David F. Moffett

The anterior stomach of larval mosquitoes (Aedes aegypti): effects of neuropeptides on transepithelial ion transport and muscular motility

SUMMARY The present investigation studied the influence of a number of neuropeptides on semi-open preparations of the isolated and perfused anterior stomach of larval Aedes aegypti. Effects of peptides were observed on the lumen negative transepithelial voltage (Vte) that is present with serotonin in the bath; this voltage most likely reflects active HCO3– secretion involved in alkalization of the larval anterior stomach. The five different A. aegypti allatostatins (allatostatin A 1–5) all affected Vte in almost identical ways, causing a 10–15% reduction of the voltage at 10–7 mol l–1. A. aegypti neuropeptide F and proctolin reduced Vte at submicromolar concentrations. At 10–6 mol l–1, neuropeptide F reduced Vte by 30% and proctolin reduced Vte by 50%. In contrast, A. aegypti allatotropin, A. aegypti head peptides I and III and A. aegypti short neuropeptide F were without effect on Vte. During the investigation it was observed that the peristaltic contractions of the preparations caused a dynamic component of Vte. Peristaltic contractions and the correlated voltage fluctuations depended on the presence of serotonin. Peristaltic activity and Vte deflections were progressively inhibited by A. aegypti head peptides I and III by A. aegypti short neuropeptide F and by A. aegypti neuropeptide F when the peptide concentrations were increased from 10–8 to 10–6 mol l–1. These observations show that physiological concentrations of some of the tested neuropeptides affect two processes that require coordination: ion transport and motility of the larval anterior stomach.

Inhibition of active K+ transport in the tobacco hornworm (Manduca sexta) midgut after ingestion of Bacillus thuringiensis endotoxin

The effect of Bacillus thuringiensis endotoxin after ingestion on the K+ transport of Manduca sexta midgut is described. Direct measurements of short circuit current and transepithelial potential demonstrate that the transport is inhibited at the end of 1 hr with many of the insects exhibiting both negative short circuit current and transepithelial potential by the end of a 4-hr ingestion period.

Intracellular K+ activities and cell membrane potentials in a K+-transporting epithelium, the midgut of tobacco hornoworm (Manduca sexta)

SummaryTransbasal electrical potential (Vb) and intraepithelial potassium chemical activity ((K+)i) were measured in isolated midgut epithelium of tobacco hornworm (Manduca sexta) using double-barrelled glass microelectrodes. Values ofVb ranging from +8 to −48 mV (relative to blood side) were recorded. For all sites, (K+)i is within a few millivolts of electrochemical equilibrium with the blood side bathing solution. Sites more negative than −20 mV show relatively high sensitivity ofVb to changes in blood side K+ concentration: 43% of these sites can be marked successfully with iontophoresed Lucifer yellow CH dye and shown to represent epithelial cells of all three types present in the midgut. In about half of successful marks, “dye-coupling” of several adjacent cells is seen. Low potential sites — those withVb less negative than −20 mV —typically do not show high sensitivity ofVb to changes of external K+, but rather (K+)i rapidly approaches the K+ activity of blood side bathing solution. These sites can seldom be marked with Lucifer yellow (4% success). The mean (K+)i of the high potential sites is 95±29 (sd)mm under standard conditions, a value which is in accord with published values for the whole tissue.

Alkalinization in the isolated and perfused anterior midgut of the larval mosquito, Aedes aegypti.

Abstract In the present study, isolated midguts of larval Aedes aegypti L. (Diptera: Culicidae) were mounted on perfusion pipettes and bathed in high buffer mosquito saline. With low buffer perfusion saline, containing m-cresol purple, transepithelial voltage was monitored and luminal alkalinization became visible through color changes of m-cresol purple after perfusion stop. Lumen negative voltage and alkalinization depended on metabolic energy and were stimulated in the presence of serotonin (0.2 µmol l-1). In some experiments a pH microelectrode in the lumen recorded pH values up to 10 within minutes after perfusion stop. The V-ATPase inhibitor concanamycin (50 µmol l-1) on the hemolymph side almost abolished Vte and inhibited luminal alkalinization. The carbonic anhydrase inhibitor, methazolamide (50 µmol l-1), on either the luminal or hemolymph-side, or the inhibitor of anion transport, DIDS (1 mmol l-1) on the luminal side, had no effect on Vte or alkalinization. Cl- substitution in the lumen or on both sides of the tissue affected Vte, but the color change of m-cresol purple was unchanged from control conditions. Hemolymph-side Na+ substitution or addition of the Na+/H+ exchange inhibitor, amiloride (200 µmol l-1), reduced Vte and luminal alkalinization. Luminal amiloride (200 µmol l-1) was without effects on Vte or alkalinization. High K+ (60 mmol l-1) in the lumen reduced Vte without affecting alkalinization. These results indicate that strong luminal alkalinization in isolated and perfused anterior midgut of larval A. aegypti depends on basolateral V-ATPase, but is apparently independent of carbonic anhydrase, apical Cl-/HCO3- exchange or apical K+/2H+ antiport.

A highly stable and selective biosensor using modified nicotinic acetylcholine receptor (nAChR)

Methods for developing stable, sensitive and selective bilayer lipid membrane (BLM)-based biosensors are discussed. Stable BLMs were formed over micromachined polyimide apertures. Selective sensors were made by incorporating nicotinic acetylcholine receptors (nAChRs) modified with bispecific antibodies (BsAbs). When two BsAbs, attached to one nAChR, encounter antigen (Ag), channels are blocked. Sensitivity to single Ag molecules would be possible by monitoring closure of individual nAChRs.

Revisiting the cellular mechanisms of strong luminal alkalinization in the anterior midgut of larval mosquitoes

SUMMARY Here we critically review two recent hypotheses about the mechanism of strong alkalinization by the anterior midgut of mosquito larvae and our tests of these hypotheses. We present experimental evidence against the major components of transport models proposed in these hypotheses. Measurements of the transapical and transbasal proton electrochemical gradients provide an indication of driving forces faced by and generated by the transport mechanisms of the tissue. These measurements confirmed that basal V-ATPase energizes alkalinization. Serotonin stimulates the V-ATPase, as indicated by the ensuing increase in proton-motive force across the basal membrane. Moreover, the neurohormone resulted in a surprisingly large increase in the intracellular pH. The results of inhibitor studies indicate that, contrary to previous proposals, carbonic anhydrase is apparently not involved in supplying acid–base equivalents to the respective transporters. Furthermore, any apical processes proposed to be involved in alkali secretion or acid absorption must be Cl– independent and insensitive to DIDS, amiloride, Zn2+ and ouabain. These results argue against the involvement of putative apical Cl–/HCO –3 exchangers, apical H+ channels, apical cation/proton exchangers and the importance of the apical Na+/K+ pump. The studies analyzed here thus provide both a limitation and direction for further studies of the mechanism of strong alkalinization in this system.

Bilayer lipid membrane (BLM) based ion selective electrodes at the meso-, micro-, and nano-scales

This paper presents a novel method for making micron-sized apertures with tapered sidewalls and nano-sized apertures. Their use in bilayer lipid membrane-based ion selective electrode design is demonstrated and compared to mesoscale bilayers and traditional PVC ion selective electrodes. Micron-sized apertures are fabricated in SU-8 photoresist films and vary in diameter from 10 to 40 microm. The tapered edges in SU-8 films are desired to enhance bilayer lipid membrane (BLM) formation and are fabricated by UV-light overexposure. Nano-apertures are made in boron diffused silicon film. The membranes are used as septa to separate two potassium chloride solutions of different concentrations. Lecithin BLMs are assembled on the apertures by ejecting lipid solution. Potassium ionophore, dibenzo-18-crown-6, is incorporated into BLMs by dissolving it in the lipid solution before membrane assembly. Voltage changes with increasing potassium ion concentrations are recorded with an A/D converter. Various ionophore concentrations in BLMs are investigated. At least a 1% concentration is needed for consistent slopes. Electrode response curves are linear over the 10(-6) to 0.1M range with a sub-Nernstian slope of 20mV per Log concentration change. This system shows high selectivity to potassium ions over potential interfering sodium ions. BLMs on the three different aperture sizes at the meso-, micro-, and nano-scales all show similar linear ranges and limits of detection (LODs) as PVC ion selective membranes.

Highly stable bilayer lipid membranes (BLMs) formed on microfabricated polyimide apertures

Abstract Microfabrication is used to realize a polyimide aperture for highly stable bilayer lipid membranes (BLMs). The microfabrication process used in this work is compatible with standard processes used in microelectronics. The physical features of the aperture, such as diameter, thickness and tapering of the edges are precisely controlled during the microfabrication process which results in high yield and reproducibility of the polyimide apertures. A study of BLM stability using precise measurements of specific capacitance shows that the BLM is stable up to 50 h. Alamethicin channels were successfully incorporated into these membranes further proving their bilayer nature. Vesicles containing reconstituted nicotinic acetylcholine receptors (nAChRs) were fused to the BLMs. The successful incorporation of functioning ion channel activity into highly stable BLMs promises a future class of miniature sensors that are very sensitive with fast response times.

Barium modifies the concentration dependence of active potassium transport by insect midgut

SummaryThe rate of active K+ transport by the isolated lepidopteran midgut shows a rectangular hyperbolic relation to [K+] over the range 20 to 70mm K+ in the absence of any divalent cation. Addition of Ba++ to the hemolymph (K+ uptake) side introduces a linear component to the concentration dependence, such that active K− transport is decreased at [K+] of 55mm or less, but increased transiently at higher [K+]. As [Ba++] is increased over the range 2 to 8mm the linear component increases and the saturating component decreases; in 8mm Ba++ the concentration dependence is dominated by the linear component. The effect of Ba++ cannot easily be accounted for by simple competition with K+ for basal membrane uptake sites. Similar effects might be exercised by other alkali earth cations, since the concentration dependence of active K+ transport possesses a substantial linear component in solutions containing 5mm Ca++ and 5mm Mg++ (the alkali earth metal concentrations of standard lepidopteran saline).

Bispecific antibody modification of nicotinic acetylcholine receptors for biosensing.

Recent results show that bispecific antibodies can be used to tailor the selectivity of nicotinic acetylcholine receptors for biosensing purposes. The nicotinic acetylcholine receptors reconstituted in bilayer lipid membranes are inactivated when two bispecific antibodies, attached to the same receptor, bind to a single antigen molecule. Experiments with patch clamp recording equipment reveal that antigen levels of 10(-8) M completely and irreversibly inactivate small numbers of nicotinic acetylcholine receptors. This approach may lead to the construction of biosensors capable of detecting individual antibody-antigen (Ab-Ag) binding events.