Neurophysiological action of centrally-acting spider toxin polypeptides derived from Hadronyche versuta and Tegenaria agrestis venoms.

Established dogma concerning the action of insecticidal arthropod-derived peptides (e.g., scorpion toxins), was that they acted on the peripheral nervous system and were excluded from the central nervous system (CNS) by barrier systems. Initial evidence for a CNS-directed toxicological effect following parenteral administration was for a novel peptide from the Hobo spider, Tegeneria agrestis. This toxin was inactive on peripheral sensory and motor nerves, but had a potent excitatory effect on the CNS of larval Musca domestica. Recently, a commercialized formulation of GS-omega/kappa-Hxtx-Hv1a (HXTX), derived from the venom of the Australian blue mountain funnel web spider (Hadronyche versuta) was introduced for use in agriculture by Vestaron Corp. Its primary mode of action was found to be central neuroexcitation via positive allosteric modulation of nicotinic acetylcholine receptors (nAchR) of cockroach neurons. In the present study, this peptide showed hyperexcitation followed by a decrease in firing of the Drosophila melanogaster larval CNS that was prevented by co-exposure to 100 nM α-bungarotoxin (α-BGTX), a classical nAchR noncompetitive antagonist. This effect was mirrored in isobologram analysis, which showed clear antagonism between the two toxins when injected into adult houseflies. Interestingly, U1-agatoxin-Ta1b-QA derived from Tegeneria agrestis (VST-7304) had a similar biphasic action, but showed increased nerve discharge when co-exposed with 100 nM α-BGTX, and had additive effects when injected together with α-BGTX in isobologram analyses. Binary mixtures of HXTX or VST-7304 with 30 nM nicotine showed clear evidence of synergized nerve block, which was also observed for mixtures of HXTX and VST-7304. Taken together, these data suggest that HXTX and VST-7304 have somewhat different and complementary modes of action.

High-throughput screening method for evaluating spatial repellency and vapour toxicity to mosquitoes.

Spatial repellents are an essential tool for personal protection against mosquitoes that bite and transmit disease pathogens to humans. Current repellent screening methods, such as olfactometers and alternative choice tests, are complex systems that require a relatively large quantity of compound (mg). The present study validates a high-throughput spatial repellent screening method using a glass tube that has the ends covered with netting, in addition to treated filters and plastic end caps. The apparatus occupies relatively little space, is easy to decontaminate, and requires small amounts of compound (µg). In a horizontal tube orientation, DEET (N,N-diethyl-meta-toluamide), citronella oil and IR3535 had 1 h half repellent concentration (EC50 ) values of 32, 32 and 298 µg/cm2 , respectively, against the Orlando strain of Aedes aegypti (L.) (Diptera: Culicidae). Vertical tube orientation increased EC50 values by approximately two-fold, except IR3535, which remained essentially unchanged. Transfluthrin showed concentration-dependent spatial repellency (1 h EC50 = 0.5 µg/cm2 ) without any knockdown, although only in vertical tubes. Transfluthrin showed 50% knockdown in 1 h at 0.5 µg/cm2 and 50% mortality at 0.15 µg/cm2 in horizontal tubes. In conclusion, this high-throughput screening method is useful for assessing vapour toxicity and the spatial repellency of candidate molecules prior to semi-field and field studies.

Determination of metabolic resistance mechanisms in pyrethroid-resistant and fipronil-tolerant brown dog ticks.

Rhipicephalus sanguineus (Latreille) (Ixodida: Ixodidae) is a three-host dog tick found worldwide that is able to complete its' entire lifecycle indoors. Options for the management of R. sanguineus are limited and its' control relies largely on only a few acaricidal active ingredients. Previous studies have confirmed permethrin resistance and fipronil tolerance in R. sanguineus populations, commonly conferred by metabolic detoxification or target site mutations. Herein, five strains of permethrin-resistant and three strains of fipronil-tolerant ticks were evaluated for metabolic resistance using synergists to block metabolic enzymes. Synergist studies were completed with triphenyl phosphate (TPP) for esterase inhibition, piperonyl butoxide (PBO) for cytochrome P450 inhibition, and diethyl maleate (DEM) for glutathione-S-transferase inhibition. Additionally, increased esterase activity was confirmed using gel electrophoresis. The most important metabolic detoxification mechanism in permethrin-resistant ticks was increased esterase activity, followed by increased cytochrome P450 activity. The inhibition of metabolic enzymes did not have a marked impact on fipronil-tolerant tick strains.

Permethrin and malathion LD90 values for Culex quinquefasciatus vary with topical application site.

Prior research in multiple insect species has demonstrated that insecticide-induced mortality varies according to the body region exposed on the insect. This variation has been demonstrated in Culex quinquefasciatus Say (Diptera: Culicidae), but has not been quantified using dose-response curves. Applications of technical permethrin or malathion to one of three body regions on Cx. quinquefasciatus resulted in dose-response curves that were not equivalent to one another. The generated LD90 values and curves for each body region were compared with previously reported LD values for analogous sites in several mosquito species, specifically the mesothorax. Based on the present results, the permethrin and malathion LD50 and LD90 concentrations required for droplets impinging on the abdomen and mesothorax of Cx. quinquefasciatus when applied through ground-based spray systems utilized by mosquito control programmes were calculated.

Acute Toxicity and Sublethal Effects of Fenpyroximate to Amblyseius swirskii (Acari: Phytoseiidae).

Knowledge about the effects of pesticides on biological control agents is required in order to successfully implement integrated pest management programs. The predatory mite Amblyseius swirskii Athias-Henriot has been used to control thrips, whiteflies, and broad mites in vegetable production; however, effects of fenpyroximate, an acaricide and insecticide used in vegetable crops, on A. swirskii have not been evaluated. The effect of four residual concentrations of fenpyroximate on A. swirskii females was measured under laboratory conditions including its effect on their fecundity and larval survival. Fresh residues of fenpyroximate were significantly toxic to adult females and larvae. Mortality increased and fecundity decreased as the concentration (0.026-0.208 ml/50 ml of water) and time after treatment (24-120 h) increased. Fifty percent of the larvae survived on the two lower concentrations (0.026 and 0.052 ml/50 ml of water) after 120 h.

Pre-treatment of Stegomyia aegypti mosquitoes with a sublethal dose of imidacloprid impairs behavioural avoidance induced by lemon oil and DEET.

The present study was conducted to determine whether imidacloprid can impair the avoidance behaviour of the mosquito Stegomyia aegypti. Laboratory investigations using a T-maze apparatus showed that St. aegypti mosquitoes present long term avoidance behaviour when they are exposed to repetitive trials with lemon oil and DEET. The present study tested the effect of a sublethal dose of imidacloprid on the avoidance behaviour of St. aegypti mosquitoes over a 48 h period. Data suggest that 0.5 ng of imidacloprid/mosquito reduces the avoidance behaviour of mosquitoes exposed to lemon oil, on the first day of exposure, after the second trial; whereas imidacloprid affected DEET repellency only the first day of exposure, after the second trial. Imidacloprid was toxic against St. aegypti mosquitoes, and at sublethal doses was able to impair the repellency induced by lemon oil and DEET. The present data were consistent with the finding that St. aegypti mosquitoes exhibit long term avoidance behaviour, and treatment of mosquitoes with a sublethal dose of imidacloprid under DEET application can affect the repellency of DEET against St. aegypti.

Consequences of manganese administration for striatal dopamine and motor behavior in 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine-exposed C57BL/6 mice.

Environmental compounds may be important contributors to Parkinson's disease etiology. Epidemiological and experimental evidence for the facilitation of parkinsonism by manganese is equivocal. This work addressed methodological concerns in the few studies of manganese modulation of 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP)-induced toxicity in C57BL/6 mice. Male, retired breeder mice received 0 or 100 mg/kg of manganese chloride (MnCl2; subcutaneously on days 1, 4 and 7) and 0 or 20 mg/kg of MPTP (intraperitoneally on day 8) and survived up to day 15 or 22. On the day of sacrificing, horizontal (grid crossing) and vertical (rearing) open field movement, swimming, grip strength and grip fatigue were examined. Striata were analyzed for dopamine and 3,4-dihydroxyphenylacetic acid (DOPAC) using high-performance liquid chromatography. MPTP produced a main effect decrease in striatal dopamine (48.8%) and DOPAC (38.1%), but there was no main effect of MnCl2 or MnCl2 x MPTP interaction. However, modulatory interactions were observed between the effects of MnCl2 and MPTP for grid crossing, rearing and grip strength. Interestingly, these interactions reduced the severity of behavioral deficits attributable to either of these compounds alone. For rearing and grip strength, the MnCl2 x MPTP interaction was dependent upon survival time. The mechanistic nature of the MnCl2 x MPTP interaction upon these behaviors, in the absence of such an interaction for striatal dopamine and DOPAC, remains to be clarified.

Developmental regulation of a turkey intestinal peptide transporter (PepT1).

A cDNA encoding a turkey intestinal peptide transporter, tPepT1, was isolated from a turkey small intestinal cDNA library. The tPepT1 cDNA encodes a 714-amino acid protein with 12 predicted transmembrane domains. The amino acid sequence of tPepT1 is 94.3% identical to chicken PepT1 and approximately 60% identical to PepT1 from rat, sheep, rabbit, and human. Using a 2-electrode voltage-clamp technique in Xenopus oocytes expressing tPepT1, Gly-Sar transport was pH dependent but was independent of Na+ and K+. For the dipeptides Gly-Sar and Met-Met, the evoked inward currents indicated that the transporter was saturable and had high affinity (0.69 +/- 0.14 mM and 0.23 +/- 0.04 mM, respectively) for these substrates. However, transport of the tetrapeptide, Met-Gly-Met-Met, exhibited apparent substrate inhibition at high substrate concentrations. To study developmental regulation of PepT1 mRNA in turkey embryos, embryos (6 males and 6 females) were sampled daily from 5 d before hatch to the day of hatch (d 0). The abundance of PepT1 mRNA in the small intestine was quantified densitometrically from Northern blots after hybridization with full-length tPepT1 cDNA as probe. A 3.2-fold increase in PepT1 mRNA was observed in intestinal tissue from 5 d before hatch to d 0. This increase in PepT1 mRNA abundance indicates that the PepT1 gene is developmentally regulated and that there may be an important role for PepT1 in the neonatal poult.

Insecticidal arylalkylbenzhydrolpiperidines: novel inhibitors of voltage-sensitive sodium and calcium channels in mammalian brain.

Using preparations derived from whole mouse brain, we have demonstrated that insecticidal arylalkylbenzhydrolpiperidines inhibit the depolarization of synaptoneurosomes as measured by rhodamine 6G fluorescence and block 22Na+ uptake into synaptosomes, when veratridine is used as the activator. These insecticides also have the ability to potently displace the binding of [3H]batrachotoxinin A 20-alpha-benzoate ([3H]BTX-B) to neuronal sodium channels in a concentration-dependent manner. The study compounds can be classified as competitive inhibitors of radioligand binding, since they decrease the affinity of [3H]BTX-B for site 2 without affecting the concentration of sites labelled by this radioligand. Our kinetic analyses revealed that at its IC50, the 4-carbomethoxyaminobenzyl-piperidine analogue reduces the rate of association of [3H]BTX-B with site 2, whereas higher concentrations were required to accelerate dissociation of the [3H]BTX-B:sodium channel complex. These results indicate an ability to interact with both non-activated and persistently activated states of the voltage-sensitive sodium channel, but higher affinity for the former. Such a profile also implies that inhibition of [3H]BTX-B binding to site 2 occurs via an allosteric mechanism. In addition, arylalkylbenzhydrolpiperidines interact with presynaptic voltage-sensitive calcium channels, since we demonstrate that they inhibit increases in [free Ca++] and 45Ca++ uptake when evoked by high KC1 concentration in mouse brain synaptosomal preparations. Such effects generally occur at concentrations that are higher than those required to inhibit sodium channels. Blockade of sodium and calcium channels may therefore contribute to the in vivo neurological effects observed in rodents exposed to these insecticides.

Pharmacological properties of the MPTP analog trans-1-methyl-4-[4-dimethylaminophenylethenyl]-1,2,3,6-tetrahydropyridine and its pyridinium metabolite in mouse brain synaptosomes: a potential visual marker for substrates of MPTP-induced neurotoxicity.

1. The tetrahydropyridine trans-1-methyl-4-[4-dimethylaminophenylethenyl]-1,2,3,6-tetrahydropyridine (t-THP), like MPTP, can undergo monoamine oxidase (MAO)-mediated conversion to a dihydropyridinium intermediate and subsequent metabolism to a pyridinium species. t-THP is also a better substrate for MAO B than MAO A. In contrast to the metabolism of MPTP, the pyridinium ion derived from t-THP is highly fluorescent. This endows t-THP with potential as an in vivo visual probe for localizing the substrates of MPTP-like neurotoxicity. As a prelude to in vivo labeling studies, we examined the metabolism and uptake kinetics of t-THP and its metabolites in mouse striatal and cortical synaptosomes. 2. T-THP was found to induce a concentration-dependent and saturable fluorescence within striatal and cortical synaptosomes that was also MAO-dependent. Like MPP+, the fluorescent pyridinium ion t-P+, derived from t-THP, inhibited the uptake and facilitated the release of monoamines from synaptosomes in a concentration-dependent fashion. The ion did not rely on sodium-dependent membrane transporters for its concentration-dependent uptake into synaptosomes, although it may have an irreversible affinity for the dopamine transporter. 3. These data suggest that t-THP could be appropriate for use as a visual marker for microenvironments where MPTP-like compounds are taken up and converted to potentially neurotoxic pyridinium species. Such a marker could be employed to address some of the issues regarding the selectivity of MPTP-induced neurotoxicity.

Expression of a cloned ovine gastrointestinal peptide transporter (oPepT1) in Xenopus oocytes induces uptake of oligopeptides in vitro.

We determined the primary structure, tissue distribution and in vitro functional characterization of a peptide transporter, oPepT1, from ovine intestine. Ovine PepT1 (oPepT1) cDNA was 2829-bp long, encoding a protein of 707 amino acid residues with an estimated molecular size of 78 kDa and an isoelectric point (pI) of 6.57. Transport function of oPepT1 was assessed by expressing oPepT1 in Xenopus oocytes using a two-electrode voltage-clamp technique. The transport process was electrogenic and pH dependent, but independent of Na+, Cl- and Ca2+. The oPepT1 displayed a broad substrate specificity for transport of neutral and charged dipeptides and tripeptides. All dipeptides and tripeptides examined evoked inward currents in a saturable manner, with an affinity constant (Kt) ranging from 27 micromol/L to 3.0 mmol/L. No responses were detected from tetrapeptides or free amino acids. Northern blot analysis demonstrated that oPepT1 was expressed in the small intestine, omasum and rumen, but was not expressed in liver and kidney. The presence of the peptide transporter in the forestomach at such levels could provide nutritionally important amino acid nitrogen to ruminants.

Neurotoxicity of the organochlorine insecticide heptachlor to murine striatal dopaminergic pathways.

Changes in biochemical status of nerve terminals in the corpus striatum, one of the primary brain regions affected in Parkinson's disease, were studied in groups of C57BL/6 mice treated by ip injection three times over a 2-week period with 3--100 mg/kg heptachlor. On average, the maximal rate of striatal dopamine uptake increased > 2-fold in mice treated at doses of 6 mg/kg heptachlor and 1.7-fold at 12 mg/kg heptachlor. Increases in maximal rate of striatal dopamine uptake were attributed to induction of the dopamine transporter (DAT) and a compensatory response to elevated synaptic levels of dopamine. Significant increase in V(max) of striatal DAT was not observed at doses > 12 mg/kg, which suggested that toxic effects of heptachlor epoxide may be responsible for loss of maximal dopamine uptake observed at higher doses of heptachlor. In support of this conclusion, polarigraphic measurements of basal synaptosomal respiration rates from mice treated with doses of heptachlor > 25 mg/kg indicated marked, dose-dependent depression of basal tissue respiration. At doses of 6 and 12 mg/kg heptachlor, which increased expression of striatal DAT, uptake of 5-hydroxytryptamine into cortical synaptosomes was unaffected. Thus, striatal dopaminergic nerve terminals were found to be differentially sensitive to heptachlor. This reduced sensitivity of serotonergic pathways was mirrored in the greater potency of heptachlor epoxide to cause release of dopamine from preloaded striatal synaptosomes in vitro compared to release of serotonin from cortical membranes. These results suggest that heptachlor, and perhaps other organochlorine insecticides, exert selective effects on striatal dopaminergic neurons and may play a role in the etiology of idiopathic Parkinson's disease.

Striatal dopaminergic pathways as a target for the insecticides permethrin and chlorpyrifos.

Because insecticide exposure has been linked to both Parkinsons disease and Gulf War illness, the neurotoxic actions of pyrethroid and organophosphate insecticides on behavior and striatal dopaminergic pathways were investigated in C57BL/6 mice treated with permethrin (three i.p. doses at 0.2-200 mg/kg) or chlorpyrifos (three s.c. doses at 25-100 mg/kg) over a 2-week period. Permethrin altered maximal [3H]dopamine uptake in striatal synaptosomes from treated mice, with changes in Vmax displaying a bell-shaped curve. Uptake was increased to 134% of control at a dose of 1.5 mg/kg. At higher doses of PM (25 mg/kg), dopamine uptake declined to a level significantly below that of control (50% of control at 200 mg/kg, P < 0.01). We also observed a small, but statistically significant decrease in [3H]dopamine uptake by chlorpyrifos, when given at a dose of 100 mg/kg. There was no significant effect on the Km for dopamine transport. Evidence of cell stress was observed in measures of mitochondrialfunction, which were reduced in mice given high-end doses of chlorpyrifos and permethrin. Although cytotoxicity was not reflected in decreased levels of striatal dopamine in either 200 mg/kg PM or 100 mg/kg CPF treatment groups, an increase in dopamine turnover at 100 mg/kg CPF was indicated by a significant increase in titers of the dopamine metabolite, 3,4-dihydroxyphenylacetic acid. Both permethrin and chlorpyrifos caused a decrease in open field behavior at the highest doses tested. Although frank Parkinsonism was not observed, these findings confirm that dopaminergic neurotransmission is affected by exposure to pyrethroid and organophosphorus insecticides, and may contribute to the overall spectrum of neurotoxicity caused by these compounds.

Heptachlor alters expression and function of dopamine transporters.

Epidemiological data support a relationship between pesticide exposure and Parkinson's disease; however, no experimental evidence has been provided to support this association. Here we report that subchronic administration of the organochlorine insecticide heptachlor (0, 3, 6, 9, or 12 mg/kg given 3 times over a 2 week period) leads to a pronounced increase in both the plasma membrane transport of dopamine and the expression of the plasma membrane dopamine transporter (DAT), as well as the vesicular monoamine transporter (VMAT2) in the striatum of C57BL mice. To address possible mechanisms of increased DAT and VMAT2 expression, we performed transport studies in cell lines expressing the human forms of either DAT or VMAT2. In a DAT expressing cell line, acute treatment with the putative toxic species of heptachlor, heptachlor epoxide, did not alter plasma membrane dopamine uptake. In a VMAT2 expressing cell line, heptachlor epoxide significantly inhibited vesicular uptake of dopamine (45% reduction at 10 microM). Since DAT has been proposed to be the molecular gateway for dopaminergic toxins, such as the parkinsonism-inducing neurotoxin MPP, and VMAT2 has been proposed to protect cells from MPP and other toxins by sequestering the toxin into vesicles, the combined effects of heptachlor could increase the susceptibility of the nigrostriatal dopamine system to neurodegeneration. We further propose that altered dopamine transport by exposure to pesticides may provide a molecular basis for the increased incidence of Parkinson's disease.

p-Fluorophenylglycine in the urine of baboons treated with HPTP, the tetrahydropyridine analog of haloperidol.

We report the presence of p-fluorophenylglycine (p-FPG) in the urine of six baboons treated with HPTP, the tetrahydropyridine dehydration product of haloperidol (HP). Oxidative N-dealkylation, the major metabolic pathway of HP, gives rise to 3-(4-fluorobenzoyl)propionic acid (p-FBPA). Subsequent beta-oxidation of p-FBPA produces p-fluorophenylacetic acid (p-FPA). The presence of p-FPA argues for the formation also of p-fluorophenylglyoxylic acid (p-FPGA) derived from beta-oxidation of p-FBPA. Plasma aminotransferases should convert p-FPGA to p-FPG. The presence of p-FPG in these animals suggest the presence of phenylglycine aminotransferases in the baboon and possibly also in other primates, including the human. Reports by other authors found that treatment with alpha-phenylglycine (alpha-PG), an "unnatural" amino acid, leads to striatal dopamine (DA) depletion in rabbits--an effect explained on the basis of alpha-PG competing with DA for the neuronal vesicular storage sites. We performed in vitro DA release assays in mouse striatal synaptosomal preparations but found that neither alpha-PG nor p-FPG released any DA. It therefore remains unclear whether p-FPG may be a contributing factor to neurologic side-effects such as tardive dyskinesia (TD) found in patients after long-term HP treatment.

Effects of haloperidol metabolites on neurotransmitter uptake and release: possible role in neurotoxicity and tardive dyskinesia.

This research explored the effects of haloperidol (HP) metabolites on biogenic amine uptake and release, and compared them to those of MPTP and its toxic metabolite, MPP+. In synaptosome preparations from mouse striatum and cortex, the HP metabolites haloperidol pyridinium (HPP+), reduced haloperidol pyridinium (RHPP+), and haloperidol tetrahydropyridine (HPTP) inhibited the presynaptic uptake of dopamine and serotonin, with greater affinity for the serotonin transporter. HPP+ was the most potent inhibitor of dopamine uptake, and HPTP of serotonin uptake, both with IC50 values in the low micromolar range. RHPP+ was less active than the other metabolites, but was more active than the parent compound, HP. Inhibition of uptake was reversed when free drug was removed by centrifugation and then resuspension of the synaptosomes in fresh buffer, suggesting that inhibition of uptake was due to interaction with the transporters and was not due to irreversible cytotoxicity. HPP+ showed noncompetitive inhibition of both serotonin and dopamine uptake, suggesting that it has a relatively slow dissociation rate for its interaction with the transporter proteins. In experiments on amine release, HPP+ and HPTP were four-fold less potent than MPP+ for releasing preloaded dopamine from striatal synaptosomes, and only MPP+-dependent release was antagonized by the uptake blocker, mazindol. In contrast, RHPP+ displayed little ability to release either amine neurotransmitter. HPTP was about two-fold more potent than MPP+ for releasing serotonin from cortical synaptosomes, whereas HPP+ was less active than MPP+. The specific serotonin transport blocker fluoxetine was only able to antagonize release induced by MPP+. These results suggest that HP metabolites bind to the transporters for dopamine and serotonin, but are not transporter substrates. In contrast to their potent effects on amine release, HPP+ and HPTP were unable to release preloaded GABA from cortical synaptosomes. The implications of these results concerning a possible role of HP metabolites in the development of tardive dyskinesia are discussed.

Novel insecticidal peptides from Tegenaria agrestis spider venom may have a direct effect on the insect central nervous system.

Fractionation of venom from an agelenid spider, Tegenaria agrestis, resulted in the isolation of a family of three peptides with potent insecticidal activity. These peptide toxins, TaITX-1, -2, and -3, whose sequences were revealed from cloned cDNAs, each consist of 50 amino acid residues, six of which are cysteines. They appear to be amidated at their C-termini and exhibit greater than 90% sequence identity. Unlike other reported spider toxins, the TaI toxins are processed from precursors containing no propeptide sequences. In lepidopteran larvae and corn rootworm beetles, the insecticidal Tegenaria toxins caused an unusual excitatory symptomatology with 50% paralytic doses ranging from 0.23 to 2.6 nmol/g. In a series of electrophysiological experiments performed in house fly larvae, these toxins caused an elevated rate of firing from central nervous system neurons. No significant effects were found when any peripheral sensory or motor systems were examined. Thus, it appears that the TaI toxins may act in a fashion not previously reported for insecticidal peptide toxins; they may act directly on the insect central nervous system.

Poly(A)+ RNA from sheep omasal epithelium induces expression of a peptide transport protein(s) in Xenopus laevis oocytes.

To verify research from this laboratory indicating that sheep omasal epithelium contains mRNA encoding for a peptide transporter(s) and to determine di- to octapeptide transport capability, we injected poly(A)+ RNA isolated from sheep omasal epithelium into Xenopus laevis oocytes. Poly(A)+ RNA was functionally expressed in Xenopus oocytes 4 to 7 d after injection. Peptide (5 di-, 10 tri-, 6 tetra-, 2 penta-, 1 hexa-, 1 hepta-, and 1 octapeptide) transport capability was measured by impaling oocytes with a microelectrode to monitor membrane potential (Vm). Oocytes were maintained in pH 5.5 buffer. Peptide transport was identified as being expressed when, in the presence of a buffered peptide substrate (1 mM), the oocyte membrane showed persistent depolarization (a more positive Vm). In the absence of peptide transport, the membrane became depolarized with the addition of buffered substrate, but it rapidly repolarized to the resting potential. Peptide transport was expressed for some di-, tri-, and tetrapeptides. Measured depolarization ranged from 9.6 mV to 42.1 mV. Larger peptides were not transported by the oocytes. When transport expression was measured with the substrates in a pH 7.5 buffer, no transport occurred, indicating that transport was dependent on a proton gradient. Thus, sheep omasal epithelium contains mRNA that codes for a protein(s) capable of proton-dependent di-, tri-, and tetrapeptide transport. Results from the present study provide further evidence that absorption of peptides from the ruminant stomach is possible.

Mode of action of an insecticidal peptide toxin from the venom of a weaving spider (Diguetia canities).

A recently discovered spider toxin (DTX9.2) induced a rapid paralysis when injected into insects. In neurophysiological experiments, DTX9.2 elevated spike discharges in sensory nerves and at the neuromuscular junction, and also caused a depolarization of the membrane potential in cockroach giant axons. All these effects were reversed by treatment with the specific sodium channel blocker, tetrodotoxin. These findings suggest that DTX9.2 acts upon the voltage-dependent sodium channels of insect nerve membrane.

Demonstration and characterization of dipeptide transport system activity in sheep omasal epithelium by expression of mRNA in Xenopus laevis oocytes.

Research from this laboratory has recently demonstrated that the omasal epithelium of sheep is capable of absorbing dipeptides. In order to express proteins potentially responsible for the mediated absorption of small peptides, size-fractionated poly(A)+RNA (RNA) isolated from omasal epithelial tissue of sheep (average BW 67.5 kg) were injected into defolliculated Xenopus laevis oocytes. The ability of oocytes injected with RNA or water to absorb [14C]glycyl-L-sarcosine (Gly-Sar) from media (usually pH 5.5) was compared. After 4 d (P < .02) of culture, specific RNA fractions induced an increased (P < .02) rate of Gly-Sar absorption, as compared with water-injected oocytes. The dependency of Gly-Sar uptake on the presence of a pH gradient was evaluated at pH 5.0, 5.5, 6.0, 6.5, and 7.5. Inducible uptake increased (P < .001) in the presence of increasing proton concentrations, whereas endogenous uptake of Gly-Sar decreased (P < .001). At pH 5.5, induced Gly-Sar uptake was saturable (Kt = .4 mM), but endogenous uptake was not. The specificity of Gly-Sar absorption was studied by the co-incubation of .1 mM Gly-Sar with 5 mM levels of competing substrates (pH 5.5). Induced uptake was inhibited (P < .05) 44% by carnosine, 94% by methionylglycine, and 91% by glycylleucine, but not by glycine. Incubation of RNA with DNA oligomers that were complementary to the rabbit intestinal transporter completely inhibited (P < .05) induced Gly-Sar uptake. These results indicate that sheep omasal epithelial cells express messenger RNA that encode for proteins that are capable of H(+)-dependent dipeptide transport activity.