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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

View larger version (99K): [in a new window]   Fig. 1. CLSM images showing accumulation of Texas Red in the lumen and/or cells of Malpighian tubules isolated from crickets (A–D), adult Drosophila (E) or larval Drosophila (F). The bathing saline (B), lumen (L) and cells (C) are indicated in each image. Tubules were bathed in 10 µmol l–1 Texas Red for 15–30 min before each image was recorded. Tubule diameter is 60 µm in A–C and 35 µm in D,E. Panel B shows the fluorescence intensity profile from the base to the tip of the 110 µm-long arrow in A. Note the presence of large (C) or small (D) spherical concretions in the lumen of the cricket tubules. Some of the concretions in D appear to accumulate high concentrations of Texas Red and appear white whereas others are dark or opaque. The presence of large numbers of small intracellular concretions prevents clear distinction of luminal from cellular compartments in Drosophila tubules (E,F).

View larger version (14K): [in a new window]   Fig. 2. Fluorescence intensity (FI) ratios for cells versus bath (circles) and lumen versus bath (triangles) as functions of the concentration of Texas Red in the saline bathing isolated cricket Malpighian tubules. An FI ratio for each site was calculated from the mean of five FI measurements in the bathing saline, cells and lumen. Each data point is the mean (± S.E.M.) ratio for 5–6 widely separated regions in three tubules.

View larger version (59K): [in a new window]   Fig. 3. Analysis of fluorescent fluid samples collected in optically flat hollow rectangle glass capillaries. (A) X–Y image of a sample of 50 µmol l–1 Texas Red collected under paraffin oil in a hollow rectangle glass capillary. The width of the image is 250 µm. (B) X–Z image of a sample of 125 µmol l–1 Texas Red. The solid line was positioned at the apparent midline of the bright band of fluorescence, corresponding to the Z-axis midpoint. For comparison, the upper and lower dotted lines indicate positions 10 µm from the apparent Z-axis midpoint. The dotted lines are clearly well away from the midline of the bright band in the X–Z image and are shown to indicate that the Z-axis midpoint can be reliably judged by eye. (C) Fluorescence intensity of X–Y images collected for the same sample at the Z-positions indicated. The curve shows that fluorescent intensity is nearly constant when the optical slice is positioned within 5 µm of the apparent Z-axis midpoint. (D) Representative calibration curve relating fluorescence intensity to the concentration of Texas Red in fluid samples collected in optically flat glass capillaries. Texas Red concentrations are plotted on the Y-axis so that the equation of the curve fit to the data by non-linear regression analysis allows calculation of the dye concentration of experimental samples from the measured fluorescence intensities. Measured fluorescence intensities were divided by 100 for convenience of the curve-fitting calculations. For the curve shown, CLSM detector gain was adjusted so that a near maximal signal (3900 on a scale of 0–4095) was obtained from the highest concentration (250 µmol l–1) of Texas Red in this series of four calibration samples.

View larger version (19K): [in a new window]   Fig. 4. Secreted fluid Texas Red concentration ([Texas Red]sf) as a function of bathing saline Texas Red concentration for cricket tubules set up in Ramsay assays. Each point shows the mean ± S.E.M. for N=6–13 tubules. The solid line represents the fit to the Michaelis–Menten equation by non-linear regression analysis. As discussed in the text, the point at 50 µmol l–1 Texas Red was excluded from the curve-fitting procedure. The inset shows the ratio of the concentration of Texas Red in the secreted fluid to that in the bathing medium (S/M) as a function of bathing medium Texas Red concentration.

View larger version (20K): [in a new window]   Fig. 5. Transepithelial flux of Texas Red as a function of bathing saline Texas Red concentration for Drosophila tubules set up in Ramsay assays. Each point shows the mean ± S.E.M. for N=7–30 tubules. The solid line represents the fit to the Michaelis–Menten equation by non-linear regression analysis. The inset shows the ratio of the concentration of Texas Red in the secreted fluid to that in the bathing medium (S/M) as a function of bathing medium Texas Red concentration. As discussed in the text, the point at 50 µmol l–1 Texas Red was excluded from the curve-fitting procedure.

View larger version (22K): [in a new window]   Fig. 6. Secreted fluid rhodamine 123 concentration as a function of bathing saline rhodamine 123 concentration for cricket tubules set up in Ramsay assays. Each point shows the mean ± S.E.M. for N=6–28 tubules. The solid line represents the fit to the Michaelis–Menten equation by non-linear regression analysis. The inset shows the ratio of the concentration of rhodamine 123 in the secreted fluid to that in the bathing medium (S/M) as a function of bathing medium rhodamine 123 concentration.

View larger version (20K): [in a new window]   Fig. 7. Secreted fluid daunorubicin concentration as a function of bathing saline daunorubicin concentration for cricket tubules set up in Ramsay assays. Each point shows the mean ± S.E.M. for N=5–8 tubules. The solid line represents the fit to the Michaelis–Menten equation by non-linear regression analysis. The inset shows the ratio of the concentration of daunorubicin in the secreted fluid to that in the bathing medium (S/M) as a function of bathing medium daunorubicin concentration.

View larger version (21K): [in a new window]   Fig. 8. Transepithelial flux of daunorubicin as a function of bathing saline daunorubicin concentration for Drosophila tubules set up in Ramsay assays. Each point shows the mean ± S.E.M. for N=5 tubules. The solid line represents the fit to the Michaelis–Menten equation by non-linear regression analysis. The inset shows the ratio of the concentration of daunorubicin in the secreted fluid to that in the bathing medium (S/M) as a function of bathing medium daunorubicin concentration.

View larger version (20K): [in a new window]   Fig. 9. Transepithelial flux of fluorescein as a function of bathing saline fluorescein concentration for isolated Drosophila tubules set up in Ramsay assays. Each point shows the mean ± S.E.M. for N=5 tubules. The solid line represents the fit to the Michaelis–Menten equation by non-linear regression analysis. The inset shows the ratio of the concentration of fluorescein in the secreted fluid to that in the bathing medium (S/M) as a function of bathing medium fluorescein concentration.

View larger version (12K): [in a new window]   Fig. 10. Effects of the MRP2 inhibitors MK-571 and probenecid (PROB) on transepithelial flux of Texas Red across isolated Drosophila tubules set up in Ramsay assays. Each bar shows the flux (mean + S.E.M.) for 7–9 tubules (MK-571) or 10 tubules (probenecid) bathed in saline containing the indicated concentration (µmol l–1) of Texas Red in the absence (filled bars) or presence (open bars) of the indicated inhibitor. Asterisks indicate significant (P<0.05) decreases in flux in the presence of each drug, relative to the corresponding controls.

View larger version (18K): [in a new window]   Fig. 11. Transepithelial flux of quinacrine as a function of bathing saline quinacrine concentration for isolated Drosophila tubules set up in Ramsay assays. Each point shows the mean ± S.E.M. for N=5 tubules. The solid line represents the fit to the Michaelis–Menten equation by non-linear regression analysis. The inset shows the ratio of the concentration of quinacrine in the secreted fluid to that in the bathing medium (S/M) as a function of bathing medium quinacrine concentration.

View larger version (16K): [in a new window]   Fig. 12. Effects of p-glycoprotein (P-gp) inhibitors on secreted fluid concentration of daunorubicin (A,B) or rhodamine 123 (C,D) for cricket tubules set up in Ramsay assays. Each panel shows the flux (mean + S.E.M.) for control tubules (filled bars) exposed to the dye alone at the indicated concentration (µmol l–1) and those treated with the same dye concentration and a P-gp inhibitor at the concentration (µmol l–1) indicated (open bars). Abbreviations: daunorubicin (DNR), verapamil (VRP), rhodamine 123 (R123); tetraethylammonium (TEA). Significant differences (P<0.05) between control and experimental groups indicated by asterisks. N=7–10 tubules for each bar.

View larger version (28K): [in a new window]   Fig. 13. Effects of p-glycoprotein (P-gp) inhibitors on transepithelial flux of daunorubicin (A–F) or rhodamine 123 (G,H) across Drosophila tubules set up in Ramsay assays. Each panel shows the flux (mean + S.E.M.) for control tubules (filled bars) exposed to the dye alone at the indicated concentration (µmol l–1) and those treated with the same dye concentration and a P-gp inhibitor at the concentration (µmol l–1) indicated (open bars). Abbreviations: daunorubicin (DNR), verapamil (VRP), quinacrine (QUIN), rhodamine 123 (R123); tetraethylammonium (TEA). Significant differences (P<0.05) between control and experimental groups indicated by asterisks. N=7–10 tubules for each bar.

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