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First published online September 16, 2005
Journal of Experimental Biology 208, 3771-3783 (2005)
Published by The Company of Biologists 2005
doi: 10.1242/jeb.01829
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Sulphonylurea sensitivity and enriched expression implicate inward rectifier K+ channels in Drosophila melanogaster renal function

Jennifer M. Evans, Adrian K. Allan, Shireen A. Davies and Julian A. T. Dow*

Division of Molecular Genetics, Faculty of Biomedical and Life Sciences, University of Glasgow, Glasgow, G11 6NU, UK



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  		Fig. 1. Gene structure of irk2. Blue bars denote exons, green lines denote the start codons, and red lines denote the stop codons. The transcripts thus differ at their 5' ends, and transcript B encodes a different protein from transcripts A and C. This display is based on the Drosophila genome annotation for irk2 (www.flybase.org).

 


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  		Fig. 2. ir expression in the principal cells of the tubule main segment and the ureter. (A) Low-power views, showing little expression in the spermatheca (SP) and rectum (R). (B) Low-power view of the tubule, showing strongest staining in the ureter (U), with further staining along the length of the main segment (MS) and lower segment (LS) but not in the initial segment (IS). (C) High-power view, showing staining in the ureter. (D) High-power view, showing staining in the main segment but no expression in the IS. (E) High-power view of tubule main segment, showing that expression is confined to principal cells but is not in the very narrow stellate cells (SC). Sense controls did not produce any staining (data not shown). Tubule diameter can be taken as 35 µm throughout.

 


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  		Fig. 3. irk2 expression in principal cells of the tubule main segment and spermatheca. (A) Low-power view, showing the expression in the spermatheca (SP), but not in other areas, including the rectum (RE). (B) Expression in the tubules along the length of the main segment (MS) but not in the initial segment (IS) of the anterior tubule. (C) High-power view of the IS showing no staining. Sense controls did not produce any staining (data not shown). Tubule diameter can be taken as 35 µm throughout.

 


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  		Fig. 4. irk3 expression is localised to the principal cells of the tubule main segment by in situ analysis. (A,B) Low-power views, showing expression in the main segment (MS), lower segment (LS) and ureter (U) of the tubule but not in the initial segment (IS) of the anterior tubule or the hindgut (HG). (C) High-power view of the anterior tubule initial segment, showing no expression. By contrast, the initial segment of the posterior tubule (D) shows staining as intense as the main segment. (E) High-power view of the lower tubule and ureter, showing expression throughout. (F) High levels of expression in nurse cells and oocytes. (G,H) High-power view of tubule main segment, showing that expression is confined to principal cells (PC) but is not in the very narrow stellate cells (SC). The two cell types can be distinguished because stellate cell nuclei are smaller than those of principal cells (arrows). Sense controls did not produce any staining (data not shown). Tubule diameter can be taken as 35 µm throughout.

 


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  		Fig. 5. Inhibition of fluid secretion by glibenclamide. (A) Typical experiment showing secretion response (means ± S.E.M., N=13) to glibenclamide. (B) Dose-response curve; inhibition relative to control was calculated 50 min after glibenclamide was added (data are expressed as mean percentage inhibition ± S.E.M., N=7).

 


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  		Fig. 6. Inhibition of secretion by a range of sulphonylureas. (A) Diazoxide, (B) minoxidil, (C) tolbutamide or (D) glibenclamide were added after 30 min. Drosophila corticotropin releasing factor-like peptide (dCRF) and Drosophila leucokinin (dLK) were added (to final concentrations of 10-7 mol l-1) after a further 30 min to stimulate the secretion rate. Secretion rates are shown as means ± S.E.M. (N=7).

 


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  		Fig. 7. Effect of diflubenzuron on secretion and glibenclamide inhibition. Diflubenzuron was added to the final concentrations shown after 30 min. Glibenclamide (final concentration 0.75 mmol l-1) was added 30 min later. Secretion rates are shown as means ± S.E.M.(N=8).

 


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  		Fig. 8. Glibenclamide blocks amaranth transport. Eight tubules were arranged radially for classical (Ramsay) secretion assays (Dow et al., 1994Go): the bathing drops are just out of shot. Droplets were collected every 10 min, and so the colour of the droplets provides a correlate of real-time amaranth transport. (A) Secreted drops before glibenclamide was added. (B) Secreted droplets 35 min after glibenclamide was added to the final concentrations (in mmol l-1) shown. Drops labelled `C' had no glibenclamide added to the bubbles. The concentration of amaranth in the reservoir bubbles was 2 µmol l-1.

 


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  		Fig. 9. Anionic dyes do not impact on glibenclamide inhibition of fluid secretion. Classical secretion assays were performed in normal medium or with the addition of phenol red (2x10-5 mol l-1) or amaranth (2x10-5 mol l-1). Glibenclamide (final concentration 10-4 mol l-1) was added after 30 min. Secretion rates are shown as means ± S.E.M. (N=6).

 


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  		Fig. 10. Transport of fluorescent glibenclamide. Tubules were dissected and incubated in medium containing Texas Red (TR)-labelled glibenclamide. (A-D) Images taken from a time series at 1 min, 15 min, 30 min and 45 min, respectively. Panels E-L show features of glibenclamide transport in specific areas of the tubule: (E) urethra; (F) surface of the main segment, showing the stellate cells; (G) main segment; (H) initial segment of anterior tubule. Images A-D were taken 40 min after the addition of 0.1 mmol l-1 TR-glibenclamide. Images I-L were taken of the same regions of the tubule as E-H but were not exposed to TR-glibenclamide. The same detector gain (0.01) and off-set amplification (500) were used for all images. Tubule diameters can be taken as 35 µm throughout.

 


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  		Fig. 11. Transport and metabolism of 125I-labelled glibenclamide. (A) Transport rates and (B) secreted:bathing ratios of [125I]glibenclamide at different bathing glibenclamide concentrations. (C) Thin-layer chromatography plate image. Lane 1, authentic [125I]glibenclamide; lane 2, secreted drop after [125I]glibenclamide was added to the bathing solution; lane 3, secreted drop where no radiolabelled glibenclamide was added. Lower and upper bars represent the origin and solvent front, respectively.

 


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  		Fig. 12. Glibenclamide mimics the effect of K+-free saline. Tubules were dissected in AARS saline (a defined control saline; Linton and O'Donnell, 1999Go), then either mounted for fluid secretion directly in 10 µl drops of AARS saline or rinsed and mounted for secretion in drops of K+-free saline (Linton and O'Donnell, 1999Go). Readings were taken every 10 min. At 30 min (arrow), glibenclamide was added (to a final concentration of 0.1 mmol l-1) to half of the AARS and half of the K+-free drops. Key: open squares, control saline without glibenclamide; filled squares, control saline with glibenclamide; open circles, K+-free saline without glibenclamide; filled circles, K+-free saline with glibenclamide. Data are shown as means ± S.E.M. (N=8).

 

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© The Company of Biologists Ltd 2005