R.G.D.M. van Kleef

Nanomolar concentrations of lead selectively block neuronal nicotinic acetylcholine responses in mouse neuroblastoma cells

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.

Novel type of ion channel activated by Pb2+, Cd2+, and Al3+ in cultured mouse neuroblastoma cells

SummarySuperfusion 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 μm Pb2+. Single-channel patch-clamp experiments have revealed that this inward current is mediated by discrete ion channels. Reversal potentials from linearI–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 1μm Pb2+ is dual exponential with time constants of 17 and 194 msec. When the Pb2+ concentration is increased from 1 to 20 μm 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 μm Cd2+ and 15 and 99 msec in the presence of 50 μm 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 ionactivated (MIA) channel in N1E-115 cells.

Modulation of human GABAA receptor function: A novel mode of action of drugs of abuse

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.

Neurotoxicity screening of (illicit) drugs using novel methods for analysis of microelectrode array (MEA) recordings

Annual prevalence of the use of common illicit drugs and new psychoactive substances (NPS) is high, despite the often limited knowledge on the health risks of these substances. Recently, cortical cultures grown on multi-well microelectrode arrays (mwMEAs) have been used for neurotoxicity screening of chemicals, pharmaceuticals, and toxins with a high sensitivity and specificity. However, the use of mwMEAs to investigate the effects of illicit drugs on neuronal activity is largely unexplored. We therefore first characterised the cortical cultures using immunocytochemistry and show the presence of astrocytes, glutamatergic and GABAergic neurons. Neuronal activity is concentration-dependently affected following exposure to six neurotransmitters (glutamate, GABA, serotonin, dopamine, acetylcholine and nicotine). Most neurotransmitters inhibit neuronal activity, although glutamate and acetylcholine transiently increase activity at specific concentrations. These transient effects are not detected when activity is determined during the entire 30min exposure window, potentially resulting in false-negative results. As expected, exposure to the GABAA-receptor antagonist bicuculline increases neuronal activity. Exposure to a positive allosteric modulator of the GABAA-receptor (diazepam) or to glutamate receptor antagonists (CNQX and MK-801) reduces neuronal activity. Further, we demonstrate that exposure to common drugs (3,4-methylenedioxymethamphetamine (MDMA) and amphetamine) and NPS (1-(3-chlorophenyl)piperazine (mCPP), 4-fluoroamphetamine (4-FA) and methoxetamine (MXE)) decreases neuronal activity. MXE most potently inhibits neuronal activity with an IC50 of 0.5μM, whereas 4-FA is least potent with an IC50 of 113μM. Our data demonstrate the importance of analysing neuronal activity within different time windows during exposure to prevent false-negative results. We also show that cortical cultures grown on mwMEAs can successfully be applied to investigate the effects of different (illicit) drugs on neuronal activity. Compared to investigating multiple single endpoints for neurotoxicity or neuromodulation, such as receptor activation or calcium channel function, mwMEAs can provide information on integrated aspects of drug-induced neurotoxicity more rapidly. Therefore, this approach could contribute to a faster insight in possible health risks and shorten the regulation process.

Potentiation of the human GABA(A) receptor as a novel mode of action of lower-chlorinated non-dioxin-like PCBs.

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.

Divalent cations activate small- (SK) and large-conductance (BK) channels in mouse neuroblastoma cells: selective activation of SK channels by cadmium.

Effects of Cd2+, Co2+, Fe2+ and Mg2+ (1 μM and 100 μM) and Pb2+ (1 μM and 90 μM) on single-channel properties of the small-conductance (SK) and large-conductance (BK) Ca2+-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+ ≈ 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 Ca2+-activated K+ channeles have distinct chemical and physical properties.

Differential effects of heavy metal ions on Ca(2+)-dependent K+ channels.

Summary1. The ability of various divalent metal ions to substitute for Ca2+ in activating distinct types of Ca2+-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 µM 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.

In vitro approaches to species and receptor diversity in cellular neurotoxicology

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.

Nicotinic acetylcholine receptors in cultured cells as targets of neurotoxic compounds

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.