Proceedings of The Physiological Society
University College London (2011) Proc Physiol Soc 24, C14 and PC14
Electrolyte transport in the perfused Drosophila larval posterior midgut is selectively inhibited by paradoxical actions of fungal toxins.
S. Shanbhag1, N. M. D'Silva1, T. V. Abraham1, S. Tripathi1
1. Tata Inst Fundamental Research, Mumbai, India.
The Drosophila midgut is a powerful model for the study of function (1-3) and cellular homeostasis (4) of gastrointestinal epithelia. The posterior midgut secretes strong base into the lumen (1) driving it intensely alkaline (pH ~11). This is accomplished by secretion of NaHCO3, and absorption of KCl and HCl into the haemolymph (2,3). One of the unique features of this epithelium is that the basal membrane (b) is an order of magnitude more resistive than the apical (a), and the overall transepithelial resistance (Rt) is very high (~1kΩ.cm2). This fortuitous situation enables excellent voltage-clamp not only of the epithelium, despite its tubular geometry, but also voltage-clamp of the basal membrane only (2). We used Nystatin, and the cyclic peptide Destruxin A, to elucidate transport pathways for electrolytes. The midgut was perfused and superfused with a 1:3 mix of Schneider medium and Ringer. Intra- and extra-cellular ionic activities were measured with ion-selective microelectrodes. Luminal perfusion with 500 nM Nystatin in Schneider-Ringer did not show changes in transepithelial potential Vt (-42.4 ± 4.2 and -41.4 ± 4.3mV), transepithelial resistance Rt (781±58 and 786±128 Ω.cm2), and current Isc (47 ± 6 and 60 ± 13 µA.cm-2), Vb (-62.3 ± 3.8 and -63.5 ± 6mV) and Va (20.1 ± 5.8 and 21.9 ± 8.1mV) (p > 0.05; n = 10). However the resistance ratio of basal to apical membranes was decreased from 4.2 ± 1.1 to 1.7 ± 2.1 (n = 10, p < 0.03). Cyclic peptides like Destruxin A are highly toxic to diptera. We verified that non-selective channels (5-600 pS, n=30) were formed in artificial bilayer membranes (5), and expected that, as in the case of Nystatin, Rt would fall. Basal Destruxin A (200 µM) decreased Vt from -37.7 ± 2.5 to -8.3 ± 1.1 mV and Isc from 66.0 ± 9.0 to 10.0 ± 1.7 µA.cm-2(n=17; p < 0.01). Rt, however, increased from 664 ± 63 to 1062 ± 128 Ω.cm2(n=18; p < 0.01) and depolarized apical (Va) and basal (Vb) membrane potentials (17.0 ± 3.2 mV to 7.4 ± 2.2 mV and -60.1 ± 3.9 to -17.0 ± 2.4 mV respectively (n=18, p<0.01). Rb/Ra ratios decreased (9.8 ± 1.7 to -3.8 ± 3.4 (n = 17, p < 0.01). The action of Destruxin A was dose-dependent; half maximal effective concentration was ~100 µM. For lower doses (≤ 50 µM) the recovery was very rapid (within 5 min) and both the basal (Vb) and apical (Va) membrane potentials hyperpolarized significantly after the washout of Destruxin A at these doses. Luminal Destruxin was without effect indicating that the effects of this toxin is probably through the tracheoles. The rise in Rt is probably due to the inactivation of channels in the apical membrane. Data reported are means and S.E.M). All procedures accorded with current national legislation/guidelines and also current local and institutional guidelines.
Where applicable, experiments conform with Society ethical requirements