Pharmacological and toxicological in vitro and in vivo effect of higher doses of oxime reactivators.

The major function of compounds with an oxime moiety attached to a quarternary nitrogen pyridinium ring is to reactivate acetylcholinesterase inhibited by organophosphorus agent (OP). However, other oxime mechanisms (e.g. modulation of cholinergic or glutamatergic receptor) may be involved in the recovery. The main disadvantage of positively charged reactivators is their low ability to penetrate into the brain although crossing the blood brain barrier could be supported via increasing the dose of administered oxime. Thus, this study presents maximal tolerated doses (MTD) for marketed oximes (TMB-4, MMB-4, LüH-6, HI-6, 2-PAM) and the most promising K-oximes (K027, K048, K203) which can be used in OP therapy in the future. No signs of sarin intoxication were observed in mice treated with 100% MTD of HI-6 in contrast to those treated with atropine and only 5% LD50 of HI-6. 100% MTD of HI-6 resulted in levels of 500 µM and 12 µM in plasma and brain, respectively. This concentration is by a far margin safe with respect to direct effects on neuronal cell viability and, on the other hand, does not have any effects on central NMDA receptors or central nACh receptors. However, a weak antimuscarinic activity in case of LüH-6 and a weak peripheral antinicotinic action in case of TMB-4 and 2-PAM could be observed at their respective 100% MTD dose. These high doses, represented by MTD, are, however, irrelevant to clinical practice since they led to mild to moderate toxic side effects. Therefore, we conclude that clinically used doses of marketed oxime reactivators have no significant direct pharmacological effect on the tested receptors.

Exploring the overlapping binding sites of ifenprodil and EVT-101 in GluN2B-containing NMDA receptors using novel chicken embryo forebrain cultures and molecular modeling.

N-methyl-d-aspartate receptors (NMDAR) are widely expressed in the brain. GluN2B subunit-containing NMDARs has recently attracted significant attention as potential pharmacological targets, with emphasis on the functional properties of allosteric antagonists. We used primary cultures from chicken embryo forebrain (E10), expressing native GluN2B-containing NMDA receptors as a novel model system. Comparing the inhibition of calcium influx by well-known GluN2B subunit-specific allosteric antagonists, the following rank order of potency was found: EVT-101 (EC 50 22 ± 8 nmol/L) > Ro 25-6981 (EC 50 60 ± 30 nmol/L) > ifenprodil (EC 50 100 ± 40 nmol/L) > eliprodil (EC 50 1300 ± 700 nmol/L), similar to previous observations in rat cortical cultures and cell lines overexpressing chimeric receptors. The less explored Ro 04-5595 had an EC 50 of 186 ± 32 nmol/L. Venturing to explain the differences in potency, binding properties were further studied by in silico docking and molecular dynamics simulations using x-ray crystal structures of GluN1/GluN2B amino terminal domain. We found that Ro 04-5595 was predicted to bind the recently discovered EVT-101 binding site, not the ifenprodil-binding site. The EVT-101 binding pocket appears to accommodate more structurally different ligands than the ifenprodil-binding site, and contains residues essential in ligand interactions necessary for calcium influx inhibition. For the ifenprodil site, the less effective antagonist (eliprodil) fails to interact with key residues, while in the EVT-101 pocket, difference in potency might be explained by differences in ligand-receptor interaction patterns.

Investigation of New Orexin 2 Receptor Modulators Using In Silico and In Vitro Methods.

The neuropeptides, orexin A and orexin B (also known as hypocretins), are produced in hypothalamic neurons and belong to ligands for orphan G protein-coupled receptors. Generally, the primary role of orexins is to act as excitatory neurotransmitters and regulate the sleep process. Lack of orexins may lead to sleep disorder narcolepsy in mice, dogs, and humans. Narcolepsy is a neurological disorder of alertness characterized by a decrease of ability to manage sleep-wake cycles, excessive daytime sleepiness, and other symptoms, such as cataplexy, vivid hallucinations, and paralysis. Thus, the discovery of orexin receptors, modulators, and their causal implication in narcolepsy is the most important advance in sleep-research. The presented work is focused on the evaluation of compounds L1⁻L11 selected by structure-based virtual screening for their ability to modulate orexin receptor type 2 (OX2R) in comparison with standard agonist orexin-A together with their blood-brain barrier permeability and cytotoxicity. We can conclude that the studied compounds possess an affinity towards the OX2R. However, the compounds do not have intrinsic activity and act as the antagonists of this receptor. It was shown that L4 was the most potent antagonistic ligand to orexin A and displayed an IC50 of 2.2 µM, offering some promise mainly for the treatment of insomnia.

Bispyridinium Compounds Inhibit Both Muscle and Neuronal Nicotinic Acetylcholine Receptors in Human Cell Lines.

Standard treatment of poisoning by organophosphorus anticholinesterases uses atropine to reduce the muscarinic effects of acetylcholine accumulation and oximes to reactivate acetylcholinesterase (the effectiveness of which depends on the specific anticholinesterase), but does not directly address the nicotinic effects of poisoning. Bispyridinium molecules which act as noncompetitive antagonists at nicotinic acetylcholine receptors have been identified as promising compounds and one has been shown to improve survival following organophosphorus poisoning in guinea-pigs. Here, we have investigated the structural requirements for antagonism and compared inhibitory potency of these compounds at muscle and neuronal nicotinic receptors and acetylcholinesterase. A series of compounds was synthesised, in which the length of the polymethylene linker between the two pyridinium moieties was increased sequentially from one to ten carbon atoms. Their effects on nicotinic receptor-mediated calcium responses were tested in muscle-derived (CN21) and neuronal (SH-SY5Y) cells. Their ability to inhibit acetylcholinesterase activity was tested using human erythrocyte ghosts. In both cell lines, the nicotinic response was inhibited in a dose-dependent manner and the inhibitory potency of the compounds increased with greater linker length between the two pyridinium moieties, as did their inhibitory potency for human acetylcholinesterase activity in vitro. These results demonstrate that bispyridinium compounds inhibit both neuronal and muscle nicotinic receptors and that their potency depends on the length of the hydrocarbon chain linking the two pyridinium moieties. Knowledge of structure-activity relationships will aid the optimisation of molecular structures for therapeutic use against the nicotinic effects of organophosphorus poisoning.

Microvesicle shedding and lysosomal repair fulfill divergent cellular needs during the repair of streptolysin O-induced plasmalemmal damage.

Pathogenic bacteria secrete pore-forming toxins that permeabilize the plasma membrane of host cells. Nucleated cells possess protective mechanisms that repair toxin-damaged plasmalemma. Currently two putative repair scenarios are debated: either the isolation of the damaged membrane regions and their subsequent expulsion as microvesicles (shedding) or lysosome-dependent repair might allow the cell to rid itself of its toxic cargo and prevent lysis. Here we provide evidence that both mechanisms operate in tandem but fulfill diverse cellular needs. The prevalence of the repair strategy varies between cell types and is guided by the severity and the localization of the initial toxin-induced damage, by the morphology of a cell and, most important, by the incidence of the secondary mechanical damage. The surgically precise action of microvesicle shedding is best suited for the instant elimination of individual toxin pores, whereas lysosomal repair is indispensable for mending of self-inflicted mechanical injuries following initial plasmalemmal permeabilization by bacterial toxins. Our study provides new insights into the functioning of non-immune cellular defenses against bacterial pathogens.

The use of organotypic hippocampal slice cultures to evaluate protection by non-competitive NMDA receptor antagonists against excitotoxicity.

There is great interest in testing neuroprotectants which inhibit the neurodegeneration that results from excessive activation of N-methyl-D-aspartate (NMDA) receptors. As an alternative to in vivo testing in animal models, we demonstrate here the use of a complex in vitro model to compare the efficacy and toxicity of NMDA receptor inhibitors. Organotypic hippocampal slice cultures were used to compare the effectiveness of the Alzheimer's disease drug, memantine, the Parkinson's disease drug, procyclidine, and the novel neuroprotectant, gacyclidine (GK11), against NMDA-induced toxicity. All three drugs are non-competitive NMDA receptor open-channel blockers that inhibit excitotoxic injury, and their neuroprotective capacities have been extensively investigated in vivo in animal models. They have also been evaluated as potential countermeasure agents against organophosphate poisoning. Quantitative densitometric image analysis of propidium iodide uptake in hippocampal regions CA1, CA3 and DG, showed that, after exposure to 10microM NMDA for 24 hours, GK11 was the most potent of the three drugs, with an IC50 of about 50nM and complete protection at 250nM. When applied at high doses, GK11 was still the more potent neuroprotectant, and also the least cytotoxic. These findings are consistent with those from in vivo tests in rodents. We conclude that the slice culture model provides valuable pre-clinical data, and that applying the model to the screening of neuroprotectants might significantly limit the use of in vivo tests in animals.

Neuronal glucose but not lactate utilization is positively correlated with NMDA-induced neurotransmission and fluctuations in cytosolic Ca2+ levels.

Although the brain utilizes glucose for energy production, individual brain cells may to some extent utilize substrates derived from glucose. Thus, it has been suggested that neurons consume extracellular lactate during synaptic activity. However, the precise role of lactate for fueling neuronal activity is still poorly understood. Recently, we demonstrated that glucose metabolism is up-regulated in cultured glutamatergic neurons during neurotransmission whereas that of lactate is not. Here, we show that utilization of glucose but not lactate correlates with NMDA-induced neurotransmitter glutamate release in cultured cerebellar neurons from mice. Pulses of NMDA at 30, 100, and 300 microM, leading to a progressive increase in both cytosolic [Ca2+] and release of glutamate, increased uptake and metabolism of glucose but not that of lactate as evidenced by mass spectrometric measurement of 13C incorporation into intracellular glutamate. In this manuscript, a cascade of events for the preferential neuronal utilization of glucose during neurotransmission is suggested and discussed in relation to our current understanding of neuronal energy metabolism.

Measurements with fluorescent probes in primary neural cultures; improved multiwell techniques.

INTRODUCTION: Fluorescence imaging techniques are valuable tools for the pharmacological characterization of CNS drugs. Dissected cerebellar granule neurons (CGN) are an important model system in the study of mechanisms of excitotoxicity, glutamate receptors and transporters. Widely applied techniques use fluorescent probes loaded in neural cells cultured on glass supports. CGN, however, require at least 7 days for differentiation and over time cells tend to cluster and loose adherence to the glass substrate. This problem is accentuated in small wells (e.g. 96-well plates). METHODS: CGN were grown on large coverslips (60 x 24 mm) and measurements made with a designed mountable multiwell in 48 regions on 4 coverslips at a time. The UV ratiometric probe fura-2 was used to measure glutamatergic calcium ([Ca(2+)](i)) responses induced by NMDA. The IC(50) of NMDA receptor antagonists was determined from inhibition curves with 6 doses and 8 parallels per experiment. RESULTS: The method was validated by comparing with published data for the dose response to NMDA and glycine and IC(50) values for ion-channel block by Mg(2+) and MK-801. DISCUSSION: Resolution is enhanced with the new technique since it allows measurement of multiple doses on cells from the same batch. It has advantages to cuvette techniques because cells have intact dendritic tree and synaptic function and it is a convenient method to obtain reliable dose-response curves for NMDA channel modulators on differentiated neural cells.

In vitro toxicity of tetrabromobisphenol-A on cerebellar granule cells: cell death, free radical formation, calcium influx and extracellular glutamate.

Tetrabromobisphenol-A (TBBPA) is one of the worlds most widely used brominated flame retardant. The present study reports effects of TBBPA on primary cultures of cerebellar granule cells (CGC). Using the trypan blue exclusion assay, we show that TBBPA induces death of CGC at low micro molar concentrations. Cell death was reduced by the NMDA receptor antagonist MK-801 (3 microM), the antioxidant vitamin E (50 microM), and in calcium-free buffer. We further demonstrate that TBBPA's toxicity was accompanied by apoptosis-like nuclear shrinkage, chromatin condensation, and DNA fragmentation. Other hallmarks of apoptosis such as caspase activity were, however, absent, indicating an atypical form of apoptosis. TBBPA increased intracellular free calcium in a concentration-dependent manner. TBBPA also induced an increase in extracellular glutamate in a time-dependent manner. TBBPA gave a concentration-dependent increase information reactive oxygen species (ROS) of measured with 2,7-dichlorofluorescein diacetate. The ROS formation was inhibited by the extracellular signal-regulated protein kinase (ERK) inhibitor U0126 (10 microM), the tyrosine kinase inhibitor erbstatin-A (25 microM), eliminating calcium from the buffer and by the superoxide dismutase inhibitor diethyldithio-carbamic acid (DDC, 100 microM). Further analysis with Western blot confirmed phosphorylation of ERK1/2 after exposure to TBBPA. We found that TBBPA induces ROS formation, increases intracellular calcium, extracellular glutamate, and death of CGC in vitro at concentrations comparable to those of polychlorinated biphenyl. These findings implicate TBBPA as a predicted environmental toxin and bring out the importance of awareness of its hazardous effects.

Organic solvent-induced cell death in rat cerebellar granule cells: structure dependence of c10 hydrocarbons and relationship to reactive oxygen species formation.

Previously, increased formation of reactive oxygen species (ROS) has been demonstrated in cultured rat cerebellar granule cells (CGCs) exposed to t-butylcyclohexane, n-decane, and n-butylbenzene (Dreiem et al. Relationship between lipophilicity of C6-10 hydrocarbon solvents and their ROS-inducing potency in rat cerebellar granule cells. Neurotoxicology 2002;23:701-9). In the present paper, we have studied the effects of these hydrocarbons on the viability of CGCs. Cell death was assessed by measurement of lactate dehydrogenase (LDH) release and trypan blue staining. t-butylcyclohexane and n-butylbenzene induced cell death in rat CGCs in a time-dependent and concentration-dependent manner. In contrast, n-decane did not cause release of LDH from rat CGCs even at 1mM. Morphological studies revealed apoptotic morphology characterized by cell shrinkage and chromatin condensation after exposure to low concentrations of t-butylcyclohexane and n-butylbenzene. However, there was no internucleosomal DNA fragmentation and no protection by the pan-caspase inhibitor Boc-D-FMK or the protein synthesis inhibitor cycloheximide. This indicates that cell death after t-butylcyclohexane and n-butylbenzene exposure is an intermediate between classical apoptosis and necrosis. Treatment with the antioxidant alpha-tocopherol ameliorated hydrocarbon-induced cell death, indicating involvement of reactive oxygen species in the mechanism of hydrocarbon toxicity. The significance of ROS formation in relation to cell death is discussed.

Nitric oxide induces resensitization of P2Y nucleotide receptors in cultured rat mesangial cells.

Receptor desensitization of G protein-coupled receptors (GPCRs), which occurs during short-term (seconds to minutes) exposure of cells to agonists, is mediated by phosphorylation and receptor endocytosis. Recycling of the receptors is a requisite for resensitization of the response. The mechanisms that attenuate signaling by GPCRs are of considerable importance to regulation of intercellular signaling and maintenance of their ability to respond to agonists over time. This study evaluates the effect of nitric oxide (NO) on P2Y nucleotide receptor resensitization in cultured rat glomerular mesangial cells. The NO production in cultured mesangial cells was measured by using confocal microscopy and the fluorescence NO indicator 4,5-diaminofluorescein diacetate (DAF-2 DA). L-arginine increased and Nomega-nitro-L-arginine methyl ester (L-NAME) decreased NO production significantly (P < 0.05). Calcium responses to ATP were measured with fura-2 and imaging techniques. Repeated stimulation with ATP results in receptor desensitization that is characterized by lower calcium peak amplitude. Desensitization was induced by challenging mesangial cells with four consecutive 2-min pulses of ATP (0.1 mM) separated by 4.5-min control perfusions. Intracellular calcium concentration ([Ca2+]i) increase evoked by second, third, and fourth ATP challenges were about 40%, 26%, and 18% of the first one. The NO precursor, L-arginine (10 mM), and the NO donors, spermine-NONOate (500 microM) and sodium nitroprusside (SNP) (1 mM), were added before and during a fourth ATP challenge. Spermine-NONOate and L-arginine induced a recovery of the [Ca2+]i response to the fourth ATP challenge (P < 0.01 and 0.05, respectively). The NO synthase inhibitor, L-NAME (5 mM), applied along with ATP, was shown to enhance desensitization. 1H-(1,2,4)oxadiazolo(4,3-alpha)quinoxalin-1-one (ODQ, 30 microM), an inhibitor of guanylate cyclase, was used along with L-arginine, SNP, or spermine-NONOate. There was no significant difference with or without ODQ. Neither ODQ nor 8-Br-cGMP, an analog of cGMP, at different concentrations showed effects on ATP-stimulated [Ca2+]i. There was no elevation of [Ca2+]i when the cells were challenged by different concentrations (1 microM, 100 microM, 1 mM, 20 mM, and 30 mM) of caffeine, caffeine plus ATP (0.1 mM), and 4-chloro-3-ethylphenol (100 microM, 500 microM, and 1 mM), a new agonist of ryanodine receptors. The results indicate that NO can increase the P2Y receptor resensitization in rat glomerular mesangial cells by acting through a cGMP-independent pathway. No evidence was found for the existence of ryanodine-sensitive intracellular calcium stores in rat mesangial cells.