Analysis of Epithelial Transport by Measurement of K⁺, Cl⁻ and pH Gradients in Extracellular Unstirred Layers: Ion Secretion and Reabsorption by Malpighian Tubules of Rhodnius Prolixus

A recurrent theme in studies of insect Malpighian tubules (MTs) is the very high rate of ion transport achieved by tubules during diuresis. The MTs of Rhodnius prolixus, for example, secrete a volume of iso-osmotic fluid equivalent to their own volume every 15 s (Maddrell, 1991). R. prolixus provides a particularly useful model system because its MTs both secrete and reabsorb ions at high rates. The rapid rates of ion transport raise the possibility of significant depletion or accumulation of ions in the unstirred layer (USL) adjacent the basolateral surface of secretory and reabsorptive segments, respectively. This paper describes direct measurements of the temporal and spatial changes in ion concentrations in the USLs of secretory and reabsorptive segments of R. prolixus MTs.

R. prolixus ingests blood meals which may equal 10–12 times its unfed mass. The MTs eliminate much of the plasma fraction of the blood meal during a dramatic post-prandial diuresis (Maddrell and Phillips, 1975). There are two morphologically distinct segments of the Malpighian tubule: the upper (distal) Malpighian tubule (UMT) and the lower (proximal) Malpighian tubule (LMT) (Wigglesworth, 1931). Fluid secreted by the UMT is an approximately equimolar mixture of NaCl and KCl and is iso-osmotic with the insect’s haemolymph (approximately 370 mosmol kg⁻¹), but hyperosmotic to the blood meal (approximately 320 mosmol kg⁻¹; Maddrell, 1969). KCl but not water is then reabsorbed by the LMT (Maddrell and Phillips, 1975; O’Donnell et al. 1982). Although the structure of the LMT is homogeneous along its length (Wigglesworth, 1931; Wigglesworth and Salpeter, 1962), KCl reabsorption is confined to the lower third of the LMT closest to the junction with the hindgut (Maddrell, 1978). Concentrations of K⁺ and Cl⁻ in fluid secreted by the UMT are approximately 70 mmol l⁻¹ and 185 mmol l⁻¹, respectively. These decrease to approximately 3 mmol l⁻¹ and 120 mmol l⁻¹, respectively, during passage through the LMT (Ramsay, 1952; Maddrell and Phillips, 1975). Overall, the process of iso-osmotic secretion followed by reabsorption of KCl preserves haemolymph osmolality and K⁺ concentration following ingestion of a hypo-osmotic blood meal (Maddrell and Phillips, 1975).

In the present paper, the gradients in ion concentration between the USL and bathing saline have been measured by means of ion-selective microelectrodes positioned at various distances from the surface of the tubules. Results obtained with UMTs and LMTs, both isolated and in situ, demonstrate that the pH and concentrations of K⁺ and Cl⁻ in the USL can be quite different from the corresponding values in the bathing saline. Measurements of USL ion concentrations can be used to indicate whether ions are secreted or reabsorbed by different regions of MTs, and the rates of ion transport can be calculated from such measurements.

Fifth-instar Rhodnius prolixus Stål from a laboratory culture maintained at 25–28 °C and 60 % relative humidity were used 3–6 weeks after the previous blood meal. After insects had been killed by crushing the head with forceps, Malpighian tubules were dissected under physiological saline which consisted of (in mmol l⁻¹): NaCl, 133.6; KCl, 4.0; MgCl₂.6H₂O, 8.5; CaCl₂.2H₂O, 2.0; glucose, 20.0; NaHCO₃, 10.2; NaH₂PO₄, 4.3; and Hepes, 8.6. Saline was titrated with NaOH to pH 7.0. Salines of varying [K⁺] were prepared by substitution with Na⁺, maintaining the total concentration of NaCl and KCl at 137.6 mmol l⁻¹, and are referred to by their respective concentrations of K⁺ (e.g. 4K for control saline). A saline with reduced buffering capacity was prepared using 1 mmol l⁻¹ Hepes (omitting NaHCO₃ and NaH₂PO₄) and 154.7 mmol l⁻¹ NaCl (for 4K saline) to maintain osmolality. Saline containing 50 % of the control level of Cl⁻ was prepared by mixing equal parts of 4K saline and Cl⁻-free saline. The latter was prepared by using sulphate salts of calcium, potassium and magnesium and substituting sodium isethionate for NaCl.

Upper and lower segments of isolated whole Malpighian tubules were placed in separate 200 μl droplets of saline (Fig. 1). Lower tubules were bathed in 4K saline because this [K⁺] approximates that found in the haemolymph (3.6 mmol l⁻¹; Maddrell et al. 1993). Upper tubules were bathed in 24K saline because the secreted fluid contains higher K⁺ levels (approximately 100 mmol l⁻¹) and because they secrete fluid at constant rates for longer periods in vitro (Maddrell et al. 1993). The ampulla of the lower tubule was pulled out of the bathing droplet and anchored on a glass pin. Serotonin (5-hydroxytryptamine, 5-HT) was added at 10⁻⁶ mol l⁻¹ to both bathing saline droplets to stimulate fluid secretion by the upper tubule and KCl reabsorption by the lower tubule. Droplets of secreted fluid were collected from the ampulla at intervals. Droplet diameters were measured with an eyepiece micrometer and droplet volume (in nl) was calculated using a standard formula (e.g. Haley and O’Donnell, 1997). Secretion rates (in nl min⁻¹) were calculated by dividing droplet volume by the time over which the droplet was formed.

Extracellular ion activity and pH were measured using liquid membrane pH- and ion-selective microelectrodes (ISMEs). The slope and selectivity of the electrodes are unaltered by passing them through the paraffin oil to reach the bathing saline droplets (e.g. Maddrell et al. 1993). K⁺-selective microelectrodes were based on the neutral carrier valinomycin (K⁺ ionophore I, Cocktail B, Fluka Chemical Corp., Ronkonkoma, NY, USA). Cl⁻-selective microelectrodes were based on the Cl⁻ exchanger IE-173 (World Precision Instruments; Sarasota, FL, USA). The pH-selective microelectrodes were based on the neutral carrier tridodecylamine (H⁺ ionophore II, Cocktail A, Fluka). ISMEs were pulled to tip diameters of less than 1 μm and were then broken back to tip diameters of approximately 10 μm. Techniques for fabrication of ISMEs and reference electrodes have been described previously (Maddrell et al. 1993). Ion-selective and reference microelectrodes were connected by chlorided silver wires to high-impedance (>10¹⁵ Ω) electrometers, which were connected in turn to a computerized data acquisition and analysis system (Axotape, Burlingame, CA, USA).

Although ion-selective electrodes measure ion activity and not concentration, data can be expressed as concentrations if it is assumed that the activity coefficient is the same in both the calibration solution and the bathing saline. Over the range of ionic strengths of fluids encountered in this study (80–180 mmol l⁻¹), errors resulting from this assumption are less than 5 % (Maddrell et al. 1993). Expressing ISME data as concentrations simplifies comparisons with previous studies in which ion concentrations were measured by flame photometry.

Ion concentrations or pH were measured at varying distances from the basolateral surface and at several positions along the tubule’s length. Distances were measured using a calibrated eyepiece micrometer in the dissecting microscope. Positions along the length of the lower tubule are expressed as percentages of its length (approximately 17 mm for a tubule from a fifth-instar R. prolixus), where 0 % corresponds to the ampulla and 100 % to the junction of the upper and lower tubules. Reference electrodes were positioned at least 5 mm away from the tubule.

Measurements of [K⁺]USL and pHUSL were also made on MTs in situ. Insects were killed by crushing the head with forceps, the dorsal cuticle of the posterior half of the abdomen was removed, and 100 μl of 4K saline was added to the haemocoel. The animal was pinned down by the legs to a dissecting dish lined with Sylgard, and the fat body and heart were pulled slightly to one side to expose the tubules and the gut. Evaporation of the saline was prevented by covering the saline surface with 50 μl of paraffin oil. The preparation was illuminated from the ventral side. Ion-selective and reference electrodes were placed in the saline/haemolymph to measure ion activity. Mean ion concentrations were measured 5 μm from the basolateral surface. The reference electrode was placed at least 5 mm away from the tubules.

Preliminary measurements indicated that [K⁺]_USL and pH_USL did not vary along the length of the UMT, and a mean value was determined for three measurements for three tubules from each animal. For the lower tubule, [K⁺]_USL and pH_USL were calculated as the mean of three measurements at the same site 20–30 % of the distance along the length of each of three tubules in each insect.

Micropipettes pulled to tip diameters of less than 1 μm were broken back to tip diameters of 3–4 μm. Each pipette was filled with paraffin oil and attached to polyethylene tubing and a 25 ml syringe filled with distilled water. Drugs were dissolved in saline and droplets of known volume were expelled under paraffin oil (see Results). The tip of the micropipette was placed into the droplet containing the dissolved drug, and the entire droplet was taken up into the micropipette by creating a slight negative pressure in the syringe. The micropipette was then placed next to the ion-selective microelectrode in the USL. While observing the saline/oil interface in the micropipette through the microscope, sufficient pressure was applied to the syringe plunger to eject the entire volume of saline within the micropipette.

All experiments were performed at room temperature, 20–25 °C. Values are reported as means ± S.E.M. Significance of differences between means was evaluated by paired or unpaired Student’s t-tests (two-tailed), using a critical value of P<0.05 for significance. Previous studies by Boutilier and Shelton (1980) justify the calculation of mean values and standard errors for pH data, and the pH measurements were not converted, therefore, to [H⁺] (mol l⁻¹) before statistical treatment.

K⁺ concentrations were measured in the USL within 5 μm of the basolateral surface at 10 %, 25 %, 50 %, 75 % and 95 % along the length of the lower tubule (inset, Fig. 2A). This minimum distance was a consequence of the wall thickness of the ISME. For unstimulated tubules, the mean [K⁺] in the fluid of the unstirred layer of the basolateral surface of the lower tubule ([K⁺]_USL) did not vary significantly along the length of the tubule (Fig. 2A), and the data were therefore pooled. [K⁺]_USL of unstimulated tubules (4.49±0.03 mmol l⁻¹; N=30 sites on six tubules) was slightly but significantly (P<0.05) above [K⁺]_Bath (4.33±0.01 mmol l⁻¹, N=6). This small difference may reflect passive leakage of K⁺ from lumen to bath in unstimulated lower tubules. In stimulated tubules, the concentration of K⁺ in the unstirred layer adjacent to the basolateral surface increased, relative to that of the bath, and these differences were most dramatic at 10 % and 25 % along the length of the lower tubules (Fig. 2A). The increase in [K⁺]_USL relative to [K⁺]_Bath is consistent with accumulation of K⁺ in the USL as K⁺ is transported from lumen to bath by the LMT. The pattern of changes in [K⁺]_USL was examined in greater detail by scanning at eight positions along the length of the lower tubule (Fig. 2B). The lower values of [K⁺]_USL at distances greater than or less than the peak at 25 % along the length of the lower tubule presumably reflect both the reabsorptive capacity of the epithelium and the luminal concentration of K⁺ (see Discussion). The differences in bathing saline [K⁺] in Fig. 2A,B reflect the gradual increase in [K⁺]_Bath with time as K⁺ is reabsorbed from lumen to bath in stimulated tubules. For tubules isolated in 200 μl droplets of 4K bathing saline, this increase was less than 2 mmol l⁻¹ over a period of 60 min. In all subsequent experiments, therefore, the duration of the experiments after addition of 5-HT was restricted to 50 min.

Previous studies have shown that equal amounts of K⁺ and Cl⁻ are reabsorbed by the lower tubule (Maddrell and Phillips, 1975). Measurement of [Cl⁻]_USL was not feasible in salines containing the control Cl⁻ concentration (148.1 mmol l⁻¹); an increase of 2 mmol l⁻¹ above the bath level, for example, would result in a voltage change of only 0.3 mV for a Cl⁻-selective microelectrode with a slope of 58 mV per 10-fold change in [Cl⁻]. For this reason, measurements of [Cl⁻]_USL near the lower tubule were made in saline containing 4 mmol l⁻¹ K⁺ and 74.1 mmol l⁻¹ Cl⁻ (i.e. 50 % of control [Cl⁻]). Preliminary results showed significant increases in [Cl⁻]_USL relative to [Cl⁻]_Bath in the lower half of stimulated but not unstimulated lower tubules (N=5, data not shown).

Simultaneous measurement of [Cl⁻] and [K⁺] with double-barrelled ISMEs showed that USL concentrations of both ions reached maximum values at 15–30 % along the length of the tubule (Fig. 2C). The maximum [Cl⁻]_USL was nearly 24 mmol l⁻¹ higher than [Cl⁻]_Bath in 74.1 mmol l⁻¹ Cl⁻ saline (N=6). Comparison of Fig. 2B and Fig. 2C shows that [K⁺]_USL was not affected by a twofold change in [Cl⁻] in the bathing saline.

Measurement with voltage-sensitive microelectrodes (Haley and O’Donnell, 1997) show no change in potential when the microelectrode is moved adjacent to the basolateral surface of the LMT prior to impalement. Previous studies have shown that extracellular voltage gradients near cells, when present, are typically in the nanovolt range, beyond the resolution of ISMEs (Kuhtreiber and Jaffe, 1990). The increase in potential when the K⁺ or Cl⁻ microelectrode is moved from the bath into the USL is due to an increase in ion concentration relative to that in the bath, therefore, and not to a voltage gradient.

The dependence of elevated [K⁺]_USL on active transport in 5-HT-stimulated lower tubules was demonstrated by inhibiting metabolism with KCN (Fig. 3). Upper and lower tubules were set up in control conditions under oil (B1 in Fig. 3) and the LMT was scanned to find the site of maximum [K⁺]_USL. [K⁺]_USL was quickly re-established, and there was no change in [K⁺] in the secreted fluid ([K⁺]_SF) after transferring tubules to fresh droplets (B2 in Fig. 3) of control saline. When the UMT remained in control saline and the LMT was moved into 4K saline containing 2 mmol l⁻¹ KCN, [K⁺] in droplets of secreted fluid ([K⁺]_SF) increased and [K⁺]_USL declined, consistent with an inhibition of K⁺ flux from lumen to bath by KCN; both changes were reversed in KCN-free saline.

The maximum [K⁺]_USL (i.e. that measured between 20 % and 30 % along the length of the LMT) exceeded [K⁺]_Bath by as much as 5.3-fold when the latter was varied from 2 to 20 mmol l⁻¹. Lower tubules were bathed in saline droplets containing 2, 4, 6, 8.6 or 20 mmol l⁻¹ K⁺; the corresponding values of [K⁺]_USL (in mmol l⁻¹, mean ± S.E.M., for the number of tubules indicated in parentheses) were: 10.6±0.8 (9), 11.8±0.4 (59), 12.0±1.0 (5), 15.4±1.3 (6) and 28.2±1.8 (5). These data show that the maximum value of [K⁺] _USL was relatively constant for a [K⁺]_Bath of 2–6 mmol l⁻¹, bracketing the normal haemolymph [K⁺] of 3.6 mmol l⁻¹ (Maddrell et al. 1993).

[K⁺]_USL associated with stimulated upper tubules was slightly but significantly lower (P<0.05) than [K⁺]_Bath in both 8.9K and 24K salines (Table 1). Reduction in [K⁺]_USL relative to [K⁺]_Bath is consistent with a depletion of K⁺ from the USL as K⁺ is transported from bath to tubule lumen by the UMT. The [K⁺]_USL of stimulated tubules was also significantly (P<0.05) lower than the [K⁺]_USL of unstimulated tubules in either saline. There were no differences along the length of the upper tubule, in contrast to the findings for the lower tubule and consistent with previous studies showing the secretory capacity of the upper tubule to be homogeneous along its length (Maddrell, 1969). Measurements were taken at evenly spaced points along the upper tubule, and a single mean value of [K⁺]_USL was calculated for each tubule.

Given the large differences between [K⁺]_Bath and [K⁺]_USL for isolated lower tubules, it was of interest to determine the extent of such gradients in situ. Measurements in situ indicated that [K⁺]_USL for stimulated lower tubules was significantly elevated above [K⁺]_Bath and [K⁺]_USL of unstimulated lower tubules (Table 1). However, the increases were less dramatic than for isolated tubules (Fig. 2). [K⁺]_USL of stimulated upper tubules in situ was significantly lower than [K⁺]_Bath or [K⁺]_USL of unstimulated tubules (Table 1).

One explanation for the difference between in situ and isolated tubules is that the saline (or haemolymph) surrounding the tubules in situ is mixed by contractions of the hindgut and midgut. These contractions increased in both frequency (from 15.8±0.8 min⁻¹ to 37.0±0.9 min⁻¹, N=9 animals) and amplitude in response to 5-HT, and may have minimised the build-up of K⁺ in unstirred layers in situ, relative to isolated tubules. In addition, the use of 24K saline for isolated upper tubules increases [K⁺] of fluid secreted by the upper tubules. More K⁺ was reabsorbed by the LMT, therefore, and the difference between [K⁺]_USL and [K⁺]_Bath increased.

pH was measured in the USL within 5 μm of the basolateral surface of upper or lower tubules. Measurements were taken at 10 %, 25 %, 50 %, 75 % and 95 % along the length of upper and lower tubules. Upper tubule measurements taken at these various points, whether the tubule was unstimulated or stimulated, showed no minimum pH (i.e. maximum [H⁺]) at a particular position along its length, and data for each upper tubule were therefore pooled. The pH_USL of unstimulated upper tubules was slightly but significantly acidic to pH_Bath in saline of reduced buffering capacity (Table 2, P<0.001). In both salines, stimulation with 5-HT resulted in significant acidification of pH_USL relative to the values measured for the same tubules prior to stimulation. The extent of acidification was 0.3 pH units in saline with reduced buffering capacity (Table 2).

Values of pH_USL for unstimulated lower tubules were not significantly acidic relative to pH_Bath (Fig. 4). Stimulation of the lower tubule alone, or both upper and lower tubules, resulted in slight but significant (P<0.05) reduction of pH_USL relative to pH_Bath (Fig. 4). As with [K⁺]_USL and [Cl⁻]_USL, the maximum change was found at a site approximately 25 % of the distance along the length of the tubule, and this site was significantly (P<0.05) more acidic than the pH_USL at 10 % along the length of the tubule. Acidification is probably a consequence of one (or both) of the following processes: (1) accumulation of metabolic CO₂ generated during active reabsorption of KCl; and (2) transfer of acidic equivalents from lumen to bath as the luminal fluid is alkalized during passage through the LMT (Haley and O’Donnell, 1997; see Discussion). Acidification of the USL in non-reabsorptive regions of the LMT (i.e. at 50 % and 75 % of the LMT’s length, Fig. 4) may reflect accumulation of metabolic CO₂ in the fluid secreted by the UMT and subsequent diffusion of CO₂ from lumen to bath across the unstimulated lower tubule.

In situ the maximum differences in pH_USL of stimulated versus unstimulated tubules bathed in saline of normal buffering capacity were 0.12 and 0.18 pH units for UMTs and LMTs, respectively (Table 2). For isolated tubules bathed in saline of normal buffering capacity, the corresponding difference was 0.05 pH units for both UMTs (Table 2) and LMTs (Fig. 4). The bases for these differences are discussed below.

Fluxes were calculated from [K⁺]_USL measured at eight sites (10 %, 20 %, 25 %, 30 %, 50 %, 60 %, 80 % and 100 %) along the lower tubule’s length. At each site, the [K⁺] was measured at distances of 5, 47, 84, 168, 210, 315 and 550 μm perpendicular to the long axis of the basolateral surface of the tubule (Fig. 5A–D). Fluxes were calculated using the Fick equation:

Fig. 6 shows measured fluxes and those estimated at increments of 5 % of tubule length by interpolation (hatched bars). For example, the K⁺ flux at 40 % was calculated as the mean of the fluxes at 30 % and 50 %, and the flux at 35 % was calculated from the mean of the fluxes at 30 % and 40 %. Separate experiments indicated that fluxes in the upper half of the lower tubule (i.e. 50–100 % along the tubule length) were negligible. The flux of 2.03 nmol cm⁻² s⁻¹ across the whole lower tubule was then calculated by summing the fluxes measured in each of the bins (where each bin equals 5 % of the LMT’s length) and dividing by the number of bins. This value is within 5 % of the flux calculated from the change in [K⁺]_SF after passage through the LMT and the surface area of the LMT, as follows. For the tubule in Fig. 5, the change in [K⁺] during passage through the lower tubule was 82.6 mmol l⁻¹, and fluid was secreted at 73.2 nl min⁻¹. The surface area of the tubules was estimated to be 0.048 cm², calculated from πdL, where d is the tubule diameter (90 μm) and L is its length (17 mm). The K⁺ secretion rate of 0.101 nmol s⁻¹[=(73.2×10⁻⁹ l min⁻¹)(82.6×10⁻³ mol l⁻¹)(1/60)] then corresponds to an area-specific flux of 2.10 nmol cm⁻² s⁻¹ [=(0.101 nmols⁻¹/0.048 cm²)].

Measurements of changes in [K⁺]_USL of the upper tubule could also be used as an assay for drugs known to inhibit MT fluid secretion, and the changes in [K⁺]_USL could be correlated with a decrease in K⁺ flux. For example, the presence of a basolateral Na⁺/K⁺/2Cl⁻ cotransporter in the Malpighian tubules of R. prolixus (O’Donnell and Maddrell, 1984), Aedes aegypti (Hegarty et al. 1991) and Formica polyctena (Leyssens et al. 1994) has been proposed on the basis of inhibition of fluid secretion by 10⁻⁵ to 10⁻⁴ mol l⁻¹ bumetanide or 10⁻⁴ mol l⁻¹ furosemide. Fluid secretion by the upper Malpighian tubules of R. prolixus is reduced by 79–80 % by 10⁻⁴ mol l⁻¹ furosemide or 10⁻⁵ mol l⁻¹ bumetanide (O’Donnell and Maddrell, 1984).

Fig. 7 shows that bumetanide (4×10⁻⁵ mol l⁻¹) significantly (P<0.05) increased [K⁺]_USL, consistent with less depletion of K⁺ from the USL (i.e. a decrease in K⁺ influx). The increase in [K⁺]_USL was reversible upon returning the tubules to bumetanide-free saline. Upper tubule K⁺ flux, calculated as the product of secretion rate (nl min⁻¹) and [K⁺]_SF (mmol l⁻¹), also declined by more than 65 % within 10 min of addition of 4×10⁻⁵ mol l⁻¹ bumetanide to the bathing saline. The effects of 10⁻⁴ mol l⁻¹ furosemide on [K⁺]_USL and K⁺ flux were similar (N=6, data not shown). These findings suggest that measurement of USL ion concentrations can provide useful information on the effects of drugs that inhibit specific ion transporters.

It was of interest to determine how rapidly changes in [K⁺]_USL occurred in response to stimulation of KCl reabsorption with 5-HT. A pulled micropipette (tip diameter approximately 3–4 μm) containing 10–15 nl of 10⁻⁷ to 10⁻⁴ mol l⁻¹ 5-HT was positioned adjacent to the ISME in the USL. Tubules were set up in control conditions, with only the UMT stimulated with 10⁻⁶ mol l⁻¹ 5-HT. 5-HT (1–1000 fmol) was then ejected from the micropipette into the USL at 25 % along the length of the LMT. A volume of 10–15 nl was sufficient to envelop 2–3 LMT cells.

[K⁺]_USL increased over 10 min following localized application of 5-HT (Fig. 8A). The mean time taken to produce 50 % of the maximal stimulation was 1.5 min (N=5). In contrast, a 10–15 nl pulse of control saline at the same site caused no sustained increase in [K⁺]_USL over 3 min (Fig. 8A). The maximum [K⁺]_USL and the duration of the rise in [K⁺]_USL increased with higher doses of 5-HT (Fig. 8B). [K⁺]_USL subsequently declined as 5-HT diffused into the 200 μl droplet of bathing saline, reducing the concentration of 5-HT to less than 5 nmol l⁻¹, which is below the threshold for stimulation of KCl reabsorption (Maddrell et al. 1993).

The results demonstrate that information about ion transport by isolated or in situ tubules can be inferred from analysis of USL ion concentrations measured using ISMEs. Importantly, we have found that it is feasible to measure the effects of ion transport by a single cell on USL ion concentrations. These measurements exploited the finding that, near the junction of the upper and lower Malpighian tubule, one or more UMT cells may be isolated from the rest of the UMT and surrounded by LMT cells (Fig. 9, inset). As noted above, the LMT cells near the junction of the LMT and UMT do not reabsorb K⁺ (Fig. 2). Isolated UMT cells are found in about 10 % of tubules (Maddrell and Overton, 1985) and have been shown to transport Na⁺ and organic acids in the same manner as UMT cells found in the upper tubule proper (Maddrell and Overton, 1985).

[K⁺]_USL was measured near isolated UMT cells and neighbouring LMT cells. Significant decreases in [K⁺]_USL for isolated UMT cells relative to that for neighbouring LMT cells were apparent after, but not before, 5-HT stimulation (Fig. 9). Measurement of [K⁺]_USL can thus provide information on MT ion transport with a spatial resolution comparable to that of the dimensions of an individual cell (100 μm).

This study demonstrates that both secretion and reabsorption of ions by Malpighian tubules from R. prolixus can be assessed both qualitatively and quantitatively by ISME measurement of ion concentrations in the USL associated with the basolateral cell surface. This technique can be used to measure anions or cations, provided that there is an appropriately selective ionophore. Also, basolateral measurements of ion transport capacity can be determined before and after putative stimulants or inhibitors are added to the bathing saline droplets. Very small differences in electrical potential (approximately 0.1–0.2 mV) can be consistently resolved with a temporal resolution of approximately 2–10 s. The spatial resolution of this technique is comparable to that of an individual cell (100 μm). Importantly, the techniques described in this paper can be used for Malpighian tubules in situ as well as in vitro.

This technique revealed several new features of ion transport in a well-characterized transporting epithelium, the Malpighian tubule of R. prolixus. The effects of 5-HT and KCN indicate that elevation of [K⁺] and [Cl⁻] within the USL is a direct consequence of 5-HT stimulation of metabolically dependent KCl reabsorption by the lower tubule. The decline in [K⁺]_USL as the electrode is moved from 30 % to 50 % of the LMT’s length is consistent with previous studies which demonstrate that KCl reabsorption is restricted to the lower third of the lower tubule (Maddrell, 1978). Moreover, our data reveal a new finding: K⁺ reabsorption within the lower third of the tubule is not constant. K⁺ flux between 20 and 30 % of the LMT’s length is higher than flux calculated from [K⁺]_USL measurements closer to the ampulla. The decline in [K⁺]_USL closer to the ampulla may reflect a decline in [K⁺] as fluid in the tubule lumen passes through the lower third of the LMT. K⁺ reabsorption becomes progressively more difficult and both [K⁺]_USL and K⁺ flux decline. Double-barrelled ISME measurements confirm that both K⁺ and Cl⁻ are reabsorbed across the same regions of the lower tubule.

The [K⁺] in the USL of the lower tubule may be more than five times greater than that in the bathing saline when [K⁺]_Bath is in the range 2–6 mmol l⁻¹, bracketing the normal haemolymph [K⁺] of 3.6 mmol l⁻¹. These findings are of relevance for the development of models of ion transport by rapidly transporting epithelia such as Malpighian tubules. Such models often involve calculations of electrochemical gradients for ion movements across epithelia, and it is usually assumed that the concentration of an ion in contact with the apical or basolateral surface is equal to the concentration in the bulk solution at a distance from the surface. Our results indicate that this assumption would lead to errors in calculated electrochemical gradients; estimates of electrochemical gradients should be made, therefore, using tubules which are superfused so as to minimize USL effects.

Measurements of [K⁺]_USL surrounding the basolateral membrane of the upper tubule confirm findings from previous studies regarding the homogeneous secretory capacity of the UMT along its length (Maddrell, 1969). Experiments with bumetanide and furosemide indicate that the effects of these drugs can be detected not just through changes in fluid secretion rates (O’Donnell and Maddrell, 1984) and K⁺ flux (this paper) but also through the decline in the K⁺ concentration gradient between the USL and the bathing saline.

Acidification of the USL of upper and lower tubules presumably reflects the transfer of acid–base equivalents into or out of the USL, and also accumulation of metabolic CO₂ and its subsequent hydration to form HCO₃⁻ and H⁺. The increase in USL acidification in situ may be related to higher rates of ion transport of in situ relative to isolated tubules (Maddrell, 1991). Experiments with microelectrodes which measure pH and P_CO? simultaneously (Bomsztyk and Calalb, 1986) will be of use in determining the contributions of various processes to USL acidification. During KCl reabsorption, the LMT alkalizes the fluid produced by the UMT by 1.2 pH units, and the secreted fluid is 1.4 pH units alkaline to the bath after passage through the LMT (Haley and O’Donnell, 1997). Acidification of the basolateral USL may be a consequence, in part, therefore of luminal alkalization; if base is transported into the lumen, then maintenance of cytoplasmic pH will require an equivalent transfer of acidic equivalents into the bathing saline or haemolymph. Acidification of the USL in unstimulated upper tubules may reflect metabolism associated with secretion of KCl at slow rates, and with homeostatic and excretory processes. The UMT secretes fluid of nearly neutral pH, and this is unaffected when pHBath is varied between 6.9 and 8.1 (Maddrell and O’Donnell, 1992). When pH_Bath is 7, therefore, there does not appear to be a requirement for transfer of acid–base equivalents from lumen to bath. Reductions in upper tubule pH_USL relative to pH_Bath are more likely, therefore, to reflect diffusion of CO₂ into the USL or acid–base exchange across the basolateral cell membrane.

Conversion of CO₂ to H⁺ and HCO₃⁻ may introduce a small error into our measurements of [Cl⁻]_USL, because the Cl⁻-selective liquid ion exchanger microelectrodes used here are only 20 times more sensitive to Cl⁻ than to HCO₃⁻ (Walker, 1971). The electrode may therefore overestimate the extent of Cl⁻ accumulation in the USL if HCO₃⁻ concentrations rise dramatically above those of the bath (10.2 mmol l⁻¹). Measurements with solid-state Cl⁻-selective microelectrodes, which are insensitive to HCO₃⁻ (e.g. Wright and O’Donnell, 1992), could resolve the extent of this error.

Previous studies have shown that a bathing saline [K⁺] above 30 mmol l⁻¹ inhibits KCl reabsorption (Maddrell and Phillips, 1977), consistent with blockade of passive efflux from cell to bath through basolateral K⁺ channels (Haley and O’Donnell, 1997). Our data indicate that the effective concentration of K⁺ at the basolateral surface of the tubule will, in fact, be much higher than this value because of accumulation of K⁺ in the USL. Our in situ measurements suggest that the convective mixing of the haemolymph by 5-HT-stimulated contractions of the midgut and hindgut minimizes the build-up of K⁺ within the USL. This decrease in the boundary layer concentrations of [K⁺] in situ was detected in both upper and lower tubules. These findings raise the possibility that, in intact animals, the contractions of the hindgut are stimulated by the hormones released at diuresis and that this increase in contraction frequency serves to minimize the establishment of large differences in [K⁺] between the haemolymph and the USL. The build-up of Cl⁻ in the USL will be much less inhibitory since the levels of Cl⁻ in the bathing fluid are high (148 mmol l⁻¹ in 4K saline) and the proportionate change in [Cl⁻]_USL relative to [Cl⁻]_Bath will be much smaller.

Flux measurements determined from measurements of [K⁺]_USL are in good agreement with those determined from measurements of fluid secretion rates and secreted fluid [K⁺]. The technique described in this paper may, therefore, be useful in characterizing Malpighian tubules of other species. This may be of particular interest for species such as the hemipteran Cenocorixa bifida (Cooper et al. 1989), in which MTs have several identifiable segments, each of which may have different transport functions, or for species having Malpighian tubules composed of heterogeneous cell types, such as the Malpighian tubules of Drosophila melanogaster, which contain stellate cells and principal cells (e.g. O’Donnell et al. 1996). Our preliminary results (K. A. Collier and M. J. O’Donnell, unpublished observations) indicate that K⁺ gradients near secretory and reabsorptive segments of D. melanogaster can be detected. This technique should also find use with other rapidly transporting epithelia, including insect salivary glands and midgut.

This study has shown that the influence of even a single cell on ion concentrations in the basolateral USL can be detected by an extracellular ISME. The technique is also useful for assessing the effects of pharmacological reagents and putative diuretic hormones on tubule functions, since small quantities of material can be applied by pressure or ionophoresis onto the tubule surface. Measurement of ion concentrations in the USL permits transport to be assessed without the use of radio-isotopes. For systems which transport more slowly, the more sensitive vibrating ion probe technique, involving slow vibration of the ISME and signal averaging, can be used (Smith et al. 1994).

We are grateful to Dr Jon Harrison for his comments on a draft of the manuscript and Dr John Machin for his suggestions regarding flux calculations.