Xilong Zhao

Differential actions of insecticides on target sites: basis for selective toxicity.

Whereas the selective toxicity of insecticides between insects and mammals has a long history of studies, it is now becoming abundantly clear that, in many cases, the differential action of insecticides on insects and mammalian target receptor sites is an important factor. In this paper, we first introduce the mechanism of action and the selective toxicity of pyrethroids as a prototype of study. Then, a more detailed account is given for fipronil, based primarily on our recent studies. Pyrethroids keep the sodium channels open for a prolonged period of time, causing elevation of the depolarizing after-potential. Once the after-potential reaches the threshold for excitation, repetitive after-discharges are produced, resulting in hyperexcitation of intoxicated animals. Only about 1% of sodium channels needs to be modified to produce hyperexcitation, indicating a high degree of toxicity amplification from sodium channels to animals. Pyrethroids were >1000-fold more potent on cockroach sodium channels than rat sodium channels, and this forms the most significant factor to explain the selective toxicity of pyrethroids in insects over mammals. Fipronil, a phenylpyrazole, is known to act on the γ-aminobutyric acid receptor to block the chloride channel. It is effective against certain species of insects that have become resistant to most insecticides, including those acting on the γ-aminobutyric acid receptor, and is much more toxic to insects than to mammals. Recently, fipronil has been found to block glutamate-activated chloride channels in cockroach neurons in a potent manner. Since mammals are devoid of this type of chloride channel, fipronil block of the glutamate-activated chloride channel is deemed responsible, at least partially, for the higher selective toxicity to insects over mammals and for the lack of cross-resistance. Human & Experimental Toxicology (2007) 26, 361-366

Fipronil modulation of glutamate-induced chloride currents in cockroach thoracic ganglion neurons.

Fipronil is the first phenylpyrazole insecticide introduced for pest control. It is effective against some insects that have become resistant to other insecticides, and exhibits low mammalian toxicity. Although fipronil is known to block GABA receptors, the mechanisms of its selective toxicity and efficacy against insects with dieldrin-resistant GABA receptors are not fully understood. We studied the effects of fipronil on the inhibitory glutamate receptor-chloride channel complex, which is found only in invertebrates. Glutamate-activated chloride currents were recorded from neurons isolated from cockroach thoracic ganglia using the whole-cell patch clamp technique. When glutamate was applied to a neuron, it evoked inward currents with an EC50 of 36.8 +/- 3.0 microM and a Hill coefficient of 1.56 +/- 0.17. The similarity between the reversal potential and the calculated chloride equilibrium potential indicated that glutamate-induced currents were carried by chloride ions. Fipronil suppressed the glutamate-induced peak currents in a dose-dependent manner with an IC50 of 0.73 +/- 0.27 microM and a Hill coefficient of 0.68 +/- 0.15. The current decay phases were greatly prolonged after fipronil application in a concentration-dependent manner. Picrotoxinin (PTX) at 100 microM slightly suppressed glutamate-induced currents to 87.8 +/- 3.7% of the control, and dieldrin at 100 microM had no effect (96.7 +/- 3.1%). AP5 and CNQX, mammalian glutamate receptor antagonists, were without effect on glutamate-induced Cl- currents. It is concluded that the potent blocking action of fipronil against glutamate-gated chloride channels may contribute to the higher toxicity against insects than mammals, as well as the efficacy against insects resistant to other insecticides.

Voltage-dependent block of sodium channels in mammalian neurons by the oxadiazine insecticide indoxacarb and its metabolite DCJW

Indoxacarb is a newly developed insecticide with high insecticidal activity and low toxicity to non-target organisms. Its metabolite, DCJW, is known to block compound action potentials in insect nerves and to inhibit sodium currents in cultured insect neurons. However, little is known about the effects of these compounds on the sodium channels of mammalian neurons. We compared the effects of indoxacarb and DCJW on tetrodotoxin-sensitive (TTX-S) and tetrodotoxin-resistant (TTX-R) sodium channels in rat dorsal root ganglion neurons by using the whole-cell patch clamp technique. Indoxacarb and DCJW at 1-10 microM slowly and irreversibly blocked both TTX-S and TTX-R sodium channels in a voltage-dependent manner. The sodium channel activation kinetics were not significantly modified by 1 microM indoxacarb or 1 microM DCJW. The steady-state fast and slow inactivation curves were shifted in the hyperpolarization direction by 1 microM indoxacarb or 1 microM DCJW indicating a higher affinity of the inactivated sodium channels for these insecticides. These shifts resulted in an enhanced block at more depolarized potentials, thus explaining voltage-dependent block, and an apparent difference in the sensitivity of TTX-R and TTX-S channels to indoxacarb and DCJW near the resting potential. Indoxacarb and its metabolite DCJW cause toxicity through their action on the sodium channels.

Post-Stroke Dementia

Abstract: Nefiracetam is a new pyrrolidone nootropic drug that is being developed for clinical use in the treatment of post‐stroke vascular‐type and Alzheimers‐type dementia. Among a few neuroreceptors that have been identified as potential targets of nootropics, neuronal nicotinic acetylcholine receptors (nnAChRs) are deemed the most important since they are related to learning, memory, and Alzheimers disease dementia. We have recently found potent stimulating action of nefiracetam on nnAChRs. Rat cortical neurons in long‐term primary culture expressed nnAChRs. Whole‐cell patch clamp experiments revealed two types of currents induced by ACh, α‐bungarotoxin (α‐BuTX)‐sensitive, rapidly desensitizing, α7‐type currents and α‐BuTX‐insensitive, slowly desensitizing, α4β2‐type currents. Although α7‐type currents were only weakly inhibited by nefiracetam, α4β2‐type currents were potently and efficaciously potentiated by nefiracetam. Nefiracetam at 0.1 nM reversibly potentiated ACh‐induced currents to 200–300% of control. Very high concentrations (about 10 μM) also potentiated these currents, but to a lesser extent, indicative of the bell‐shaped dose‐response relationship known to occur for nefiracetam, even in animal behavior experiments. Three specific inhibitors of each of PKA and PKC did not prevent nefiracetam from potentiating ACh‐induced currents, indicating that these protein kinases are not involved in nefiracetam action. Pretreatment with pertussis toxin did not alter nefiracetam potentiation, indicating Gi/Go proteins are not involved. Pretreatment with cholera toxin did abolish nefiracetam potentiation. Thus, nefiracetam potentiation is mediated via Gs proteins. In conclusion, nefiracetam stimulates α4β2‐type nnAChRs via Gs proteins at nanomolar concentrations. The potentiation of α4β2‐type nnAChRs is thought to be at least partially responsible for cognitive enhancing action.

In vitro galantamine-memantine co-application: Mechanism of beneficial action

Several drugs are in clinical use for symptomatic treatment of Alzheimers disease patients. Since Alzheimers disease is known to be associated with down-regulation of the cholinergic and N-methyl-D-aspartate (NMDA) systems, most of these drugs inhibit acetylcholinesterase, potentiate the activity of nicotinic acetylcholine receptors (nAChRs), or modulate NMDA receptors. Galantamine is an anticholinesterase and allosterically potentiates the activity of the nicotinic receptors. We have recently found that galantamine potentiates the activity of NMDA receptors as well. Memantine is unique in that it inhibits the NMDA receptors. We have developed a hypothesis that combining galantamine and memantine will be more effective for improving the patients conditions than monotherapy with either drug. Patch clamp and intracellular Ca(2+) imaging experiments using rat cortical and hippocampal neurons clearly provided the in vitro bases for our hypothesis. Memantine blocked the extrasynaptic NMDA receptor 100 times more potently than the synaptic NMDA receptor at negative membrane potentials and the block of both types of NMDA receptors was attenuated with depolarization. However, galantamine potentiation of the NMDA receptors was not voltage dependent. Thus, co-application of memantine with galantamine prevented the galantamine potentiation and the activation of extrasynaptic NMDA receptors, but membrane depolarization revealed the galantamine potentiation. Therefore, cell death is expected to be prevented by memantine near the resting potential while the NMDA-mediated synaptic transmission, which is down-regulated in the patients, is maintained and potentiated by galantamine. These results provide in vitro bases for the beneficial actions of galantamine and memantine combinations.

Nefiracetam Potentiates N-Methyl-D-aspartate (NMDA) Receptor Function via Protein Kinase C Activation and Reduces Magnesium Block of NMDA Receptor

Nicotinic acetylcholine receptors and N-methyl-d-aspartate (NMDA) receptors are known to be down-regulated in the brain of Alzheimers disease patients. We have previously demonstrated that the nootropic drug nefiracetam potentiates the activity of both nicotinic acetylcholine and NMDA receptors and that nefiracetam modulates the glycine binding site of the NMDA receptor. Because the NMDA receptor is also modulated by Mg2+ and protein kinases, we studied their roles in nefiracetam action on the NMDA receptor by the whole-cell patch-clamp technique and immunoblotting analysis using rat cortical or hippocampal neurons in primary culture. The nefiracetam potentiation of NMDA currents was inhibited by the protein kinase C (PKC) inhibitor chelerythrine, but not by the protein kinase A (PKA) inhibitor N-[2-(4-bromocinnamylamino)ethyl]-5-isoquinoline (H89). In immunoblotting analysis, nefiracetam treatment increased the PKCα activity with a bell-shaped dose-response relationship peaking at 10 nM, thereby increasing phosphorylation of PKC substrate and NMDA receptor. Such an increase in PKCα-mediated phosphorylation was prevented by chelerythine. Nefiracetam treatment did not affect the PKA activity. Analysis of the current-voltage relationships revealed that nefiracetam at 10 nM largely eliminated voltage-dependent Mg2+ block and that this action of nefiracetam was sensitive to PKC inhibition. It was concluded that nefiracetam potentiated NMDA currents not by acting as a partial agonist but by interacting with PKC, allosterically enhancing glycine binding, and attenuating voltage-dependent Mg2+ block.

Modulation of tetrodotoxin-resistant sodium channels by dihydropyrazole insecticide RH-3421 in rat dorsal root ganglion neurons

The effects of the dihydropyrazole insecticide RH-3421 on the retrodotoxin-resistant (TTX-R) voltage-gated sodium channels in rat dorsal root ganglion (DRG) neurons were studied using the whole-cell patch clamp technique. RH-3421 at 10 nM to 1 microM completely blocked action potentials. The sodium currents were irreversibly suppressed by 1 microM RH-3421 in a time- and a dose-dependent manner and the IC50 value of RH-3421 was estimated to be 0.7 microM after 10 min of application. RH-3421 blocked the sodium currents to the same extent over the entire range of test potentials. The sodium conductance-voltage curve was not shifted along the voltage axis by 1 microM RH-3421 application In contrast, both fast and slow steady-state sodium channel inactivation curves were shifted in the hyperpolarizing direction in the presence of 1 microM RH-3421. It was concluded that RH-3421 bound to the resting and inactivated sodium channels to cause block with a higher affinity for the latter state.

Nefiracetam and galantamine modulation of excitatory and inhibitory synaptic transmission via stimulation of neuronal nicotinic acetylcholine receptors in rat cortical neurons

The cholinergic and glutamatergic systems are known to be downregulated in the brain of Alzheimers disease patients. Galantamine and nefiracetam have been shown to potentiate the phasic activity of nicotinic acetylcholine receptors (nAChRs) in the brain. Stimulation of nAChRs is also known to cause release of various neurotransmitters including glutamate and gamma-aminobutyric acid (GABA). We have previously reported that nefiracetam and galantamine potentiate the activity of nAChRs. Therefore, nefiracetam and galantamine are hypothesized to cause stimulations of the glutamate and GABA systems via stimulation of nAChRs. The present study was set out to test this hypothesis by measuring the effects of these drugs on spontaneous miniature excitatory postsynaptic currents (mEPSCs) and spontaneous miniature inhibitory postsynaptic currents (mIPSCs) recorded by the whole-cell patch clamp technique from rat cortical neurons in primary cultures. Acetylcholine (ACh) at 30 nM generated a steady inward current and increased the frequency of mEPSCs and mIPSCs. Nefiracetam at 10 nM plus 30 nM ACh increased the frequency of mEPSCs and mIPSCs beyond the levels increased by ACh alone. The potentiating action of nefiracetam was abolished by dihydro-beta-erythroidine. None of these treatments affected the amplitude of mEPSCs or mIPSCs. Galantamine at 1 muM plus ACh did not significantly potentiate the frequency. Nefiracetam at 10 nM had no effect on neurons that did not respond to 30 nM ACh. It was concluded that the nefiracetam released glutamate via stimulation of the alpha4beta2 nAChRs.