+) channels. EXPERIMENTAL APPROACH: Sodium (I(Na)) and potassium (I(K)) currents in amphibian skeletal muscle fibres were recorded using 'loose-patch' and two-microelectrode voltage clamp techniques (2-MVC). Action potentials were generated using current clamp. KEY RESULTS: Under 'loose patch' clamp conditions, the IC(50) for I(Na) was 23.2 microM with Hill-coefficient h=1.21. For I(K), IC(50) was 9.2 microM, h=0.87. Clinically relevant ajmaline concentrations (1-3 microM) reduced peak I(Na) by approximately 5% but outward I(K) values were reduced by approximately 20%. Na(+) channel steady-state activation and fast inactivation were concentration-dependently shifted towards hyperpolarized potentials ( approximately 10 mV at 25 microM). Inactivation curves were markedly flattened by ajmaline. Peak-I(K) under maintained depolarisation was reduced to approximately 30% of control values by 100 microM ajmaline. I(K) activation time constants were increased at least two-fold. Lower concentrations (10 or 25 microM) reduced steady-state-I(K) slightly but peak-I(K) significantly. Action potential generation threshold was increased by 10 microM ajmaline and repolarisation prolonged. CONCLUSIONS AND IMPLICATIONS: Ajmaline acts differentially on Na(+) and K(+) channels in skeletal muscle. This suggests at least multiple sites of action including the S4 subunit. Our data may provide a first insight into specific mechanisms of ajmaline-ion channel interaction in tissues other than cardiac muscle and could suggest possible side-effects that need to be further evaluated.     

Selective block of late Na(+) current by local anaesthetics in rat large sensory neurones

The actions of lignocaine and benzocaine on transient and late Na(+) current generated by large diameter (> or =50 microm) adult rat dorsal root ganglion neurones, were studied using patch-clamp techniques. Both drugs blocked whole-cell late Na(+) current in a concentration-dependent manner. At 200 ms following the onset of a clamp step from -110 to -40 mV, the apparent K for block of late Na(+) current by lignocaine was 57.8+/-15 microM (mean+/-s.e.mean, n = 4). The value for benzocaine was 24.9+/-3.3 microM, (mean+/-s.e. mean, n = 3). The effect of lignocaine on transient current, in randomly selected neurones, appeared variable (n = 8, half-block from approximately 50 to 400 microM). Half-block by benzocaine was not attained, but both whole-cell (n = 11) and patch data suggested a high apparent K,>250 microM. Transient current always remained after late current was blocked. The voltage-dependence of residual late current steady-state inactivation was not shifted by 20 microM benzocaine (n = 3), whereas 200 microM benzocaine shifted the voltage-dependence of transient current steady-state inactivation by -18.7+/-5.9 mV (mean+/-s.e.mean, n = 4). In current-clamp, benzocaine (250 microM) could block subthreshold, voltage-dependent inward current, increasing the threshold for eliciting action potentials, without preventing their generation (n = 2). Block of late Na(+) current by systemic local anaesthetic may play a part in preventing ectopic impulse generation in sensory neurones.     

Coupling of metabotropic glutamate receptors to phosphoinositide mobilisation and inhibition of cyclic AMP generation in the guinea-pig cerebellum.

1. The effects of metabotropic glutamate receptor (mGluR) agonists on cyclic nucleotide and phosphoinositide turnover were investigated in adult guinea-pig cerebellar slices by use of radioactive precursors. 2.L-Glutamate, 1-aminocyclopentane-1S,3R-dicarboxylate (1S,3R-ACPD) and RS-3,5-dihydroxyphenylglycine (DHPG) evoked concentration-dependent increases in the accumulation of [3H]-inositol phosphates with pEC50 values of 2.98 +/- 0.02, 4.45 +/- 0.06 and 4.47 +/- 0.07, respectively. Maximal responses to these agents were 43 +/- 8, 52 +/- 12 and 84 +/- 11% of the response to 1 mM histamine, respectively. 3. The phosphoinositide response to 1S,3R-ACPD was antagonized in the presence of (+)-alpha-methyl-4-carboxyphenylglycine, with a calculated pKi value of 3.55 +/- 0.03. 4. Forskolin-stimulated accumulation of [3H]-cyclic AMP was not significantly altered in the presence of 10 microM DCG-IV or 300 microM 1S,3R-ACPD. Similarly, 300 microM 1S,3R-ACPD failed to alter isoprenaline-(1 microM) or 2-chloroadenosine (2-CA, 30 microM)-stimulated accumulation of [3H]-cyclic AMP. 5. Forskolin-stimulated accumulation of [3H]-cyclic AMP was concentration-dependently inhibited in the presence of L-glutamate and L-serine-O-phosphate (L-SOP) with pIC50 values of 2.91 +/- 0.17 and 2.86 +/- 0.04 with maximal inhibitions of 47 +/- 2 and 92 +/- 3%, respectively. L-2-Amino-4-phosphonobuty-rate (L-AP4) inhibited the forskolin response without saturating, evoking an inhibition of 71 +/- 7% at 3 mM. 6. 2-CA-evoked accumulation of [3H]-cyclic AMP was also inhibited by L-glutamate and L-SOP with pIC50 values of 2.71 +/- 0.03 and 2.72 +/- 0.08 and maximal inhibitions of 51 +/- 5 and 99 +/- 0%, respectively. L-AP4 inhibited the 2-CA response without saturating, evoking an inhibition of 68 +/- 1% at 3 mM. 7. Isoprenaline-evoked accumulation of [3H]-cyclic AMP was inhibited by L-glutamate and L-SOP with pIC50 values of 3.21 +/- 0.01 and 2.96 +/- 0.08 and maximal inhibitions of 88 +/- 2 and 93 +/- 3%, respectively. 8. These results suggest that the guinea-pig cerebellum expresses Group I and Group III mGluRs coupled to phosphoinositide turnover and inhibition of cyclic AMP generation, respectively.     

Induction of Na+/K(+)-ATPase activity by long-term stimulation of nicotinic acetylcholine receptors in C2C12 myotubes.

1. To investigate the role of long-term stimulation of nicotinic acetylcholine receptors (AChRs) on the regulation of membrane potential, non-contracting C2C12 myotubes were stimulated for 1-4 days with carbachol (10 microM) and membrane potentials were measured by the intracellular microelectrode technique after washing out of the drug. 2. The membrane potential (-45.7 mV) gradually increased by 10.1 mV to -55.8 mV during 4 days treatment, which was caused by enhanced electrogenic Na+/K(+)-pumping. 3. The concentration-dependent enhancement of Na+/K(+)-ATPase activity in long-term carbachol-treated myotubes (4 days, EC50 = 5.3 microM) was prevented by co-treatment with the competitive nicotinic AChR antagonist, pancuronium but not by the muscarinic antagonist, atropine. 4. Enhanced Na+/K(+)-ATPase activity still developed in carbachol-stimulated myotubes during co-treatment (4 days) with the nicotinic AChR-channel blocker, chlorpromazine (1 microM). Membrane depolarization as such, obtained by incubation in high K+ medium (40 mM, 4 days) did not enhance Na+/K(+)-ATPase activity. 5. Non-treated myotubes possessed a high-affinity ouabain binding site (Kd = 119 nM) in association with the low Na+/K(+)-pumping activity. Long-term stimulation of myotubes (4 days) with carbachol or with a combination of carbachol and chlorpromazine was accompanied by the development of an additional low-affinity ouabain binding site (Kd = 13 microM). 6. Binding of monoclonal antibodies directed against either alpha 1- or alpha 2-subunit of Na+/K(+)-ATPase were both increased in myotubes treated with carbachol (4 days). 7. These results support the concept that nicotinic AChRs regulate Na+/K(+)-ATPase activity, independent of the functionality of the receptor-operated ion-channel.