Analysis of epithelial K+ transport in Malpighian tubules of Drosophila melanogaster: evidence for spatial and temporal heterogeneity
Department of Biology, McMaster University, 1280 Main Street West, Hamilton, Ontario, Canada L8S 4K1
*e-mail: rheaulmr{at}mcmaster.ca
Accepted April 30, 2001
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Summary |
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Key words: Drosophila melanogaster, Malpighian tubule, K+ transport, unstirred layer, self-referencing ion-selective microelectrode.
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Introduction |
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The Malpighian tubule of D. melanogaster is divided into morphologically distinct distal, main and lower segments (Fig.1). In addition, molecular genetic analysis suggests that morphologically similar cells within each segment may have different functions (Sözen et al., 1997). The main segment secretes both KCl and water, and the lower segment reabsorbs KCl, but not water, while acidifying the lumen and secreting Ca2+. The K+ concentration of the secreted fluid is reduced from approximately 120 mmoll-1 to approximately 105 mmoll-1 during passage through the lower tubule (ODonnell and Maddrell, 1985). The distal segment has been shown to be non-secretory (Dow et al., 1994a), but nothing is known about its reabsorptive capability.
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Materials and methods |
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Dissection and secretion assay
Procedures for dissection of Malpighian tubules and fluid secretion assays have been described previously (Dow et al., 1994a). Briefly, pairs of Malpighian tubules joined by a common ureter were dissected out under a Drosophila saline consisting of (in mmoll-1): 117.5 NaCl, 20 KCl, 2 CaCl2, 8.5 MgCl2.6H2O, 20 glucose, 10.2 NaHCO3, 4.3 NaH2PO4 and 8.6 Hepes. The saline was titrated with NaOH to pH7. Pairs of isolated tubules were transferred on fine glass probes from the dissecting saline to 10µl droplets of standard bathing medium (SBM), under paraffin oil. SBM was a 1:1 mixture of standard Drosophila saline and Schneiders Drosophila medium (Sigma). One tubule of each pair was pulled out of the bathing droplet and wrapped around a fine steel pin until the common ureter of the tubules was positioned within the oil just outside the bathing droplet.
Droplets secreted by the Malpighian tubules formed at the end of the ureter and were collected with a glass probe under paraffin oil. Droplet diameters (d) were measured using an ocular micrometer, and droplet volume (nl) was calculated as d3/6. Secretion rate (nlmin-1) was calculated by dividing the droplet volume by the time (min) over which the droplet was formed.
For each experiment, up to 18 tubules were allocated at random to two groups, experimental and control. In some experiments, tubule fluid secretion was stimulated by the addition of cyclic AMP (cAMP) or cGMP, or inhibited by the addition of NaCN. Secreted droplets were collected for the first 30min to establish a baseline rate of secretion. After 30min, drugs were added, and secreted droplets were collected over 10min intervals for a further 30min.
Construction of K+-selective microelectrodes
The construction of liquid membrane ion-selective microelectrodes has been described in detail (Kühtreiber and Jaffe, 1990; Piñeros et al., 1998; Smith et al., 1994). Briefly, 1.5 mm diameter non-filamented glass capillary tubes (TW150-4; World Precision Instruments Inc., Sarasota, FL, USA) were first cleaned by washing in nitric acid. Capillaries were then pulled on a programmable horizontal puller (P-97 Flaming-Brown, Sutter instrument Co., Novato, CA, USA) using a three-stage pulling procedure. The resulting microelectrode had a shank of approximately 4 mm and a tip diameter of approximately 24µm. Microelectrodes were heated (200°C, 30min), silanized by vapour phase treatment with N,N-dimethyltrimethylsilylamine (200°C, 30min), cooled and then stored in an air-tight chamber over desiccant until use. Immediately prior to use, microelectrodes were back-filled with 100 mmoll-1 KCl to a column length of approximately 1.5 cm. The KCl solution was forced to the tip by application of air pressure. The microelectrode tip was then front-filled with a short column length (180200µm) of potassium ionophore (K+ ionophore ICocktail B; Fluka Chemical Co., Ronkonkoma, NY, USA). Electrical contact between the microelectrode and the head stage of the self-referencing probe apparatus was made through a chlorided silver wire (WPI EHBI; World Precision Instruments, Sarasota, FL, USA). The reference electrode consisted of a 10 cm long, 1.5 mm diameter glass capillary tube (TW150-4) filled with a mixture of 3moll-1 KCl and 1% agar and inserted into a microelectrode holder half-cell filled with 3moll-1 KCl (WPI MEH3S; World Precision Instruments, Sarasota, FL, USA).
Self-referencing ion-selective microelectrode (SeRIS) systems
Technical and theoretical aspects of SeRIS microelectrodes have been described previously (Kühtreiber and Jaffe, 1990; Kochian et al., 1992; Smith et al., 1994; Piñeros et al., 1998). Briefly, the system used in this study utilized an orthogonal array of computer-controlled stepper motors (CMC-4, Applicable Electronics Inc., Forrestdale, MA, USA) fitted to a set of translator stages (Newport Corp., Fountain Valley, CA, USA). The stepper motors accomplished both coarse positioning and vibration of the microelectrode in three dimensions with submicrometre accuracy and repeatability. At each measurement site, the electrode was vibrated perpendicular to the Malpighian tubule surface between two positions separated by 100µm (Fig.1). Voltage measurements were taken at each extreme of the vibration and amplified using an IPA-2 ion/polarographic amplifier (Applicable Electronics Inc., Forrestdale, MA, USA). The signal first undergoes a 10-fold followed by a 100-fold amplification step for a total signal amplification of 1000-fold. A voltage difference was calculated from the signals at each position. Voltage differences were then converted into K+ concentration differences using a standard microelectrode calibration curve that related voltage output to K+ concentration in solution. Although K+-selective electrodes measure K+ activity and not concentration, data can be expressed in terms of concentrations if it is assumed that the K+ activity coefficient is the same in the calibration solutions and in the bathing saline. The highly sensitive self-referencing system allowed the resolution of voltage differences as small as 10µV, corresponding to differences in K+ concentration (in bathing medium containing 20 mmoll-1 K+) as small as 0.04%.
The SeRIS microelectrode was viewed using an inverted microscope (TMS, Nikon) equipped with a video camera. A Pentium PC computer running automated scanning electrode technique (ASET) software (Sciencewares, East Falmouth, MA, USA) controlled the movement of the microelectrode for both electrode positioning and vibrational amplitude. The software also allowed for the visual display of the voltage differences and the programming of a user-defined automated scanning protocol. Measurements were made by moving the probe to the site of interest, then by vibrating the electrode at a frequency of 0.14Hz with an amplitude of 100µm using the Move, Wait and Sample parameters of the ASET software. First, the probe moved to one extreme of the 100µm excursion. The probe then remained stationary during the wait period to allow ion gradients near the tubule to re-establish after the localized stirring during the movement period. No data were collected during the wait period. Lastly, the probe voltage was recorded during the sampling period. The probe was then moved to the other extreme of the 100µm excursion, followed by another wait and sample period. Each move, wait and sample cycle at each extreme of probe excursion was complete in 7s. A flux measurement requires measurement at both extremes of probe excursion, for a total of 14s.
Calculation of SeRIS electrode efficiency
A potassium source was used to generate a K+ gradient to test the efficiency of a self-referencing K+-selective microelectrode. The source was constructed by filling a blunt micropipette (tip diameter approximately 10µm) with 100 mmoll-1 KCl plus 0.5% (w/v) agar and placing this in a 35 mm Petri dish filled with SBM containing a background K+ concentration of 20 mmoll-1. The dish was left undisturbed for 30min before any measurements were taken to permit K+ diffusion between the source and the bathing medium to reach a steady state. Agar was included in the source pipette to minimize bulk water flow into the pipette. Convective disturbances were minimized by placing the tips of both source and measuring electrodes within 50µm of the bottom of the dish.
Theoretical values for the K+ gradient generated at the tip of the source pipette were calculated according to the following equation (Piñeros et al., 1998):
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where V is the change (in mV) over the vibration excursion of the electrode, S is the slope of the electrode calibration, r is the distance from the source,
r is the amplitude of vibration, CB is the background concentration of K+ and U is an empirical constant.
The constant U was calculated by generating a calibration curve for the microelectrode to characterize its response. A series of static K+ electrode voltage readings were taken at known distances from the K+ source. The term static is used to indicate that the electrode was not vibrated at each site. The millivolt readings were converted to K+ activity values using the calibration curve. Plotting these activity values (C) versus the inverse of the distance from the K+ source (1/r) resulted in a line with a slope of U, according to the equation:
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The empirical constant U was then substituted into equation 1 and used to calculate the theoretical voltage change over a vibrational distance at a known distance from the source. Actual experimental measurements of the voltage using the self-referencing K+ microelectrode at the same distances were plotted. Using the method of Piñeros et al. (Piñeros et al., 1998), electrode efficiency was calculated as the ratio of the slope of the experimental data to the slope of the theoretical data.
Measurement of ion fluxes
Ion fluxes were calculated according to Ficks first law of diffusion:
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where JK is the net flux of K+ (pmolcm-2s-1), DK is the diffusion constant for K+ (1.9x10-5cm-2s-1) (Robinson and Stokes, 1968), C1 and C2 are the K+ concentrations (pmolcm-3) at the two extremes of the vibration, and x is the amplitude of the vibration (cm).
For transport studies, Malpighian tubules were dissected as described above for fluid secretion experiments. Tubules were then transferred to a 35 mm Petri dish and bathed in 2ml of SBM. To improve the optics, a 1.25 cm diameter hole was cut out of the bottom of the dish and a 22 mm2 glass coverslip (VWR Scientific Inc, thickness no. 1) was sealed in place using paraffin wax. The glass slide was coated with poly-L-lysine (125µgml-1) to facilitate tubule adhesion. Anterior tubules were positioned such that the pair of tubules spanned a straight line at right angles to the ureter. Flux measurements were carried out at different positions along the length of the tubule. Microelectrodes were vibrated perpendicular to the surface of the tubule with an amplitude of 100µm such that the extremes of the vibration were at 10 and 110µm from the tubule surface. All experiments were conducted at room temperature (2125°C). Note that all reported flux measurements refer to free solution flux and are not direct measurements of transepithelial flux.
Chemicals
Stock solutions of cAMP, cGMP and NaCN (Sigma) were prepared in saline and diluted in a 1:1 mixture of standard Drosophila saline and Schneiders insect medium (Sigma).
Data analysis and statistical analyses
Data were analyzed using Microsoft Excel. Values are reported as means ± S.E.M.; N is the number of tubules for fluid secretion studies or K+ flux measurements. Where error bars are not visible in the figures, they are smaller than the symbol used. Where appropriate, the significance of differences between control and experimental groups or between different treatments was assessed using Students t-tests (two-tailed) with P=0.05 as the critical level.
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Results |
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Temporal analysis of K+ fluxes in unstimulated tubules
Scans of single sites in the lower (reabsorbtive), main (secretory) and distal (non-secretory) segments of the Malpighian tubule are shown in Fig.4. The voltage difference was positive when the self-referencing K+-selective microelectrode was moved into the unstirred layer near the surface of the lower segment (Fig.4A), indicating accumulation of K+ in the unstirred layer adjacent to the basolateral membrane of the tubule. This accumulation is consistent with an efflux of K+ from the tubule into the unstirred layer and, therefore, with K+ reabsorption from lumen to bath. The voltage difference was negative when the K+-selective microelectrode was moved into the unstirred layer near the surface of the main segment (Fig.4B), indicating a reduction of [K+] in the unstirred layer adjacent to the tubule. This reduction is consistent with an influx of K+ into the tubule and, therefore, with K+ secretion by the main segment. Repeated scans of the distal segment of tubules showed no significant voltage difference when the electrode was moved into the unstirred layer near the tubule surface (Fig.4C). Accordingly, no significant influx or efflux and, therefore, no secretion or reabsorption, was evident in the distal segment of anterior Malpighian tubules.
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Spatial analysis of K+ flux in unstimulated tubule segments
In these experiments, the spatial distribution of K+ flux along the lower, main and distal segments of unstimulated tubules was studied. Each of 48 sites in each segment was separated from the previously measured site by approximately 100µm.
Voltage differences in scans of all points in the lower segment were surprisingly variable, ranging from 32±21µV at site 7 of Malpighian tubule 1 to 343±49µV at site 2 of Malpighian tubule 2 (Fig.5). The highest K+ efflux averaged over all seven sites in this segment was seen in Malpighian tubule 2 and the smallest in Malpighian tubule 3. Average K+ reabsorption in lower segments of four other Malpighian tubules were between those of Malpighian tubule 2 and Malpighian tubule 3. Malpighian tubule 1 showed the greatest variation within a single Malpighian tubule. Variation within the lower segments of four other Malpighian tubules (not shown) was significant, but less dramatic than for Malpighian tubule 1.
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In contrast to the lower and main segments, the voltage differences measured adjacent to the basolateral membrane of the distal segment were not significantly different from 0mV (Fig.5; Malpighian tubules 79), where 0mV was the voltage difference measured in the bathing medium at a distance of 1000µm from the tubule. Similar results were found for distal segments of eight other Malpighian tubules (results not shown).
Averaging of all points across each segment for all tubules yields a global mean voltage difference of 137±10µV for K+ reabsorption in the lower segment, -218±7µV for K+ secretion in the main segment and 20±7µV in the non-secretory distal segment. The latter value was not significantly different from zero. The voltage differences correspond to changes in K+ concentration of +0.7% for the lower segment and -1% for the main segment. Corresponding fluxes are 255pmolcm-2s-1 for the lower segment and -406pmolcm-2s-1 for the main segment (Fig.6).
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In the distal segment, K+ flux was negligible before and after the addition of NaCN. Stimulation with cAMP or cGMP caused no increase or decrease in K+ flux in the distal segment (Fig.8C).
K+ fluxes for unstimulated and stimulated principal cells and stellate cells
Previous studies indicate that stellate cells are involved in transepithelial Cl- secretion and are not involved in K+ transport (ODonnell et al., 1996; ODonnell et al., 1998).
Prior to stimulation, there was no significant difference between K+ fluxes at sites adjacent to principal cells and that at sites adjacent to nearby stellate cells in the same tubule (Fig.9). After stimulation with 1 mmoll-1 cAMP, K+ flux adjacent to principal cells increased significantly by 20% (Fig.9). K+ flux adjacent to nearby stellate cells did not increase significantly.
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Discussion |
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Accuracy of the SeRIS measurements of K+ flux
The accuracy of the SeRIS technique for K+ flux measurement was assessed by comparing K+ fluxes calculated from fluid secretion data and those directly measured using the SeRIS system. Rates of fluid secretion by the Malpighian tubules of D. melanogaster were similar in this study to those observed previously (Dow et al., 1994a; Dow et al., 1994b; ODonnell and Maddrell, 1995). K+ fluxes can be calculated by multiplying the measured fluid secretion rate (nlmin-1) by the K+ concentrations (mmoll-1) in the secreted fluid droplet. This value is then divided by the estimated surface area (cm2) of the tubule to yield a K+ flux. Tubule surface area is estimated using the formula for the surface area of a cylinder (dl, where d is diameter and l is length). Previous studies (ODonnell and Maddrell, 1995), in which secretion rates were similar to those observed in the present study, indicated that the K+ flux into the main segment is 254±53pmolcm-2s-1 and that the K+ flux out of the lower segment is 152±41pmolcm-2s-1. The lower segment thus reabsorbs 60% of the K+ secreted by the main segment. In the present study, direct measurement of K+ fluxes using the K+-selective microelectrode yielded a K+ flux into the main segment of 406±14pmolcm-2s-1 and a K+ flux out of the lower segment of 255±19pmolcm-2s-1. This corresponds to a 63% recovery of K+, which is very close to the value calculated by the previous method.
It should be noted that K+ flux measured by the SeRIS technique for the main segment is 1.6 times that calculated from measurements of fluid secretion rate and secreted fluid K+ concentration. Similarly, the K+ flux measured by SeRIS microelectrodes for the lower segment is 1.7 times that calculated from fluid secretion rate and secreted fluid K+ concentration. The discrepancy between the two techniques indicates higher transport rates in the tubules used for SeRIS measurements. The discrepancy may simply reflect the approximations inherent in previous calculations of tubule surface area (ODonnell and Maddrell, 1995). An alternative explanation is that O2 and metabolites such as glucose may have greater access to the Malpighian tubule cells during SeRIS measurements than during fluid secretion experiments. As a result of the move, wait and sample protocol employed by the SeRIS technique, a small amount of localized stirring is created whenever the probe moves to a new site. This movement mixes the unstirred layer slightly and may thus increase the concentration of metabolites in the unstirred layer adjacent to the metabolically active tubules. This argument is supported by the finding that both the K+ flux into the main segment and that out of the lower segment increase by a similar factor, resulting in higher absolute values for flux but similar ratios to those of fluid secretion studies. K+ fluxes measured by SeRIS microelectrodes may also be higher because the large surface area of bathing media may permit higher oxygen tensions to be maintained than is the case for small droplets under paraffin oil.
K+ flux in the distal segment of Malpighian tubules
Previous studies (Dow et al., 1994b) have shown that the distal segment of the Malpighian tubules of D. melanogaster is non-secretory. The self-referencing K+-selective microelectrode has provided the first direct evidence that the distal segment is not only non-secretory but is also non-reabsorptive. This finding could not have been revealed using Ramsay secretion assays (Ramsey, 1952). The distal segment was also unaffected by putative stimulators and inhibitors of fluid secretion. Although these data support the hypothesis that the distal segment does not transport K+, they do not rule out the possibility that this segment may perform some other vital physiological role such as transport or sequestration of other ions or organic solutes (Wessing et al., 1988; Dube et al., 2000). For example, the distal segment transports Ca2+ at very high rates (Dube et al., 2000).
Temporal analysis of K+ flux in Malpighian tubules
Temporal scans of the lower segment showed oscillations in SeRIS microelectrode voltage that reflected oscillations in K+ flux (Fig.4A). Our tentative explanation for the oscillations in flux is based upon the action of the ureter as a muscular sphincter. When this sphincter is closed, K+ reabsorption by the lower tubule progressively reduces the K+ concentration of the luminal fluid. Over time, the rate of reabsorption will decline, because the source of K+ in the lumen is being depleted. There is, therefore, a gradual slowing in the rate of K+ reabsorption, and this is seen as a reduction in K+ efflux measured by the SeRIS microelectrode. When the ureter opens, fluid within the lumen of the lower segment is released into the bath and replaced by K+-rich fluid moving downstream from the main segment. The rate of K+ reabsorption thus increases until the next closing of the ureter. Validation of this hypothesis is complicated by the high frequency of observed ureter contractions relative to the time required for K+ flux measurements using the SeRIS technique. Resolution of an oscillation in K+ flux required measurements at two successive troughs and the intervening peak, for example. Each flux measurement required 14s, so one oscillation in flux could be measured every 42s. Accordingly, the maximum resolvable frequency was 1.4oscillationsmin-1. The frequency of 1.2 oscillations in flux per minute observed was very close to this predicted maximum. However, it should be noted that, because of the time required for acquisition of voltage measurements, the SeRIS technique may underestimate the frequency of oscillations in K+ flux.
Spatial analysis of K+ flux in Malpighian tubules
Spatial scans of K+ flux in both unstimulated and stimulated tubules indicated pronounced heterogeneity of K+ transport across segments that are morphologically and ultrastructurally homogeneous. Unstimulated tubules showed a highly variable pattern of K+ flux adjacent to the lower segment. Variations in K+ flux adjacent to the main segment were evident, albeit less dramatic. These findings were unexpected for a number of reasons. First, one would expect that a tubule region that is morphologically homogeneous would be physiologically homogeneous. However, there is a precedent for physiological discontinuities in epithelia of apparently uniform ultrastructure. Reabsorption of KCl by the lower segment of the Malpighian tubule of Rhodnius prolixus has been shown to be restricted to the lower third of the length of the lower segment (Maddrell, 1978). Osmotic permeability also varies along the length of the lower segment (ODonnell et al., 1982). What is noteworthy, however, is that previous studies (Collier and ODonnell, 1997; Maddrell, 1978) showed a predictable pattern of K+ reabsorption along the length of the lower segment of the Malpighian tubules of Rhodnius prolixus. In contrast, our study revealed that both the lower and main segments of the Malpighian tubules of D. melanogaster exhibit highly variable patterns of K+ flux along their lengths.
One possible explanation for variability in K+ transport within main and lower segments may be found in previous work (Sözen et al., 1997), which demonstrated that morphologically homogeneous cells of D. melanogaster tubules show genetic heterogeneity. This finding is important because it suggests that the variability in physiology observed in our study may reflect genetic heterogeneity. In other words, not all cells within each segment have the same K+ transport capability. Further evidence for physiological heterogeneity was evident in responses to stimulation with cAMP. Measurements of K+ flux indicated that not all the principal cells in the main segment responded equally to cAMP stimulation.
Effects of cyclic nucleotides and metabolic inhibition on K+ flux
Inhibition of K+ flux in the main segment by addition of NaCN, a metabolic inhibitor, mirrored the effect of NaCN on fluid secretion. The ability of NaCN to completely block active fluid secretion and K+ flux can be traced back to the principal cells of the main segment. The current model, now generally accepted, is that the central role in active transport of cations is accomplished by an apical vacuolar type H+-ATPase (Bertram et al., 1991; Bowman et al., 1988) confined to the principal cells (Sözen et al., 1997). The proton gradient maintained by this H+-ATPase provides the energy source for the secondary active transport of K+ from cell to lumen through a K+/H+ antiporter. It is apparent that, if cell metabolism is inhibited by NaCN, both fluid secretion and K+ flux should be inhibited, as observed in our study. The blocking of K+ reabsorption in the lower segment by NaCN is consistent with previous studies showing that reabsorption is also a process of active transport (ODonnell and Maddrell, 1995).
A comparison of the effects of cAMP on K+ flux and fluid secretion rates reveals an unexpectedly low correlation. Cyclic AMP increased fluid secretion rates by 70% but increased K+ influx in the main segment by only 36%. The difference in these values may be due to transport of ions or osmolytes other than K+. Such transport would contribute to the secretion of osmotically obliged water, but would not affect the K+-selective flux measurements. These results raise the intriguing possibility that cAMP may modulate V-ATPase activity in principal cells (ODonnell et al., 1996) and may also preferentially stimulate the transport of organic or inorganic osmolytes other than K+. The first messenger leading to cAMP production in principal cells is unknown.
Cyclic GMP raised the secretion rate by 29%, closely mirroring the 24% increase in K+ influx into the main segment. This result combined with the effect of cAMP above supports the currently held opinion that cGMP is the natural second messenger that responds to the endogenous peptide CAP2b (Davies et al., 1995). Our results also show that neither cAMP nor cGMP altered the extent of K+ reabsorption by the lower tubule. At present, it is not known whether K+ reabsorption can be modulated by hormonal signals.
Although stellate cells have been proposed as the transcellular routes of Cl- conductance (ODonnell et al., 1998), the K+-selective microelectrode detected an apparent K+ influx adjacent to stellate cells. The explanation may reside in the relative dimensions of the cells and the unstirred layer K+ gradient produced by active transport. In the present study, we found that gradients in K+ activity could be measured as far as 200µm from the basolateral surface of the tubule. As a result, there is extensive overlap of concentration gradients produced by adjacent K+-transporting principal cells, each of which is approximately 30µm in length and width. In other words, the apparent K+ influx associated with a stellate cell may be due to K+ transport of the surrounding principal cells. It is possible that, when K+ transport is stimulated by cAMP, the K+ concentration gradient is steeper and more sharply defined spatially, so that a 20% increase in K+ flux over principal cells is detectable. In contrast, this smaller increase may not be detectable over stellate cells because of a blunting of the K+ activity gradient as a result of the presence of a cell not itself contributing to K+ transport.
To summarise, this paper provides evidence for temporal and spatial heterogeneity in K+ transport by the Malpighian tubules of D. melanogaster. Not all cells in the main segment secrete K+ at the same rate, nor do all cells in the lower segment contribute equally to K+ reabsorption. It will be of interest to determine whether such heterogeneity is apparent in the transport of other ions. Moreover, our results raise the possibility that ion transport by other epithelia is also heterogeneous and that such heterogeneity can be revealed using the SeRIS microelectrode technique.
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Acknowledgments |
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