Use-dependent Inhibition Research Articles

Inhibition of Neuronal Na + Channels by the Novel Antiepileptic Compound DCUKA: Identification of the Diphenylureido Moiety as an Inactivation Modifier

In a previous analysis of existing antiseizure compounds, we suggested that a common diphenylureido moiety was responsible for the activity-dependent, Na + channel blocking actions of these drugs (L. D. Snell et al., 2000, J. Pharmacol. Exp. Ther. 292: 215–227). Thus the novel diphenylureido compound [ N,N-(diphenyl)-4-ureido-5,7-dichloro-2-carboxyquinoline] DCUKA was developed to incorporate the diphenylureido pharmacophore into a structure that also acted as an NMDA receptor antagonist. DCUKA has previously been shown to have antiepileptic properties in animals, and in the present study the actions of DCUKA on Na + currents were characterized using transfected cells that stably expressed the rat brain Na v1.2 channel isoform. In whole-cell voltage-clamp recordings, DCUKA reduced Na + currents in a dose- and membrane potential-dependent fashion, with an apparent 1:1 stoichiometry of drug:channel interaction. Characterization of the effects of DCUKA on Na + channel function strongly suggested that DCUKA acts by enhancing Na + channel inactivation. Thus in the presence of DCUKA, Na v1.2 channels showed reduced availability in steady-state inactivation protocols, displayed use-dependent inhibition, and were slower to recover from inactivation than untreated channels, while DCUKA showed no significant interaction with the open state of the channel. As previously postulated for the anticonvulsants carbamazepine and phenytoin, these results could be well explained by a model in which the drug preferentially interacts with the fast inactivated state of the channel. Finally, DCUKA was generally more efficacious than carbamazepine in modifying sodium channel behavior. Thus the diphenylureido moiety identified by a structural analysis of classic anticonvulsants appears to be important to the inactivation-specific Na + channel inhibition by this class of antiseizure agents.

Cocaine binds to a common site on open and inactivated human heart (Na(v)1.5) sodium channels.

The inhibition by cocaine of the human heart Na+ channel (Na(v)1.5) heterologously expressed in Xenopus oocytes was investigated. Cocaine produced little tonic block of the resting channels but induced a characteristic, use-dependent inhibition during rapid, repetitive stimulation, suggesting that the drug preferentially binds to the open or inactivated states of the channel. To investigate further the state dependence, depolarizing pulses were used to inactivate the channels and promote cocaine binding. Cocaine produced a slow, concentration-dependent inhibition of inactivated channels, which had an apparent K(D) of 3.4 microM. Mutations of the interdomain III-IV linker that remove fast inactivation selectively abolished this high-affinity component of cocaine inhibition, which appeared to be linked to the fast inactivation of the channels. A rapid component of cocaine inhibition persisted in the inactivation-deficient mutant that was enhanced by depolarization and was sensitive to changes in the concentration of external Na+, properties that are consistent with a pore-blocking mechanism. Cocaine induced a use-dependent inhibition of the non-inactivating mutant and delayed the repriming at hyperpolarized voltages, indicating that the drug slowly dissociated when the channels were closed. Mutation of a conserved aromatic residue (Y1767) of the D4S6 segment weakened both the inactivation-dependent and the pore-blocking components of the cocaine inhibition. The data indicate that cocaine binds to a common site located within the internal vestibule and inhibits cardiac Na+ channels by blocking the pore and by stabilizing the channels in an inactivated state.

Mechanisms of Action of Antiarrhythmic Agent Bertosamil on hKv1.5 Channels and Outward Potassium Current in Human Atrial Myocytes

We analyzed the mechanism of action of the antiarrhythmic agent bertosamil on hKv1.5 channels expressed in Chinese hamster ovary cells (I(hKv1.5)) and on the outward current (I(o)) of human atrial myocytes (HAMs) by using the whole cell patch-clamp technique to record current. External application of 10 microM bertosamil inhibited I(hKv1.5), accelerated its time-dependent decay, and slowed its deactivation. When bertosamil was applied at rest or intracellularly (50 microM), it accelerated the rate of I(hKv1.5) inactivation without change of the peak amplitude. At the steady-state effect of intracellular bertosamil, external drug application only inhibited I(hKv1.5). When cesium was the charge carrier, bertosamil inhibited I(hKv1.5) but had no effect on its time course. Intracellular tetraethylammonium inhibited I(hKv1.5), suppressed its inactivation, and prevented bertosamil effects. Bertosamil-treated I(hKv1.5) became highly sensitive to the rate of membrane stimulation and to cumulative inactivation phenomenon. In HAMs, bertosamil also increased the rate and extent of I(o) inactivation and slowed its recovery from inactivation, whereas after drug application I(o) became highly sensitive to cumulative inactivation phenomenon. In conclusion, bertosamil 1) causes a use-dependent inhibition of the current upon external drug application, and 2) accelerates the rate of current inactivation when applied at rest or intracellularly. These effects result from both an open-channel block and acceleration of the rate of channel inactivation and contribute to the modulation by bertosamil of I(o) in HAM.

State-Dependent Inhibition of Inactivation-Deficient Ca V 1.2 and Ca V 2.3 Channels By Mibefradil

The structural determinants of mibefradil inhibition were analyzed using wild-type and inactivation-modified Ca(V)1.2 (alpha1C) and Ca(V)2.3 (alpha1E) channels. Mibefradil inhibition of peak Ba2+ currents was dose- and voltage-dependent. An increase of holding potentials from -80 to -100 mV significantly shifted dose-response curves toward higher mibefradil concentrations, namely from a concentration of 108 +/- 21 microm (n = 7) to 288 +/- 17 microm (n = 3) for inhibition of half of the Ca(V)1.2 currents (IC(50)) and from IC(50) = 8 +/- 2 microm (n = 9) to 33 +/- 7 microm (n = 4) for Ca(V)2.3 currents. In the presence of mibefradil, Ca(V)1.2 and Ca(V)2.3 experienced significant use-dependent inhibition (0.1 to 1 Hz) and slower recovery from inactivation suggesting mibefradil could promote transition(s) to an absorbing inactivated state. In order to investigate the relationship between inactivation and drug sensitivity, mibefradil inhibition was studied in inactivation-altered Ca(V)1.2 and Ca(V)2.3 mutants. Mibefradil significantly delayed the onset of channel recovery from inactivation in CEEE (Repeat I + part of the I-II linker from Ca(V)1.2 in the Ca(V)2.3 host channel), in EC(AID)EEE (part of the I-II linker from Ca(V)1.2 in the Ca(V)2.3 host channel) as well as in Ca(V)1.2 E462R, and Ca(V)2.3 R378E (point mutation in the beta-subunit binding motif) channels. Mibefradil inhibited the faster inactivating chimera EC(IS1-6)EEE with an IC(50) = 7 +/- 1 microm (n = 3), whereas the slower inactivating chimeras EC(AID)EEE and CEEE were, respectively, inhibited with IC(50) = 41 +/- 5 microm (n = 4) and IC(50) = 68 +/- 9 microm (n = 5). Dose-response curves were superimposable for the faster EC(IS1-6)EEE and Ca(V)2.3, whereas intermediate-inactivating channel kinetics (CEEE, Ca(V)1.2 E462R, and Ca(V)1.2 E462K) were inhibited by similar concentrations of mibefradil with IC(50) approximately 55-75 microm. The slower Ca(V)1.2 wild-type and Ca(V)1.2 Q473K channels responded to higher doses of mibefradil with IC(50) approximately 100-120 microm. Mibefradil was also found to significantly speed up the inactivation kinetics of slower channels (Ca(V)1.2, CEEE) with little effect on the inactivation kinetics of faster-inactivating channels (Ca(V)2.3). A open-channel block model for mibefradil interaction with high-voltage-activated Ca2+ channels is discussed and shown to qualitatively account for our observations. Hence, our data agree reasonably well with a "receptor guarded mechanism" where fast inactivation kinetics efficiently trap mibefradil into the channel.

Ketamine and its preservative, benzethonium chloride, both inhibit human recombinant alpha7 and alpha4beta2 neuronal nicotinic acetylcholine receptors in Xenopus oocytes.

1. Ketamine is a dissociative anaesthetic that is formulated as Ketalar, which contains the preservative benzethonium chloride (BCl). We have studied the effects of pure racemic ketamine, the preservative BCl and the Ketalar mixture on human neuronal nicotinic acetylcholine receptors (nAChRs) composed of the alpha7 subunit or alpha4 and beta2 subunits expressed in Xenopus laevis oocytes. 2. Ketamine inhibited responses to 1 mM acetylcholine (ACh) in both the human alpha7 and alpha4beta2 nAChRs, with IC(50) values of 20 and 50 microM respectively. Inhibition of the alpha7 nAChRs occurred within a clinically relevant concentration range, while inhibition of the alpha4beta2 nAChR was observed only at higher concentrations. The Ketalar formulation inhibited nAChR function more effectively than was expected given its ketamine concentration. The surprising increased inhibitory potency of Ketalar compared with pure ketamine appeared to be due to the activity of BCl, which inhibited both alpha7 (IC(50) value of 122 nM) and alpha4beta2 (IC(50) value of 49 nM) nAChRs at concentrations present in the clinical formulation of Ketalar. 3. Ketamine is a noncompetitive inhibitor at both the alpha7 and alpha4beta2 nAChR. In contrast, BCl causes a parallel shift in the ACh dose-response curve at the alpha7 nAChR suggesting competitive inhibition. Ketamine causes both voltage-dependent and use-dependent inhibition, only in the alpha4beta2 nAChR. 4. Since alpha7 nAChRs are likely to be inhibited during clinical use of Ketalar, the actions of ketamine and BCl on this receptor subtype may play a role in the profound analgesia, amnesia, immobility and/or autonomic modulation produced by this anaesthetic.

Effects of norfluoxetine, the major metabolite of fluoxetine, on the cloned neuronal potassium channel Kv3.1

The effects of fluoxetine and its major metabolite, norfluoxetine, were studied using the patch-clamp technique on the cloned neuronal rat K + channel Kv3.1, expressed in Chinese hamster ovary cells. In whole-cell recordings, fluoxetine and norfluoxetine inhibited Kv3.1 currents in a reversible concentration-dependent manner, with an IC 50 value and a Hill coefficient of 13.11±0.91 μM and 1.33±0.08 for fluoxetine and 0.80±0.06 μM and 1.65±0.08 for norfluoxetine at +40 mV, respectively. In inside-out patches, norfluoxetine applied to the cytoplasmic surface inhibited Kv3.1 with an IC 50 value of 0.19±0.01 μM. The inhibition of Kv3.1 currents by both drugs was characterized by an acceleration in the apparent rate of current decay, without modification of the activation time course and with relatively fewer effects on peak amplitude. The degree of inhibition of Kv3.1 by norfluoxetine was voltage-dependent. The inhibition increased steeply between 0 and +30 mV, which corresponded with the voltage range for channel opening. In the voltage range positive to +30 mV, inhibition displayed a weak voltage dependence, consistent with an electrical distance δ of 0.31±0.05. The association ( k +1) and dissociation ( k −1) rate constants for norfluoxetine-induced inhibition of Kv3.1 were 21.70±3.39 μM −1 s −1 and 14.68±3.94 s −1, respectively. The theoretical K D value derived by k −1/ k +1 yielded 0.68 μM. Norfluoxetine did not affect the ion selectivity of Kv3.1. The reversal potential under control conditions was about −85 mV and was not affected by norfluoxetine. Norfluoxetine slowed the deactivation time course, resulting in a tail crossover phenomenon when the tail currents, recorded in the presence and absence of norfluoxetine, were superimposed. The voltage dependence of steady-state inactivation was not changed by the drug. Norfluoxetine produced use-dependent inhibition of Kv3.1 at a frequency of 1 Hz and slowed the recovery from inactivation. It is concluded that at clinically relevant concentrations, both fluoxetine and its major metabolite norfluoxetine inhibit Kv3.1, and that norfluoxetine directly inhibits Kv3.1 as an open channel blocker.

Disparate Role of Na+ Channel D2-S6 Residues in Batrachotoxin and Local Anesthetic Action

Batrachotoxin (BTX) stabilizes the voltage-gated Na(+) channels in their open conformation, whereas local anesthetics (LAs) block Na(+) conductance. Site-directed mutagenesis has identified clusters of common residues at D1-S6, D3-S6, and D4-S6 segments within the alpha-subunit Na(+) channel that are critical for binding of these two types of ligands. In this report, we address whether segment D2-S6 is similarly involved in both BTX and LA actions. Thirteen amino acid positions from G783 to L795 of the rat skeletal muscle Na(+) channel ((mu)1/Skm1) were individually substituted with a lysine residue. Four mutants (N784K, L785K, V787K, and L788K) expressed sufficient Na(+) currents for further studies. Activation and/or inactivation gating was altered in mutant channels; in particular, mu1-V787K displays enhanced slow inactivation and exhibited use-dependent inhibition of peak Na(+) currents during repetitive pulses. Two of these four mutants, (mu)1-N784K and (mu)1-L788K, were completely resistant to 5 microM BTX. This BTX-resistant phenotype could be caused by structural perturbations induced by a lysine point mutation in the D2-S6 segment. However, these two BTX-resistant mutants remained quite sensitive to bupivacaine block with affinity for inactivated Na(+) channels (K(I)) of approximately 10 microM or less, which suggests that (mu)1-N784 and (mu)1-L788 residues are not in close proximity to the LA binding site.

Blockade of U50488H on potassium currents of acutely isolated mouse hippocampal CA3 pyramidal neurons

The actions of the opioid agonist U50488H on I A and I K were examined in acutely isolated mouse hippocampal CA3 pyramidal neurons using the whole-cell patch clamp technique. U50488H caused a concentration dependent, rapidly developing and reversible inhibition of voltage-activated I A and I K. The inhibitory actions were still observed in the presence of 30 μM naloxone or 5 μM nor-binaltorphimine dihydrochloride. The IC 50 values for the blockade of I A and I K were calculated as 20.1.9 and 3.7 μM, respectively. In the presence of 3.3 μM U50488H, repetitive stimulation induced use-dependent inhibition of I A and I K. A 10 μM concentration of U50488H positively shifted the half-activation membrane potential of I A by +11 mV, but negatively shifted I K by −14 mV. These results demonstrate that U50488H can directly inhibit neuronal I A and I K without involvement of the activation of κ-opioid receptors.

Effects of spinorphin and tynorphin on synaptic transmission in rat hippocampal slices

Spinorphin has been isolated from the bovine spinal cord as an endogenous inhibitor of enkephalin-degrading enzymes (aminopeptidase, dipeptidyl aminopeptidase III, angiotensin-converting enzyme and enkephalinase), and tynorphin has been synthesized as a more potent inhibitor of dipeptidyl aminopeptidase III. In this study, the effects of spinorphin and tynorphin on synaptic transmission were studied in rat isolated hippocampal slices. Field potentials were recorded from the CA1 region after stimulation of Schaffer collaterals. Spinorphin (1 μM), which alone had no effect, potentiated the facilitatory effects of enkephalin on the filed potentials at a stimulation interval of 15 s. At a stimulation interval of 10–4 s, spinorphin alone frequency dependently inhibited the field potential. On the other hand, tynorphin (1 μM), which alone had no effect at any stimulus interval, tended to potentiate the facilitatory effects of enkephalin. Spinorphin blocked long-term potentiation induced by tetanic stimulation (100 Hz, 1 s), whereas tynorphin had no effect on long-term potentiation. These results suggest that, at a low stimulation frequency, spinorphin potentiates the facilitatory effects of enkephalin by preventing degradation of enkephalin, whereas at a high stimulation frequency spinorphin use dependently inhibits synaptic transmission independently of enkephalin. On the other hand, tynorphin tends to potentiate the facilitatory effects of enkephalin without use-dependent inhibition.

High affinity interaction of mibefradil with voltage-gated calcium and sodium channels.

Mibefradil is a novel Ca(2+) antagonist which blocks both high-voltage activated and low voltage-activated Ca(2+) channels. Although L-type Ca(2+) channel block was demonstrated in functional experiments its molecular interaction with the channel has not yet been studied. We therefore investigated the binding of [(3)H]-mibefradil and a series of mibefradil analogues to L-type Ca(2+) channels in different tissues. [(3)H]-Mibefradil labelled a single class of high affinity sites on skeletal muscle L-type Ca(2+) channels (K(D) of 2.5+/-0.4 nM, B(max)=56.4+/-2.3 pmol mg(-1) of protein). Mibefradil (and a series of analogues) partially inhibited (+)-[(3)H]-isradipine binding to skeletal muscle membranes but stimulated binding to brain L-type Ca(2+) channels and alpha1C-subunits expressed in tsA201 cells indicating a tissue-specific, non-competitive interaction between the dihydropyridine and mibefradil binding domain. [(3)H]-Mibefradil also labelled a heterogenous population of high affinity sites in rabbit brain which was inhibited by a series of nonspecific Ca(2+) and Na(+)-channel blockers. Mibefradil and its analogue RO40-6040 had high affinity for neuronal voltage-gated Na(+)-channels as confirmed in binding (apparent K(i) values of 17 and 1.0 nM, respectively) and functional experiments (40% use-dependent inhibition of Na(+)-channel current by 1 microM mibefradil in GH3 cells). Our data demonstrate that mibefradil binds to voltage-gated L-type Ca(2+) channels with very high affinity and is also a potent blocker of voltage-gated neuronal Na(+)-channels. More lipophilic mibefradil analogues may possess neuroprotective properties like other nonselective Ca(2+)-/Na(+)-channel blockers.

Use-dependent inhibition by spinorphin of synaptic transmission in rat hippocampal slices

Use-dependent inhibition by spinorphin of synaptic transmission in rat hippocampal slices

Oita International Electrocardiology Symposium 2000 “Electrophysiology and Management of Lethal Arrhythmias in the New Millennium: From Genes to Bedside”

The effects of calcium (Ca2+) channel antagonists on the T-type Ca2+ channel current were investigated in Human Embryonic Kidney (HEK-293) cells heterologously expressing the a subunit of the human cardiac T-type Ca2+ channel. Ca2+ current were elicited by depolarization in 1.8 mM Ca2+ and recorded in the whole-cell configuration of the patch-clamp technique. The T-type Ca2+ current was inhibited by the following calcium channel antagonists: mibefradil (IC50=0.89μM), efonidipine (IC50=0.43μM) and nifedipine (IC50=1.91μM) . However, the agents differed in the use dependence of their effects. Mibefradil and efonidipine exhibit a strong use-dependent inhibition of the current that is due to selective interaction with the open state and the inactivated state of the channel, rather than with the resting state. Voltage dependence of inactivation and activation were modified in the presence of these agents: both curves were shifted in the hyperpolarized potentials by 5-10 mV by application of these Ca2+ channel antagonists at the concentration of IC50, respectively.

Target pharmacology of topiramate, a new antiepileptic drug

Topiramate is a novel antiepileptic drug, a fructopyranose derivative. In animal studies, topiramate suppresses maximal electroshock seizures, whereas it does not exert inhibitory effects on pentylenetetrazol-induced seizures. Since topiramate hardly affects the threshold of the seizure, topiramate has been believed to be a type of antiepileptic drug that blocks spread of seizures. Thus far, the mechanisms of its actions have been proven to include use-dependent inhibition of voltage-dependent Na+ channels in neurons, potentiation of GABA (gamma-amino-butyric acid)-induced Cl- influx, and inhibitory effects on inward currents by antagonizing kainate/alpha-amino-3-hydroxy-5-methylisoxazole-4-propionic acid (AMPA) receptors. In clinical studies conducted overseas, topiramate has been demonstrated to be effective in the treatment of partial seizures etc. In 55 countries including UK and USA, topiramate has been already approved for the clinical use as a drug for partial seizures, while a phase III study has been planned in Japan, using patients with symptomatic localization-related epilepsies.

The anticonvulsant BW534U87 depresses epileptiform activity in rat hippocampal slices by an adenosine-dependent mechanism and through inhibition of voltage-gated Na+ channels.

1. The cellular and molecular actions of BW534U87 were studied using intracellular and extracellular recordings from the CA1 region of rat hippocampal slices and whole-cell voltage-clamp recordings of recombinant human brain type IIA Na+ channels expressed in Chinese hamster ovary (CHO) cells. 2. Normal excitatory and inhibitory postsynaptic potentials evoked in hippocampal slices were unaffected by BW534U87 or the adenosine deaminase inhibitor EHNA. However, epileptiform activity was depressed by BW534U87 (50 micronM) and this inhibition was reversed by the adenosine receptor antagonist 8-phenyl theophylline (8-PT, 30 micronM). EHNA (10 micronM) mimicked the effects of BW534U87. Furthermore, BW534U87 enhanced the inhibitory effects of exogenous adenosine on evoked synaptic potentials. BW534U87 (50 micronM) also voltage- and use-dependently inhibited action potentials elicited by current injection, independent of the adenosine system, since it was not affected by 8-PT. 3. In CHO cells expressing the recombinant human brain Na+ channel, BW534U87 produced a concentration- and voltage-dependent inhibition of Na+ currents with a half-maximal inhibitory concentration of 10 micronM at a Vh of -60 mV. Use-dependent inhibition was evident at high-frequencies (20x20 ms pulse train at 10 Hz). 4 In conclusion, BW534U87 blocks hippocampal epileptiform activity by a dual mechanism. The first action is similar to that produced by EHNA and is dependent on endogenous adenosine probably by inhibition of adenosine deaminase. Secondly, BW534U87 directly inhibits voltage-gated Na+ channels in a voltage- and frequency-dependent manner. Both actions of BW534U87 are activity-dependent and may synergistically contribute to its overall anticonvulsant effects in animal models of epilepsy.

Inactivation determinant in the I-II loop of the Ca2+ channel alpha1-subunit and beta-subunit interaction affect sensitivity for the phenylalkylamine (-)gallopamil.

1. The role of calcium (Ca2+) channel inactivation in the molecular mechanism of channel block by phenylalkylamines (PAAs) was analysed in a PAA-sensitive rabbit brain class A Ca2+ channel mutant (alpha1A-PAA). Use-dependent barium current (IBa) inhibition of alpha1A-PAA by (-)gallopamil and Ca2+ channel recovery from inactivation and block were studied with two-microlectrode voltage clamp after expression of alpha1A-PAA and auxiliary alpha2-delta- and beta1a- or beta2a-subunits in Xenopus oocytes. 2. Mutation Arg387Glu (alpha1A numbering) in the intracellular loop connecting domains I and II of alpha1A-PAA slowed the inactivation kinetics and reduced use-dependent inhibition (100 ms test pulses at 0.2 Hz from -80 to 20 mV) of the resulting mutant alpha1A-PAA/R-E/beta1a channels by 100 microM (-)gallopamil (53 +/- 2 %, alpha1A-PAA/beta1a vs. 31 +/- 2 %, alpha1A-PAA/R-E/beta1a, n >= 4). This amino acid substitution simultaneously accelerated the recovery of channels from inactivation and from block by (-)gallopamil. 3. Coexpression of alpha1A-PAA with the beta2a-subunit reduced fast IBa inactivation and induced a substantial reduction in use-dependent IBa inhibition by (-)gallopamil (25 +/- 4 %, alpha1A-PAA/beta2a; 13 +/- 1 %, alpha1A-PAA/R-E/beta2a). The time constant of recovery from block at rest was not significantly affected. 4. These results demonstrate that changes in channel inactivation induced by Arg387Glu or beta2a-alpha1-subunit interaction affect the drug-channel interaction.

The position of the fast-inactivation gate during lidocaine block of voltage-gated Na+ channels.

Lidocaine produces voltage- and use-dependent inhibition of voltage-gated Na+ channels through preferential binding to channel conformations that are normally populated at depolarized potentials and by slowing the rate of Na+ channel repriming after depolarizations. It has been proposed that the fast-inactivation mechanism plays a crucial role in these processes. However, the precise role of fast inactivation in lidocaine action has been difficult to probe because gating of drug-bound channels does not involve changes in ionic current. For that reason, we employed a conformational marker for the fast-inactivation gate, the reactivity of a cysteine substituted at phenylalanine 1304 in the rat adult skeletal muscle sodium channel alpha subunit (rSkM1) with [2-(trimethylammonium)ethyl]methanethiosulfonate (MTS-ET), to determine the position of the fast-inactivation gate during lidocaine block. We found that lidocaine does not compete with fast-inactivation. Rather, it favors closure of the fast-inactivation gate in a voltage-dependent manner, causing a hyperpolarizing shift in the voltage dependence of site 1304 accessibility that parallels a shift in the steady state availability curve measured for ionic currents. More significantly, we found that the lidocaine-induced slowing of sodium channel repriming does not result from a slowing of recovery of the fast-inactivation gate, and thus that use-dependent block does not involve an accumulation of fast-inactivated channels. Based on these data, we propose a model in which transitions along the activation pathway, rather than transitions to inactivated states, play a crucial role in the mechanism of lidocaine action.

The effects of Aconitum alkaloids on the central nervous system

Preparations of Aconitum roots are employed in Chinese and Japanese medicine for analgesic, antirheumatic and neurological indications. The recent surge in use of phytomedicine derived from traditional Chinese medicine as well as increasing concerns about possible toxic effects of these compounds have inspired a great deal of research into the mechanisms by which certain Aconitum alkaloids may act on the central nervous system. The pharmacological effects of preparations of Aconitum roots are attributed to several diterpenoid alkaloids. The main alkaloid of these plants is aconitine, a highly toxic diterpenoid alkaloid which is known to suppress the inactivation of voltage-dependent Na + channels by binding to neurotoxin binding site 2 of the α-subunit of the channel protein. In this article the pharmacology of several structurally related Aconitum alkaloids is highlighted and their therapeutic vs toxic potential is discussed. Neurochemical and neurophysiological studies will be reviewed with emphasis on the effects of the alkaloids in regions of the brain that have been implicated in pain transmission and generation of epileptic activity. Considering the chemical structure of the Aconitum alkaloids as well as their mechanism of action, a subdivision in three groups becomes obvious: the first group comprises such alkaloids which possess high toxicity due to two ester boundings at the diterpene skeleton. The members of this group activate voltage-dependent sodium channels already at resting potential and inhibit noradrenaline reuptake. Activation of sodium channels and in consequence excessive depolarization with final inexcitability and suppression of pain transmission account for their antinociceptive properties. The second group comprises less toxic monoesters which have been shown to possess strong antinociceptive, antiarrhythmic and antiepileptiform properties due to a blockade of the voltage-dependent sodium channel. Electrophysiological studies have revealed a use-dependent inhibition of neuronal activity by these alkaloids. They seem to be competitive antagonists of the group I-alkaloids. The third group of Aconitum alkaloids are lacking an ester side chain in the molecule. Toxicity is markedly reduced when compared with the two other groups. They fail to affect neuronal activity, but are reported to have antiarrhythmic actions suggesting that they may have different affinities to various subtypes of the α-subunit of the Na + channel in brain and heart.

Characteristics of Ca 2+ channel blockade by oxodipine and elgodipine in rat cardiomyocytes

The two novel dihydropyridines, oxodipine and elgodipine greatly depressed the KCl-induced contraction of rabbit aorta and decreased the cardiac force of contraction of rat ventricular strips with lower potency. Both compounds markedly shortened cardiac action potentials. In rat cultured neonatal ventricular myocytes, oxodipine and elgodipine decreased the L-type Ca 2+ current ( I CaL) with IC 50 of 0.24 and 0.33 μM respectively while oxodipine was slightly more potent on the T-type Ca 2+ current ( I CaT) than elgodipine (IC 50=0.41 vs. 2.18 μM). Both compounds were less potent in inhibiting I CaL of adult cardiomyocytes. Oxodipine exhibited mostly a tonic block of both currents while elgodipine induced mainly a use-dependent block. Oxodipine and elgodipine increased by at least one order of magnitude their inhibitory potency on I CaT and I CaL when the cells were partially depolarized. We conclude that the mechanisms of inhibition of Ca 2+ channels by these two dihydropyridines are different and suggest that the underlying mechanism of vascular selectivity is the voltage-dependent block of I CaL, with the use-dependent inhibition of Ca 2+ currents by elgodipine further contributing to this selectivity.

Voltage- and use-dependent inhibition by amphetamine of field potentials and Na + current in rat nucleus accumbens neurons

The psychostimulant amphetamine (AMPH) is known to act as an indirect dopamine agonist by promoting dopamine release. Here we demonstrate direct AMPH inhibition of field potentials and Na + currents in rat nucleus accumbens (NAc) neurons. The experiments were done with field potential recordings from NAc slices and whole-cell recordings from isolated NAc neurons. In NAc slices, AMPH inhibited the field potentials. The inhibition increased when the NAc neurons were depolarized with higher extracellular K + or when the field potentials were evoked at a higher rate. In isolated NAc neurons, AMPH inhibited the Na + currents. The inhibition increased when Na + currents were activated from more depolarized holding potentials or were activated more frequently. The voltage- and use-dependent inhibition of field potentials and Na + currents by AMPH suggests a similar mechanism of AMPH action with local anesthetics and antiarrhythmic drugs.

Na + channel blocking effects of cibenzoline on guinea-pig ventricular cells

The effects of cibenzoline on transmembrane action potentials were examined in right ventricular papillary muscles and in single ventricular myocytes isolated from guinea-pig hearts. In papillary muscles, cibenzoline ≥3 μM caused a significant decrease in the maximum upstroke velocity ( V max) of the action potential without affecting the action potential duration. The inhibition of V max was enhanced at higher stimulation frequencies. In the presence of cibenzoline, trains of stimuli at rates ≥0.2 Hz led to a use-dependent inhibition of V max. The time constant for V max recovery ( τ R) from the use-dependent block was 26.2 s. The use-dependent block of V max with cibenzoline was enhanced and τ R was shortened when the resting potential was depolarized by high (8, 10 mM) [K +] o. The curve relating membrane potential and V max in single myocytes was shifted by cibenzoline (10 μM) in a hyperpolarizing direction by 7.1 mV. In myocytes treated with cibenzoline (10 μM), a 10-ms conditioning clamp to 0 mV caused a significant decrease in V max of the subsequent test action potential; the V max inhibition was enhanced modestly in association with a prolongation of the 0 mV clamp pulse duration. In the presence of cibenzoline (3 μM), application of a train of depolarizing pulses (10 ms, 200 ms) to myocytes from the resting level (−80 mV) to 0 mV resulted in a progressive V max reduction in a pulse number-dependent manner. Unlike glibenclamide (30 μM), cibenzoline (10 μM) did not prevent the hypoxia-induced shortening of action potential duration in papillary muscles. These findings indicate that the onset and offset kinetics of use-dependent Na + channel block by cibenzoline are slow. Given its state dependence, cibenzoline may be a blocker of activated Na + channels. The inhibitory action of this compound on the ATP-sensitive K + current ( I K, ATP) would be minimal or negligible at concentrations causing sufficient Na + channel block.