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Analysis of epithelial K⁺ transport in Malpighian tubules of Drosophila melanogaster: evidence for spatial and temporal heterogeneity

Mark R. Rheault^* and Michael J. O’Donnell

Department of Biology, McMaster University, 1280 Main Street West, Hamilton, Ontario, Canada L8S 4K1

View larger version (16K): [in a new window]   Fig. 1. Schematic diagram (not to scale) showing the self-referencing ion-selective (SeRIS) microelectrode for the study of K+ transport by isolated Malpighian tubules. The individual segments of the Malpighian tubule and their relationship to the ureter are shown. At all sites, the SeRIS microelectrode was vibrated over a distance of 100µm near the basolateral surface of the tubule.

View larger version (12K): [in a new window]   Fig. 2. Calibration curve for K+ microelectrodes in KCl solutions of 1–600 mmoll-1. Data points are mean responses ± S.E.M. of four different electrodes; standard errors are smaller than the symbol used. The solid line (slope 52.7mV per decade) is the linear regression (r2=0.992, P=0.0002).

View larger version (17K): [in a new window]   Fig. 3. K+ microelectrode voltage difference as a function of distance from a K+ source. Experimental measurements (open circles) were made by vibrating the microelectrode through an amplitude of 100µm at each distance. Theoretical values (filled circles) were calculated according to the following equation: V=S[(-U)/(CBr2+Ur)]/2.3, where V is the change (mV) over the vibration excursion, S is the slope of the electrode, r is the distance from the source, r is the amplitude of the vibration, CB is the background activity of K+ and U is an empirical constant. The inset shows the calculation of the empirical constant U. Static measurements were made at a series of distances from the source, and the millivolt outputs were then converted to activity values. A plot of these activity (C) values versus the inverse of the distance from the K+ source (1/r) yields a line with a slope of U, according to the equation: C=CB+U/r, where CB is the background activity of K+ (20 mmoll-1) and U (µmolcm-2) defines the diffusion characteristics of the gradient source (r2=0.969, P<0.0001). Using the method of Piñeros et al. (Piñeros et al., 1998), electrode efficiency (85%) was calculated as the ratio of the slope of the experimental data (dashed line) to the slope of the theoretical data (solid line).

View larger version (23K): [in a new window]   Fig. 4. Representative examples of K+ microelectrode voltage difference at single sites in the lower (A), main (B) and distal (C) segments of Malpighian tubules of Drosophila melanogaster. Bathing medium K+ concentration was 20 mmoll-1 in all experiments. The electrode was vibrated through an amplitude of 100µm and positioned so that its point of closest approach was approximately 10µm from the tubule surface (open horizontal bar) at each site. Hatched horizontal bars denote the background signal measured when the vibrating electrode was positioned 1000µm from the tubule surface. Note that positive voltages denote increases in [K+] of the unstirred layer relative to the bath and negative voltages denote decreases in [K+] of the unstirred layer relative to the bath.

View larger version (27K): [in a new window]   Fig. 5. Representative spatial scans of K+ microelectrode voltage difference along the lower (MT1–3), main (MT4–6) and distal segments (MT7–9) of the Malpighian tubules (MT) of Drosophila melanogaster. Segments were divided into 4–8 sites 100µm apart, with site number 1 in all cases closest to the ureter. Note that positive voltages denote increases in [K+] of the unstirred layer relative to the bath and negative voltages denote decreases in [K+] of the unstirred layer relative to the bath.

View larger version (7K): [in a new window]   Fig. 6. Global mean K+ flux values calculated from the voltage differences for the lower, main and distal segments. Only the flux values for the lower and main segments are significantly different from zero (P>0.05). Values are means ± S.E.M., N=34–56 sites on 6–11 tubules for each segment.

View larger version (13K): [in a new window]   Fig. 7. Representative effects of 1 mmoll-1 cAMP on K+ flux in the main segment of a Malpighian tubule of Drosophila melanogaster. Filled columns represent flux values prior to cAMP stimulation. Open columns represent flux values after the addition of cAMP. K+ flux rates were significantly increased by cAMP at sites 1–5 (open versus closed columns) but not at site 6. Similar patterns were seen for all other tubules studied. Values are means ± S.E.M., N=27 sites on five tubules (P>0.05).

View larger version (13K): [in a new window]   Fig. 8. Effects of 1 mmoll-1 NaCN, cAMP and cGMP on voltage differences measured by self-referencing K+ microelectrodes. (A) In the lower segment, only NaCN significantly altered K+ microelectrode voltage (N=18–24 sites on 5–6 tubules). (B) In the main segment, K+ microelectrode voltage was altered significantly (P>0.05) by NaCN, cGMP and cAMP (N=16–27 sites on 4–8 tubules). (C) In the distal segment, none of the stimulants or inhibitors had a significant effect on K+ microelectrode voltage (N=10–15 sites on 3–5 tubules). Values are means ± S.E.M. Note that positive voltages denote increases in [K+] of the unstirred layer relative to the bath and negative voltages denote decreases in [K+] of the unstirred layer relative to the bath.

View larger version (11K): [in a new window]   Fig. 9. Effects of 1 mmoll-1 cAMP on K+ fluxes on adjacent principal and stellate cells in the main segment of the Malpighian tubules of Drosophila melanogaster. The effects before (filled columns) and after (open columns) treatment with cAMP are shown. There is a significant (P<0.05) effect of cAMP only on principal cells. Values are means ± S.E.M.; N=19 principal cells and five stellate cells in five tubules.