Toshio Narahashi - US grants

The specific aim of the proposed project is to elucidate the mechanism whereby the kinetics of the gating mechanism of nerve membrane sodium channels are modified by specific chemical agents which are used as probes. This will be a step toward accomplishing our long-term goal which calls for characterization and identification of sodium channels. Kinetics of opening and closing of sodium channels as affected by these chemical agents will be analyzed using internally perfused, voltage-clamped squid and crayfish giant axons. Furthermore, the activity of single channels will be studied by patch voltage clamp techniques with cultured neuroblastoma cells. The specific chemical agents to be studied are classified into two large groups, both modifying the channel kinetics drastically. One group may be called sodium channel modulators including grayanotoxins, batrachotoxin, veratridine and aconitine, all of which modify a population of sodium channels to give rise to slow opening and closing presumably through binding to open and/or closed sodium channels. The other group is represented by sodium inactivation inhibitors, including the sea anemone toxin anthopleurin-A, N-bromoacetamide, and high and low internal pH. The specific projects are aimed at the process of channel modification, the properties of the modified channels including cation selectivity, cation binding, voltage dependence and single channel properties, the site of action of the specific agents within the sodium channel, and the gating current in the modified channel. This study is expected to determine normal physiological functioning and topography of verve membrane sodium channels which are the bases for excitation.

The long-term objectives of the proposed study are to elucidate the mechanism of action of environmentally important insecticides on the nerve membrane which is the most critical target site of various insecticides. The knowledge obtained through this research will have direct impact on our environmental concern, i.e., developments of effective therapeutic means for insecticide intoxication, and of newer insecticides which are more effective against insects yet safer for humans. The specific aims are to clarify the nature of interactions of pyrethroid and DDT-type insecticides with nerve membrane ion channels which have been demonstrated to be mainly responsible for development of the symptoms of poisoning in animals. Advanced electrophysiological techniques developed in our laboratory over many years will be fully utilized, including intracellular and extracellular microelectrode, voltage clamp, patch clamp for whole cell and single channel recording, and intracellular perfusion. These techniques will be applied to cultured neuroblastoma cells, squid and crayfish giant axons, rat and crayfish neuromuscular junctions, and hippocampus slices from guinea-pigs. Six specific projects will be investigated: 1) Modification of kinetics of sodium channels by the insecticides will be analyzed in detail; 2) pyrethroids and DDT appear to bind to a membrane site near the gating machinery as approached through the lipid phase, and this hypothesis will be demonstrated by use of various chemical agents; 3) structure-activity relationship will be determined with special reference to stereospecificity; 4) the mechanism underlying the profound negative temperature coefficient of action of the insecticides will be elucidated at the channel level; 5) the mechanism underlying the drastic effects of the pyrethroids on calcium channels will be determined; and 6) controversial results concerning the effects of pyrethroids on neuroreceptors such as GABA receptor-channel complex will be re-examined and resolved. These studies are expected to provide the basis for the molecular mechanism of action of insecticides on the nervous system.

The long-term objectives of the proposed study are to elucidate the mechanisms by which alcohols exert their toxic actions on the nervous system. Specific attention is focused on the mechanisms underlying the interactions of ethanol and longer-chain alcohols with ion channels by using patch clamp techniques as applied to mammalian neurons. Our previous studies as well as those by other investigators have laid out the groundwork along this line, establishing the phenomenological aspects of alcohol interactions with certain types of ion channels including those activated by GABA, excitatory amino acid (EAA), acetylcholine (ACh) and 5-hydroxytryptamine (5-HT), and voltage-activated sodium, potassium and calcium channels. However, conflicting data have been obtained for certain types of ion channels, and in many cases no detailed mechanisms of alcohol action have been elucidated. The proposed study is aimed at solving some of these problems. Thus the proposed projects will focus on elucidation of the mechanisms of ethanol and longer-chain alcohols on the GABA/A receptor-channel complex. Both whole-cell and single-channel patch clamp techniques will be applied to rat dorsal root gang lion, hippocampal and cortical neurons, and cerebellar Purkinje and granular layer neurons. The specific aims will be concerned with the modulation of open and/or closed channels, dependence of alcohol action on neuron type, animal age and experimental temperature, the role of intracellular components in alcohol action, and the comparison of subtypes and subunits of receptor-channel complex. For the GABA receptor subunit study, human embryonic kidney (HEK-293) cells, in which various combinations of GABA subunits have been transfected, will be used to determine the role of each subunit in alcohol action. Attention will be focused not only on changes in tee amplitude of GABA-induced currents, but also on changes in the rate of desensitization of the currents. The results of the proposed study will provide the basis for the mechanisms of alcoholism and for approaches to prevention and cure of the disorder.

The long-term objectives of the proposed study are to elucidate the mechanism of action of environmentally important insecticides on the nerve membrane which is the most critical target site of various insecticides. The knowledge obtained through this research will have direct impact on our environmental concern, i.e., developments of effective therapeutic means for insecticide intoxication, and of newer insecticides which are more effective against insects yet safer for humans. The specific aims are to clarify the nature of interactions of pyrethroid and DDT-type insecticides with nerve membrane ion channels which have been demonstrated to be mainly responsible for development of the symptoms of poisoning in animals. Advanced electrophysiological techniques developed in our laboratory over many years will be fully utilized, including intracellular and extracellular microelectrode, voltage clamp, patch clamp for whole cell and single channel recording, and intracellular perfusion. These techniques will be applied to cultured neuroblastoma cells, squid and crayfish giant axons, rat and crayfish neuromuscular junctions, and hippocampus slices from guinea-pigs. Six specific projects will be investigated: 1) Modification of kinetics of sodium channels by the insecticides will be analyzed in detail; 2) pyrethroids and DDT appear to bind to a membrane site near the gating machinery as approached through the lipid phase, and this hypothesis will be demonstrated by use of various chemical agents; 3) structure-activity relationship will be determined with special reference to stereospecificity; 4) the mechanism underlying the profound negative temperature coefficient of action of the insecticides will be elucidated at the channel level; 5) the mechanism underlying the drastic effects of the pyrethroids on calcium channels will be determined; and 6) controversial results concerning the effects of pyrethroids on neuroreceptors such as GABA receptor-channel complex will be re-examined and resolved. These studies are expected to provide the basis for the molecular mechanism of action of insecticides on the nervous system.

The long-term objectives of the proposed study are to elucidate the mechanisms whereby various therapeutic drugs exert their effects on neuroreceptor/channel systems. Emphasis is placed on their actions on voltage-activated channels and transmitter receptor-channel complexes. Patch clamp techniques will be used to record whole cell and single channel currents. Several classes of drugs and chemicals will be studied. Opioids have been demonstrated to block certain types of voltage-activated calcium channels through their action on opioid receptors. Agonist-receptor-calcium channel relationship, which has been controversial somewhat, will be established using different opioids and different preparations which include mouse neuroblastoma-glioma hybrid cells (NG108-15), human neuroblastoma cells (SH-SY5Y), and rat dorsal root ganglion neurons. Available evidence suggests that G proteins and/or second messengers are involved in the action of opioids. This problem will be studies by combining appropriate inhibitors such as pertussis toxin and staurosporine with calcium channel current recording. Calcium channel currents have been found to exhibit a gradual decrease and a large increase (rebound) during and after application of opioid, respectively. These mimic tolerance and withdrawal symptoms. Single channel analyses will be performed to elucidate the mechanisms underlying these phenomena. The excitatory amino acid (EAA) receptor-channel system is an important site of action of various therapeutic agents. Patch clamp analyses will be performed to elucidate the mechanism of action of certain antidepressants, psychotropic drugs, polyamines, and polyvalent cations on EAA-induced currents. These drugs include imipramine, desmethylimipramine, haloperidol, barbiturates and chlorpromazine. Attention will be focused on their mode of interactions with channel currents at both whole cell and single channel levels, their sites of action on the receptor-channel complex, and the interactions between these drugs and polyvalent cations. Some divalent cations such as Mg2+ and Zn2+ have been studied extensively and are useful as tolls for the study of drug action. Polyamines such as spermine, spermidine and putrescine have been suggested to modulate the EAA system and will be subjected to single channel analyses. These are tetrodotoxin (TTX)- resistant sodium channels in rat dorsal root ganglion neurons. Their physiological and pharmacological properties have been found to be considerably different from those of TTX-sensitive sodium channels. Whole cell and single channel analyses will be conducted to characterize then in comparison with TTX-sensitive channels. These differences are of particular importance from the pharmacological point of view as various therapeutic drugs are known to act on sodium channels.

The long-term objectives of the proposed study are to elucidate the mechanisms by which insecticides exert their toxic actions on mammals. Specific attention is focused on the mechanisms underlying the interactions of neuroactive insecticides with ion channels by using patch clamp techniques as applied to mammalian neurons which include mouse neuroblastoma cells, rat dorsal root ganglion neurons, and rat hippocampal and cortical neurons. Two classes of insecticides will be studied. One class includes pyrethroids and DDT which are different in chemical structure but are similar in the mode of action. The other class includes cyclodienes and lindane. Pyrethroids and DDT are known to act on the neuronal sodium channels thereby causing hyperactivity and convulsions in animals. Detailed mechanisms whereby these insecticides modify the sodium channel activity will be studied with special reference to the negative temperature dependence, tetrodotoxin-resistant and tetrodotoxin-sensitive sodium channels, comparison between pyrethroids and DDT, and selective block of pyrethroid-modified sodium channels by certain local anesthetics. The effects of pyrethroids on calcium channels, GABA receptor-channel complex and excitatory amino acid complex have been controversial, and detailed patch clamp experiments will be performed to clarify the significance in toxicity. Cyclodienes and lindane are known to block the GABA receptor-channel complex, yet no detailed mechanisms have been elucidated. The study will address the questions of single channel modification, exact site of action on the receptor-channel complex, comparison of lindane isomers, voltage and use dependence of action, interactions of polyvalent cations, and tissue specificity. The proposed study will provide useful and critical information for development of newer and safer insecticides and for treatment of insecticide intoxication in humans.

DESCRIPTION: The long-term goal of the proposed study is to elucidate the mechanism by which neuroactive insecticides exert their toxic actions on mammals. Specifically, the interactions of the insecticides with ion channels will be studied by using patch clamp techniques as applied to mammalian neurons isolated from the dorsal root ganglion, cerebellum, and hippocampus of the rat. The major target sites of pyrethroids and dieldrin/lindane are the voltage-gated sodium channel and the GABA-A receptor-channel, respectively. Several important breakthroughs accomplished recently have raised new critical questions regarding the mechanisms by which these insecticides modulate the respective target site. Type II (alpha-cyano) pyrethroids act on the sodium channel in a manner somewhat different from type I (no alpha-cyano) pyrethroids. Differential actions of type II pyrethroids on tetrodotoxin-sensitive (TTX-S) and tetrodotoxin-resistant (TTX-R) sodium channels will be analyzed with respect to the percentages of the modified sodium channels (project Aa). Negative temperature dependence of pyrethroids is a critical factor for the selective toxicity between mammals and invertebrates, and the mechanisms will be elucidated in terms of single sodium channel kinetics for both type I and type II pyrethroids (projects Ab and Ac). The discovery of the selective vitamin E block of the sodium channel modified by type I pyrethroids has opened the door for development of antidotes, and the underlying mechanisms for both types of pyrethroids will be explored with special emphasis on the open channel block by vitamin E (project Ad). The newly discovered dual action (stimulation and suppression) of dieldrin and lindane on the GABA receptor channel has raised important questions. Channel state dependence of action will be studied by analyzing use-dependent onset and recovery (project Ba). The dual effect is likely to due to the direct stimulating action and desensitization. The mechanism of desensitization caused by dieldrin and lindane will be studied by analyzing the drug dissociation rate from the binding site and single-channel kinetics (project Bb). The roles of GABA receptor subunits, especially the alpha1, alpha6, gamma2 and delta subunits, in the dual action of dieldrin and lindane will be determined by using the human embryonic cell line expressing various subunits with due consideration of subconductance states of single channels (project Bc). The GABA receptor subunits undergo developmental changes, and therefore, the actions of dieldrin and lindane are expected to change. This hypothesis will be studied during in vitro cell development and in vivo animal development (project Bd). The proposed study will provide useful and critical information for development of newer and safer insecticides and of prophylactic and treatment methods for insecticide intoxication.

chloride channels; membrane channels; neurotoxins; ethanol; electrophysiology; age difference; excitatory aminoacid; active sites; protein kinase; neurons; spinal ganglion; serotonin; hippocampus; acetylcholine; dorsal root; phosphorylation; GABA receptor; cyclic AMP; temperature; tissue /cell culture; kidney cell; human fetus tissue; laboratory rat; voltage /patch clamp; transfection;

The long-term goal of the proposed study is to elucidate the mechanism by which neuroactive insecticides exert their toxic actions. The specific aims of the proposed renewal application are to elucidate the physiological mechanisms that underlie the selective toxicity of several selected newer insecticides between mammals and insects. Most insecticides are much more toxic to insects than to mammals, and the mechanism of selective toxicity lies in many cases in differential actions on the target neuroreceptors/ion channels. Although recent developments and applications of molecular biology and genetics techniques have identified the molecular structures such as amino acid compositions of target receptors/channels that are deemed responsible for differential actions, almost nothing is known about how the differential actions are brought about as a result of the difference in molecular structures. Our working hypothesis is that the differential actions of insecticides on the target receptor/channels of mammals and insects could be caused by some difference in the kinetics of receptors/channels. For example, insecticide modification of the channel may be dependent upon the channel open or closed state, the kinetics of insecticide binding and unbinding, the temperature coefficient, etc. In order to elucidate the physiological mechanisms of selective toxicity, patch clamp data on the kinetics of receptors/channels and those of insecticide modification will be compared between rat and cockroach neurons for fipronil modulation of GABA receptors, imidacloprid modulation of neuronal nicotinic acetylcholine receptors (nnAChRs), spinosad modulation of nnAChRs and GABA receptors, and indoxacarb modulation of sodium channels, nnAChRs and GABA receptors. The results thus obtained are expected to answer the question of how selective toxicity between mammals and insects can be explained in terms of the differential actions on the target receptors/channels. This information will significantly contribute to the development of newer therapeutic means of insecticide intoxication of humans and of more effective and safer insecticides.

DESCRIPTION (provided by applicant): Abstract: The mechanisms of action of alcohol (ethanol) and nicotine on the nervous system have been studied extensively. However, it was not until a few years ago that microglia were recognized as an important site of action of alcohol and nicotine;alcohol for toxic effects and nicotine for neuroprotective effects. Heavy drinkers tend to be heavy smokers. We propose a hypothesis that microglia are a site where the drinking-smoking correlation resides. When activated by proinflammatory factors, microglia produce reactive oxygen species (ROS) such as NO and toxic cytokines such as TNF1. Electrons are moved across the membrane from intracellular NADPH to extracellular O2 to generate O2-*. This charge movement is compensated for by voltage-gated proton channels. Thus, both proton currents and electron currents are generated. The long-term goal of this project is to develop assessment of microglial function when alcohol and nicotine interact with each other. Specifically, we will elucidate the mechanism of alcohol-nicotine interaction through microglia. The results of this study will lay the foundation for understanding of alcohol and nicotine interaction. The proposed study comprises two parts: electrophysiological experiments and biochemical experiments. Proton currents are recorded from BV-2 microglial cell line as a measure of activation of microglia, and the effects of ethanol, nicotine and alcohol plus nicotine will be studied. Parallel biochemical experiments will be performed to measure ROS and toxic cytokines. We expect that ethanol augments the proinflammatory stimulation of microglia resulting in increases in proton currents, ROS and cytokines, and that nicotine suppresses the augmentation. PUBLIC HEALTH RELEVANCE: The proposed study of the interactive effects of ethanol and nicotine on microglia will have significant impact on preventing damages caused by drinking-smoking combination, and will lay the foundation of further mechanism of toxic effects of ethanol.