First published online May 13, 2004
Journal of Experimental Biology 207, 2173-2184 (2004)
Published by The Company of Biologists 2004
doi: 10.1242/jeb.01003
Organic cation transport by Malpighian tubules of Drosophila melanogaster: application of two novel electrophysiological methods
Mark R. Rheault* and
Michael J. O'Donnell
Department of Biology, McMaster University, 1280 Main Street West,
Hamilton, Canada L8S 4K1
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Fig. 1. (A) Representative scan of tetraethylammonium (TEA) flux at locations along
the secretory segment of the Malpighian tubule (MT) and the lower MT (LMT).
Tubules were bathed in saline containing 100 µmol l–1 TEA.
The common ureter and part of the other LMT of the pair are shown. The tip of
the TEA-SeR microelectrode is located just above the asterisk. The image is a
collage formed from two images. At each site, indicated by arrowheads, ASET
software calculated the TEA-specific signal differences ( V;
µV) between the two limits of microelectrode excursion by subtracting the
voltage at the outer limit of the excursion from that measured at the inner
limit. The length of each arrow corresponds to the magnitude of TEA influx.
(B) TEA influx as a function of distance from the ureter along lower, main and
distal segments of the MT. An influx of TEA reduces TEA concentration in the
unstirred layer adjacent to the surface of the tissue, and the corresponding
voltage difference is therefore negative. Distance 0 on the abscissa
corresponds to the junction of the ureter and the LMT. Both the differential
signal recorded by the TEA-SeR microelectrode (right ordinate) and the
calculated TEA influx (left ordinate) are shown (N=4).
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Fig. 2. Temporal stability of tetraethylammonium (TEA) influx recorded at a single
site in the lower Malpighian tubule (LMT) on each of two tubules (circles,
squares). Tubules were bathed in saline containing 100 µmol
l–1 TEA. Each point is the mean of three measurements at the
same site at intervals of 2.5 min.
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Fig. 3. (A) Representative scan of tetraethylammonium (TEA) flux at locations along
the lower Malpighian tubule (LMT) and ureter. Tubules were bathed in saline
containing 100 µmol l–1 TEA. The length of each arrow
corresponds to the magnitude of TEA influx and the axis of each arrow
indicates the axis of the TEA-SeR microelectrode's movement. (B) TEA influx
(left ordinate) and differential signal (right ordinate) in the LMT, distal
ureter and proximal ureter. The proximal ureter is defined as the 50% of the
ureter length closest to the gut. Each bar shows mean +
S.E.M. for N=6 preparations.
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Fig. 4. Concentration–response curves for tetraethylammonium (TEA) influx in
the (A) lower Malpighian tubule (LMT), (B) Malpighian tubule main segment and
(C) midgut. Each point is the mean ±
S.E.M. of N=4–7 preparations.
Values for Jmax and Kt were determined
by non-linear regression analysis as described in the Materials and
methods.
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Fig. 5. Effects of bathing saline potassium concentration on tetraethylammonium
(TEA) influx (left ordinate) and differential signal (right ordinate) in the
(A) lower Malpighian tubule (LMT) and (B) Malpighian tubule main segment. LMTs
were bathed in saline containing 10 or 100 mmol l–1
K+. Main segments were bathed in salines containing 20 or 2 mmol
l–1 K+. All salines contained 100 µmol
l–1 TEA. The height of each bar represents the mean +
S.E.M. (N=4 main segments;
N=7 LMTs). Asterisks denote significant differences
(P<0.05) from the value of the bar to the immediate left.
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Fig. 6. Effects of verapamil on tetraethylammonium (TEA) flux (left ordinate) and
differential signal (right ordinate) in the lower, main and distal segments of
the Malpighian tubule. Mean values + S.E.M.
(N=5) are shown for the same tubules before (filled bars) and
20–30 min after (open bars) the addition of verapamil (5 µmol
l–1) to saline containing 100 µmol l–1
TEA. The asterisk indicates a significant (P<0.05) reduction in
TEA influx after the addition of verapamil.
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Fig. 7. Sample recording showing the change in electrical potential of a
tetraethylammonium (TEA)-selective microelectrode positioned in droplets of
secreted fluid or calibration solutions. Microelectrode voltage was sampled at
3 Hz by the data acquisition system. The labels 10, 1 and 0.1 refer to
calibration solutions containing 10, 1 or 0.1 mmol l–1 TEA
chloride, respectively, in Drosophila saline. BG refers to the
background voltage recorded due to endogenously secreted compounds in the
absence of TEA. W1 and W2 refer to secreted droplets collected from a pair of
whole tubules after the addition of TEA. M1 and M2 refer to secreted droplets
collected from the main segment of one of the same pair of tubules after the
addition of TEA. All tubules were exposed to 100 µmol l–1
TEA in the bathing saline.
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Fig. 8. Concentration–response curves for secreted fluid TEA concentration as
a function of bathing saline TEA concentration for the main segment (A) and
whole tubule (B). Concentration–response curves for TEA flux as a
function of bathing saline TEA concentration for the main segment (C) and
whole tubule (D). Each point shows the mean ±
S.E.M. for N=6–16 main
segments or 3–8 whole tubules. The insets in the upper panels reflect
the corresponding arrangements of the tubules and bathing droplets. A droplet
of secreted fluid (SF) is indicated by the open circle. Abbreviations U,
ureter; D, distal segment; M, main segment; L, lower segment.
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Fig. 9. Effects of (A) 0.1 mmol l–1 and (B) 1.0 mmol
l–1 cimetidine on secreted fluid [TEA] for whole Malpighian
tubules. Tubules were bathed in saline alone for the first 20 min. Either 0.1
mmol l–1 TEA (open symbols) or cimetidine (filled symbols)
was added at 20 min. At 60 min either cimetidine (open symbols) or 0.1 mmol
l–1 TEA (filled symbols) was added.
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© The Company of Biologists Ltd 2004
