Kristopher Silver

Voltage‐Gated Sodium Channels as Insecticide Targets

Voltage-gated sodium channels are critical for the generation and propagation of action potentials. They are the primary target of several classes of insecticides, including DDT, pyrethroids and sodium channel blocker insecticides (SCBIs). DDT and pyrethroids preferably bind to open sodium channels and stabilize the open state, causing prolonged currents. In contrast, SCBIs block sodium channels by binding to the inactivated state. Many sodium channel mutations are associated with knockdown resistance (kdr) to DDT and pyrethroids in diverse arthropod pests. Functional characterization of kdr mutations together with computational modelling predicts dual pyrethroid receptor sites on sodium channels. In contrast, the molecular determinants of the SCBI receptor site remain largely unknown. In this review, we summarize current knowledge about the molecular mechanisms of action of pyrethroids and SCBIs, and highlight the differences in the molecular interaction of these insecticides with insect versus mammalian sodium channels.

Expression of pleiotrophin, an important regulator of cell migration, is inhibited in intestinal epithelial cells by treatment with non-steroidal anti-inflammatory drugs

Non-steroidal anti-inflammatory drugs (NSAIDs) are among the most widely used drugs for the suppression of inflammation and pain. However, the analgesic properties of NSAIDs are also associated with significant negative side effects, most notably in the gastrointestinal (GI) tract. Increasingly, evidence indicates that the ulcerogenic properties of some NSAIDs are not exclusively the result of inhibition of cyclooxygenase isoforms in the GI tract, and other mechanisms, including inhibition of cell migration and epithelial restitution, are being explored. Recently, microarray analysis was used to identify potential novel targets of NSAID activity in intestinal epithelial cells. Treated cells exhibited significant reductions in the gene expression of pleiotrophin (PTN), a cytokine and growth factor known to participate in angiogenesis and bone growth. This report aimed to confirm the microarray results reported previously, and to measure protein expression of PTN in intestinal epithelial cells. Furthermore, we also examined the effects of exogenous PTN on cell migration in the presence and absence of either NSAIDs with variable ulcerogenic potential or PTN-specific siRNA. Our results demonstrated that indomethacin and NS-398, two NSAIDs with ulcerogenic potential significantly decrease both gene and protein expressions of PTN in IEC-6 cells and protein expression in IEC-6-Cdx2 cells. Additionally, cell migration experiments with PTN siRNA showed that PTN is an important mediator of IEC-6 cell migration, and addition of exogenous PTN partially restores the deficits in cell migration caused by treatment with indomethacin and NS-398. Finally, measurement of PTN protein expression in the GI tract of horses treated with phenylbutazone showed that PTN expression is reduced by NSAIDs in vivo. Our results show that PTN is an important mediator of cell migration in IEC-6 cells, and PTN is a potential target through which NSAIDs may inhibit cell migration, epithelial restitution, and wound healing in the GI tract.

Role of the sixth transmembrane segment of domain IV of the cockroach sodium channel in the action of sodium channel blocker insecticides

Sodium channel blocker insecticides (SCBIs), such as indoxacarb and metaflumizone, are a new class of insecticides with a mechanism of action different from those of other insecticides that target sodium channels. SCBIs block sodium channels in a manner similar to local anesthetics (LAs) such as lidocaine. Several residues, particularly F1579 and Y1586, in the sixth transmembrane segment (S6) of domain IV (IV) of rat Na(v)1.4 sodium channels are required for the action of LAs and SCBIs and may form part of overlapping receptor sites. However, the binding site for SCBIs in insect sodium channels remains undefined. We used site-directed mutagenesis, the Xenopus laevis oocyte expression system, and the two-electrode voltage clamp technique to study the effects on SCBI activity of mutating F1817 and Y1824 (analogous to those residues identified in mammalian sodium channels) to alanine, in the voltage-sensitive sodium channel of the German cockroach, Blattella germanica. The mutant channels showed no effect or a marked increase in channel sensitivity to both DCJW (the active metabolite of indoxacarb) and metaflumizone. Thus, it appeared that although the F1817 residue plays a role in the action of SCBIs and that both residues are involved in LA activity in mammalian sodium channels, neither F1817 nor Y1824 are integral determinants of SCBI binding on insect sodium channels. Our results suggest that the receptor site of SCBIs on insect sodium channels may be significantly different from that on mammalian sodium channels.

Drug-Induced Alterations to Gene and Protein Expression in Intestinal Epithelial Cell 6 Cells Suggest a Role for Calpains in the Gastrointestinal Toxicity of Nonsteroidal Anti-Inflammatory Agents

Nonsteroidal anti-inflammatory drugs (NSAIDs) are used extensively as therapeutic agents, despite their well documented gastrointestinal (GI) toxicity. At this time, the mechanisms responsible for NSAID-associated GI damage are incompletely understood. In this study, we used microarray analysis to generate a novel hypothesis about cellular mechanisms that underlie the GI toxicity of NSAIDs. Monolayers of intestinal epithelial cells (IEC-6) were treated with NSAIDs that either exhibit (indomethacin, NS-398 [N-[2-(cyclohexyloxy)-4-nitrophenyl]-methanesulfonamide]) or lack (SC-560 [5-(4-chlorphenyl)-1-(4-methoxyphenyl)-3-(trifluoromethyl)-1H-pyrazole]) inhibitory effects on IEC-6 migration. Bioinformatic analysis of array data identified the calpain cysteine proteases and their endogenous inhibitor calpastatin as potential targets of NSAIDs shown previously to retard IEC-6 migration. Accordingly, quantitative real-time reverse transcription polymerase chain reaction and immunoblotting were performed to assess the effects of NSAIDs on the expression of mRNA and protein for calpain 8, calpain 2, calpain 1, and calpastatin. In treated IEC-6 monolayers, NS-398 decreased the expression of mRNA for calpain 2 and calpain 8. Both NS-398 and indomethacin decreased the protein expression of calpains 8, 2, and 1. None of the NSAIDs affected expression of calpastatin mRNA or protein. The calpain inhibitors, N-acetyl-Leu-Leu-methioninal and N-acetyl-Leu-Leu-Nle-CHO, retarded IEC-6 cell migration in a concentration-dependant fashion, and these inhibitory effects were additive with those of indomethacin and NS-398. Our experimental results suggest that the altered expression of calpain proteins may contribute to the adverse effects of NSAIDs on intestinal epithelial restitution.

Analysis of the action of lidocaine on insect sodium channels.

A new class of sodium channel blocker insecticides (SCBIs), which include indoxacarb, its active metabolite, DCJW, and metaflumizone, preferably block inactivated states of both insect and mammalian sodium channels in a manner similar to that by which local anesthetic (LA) drugs block mammalian sodium channels. A recent study showed that two residues in the cockroach sodium channel, F1817 and Y1824, corresponding to two key LA-interacting residues identified in mammalian sodium channels are not important for the action of SCBIs on insect sodium channels, suggesting unique interactions of SCBIs with insect sodium channels. However, the mechanism of action of LAs on insect sodium channels has not been investigated. In this study, we examined the effects of lidocaine on a cockroach sodium channel variant, BgNa(v)1-1a, and determined whether F1817 and Y1824 are also critical for the action of LAs on insect sodium channels. Lidocaine blocked BgNa(v)1-1a channels in the resting state with potency similar to that observed in mammalian sodium channels. Lidocaine also stabilized both fast-inactivated and slow-inactivated states of BgNa(v)1-1a channels, and caused a limited degree of use- and frequency-dependent block, major characteristics of LA action on mammalian sodium channels. Alanine substitutions of F1817 and Y1824 reduced the sensitivity of the BgNa(v)1-1a channel to the use-dependent block by lidocaine, but not to tonic blocking and inactivation stabilizing effects of lidocaine. Thus, similar to those on mammalian sodium channels, F1817 and Y1824 are important for the action of lidocaine on cockroach sodium channels. Our results suggest that the receptor sites for lidocaine and SCBIs are different on insect sodium channels.

Non-steroidal Anti-inflammatory Drugs Inhibit Calpain Activity and Membrane Localization of Calpain 2 Protease

Non-steroidal anti-inflammatory drugs (NSAIDs) are used frequently worldwide for the alleviation of pain despite their capacity to cause adverse gastrointestinal (GI) side effects. GI toxicity, once thought to be the result of non-specific inhibition of cyclooxegenase (COX) enzymes, is now hypothesized to have multiple other causes that are COX independent. In particular, NSAIDs inhibit intestinal epithelial restitution, the process by which barrier function in intestinal mucosa is restored at sites of epithelial wounds within hours through cell spreading and migration. Accordingly, recent evidence indicates that the expression of calpain proteases, which play a key role in cell migration, is decreased by NSAIDs that inhibit cell migration in intestinal epithelial cells (IEC). Here, we examine the effect of NSAIDs on calpain activity and membrane expression in IEC-6 cells. Indomethacin, NS-398, and SC-560 inhibited calpain activity and decreased expression of calpain 2 in total membrane fractions and in plasma membranes involved in cell attachment to the substrate. Additionally, we demonstrated that inhibition of calpain activity by NSAIDs or ALLM, a calpain inhibitor, limits cell migration and in vitro wound healing of IEC-6 cells. Our results indicate that NSAIDs may inhibit cell migration by decreasing calpain activity and membrane-associated expression of calpain 2. Our results provide valuable insight into the mechanisms behind NSAID-induced GI toxicity and provide a potential pathway through which these negative side effects can be avoided in future members of the NSAID class.

The Tribolium castaneum cell line TcA: a new tool kit for cell biology

The red flour beetle, Tribolium castaneum, is an agriculturally important insect pest that has been widely used as a model organism. Recently, an adherent cell line (BCIRL-TcA-CLG1 or TcA) was developed from late pupae of the red flour beetle. Next generation transcriptome sequencing of TcA cells demonstrated expression of a wide variety of genes associated with specialized functions in chitin metabolism, immune responses and cellular and systemic RNAi pathways. Accordingly, we evaluated the sensitivity of TcA cells to dsRNA to initiate an RNAi response. TcA cells were highly sensitive to minute amounts of dsRNA, with a minimum effective dose of 100 pg/mL resulting in significant suppression of gene expression. We have also developed a plasmid containing two TcA-specific promoters, the promoter from the 40S ribosomal protein subunit (TC006550) and a bi-directional heat shock promoter (TcHS70) from the intergenic space between heat shock proteins 68a and b. These promoters have been employed to provide high levels of either constitutive (TC006550) or inducible (TcHS70) gene expression of the reporter proteins. Our results show that the TcA cell line, with its sensitivity to RNAi and functional TcA-specific promoters, is an invaluable resource for studying basic molecular and physiological questions.

Inhibition of Kv channel expression by NSAIDs depolarizes membrane potential and inhibits cell migration by disrupting calpain signaling

Clinical use of non-steroidal anti-inflammatory drugs (NSAIDs) is well known to cause gastrointestinal ulcer formation via several mechanisms that include inhibiting epithelial cell migration and mucosal restitution. The drug-affected signaling pathways that contribute to inhibition of migration by NSAIDs are poorly understood, though previous studies have shown that NSAIDs depolarize membrane potential and suppress expression of calpain proteases and voltage-gated potassium (Kv) channel subunits. Kv channels play significant roles in cell migration and are targets of NSAID activity in white blood cells, but the specific functional effects of NSAID-induced changes in Kv channel expression, particularly on cell migration, are unknown in intestinal epithelial cells. Accordingly, we investigated the effects of NSAIDs on expression of Kv1.3, 1.4, and 1.6 in vitro and/or in vivo and evaluated the functional significance of loss of Kv subunit expression. Indomethacin or NS-398 reduced total and plasma membrane protein expression of Kv1.3 in cultured intestinal epithelial cells (IEC-6). Additionally, depolarization of membrane potential with margatoxin (MgTx), 40mM K(+), or silencing of Kv channel expression with siRNA significantly reduced IEC-6 cell migration and disrupted calpain activity. Furthermore, in rat small intestinal epithelia, indomethacin and NS-398 had significant, yet distinct, effects on gene and protein expression of Kv1.3, 1.4, or 1.6, suggesting that these may be clinically relevant targets. Our results show that inhibition of epithelial cell migration by NSAIDs is associated with decreased expression of Kv channel subunits, and provide a mechanism through which NSAIDs inhibit cell migration and may contribute to NSAID-induced gastrointestinal (GI) toxicity.

Molecular Mechanism of Action and Selectivity of Sodium Channel Blocker Insecticides

Sodium channel blocker insecticides (SCBIs) are a relatively new class of insecticides that are represented by two commercially registered compounds, indoxacarb and metaflumizone. SCBIs, like pyrethroids and DDT, target voltage-gated sodium channels (VGSCs) to intoxicate insects. In contrast to pyrethroids, however, SCBIs inhibit VGSCs at a distinct receptor site that overlaps those of therapeutic inhibitors of sodium channels, such as local anesthetics, anticonvulsants and antiarrhythmics. This review will recount the development of the SCBI insecticide class from its roots as chitin synthesis inhibitors, discuss the symptoms of poisoning and evidence supporting inhibition of VGSCs as their mechanism of action, describe the current model for SCBI-induced inhibition of VGSCs, present a model for the receptor for SCBIs on VGSCs, and highlight differences between data collected from mammalian and insect experimental models.

Molecular mechanisms influencing efficiency of RNA interference in insects: Molecular mechanisms influencing the efficiency of RNAi in insects

RNA interference (RNAi) is an endogenous, sequence-specific gene-silencing mechanism elicited by small RNA molecules. RNAi is a powerful reverse genetic tool, and is currently being utilized for managing insects and viruses. Widespread implementation of RNAi-based pest management strategies is currently hindered by inefficient and highly variable results when different insect species, strains, developmental stages, tissues, and genes are targeted. Mechanistic studies have shown that double-stranded ribonucleases (dsRNases), endosomal entrapment, deficient function of the core machinery, and inadequate immune stimulation contribute to limited RNAi efficiency. However, a comprehensive understanding of the molecular mechanisms limiting RNAi efficiency remains elusive. Recent advances in dsRNA stability in physiological tissues, dsRNA internalization into cells, the composition and function of the core RNAi machinery, as well as small-interfering RNA/double-stranded RNA amplification and spreading mechanisms are reviewed to establish a global understanding of the obstacles impeding wider understanding of RNAi mechanisms in insects.