Interactions of DEET and Novel Repellents With Mosquito Odorant Receptors.

The carboxamide N,N-di-ethyl-meta-toluamide (DEET) is the most effective and widely used insect repellent today. However, drawbacks concerning the efficacy and the safety of the repellent have led to efforts to design new classes of insect repellents. Through quantitative structure-activity relationships, chemists have discovered two chemical groups of novel repellents: the acylpiperidines and the carboxamides, with the acylpiperidines generally more potent in biological assays. Although the exact mechanism of action of DEET and other repellents has not yet been thoroughly elucidated, previous research shows that the activity of insect odorant receptors are inhibited in the presence of repellents. The present electrophysiological study employs two-electrode voltage clamp with Xenopus laevis oocytes expressing AgOR2/AgOrco and AgOR8/AgOrco receptors to assess the effects of the novel repellents on Anopheles gambiae Giles (Insecta: Diptera: Culicidae) mosquito odorant receptors. The novel acylpiperidines and carboxamides reversibly inhibited (12-91%) odorant-evoked currents from both AgOR2/AgOrco and AgOR8/AgOrco receptors in a dose-dependent manner at all tested concentrations (30 µM to 1 mM). Furthermore, all the novel agents were more potent inhibitors of the receptors than DEET, with the acylpiperidines producing on average greater inhibition than the carboxamides. Interestingly, there was a correlation (r2 = 0.72) between the percentage inhibition of AgOR2/AgOrco receptor currents and protection times of the acylpiperidines. Our results add to existing evidence that the repellency of a compound is linked to its ability to disrupt the insect olfactory system and that the acylpiperidines could represent a class of more effective alternatives to the current gold standard, DEET.

Alkylphenol inverse agonists of HCN1 gating: H-bond propensity, ring saturation and adduct geometry differentially determine efficacy and potency.

BACKGROUND AND PURPOSE: In models of neuropathic pain, inhibition of HCN1 is anti-hyperalgesic. 2,6-di-iso-propyl phenol (propofol) and its non-anesthetic congener, 2,6-di-tert-butyl phenol, inhibit HCN1 channels by stabilizing closed state(s). EXPERIMENTAL APPROACH: Using in vitro electrophysiology and kinetic modeling, we systematically explore the contribution of ligand architecture to alkylphenol-channel coupling. KEY RESULTS: When corrected for changes in hydrophobicity (and propensity for intra-membrane partitioning), the decrease in potency upon 1-position substitution (NCO∼OH >> SH >>> F) mirrors the ligands' H-bond acceptor (NCO > OH > SH >>> F) but not donor profile (OH > SH >>> NCO∼F). H-bond elimination (OH to F) corresponds to a ΔΔG of ∼4.5 kCal mol-1 loss of potency with little or no disruption of efficacy. Substitution of compact alkyl groups (iso-propyl, tert-butyl) with shorter (ethyl, methyl) or more extended (sec-butyl) adducts disrupts both potency and efficacy. Ring saturation (with the obligate loss of both planarity and π electrons) primarily disrupts efficacy. CONCLUSIONS AND IMPLICATIONS: A hydrophobicity-independent decrement in potency at higher volumes suggests the alkylbenzene site has a volume of ≥800 Å3. Within this, a relatively static (with respect to ligand) H-bond donor contributes to initial binding with little involvement in generation of coupling energy. The influence of π electrons/ring planarity and alkyl adducts on efficacy reveals these aspects of the ligand present towards a face of the channel that undergoes structural changes during opening. The site's characteristics suggest it is "druggable"; introduction of other adducts on the ring may generate higher potency inverse agonists.

Differential Potency of 2,6-Dimethylcyclohexanol Isomers for Positive Modulation of GABAA Receptor Currents.

GABAA receptors meet all of the pharmacological requirements necessary to be considered important targets for the action of general anesthetic agents in the mammalian brain. In the following patch-clamp study, the relative modulatory effects of 2,6-dimethylcyclohexanol diastereomers were investigated on human GABAA (α1ß3γ2s) receptor currents stably expressed in human embryonic kidney cells. Cis,cis-, trans,trans-, and cis,trans-isomers were isolated from commercially available 2,6-dimethylcyclohexanol and were tested for positive modulation of submaximal GABA responses. For example, the addition of 30 µM cis,cis-isomer resulted in an approximately 2- to 3-fold enhancement of the EC20 GABA current. Coapplications of 30 µM 2,6-dimethylcyclohexanol isomers produced a range of positive enhancements of control GABA responses with a rank order for positive modulation: cis,cis > trans,trans ≥ mixture of isomers > > cis,trans-isomer. In molecular modeling studies, the three cyclohexanol isomers bound with the highest binding energies to a pocket within transmembrane helices M1 and M2 of the ß3 subunit through hydrogen-bonding interactions with a glutamine at the 224 position and a tyrosine at the 220 position. The energies for binding to and hydrogen-bond lengths within this pocket corresponded with the relative potencies of the agents for positive modulation of GABAA receptor currents (cis,cis > trans,trans > cis,trans-2,6-dimethylcyclohexanol). In conclusion, the stereochemical configuration within the dimethylcyclohexanols is an important molecular feature in conferring positive modulation of GABAA receptor activity and for binding to the receptor, a consideration that needs to be taken into account when designing novel anesthetics with enhanced therapeutic indices.

Microarray analyses of genes regulated by isoflurane anesthesia in vivo: a novel approach to identifying potential preconditioning mechanisms.

BACKGROUND: Although general anesthetics are recognized for their potential to render patients unconscious during surgery, exposure can also lead to long-term outcomes of both cellular damage and protection. As regards the latter, delayed anesthetic preconditioning is an evolutionarily conserved physiological response that has the potential for protecting against ischemic injury in a number of tissues. Although it is known that delayed preconditioning requires de novo protein synthesis, knowledge of anesthetic-regulated genes is incomplete. In this study, we used the conserved nature of preconditioning to analyze differentially regulated genes in 3 different rat tissues. We hypothesized that by selecting those genes regulated in multiple tissues, we could develop a focused list of gene candidates potentially involved in delayed anesthetic preconditioning. METHODS: Young adult male Sprague-Dawley rats were anesthetized with a 2% isoflurane/98% air mixture for 90 minutes. Immediately after anesthetic exposure, animals were euthanized and liver, kidney, and heart were removed and total RNA was isolated. Differential gene expression was determined using rat oligonucleotide gene arrays. Array data were analyzed to select for genes that were significantly regulated in multiple tissues. RESULTS: All 3 tissues showed differentially regulated genes in response to a clinically relevant exposure to isoflurane. Analysis of coordinately regulated genes yielded a focused list of 34 potential gene candidates with a range of ontologies including regulation of inflammation, modulation of apoptosis, regulation of ion gradients, and maintenance of energy pathways. CONCLUSIONS: Through using an analysis approach focusing on coordinately regulated genes, we were able to generate a focused list of interesting gene candidates with potential to enable future preconditioning studies.

Cyclohexanol analogues are positive modulators of GABA(A) receptor currents and act as general anaesthetics in vivo.

GABA(A) receptors meet all the pharmacological criteria required to be considered important general anaesthetic targets. In the following study, the modulatory effects of various commercially available and novel cyclohexanols were investigated on recombinant human γ-aminobutyric acid (GABA(A), α(1)ß(2)γ(2s)) receptors expressed in Xenopus oocytes, and compared to the modulatory effects on GABA currents observed with exposures to the intravenous anaesthetic agent, propofol. Submaximal EC(20) GABA currents were typically enhanced by co-applications of 3-300 µM cyclohexanols. For instance, at 30 µM 2,6-diisopropylcyclohexanol (a novel compound) GABA responses were increased ~3-fold (although similar enhancements were achieved at 3 µM propofol). As regards rank order for modulation by the cyclohexanol analogues at 30 µM, the % enhancements for 2,6-dimethylcyclohexanol~2,6-diethylcyclohexanol~2,6-diisopropylcyclohexanol~2,6-di-sec-butylcyclohexanol ≫2,6-di-tert-butylcyclohexanol~4-tert-butylcyclohexanol>cyclohexanol~cyclopentanol~2-methylcyclohexanol. We further tested the potencies of the cyclohexanol analogues as general anaesthetics using a tadpole in vivo assay. Both 2,6-diisopropylcyclohexanol and 2,6-dimethylcyclohexanol were effective as anaesthetics with EC(50)s of 14.0 µM and 13.1 µM respectively, while other cyclohexanols with bulkier side chains were less potent. In conclusion, our data indicate that cyclohexanols are both positive modulators of GABA(A) receptors currents and anaesthetics. The positioning and size of the alkyl groups at the 2 and 6 positions on the cyclohexanol ring were critical determinants of activity.

Assessment of general anaesthetic cytotoxicity in murine cortical neurones in dissociated culture.

General anaesthetics are proposed to cause unconsciousness by modulating neuronal excitability in the mammalian brain through mechanisms that include enhancement of inhibitory GABA(A) receptor currents and suppression of excitatory glutamate receptor responses. Both intravenous and volatile agents may produce neurotoxic effects during early postnatal rodent brain development through similar mechanisms. In the following study, we investigated anaesthetic cytotoxicity in primary cortical neurones and glia from postnatal day 2-8 mice. Cultures at 4-20 days in vitro were exposed to combinations of ketamine (100 µM to 3 mM), nitrous oxide (75%, v/v) and/or isoflurane (1.5-5%, v/v) for 6-12 h. Neuronal survival and cell death were measured via microtubule associated protein 2 immunoassay and lactate dehydrogenase release assays, respectively. Clinically relevant anaesthetic concentrations of ketamine, nitrous oxide and isoflurane had no significant neurotoxic effects individually or when given as anaesthetic cocktails, even with up to 12 h exposure. This lack of neurotoxicity was observed regardless of whether cultures were prepared from postnatal day 0-2 or day 8 mice, and was also unaffected by number of days in vitro (DIV 4-20). Significant neurotoxic effects were only observed at supraclinical concentrations (e.g. 1-3 mM ketamine). Our study suggests that neurotoxicity previously reported in vivo is not due to direct cytotoxicity of anaesthetic agents, but results from other impacts of the anaesthetised state during early brain development.

Synthesis and characterization of a redox-active ion channel supporting cation flux in lipid bilayers.

The synthesis, cation binding and transmembrane conductive properties of a novel synthetic ion channel containing a redox-active ferrocene unit are described. Fluorescence spectroscopy was used to demonstrate that the channel supports multiple ion coordination and association constants for 1:1 and 1:2 (channel:cation) coordination for both Na(+) and K(+) were evaluated. Experiments using a black lipid membrane preparation revealed that this compound functioned effectively as an ion channel for both Na(+) and K(+). Concomitant (23)Na NMR spectroscopy studies supported this finding and revealed a Na(+) flux, at least 5 times higher than ion transport rates by monensin. Furthermore, oxidation of the redox-active centre (Fe(2+) to Fe(3+)) effectively inhibited ion transport.

Role for metallothioneins-I/II in isoflurane preconditioning of primary murine neuronal cultures.

BACKGROUND: Pretreatment with inhaled anesthetics, including isoflurane, can induce long-lasting cellular protection against ischemia-derived toxicity in multiple tissues, including brain tissue. Metal-regulatory genes, metallothioneins-I/II (MT-I/II), have been shown to protect against oxidative damage in multiple tissues. Furthermore, MT have been found to be differentially regulated in response to isoflurane and ischemic preconditioning. In this study, we assess the role of MT-I/II in mediating isoflurane preconditioning in primary neuronal-glial cultures. METHODS: Primary mouse neuronal-glial cultures were preconditioned with isoflurane (3 h, 1.5%) 24-96 h before 3-h oxygen-glucose deprivation (OGD, ischemic model). After OGD, isoflurane protection and responsiveness of MT-I/II knockdown and knockout cultures to preconditioning were assessed by lactate dehydrogenase release. Immunoassays for microtubule associated protein 2 and glial fibrillary acidic protein determined neuronal-glial sensitivity to preconditioning. MT-I/II messenger RNA was assessed by quantitative reverse transcriptase-polymerase chain reaction. Cultures transfected with exogenous MT-I/II were analyzed for protection against OGD toxicity. RESULTS: Isoflurane preconditioning reduced OGD-mediated toxicity by 11.6 +/- 7.9% at 24 h, with protection increasing to 37.5 +/- 2.5% at 72 h after preconditioning. Immunolabeling showed that neurons were more sensitive to OGD and more responsive to isoflurane preconditioning compared to glia. Quantitative reverse transcriptase-polymerase chain reaction showed MT-I/II messenger RNA were upregulated (approximately 2.5-fold) by isoflurane treatments. Also MT-I/II protein transfection significantly decreased OGD-mediated toxicity. Finally, knockdown and knockout of MT-I/II diminished and abolished isoflurane-mediated protection, respectively. CONCLUSIONS: MT-I/II play an important role in isoflurane-mediated delayed preconditioning against OGD toxicity of neuronal and glial cells in vitro.

Menthol shares general anesthetic activity and sites of action on the GABA(A) receptor with the intravenous agent, propofol.

Menthol and related compounds were investigated for modulation of recombinant human gamma-aminobutyric acid type A (GABA(A), alpha(1)beta(2)gamma(2s)) receptor currents expressed in Xenopus oocytes. Sub-maximal (EC(20)) GABA currents were typically enhanced by co-applications of 3-300 microM (+)-menthol (e.g. by approximately 2-fold at 50 microM) > isopulegol > isomenthol> alpha-terpineol >> cyclohexanol. We studied menthol's actions on GABA(A) receptors compared to sedatives (benzodiazepines) and intravenous anesthetics (barbiturates, steroids, etomidate and propofol). Flumazenil (a benzodiazepine antagonist) did not inhibit menthol enhancements while currents directly activated by 50 microM propofol were significantly inhibited (by 26+/-3%) by 50 microM (+)-menthol. GABA(A) receptors containing beta(2) subunits with either a point mutation in a methionine residue to a tryptophan at the 286 position (in transmembrane domain 3, TM-3) or a tyrosine to a tryptophan at the 444 position (TM-4) are insensitive to modulation by propofol. Enhancements of GABA EC(20) currents by menthol were equally abolished in GABA(A) alpha(1)beta(2)(M286W)gamma(2s) and alpha(1)beta(2)(Y444W)gamma(2s) receptors while positive modulations by benzodiazepines, barbiturates and steroids were unaffected. Menthol may therefore exert its actions on GABA(A) receptors via sites distinct from benzodiazepines, steroids and barbiturates, and via sites important for modulation by propofol. Finally, using an in vivo tadpole assay, addition of (+)-menthol resulted in a loss of righting reflex with an EC(50) of 23.5+/-4.7 microM (approximately10-fold less potent anesthesia than propofol). Thus, menthol and analogs share general anesthetic action with propofol, possibly via action at similar sites on the GABA(A) receptor.

Subunit-dependent block by isoflurane of wild-type and mutant alpha(1)S270H GABA(A) receptor currents in Xenopus oocytes.

The volatile anesthetic isoflurane both prolongs and reduces the amplitude of GABA-mediated inhibitory postsynaptic currents (IPSCs) recorded in neurons. To explore the latter effect, we investigated isoflurane-induced inhibition of steady-state desensitized GABA currents in Xenopus oocytes expressing wild-type alpha(1)beta(2), alpha(1)beta(2)gamma(2s), mutant alpha(1)(S270H)beta(2) (serine to histidine at residue 270) or alpha(1)(S270H)beta(2)gamma(2s) receptors. The alpha(1) serine 270 site in TM2 (second transmembrane domain of the subunit) is postulated as a binding site for some volatile agents and is critical for positive modulation of sub-maximal GABA responses by isoflurane. For all receptor combinations, at < or =0.6 mM isoflurane (< or =2 minimum alveolar concentration (MAC)) current inhibitions were not pronounced ( approximately 10%) with block reaching half-maximal levels at supraclinical concentrations ( approximately 2 mM isoflurane, 6 MAC). Comparisons with other GABA(A) receptor blockers indicated that isoflurane blocks in a similar manner to picrotoxin, possibly via the pore of the receptor. The extent of isoflurane-induced inhibition was significantly attenuated by inclusion of the gamma(2s)-subunit but was unaffected by introduction of the S270H mutation in the alpha(1)-subunit. In conclusion, isoflurane binds with low affinity and with subunit-specificity to an inhibitory site on the GABA(A) receptor that is distinct from the site that facilitates positive modulation at the extracellular end of the pore.

Modulation of human GABAA and glycine receptor currents by menthol and related monoterpenoids.

Effects of common monoterpenoid alcohols and ketones were investigated on recombinant human gamma-aminobutyric acid A (GABAA; alpha1beta2gamma2s) and glycine (alpha1 homomers) receptors expressed in Xenopus oocytes. GABA currents were enhanced by coapplications of 10-300 microM: (+)-menthol>(-)-menthol>(-)-borneol>>(-)-menthone=camphor enantiomers>carvone enantiomers, with menthol acting stereoselectively. By contrast, thujone diastereomers inhibited GABAA receptor currents while glycine currents were only markedly potentiated by menthol. Positive modulation by (+)-menthol was explored given its pronounced effects (e.g., at 100 microM, GABA and glycine EC20 responses increased by 496+/-113% and 135+/-56%, respectively). (+)-Menthol, 100 microM, reduced EC50 values for GABA and glycine from 82.8+/-9.9 to 25.0+/-1.8 microM, and from 98.7+/-8.6 to 75.7+/-9.4 microM respectively, with negligible effects on maximal currents. This study reveals a novel neuroactive role for menthol as a stereoselective modulator of inhibitory ligand-gated channels.

The effects of isoflurane on desensitized wild-type and alpha 1(S270H) gamma-aminobutyric acid type A receptors.

UNLABELLED: gamma-aminobutyric acid type A receptors (GABA(A)-R) mediate synaptic inhibition and meet many pharmacological criteria required of important general anesthetic targets. During synaptic transmission GABA release is sufficient to saturate, maximally activate, and transiently desensitize postsynaptic GABA(A)-Rs. The resulting inhibitory postsynaptic currents (IPSCs) are prolonged by volatile anesthetics like isoflurane. We investigated the effects of isoflurane on maximally activated and desensitized GABA(A)-R currents expressed in Xenopus oocytes. Wild-type alpha(1)beta(2) and alpha(1)beta(2)gamma(2s) receptors were exposed to 600 microM GABA until currents reached a steady-state desensitized level. At clinical concentrations (0.02-0.3 mM), isoflurane produced a dose-dependent enhancement of steady-state desensitized current in alpha(1)beta(2) receptors, an effect that was less apparent in receptors including a gamma(2s)-subunit. When serine at position 270 is mutated to histidine (alpha(1)(S270H)) in the second transmembrane segment of the alpha(1)-subunit, the currents evoked by sub-saturating concentrations of GABA became less sensitive to isoflurane enhancement. In addition, isoflurane enhancements of desensitized currents were greatly attenuated by this mutation and were undetectable in alpha(1)(S270H)beta(2)gamma(2s) receptors. In conclusion, isoflurane enhancement of GABA(A)-R currents evoked by saturating concentrations of agonist is subunit-dependent. The effects of isoflurane on desensitized receptors may be partly responsible for the prolongation of IPSCs during anesthesia. IMPLICATIONS: Isoflurane enhances desensitized gamma-aminobutyric acid type A receptor (GABA(A)-R) currents, an effect that is subunit-dependent and attenuated by a mutation in an alpha(1)-subunit pore residue of the GABA(A)-R. As GABA release at inhibitory synapses is typically saturating, isoflurane modulation of desensitized receptors may be partly responsible for prolongation of inhibitory postsynaptic currents during anesthesia.

Cation transport by a redox-active synthetic ion channel.

The synthesis, cation binding and transmembrane conductive properties of a novel group of synthetic ion channels containing a redox-active centre are described. Experiments using a black lipid membrane preparation revealed that these compounds function effectively as ion channels. Subsequent 23Na NMR spectroscopy studies focused on a synthesized ion channel with a ferrocene centre. When incorporated in vesicular bilayers, this channel was demonstrated to support a Na+ flux that was at least six times faster than ion transport by monensin. Since oxidation of the ferrocene moiety completely inhibited the Na+ transport, the redox-active centre provides a potential mechanism for controlling ion flux.

Experimental methods in neuroscience: an undergraduate neuroscience laboratory course for teaching ethical issues, laboratory techniques, experimental design, and analysis.

We have developed and recently taught a 200 level undergraduate course entitled, 'Experimental Methods in Neuroscience'. This is a required course in an increasingly popular Neuroscience major at Smith College. Students are introduced initially to issues of animal ethics and experimentation, and are familiarized with our Animal Care Facility. Using an open field and rotarod apparatus, and the elevated plus and Barnes mazes, they conduct behavioral testing of two strains of mice, C57/BL/6J and 129S1/SvImJ, known to exhibit distinct behavioral traits. The group then employs histological techniques to prepare brain sections for observing neuroanatomical variation between strains (for example, 129S1/SvImJ mice are occasionally acallosal). In the final laboratory exercise, they assay the acetylcholinesterase activity in fore- and hindbrains from each strain. The experiments enable the students to gain confidence in collecting data, compiling large data sets, handling spreadsheets and graphing, applying appropriate statistics, and writing accurate and concise scientific reports in journal article format. The course concludes with pairs of students conducting self-designed independent projects using the acquired behavioral, histological or neurochemical techniques. Experimental Methods in Neuroscience is proving particularly successful as it is relatively straightforward for students to design interesting experiments, gain experience in neuroscience experimentation without excessive use of animals, gather substantial data sets, and develop skills in scientific report writing and presentation at an early stage in their neuroscience curricula. Furthermore, the course has emerged as a centralizing focus for our neuroscience program and is suitable for transfer to and adaptation by other institutions.