Electromagnetic modelling of current flow in the heart from TASER devices and the risk of cardiac dysrhythmias.

Increasing use by law enforcement agencies of the M26 and X26 TASER electrical incapacitation devices has raised concerns about the arrhythmogenic potential of these weapons. Using a numerical phantom constructed from medical images of the human body in which the material properties of the tissues are represented, computational electromagnetic modelling has been used to predict the currents arising at the heart following injection of M26 and X26 waveforms at the anterior surface of the chest (with one TASER 'barb' directly overlying the ventricles). The modelling indicated that the peak absolute current densities at the ventricles were 0.66 and 0.11 mA mm(-2) for the M26 and X26 waveforms, respectively. When applied during the vulnerable period to the ventricular epicardial surface of guinea-pig isolated hearts, the M26 and X26 waveforms induced ectopic beats, but only at current densities greater than 60-fold those predicted by the modelling. When applied to the ventricles in trains designed to mimic the discharge patterns of the TASER devices, neither waveform induced ventricular fibrillation at peak currents >70-fold (for the M26 waveform) and >240-fold (for the X26) higher than the modelled current densities. This study provides evidence for a lack of arrhythmogenic action of the M26 and X26 TASER devices.

Interaction of the novel anticonvulsant, BIA 2-093, with voltage-gated sodium channels: comparison with carbamazepine.

PURPOSE: BIA 2-093 [(S)-(-)-10-acetoxy-10,11-dihydro-5H-dibenz/b,f/azepine-5-carboxamide] is endowed with an anticonvulsant potency similar to that of carbamazepine (CBZ), but produces less cognitive and motor impairment. This study evaluated whether voltage-gated sodium channels (VGSCs) are a primary locus for the action of BIA 2-093. METHODS: We used the whole-cell voltage-clamp technique in the mouse neuroblastoma cell line N1E-115 to investigate the effects of BIA 2-093 and CBZ on VGSCs, displacement of [3H]-batrachotoxinin A 20-alpha-benzoate ([3H]-BTX), and [3H]-saxitoxin to define their relative potency to bind to rat brain sodium channels, and inhibition of uptake of 22Na by rat brain cortical synaptosomes stimulated by veratridine as a measure of sodium entry. RESULTS: The inhibitory potencies of BIA 2-093 and CBZ increased as the holding potential was made less negative (-100, -90, -80, and -70 mV) with median inhibitory concentration (IC50) values (in microM) of, respectively, 4,337, 618, 238, and 139 for BIA 2-093, and 1,506, 594, 194, and 101 for CBZ. BIA 2-093 displayed a similar potency in displacing [3H]-BTX (IC50 values, 222 vs. 361 microM; p > 0.05) and inhibiting the uptake of 22Na (IC50 values, 36 vs. 138 microM; p > 0.05). Both drugs failed to displace [3H]-saxitoxin in concentrations up to 300 microM. CONCLUSIONS: BIA 2-093, like CBZ, inhibits sodium currents in a voltage-dependent way by an interaction predominantly with the inactivated state of the channel and interacts with neurotoxin receptor site 2, but not with receptor site 1. BIA 2-093 displayed a potency blocking VGSCs similar to that of CBZ.

A comparison of the effects of SCA40, NS 004 and NS 1619 on large conductance Ca(2+)-activated K+ channels in bovine tracheal smooth muscle cells in culture.

1. The effects of imidazopyrazine derivative, SCA40, on the activity of single large conductance, Ca(2+)-activated K+ (BKCa) channels in inside-out and outside-out patches from bovine tracheal smooth muscle (BTSM) cells in culture have been compared with those of two established BKCa channel openers, NS 004 and NS 1619. 2. The presence of BKCa channels on inside-out patches of BTSM membranes was confirmed by the single channel conductance (240 pS), selectivity for K+, dependence of channel activity on [Ca2+]i, and sensitivity to the selective BKCa channel blocker, iberiotoxin. 3. NS 004 and ND 1619 (3-30 microM) induced concentration-related increases in open state probability of BKCa channels when applied to either inside-out or outside-out BTSM patches, thus confirming that these compounds are activators of the BKCa channel in this preparation. 4. SCA40 (0.1-10 microM) had no effect on the activity of BKCa channels when applied to either inside-out or outside-out patches which subsequently responded to the application of NS 004 (10-20 microM). 5. It is concluded that SCA40 does not have a direct effect on BKCa channel activity in BTSM patches and that the previously reported relaxant action of SCA40 on tracheal smooth muscle is unlikely to be mediated by this mechanism.

[3H]-lifarizine, a high affinity probe for inactivated sodium channels.

1. [3H]-lifarizine bound saturably and reversibly to an apparently homogeneous class of high affinity sites in rat cerebrocortical membranes (Kd = 10.7 +/- 2.9 nM; Bmax = 5.10 +/- 1.43 pmol mg-1 protein). 2. The binding of [3H]-lifarizine was unaffected by sodium channel toxins binding to site 1 (tetrodotoxin), site 3 (alpha-scorpion venom) or site 5 (brevetoxin), Furthermore, lifarizine at concentrations up to 10 microM had no effect on [3H]-saxitoxin (STX) binding to toxin site 1. Lifarizine displaced [3H]-batrachotoxinin-A 20-alpha-benzoate (BTX) binding with moderate affinity (pIC50 7.31 +/- 0.24) indicating an interaction with toxin site 2. However, lifarizine accelerated the dissociation of [3H]-BTX and decreased both the affinity and density of sites labelled by [3H]-BTX, suggesting an allosteric interaction with toxin site 2. 3. The binding of [3H]-lifarizine was voltage-sensitive, binding to membranes with higher affinity than to synaptosomes (pIC50 for cold lifarizine = 7.99 +/- 0.09 in membranes and 6.68 +/- 0.14 in synaptosomes). Depolarization of synaptosomes with 130 mM KCl increased the affinity of lifarizine almost 10 fold (pIC50 = 7.86 +/- 0.25). This suggests that lifarizine binds selectively to inactivated sodium channels which predominate both in the membrane preparation and in the depolarized synaptosomal preparation. 4. There was negligible [3H]-lifarizine and [3H]-BTX binding to solubilized sodium channels, although [3H]-STX binding was retained under these conditions. 5. The potencies of a series of compounds in displacing [3H]-lifarizine from rat cerebrocortical membranes correlated well with their affinities for inactivated sodium channels estimated from whole-cell voltage clamp studies in the mouse neuroblastoma cell line, NIE-115 (r=0.96).6. These results show that [3H]-lifarizine is a high affinity ligand for neuronal sodium channels which potently and selectively labels a site, allosterically linked to toxin binding site 2, associated within activated sodium channels.

Electrophysiological actions of phenytoin on N-methyl-D-aspartate receptor-mediated responses in rat hippocampus in vitro.

1. The effects of the anticonvulsant, phenytoin, have been examined on N-methyl-D-aspartate (NMDA) receptor-mediated population spikes in the CA1 region of the rat hippocampus in vitro. 2. The 'conventional' (AMPA receptor-mediated) CA1 population spike, evoked by electrical stimulation of the Schaffer collateral/commissural pathway, was abolished by 5 min treatment with 5 x 10(-6) M 6-cyano-7-nitroquinoxaline-2,3-dione (CNQX), after which superfusion with a nominally Mg(2+)-free Krebs solution (containing 5 x 10(-6) M CNQX) led to the appearance of an epileptiform population spike which was fully developed by 30-40 min. 3. The epileptiform population spike was abolished by the non-competitive NMDA antagonist, dizocilpine (1 x 10(-6) M, 20-30 min) and inhibited by the competitive NMDA receptor antagonist, D-CPP (IC50 for reducing the amplitude of the first spike in the train = 8.3 x 10(-7) M), demonstrating that the response was mediated by activation of NMDA receptors and validating its use as an assay for antagonists acting at the NMDA receptor/channel complex. 4. Phenytoin (0.1, 0.3 and 1 x 10(-4) M applied cumulatively for 30 min at each concentration) failed to inhibit the NMDA receptor-mediated epileptiform population response (n = 7 slices). 5. Phenytoin (3 x 10-6 M to 1 x 10-4M) attenuated the effects of the sodium channel activator,veratridine (2 x 10-6 M), on the CAl population spike amplitude (recorded in normal Krebs solution),indicating that the previously observed lack of effect of phenytoin on the NMDA receptor-mediated response was not due to impaired access of phenytoin to the biophase.6. These data support the conclusion that antagonism of NMDA receptor-mediated events is not a pharmacological property of phenytoin and that such an action is therefore unlikely to contribute to the anticonvulsant activity of this drug.

Neuroprotective profile of lifarizine (RS-87476) in rat cerebrocortical neurones in culture.

1. The ability of the neuroprotective agent, lifarizine (RS-87476), to mitigate veratridine-, cyanide- and glutamate-induced toxicity in rat embryonic cerebrocortical neurones in primary culture has been compared with that of tetrodotoxin (TTX), nitrendipine, (+)-MK-801 and (-)-MK-801. Lactate dehydrogenase (LDH) released into the culture medium was used as the indicator of cell viability. 2. Incubation of cultures for 16 h in a medium containing veratridine (10(-4) M), sodium glutamate (10(-3) M) or sodium cyanide (10(-3) M) resulted in consistent elevations of LDH activity in the culture medium. The ability of compounds to attenuate these elevations was expressed as the concentration required to inhibit the increases in LDH release by 50% (IC50). 3. Neurotoxicity induced by veratridine was inhibited by lifarizine (IC50 = 4 x 10(-7) M), TTX (IC50 = 3 x 10(-8) M) and nitrendipine (IC50 = 3 x 10(-5) M). In contrast, (+)-MK-801 (up to 3 x 10(-5) M) was ineffective against this insult. 4. Glutamate-induced neurotoxicity was inhibited by (+)-MK-801 (IC50 = 1.4 x 10(-8) M) and to a lesser extent by (-)-MK-801 (IC50 = 1 x 10(-7) M), but was unaffected by lifarizine, TTX or nitrendipine (up to 10(-6) M). 5. (+)-MK-801 was effective against sodium cyanide-induced neurotoxicity (IC50 = 1.9 x 10(-8) M), whereas lifarizine and TTX (up to 10(-6) M) and nitrendipine (up to 3 x 10(-6) M) were without protective activity against this insult. 6. The results demonstrate that lifarizine potently protects rat cortical neurones in vitro against a neurotoxic insult that requires activation of sodium channels for its expression, and that the compound is ineffective against insults mediated by N-methyl-D-aspartate receptor activation. The weak efficacy of nitrendipine against veratridine-induced cell death argues against the involvement of L-type calcium channels in this insult. These data are consistent with the notion that the neuroprotective activity oflifarizine observed in vivo may be mediated by inhibition of neuronal sodium currents.

Actions of the novel neuroprotective agent, lifarizine (RS-87476), on voltage-dependent sodium currents in the neuroblastoma cell line, N1E-115.

1. The actions of the neuroprotective agent, lifarizine (RS-87476-190), on voltage-dependent Na+ currents have been examined in the neuroblastoma cell line, N1E-115, using the whole-cell variant of the patch clamp technique. 2. At a holding potential of -80 mV, lifarizine reduced the peak Na+ current evoked by a 10 ms depolarizing step with an IC50 of 1.3 microM. At holding potentials of -100 and -60 mV the IC50 concentrations of lifarizine were 7.3 microM and 0.3 microM, respectively. 3. At a holding potential of -100 mV, most channels were in the resting state and the IC50 value for inhibition of Na+ current should correspond to the dissociation constant of lifarizine for resting channels (KR). KR was therefore estimated to be 7.3 microM. 4. In the absence of lifarizine, recovery from inactivation following a 20 s depolarization from -100 mV to 0 mV was complete within 2 s. However, in the presence of 3 microM lifarizine recovery took place in a biexponential fashion with time constants of 7 s and 79 s. 5. Lifarizine (1 microM) had no effect on steady-state inactivation curves when conditioning pre-pulses of 1 s duration were used. However, when pre-pulse durations of 1 min were used the curves were shifted to the left by lifarizine by about 10 mV. Analysis of the shifts induced by a range of lifarizine concentrations revealed that the apparent affinity of lifarizine for the inactivated state of the channel (K1) was 0.19 microM. 6. Lifarizine (1 microM) had no effect on chloramine-T-modified Na+ currents, suggesting no significant open channel interaction. 7. Taken together, these data show that lifarizine is a potent voltage-dependent inhibitor of Na+currents in NIE-115 cells and that the voltage-dependence arises from an interaction of the compound with the inactivated state of the channel. The possible contribution of Na+ current inhibition to the neuroprotective actions of lifarizine is discussed.

Presynaptic M1 muscarinic cholinoceptors mediate inhibition of excitatory synaptic transmission in the hippocampus in vitro.

The effects of the cholinoceptor agonist, carbachol (CCh), were examined in the rat hippocampal slice preparation. Intracellular recordings from CA1 pyramidal neurones revealed that CCh (1-3 microM) inhibited excitatory postsynaptic responses evoked by stimulation of the Schaffer collateral/commissural pathway while, at the same time, direct excitability was enhanced. Extracellularly, CCh produced a concentration-dependent reduction of the amplitude of the field excitatory postsynaptic potential (field EPSP) recorded in the CA1 apical dendritic region. The muscarinic receptor antagonist, pirenzepine, competitively antagonized the effects of CCh on the field EPSP with a pA2 of 7.4. These results confirm earlier reports of a presynaptic inhibitory action of CCh in the hippocampal CA1 region and provide strong evidence that this effect is mediated by muscarinic receptors of the M1 subtype.

Involvement of M1-muscarinic receptors in the excitation of neocortical neurones by acetylcholine.

The technique of microelectrophoresis was used to investigate the cholinoceptor pharmacology of spontaneously active single neurones in the parietal cortex of the rat. Acetylcholine, carbachol and the selective M1-muscarinic receptor agonist, McN-A-343, were each potent excitants (rank order of apparent potency: carbachol greater than acetylcholine greater than McN-A-343). When measured in vitro, the apparent mobilities of carbachol and acetylcholine were similar although significantly less than that of McN-A-343, suggesting that the lower potencies of acetylcholine and McN-A-343 probably reflect a genuine biological phenomenon. In addition to excitation, carbachol also evoked biphasic (excitation/depression) and depressant responses. In contrast to the other cholinoceptor agonists, nicotine produced weak and inconsistent excitations. Excitatory responses to acetylcholine and carbachol were significantly attenuated by the selective M1-muscarinic receptor antagonist, pirenzepine, at a time when the excitatory response to McN-A-343 was also significantly reduced. Responses to phenylephrine were not diminished. On several cells an excitatory response to carbachol was converted to a depression by pirenzepine. These results suggest that the excitatory responses of cortical neurones to cholinoceptor agonists are mediated predominantly by M1-muscarinic receptors. The identity of the receptor mediating the depressant response to carbachol remains uncertain, although nicotinic cholinoceptors do not appear to be involved.

Excitatory neuronal responses to dopamine in the cerebral cortex: involvement of D2 but not D1 dopamine receptors.

The technique of microelectrophoresis was used to evaluate the relative contribution of D1 and D2 dopamine receptors towards the mediation of the excitatory response of single neurones to dopamine in the somatosensory cortex of the rat. The selective D1 dopamine receptor agonist, SKF 38393, failed to excite any of the cells to which it was applied. In contrast, the selective D2 dopamine receptor agonist, LY 171555, excited the majority of cells tested. The apparent potency of LY 171555 was significantly lower than that of dopamine. When the mobilities of SKF 38393 and LY 171555 were assessed by an in vitro method, they were found to be at least as great as those of dopamine and phenylephrine, suggesting that the lack of effect of SKF 38393 and the lower apparent potency of LY 171555 compared to dopamine reflect genuine biological phenomena. The alpha 1-adrenoceptor antagonist, prazosin, discriminated between excitatory responses to the alpha 1-adrenoceptor agonist, phenylephrine, and LY 171555: responses to phenylephrine were more susceptible to antagonism than were those to LY 171555. The dopamine receptor antagonist, haloperidol, produced the reverse discrimination: responses to LY 171555 were more affected than were those to phenylephrine. Neither antagonist reduced the response to the control agonist, acetylcholine. When applied continuously with low ejecting currents, LY 171555 antagonized the excitatory response to dopamine while the response to phenylephrine was relatively preserved. The response to acetylcholine was unaffected. When similarly applied, SKF 38393 had no selective action on the response to dopamine. 6 These results suggest that D2 dopamine receptors are involved in mediating the excitatory neuronal response to dopamine in the cerebral cortex, whereas DI dopamine receptors are unlikely to be involved. LY 171555 appears to act as a partial agonist at D2 dopamine receptors in this test system.

Neuronal responses to noradrenaline in the cerebral cortex: evidence against the involvement of alpha 2-adrenoceptors.

The technique of microelectrophoresis was used to test the hypothesis that alpha 2-adrenoceptors are involved in mediating the excitatory responses of single neurones to noradrenaline in the somatosensory cerebral cortex of the rat. In the first series of experiments the effects of two alpha 2-adrenoceptor antagonists, yohimbine and idazoxan (RX-781094), were compared on excitatory responses to noradrenaline, phenylephrine and acetylcholine. The response to noradrenaline was not more susceptible to antagonism by these drugs than the response to the alpha 1-adrenoceptor stimulant, phenylephrine. Yohimbine antagonized responses to all three agonists equally, while idazoxan antagonized responses to noradrenaline and phenylephrine equally with relative preservation of responses to acetylcholine. In the second series of experiments the effects of the selective alpha 2-adrenoceptor stimulant, UK-14304, were examined. UK-14304 produced weak and inconsistent excitations on a small number of cells; however, most of the cells did not respond to this drug. When applied continuously using low ejection currents, UK-14304 selectively and reversibly antagonized responses to noradrenaline and phenylephrine without affecting responses to acetylcholine. These results suggest that, in the somatosensory cortex of the rat, neuronal excitation to noradrenaline is unlikely to be mediated either wholly or partly by alpha 2-adrenoceptors. The antagonism of neuronal responses to noradrenaline and phenylephrine by idazoxan probably reflects the alpha 1-adrenoceptor antagonistic properties of the drug which is known to occur at higher concentrations. The low agonistic potency of UK-14304 and the antagonism of responses to noradrenaline and phenylephrine by UK-14304 suggest that this drug, like clonidine, may act as a partial agonist at alpha-adrenoceptors.