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There is an increasing demand for in vitro test systems to detect neurotoxicity for use in chemical risk assessment. In this study, we evaluated the applicability of rat primary cortical cultures grown on multi-well micro-electrode arrays (mwMEAs) to detect effects of chronic 14-day exposure to structurally different insecticides or methylmercury on neuronal activity (mean spike rate; MSR). Effects of chronic exposure to α-cypermethrin, endosulfan, carbaryl, chlorpyrifos(-oxon), methylmercury or solvent control [14days exposure, initiated after baseline recording at day in vitro (DIV)7] were studied in five successive recordings between DIV10 and DIV21. The results were compared to effects of acute exposure to these same compounds (activity recorded immediately after the start of exposure after baseline recording at DIV10-11). Chronic 14-day exposure to methylmercury, chlorpyrifos and α-cypermethrin inhibited MSR, all with a lowest-observed effect concentration (LOEC) of 0.1μM, while exposure to endosulfan increased MSR [LOEC: 1μM]. No significant effects were observed for chlorpyrifos-oxon and carbaryl. Similar to the observations in the chronic 14-day exposure studies, MSR was inhibited by acute 30-min exposure to methylmercury, chlorpyrifos, and α-cypermethrin [LOECs: 1μM, 10μM, and 1μM, respectively], whereas endosulfan increased MSR [LOEC: 0.3μM]. While not observed in the chronic 14-day exposure study, acute exposure to chlorpyrifos-oxon and carbaryl resulted in inhibition of MSR [LOECs: 10μM, and100 μM, respectively]. Effects on median interspike intervals (mISI; a measure for neuronal firing pattern) were not detected following chronic 14-day or acute 30-min exposure, except for increased mISI at acute chlorpyrifos and α-cypermethrin exposures at concentrations that also inhibited MSR. These data indicate that the effects of chronic 14-day exposures to methylmercury and insecticides at low concentrations on spontaneous neuronal activity in vitro can be predicted in rapid acute screening studies using mwMEAs., (Copyright © 2016 Elsevier B.V. All rights reserved.)

Exposure to 50-60 Hz extremely low-frequency electromagnetic fields (ELF-EMFs) has increased considerably over the last decades. Several epidemiological studies suggested that ELF-EMF exposure is associated with adverse health effects, including neurotoxicity. However, these studies are debated as results are often contradictory and the possible underlying mechanisms are unknown. Since the developing nervous system is particularly vulnerable to insults, we investigate effects of chronic, developmental ELF-EMF exposure in vitro. Primary rat cortical neurons received 7 days developmental exposure to 50 Hz block-pulsed ELF-EMF (0-1000 μT) to assess effects on cell viability (Alamar Blue/CFDA assay), calcium homeostasis (single cell fluorescence microscopy), neurite outgrowth (β(III)-Tubulin immunofluorescent staining), and spontaneous neuronal activity (multi-electrode arrays). Our data demonstrate that cell viability is not affected by developmental ELF-EMF (0-1000 μT) exposure. Depolarization- and glutamate-evoked increases in intracellular calcium concentration ([Ca(2+)]i) are slightly increased at 1 μT, whereas both basal and stimulation-evoked [Ca(2+)]i show a modest inhibition at 1000 μT. Subsequent morphological analysis indicated that neurite length is unaffected up to 100 μT, but increased at 1000 μT. However, neuronal activity appeared largely unaltered following chronic ELF-EMF exposure up to 1000 μT. The effects of ELF-EMF exposure were small and largely restricted to the highest field strength (1000 μT), ie, 10 000 times above background exposure and well above current residential exposure limits. Our combined data therefore indicate that chronic ELF-EMF exposure has only limited (developmental) neurotoxic potential in vitro., (© The Author 2015. Published by Oxford University Press on behalf of the Society of Toxicology. All rights reserved. For Permissions, please e-mail: journals.permissions@oup.com.)

Piperazine derivatives are a class of psychoactive substances applied in prescription medicines like antidepressants as well as in drugs of abuse. They are known to increase brain levels of catecholamines, likely via reversal of reuptake transporters. However, other mechanisms could also contribute to increased neurotransmitter levels, e.g., reduced inhibitory inputs on catecholaminergic neurons. Inhibition of the main inhibitory input in the brain, the GABAergic system, by piperazine derivatives could contribute to increased neurotransmitter levels. Our previous studies support this by demonstrating that 1-(3-chlorophenyl)piperazine (3CPP/mCPP) is an antagonist of the human α1β2γ2 GABAA receptor (GABAA-R). We therefore investigated the effect of 12 additional piperazine derivatives on the function of the human α1β2γ2 GABAA-R expressed in Xenopus oocytes, using the two-electrode voltage-clamp technique. Tested derivatives included benzylpiperazine (BZP), methylbenzylpiperazines (2/3MBP), phenylpiperazine (PP), methoxyphenylpiperazines (2/3/4MPP/MeOPP), chlorophenylpiperazines (2/4CPP) and fluorophenylpiperazines (4FPP/TFMPP). All derivatives concentration-dependently inhibited the GABA-evoked ion current. Chlorophenylpiperazines were the most potent GABAA-R antagonists; the IC20 value for 1-(2-chlorophenyl)piperazine (2CPP) was 46μM and 2CPP induced a maximum inhibition of ∼ 90% at 1mM. Derivatives can be ranked as follows from highest to lowest potency based on IC20 values: 2CPP>3MPP>4CPP>4MPP>2MBP>3CPP>PP>4FPP>2MPP>TFMPP>3MBP>BZP. This study demonstrates a novel mode of action of piperazine derivatives, i.e., antagonism of the GABAA-R. This mechanism can result in increased catecholamine levels that indirectly contribute to toxicity, e.g., adverse effects during overdoses. Therefore, this important mode of action is not only relevant for therapeutic psychiatric interventions, but could also proof valuable for therapeutic interventions in intoxications., (Copyright © 2015 Elsevier Inc. All rights reserved.)

Scope: At the European level, detection of marine neurotoxins in seafood is still based on ethically debated and expensive in vivo rodent bioassays. The development of alternative methodologies for the detection of marine neurotoxins is therefore of utmost importance. We therefore investigated whether and to what extent a multielectrode array (MEA) approach can be used as an in vitro alternative for screening of marine neurotoxins potentially present in seafood., Methods: This MEA approach utilizes rat cortical neurons comprising a wide range of ion channels/pumps and neurotransmitter receptors targeted by marine neurotoxins. We tested the effects of neurotoxic model compounds, pure marine neurotoxins, and extracts from contaminated seafood on neuronal activity of rat cortical neurons cultured on commercial 48-well plates to increase throughput., Conclusion: We demonstrate that the MEA approach has a sensitivity of 88% (7/9 model compounds, 6/6 pure marine neurotoxins, and 2/2 marine neurotoxins present in seafood extracts were correctly identified) and a good reproducibility compared to existing in vitro alternatives. We therefore conclude that this MEA-based approach could be a valuable tool for future food safety testing., (© 2014 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.)

Yearly, exposure to drugs of abuse results in ∼1 million emergency department visits in the US. In ∼50% of the visits, stimulant drugs like cocaine and amphetamine-like substances (e.g. 3,4-methylenedioxymethamphetamine (MDMA, the main active ingredient of ecstasy)) are involved, whereas in ∼60% multiple drugs are involved. These drugs induce higher dopamine and serotonin levels resulting in drug-induced toxicity. Since GABA receptors (GABA-R) provide the main inhibitory input on dopaminergic and serotonergic neurons, drug-induced inhibition of GABA-R could contribute to higher neurotransmitter levels and thus toxicity. We therefore investigated the effects of combinations of commonly abused stimulant drugs (cocaine, MDMA, 3,4-methylenedioxyamphetamine (MDA) and meta-chlorophenylpiperazine (mCPP)) on the function of the human α1β2γ2 GABAA receptor (hGABAA-R), expressed in Xenopus oocytes, using the two-electrode voltage-clamp technique. These drugs concentration-dependently inhibited the GABA-evoked current (mCPP>cocaine>MDMA>MDA). Most drug combinations decreased the GABA-evoked current stronger than the single drug. Additivity was observed during combined exposure to low concentrations of cocaine and mCPP as well as during combined exposure to MDA with cocaine or mCPP. However, combinations containing MDMA mainly resulted in sub-additivity or no additivity. At drug concentrations relevant for clinical toxicology, co-exposure to ≥2 drugs can decrease the GABA-evoked current in an additive manner. Thus, in patients exposed to multiple drugs, inhibitory GABA-ergic input is reduced more prominently, likely resulting in higher brain dopamine levels. As this will increase the risk for drug-induced toxicity, treatment of drug-intoxicated patients with drugs that enhance GABA-ergic input should be further optimized., (Copyright © 2012 Elsevier Inc. All rights reserved.)

Brominated flame retardants (BFRs) are abundant persistent organic pollutants with well-studied toxicity. The toxicological and ecological concern associated with BFRs argues for replacement by safer alternatives. However, the (neuro)toxic potential of alternative halogen-free flame retardants (HFFRs) is unknown. Previous research identified the nervous system as a sensitive target organ for BFRs, with modulation of excitatory nicotinic acetylcholine (nACh) receptors as one of the modes of action. Since it is essential to assess the (neuro)toxic potential of HFFRs before large scale use, we measured the effects of three BFRs and 13 HFFRs on the function of human α(4)β(2) nACh receptors, expressed in Xenopus oocytes, using the two-electrode voltage-clamp technique. The results demonstrate that some BFRs (TBBPA and to a lesser extent BDE-209) and HFFRs (TPP, Alpi, APP, MMT and to a lesser extent ATH, ATO, MHO, MPP, RDP and ZHS) act as nACh receptor antagonists. Contrary, BPS, BDP, DOPO and ZS were unable to modulate nACh receptors. Despite the lack of toxicological data on HFFRs and the need for additional studies to perform a full (neuro)toxic risk assessment, the current data on antagonistic effects on nACh receptors could be an important step in prioritizing viable HFFRs for substitution of BFRs., (Copyright © 2012 Elsevier Ireland Ltd. All rights reserved.)

Neurotoxicological data on the widely used brominated flame retardant tetrabromobisphenol-A (TBBPA) is limited. Since recent studies indicated that inhibitory GABA(A) and excitatory α(4)β(2) nicotinic acetylcholine (nACh) receptors are sensitive targets for persistent organic pollutants, we investigated the effects of TBBPA on these receptors, expressed in Xenopus oocytes, using the two-electrode voltage-clamp technique. Our results demonstrate that TBBPA acts as full (≥ 10 μM) and partial (≥ 0.1 μM) agonist on human GABA(A) receptors, whereas it acts as antagonist (≥ 10 μM) on human α(4)β(2) nACh receptors. Next, neuronal B35 cells were used to further study the effects of TBBPA on calcium-permeable nACh receptors using single-cell fluorescent calcium imaging. These results demonstrate that TBBPA (≥ 1 μM) inhibits acetylcholine (ACh) receptors as evidenced by a reduction in the ACh-evoked increases in the intracellular calcium concentration ([Ca(2+)](i)). Additionally, TBBPA (> 1 μM) induced a strong and concentration-dependent increase in basal [Ca(2+)](i) in B35 cells. Similarly, TBBPA (> 1 μM) increases basal [Ca(2+)](i) in dopaminergic PC12 cells. This increase is also evident under calcium-free conditions, indicating it originates from intracellular calcium stores. Moreover, depolarization-evoked increases in [Ca(2+)](i) are strongly reduced by TBBPA (≥ 1 μM), indicating TBBPA-induced inhibition of voltage-gated calcium channels. Our in vitro studies thus demonstrate that TBBPA exerts several adverse effects on functional neurotransmission endpoints with effect concentrations that are only two orders of magnitude below the highest cord serum concentrations. Although epidemiological proof for adverse TBBPA effects is lacking, our data justify the quest for flame retardants with reduced neurotoxic potential.

Drugs of abuse are known to mainly affect the dopaminergic and serotonergic system, although behavioral studies indicated that the GABA-ergic system also plays a role. We therefore investigated the acute effects of several commonly used drugs of abuse (methamphetamine, amphetamine, 3,4-methylenedioxymethamphetamine (MDMA), 3,4-methylenedioxyamphetamine (MDA) and meta-chlorophenylpiperazine (mCPP)) on the function of the human α(1)β(2)γ(2) GABA(A) receptor (hGABA(A)-R), expressed in Xenopus oocytes, using the two-electrode voltage-clamp technique. Although none of the tested drugs acted as full agonist on the hGABA(A)-R, some drugs induced differential modulation of hGABA(A)-R function, depending on the degree of receptor occupancy. Methamphetamine did not affect the GABA-evoked current at high receptor occupancy, but induced a minor inhibition at low receptor occupancy. Its metabolite amphetamine slightly potentiated the GABA-evoked current. MDMA and its metabolite MDA both inhibited the current at low receptor occupancy. However, MDMA did not affect the current at high occupancy, whereas MDA induced a potentiation. mCPP induced a strong inhibition (max. ∼ 80%) at low receptor occupancy, but ∼ 25% potentiation at high receptor occupancy. Competitive binding to one of the GABA-binding sites could explain the drug-induced inhibitions observed at low receptor occupancy, whereas an additional interaction with a positive allosteric binding site may play a role in the observed potentiations at high receptor occupancy. This is the first study to identify direct modulation of hGABA(A)-Rs as a novel mode of action for several drugs of abuse. Consequently, hGABA(A)-Rs should be considered as target for psychiatric pharmaceuticals and in developing treatment for drug intoxications., (Copyright © 2011 Elsevier Inc. All rights reserved.)

The neurotoxic potential of non-dioxin-like polychlorinated biphenyls (NDL-PCBs) is characterized by disruption of presynaptic processes, including calcium homeostasis and neurotransmitter transport. Recently, using a limited set of congeners, we demonstrated that PCB28 and PCB52 can potentiate postsynaptic GABA(A) receptors. In the present study, effects of 20 NDL-PCBs and 2 dioxin-like PCBs, selected based on their chemical variation and abundance in the environment, on human GABA(A) receptors were investigated. GABA(A) receptors were expressed in Xenopus oocytes, and NDL-PCB effects were determined using the two-electrode voltage-clamp technique. Results demonstrate that lower chlorinated PCB19, PCB28, PCB47, PCB51, PCB52, PCB95, and PCB100 act as a partial agonists (at low receptor occupancy), i.e., potentiating the receptor response during coapplication with GABA (at EC(20)). Importantly, PCB19, PCB47, PCB51, and PCB100 can also act as full agonist, i.e., activate the GABA(A) receptor in the absence of GABA. Potentiation and activation of the GABA(A) receptor is concentration dependent and limited to NDL-PCBs that have 3-5 chlorine atoms, 1-3 ortho-substitutions, an equal number (0-1) of meta-substitutions on both phenyl rings, and do not have an adjacent para- and meta-substitution on the same phenyl ring. Activation and potentiation of the GABA(A) receptor by PCB47, the most potent congener (lowest observed effect concentration of 10nM), is attenuated when coapplied with PCB19, PCB28, PCB153, or PCB180, indicative for competitive binding. Considering the importance of GABA-ergic signaling for brain development, motor coordination, learning, and memory, this mode of action can contribute to the previously observed NDL-PCB-induced neurobehavioral and neurodevelopmental effects and should be included in human risk assessment.

PCBs are still ubiquitous pollutants despite the ban on their industrial and commercial use. To date, risk characterization and assessment of non-dioxin-like PCBs (NDL-PCBs), especially with respect to neurotoxicity, is hampered by a lack of data. Therefore, the effects of six common NDL congeners (PCB28, 52, 101, 138, 153 and 180) on human GABA(A) receptors, expressed in Xenopus oocytes, were investigated using the two-electrode voltage-clamp technique. When coapplied with GABA (at EC(20)), PCB28 and PCB52 concentration-dependently potentiate the GABA(A) receptor-mediated ion current. Though the LOEC for both PCB28 and PCB52 is 0.3 microM, PCB28 is more potent than PCB52 (maximum potentiation at 10 muM amounting to 98.3 +/- 12.5% and 25.5 +/- 1.4%, respectively). Importantly, coapplication of PCB28 (0.3 microM) and PCB52 (10 microM) resulted in an apparently additive potentiation of the GABA(A) response, whereas coapplication of PCB28 (0.3 microM) and PCB153 (10 microM) attenuated the PCB28-induced potentiation. The present results suggest that the potentiation of human GABA(A) receptor function is specific for lower-chlorinated NDL-PCBs and that higher molecular weight PCBs may attenuate this potentiation as a result of competitive binding to human GABA(A) receptors. Nonetheless, this novel mode of action could (partly) underlie the previously recognized NDL-PCB-induced neurobehavioral alterations.

Ethylbenzene and para-xylene (p-xylene), but not the chemically closely related organic solvents ortho-xylene (o-xylene) and meta-xylene (m-xylene), are known to cause ototoxicity and irreversible hearing loss, though the underlying mechanisms are still unknown. In this study, effects of ethylbenzene and of p-, o-, and m-xylene on human heteromeric alpha9alpha10 nicotinic acetylcholine receptors (nAChRs) expressed in Xenopus oocytes were investigated using the two-electrode voltage clamp technique. ACh dose-dependently evoked an alpha9alpha10 nAChR-mediated ion current with an EC(50) of 137 microM. When ACh is applied at a low concentration (10 microM), the nAChR-mediated ion current is inhibited by a low concentration (10 microM) of ethylbenzene and p-xylene, but not by the same concentration of the non-ototoxic solvents. At a high solvent concentration (300 microM), all solvents cause inhibition of the ion currents evoked by 10 microM ACh. Ion currents evoked by a near maximum-effective concentration ACh (1mM) are inhibited by the selected organic solvents only at 300 microM. These results demonstrate that low concentrations of the known ototoxic solvents ethylbenzene and p-xylene inhibit alpha9alpha10 nAChR-mediated ion currents, whereas the structurally related, non-ototoxic solvents m-xylene and o-xylene do not, indicating that the alpha9alpha10 nAChR is a potential target for solvent-induced ototoxicity.

Background: Oxidative metabolism, resulting in the formation of hydroxylated polybrominated diphenyl ether (PBDE) metabolites, may enhance the neurotoxic potential of brominated flame retardants., Objective: Our objective was to investigate the effects of a hydroxylated metabolite of 2,2',4,4'-tetra-bromodiphenyl ether (BDE-47; 6-OH-BDE-47) on changes in the intracellular Ca2+ concentration ([Ca2+]i) and vesicular catecholamine release in PC12 cells., Methods: We measured vesicular catecholamine release and [Ca2+]i using amperometry and imaging of the fluorescent Ca2+-sensitive dye Fura-2, respectively., Results: Acute exposure of PC12 cells to 6-OH-BDE-47 (5 microM) induced vesicular catecholamine release. Catecholamine release coincided with a transient increase in [Ca2+]i, which was observed shortly after the onset of exposure to 6-OH-BDE-47 (120 microM). An additional late increase in [Ca2+]i was often observed at > or =1 microM 6-OH-BDE-47. The initial transient increase was absent in cells exposed to the parent compound BDE-47, whereas the late increase was observed only at 20 microM. Using the mitochondrial uncoupler carbonyl cyanide 4-(trifluoromethoxy)phenylhydrazone (FCCP) and thapsigargin to empty intracellular Ca2+ stores, we found that the initial increase originates from emptying of the endoplasmic reticulum and consequent influx of extracellular Ca2+, whereas the late increase originates primarily from mitochondria., Conclusion: The hydroxylated metabolite 6-OH-BDE-47 is more potent in disturbing Ca2+ homeostasis and neurotransmitter release than the parent compound BDE-47. The present findings indicate that bioactivation by oxidative metabolism adds considerably to the neurotoxic potential of PBDEs. Additionally, based on the observed mechanism of action, a cumulative neurotoxic effect of PBDEs and ortho-substituted polychlorinated biphenyls on [Ca2+]i cannot be ruled out.

Background: Increasing environmental levels of brominated flame retardants raise concern about possible adverse effects, particularly through early developmental exposure., Objective: The objective of this research was to investigate neurodevelopmental mechanisms underlying previously observed behavioral impairments observed after neonatal exposure to polybrominated diphenyl ethers (PBDEs)., Methods: C57Bl/6 mice received a single oral dose of 2,2',4,4'-tetrabromodiphenyl ether (BDE-47) on postnatal day (PND) 10 (i.e., during the brain growth spurt). On PND17-19, effects on synaptic plasticity, levels of postsynaptic proteins involved in long-term potentiation (LTP), and vesicular release mechanisms were studied ex vivo. We investigated possible acute in vitro effects of BDE-47 on vesicular catecholamine release and intracellular Ca(2+) in rat pheochromocytoma (PC12) cells., Results: Field-excitatory postsynaptic potential (f-EPSP) recordings in the hippocampal CA1 area demonstrated reduced LTP after exposure to 6.8 mg (14 micromol)/kg body weight (bw) BDE-47, whereas paired-pulse facilitation was not affected. Western blotting of proteins in the postsynaptic, triton-insoluble fraction of hippocampal tissue revealed a reduction of glutamate receptor subunits NR2B and GluR1 and autophosphorylated-active Ca(2+)/calmodulin-dependent protein kinase II (alphaCaMKII), whereas other proteins tested appeared unaffected. Amperometric recordings in chromaffin cells from mice exposed to 68 mg (140 micromol)/kg bw BDE-47 did not reveal changes in catecholamine release parameters. Modest effects on vesicular release and intracellular Ca(2+) in PC12 cells were seen following acute exposure to 20 microM BDE-47. The combined results suggest a post-synaptic mechanism in vivo., Conclusion: Early neonatal exposure to a single high dose of BDE-47 causes a reduction of LTP together with changes in postsynaptic proteins involved in synaptic plasticity in the mouse hippocampus.

Effects of cholinergic drugs on human alpha4beta2 nicotinic acetylcholine receptors expressed in Xenopus oocytes have been investigated in electrophysiological and ligand binding experiments. Atropine, scopolamine, physostigmine, and tacrine combine potentiation of ion current induced by low concentrations of acetylcholine with inhibition of ion current evoked by high concentrations of acetylcholine. Rivastigmine, galanthamine, and dichlorvos cause only inhibition of ion current evoked by low concentrations of acetylcholine. Binding experiments show that the potentiating cholinergic drugs atropine, scopolamine, and physostigmine are competitive ligands of human alpha4beta2 nicotinic acetylcholine receptors. Conversely, the inhibitory cholinergic drugs galanthamine and rivastigmine are non-competitive. The non-competitive drugs are not allosteric, since they do not affect the saturation curve of the radioligand [3H]cytisine. Effects of potentiating cholinergic drugs on nicotinic acetylcholine receptors are consistent with and predicted by a model comprising competitive drug effects at two equivalent agonist recognition sites on the nicotinic acetylcholine receptor combined with non-competitive ion channel block.

Chronic and acute exposure to organophosphate (OP) pesticides may lead to persistent neurological and neurobehavioral effects, which cannot be explained by acetylcholinesterase (AChE) inhibition alone. It is suggested that other brain proteins are involved. Effects of commonly used organophosphate pesticides on rat neuronal alpha4beta2 nicotinic acetylcholine receptors (nAChRs) expressed in Xenopus laevis oocytes have been investigated using the two-electrode voltage clamp technique. Several OP pesticides, e.g., parathion-ethyl, chlorpyrifos and disulfoton, inhibited the ACh-induced ion current with potencies in the micromolar range. The potency of inhibition increased with increasing concentrations of the agonist ACh. Comparison of the potency of nAChR inhibition with the potency of AChE inhibition demonstrated that some OPs inhibit nAChRs more potently than AChE. Binding experiments on alpha4beta2 nAChRs showed that the OPs noncompetitively interact with nAChRs. The inhibitory effects on nAChRs are adequately described and explained by a sequential two-step mechanism, in which rapidly reversible OP binding to a separate binding site leads to inhibition followed by a stabilization of the blocked state or receptor desensitization. It is concluded that OPs interact directly with neuronal alpha4beta2 nAChRs to inhibit the agonist-induced response. This implicates that neuronal alpha4beta2 nAChRs are additional targets for some OP pesticides.

The mechanism by which carbamate pesticides inhibit rat alpha4beta2 nicotinic acetylcholine (ACh) receptors (nAChRs) expressed in Xenopus laevis oocytes has been investigated using the two-electrode voltage clamp technique. Carbaryl, S-ethyl N,N-dipropylthiocarbamate (EPTC), and fenoxycarb inhibit ACh-induced ion currents in a concentration-dependent way. EPTC and fenoxycarb inhibit ion currents induced by 1 mM ACh with 3-fold to 5-fold higher potency than ion currents induced by 1 microM ACh. The potency of carbaryl appears to be independent of ACh concentration. Fenoxycarb displaces (3)H-epibatidine bound to alpha4beta2 (nAChRs) with a K(i) of 750 microM, which is much higher than the functional IC(50) of 2.3-11 microM. This shows that the inhibition of ion current by the carbamate is a noncompetitive effect. Inhibition by fenoxycarb is independent of the state of the ion channel. The rate of onset of inhibition is enhanced, and the rate of reversal of inhibition is reduced, when the concentration of fenoxycarb is increased. The rate of reversal of inhibition is also reduced when the period of exposure to fenoxycarb is increased. The time- and concentration-dependent inhibition of nAChR-mediated ion current by fenoxycarb is accounted for by a two-step mechanism involving a rapid blocked state and a sequential more stably blocked or desensitized state.

Effects of commonly used carbamate pesticides on rat neuronal nicotinic acetylcholine receptors expressed in Xenopus laevis oocytes have been investigated using the two-electrode voltage clamp technique. The potencies of these effects have been compared to the potencies of the carbamates to inhibit rat brain acetylcholinesterase. The potency order of six carbamates to inhibit alpha4beta4 nicotinic receptors is fenoxycarb > EPTC > carbaryl, bendiocarb > propoxur > aldicarb with IC50 values ranging from 3 microM for fenoxycarb to 165 microM for propoxur and >1 mM for aldicarb. Conversely, the potency order of these carbamates to inhibit rat brain acetylcholinesterase is bendiocarb > propoxur, aldicarb > carbaryl > EPTC, fenoxycarb with IC50 values ranging from 1 microM for bendiocarb to 17 microM for carbaryl and > mM for EPTC and fenoxycarb. The alpha4beta2, alpha3beta4, and alpha3beta2 nicotinic acetylcholine receptors are inhibited by fenoxycarb, EPTC, and carbaryl with potency orders similar to that for alpha4beta4 receptors. Comparing the potencies of inhibition of the distinct subtypes of nicotinic acetylcholine receptors shows that the alpha3beta2 receptor is less sensitive to inhibition by fenoxycarb and EPTC. The potency of inhibition depends on the carbamate as well as on a combination of alpha and beta subunit properties. It is concluded that carbamate pesticides affect different subtypes of neuronal nicotinic receptors independently of acetylcholinesterase inhibition. This implicates that neuronal nicotinic receptors are additional targets for some carbamate pesticides and that these receptors may contribute to carbamate pesticide toxicology, especially after long-term exposure.

The effects of the acetylcholinesterase inhibitors physostigmine and tacrine on alpha4beta2 and alpha4beta4 subtypes of neuronal nicotinic acetylcholine (ACh) receptors, expressed in Xenopus laevis oocytes, have been investigated. In voltage-clamp experiments low concentrations of physostigmine and tacrine potentiate ion currents induced by low concentrations of ACh, whereas at high concentrations they inhibit ACh-induced ion currents. These dual effects result in bell-shaped concentration-effect curves. Physostigmine and tacrine, by themselves, do not act as nicotinic receptor againsts. The larger potentiation is observed with 10 microM: physostigmine on alpha4beta4 nicotinic receptors and amounts to 70% at 1 microM: ACh. The mechanism underlying the effects of physostigmine on alpha4beta4 ACh receptors has been investigated in detail. Potentiation of ACh-induced ion current by low concentrations of physostigmine is surmounted at elevated concentrations of ACh, indicating that this is a competitive effect. Conversely, inhibition of ACh-induced ion current by high concentrations of physostigmine is not surmounted at high concentrations of ACh, and this effect appears mainly due to noncompetitive, voltage-dependent ion channel block. Radioligand binding experiments demonstrating displacement of the nicotinic receptor agonist (125)I-epibatidine from its recognition sites on alpha4beta4 ACh receptors by physostigmine confirm that physostigmine is a competitive ligand at these receptors. A two-site equilibrium receptor occupation model, combined with noncompetitive ion channel block, accounts for the dual effects of physostigmine and tacrine on ACh-induced ion currents. It is concluded that these acetylcholinesterase-inhibiting drugs interact with the ACh recognition sites and are coagonists of ACh on alpha4-containing nicotinic ACh receptors.

The effects of the carbamate physostigmine and of the organophosphates (OPs) parathion, paraoxon and phenyl saligenin cyclic phosphate (PSP) were examined on different subtypes of neuronal nicotinic acetylcholine receptors (nAChR). Stimulation with 1 mM ACh induced transient nicotinic inward currents in mouse N1E-115 and human SH-SY5Y neuroblastoma and in locust thoracic ganglion cells. All four acetylcholinesterase (AChE) inhibitors reduced the nicotinic currents in a concentration-dependent manner. Parathion is about 50 times more potent in blocking nAChR, compared to its active AChE inhibiting metabolite paraoxon. The relative blocking potency of the different AChE inhibitors was the same in all cell types, and followed the order parathion > physostigmine > PSP > paraoxon. In N1E-115 cells the IC50 values of block amounted to 2 microM, 30 microM, 39 microM and 96 microM for parathion, physostigmine, PSP and paraoxon, respectively. In all cell types, the nicotinic currents were equally blocked by parathion. Human nAChR in SH-SY5Y cells appeared more sensitive to block by physostigmine, PSP and paraoxon, while these AChE inhibitors similarly inhibited nicotinic currents in insect cells and in mouse neuroblastoma cells. The observation that the concentration-dependence of block is different from that of AChE inhibition, indicates a distinct interaction of AChE inhibitors with nAChR. Only in locust cells physostigmine induced a non-desensitizing inward current, that appeared to originate from nAChR activation. Occasionally, the OPs were able to activate slow ionic currents in mouse, but not in human and locust cells. As the OP-induced agonistic activity in mouse cells was not associated with the blocking action, the target site appeared to be distinct from nAChR. These results show that AChE inhibitors block nAChR with different potencies, dependent on the compound and the receptor subtype, and may activate distinct ion currents in neuronal cells of different species origin.

The effects of acetylcholine (ACh) and physostigmine (PHY) on thoracic ganglion neurons of Locusta migratoria were investigated using whole-cell and cell-attached voltage clamp. ACh activated whole-cell currents with variable amplitudes, time course and ion channel block between cells, suggesting differential expression of nicotinic acetylcholine receptor (nAChR) subtypes. This was supported by selective block of the peak of the currents by the alpha7-specific alpha-conotoxin ImI. PHY at 100 microM evoked smaller whole-cell currents with variable amplitudes and marginal desensitization. The PHY/ACh amplitude ratio varied between cells, and was positively related to the time constant of decay of the ACh response. EC50 values for the peak amplitude of the ACh- and PHY-induced currents were 50 microM and 3 microM, respectively. Both agonists activated nAChR, indicated by equal voltage-dependence and reversal potentials and the same pharmacological properties of ACh and PHY responses. In addition, PHY and ACh induced ion channel block. Co-application and cross-desensitization experiments showed that ACh and PHY activate the same nAChR subpopulations. Both agonists activated nicotinic single channels with three conductance levels, which were equal for ACh and PHY, indicating activation of the same nAChR subtypes by both agonists. However, for all levels PHY displayed a lower open probability than ACh. Taken together, different whole-cell responses appear to originate from differential activation, desensitization and ion channel block by ACh and PHY of distinct nAChR populations., (Copyright 1998 Elsevier Science B.V.)

Effects of selective and non-selective neurotoxic compounds on membrane currents mediated by nicotinic acetylcholine receptors (nAChR), natively expressed in cultured cells and artificially expressed in Xenopus oocytes, have been investigated in vitro using voltage clamp techniques. Mammalian neuronal nAChR in cultured mouse N1E-115 cells, muscle type nAChR in cultured mouse BC3H1 cells and insect neuronal nAChR in dissociated locust thoracic ganglion neurons show interspecies differences in sensitivity to neurotoxic compounds. The nitromethylene heterocyclic insecticide WL145004 and physostigmine selectively agonize the insect type nAChR. The mouse neuronal and muscle types of nAChR are much less sensitive to and are partially inhibited by WL 145004. Intraspecies differences have been investigated for the effects of Pb(2+) on subtypes of nAChR expressed in Xenopus oocytes. The nature of the effect of the heavy metal Pb(2+) depends on the combination of mammalian neuronal a and beta nAChR subunits. Ion currents mediated by alpha4beta2 and alpha3beta4 nAChR are inhibited and those mediated by alpha3beta2 nAChR are potentiated by Pb(2+). Distinct sensitivities of subtypes of mammalian neuronal nAChR to agonists and antagonists are employed to characterize native nAChR in N 1E-115 cells. Implications of receptor diversity for neurotoxicology are discussed.

In cultured mouse neuroblastoma N1E-115 cells, inorganic lead (Pb2+) affects inward currents induced by activation of neuronal type nicotinic acetylcholine receptors (nAChR) in a biphasic manner. At nanomolar concentrations a blocking action is observed, while at submillimolar concentrations this blocking effect is reversed, resulting in a U-shaped concentration-effect curve. Maximal block by 90% is observed at 1-3 microM Pb2+. The interactions of Pb2+ with nAChR were examined at the blocking concentration of 10 nM Pb2+ and at 10 microM Pb2+, presenting the reversal of block. The fitted Emax for nAChR receptor activation by ACh was attenuated at both high and low Pb2+ concentrations by 24% and 54%, respectively. The EC50 values of the activation curves were not significantly altered; amounting to 53 microM, 64 microM and 86 microM ACh in the control situation and in the presence of 10 nM and 10 microM Pb2+, respectively. Further, receptor desensitization and ion channel block by ACh were also not affected by Pb2+. The results indicate that Pb2+ affects nAChR in a dual manner that involves inhibition and potentiation, both by non-competitive interactions. Neuronal nAChR expressed in N1E-115 cells resemble a combination of alpha 4 and beta 2 subunits, that constitute the predominant subunits in the central nervous system. The differential inhibition and potentiation of nAChR, together with the high sensitivity, are of interest with respect to Pb2+ neurotoxicity.

Among persistent neurotoxicants inorganic lead (Pb2+) is known to cause a variety of cellular effects associated with alterations in neuronal performance. Here we describe differential effects of Pb2+ on distinct subtypes of neuronal nicotinic acetylcholine receptors (nAChR) in Xenopus oocytes expressing various combinations of alpha and beta receptor subunits. Transient inward currents evoked by ACh in voltage clamped oocytes expressing alpha4beta2 and alpha3beta4 nAChR are inhibited by external Pb2+ at concentrations in the range of 1 nM-200 microM. The sensitivities of oocytes expressing alpha4beta2 and alpha3beta4 nAChR vary greatly. Additional experiments suggest that this variation may be due to receptor heterogeneity, which is demonstrated for alpha4beta2 nAChR in cell-attached patch clamp experiments. Conversely, the inward current evoked by ACh in oocytes expressing alpha3beta2 nAChR is potentiated by Pb2+ at concentrations in the range of 1-250 microM. The potentiating effect shows little variation between oocytes and is similar for inward currents evoked by low and high concentrations of ACh. The distinct effects on alpha3beta2 nAChR as compared to alpha4beta2 and alpha3beta4 nAChR suggest that Pb2+ interacts with both alpha and beta subunits of the neuronal nAChR. The effects of Pb2+ on defined subtypes of nAChR in oocytes are compared to those on ligand-gated ion channels in intact cells.

Effects of inorganic lead (Pb2+) on defined subtypes of neuronal nicotinic acetylcholine receptors have been investigated. Voltage clamp experiments have been performed on Xenopus oocytes expressing alpha 3 beta 2, alpha 3 beta 4 and alpha 4 beta 2 neuronal nicotinic acetylcholine receptor subunit combinations. In oocytes expressing the alpha 3 beta 2 subunit combination Pb2+ enhances the peak amplitude of nicotinic acetylcholine receptor-mediated inward currents evoked by superfusion with 100 microM acetylcholine. At concentrations of 1-250 microM, Pb2+ potentiates alpha 3 beta 2 receptor-mediated inward current concentration dependently by a factor of 1.1-11.0. Inward currents evoked by low (3 microM) and high (1 mM) concentrations of acetylcholine are potentiated to a similar extent. Conversely, in oocytes expressing the alpha 3 beta 4 subunit combination Pb2+ inhibits the nicotinic receptor-mediated inward currents evoked with 100 microM acetylcholine. Inhibitory effects are observed in the concentration range of 1 nM-100 microM Pb2+ but the degree of inhibition varies between oocytes. A similar inhibition of the alpha 4 beta 2 nicotinic receptor-mediated inward current by Pb2+ indicates that alpha as well as beta subunits are involved in the potentiating and inhibitory effects. Possible reasons for the variation in the inhibitory effects of Pb2+ on alpha 3 beta 4 and alpha 4 beta 2 nicotinic receptor-mediated inward currents have been investigated and are discussed. The divalent cations Ca2+ and Mg2+ potentiate both alpha 3 beta 2 and alpha 3 beta 4 nicotinic receptor-mediated inward currents. The distinct modulation of receptor function by Pb2+ and by Ca2+ and Mg2+ and the dependence of the modulatory effect of Pb2+ on subunit composition suggest that Pb2+ interacts with multiple sites on the alpha and beta subunits of neuronal nicotinic acetylcholine receptors.

1. Pb2+ affects neuronal nicotinic acetylcholine receptors (nAChR) in N1E-115 neuroblastoma cells in a dual manner. At nanomolar concentrations a blockade is observed, while at submillimolar concentrations this blocking effect is reversed. 2. The Xenopus oocyte expression system was used to examine whether the dual effect of Pb2+ is related to a differential action on nAChR subtypes. Effects of Pb2+ were investigated in oocytes expressing nAChR after co-injection of alpha 3 and beta 2 or alpha 3 and beta 4 cDNA. 3. At 1-250 mumol/L, Pb2+ causes a 10-1000% increase of the response mediated by the alpha 3 beta 2 nAChR. 4. Pb2+ blocks ACh-induced inward currents mediated by alpha 3 beta 4 nAChR. The inhibitory potency of Pb2+ greatly varies between cells. In 50% of the cells concentrations < or = 1 mumol/L Pb2+ blocked the nicotinic response by 31-93%. In the other cells even at higher concentrations Pb2+ caused only 0-65% inhibition. 5. These results show that Pb2+ may both potentiate and block nAChR, depending on the type of nAChR subunit expressed.

1. The ability of various divalent metal ions to substitute for Ca2+ in activating distinct types of Ca(2+)-dependent K+ [K+(Ca2+)] channels has been investigated in excised, inside-out membrane patches of human erthrocytes and of clonal N1E-115 mouse neuroblastoma cells using the patch clamp technique. The effects of the various metal ions have been compared and related to the effects of Ca2+. 2. At concentrations between 1 and 100 microM Pb2+, Cd2+ and Co2+ activate intermediate conductance K+(Ca2+) channels in erythrocytes and large conductance K+(Ca2+) channels in neuroblastoma cells. Pb2+ and Co2+, but not Cd2+, activate small conductance K+(Ca2+) channels in neuroblastoma cells. Mg2+ and Fe2+ do not activate any of the K+(Ca2+) channels. 3. Rank orders of the potencies for K+(Ca2+) activation are Pb2+, Cd2+ > Ca2+, Co2+ >> Mg2+, Fe2+ for the intermediate erythrocyte K+(Ca2+) channel, and Pb2+, Cd2+ > Ca2+ > Co2+ >> Mg2+, Fe2+ for the small, and Pb2+ > Ca2+ > Co2+ >> Cd2+, Mg2+, Fe2+ for the large K+(Ca2+) channel in neuroblastoma cells. 4. At high concentrations Pb2+, Cd2+, and Co2+ block K+(Ca2+) channels in erythrocytes by reducing the opening frequency of the channels and by reducing the single channel amplitude. The potency orders of the two blocking effects are Pb2+ > Cd2+, Co2+ >> Ca2+, and Cd2+ > Pb2+, Co2+ >> Ca2+, respectively, and are distinct from the potency orders for activation. 5. It is concluded that the different subtypes of K+(Ca2+) channels contain distinct regulatory sites involved in metal ion binding and channel opening. The K+(Ca2+) channel in erythrocytes appears to contain additional metal ion interaction sites involved in channel block.

Effects of Pb and several other metal ions on various distinct types of voltage-, receptor- and Ca-activated ion channels have been investigated in cultured N1E-115 mouse neuroblastoma cells. Experiments were performed using the whole-cell voltage clamp and single-channel patch clamp techniques. External superfusion of nanomolar to submillimolar concentrations of Pb causes multiple effects on ion channels. Barium current through voltage-activated Ca channels is blocked by micromolar concentrations of Pb, whereas voltage-activated Na current appears insensitive. Neuronal type nicotinic acetylcholine receptor-activated ion current is blocked by nanomolar concentrations of Pb and this block is reversed at micromolar concentrations. Serotonin 5-HT3 receptor-activated ion current is much less sensitive to Pb. In addition, external superfusion with micromolar concentrations of Pb as well as of Cd and aluminum induces inward current, associated with the direct activation of nonselective cation channels by these metal ions. In excised inside-out membrane patches of neuroblastoma cells, micromolar concentrations of Ca activate small (SK) and big (BK) Ca-activated K channels. Internally applied Pb activates SK and BK channels more potently than Ca, whereas Cd is approximately equipotent to Pb with respect to SK channel activation, but fails to activate BK channels. The results show that metal ions cause distinct, selective effects on the various types of ion channels and that metal ion interaction sites of ion channels may be highly selective for particular metal ions.

Ca(2+)-activated K+ [K(Ca)] channels were studied in excised patches of resting and activated human peripheral blood T lymphocytes. The K(Ca) channel had a single-channel conductance of 50 +/- 6 pS in symmetrical high-K+ solutions in the potential range of -100 to -10 mV and was inwardly rectifying at more depolarized potentials. The channel was sensitive to block by charybdotoxin (10 nM) and insensitive to apamin (3 nM). Half-maximum activation occurred at an internal free Ca2+ concentration of 360 +/- 110 nM. The concentration-effect curve had a slope factor of 0.83 +/- 0.12, suggesting a 1:1 interaction of Ca2+ ions with the channel. Ca2+ affects the open time probability of the K(Ca) channels, mainly by modulating the frequency of channel opening. The open probability did not show voltage dependence. The kinetics of the channel could be described assuming one open state and two closed states. The time constant of the exponential describing the open time distribution amounted to 2.8 +/- 1.2 ms, whereas the closed time distribution could be described with two exponentials with time constants of 0.2 +/- 0.05 ms and 8.0 +/- 2.1 ms, respectively. Resting T lymphocytes expressed a low number of channels but the density of channels increased dramatically during chronic phytohaemagglutinin stimulation.

In order to resolve single channel events underlying the 5-HT3 receptor-gated ion current in N1E-115 neuroblastoma cells patch clamp experiments have been performed on excised outside-out membrane patches under optimized experimental conditions. When the driving force for Na+ through the 5-HT3 receptor-gated ion channel is enhanced by raising the external Na+ concentration to 180 mM and by substituting internal K+ with the relatively impermeable cation N-methyl-D-glucamine, a single conductance level is observed in the presence of 5-HT. Single channel activity is observed only in the presence of the agonist and is blocked by 50 nM of the selective 5-HT3 receptor antagonist MDL 72222 in a reversible manner. At membrane potentials more negative than -60 mV discrete single channel events can be resolved with a conductance of 5.6 +/- 1.2 pS.

Using electrophysiological techniques, effects of neurotoxicants were studied on mammalian neuronal and endplate type nicotinic acetylcholine receptors (nAChR) in N1E-115 cells and in BC3H(1) cells, respectively, and insect nAChR in locust neurons. Neuronal nAChR are highly sensitive to inorganic lead (Pb(2+)). Between 1 nm and 3 mum-Pb(2+) the ACh-induced inward current is blocked in a concentration-dependent manner (IC(50) = 19 nm; maximal effect (E(max)) = 90%). In contrast, the serotonin 5-HT(3) receptor is far less sensitive to Pb(2+) (IC(50) = 49 mum). Surprisingly, between 10 mum and 100 mum Pb(2+) the blocking effect on the nAChR is reversed, and the kinetics of the ACh-induced inward current are delayed. Nitromethylene heterocyclic (NMH) compound constitute a new class of selective insecticides, that presumably affect insect nAChR. The effect of the NMH compound 1-(pyridin-3-yl-methyl)-2-nitromethylene-imidazolidine (PMNI) on the different subtypes of nAChR has been analysed. Distinct agonistic effects of PMNI on nAChR are observed on insect neurons only. Further, PMNI blocks nicotinic responses mediated by the different subtypes of nAChR in the following potency order: locust å neuronal type endplate type nAChR. These results demonstrate that the analysis of electrophysiological endpoints in cultured cells is a valuable approach to the investigation of target site selectivity and species specificity of neurotoxic compounds.

Mouse neuroblastoma cells of the clone N1E-115 express a variety of ion channels and receptors, including a number that is also involved in neurotransmission. Effects of Pb2+ on several of these ion channels have been investigated under experimental conditions that allow electrophysiological recording of membrane current carried by distinct types of ion channels. In whole-cell voltage clamp experiments voltage-dependent calcium channels are blocked by Pb2+ at micromolar concentrations, while voltage-dependent sodium channels are not affected by Pb2+. The neuronal type nicotinic acetylcholine (ACh) receptor-ion channel complex is sensitive to low concentrations of Pb2+. At 1 nM-3 microM, Pb2+ reduces the peak amplitude of the ACh-induced inward current to 74%-10% of the control value in a concentration-dependent manner. However, at Pb2+ concentration between 10 and 100 microM this blocking effect is reduced and kinetics of decay of the ACh-induced inward current are slowed. The effects of Pb2+ on the nicotinic receptor-mediated inward current amplitude can be described by the sum of two sigmoidal concentration-effect curves with an IC50 value of 19 nM and an EC50 of 21 microM. The serotonin 5-HT3 receptor-ion channel complex is less sensitive to Pb2+. The serotonin-induced inward current is blocked by Pb2+ with an IC50 value of 49 microM. In single channel patch clamp experiments internal Pb2+ causes activation of calcium-activated potassium channels in N1E-115 cells. The two types of calcium-activated potassium channels show differential sensitivity: the low conductance (SK) channel is more sensitive to Pb2+ than the high conductance (BK) channel. At micromolar concentrations Pb2+ also induces an ion current mediated by metal ion-activated ion channels. Opening of these channels, which have a single channel conductance of 24 pS and a reversal potential of 0 mV, depends on Pb2+ concentration. These effects of Pb2+ support the hypothesis that Pb2+ affects synaptic transmission by blocking presynaptic voltage-dependent calcium channels. On the other hand, effects on other sensitive target sites, the neuronal nicotinic ACh receptor in particular, clearly indicate that other targets may be involved in the toxic effects of Pb2+ on the nervous system.

Effects of Cd2+, Co2+, Fe2+ and Mg2+ (1 microM and 100 microM) and Pb2+ (1 microM and 90 microM) on single-channel properties of the small-conductance (SK) and large-conductance (BK) Ca(2+)-activated K+ channels were investigated in inside-out patches of N1E-115 mouse neuroblastoma cells. Cd2+, Co2+ and Pb2+, but not Fe2+ and Mg2+, cause SK channel opening. The potency of the metals in enhancing the SK channel-open probability follows the sequence Cd2+ approximately Pb2+ > Ca2+ > Co2+ >> Mg2+, Fe2+. The four metals that cause SK channel opening are equipotent in enhancing the opening frequency of SK channels. The BK channel is activated by Pb2+ and Co2+, whereas Cd2+, Fe2+ and Mg2+ are ineffective. The potency of the metals in enhancing BK channel-open probability, open time and opening frequency follows the sequence Pb2+ > Ca2+ > Co2+ >> Cd2+, Mg2+, Fe2+. The results show that SK channels are much more sensitive to Cd2+ than BK channels and indicate that Cd2+ is a selective agonist of SK channels. It is concluded that the various metal ions bind to the same regulatory site(s) at which Ca2+ activates the SK and BK channels under physiological conditions. The different potency sequences of metal ions with respect to BK and SK channel activation indicate that the regulatory sites of these Ca(2+)-activated K+ channels have distinct chemical and physical properties.

Using the patch-clamp technique single-channel properties of Ca(2+)-activated K+ (CaK) channels were investigated in inside-out membrane patches of human erythrocytes. In a physiological K+ gradient (5 mM K+ externally: 150 mM K+ internally) the single CaK channel conductance is 15 pS in the membrane potential range of -40 to +40 mV. The channel open probability, opening frequency and open and closed time distributions are voltage-independent. The open probability and the opening frequency of the CaK channel depend on [Ca2+]i and increase between 0.5 and 60 microM Ca2+ from approx. 10% to 90% of the maximum value obtained at 115 microM. The relation between open probability and [Ca2+]i can be described by a sigmoid concentration-effect curve with an EC50 of 4.7 microM and a slope factor of 1. Independent of [Ca2+]i open time distributions yield two time constants of 5.3 and 22 ms. The relative amplitudes of the fast and slow components of the open time histogram as well as the maximum open probability and the maximum opening frequency of CaK channels vary considerably. In addition, CaK channels in multiple channel patches are highly interdependent. It is concluded that the Ca(2+)-dependence of CaK channels in human erythrocytes is due to the modulation of opening frequency by internal Ca2+. The results are consistent with a classical receptor-agonist model in which ligand interaction kinetics are much faster than channel gating.

Effects of Cd2+, Co2+, Pb2+, Fe2+ and Mg2+ (1-100 microM) on single-channel properties of the intermediate conductance Ca(2+)-activated K+ (CaK) channels were investigated in inside-out patches of human erythrocytes in a physiological K+ gradient. Cd2+, Co2+ and Pb2+, but not Fe2+ and Mg2+, were able to induce CaK channel openings. The potency of the metals to open CaK channels in human erythrocytes follows the sequence Pb2+, Cd2+ > Ca2+ > or = Co2+ >> Mg2+, Fe2+. At higher concentrations Pb2+, Cd2+ and Co2+ block the CaK channel by reducing the opening frequency and the single-channel current amplitude. The potency of the metals to reduce CaK channel opening frequency follows the sequence Pb2+ > Cd2+, Co2+ >> Ca2+, which differs from the potency sequence Cd2+ > Pb2+, Co2+ >> Ca2+ to reduce the unitary single-channel current amplitude. Fe2+ reduced the channel opening frequency and enhanced the two open times of CaK channels activated by Ca2+, whereas up to 100 microM Mg2+ had no effect on any of the measured single-channel parameters. It is concluded that the activation of CaK channels of human erythrocytes by various metal ions occurs through an interaction with the same regulatory site at which Ca2+ activates these channels. The different potency orders for the activating and blocking effects suggest the presence of at least one activation and two blocking sites. A modulatory binding site for Fe2+ exists as well. In addition, the CaK channels in human erythrocytes are distinct from other subtypes of Ca(2+)-activated K+ channels in their sensitivity to the metal ions.

Effects of local anesthetics and anticonvulsants on the pyrethroid-modified sodium current in cultured mouse neuroblastoma cells have been investigated using the suction pipette voltage clamp technique. In the presence of 10 microM of the pyrethroid deltamethrin the sodium current consists of an enhanced peak current during membrane depolarization and a slowly decaying, deltamethrin-induced tail current remaining after repolarization. At the onset of block the local anesthetics tetracaine, lidocaine and QX 314 reduced the deltamethrin-induced tail current more effectively than the peak current. Lidocaine, but not phenytoin, caused a time-dependent block of tail currents evoked by membrane depolarizations lasting 10-1000 ms. Both lidocaine- and phenytoin-induced blocks were independent of the membrane potential during the tail current. The anticonvulsants phenytoin, phenobarbital and valproate blocked the tail and the peak sodium current to the same extent, but diazepam, mephenesin and urethane blocked the peak current more effectively. Vitamin E, which suppresses pyrethroid-induced paresthesia of the skin, had no effect on the voltage-dependent sodium current. It is concluded that indirect effects of anticonvulsants on pyrethroid-induced toxic symptoms predominate, whereas local anesthetics preferentially block the pyrethroid-induced tail current. Therefore, local anesthetics are potentially useful pyrethroid antidotes.

The effects of inorganic lead (Pb2+) on the ion currents mediated by (1) neuronal nicotinic acetylcholine (ACh) receptors, (2) serotonin 5-HT3 receptors, as well as (3) voltage-dependent Ca2+ and Na+ channels have been investigated in voltage clamped mouse neuroblastoma cells. The nicotinic ACh receptor-ion channel complex appeared more sensitive to Pb2+ than the other ion channels investigated. Low concentrations of Pb2+ (1 nM - 3 microM) reduced the peak amplitude of the ACh-induced inward current to 74%-10% of the control value in a concentration-dependent manner. However, between 10 microM and 100 microM Pb2+ the blocking effect was reversed, while the decay of the ACh-induced inward current was delayed. These effects of Pb2+ on the nicotinic receptor-mediated inward current can be described by the sum of two sigmoidal concentration-effect curves with an IC50 value of 19 nM and an EC50 of 21 microM and with slope factors of -0.5 and 0.8, respectively. The current mediated by 5-HT3 receptors was less potently blocked by Pb2+ (IC50 = 49 microM; slope factor = -0.3). In addition, Pb2+ blocked the ion current through voltage-dependent Ca2+ channels. The IC50 value of the concentration-effect curve of block of transient type Ca2+ channels by Pb2+ is 4.8 microM and the slope factor is -0.9. Voltage-dependent Na+ channels were not affected by Pb2+ up to 100 microM. At concentrations greater than 1 microM, Pb2+ also induced a noninactivating inward current. The present results show that modification of neuronal nicotinic receptor function may contribute to neurotoxic effects of Pb2+ poisoning.

Superfusion with Pb2+ induces a slow, noninactivating and reversible inward current in voltage-clamped N1E-115 neuroblastoma cells. The amplitude of this inward current increases in the range of 1-200 microM Pb/+. Single-channel patch-clamp experiments have revealed that this inward current is mediated by discrete ion channels. Reversal potentials from linear I-V relationships are close to 0 mV for whole-cell and single-channel currents and the single-channel conductance amounts to 24 pS. The Pb2(+)-induced membrane current is not mediated by various known types of ion channels, since it is not blocked by external tetrodotoxin, tetraethylammonium, D-tubocurarine, atropine, ICS 205-930 and by internal EGTA. In Na(+)-free solutions superfusion with Pb2+ neither evokes a whole-cell inward current, nor single-channel openings. At -80 mV the open-time distribution of the single channels activated by 1 microM Pb2+ is dual exponential with time constants of 17 and 194 msec. When the Pb2+ concentration is increased from 1 to 20 microM these time constants decrease to 2 and 13 msec, but the amplitude of single-channel currents remains -1.9 nA. Cd2+ and Al3+ induce inward currents and single-channel openings similar to Pb2+. Time constants fitted to the open-time distribution of single channels are 14 and 135 msec in the presence of 1 microM Cd2+ and 15 and 99 msec in the presence of 50 microM Al3+. Conversely, Cu2+ induces an irreversible inward current in neuroblastoma cells. Single-channel openings are undetected in the presence of Cu2+ and in Na(+)-free solutions Cu2+ is still able to induce an inward current. It is concluded that Pb2+, Cd2+ and possibly Al3+ activate a novel type of metal ion-activated (MIA) channel in N1E-115 cells.

(1) The interaction of a series of pyrethroid insecticides with the Na+ channels in myelinated nerve fibres of the clawed frog, Xenopus laevis, was investigated using the voltage clamp technique. (2) Out of 11 pyrethroids 9 insecticidally active compounds induce a slowly decaying Na+ tail current on termination of a step depolarization, whereas the Na+ current during depolarization was hardly affected. These tail currents are most readily explained by a selective reduction of the rate of closing of the activation gate in a fraction of the Na+ channels that have opened during depolarization. (3) The rate of decay of the Na+ tail current varies considerably with pyrethroid structure. After alpha-cyano pyrethroids the decay is at least one order of magnitude slower than after non-cyano pyrethroids. The decay always follows a single-exponential time course and is reversibly slowed when the temperature is lowered from 25 to 0 degrees C. Arrhenius plots in this temperature range are linear. (4) These results indicate that the relaxation of the activation gate in pyrethroid-affected Na+ channels is governed by an apparent first order, unimolecular process and that the rate of relaxation is limited by a single energy barrier. Application of transition state theory shows that after alpha-cyano pyrethroids this energy barrier is 9.6 kJ/mol higher than after non-cyano pyrethroids. (5) Differences in rate of decay of the Na+ tail current account for the reported differences in repetitive nerve activity induced by various pyrethroids. In addition, the effect of temperature on the rate of decay explains the increase in repetitive activity with cooling.