Genetic analysis of the xenobiotic resistance-associated ABC gene subfamilies of the Lepidoptera.

Some ATP-binding cassette (ABC) transporters of subfamilies B, C and G confer resistance to xenobiotics including insecticides. We identified genes of these subfamilies expressed by the lepidopterans Trichoplusia ni and Bombyx mori. The B. mori genome includes eight, six and 13 ABC-B, -C and -G genes, respectively, which encode P-glycoprotein, multidrug resistance protein, MRP, and breast cancer resistance protein, BCRP, homologues. Among the ABC-C and -G subfamilies, gene duplication contributes to protein diversity. We have identified three ABC-B and two ABC-C T. ni genes. Analyses of the T. ni MRP (TrnMRP) revealed unique features, including the potential for TrnMRP4 hyperglycosylation and the alternative splicing of TrnMRP1. Taken together, these attributes of moth multidrug resistance-associated ABCs may confer distinct functional capacities to xenobiotic efflux.

Insecticidal activity of glufosinate through glutamine depletion in a caterpillar.

The herbicide glufosinate-ammonium (GLA) is a competitive inhibitor of glutamine synthetase (GS), an enzyme converting glutamate to glutamine in both plants and animals. Because GS is essential for ammonia detoxification in plants, GLA treatment disrupts photorespiration by causing a build-up of ammonia and a loss of glutamine in plant tissues. This study reports that GLA applied to leaf surfaces is also toxic to 5th-instar caterpillars of the skipper butterfly Calpodes ethlius (LD50 = 400 mg kg-1). After ingesting GLA, caterpillars stopped feeding and became dehydrated through a loss of rectal function. Caterpillars showed symptoms of neurotoxicity, such as proleg tremors, body convulsions and complete paralysis before death. Incubation of several tissues isolated from normal feeding-stage caterpillars with the GS substrates glutamate and ammonium showed that GLA inhibited GS activity in vitro. Within 24 h of ingesting GLA, caterpillars had a greatly reduced glutamine content and the ammonium ion levels had more than doubled. Injection of ammonium chloride into non-GLA-treated caterpillars had no deleterious effect, suggesting that glutamine depletion, and not a rise in body ammonium, was the primary cause of GLA toxicity following GS inhibition. This was supported by the observation that the onset of the symptoms of GLA poisoning could be postponed by giving GLA-fed caterpillars several subsequent daily injections of glutamine. The effective GLA dose fed to 5th-instar caterpillars in this study was comparable to the amount that might realistically by acquired from feeding on GLA-treated crops.

New observations on the secondary chemistry of world Ephedra (Ephedraceae).

For several millennia, stem extracts of Ephedra (Ephedraceae, Gnetales) have been used as folk medicines in both the Old and New World. Some species were used in treatments of questionable efficacy for venereal disease in North America during the last century. Many Eurasian species produce phenylethylamine alkaloids, mostly ephedrine and pseudoephedrine, that interact with adrenergic receptors in the mammalian sympathetic nervous system. Asian Ephedra have been used recently in the clandestine manufacture of a street drug, methamphetamine. Although ephedrine alkaloids are not detectable in New World species of Ephedra, together with Asian species they contain other nitrogen-containing secondary metabolites with known neuropharmacological activity. Many mesic and particularly xeric species worldwide accumulate substantial amounts of quinoline-2-carboxylic acids, or kynurenates, in their aerial parts. Many species of Ephedra accumulate cyclopropyl amino acid analogues of glutamate and proline in their stems and roots, and particularly in the seed endosperm. Mesic species synthesize substantial amounts of three L-2-(carboxycyclopropyl)glycine stereomers rarely seen in nature. A cyclopropyl analogue of proline with known antimicrobial activity, cis-3,4-methanoproline, is found in large amounts in the stems and seeds of many Ephedra species. The ability to synthesize cyclopropyl amino acids may be an ancestral feature in the taxon. The natural function in the taxon of these three groups of secondary compounds remains to be established.

L-Glutamate retrieved with the moulting fluid is processed by a glutamine synthetase in the pupal midgut of Calpodes ethlius.

From apolysis until pupal ecdysis, the pharate pupa of the Brazilian Skipper (Calpodes ethlius) lies wrapped in a prepupal shell composed of the larval cuticle and an ecdysial space (ES) filled with enzyme-rich moulting fluid (MF). In the 4h before ecdysis the pharate pupa drinks the moulting fluid through its mouth and anus, and transfers the cuticular degradation products to its midgut (MG). At the same time, extra fluid passes across the body wall of the pharate pupa and flushes out the ES. The MF is recovered at an overall rate of 70µl/h and reabsorbed across the pharate pupal midgut at about 26µl/h. L-Glutamate was found to be the dominant amino acid in the moulting fluid. Total MF glutamate peaked at 850nmol about 8h before pupal ecdysis (P-8), but by ecdysis it had dropped to nearly zero as the MF became diluted with new fluid and was consumed. The drop in glutamate in the ES coincided with a rise in the glutamine content of the fluid in the midgut lumen. The highest rate of glutamine synthesis occurred in midguts isolated from pharate pupae actively drinking MF (P

Substrate-stereoselectivity of a high-affinity glutamate transporter cloned from the CNS of the cockroach Diploptera punctata.

A cDNA encoding a Na(+)-dependent glutamate transporter has been cloned from the brain of the cockroach Diploptera punctata. The cDNA encodes a transporter protein of 481 amino acids, designated DipEAAT1, which when expressed in baculovirus infected insect cells, resulted in a 40-50 fold increase in [(3)H]L-glutamate uptake. DipEAAT1 mRNA is expressed in the brain, as is the RNA encoding TrnEAAT1, a related transporter recently isolated from the caterpillar Trichoplusia ni. The affinity of these transporters for L-glutamate and several structural analogues was compared. Both have a high affinity for L-glutamate, their presumed primary substrate, but quite different affinities for D-aspartate. TrnEAAT1 was found to be similar to other glutamate transporters in that its ability to transport [(3)H]L-glutamate into cells was inhibited strongly by D- and L- isomers of aspartate and its analogues. DipEAAT1, by contrast, was inhibited weakly by all D- isomers tested. The affinity of DipEAAT1 for [(3)H]D-aspartate was found to be an order of magnitude lower than that of TrnEAAT1, revealing an unusual stereoselectivity for aspartate substrates by the cockroach transporter. The activity of DipEAAT1 was also unaffected by the presence of Zn(++) in the bathing solution, despite the presence of a putative Zn(++)-binding motif conferring Zn(++)-sensitivity on some mammalian glutamate transporters.

Molecular cloning and functional characterization of a GABA transporter from the CNS of the cabbage looper, Trichoplusia ni.

A cDNA encoding a GABA transporter in the caterpillar Trichoplusia ni has been cloned and expressed in baculovirus-infected insect cells. The cDNA contains an ORF encoding a 608-residue protein, designated TrnGAT. Hydropathy analysis of the deduced amino acid sequence suggests 12 transmembrane domains, a structure similar to that of all other cloned Na+/Cl(-)-dependent GABA transporters. The deduced amino acid sequence shows high identity with a GABA transporter (MasGAT) expressed in the embryo of Manduca sexta. Expression of TrnGAT mRNA was detected only in the brain. Sf21 cells infected with recombinant baculovirus exhibited a 20- to 30-fold increase in [3H]GABA uptake compared to control-infected cells. Several blockers of GABA uptake were used to determine the pharmacological profile of TrnGAT. Although most similar to mammalian neuronal GABA transporter GAT-1 in its kinetic properties, stoichiometry of ionic dependence and pharmacological properties, TrnGAT may be distinguished from mammalian GAT-1 by the inability of cyclic GABA analogues, such as nipecotic acid and its derivatives, to inhibit GABA uptake by the insect protein. The unique pharmacology of TrnGAT suggests that the GABA transport system in the lepidopteran CNS could be a useful target in the future development of rapidly-acting neuroactive agents used to control agriculturally-important insects.

Characterization of a chloride current in the larval epidermis of the beetle Tenebrio molitor.

Voltage-clamp analysis of single cuticle-attached epidermal cells dissected from the newly-ecdysed mealworm revealed the presence of a large inwardly-rectifying anion (i.e. outwardly-going) current. In many cells this current formed spontaneously on breaking into the cell with the patch pipette when the bath solution was isoosmotic with the pipette solution (415 mosmol/l). The current was evoked rapidly by electrical stimulation or by bathing the cells in hyposmotic saline (335 mosmol/l). The reversal potential of the activated current shifted in agreement with the Nernst prediction for Cl(-) when the transmembrane chloride gradient was altered by partially substituting bath or patch pipette Cl(-) with gluconate(-). Substitution of Na(+) with choline(+) or K(+) with TEA and Ba(+) in the bath or pipette solutions did not alter the reversal potential. Addition of 200 &mgr;mol/l cyclic AMP or 1 mmol/l cyclic GMP to the pipette solution increased the initial current strength and reduced the time taken to reach half peak amplitude from 117 sec to 49 sec and 41 sec, respectively. Cyclic AMP also raised the threshold at which the current developed under hyperosmotic conditions by about 20 mosmol/l. Addition of the Cl(-) channel blockers diphenylamine-2-carboxylic acid (200 &mgr;mmol/l) and diisothiocyanostilbene-2,2'-disulphonic acid (250 &mgr;mol/l) to the bath solution reduced the inwardly-rectifying anion current by 50%. This current was barely detectable in cells prepared from the mid-instar integument. This non-constitutive pattern of expression suggests that cellular Cl(-) efflux (and that of other anions) may be required during moult-cycle specific processes such as moulting fluid formation and cell volume regulation. As the strength of the epidermal anion current could be raised by the exogenous application of cytosolic cyclic nucleotides, the activity of the anion channels responsible for this current may normally be regulated by yet-to-be-identified hormone(s) or neuropeptide(s) acting on this tissue.

Preparation and application of an isolated superior mesenteric arterial vascular preparation.

Our laboratory developed an isolated perfused superior mesenteric arterial vascular bed preparation to study and correlate vascular smooth-muscle mechanics with associated biochemical events. This preparation provides consistent dose-dependent contractile responses, contains most of the superior mesenteric artery as well as first-, second-, and third-generation arterioles, and has been used for concurrent functional and biochemical analysis of vascular smooth muscle. Preparations isolated from Sprague-Dawley rats produced rapid, dose-related vasoconstrictor responses to norepinephrine (NE) and KCl, while appearing to be unresponsive to periarterial nerve stimulation. Endothelial relaxations to bolus doses of acetylcholine (ACh) in the presence of a constant infusion of NE (10 microM) were limited, producing reductions of perfusion pressures of <25%. Receptor-binding studies conducted to evaluate alpha1-adrenoceptor subtypes revealed high- and low-affinity binding sites composing 91 and 9% of the overall population, respectively. A 60-s time course for contractile response and inositol 1,4,5-triphosphate (IP3) production revealed a significant but transient increase of IP3 that paralleled the contractile response generated by using bolus injections of NE (30 microg). This preparation offers the capacity to conduct perfusion studies investigating vasoconstrictor responses, as well as biochemical studies including receptor-binding and second-messenger assays in the same tissue.

Faecal firing in a skipper caterpillar is pressure-driven

Many leaf-rolling caterpillars have a rigid anal comb attached to the lower surface of the anal plate (or shield) situated above the anus. This comb is widely assumed to be a lever used to 'flick' away frass pellets. An alternative mechanism to explain pellet discharge is proposed on the basis of observations on the caterpillar of the skipper Calpodes ethlius. The model proposes that the underside of the anal plate serves as a blood-pressure-driven surface for the ejection of faecal pellets. Rather than acting as a lever, the anal comb serves as a latch to prevent the premature distortion of the lower wall of the anal plate until the anal haemocoel compartment is fully pressurized. The anal comb is swung into position during pellet extrusion by retractor muscles attached at its base and held in place by a catch formed by a blood-swollen torus of everted rectal wall. As the caterpillar raises the blood pressure in its anal compartment by contracting its anal prolegs, the comb eventually slips over the toral catch. This causes the underside of the anal plate to move rapidly backwards as the blood pressure is released, projecting the pellet resting against it through the air. Simulation suggests that a local blood pressure of at least 10 kPa (75 mmHg) would be required to accelerate the lower surface of the anal plate outwards at a rate fast enough to discharge a 10 mg pellet at an observed mean velocity of 1.3 m s-1.

Molecular cloning and functional expression of an insect high-affinity Na+-dependent glutamate transporter.

Excitatory amino acid transporters in the central and peripheral nervous systems of insects are thought to assist in maintaining glutamate concentrations in the resting synapse below the activation threshold of glutamate receptors. We have isolated a cDNA from the caterpillar Trichoplusia ni which encodes a high-affinity Na+-dependent glutamate transporter, designated TrnEAAT1. The deduced amino acid sequence shows strong identity with known members of the vertebrate Na+- and K+-dependent amino acid transporter family. Expression of the insect transporter mRNA was predominantly localized in the caterpillar brain. The function of the TrnEAAT1 protein was analyzed in cultured insect cells using a baculovirus expression system. Cells infected with the recombinant virus were found to exhibit a 50-fold increase in ability to accumulate labeled L-glutamate compared to mock-infected cultures, and this activity was shown to be Na+-dependent. Transport activity was further demonstrated by chromatographic identification of various glutamate analogues accumulated by infected cells. Various glutamate uptake inhibitors were used to outline the pharmacological properties of the cloned transporter and to compare it with known mammalian transporters. Despite the significant differences between insect and vertebrate physiology, the characteristics of the respective transporters were found to be remarkably similar.

Examination by radioligand binding of the alpha1 adrenoceptors in the mesenteric arterial vasculature during the development of salt-sensitive hypertension.

Previous experiments have suggested that the vascular smooth muscle of Dahl salt-sensitive (DS) rats may possess a difference in the alpha1-adrenoceptor population or its transduction processes compared to Dahl salt-resistant (DR) rats. The purpose of the current research is to study the role of alpha1-adrenoceptors in the specific supersensitivity to norepinephrine (NE) seen prior to and early in the development of hypertension in the DS rat. Experiments in isolated perfused superior mesenteric arterial vasculature from DS rats chronically fed a high (7%) salt diet for 5 days or 3 weeks, in the absence or presence of an elevation in systolic blood pressure, respectively, demonstrated a specific supersensitivity to NE relative to DR rats. The enhanced responsiveness was specific to NE after 5 days of high salt since no differences in sensitivity of these preparations was observed to either KCl or 5-HT. A small but significant elevation in sensitivity to KCl following 3 weeks of treatment suggests that multiple factors may contribute to tissue responsiveness at this time. Radioligand binding experiments were performed using [125I]-HEAT to study the alpha1-adrenoceptor population and its subtypes. Saturation experiments using membranes prepared from the superior mesenteric arterial vasculature or mesenteric arterial branches showed no significant differences in overall alpha1-adrenoceptor population between DS and DR rats fed a high-salt diet for 5 days or 3 weeks. Competition experiments using membranes prepared from the superior mesenteric arterial branches in the presence of the alpha1A-subtype selective antagonist 5-methylurapidil showed two binding sites (high and low affinity) in these resistance vessels but no significant differences in nature or ratio of these sites between the DS and DR groups. These results suggest that changes in the alpha1-adrenoceptor population are not responsible for the specific supersensitivity to NE, which may be an early event in the induction and development of hypertension.

Normal development of preimplantation mouse embryos deficient in gap junctional coupling.

The connexin multigene family (13 characterized members in rodents) encodes the subunits of gap junction channels. Gap junctional intercellular coupling, established during compaction of the preimplantation mouse embryo, is assumed to be necessary for development of the blastocyst. One member of the connexin family, connexin43, has been shown to contribute to the gap junctions that form during compaction, yet embryos homozygous for a connexin43 null mutation develop normally, at least until implantation. We show that this can be explained by contributions from one or more additional connexin genes that are normally expressed along with connexin43 in preimplantation development. Immunogold electron microscopy confirmed that roughly 30% of gap junctions in compacted morulae contain little or no connexin43 and therefore are likely to be composed of another connexin(s). Confocal immunofluorescence microscopy was then used to demonstrate that connexin45 is also assembled into membrane plaques, beginning at the time of compaction. Correspondingly, embryos homozygous for the connexin43 null mutation were found to retain the capacity for cell-to-cell transfer of fluorescent dye (dye coupling), but at a severely reduced level and with altered permeability characteristics. Whereas mutant morulae showed no evidence of dye coupling when tested with 6-carboxyfluorescein, dye coupling could be demonstrated using 2',7'-dichlorofluorescein, revealing permeability characteristics previously established for connexin45 channels. We conclude that preimplantation development in the mouse can proceed normally even though both the extent and nature of gap junctional coupling have been perturbed. Despite the distinctive properties of connexin43 channels, their role in preimplantation development can be fulfilled by one or more other types of gap junction channels.

Double whole-cell patch-clamp characterization of gap junctional channels in isolated insect epidermal cell pairs.

Double whole-cell patch-clamp methods were used to characterize junctional membrane conductances in epidermal cell pairs isolated from the prepupal integument of the flour beetle, Tenebrio molitor. The mean initial junctional conductance in 267 cell pairs was 9.5 +/- 1.0 nS (range 0-95 nS). Well-coupled cell pairs uncoupled spontaneously with a half-time of 7.6 min. Adding 5 mM ATP to the pipette solution stabilized coupling with less than a 50% drop occurring after 30 min. Nonjunctional membrane potential was the major determinant of junctional conductance with transjunctional potential playing a minor role. Junctional conductance approached 0 pA at nonjunctional membrane potentials greater than 0 mV and increased with hyperpolarization. The voltage at half-maximal conductance was -26 mV. The time course of the reversible changes in junctional conductance were slow (< or = 30 sec) with time-dependent decay occurring faster and recovery occurring slower with increasing depolarization. Single gap junctional channel activity was recorded in uncoupling cell pairs and in poorly coupled ATP-stabilized cell pairs. One main single channel conductance was observed in each cell pair. The mean single channel conductances from all cell pairs in this study ranged from 197-347 pS (mean 248 pS). Single channel conductance was linear over the +/- 60 mV transjunctional voltage range tested. A broad range of subconductance states of the main state representing 5% of the total open time of measurable main state events was observed. Single channel activity was strongly dependent on the nonjunctional membrane potential, increasing with hyperpolarization.

Growth retardation in glioma cells cocultured with cells overexpressing a gap junction protein.

To examine the role of gap-junctional intercellular communication in controlling cell proliferation, we have transfected C6 glioma cells with connexin 43 cDNA. The growth of transfected clones was dramatically reduced compared with nontransfected glioma cells. To further characterize the role of gap junctions in controlling proliferation, we have examined the growth of C6 cells cocultured with transfected cells overexpressing connexin 43. Although C6 cells grew at their normal rate when cocultured with nontransfected C6 cells, when cocultured with connexin 43-overexpressing cells they displayed a dramatic reduction in growth rate. Furthermore, a significant, dose-dependent reduction in cell proliferation was noted when C6 cells were cultured in medium conditioned by transfected cells. This effect correlated with the level of connexin 43 expression. These results suggest that the decreased cell proliferation rate of transfected cells and C6 cells cultured with them is due to the secretion of a growth inhibitory factor(s) and that the secretion of this factor may be linked to the level of gap junctional intercellular communication.

Expression of gap junction genes in astrocytes and C6 glioma cells.

The expression of the gap junction genes coding for the liver-type connexin32 and the heart-type connexin43 was examined in primary cultures of astrocytes and in cultures of C6 glioma cells. In both cell types, only connexin43 mRNA was detectable. However, the level of this mRNA was greatly reduced in C6 glioma cells compared to astrocytes. This was consistent with the further observation that astrocytes in primary culture were extensively dye-coupled, whereas such coupling was very restricted in cultures of C6 glioma cells. Connexin43 was immunocytochemically localized in astrocytes, but was not readily detected in C6 cells.

Transfection of C6 glioma cells with connexin 43 cDNA: analysis of expression, intercellular coupling, and cell proliferation.

C6 glioma cells express low levels of the gap junction protein connexin 43 and its mRNA and display very weak dye coupling. When implanted into the rat cerebrum, these cells quickly give rise to a large glioma. To investigate the role of gap junctions in the tumor characteristics of these cells, we have used Lipofectin-mediated transfection to introduce a full-length cDNA encoding connexin 43. Several transfected clones were obtained that exhibited various amounts of connexin 43 mRNA transcribed from the inserted cDNA. Immunocytochemical analysis revealed an increase in the amount of connexin 43 immunoreactivity in the transfected cells, being localized at areas of intercellular contact as well as in the cytoplasm. The level of dye coupling was also assessed and found to correlate with the amount of connexin 43 mRNA. When cell proliferation was followed over several days, cells expressing the transfected cDNA grew more slowly than non-transfected cells. These transfected cells will be useful in examining the role of gap junctions in tumorigenesis.

Engrailed gene expression in the abdominal segment of Oncopeltus: gradients and cell states in the insect segment.

A monoclonal antibody that recognizes the product of the segmental gene, engrailed (en), of Drosophila has been used to analyse expression of the homologous gene of Oncopeltus. engrailed expression in the abdominal segment of larval Oncopeltus is confined to a narrow band of epidermal cells localized immediately anterior to the segment border. Expression varies in intensity during postembryonic development: no gene product is detectable in newly moulted larvae, but reappears soon after initiation of intermoult activities. One possible function of en in this system is revealed by a series of operations confronting cells from different anteroposterior levels in the segment. New segment borders are generated only when en-expressing cells confront cells from the anteriormost region of the segment. All other combinations result in intercalation of intermediate intrasegmental levels. It is therefore suggested that the most important function of en is the establishment of new, and presumably the maintenance of existing, segment borders.

Relative roles of gap junction channels and cytoplasm in cell-to-cell diffusion of fluorescent tracers.

Intercellular (tissue) diffusion of molecules requires cytoplasmic diffusion and diffusion through gap junctional (or cell-to-cell) channels. The rates of tissue and cytoplasmic diffusion of fluorescent tracers, expressed as an effective diffusion coefficient, De, and a cytoplasmic diffusion coefficient, Dcyt, have been measured among the developing epidermal cells of a larval beetle, Tenebrio molitor L., to determine the contribution of the junctional channels to intercellular diffusion. Tracer diffusion was measured by injecting fluorescent tracers into cells and quantitating the rate of subsequent spread into adjacent cells. Cytoplasmic diffusion was determined by fluorescence photobleaching. These experiments show that gap junctional channels constitute approximately 70-80% of the total cell-to-cell resistance to the diffusion of organic tracers at high concentrations in this tissue. At low concentrations, however, the binding of tracer to cytoplasm slows down the cytoplasmic diffusion, which may limit intercellular diffusion.