Emmy Van Kerkhove

Electrophysiological evidence for the presence of an apical H+-ATPase in Malpighian tubules of Formica polyctena: intracellular and luminal pH measurements

Cellular and luminal pH of isolated ant Malpighian tubules were measured in different bath K+ concentrations using double-barrelled pH microelectrodes. The electrochemical gradient for H+ across the basolateral and the apical cell membranes was estimated. In control Ringer (51 mmol/l K+) cell and luminal pH were alkaline with respect to the basolateral solution: 7.77 and 7.36, respectively, versus 7.25. On lowering basolateral K+ concentration to 5 mmol/l or increasing it to 113 mmol/l, luminal pH and to a lesser extent cell pH followed: luminal pH changed to 7.14 and 7.43 and cell pH to 7.69 and 7.82, respectively. In all conditions a cell inward electrochemical gradient for protons across both membranes was observed. Increasing basolateral K+ concentration, which was positively correlated with secretion rate, decreased the cell inwardly directed apical proton gradient; moreover, the apical membrane potential difference decreased as well, from −93 mV in 5 mmol/l K+ to −65 mV in 113 mmol/l K+. Therefore the turnover rate of the electrogenic active proton pump at the apical membrane is facilitated in a high basolateral K+ concentration. The calculated electromotive force of this pump is −159 mV. Comparing the proton with the K+ electrochemical gradient, taken from another study in the same experimental conditions, we find that the apical proton electrochemical gradient can drive K+ extrusion into the lumen for each value of secretion rate.

Effect of Bacillus thuringiensis Cry1 Toxins in Insect Hemolymph and Their Neurotoxicity in Brain Cells of Lymantria dispar

ABSTRACT Little information is available on the systemic effects ofBacillus thuringiensis toxins in the hemocoel of insects. In order to test whether B. thuringiensis-activated toxins elicit a toxic response in the hemocoel, we measured the effect of intrahemocoelic injections of several Cry1 toxins on the food intake, growth, and survival ofLymantria dispar (Lepidoptera) and Neobellieria bullata (Diptera) larvae. Injection of Cry1C was highly toxic to the Lymantria larvae and resulted in the complete inhibition of food intake, growth arrest, and death in a dose-dependent manner. Cry1Aa and Cry1Ab (5 μg/0.2 g [fresh weight] [g fresh wt]) also affected growth and food intake but were less toxic than Cry1C (0.5 μg/0.2 g fresh wt). Cry1E and Cry1Ac (5 μg/0.2 g fresh wt) had no toxic effect upon injection. Cry1C was also highly toxic toN. bullata larvae upon injection. Injection of 5 μg/0.2 g fresh wt resulted in rapid paralysis, followed by hemocytic melanization and death. Lower concentrations delayed pupariation or gave rise to malformation of the puparium. Finally, Cry1C was toxic to brain cells of Lymantria in vitro. The addition of Cry1C (20 μg/ml) to primary cultures of Lymantria brain cells resulted in rapid lysis of the cultured neurons.

An antidiuretic factor in the forest ant: purification and physiological effects on the Malpighian tubules.

Formica polyctena antidiuretic factor (FopADF) was purified from a 15% trifluoroacetic acid (TFA) extract of the abdomens of 150,000 worker ants. After solid phase extraction of the crude extract and reversed-phase HPLC on two C(18) columns, an antidiuretic factor was isolated. Tested at a concentration of 1.0 ant-equivalents/µl (ant-eq/µl), the factor reversibly inhibited fluid secretion of isolated Malpighian tubules to 29+/-5% (mean+/-SE, n=24) of the control value. The same concentration of FopADF reversibly depolarized both the basolateral membrane potential (V(bl)), from -21+/-2 mV to -3+/-1 mV (n=5), and the apical membrane potential (V(ap)), from -65+/-5 mV to -20+/-5 mV (n=5). Similar effects on fluid secretion and V(ap) were caused by a TFA extract of the haemolymph of ants with non-secreting tubules. Unfortunately, further purification of FopADF on a C(4) column led to a loss of activity in the fluid secretion assay. This is the first time an endogenous antidiuretic factor acting directly on Malpighian tubules has been partially purified and shown to depolarize the tubule cell membranes.

MAXI K+ CHANNELS IN THE BASOLATERAL MEMBRANE OF THE EXOCRINE FROG SKIN GLAND REGULATED BY INTRACELLULAR CALCIUM AND PH

With the single-channel patch-clamp technique we have identified Ca2+-sensitive, high-conductance (maxi) K+ channels in the basolateral membrane (BLM) of exocrine gland cells in frog skin. Under resting conditions, maxi K+ channels were normally quiescent, but they were activated by muscarinic agonists or by high serosal K+. In excised inside-out patches and with symmetrical 140 mmol/l K+, single-channel conductance was 200 pS and the channel exhibited a high selectivity for K+ over Na+. Depolarization of the BLM increased maxi K+ channel activity. Increasing cytosolic free Ca2+ (by addition of 100 nmol/l thapsigargin to the bathing solution of cell-attached patches also increased channel activity, whereas thapsigargin had no effect when added to excised inside-out patches. An increase in cytosolic free Ca2+ directly activated channel activity in a voltage-dependent manner. Maxi K+ channel activity was sensitive to changes in intracellular pH, with maximal activity at pH 7.4 and decreasing activities following acidification and alkalinization. Maxi K+ channel outward current was reversibly blocked by micromolar concentrations of Ba2+ from the cytosolic and extracellular site, and was irreversibly blocked by micromolar concentrations of charybdotoxin and kaliotoxin from the extracellular site in outside-out patches.

Partial identification of a peptide that stimulates the primary urine production of single isolated Malpighian tubules of the forest ant, Formica polyctena

A peptide was purified from a 10% trifluoroacetic acid (TFA) head/thorax extract of 300,000 ants with high performance liquid chromatography (HPLC). Fluid secretion assay of single isolated Malpighian tubules was used as a bioassay. The purity of F. polyctena diuretic peptide (FopDP) after a two step HPLC protocol was confirmed by means of mass spectrometry and revealed a molecular mass of 7514 daltons. Due to lack of material, no enzymatic digestion could be performed and the sequence of only the first 25 amino acids could be determined: VPKYENCVSEVLPAGDRQRCVKVTC. A computer search of sequence data banks did not reveal any significant similarity between FopDP and other known insect diuretic peptides.FopDP had no effect on the basolateral membrane potential and depolarised the apical membrane potential of the Malpighian tubule cells. This effect as well as the stimulatory effect on the primary urine formation in the Malpighian tubule of the ant, could be mimicked with A23187, a calcium ionophore, and by thapsigargin, an inhibitor of the endoplasmic reticulum calcium ATPase. FopDP did not stimulate the cAMP content. The results suggest that FopDP uses an increase of intracellular calcium as cellular transduction mechanism.

Body extracts from the forest ant and honeybee: chromatographic comparison of factors affecting the ant Malpighian tubule

Abstract.  Crude trifluoroacetic acid extracts were prepared from different body parts of the forest ant, Formica polyctena Foerster, Hymenoptera, Formicidae, and the honeybee, Apis mellifera carnica Pollmann, Hymenoptera, Apidae. Extracts were prepurified by means of solid phase extraction over reversed‐phase cartridges. The effects of the resulting fractions on fluid secretion rates were tested on single preparations of ant Malpighian tubules. Diuretic and antidiuretic factors were shown to be present. High‐performance liquid chromatography allowed for the identification of a diuretic factor from the bee‐head extract which had a similar elution profile to a previously purified diuretic peptide from ant‐head extracts. An antidiuretic factor was also identified from the bee abdominal extract. Similar to an antidiuretic peptide, which was purified from ant abdominal extracts, this antidiuretic factor inhibited fluid secretion and depolarized the transepithelial potential of ant Malpighian tubules.