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First published online November 17, 2005
Journal of Experimental Biology 208, 4363-4376 (2005)
Published by The Company of Biologists 2005
doi: 10.1242/jeb.01911

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Transepithelial transport of fluorescent p-glycoprotein and MRP2 substrates by insect Malpighian tubules: confocal microscopic analysis of secreted fluid droplets

J. P. Leader and M. J. O'Donnell^*

Department of Physiology, University of Otago, New Zealand

^* Author for correspondence at present address: Department of Biology, McMaster University, 1280 Main Street West, Hamilton, ON, Canada L8S 4K1 (e-mail: odonnell{at}mcmaster.ca)

Accepted 3 October 2005

Transport of fluorescent substrates of p-glycoprotein (P-gp) and multidrug resistance-associated protein 2 (MRP2) by insect Malpighian tubules was examined using confocal laser scanning microscopy (CLSM). Isolated tubules of the cricket Teleogryllus commodus accumulated the MRP2 substrate Texas Red in the cells and lumen at concentrations up to 20 and 40 times, respectively, those in the bathing medium. Quantitative CLSM analysis of fluorochrome transport in some cricket tubules and all Drosophila tubules was not practical because of interfering effects of concretions in the cells and lumen. Samples of fluid secreted by tubules set up in Ramsay assays were therefore collected in hollow rectangle glass capillaries. Transepithelial dye flux was calculated as the product of fluid secretion rate (measured in the Ramsay assay) and dye concentration (measured by CLSM of the fluid samples). Dose–response curves for transport and the ratio of dye concentration in the secreted fluid to that in the bathing medium (S/M) were determined for Texas Red as well as for P-gp substrates (rhodamine 123, daunorubicin), the organic anion fluorescein and the organic cation quinacrine. Transepithelial transport of Texas Red was reduced by the MRP2 inhibitors MK571 and probenecid. Transport of daunorubicin was reduced by the P-gp inhibitors verapamil and quinacrine and also by the organic cation tetraethylammonium. The results indicate the presence of P-gp-like and MRP2-like transporters in the Malpighian tubules of both species.

Key words: p-glycoprotein, multidrug resistance associated protein, Malpighian tubule, confocal microscopy

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