Yasco Aracava

A possible involvement of cyclic AMP in the expression of desensitization of the nicotinic acetylcholine receptor: A study with forskolin and its analogs

Forskolin, an activator of adenylate cyclase, and its analogs were studied on the nicotinic acetylcholine receptor‐ion channel complex (AChR) of rat and frog skeletal muscles. At nanomolar concentrations, forskolin caused desensitization of the AChR located at the junctional region of innervated and the extrajunctional region of chronically denervated rat soleus muscles. The desensitization of the AChR occurred without alteration of the conducting state (channel lifetime, conductance or bursting) as shown by single channel currents. Accordingly, forskolin decreased the peak amplitude of the repetitive evoked endplate currents in frog sartorius muscles. These findings taken together with the good correlation found between the effects of forskolin and its analogs on the desensitization of the nicotinic AChR and their ability to activate adenylate cyclase suggested a possible involvement of phosphorylation of AChR via cyclic AMP on the desensitization process.

Effective countermeasure against poisoning by organophosphorus insecticides and nerve agents

The nerve agents soman, sarin, VX, and tabun are deadly organophosphorus (OP) compounds chemically related to OP insecticides. Most of their acute toxicity results from the irreversible inhibition of acetylcholinesterase (AChE), the enzyme that inactivates the neurotransmitter acetylcholine. The limitations of available therapies against OP poisoning are well recognized, and more effective antidotes are needed. Here, we demonstrate that galantamine, a reversible and centrally acting AChE inhibitor approved for treatment of mild to moderate Alzheimer’s disease, protects guinea pigs from the acute toxicity of lethal doses of the nerve agents soman and sarin, and of paraoxon, the active metabolite of the insecticide parathion. In combination with atropine, a single dose of galantamine administered before or soon after acute exposure to lethal doses of soman, sarin, or paraoxon effectively and safely counteracted their toxicity. Doses of galantamine needed to protect guinea pigs fully against the lethality of OPs were well tolerated. In preventing the lethality of nerve agents, galantamine was far more effective than pyridostigmine, a peripherally acting AChE inhibitor, and it was less toxic than huperzine, a centrally acting AChE inhibitor. Thus, a galantamine-based therapy emerges as an effective and safe countermeasure against OP poisoning.

Nicotinic acetylcholine receptors in cultured neurons from the hippocampus and brain stem of the rat characterized by single channel recording

Single channel recording techniques have been applied to neurons cultured from the hippocampus and the respiratory area of the brain stem of fetal rats in order to search for nicotinic acetylcholine receptors (nAChR) in the central nervous system. In addition to acetylcholine (ACh), the potent and specific agonist (+)‐anatoxin‐a was also used to characterize nicotinic channels. nAChRs were concentrated on the somal surface near the base of the apical dendrite, and in some patches their density was sufficient to record 2 or more channel openings simultaneously. Although a multiplicity of conductance states was also evident, the predominant population showed a single channel conductance of 20 pS at 10°C. Thus, these neuronal nAChRs resembled the embryonic or denervated‐type nAChRs in muscle. However, channel opening and closing kinetics were faster than reported for similar conductance channels in muscle. Therefore the nicotinic channels described here are similar but not identical to those of the well‐characterized muscle nAChR, in agreement with biochemical, pharmacological, and molecular genetic studies on brain AChR.

Multiple actions of anticholinesterase agents on chemosensitive synapses: Molecular basis for prophylaxis and treatment of organophosphate poisoning☆

The present study demonstrates that the reversible and irreversible anti-ChE agents have direct actions on the nicotinic acetylcholine receptor-ionic channel (AChR) and on the locust glutamatergic neuromuscular junction. In addition, the prophylaxis of lethality of organophosphorus anti-ChE compounds was studied. The lethality of VX and sarin was diminished when the rats were pretreated with physostigmine and atropine. The effectiveness of this protection, however, was markedly increased when a ganglionic blocker, either mecamylamine or chlorisondamine, was added, such that all the animals survived after receiving four times a lethal dose of VX. Pretreated animals receiving sarin showed significant recovery of morphological and functional properties of the neuromuscular junction as compared to the damage of structures from animals without pretreatment. Blood ChE inhibition was slightly decreased while brain and muscle AChE levels were significantly recovered (from 98 and 70% to 56 and 32%, respectively) by the pretreatment. This effect may partially explain the protection given by physostigmine but not that afforded by addition of a non-anti-ChE agent. Physostigmine, at concentrations greater than 20 microM, showed both a marked depression of the peak amplitudes of the endplate current (EPC) and a shortening of the decay time constants tau EPC. These effects were mostly due to a direct drug interaction with the nicotinic AChR blocking the ionic channel in its open conformation. Single-channel recordings showed that physostigmine decreases conductance and open times of the channels activated in the presence of ACh and in addition has an agonistic property on the nicotinic AChR. VX, on the other hand, only shortened the open times of ACh-activated channels without affecting the conductance. No agonist property was detected with VX. On glutamatergic synapses, the ChE inhibitors generated spontaneous firing of end-plate potentials (EPPs) and action potentials (APs). This effect was blocked in the presence of low external Ca2+ concentration or tetrodotoxin. It seems that the spontaneous EPP and AP firing resulted from an increased transmitter release induced by an increase in Na+ influx at the presynpatic nerve terminal. Physostigmine and some irreversible ChE inhibitors (VX and DFP) also blocked the postjunctional glutamate receptors. Similar to the nicotinic AChR, this effect was mostly related to a blockade of the open channels. In conclusion, the present studies showed significant protection of rats by physostigmine in combination with some ganglionic antagonists against lethality by organophosphate agents.(ABSTRACT TRUNCATED AT 400 WORDS)

Direct inhibition of the N-methyl-D-aspartate receptor channel by dopamine and (+)-SKF38393

Dopamine is known to modulate glutamatergic synaptic transmission in the retina and in several brain regions by activating specific G‐protein‐coupled receptors. We have examined the possibility of a different type of mechanism for this modulation, one involving direct interaction of dopamine with ionotropic glutamate receptors. Ionic currents induced by fast application of N‐methyl‐D‐aspartate (NMDA) were recorded under whole‐cell patch‐clamp in cultured striatal, thalamic and hippocampal neurons of the rat and in retinal neurons of the chick. Dopamine at concentrations above 100 μM inhibited the NMDA response in all four neuron types, exhibiting an IC50 of 1.2 mM in hippocampal neurons. The time course of this inhibition was fast, developing in less than 100 ms. The D1 receptor agonist (+)‐SKF38393 mimicked the effect of dopamine, with an IC50 of 58.9 μM on the NMDA response, while the enantiomer (−)‐SKF38393 was ineffective at 50 μM. However, the D1 antagonist R(+)‐SCH23390 did not prevent the inhibitory effect of (+)‐SKF38393. The degree of inhibition by dopamine and (+)‐SKF38393 depended on transmembrane voltage, increasing 2.7 times with a hyperpolarization of about 80 mV. The voltage‐dependent block by dopamine was also observed in the presence of MgCl2 1 mM. Single‐channel recordings showed that the open times of NMDA‐gated channels were shortened by (+)‐SKF38393. These data suggested that the site to which the drugs bound to produce the inhibitory effect was distinct from the classical D1‐type dopamine receptor sites, possibly being located inside the NMDA channel pore. It is concluded that dopamine and (+)‐SKF38393 are NMDA channel ligands.

Animal models that best reproduce the clinical manifestations of human intoxication with organophosphorus compounds.

The translational capacity of data generated in preclinical toxicological studies is contingent upon several factors, including the appropriateness of the animal model. The primary objectives of this article are: 1) to analyze the natural history of acute and delayed signs and symptoms that develop following an acute exposure of humans to organophosphorus (OP) compounds, with an emphasis on nerve agents; 2) to identify animal models of the clinical manifestations of human exposure to OPs; and 3) to review the mechanisms that contribute to the immediate and delayed OP neurotoxicity. As discussed in this study, clinical manifestations of an acute exposure of humans to OP compounds can be faithfully reproduced in rodents and nonhuman primates. These manifestations include an acute cholinergic crisis in addition to signs of neurotoxicity that develop long after the OP exposure, particularly chronic neurologic deficits consisting of anxiety-related behavior and cognitive deficits, structural brain damage, and increased slow electroencephalographic frequencies. Because guinea pigs and nonhuman primates, like humans, have low levels of circulating carboxylesterases—the enzymes that metabolize and inactivate OP compounds—they stand out as appropriate animal models for studies of OP intoxication. These are critical points for the development of safe and effective therapeutic interventions against OP poisoning because approval of such therapies by the Food and Drug Administration is likely to rely on the Animal Efficacy Rule, which allows exclusive use of animal data as evidence of the effectiveness of a drug against pathologic conditions that cannot be ethically or feasibly tested in humans.

Magnetic resonance imaging reveals that galantamine prevents structural brain damage induced by an acute exposure of guinea pigs to soman

Galantamine, a drug used to treat Alzheimers disease, has recently emerged as a potential medical countermeasure against the toxicity of organophosphorus (OP) compounds, including the nerve agent soman. Here, magnetic resonance imaging (MRI) was used to characterize the neurotoxic effects of soman and the ability of galantamine to prevent these effects in guinea pigs, the best non-primate model to predict the effectiveness of antidotes against OP toxicity in humans. The brains of treated and untreated guinea pigs were imaged using a clinical 3.0 Tesla MRI scanner at 48 h before and 6-7 h, 48 h and 7 days after their challenge with 1.0xLD50 soman (26.6 microg/kg, sc). Significant brain atrophy was observed among all untreated animals at 7 days after their challenge with soman. In mildly intoxicated animals, significant shortening of spin-spin relaxation times (T2) was observed in the thalamus and amygdala at 7h after the challenge. In severely intoxicated animals, T2 values and T2-weighted signal intensities increased significantly in the piriform cortex, hippocampus, thalamus and amygdala; in most regions, changes were long-lasting. Voxel-based morphometric analysis of the images revealed that other brain regions were also damaged in these animals. Neuronal loss was confirmed histopathologically. In animals that were treated with galantamine (8 mg/kg, im) 30 min prior to the exposure to soman, T2, T2-weighted signal intensities, and CSF volumes were largely unaffected. It is, therefore, concluded that galantamine can effectively prevent the structural brain damage induced by an acute exposure to soman.

Acute Toxicity of Organophosphorus Compounds in Guinea Pigs Is Sex- and Age-Dependent and Cannot Be Solely Accounted for by Acetylcholinesterase Inhibition

This study was designed to test the hypothesis that the acute toxicity of the nerve agents S-[2-(diisopropylamino)ethyl]-O-ethyl methylphosphonothioate (VX), O-pinacolyl methylphosphonofluoridate (soman), and O-isopropyl methylphosphonofluoridate (sarin) in guinea pigs is age- and sex-dependent and cannot be fully accounted for by the irreversible inhibition of acetylcholinesterase (AChE). The subcutaneous doses of nerve agents needed to decrease 24-h survival of guinea pigs by 50% (LD50 values) were estimated by probit analysis. In all animal groups, the rank order of LD50 values was sarin > soman > VX. The LD50 value of soman was not influenced by sex or age of the animals. In contrast, the LD50 values of VX and sarin were lower in adult male than in age-matched female or younger guinea pigs. A colorimetric assay was used to determine the concentrations of nerve agents that inhibit in vitro 50% of AChE activity (IC50 values) in guinea pig brain extracts, plasma, red blood cells, and whole blood. A positive correlation between LD50 values and IC50 values for AChE inhibition would support the hypothesis that AChE inhibition is a major determinant of the acute toxicity of the nerve agents. However, such a positive correlation was found only between LD50 values and IC50 values for AChE inhibition in brain extracts from neonatal and prepubertal guinea pigs. These results demonstrate for the first time that the lethal potencies of some nerve agents in guinea pigs are age- and sex-dependent. They also support the contention that mechanisms other than AChE inhibition contribute to the lethality of nerve agents.

An Analysis of Low Level Doses of Cholinesterase Inhibitors in Cultured Neurons and Hippocampal Slices of Rats

Recent data indicate that the neurotoxic effects of organophosphate compounds, including those of the nerve agents VX and sarin, are not solely due to irreversible cholinesterase inhibition. In this study we applied the patch clamp technique to hippocampal neurons in culture and slices to investigate the effects of VX, sarin and huperzine A on transmitter release and the mechanisms related with such effects. The nerve agents VX and sarin at very low concentrations significantly reduced the evoked release of GABA and glutamate. This effect was dependent of the activation of muscarinic receptors. In the presence or absence of the Na(+)-channel blocker tetrodotoxin (TTX), VX increased the frequency of spontaneous glutamate and GABA-induced postsynaptic currents. The effect of VX on TTX-insensitive spontaneous currents appears to be unrelated to cholinesterase inhibition, because it could be detected even after cholinesterase was blocked by high concentrations of the nerve agent soman. The ability of the nerve gases to decrease evoked release of GABA and increase spontaneous transmitter release may underlie some of the neurotoxic effects of the compounds. Huperzine A did not affect spontaneous or evoked release of GABA and glutamate, suggesting that this compound may be a pure cholinesterase inhibitor and had no effect on postsynaptic GABAA or AMPA receptors.

The Molecular Basis of Anticholinesterase Actions on Nicotinic and Glutamatergic Synapsesa

In the last 15 years, our knowledge of receptor function has been advanced considerably by studies of the acetylcholine-receptor-ion-channel complex (AChR) of the neuromuscular junction. The Occurrence of nicotinic AChRs at very high densities in Torpedo and Electrophorus electric organs made this membrane receptor easily available for study. In addition, specific chemical probes for the different active sites have contributed significantly to our understanding of the morphology and function of this receptor. In the early 19709, a-bungarotoxin (a-BGT) was isolated from snake venoms and was found to bind irreversibly and specifically to the acetylcholine (ACh) recognition site on the nicotinic AChR.’ The availability of such a highly selective probe allowed the isolation, purification, functional reconstitution into artificial lipid membranes, and, ultimately, cloning of the different subunits that comprise the nicotinic AChR.2” The pharmacological characterization of another class of toxins, the histrionicotoxins (HTX), isolated from skin secretions of frogs of the family Dendrobatidae,6*’ disclosed an important new class of sites on the nicotinic AChR. These sites, distinct from the agonist recognition site and most likely located on the ion channel component of the AChR, are responsible for allosteric alterations or noncompetitive blockade of neuromuscular transmission. Drugs with distinct and well-