Effects of dextrorotatory morphinans on brain Na+ channels expressed in Xenopus oocytes

We previously demonstrated that dextromethorphan (DM; 3-methoxy-17-methylmorphinan) analogs have neuroprotective effects. 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 Na(+) channel activity. We used the two-microelectrode voltage-clamp technique to test the effects of DM, DF, AM, CM and HM on Na(+) currents (I(Na)) in Xenopus oocytes expressing cRNAs encoding rat brain Nav1.2 alpha and beta1 or beta2 subunits. In oocytes expressing Na(+) channels, DM, DF, AM and CM, but not HM, induced tonic and use-dependent inhibitions of peak I(Na) following low- and high-frequency stimulations. The order of potency for the inhibition of peak I(Na) was AM-CM > DM=DF. The DM, DF, AM and CM-induced tonic inhibitions of peak I(Na) were voltage-dependent, dose-dependent and reversible. The IC(50) values for DM, DF, AM and CM were 116.7+/-14.9, 175.8+/-16.9, 38.6+/-15.5, and 42.5+/-8.5 microM, respectively. DM and its analogs did not affect the steady-state activation and inactivation voltages. AM and CM, but not DM and DF, inhibited the plateau I(Na) more effectively than the peak I(Na) in oocytes expressing inactivation-deficient I1485Q-F1486Q-M1487Q (IFMQ3) mutant channels; the IC(50) values for AM and CM in this system were 8.4+/-1.3 and 8.7+/-1.3 microM, respectively, for the plateau I(Na) and 43.7+/-5.9 and 32.6+/-7.8 microM, respectively, for the peak I(Na). These results collectively indicate that DM and its analogs could be novel Na(+) channel blockers acting on the resting and open states of brain Na(+) channels.     

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.     

Ginsenoside Rg(3) decelerates hERG K(+) channel deactivation through Ser631 residue interaction

The human ether-a-go-go-related gene (hERG) cardiac K(+) channels are one of the representative pharmacological targets for development of drugs against cardiovascular diseases such as arrhythmia. Panax ginseng has been known to have cardio-protective effects. However, little is known about the molecular mechanisms of how ginsenosides, the active ingredients in Panax ginseng, interact with hERG K(+) channel proteins. In the present study, we first examined the effects of various ginsenosides on hERG K(+) channel activity by expressing human α subunits in Xenopus oocytes. Among them ginsenoside Rg(3) (Rg(3)) most potently enhanced outward I(hERG) and peak I(tail). Rg(3) induced a large persistent deactivating-tail current (I(deactivating-tail)) and profoundly decelerated deactivating current decay in both concentration- and voltage-dependent manners. The EC(50) for steady-state I(hERG), peak I(tail), and persistent I(deactivating-tail) was 0.41±0.05, 0.61±0.11, and 0.36±0.04μM, respectively. Rg(3) actions were blocked by bepridil, a hERG K(+) channel antagonist. Site-directed mutation of S631, which is located at the channel pore entryway, to S631C in hERG K(+) channel abolished Rg(3) actions on hERG K(+) channels. These results indicate that S631 residue of hERG K(+) channel plays an important role in Rg(3)-mediated induction of a persistent I(deactivating-tail) and in a deceleration of hERG K(+) channel deactivation.     

Phenol derivatives accelerate inactivation kinetics in one inactivation-deficient mutant human skeletal muscle Na(+) channel

Altered inactivation kinetics in skeletal muscle Na(+) channels due to mutations in the encoding gene are causal for the alterations in muscle excitability in nondystrophic myotonia. Na(+) channel blockers like lidocaine and mexiletine, suggested for therapy of myotonia, do not reconstitute inactivation in channels with defective inactivation in vitro. We examined the effects of four methylated and/or halogenated phenol derivatives on one heterologously expressed inactivation-deficient Paramyotonia congenita-mutant (R1448H) muscle Na(+) channel in vitro. All these compounds accelerated delayed inactivation of R1448H-whole-cell currents during a depolarization and delayed accelerated recovery from inactivation. The potency of these effects paralleled the potency of the drugs to block the peak current amplitude. We conclude that the investigated phenol derivatives affect inactivation-deficient Na(+) channels more specifically than lidocaine and mexiletine. However, for all compounds, the effect on inactivation was accompanied by a substantial block of the peak current amplitude.     

Inhibition of tetrodotoxin (TTX)-resistant and TTX-sensitive neuronal Na(+) channels by the secretolytic ambroxol

Ambroxol has a long history for the treatment of airway diseases because of its beneficial effects on surfactant synthesis and mucus-modifying properties. Some findings, however, point to an additional effect on neuronal signal transduction: ambroxol can suppress reflexes such as the cough or the corneal reflex. The airways and the cornea are innervated by C-fibers, which express voltage-gated Na(+) channels with and without sensitivity to tetrodotoxin (TTX). In this study, we performed voltage-clamp experiments to investigate whether ambroxol affects these channel types. In rat dorsal root ganglia, TTX-resistant Na(+) currents were suppressed in a concentration-dependent manner with IC(50) values of 35.2 and 22.5 microM for tonic and phasic block, respectively. Depolarizing prepulses increased the potency of ambroxol, and steady-state inhibition curves were shifted to more negative values. The inhibition was not frequency-dependent. TTX-sensitive currents were inhibited with lower potency (approximately 50% inhibition with 100 microM). Recombinant rat brain IIA channels in Chinese hamster ovary cells were blocked with IC(50) values of 111.5 and 57.6 microM for tonic and phasic block, respectively; in contrast to TTX-resistant channels the block was frequency-dependent. Thus, ambroxol indeed blocks neuronal voltage-gated Na(+) channels, and TTX-resistant channels in sensory neurons were more sensitive than TTX-sensitive. Compared with known local anesthetics (e.g., lidocaine or benzocaine), the potency for Na(+) channel block was relatively high. A recent clinical trial has further confirmed that ambroxol relieved pain and was beneficial in patients who suffered from sore throat.     

Unexpected mexiletine responses of a mutant cardiac Na+ channel implicate the selectivity filter as a structural determinant of antiarrhythmic drug access

Gating properties of Na(+) channels are the critical determinants for the state-dependent block by class I antiarrhythmic drugs; however, recent site-directed mutagenesis studies have shown that the Na(+) channel selectivity filter region controls drug access to and dissociation from the binding site. To validate these observations, we have exploited a naturally occurring cardiac Na(+) channel mutation, S1710L, located next to the putative selectivity filter residue of domain 4, and evaluated the pharmacological properties to mexiletine using whole-cell, patch-clamp recordings. Consistent with the large negative shift of steady-state inactivation and the enhanced slow inactivation, the S1710L channel showed greater mexiletine tonic block than wild-type (WT) channel. In contradiction, S1710L showed attenuated use-dependent block by mexiletine and accelerated recovery from block, suggesting that the drug escape though the external access path is facilitated. Extracellularly applied QX-314, a membrane-impermeant derivative of lidocaine, elicited significantly enhanced tonic block in S1710L similar to mexiletine. However, recovery from internally applied QX-314 was accelerated by 4.4-fold in S1710L compared with WT. These results suggest that the drug access to and dissociation from the binding site through the hydrophilic path are substantially altered. Moreover, K(+) permeability was 1.9-fold increased in S1710L, verifying that the mutated residue is located in the ion-conducting pore. We propose that the Na(+) channel selectivity filter region is a structural determinant for the antiarrhythmic drug sensitivity in addition to gating properties of the indigenous Na(+) channels that govern the state-dependent drug block.     

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.     

Differential modulation of Nav1.7 and Nav1.8 peripheral nerve sodium channels by the local anesthetic lidocaine

1 Voltage-gated Na+ channels are transmembrane proteins that are essential for the propagation of action potentials in excitable cells. Nav1.7 and Nav1.8 dorsal root ganglion Na+ channels exhibit different kinetics and sensitivities to tetrodotoxin (TTX). We investigated the properties of both channels in the presence of lidocaine, a local anesthetic (LA) and class I anti-arrhythmic drug. 2 Nav1.7 and Nav1.8 Na+ channels were coexpressed with the beta1-subunit in Xenopus oocytes. Na+ currents were recorded using the two-microelectrode voltage-clamp technique. 3 Dose-response curves for both channels had different EC50 (dose producing 50% maximum current inhibition) (450 microm for Nav1.7 and 104 microm for Nav1.8). Lidocaine enhanced current decrease in a frequency-dependent manner. Steady-state inactivation of both channels was also affected by lidocaine, Nav1.7 being the most sensitive. Only the steady-state activation of Nav1.8 was affected while the entry of both channels into slow inactivation was affected by lidocaine, Nav1.8 being affected to a larger degree. 4 Although the channels share homology at DIV S6, the LA binding site, they differ in their sensitivity to lidocaine. Recent studies suggest that other residues on DI and DII known to influence lidocaine binding may explain the differences in affinities between Nav1.7 and Nav1.8 Na+ channels. 5 Understanding the properties of these channels and their pharmacology is of critical importance to developing drugs and finding effective therapies to treat chronic pain.     

Indoxacarb, an oxadiazine insecticide, blocks insect neuronal sodium channels

1. Decarbomethoxyllated JW062 (DCJW), the active component of a new oxadiazine insecticide DPX-JW062 (Indoxacarb), was tested on action potentials and the inward sodium current recorded from short-term cultured dorsal unpaired median neurones of the cockroach Periplaneta americana. 2. Under whole-cell current-clamp conditions, 100 nM DCJW reduced the amplitude of action potentials and induced a large hyperpolarization of the resting membrane potential associated with a 41% increase in input resistance. 3. In voltage-clamp, DCJW resulted in a dose-dependent inhibition (IC(50) 28 nM) of the peak sodium current. Based on IC(50) values, the effect of DCJW was about 10 fold less potent than tetrodotoxin (TTX) but 1000 fold more potent than the local anaesthetic lidocaine. DCJW (100 nM) was without effect on activation properties of the sodium current, reversal potential, voltage dependence of sodium conductance and on both fast and slow steady-state inactivations. 4. TTX (2 nM) resulted in 48% inhibition of the peak inward sodium current. Co-application of TTX (2 nM) with various concentrations of DCJW produced an additional inhibition of the peak inward current, indicating that the blocking actions of DCJW and TTX were distinct. Co-application of lidocaine (IC(50) 30 microM) with various concentrations of DCJW produced a reduction of the apparent potency of DCJW, suggesting that DCJW and lidocaine acted at the same site. 5. DCJW (100 nM) did not affect inward calcium or outward potassium currents. 6. This study describes, for the first time, the action on insect neuronal voltage-dependent sodium channels of Indoxacarb, a new class of insecticides.     

Lidocaine: a foot in the door of the inner vestibule prevents ultra-slow inactivation of a voltage-gated sodium channel

After opening, Na(+) channels may enter several kinetically distinct inactivated states. Whereas fast inactivation occurs by occlusion of the inner channel pore by the fast inactivation gate, the mechanistic basis of slower inactivated states is much less clear. We have recently suggested that the inner pore of the voltage-gated Na(+) channel may be involved in the process of ultra-slow inactivation (I(US)). The local anesthetic drug lidocaine is known to bind to the inner vestibule of the channel and to interact with slow inactivated states. We therefore sought to explore the effect of lidocaine binding on I(US). rNa(V) 1.4 channels carrying the mutation K1237E in the selectivity filter were driven into I(US) by long depolarizing pulses (-20 mV, 300 s). After repolarization to -120 mV, 53 +/- 5% of the channels recovered with a very slow time constant (tau(rec) = 171 +/- 19 s), typical for recovery from I(US). After exposure to 300 microM lidocaine, the fraction of channels recovering from I(US) was reduced to 13 +/- 4% (P < 0.01, n = 6). An additional mutation in the binding site of lidocaine (K1237E + F1579A) substantially reduced the effect of lidocaine on I(US), indicating that lidocaine has to bind to the inner vestibule of the channel to modulate I(US). We propose that I(US) involves a closure of the inner vestibule of the channel. Lidocaine may interfere with this pore motion by acting as a "foot in the door" in the inner vestibule.     

Closing and inactivation potentiate the cocaethylene inhibition of cardiac sodium channels by distinct mechanisms

Cocaethylene, a metabolite of cocaine and alcohol, is a potent inhibitor of the cardiac (Nav1.5) sodium channel heterologously expressed in Xenopus laevis oocytes. Cocaethylene produces minimal tonic block under resting conditions but causes a potent use-dependent inhibition during repetitive depolarization and a hyperpolarizing shift in the steady-state inactivation. The data are consistent with a state-dependent binding mechanism, which has high affinity for inactivated channels (KI = 17 microM) and low affinity for resting channels (KR = 185 micro). Mutations of the interdomain D3-D4 linker eliminated rapid inactivation and weakened the cocaethylene inhibition, consistent with an important role for fast inactivation in cocaethylene binding. A rapid component of cocaethylene inhibition was observed in a noninactivating mutant of Nav1.5 that was tightly linked to channel opening and displayed properties consistent with a pore blocking mechanism. Hyperpolarization caused the noninactivating mutant channel to close, trapping cocaethylene and slowing the recovery. Mutation of a conserved isoleucine (I1756C) located near the extracellular end of the D4S6 segment accelerated the recovery of the noninactivating channel, suggesting that this mutation facilitates cocaethylene untrapping, which seems to be the rate-limiting step in the recovery when the channel is closed. This contrasts with the rapidly inactivating channel, where the I1756C mutation did not alter the recovery from cocaethylene inhibition. The data suggest that additional mechanisms, such as more stable cocaethylene binding, may be a more important determinant of recovery kinetics when the channels are inactivated. The data indicate that deactivation and inactivation slow the recovery and potentiate the cocaethylene inhibition of the Nav1.5 channel by distinct mechanisms.     

Lidocaine block of neonatal Nav1.3 is differentially modulated by co-expression of beta1 and beta3 subunits

The effects of lidocaine on neonatal Na(v)1.3 (Na(v)1.3n) expressed alone and in combination with beta1 and beta3 subunits in Xenopus oocytes were examined. Lidocaine reversibly inhibited the peak Na(v)1.3n current, shifted the steady-state inactivation curve to hyperpolarized potentials and delayed recovery from inactivation. These effects were attenuated by the co-expression of the beta subunits, with greater attenuating effects being observed in oocytes co-expressing beta1 compared to those co-expressing beta3. Use-dependent block by lidocaine was assessed at 1 Hz train frequency for 60 pulses. Lidocaine caused similar use-dependent block of current amplitude at pulse 60 for Na(v)1.3n and Na(v)1.3n+beta3. In oocytes co-expressing beta1, these use-dependent actions were reduced. In conclusion, the effects of lidocaine on Na(v)1.3n are differentially modulated by beta1 and beta3 subunits. Since these subunits exhibit a complementary distribution, this finding may have importance in our understanding of lidocaine action.     

Use-dependent blockade of sodium channels by local anaesthetics and antiarrhythmic drugs. Effects of chloramine-T and calcium ions

Voltage clamp studies were carried out of the effects of chloramine-T(CT) and external Ca++ on the blocking interactions of local anaesthetics (LAs) and antiarrhythmic drugs (lidocaine, tetracaine, N-propyl ajmaline, compound KC 3791) with Na+ channels in frog Ranvier nodes. The results obtained provided direct evidence for the notion that: LAs interact preferentially with inactivated Na+ channels and stabilize their inactivated conformation ("drug-induced slow inactivation": SI); and SI underlies the cumulative inhibition of INa during repetitive membrane stimulation. Normal inactivation is not indispensable, but plays an auxiliary role in the mechanism of cumulative inhibition of INa by drugs interacting with open Na+ channels. This block results mainly from accumulation of the channels in the resting blocked state (due to the inability of charged drugs to leave the channel via a "hydrophobic pathway"). The contribution of the blockade-inactivated state to this type of block may depend on some properties of the drug and the holding membrane potential. The problem of the location of the binding site responsible for LA-induced SI requires further investigation in view of the fact that in the myocardium, along with LA, the lipid-insoluble tetrodotoxin (TTX) induces a pronounced SI.     

Electrophysiologic properties of lidocaine, cocaine, and n-3 fatty-acids block of cardiac Na+ channels

Lidocaine and cocaine, two local anesthetics, and n-3 polyunsaturated fatty acids in fish oils, inhibit the voltage-gated Na(+) channels of cardiomyocytes. This inhibition by lidocaine and n-3 fish oil is associated with antiarrhythmic effects, whereas with cocaine lethal arrhythmias may occur. These electrophysiologic studies show that at the concentrations tested, the n-3 fish oil fatty acids and lidocaine share three actions on I(Na): a potent inhibition of I(Na); a strong voltage-dependence of this inhibition; and a large shift of the steady-state inactivation to hyperpolarized potentials. By contrast cocaine shares only the potent inhibition of I(Na). The voltage-dependence of the inhibition is much decreased with cocaine, which produces only a very small leftward shift of the voltage-dependence of inactivation. The large leftward shift of the steady-state inactivation seems very important in the prevention of fatal arrhythmias by the n-3 fatty acids. Thus, we suggest that it is lack of this effect by cocaine, which is one factor, that eliminates its ability to prevent fatal cardiac arrhythmias. Further we report that in cultured neonatal rat cardiomyocytes n-3 fish oil fatty acids terminate the tachycardia induced by the alpha(1) adrenergic agonist, phenylephrine, whereas cocaine accelerates the tachycardia and causes bouts of tachyarrhythmias.     

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.     

Role of the local anesthetic receptor in the state-dependent inhibition of voltage-gated sodium channels by the insecticide metaflumizone

Sodium channel inhibitor (SCI) insecticides selectively target voltage-gated sodium (Na(v)) channels in the slow-inactivated state by binding at or near the local anesthetic receptor within the sodium channel pore. Metaflumizone is a new insecticide for the treatment of fleas on domesticated pets and has recently been reported to block insect sodium channels in the slow-inactivated state, thereby implying that it is also a member of the SCI class. Using the two-electrode voltage-clamp technique, we examined metaflumizone inhibition of rat Na(v)1.4 sodium channels expressed in Xenopus laevis oocytes. Metaflumizone selectively inhibited Na(v)1.4 channels at potentials that promoted slow inactivation and shifted the voltage dependence of slow inactivation in the direction of hyperpolarization. Metaflumizone perfusion at a hyperpolarized holding potential also shifted the conductance-voltage curve for activation in the direction of depolarization and antagonized use-dependent lidocaine inhibition of fast-inactivated sodium channels, actions not previously observed with other SCI insecticides. We expressed mutated Na(v)1.4/F1579A and Na(v)1.4/Y1586A channels to investigate whether metaflumizone shares the domain IV segment S6 (DIV-S6) binding determinants identified for other SCI insecticides. Consistent with previous investigations of SCI insecticides on rat Na(v)1.4 channels, the F1579A mutation reduced sensitivity to block by metaflumizone, whereas the Y1586A mutation paradoxically increased the sensitivity to metaflumizone. We conclude that metaflumizone selectively inhibits slow-inactivated Na(v)1.4 channels and shares DIV-S6 binding determinants with other SCI insecticides and therapeutic drugs. However, our results suggest that metaflumizone interacts with resting and fast-inactivated channels in a manner that is distinct from other compounds in this insecticide class.     

Esmolol inhibits Na+ current in rat ventricular myocytes

Esmolol is a unique cardioselective, intravenous, ultra-short acting, beta1-adrenergic blocking agent. It has been widely applied in treating ventricular and supraventricular arrhythmias, especially in emergency situations. In this study the effects of esmolol on sodium current (I(Na)) were investigated by the whole cell patch-clamp recording technique in isolated adult rat ventricular myocytes. The results indicated that esmolol reversibly inhibited I(Na) in a concentration-dependent manner, with an IC50 of 74.2 +/- 0.60 micromol l(-1) with a Hill coefficient of 1.02 +/- 0.04. This inhibition was voltage- and frequency-dependent. Esmolol decreased the peak of the I-V relationship curve at -35 mV from 16.97 +/- 1.68 pA/pF to 6.96 +/- 0.51 pA/pF. The steady-state inactivation curve of I(Na) was shifted to more negative potentials, the voltage at half-inactivation changing from -78.75 +/- 2.3 mV in control to -85.94 +/- 3.2 mV in the presence of esmolol. The development of resting inactivation from closed states was accelerated by esmolol, the time constant was shortened from 62.75 +/- 3.21 ms to 24.93 +/- 2.43 ms, whereas the activation curve was not altered. I(Na) from inactivation could not be recovered completely in the presence of esmolol. These results suggest that esmolol inhibits I(Na) through sodium channel in rat ventricular myocytes by mechanisms involving preferential interaction with the inactivated state and acceleration of the development of inactivation directly from resting state. Therefore, the effect of inhibitory sodium of esmolol may play a vital role in its antiarrhythmic efficacy.     

Differential effect of bovine serum albumin on ginsenoside metabolite-induced inhibition of α3β4 nicotinic acetylcholine receptor expressed in<Emphasis Type="Italic">Xenopus</Emphasis> oocytes

Ginsenosides, major active ingredients of Panax ginseng, that exhibit various pharmacological and physiological actions are transformed into compound K (CK) or M4 by intestinal microorganisms. CK is a metabolite derived from protopanaxadiol (PD) ginsenosides, whereas M4 is a metabolite derived from protopanaxatriol (PT) ginsenosides. Recent reports shows that ginsenosides might play a role as pro-drugs for these metabolites. In present study, we investigated the effect of bovine serum albumin (BSA), which is one of major binding proteins on various neurotransmitters, hormones, and other pharmacological agents, on ginsenoside Rg2-, CK-, or M4-induced regulation of alpha3beta4 nicotinic acetylcholine (ACh) receptor channel activity expressed in Xenopus oocytes. In the absence of BSA, treatment of ACh elicited inward peak current (I(ACh)) in oocytes expressing alpha3beta4 nicotinic ACh receptor. Co-treatment of ginsenoside Rg2, CK, or M4 with ACh inhibited I(ACh) in oocytes expressing (alpha3beta4 nicotinic ACh receptor with reversible and dose-dependent manner. In the presence of 1% BSA, treatment of ACh still elicited I(ACh) in oocytes expressing alpha3beta4 nicotinic ACh receptor and co-treatment of ginsenoside Rg2 or M4 but not CK with ACh inhibited I(ACh) in oocytes expressing alpha3beta4 nicotinic ACh receptor with reversible and dose-dependent manner. These results show that BSA interferes the action of CK rather than M4 on the inhibitory effect of I(ACh) in oocytes expressing alpha3beta4 nicotinic ACh receptor and further suggest that BSA exhibits a differential interaction on ginsenoside metabolites.     

Effects of a Lasiodora spider venom on Ca2+ and Na+ channels.

The venom of a Brazilian spider, Lasiodora sp (Mygalomorphae, Theraphosidae), was screened for activity against ion channels using Ca2+ imaging and whole-cell patch clamp in GH3 cells. When tetrodotoxin (TTX) was present to block Na+ channels, the venom abolished the Ca2+ oscillations that are normally present in these cells and reduced the basal level of intracellular Ca2+. Under patch clamp, the venom reduced the L-type Ca2+ channel conductance and caused a positive shift in its voltage dependence of activation. In addition to these effects, when applied without TTX, the venom also caused a slow and noisy increase in intracellular Ca2+. The sensitivity of this second effect to TTX suggested an effect on Na+ channels, which was tested using patch clamp. Control Na+ currents inactivated completely as a single exponential. Treatment with the venom did not affect the amplitude of I(Na), but caused it to divide in two slower exponential components plus a sustained component, all of which were suppressed by TTX. The venom also caused a negative shift in the voltage dependence of activation and steady-state inactivation of I(Na). The observed effects of this venom on whole-cell currents explain the changes it causes in intracellular Ca2+ in GH3 cells and demonstrate that the venom of this spider is a source of toxins active against ion channels.     

Oxaliplatin induces hyperexcitability at motor and autonomic neuromuscular junctions through effects on voltage-gated sodium channels

Oxaliplatin, an effective cytotoxic treatment in combination with 5-fluorouracil for colorectal cancer, is associated with sensory, motor and autonomic neurotoxicity. Motor symptoms include hyperexcitability while autonomic effects include urinary retention, but the cause of these side-effects is unknown. We examined the effects on motor nerve function in the mouse hemidiaphragm and on the autonomic system in the vas deferens. In the mouse diaphragm, oxaliplatin (0.5 mM) induced multiple endplate potentials (EPPs) following a single stimulus, and was associated with an increase in spontaneous miniature EPP frequency. In the vas deferens, spontaneous excitatory junction potential frequency was increased after 30 min exposure to oxaliplatin; no changes in resting Ca(2+) concentration in nerve terminal varicosities were observed, and recovery after stimuli trains was unaffected.In both tissues, an oxaliplatin-induced increase in spontaneous activity was prevented by the voltage-gated Na(+) channel blocker tetrodotoxin (TTX). Carbamazepine (0.3 mM) also prevented multiple EPPs and the increase in spontaneous activity in both tissues. In diaphragm, beta-pompilidotoxin (100 microM), which slows Na(+) channel inactivation, induced multiple EPPs similar to oxaliplatin's effect. By contrast, blockers of K(+) channels (4-aminopyridine and apamin) did not replicate oxaliplatin-induced hyperexcitability in the diaphragm. The prevention of hyperexcitability by TTX blockade implies that oxaliplatin acts on nerve conduction rather than by effecting repolarisation. The similarity between beta-pompilidotoxin and oxaliplatin suggests that alteration of voltage-gated Na(+) channel kinetics is likely to underlie the acute neurotoxic actions of oxaliplatin.