In the presence of 6 mmol l−1 Ba2+, known to block the K+ channels in the basal membrane, a rise in bath [K+] ([K+]bl) induced an increase in intracellular K+ concentration ([K+]i) similar in amount and in time course to that obtained in the absence of Ba2+. The presence of active and passive (other than through K+ channels) K+ uptake mechanisms across the basal membrane was investigated in different bath K+ concentrations. Dihydro-ouabain (10−3 mol l−1), a blocker of the Na+/K+-ATPase, tested in low bath [K+], and Sch28080 (10−4 mol l−1), a K+/H+-ATPase inhibitor, were without effect on fluid secretion. Dihydro-ouabain was also without effect on electrical potential differences either in the absence or in the presence of Ba2+. Vanadate (10−3 mol l−1), in contrast, strongly reduced fluid secretion not only in control solution but also in high-K+, Na+-free medium and reduced the transepithelial and the apical membrane potential differences but not the basal membrane potential difference or [K+]i. Omitting Na+ from the bathing medium, replacing Cl-by Br or applying bumetanide (10−5 mol l−1) inhibited fluid secretion only in a low-K+ (10 mmol l−1) medium. In 51 mmol l−1 [K+]bl, omitting Na+ was without effect and 10-4 mol l−1 bumetanide was needed to inhibit secretion. Replacing Clby Brstimulated fluid secretion at this K+ concentration. Bumetanide (10−4 mol l−1) had no effect in 113 mmol l−1 [K+]bl. Bumetanide (10−4 mol l−1) in 51 mmol l−1 [K+]bl did not affect membrane potentials, did not lower [K+]i and did not affect the rise in [K+]i observed on an increase in [K+]bl. The results were summarized in a model proposing that K+ channels play a dominant role in high-K+ (113 mmol l−1) bathing medium. A K+/Clcotransporter may become more important in 51 mmol l−1 [K+]bl and a K+/Na+/2Cl cotransporter may gain in importance in 10 mmol l−1 [K+]bl. Active mechanisms for K+ uptake across the basal membrane seem to play no detectable role in sustaining fluid secretion. The response to vanadate might be due to an effect on the apical electrogenic H+ pump.

Malpighian tubules of forest ants (Formica polyctena) secrete primary urine. As in many insect species, they preferentially transport K+ into the lumen in control conditions, Cland water following passively (see Phillips, 1981, for a review). The fluid secretion rate is strongly dependent on the bath K+concentration (Van Kerkhove et al. 1989).

Since K+is always transported across the epithelium against its electrochemical gradient, K+transport must occur transcellularly. The active step is primarily situated in the apical membrane, where the extrusion of K+into the lumen is believed to be realized through the combination of an electrogenic H+pump and an electroneutral K+/H+antiporter acting in parallel (Weltens et al. 1992; Zhang et al. 1994; Dijkstra, 1993; Leyssens et al. 1993a,b). In Malpighian tubules of Formica polyctena, it was found that the bath K+concentration determines the intracellular K+content available for apical K+transport. This may be an intrinsic regulatory mechanism of fluid secretion (Leyssens et al. 1992, 1993a).

K+transport systems must exist across the basal membrane to ensure a sufficiently large K+uptake to maintain the rate of fluid secretion observed in the different bathing K+concentrations and to account for the change in intracellular K+concentration in response to the surrounding bath K+level. K+entry may occur passively through K+channels. Ion substitution and K+-selective measurements have demonstrated the appreciable K+permeability of the basal membrane (Leyssens et al. 1992, 1993a). The hyperpolarization of the basal membrane potential difference (Vbl) observed at all bath K+concentrations when Ba2+was used to block the K+channels (Weltens et al. 1992) suggested the existence of an electrochemical K+gradient favourable for K+uptake. However, the calculated electrochemical gradient for K+across the basal membrane was very small when the bath K+concentration was decreased and could even reverse (Leyssens et al. 1993a). The fluid secretion was also drastically, but not completely, blocked after 30 min in the presence of Ba2+(Weltens et al. 1992). This suggested that, as in other K+-transporting Malpighian tubules (for a review, see Nicolson, 1993), alternative pathways for K+uptake may be present in the basal membrane and that the relative rates of these hypothetical K+transport systems may vary under different transport conditions.

The present study focuses on these alternative routes for K+transport across the basal membrane at different bath K+concentrations. The effects of ion substitutions and blockers of primary and secondary active transport systems on fluid secretion, on intracellular and luminal K+concentration and on electrical potential differences were investigated.

It is shown that the rise in intracellular K+ concentration when the bath [K+] is increased still occurs after the basal K+ channels have been blocked. The existence of a basal coupled entry mechanism for Cl and K+ is demonstrated and its contribution to K+ uptake under different transport conditions is discussed. The role of Na+in transcellular ion transport is also investigated. The presence of a primary active K+ uptake system is critically evaluated. On the basis of these and previous findings, a transport model for transepithelial ion transport in the Malpighian tubule of Formica polyctena is proposed.

Preparation

Worker ants of the species Formica polyctena Förster were collected from natural nests at the periphery of woods and kept in an artificial nest at a constant temperature of 20°C. The animals were fed with sugar and water.

After dissection, a single Malpighian tubule (1–2 mm) was transferred to the stage of an inverted microscope into a bathing droplet (50 μl). The bathing droplet was covered with paraffin oil to avoid evaporation (see Van Kerkhove et al. 1989).

Experimental arrangement

Fluid secretion experiments

The technique used for measuring fluid secretion rate has been described in detail by Van Kerkhove et al. (1989). A fluid secretion experiment consisted of a control (three times 10 min), a test and a wash-out period (3–4 times 10 min each). The bathing fluid could be renewed using a perfusion and suction pipette. When a change of bathing solution was needed, the new solution was added via the perfusion pipette at a rate of 150 μl min−1over a 3 min period. Perfusion was then stopped until the next change in bath solution.

The effects of ions and inhibitors on fluid secretion rate were tested in 143 mmol l−1ClRinger’s solutions, containing varying K+concentrations (see Artificial salines and Results). As described previously (Weltens et al. 1992), in each experiment, secretion rate was expressed as a relative rate, i.e. as a percentage of the rate during the last control period before the change to that experimental solution. Furthermore, to account for a decrease in fluid secretion rate with time, for each collection period the ratio of the mean value of the experimental series over the mean value of the same period of fluid secretion experiments was calculated, where the tubules were bathed continuously in the control solution.

In order to test the statistical significance of the effect of a drug, the experimental relative rate was compared with the control relative rate at the same moment (unpaired Student’s t-test). The drug effect was considered to be fully reversible when no statistically significant difference (unpaired Student’s t-test) was found between the relative secretion rate during a wash-out period and the corresponding control period. Although we realize that this method was not ideal (the change in fluid secretion with time in the absence of a test solution had to be evaluated on a different set of tubules), it seemed to be the most suitable way to distinguish drug effects from time effects. Corrected results, taking into account the spontaneous decline of the fluid secretion rate with time are shown in the figures and tables (see Results).

Electrical potential difference measurements

The method has been described in detail previously (Leyssens et al. 1992). Measurements were performed with microelectrodes filled with 0.1 mol l−1KCl and connected to a high-impedance electrometer (Duo 773, WPI) via a Ag/AgCl wire. The reference electrode was a low-resistance (1 MΩ) 3 mol l−1KCl electrode earthed via a Ag/AgCl wire. In order to facilitate impalements, the Malpighian tubule was immobilized in the bathing droplet by two holding pipettes. The secreted fluid remained in the tubule. The measurement of the basal membrane potential difference (Vbl) was accepted if a sudden negative potential deflection occurred and was stable for at least a few minutes and if the electrode potential differed maximally by 3 mV from the baseline after electrode withdrawal. The transepithelial potential difference (Vte) was measured by advancing the microelectrode through the cell layer into the lumen of the tubule; Vbl and Vte were expressed with reference to the bath side. The apical potential difference (Vap) was calculated as the difference between the transepithelial and transbasal potential differences and expressed with reference to the lumen [Vap=-(Vte-Vbl)].

The bathing droplet was continuously perfused at a constant rate of 150 μl min−1.

K+-selective measurements

The technique used for the construction of double-barrelled ion-sensitive electrodes has been described previously (Leyssens et al. 1993a). The tip of the ion-selective barrel was filled with a short column of the ion-selective liquid K+exchanger (Corning 477317), and the rest of the barrel was backfilled with 1 mol l−1KCl. The reference barrel was backfilled with 1 mol l−1sodium acetate + 10 mmol l−1KCl. The filled electrodes were bevelled in a rotating polishing alumina solution. After bevelling, the ion-sensitive and reference barrels had tip resistances ranging from 50 to 100 GΩ and from 0.5 to 1 GΩ, respectively. Three tracings were recorded on a pen recorder (Sefram or Linseis): the potential of the reference barrel, the potential of the ion-selective barrel and the difference between them, representing the K+-sensitive signal.

The K+-sensitive signal was calibrated in a 143 mmol l−1ClRinger’s solution, in which the K+concentration was varied from 5 to 113 mmol l−1, using Na+as a substitute. Interference by Na+can be ignored (see Leyssens et al. 1993a). The measured intracellular ([K+]i) or luminal ([K+]l) K+‘signal’ was always expressed as the K+concentration that would be needed in a 143 mmol l−1ClRinger’s solution to result in the same reading of the K+-selective barrel. It should be kept in mind that the value for [K+]i should, in fact, be corrected if the activity coefficient in the cell was different from that in the bath. Once the electrode has impaled the cell, it should, however, be possible to observe changes in [K+]i without too much additional error. Other possible errors arising from tip and liquid junction potentials of the reference electrode have been discussed by Leyssens et al. (1993a). Since the calibration curve has a logarithmic scale, in the high concentration range (>100 mmol l−1), small changes in intracellular and luminal concentrations (i.e. less than 10 mmol l−1) correspond to minor changes in the K+-sensitive signal (i.e. 1–2 mV). On evaluating the results, one has to take into account that this approaches the limit of detection.

The ion-sensitive electrodes were accepted for use if a stable potential signal was obtained for both the ion-sensitive and the reference barrel, if the K+-sensitivity of the reference barrel was less than 1 mV and if the calibration curve could be fitted by a straight line with a slope of at least 48 mV per decade. Except when the effect of a certain test solution on the fluid secretion rate or on the electrical potentials or concentrations was not clearly reversible, the value of a specific electrophysiological variable, registered before and after ion substitution or drug application, was averaged and taken as the control value. In this way, possible time-dependent changes in potentials or concentrations were minimized.

Artificial salines

The different control solutions containing varying K+and Na+concentrations (see Results) were obtained by mixing a K+-containing, Na+-free and a Na+-containing, K+-free standard Ringer’s solution (2 mmol l−1CaCl2, 13 mmol l−1MgCl2, 12.1 mmol l−1Hepes, 2.8 mmol l−1alanine, 10.6 mmol l−1trehalose, 11.7 mmol l−1maltose, 139 mmol l−1glucose and 113 mmol l−1KCl or NaCl, respectively; pH was adjusted to 7.20 with KOH or NaOH, respectively; the osmolality of the solution was 375 mosmol kg−1H2O).

Solutions were freshly prepared each week, filtered through 0.45 μm Millipore filters and kept at 2°C until use.

In some experiments, the following alterations in ion composition were made: 6 mmol l−1BaCl2 was added to the bath solution; Clwas completely replaced by Br; Na+was completely replaced by N-methyl-D-glucamine (NMDG+, Sigma). On replacing NaCl by NMDG+, the alkaline solution was titrated with an appropriate amount of HCl for pH adjustment; as a result, the final Clconcentration remained virtually unchanged (i.e. 143 mmol l−1).

The following pharmacological substances were tested: dihydro-ouabain (Sigma), sodium orthovanadate (Janssen Chimica), bumetanide (Leo Pharmaceutical Products), Schering 28080 (Schering-Plough; solvent, 0.05% methanol and 0.05% dimethylsulphoxide without any effect on fluid secretion; unpublished results).

Statistics

Results are presented as mean values ± S.E.M. Statistical computations were made using Statview II (Abacus Concepts Inc., Berkeley, CA, USA, 1987). The fluid secretion measurements and electrophysiological measurements were evaluated using unpaired and paired two-tailed Student’s t-test, respectively, except when indicated differently. A value of P<0.05 was accepted as indicating statistical significance.

Effects of Ba2+

K+-selective measurements

Ba2+is an effective blocker of the K+channels in the basal membrane of the Malpighian tubule cells of Formica polyctena. It probably reduces an inward K+current and, because of the activity of the electrogenic apical H+pump, both apical and basal membranes hyperpolarize (Weltens et al. 1992). In the present study, we compared the effects of a change in the bath K+concentration ([K+]bl) on [K+]i in the absence and presence of 6 mmol l−1Ba2+. An example is shown in Fig. 1. In the absence of Ba2+, an increase in bath K+concentration ([K+]bl) from 5 to 113 mmol l−1resulted in a prompt depolarization of Vbl and a concomitant rise in [K+]i (see also Leyssens et al. 1993a).

Fig. 1.

Effect of a change in bath K+ concentration on the basal membrane potential (Vbl, upper trace) and the intracellular K+ concentration ([K+]i, lower trace) in the absence and presence of 6 mmol l−1 Ba2+. On changing the bath solution, electrical disturbances were recorded by the K+-selective (i.e. the most sensitive) barrel and these have been omitted from the figure for clarity.

Fig. 1.

Effect of a change in bath K+ concentration on the basal membrane potential (Vbl, upper trace) and the intracellular K+ concentration ([K+]i, lower trace) in the absence and presence of 6 mmol l−1 Ba2+. On changing the bath solution, electrical disturbances were recorded by the K+-selective (i.e. the most sensitive) barrel and these have been omitted from the figure for clarity.

When [K+]bl was decreased to 51 mmol l−1[K+]bl, Vbl hyperpolarized but [K+]i remained at the same value. On returning to 5 mmol l−1, Vbl and [K+]i changed: [K+]i decreased but to a slightly higher value than the control and Vbl was also slightly more negative than at the start of the experiment. In the presence of 6 mmol l−1Ba2+, the response of Vbl to an increase in [K+]bl was greatly reduced: Vbl depolarized by only 7 mV for an increase in [K+]bl from 5 to 113 mmol l−1. In contrast, the rise in [K+]i on increasing [K+]bl was comparable to the response of [K+]i to a change in [K+]bl in the absence of Ba2+.

In the experiment shown, the addition of 6 mmol l−1Ba2+itself caused a considerable decrease of [K+]i in both 5 and 113 mmol l−1[K+]bl. This type of behaviour was observed in two out of the four experiments. In the other two, [K+]i increased. On average, in both 5 and 113 mmol l−1K+, [K+]i was not altered significantly on adding Ba2+to the bath solution (Table 1). Furthermore, the summarized results demonstrate that, although the K+-sensitivity of Vbl was lost under Ba2+treatment, suggesting a blockage of basal K+channels, the rise in [K+]i on increasing [K+]bl was not affected.

Table 1.

Effect of a change in bath [K+] on the basal membrane potential and the intracellular K+ concentration in the presence and absence of 6 mmol l−1 Ba2+

Effect of a change in bath [K+] on the basal membrane potential and the intracellular K+ concentration in the presence and absence of 6 mmol l−1 Ba2+
Effect of a change in bath [K+] on the basal membrane potential and the intracellular K+ concentration in the presence and absence of 6 mmol l−1 Ba2+

Effects of substitution of Na+by NMDG+ Fluid secretion measurements

In order to detect any Na+-dependent transport system (the Na+site of the Na+/K+/2Clcotransporter, for instance, cannot transport large cations such as NMDG+, see Hedge and Palfrey, 1992), Na+was completely replaced by NMDG+. In 51 mmol l−1[K+]bl, there was no significant effect (Table 2A). In a low-K+solution (10 mmol l−1[K+]bl), the substitution of Na+by NMDG+caused a small but reversible inhibition of fluid secretion (Table 2A).

Table 2.

Effect of ion substitution and drugs on fluid secretion

Effect of ion substitution and drugs on fluid secretion
Effect of ion substitution and drugs on fluid secretion

Effects of substitution of Cl-by Br-Fluid secretion experiments

It is known that the Na+/K+/2Cland K+/Clcotransport systems have a different anion-dependence (Palfrey and Greengard, 1981; Ellory and Hall, 1988). When Clwas omitted from the bath solution and replaced by Br, fluid secretion rate was significantly stimulated in 51 mmol l−1[K+]bl but inhibited in 10 mmol l−1[K+]bl containing a high Na+concentration (Table 2A).

Effects of dihydro-ouabain

Fluid secretion experiments

As a high K+concentration is competitive with ouabain (see Baker and Willis, 1970), the effect of dihydro-ouabain (DHO) was tested in a low [K+]bl (5 mmol l−1). No significant effect of a high dose of dihydro-ouabain (10−3 mol l−1) was observed during the 30 min application of the drug (Table 2B). However, in this low [K+]bl, fluid secretion rate was very slow before DHO was applied (i.e. 76±19 pl min−1, N=8) and the time course was highly variable. Consequently, it was difficult to distinguish a small drug effect from the time-dependent changes.

Electrical potential difference measurements

The effect of dihydro-ouabain was also tested on Vbl (Fig. 2A): if dihydro-ouabain were to reduce [K+]i, a depolarization of Vbl would be expected. However, in 5 mmol l−1[K+]bl, ouabain treatment (10−3 mol l−1) for at least 10 min did not depolarize Vbl. If the putative pump were electrogenic, its effect on Vbl might be masked by the high K+permeability of the basal membrane. Therefore, the potential measurements were also performed in the presence of 6 mmol l−1Ba2+(Fig. 2B) (for theoretical considerations, see also Weltens et al. 1992). The addition of 6 mmol l−1Ba2+hyperpolarized Vbl. In the presence of Ba2+and following exposure to 10−3 mol l−1dihydro-ouabain, Vbl decreased slightly; but after wash-out, Vbl declined a further 1 mV, suggesting a small time-dependent decay of Vbl. After wash-out of Ba2+, Vbl depolarized to approximately its initial control value. The potential measurements are summarized in Table 3A. Significant effects of 10−3 mol l−1dihydro-ouabain on Vbl were detected neither in the absence nor in the presence of 6 mmol l−1Ba2+.

Table 3.

Effect of drugs on electrophysiological variables

Effect of drugs on electrophysiological variables
Effect of drugs on electrophysiological variables
Fig. 2.

Effect of 10−3 mol l−1 dihydro-ouabain (DHO) on Vbl in the absence (A) and presence (B) of 6 mmol l−1 Ba2+ (5 mmol l-1 [K+]bl).

Fig. 2.

Effect of 10−3 mol l−1 dihydro-ouabain (DHO) on Vbl in the absence (A) and presence (B) of 6 mmol l−1 Ba2+ (5 mmol l-1 [K+]bl).

Effects of Sch28080

Fluid secretion experiments

At 10−4 mol l−1, Sch28080 would only affect a K+/H+-ATPase, if present, or a V-type H+-ATPase and not the activity of a Na+/K+-ATPase (see Froissart et al. 1992). Its inhibitory action increases at lower bath K+concentrations (see Scott et al. 1987). In our hands, Sch28080 (10−4 mol l−1) had no effect on fluid secretion rate either in a high (51 mmol l−1) or in a low (10 mmol l−1) [K+]bl (Table 2B).

Effects of vanadate

Fluid secretion experiments

In 51 mmol l−1[K+]bl, 10−4 mol l−1vanadate, known to be an inhibitor of E1E2 (P-type) ATPases (i.e. the Na+/K+- and K+/H+-ATPase) (see Nechay et al. 1986), did not affect the fluid secretion rate; but at 10−3 mol l−1, fluid secretion was almost completely abolished within 40 min (Fig. 3A, Table 2B). In a high-K+, Na+-free solution, vanadate (10−3 mol l−1) caused a similar inhibitory effect on fluid secretion (Fig. 3B, Table 2B). There was no statistically significant difference between the inhibitory effects in 51 or 113 mmol l−1[K+]bl. In both cases, the effect of vanadate was reversible.

Fig. 3.

Effect of vanadate on the fluid secretion rate (A) in 51 mmol l−1 [K+]bl [at 10−4 (●) and 10−3 (e) mol l−1] and (B) in 113 mmol l−1 [K+]bl (Na+-free) (at 10−3 mol l−1) [K+]bl. Mean values ± S.E.M. *Statistically significant difference between the experimental relative fluid secretion rate and the rate in the control series at the same moment (unpaired Student’s t-test, P<0.05). There was no statistically significant difference between the inhibitory effect of vanadate in 51 and 113 mmol l−1 [K+]bl (unpaired Student’s t-test, P>0.05). (C) Effect of 10−3 mol l−1 vanadate on Vbl in the presence of 6 mmol l−1 Ba2+ (51 mmol l−1 [K+]bl). (D) Effect of 10−3 mol l−1 vanadate on Vbl and [K+]i and (E) on Vte and the luminal K+ concentration ([K+]l) in 51 mmol l−1 [K+]bl. The intracellular and luminal K+-selective measurements were performed on different tubules.

Fig. 3.

Effect of vanadate on the fluid secretion rate (A) in 51 mmol l−1 [K+]bl [at 10−4 (●) and 10−3 (e) mol l−1] and (B) in 113 mmol l−1 [K+]bl (Na+-free) (at 10−3 mol l−1) [K+]bl. Mean values ± S.E.M. *Statistically significant difference between the experimental relative fluid secretion rate and the rate in the control series at the same moment (unpaired Student’s t-test, P<0.05). There was no statistically significant difference between the inhibitory effect of vanadate in 51 and 113 mmol l−1 [K+]bl (unpaired Student’s t-test, P>0.05). (C) Effect of 10−3 mol l−1 vanadate on Vbl in the presence of 6 mmol l−1 Ba2+ (51 mmol l−1 [K+]bl). (D) Effect of 10−3 mol l−1 vanadate on Vbl and [K+]i and (E) on Vte and the luminal K+ concentration ([K+]l) in 51 mmol l−1 [K+]bl. The intracellular and luminal K+-selective measurements were performed on different tubules.

Electrical potential difference measurements

The effect of vanadate was tested on intracellular and luminal potentials in a different series of tubules (Table 3A). In 5 and 51 mmol l−1[K+]bl, at a concentration of 10−3 mol l−1, vanadate did not affect Vbl but significantly depolarized Vte; within 5 min, a steady state was reached. 10−4 mol l−1vanadate had no significant effect on Vte. The depolarizing effect of 10−3 mol l−1vanadate on Vte suggested the inhibition of an electrogenic transport system (for theoretical considerations, see also Weltens et al. 1992). Therefore, it was of interest to examine the effects of vanadate on the Ba2+-induced hyperpolarization of Vbl. A typical example is shown in Fig. 3C. Vanadate caused a depolarization (i.e. a reduction of the Ba2+-induced hyperpolarization) of Vbl within 5 min. The effect was statistically significant (Table 3A). The effects of vanadate on electrical potentials were not easily reversible within the experimental period (i.e. approximately 15 min wash-out).

K+-selective measurements

Since 10−3 mol l−1vanadate did not affect Vbl in the absence of Ba2+within 5 min, a drastic effect on [K+]i was not expected. This was verified in 51 mmol l−1[K+]bl. Examples of intracellular and luminal measurements are given in Fig. 3D,E. The addition of 10−3 mol l−1vanadate did not cause significant changes in Vbl or [K+]i. In the lumen of another tubule, the reversible response of Vte and [K+]l to a decrease in [K+]bl in control conditions was first confirmed. Upon administation of 10−3 mol l−1vanadate, Vte began to decrease within the first minute; after 6 min, Vte had decreased by 18 mV. Within the same period, [K+]l was not affected. The experimental data are summarized in Table 3B. There was no significant effect on [K+]i and [K+]l. A similar lack of effect on [K+]i (N=2) and [K+]l (N=3) was observed (not shown) in 5 mmol l−1[K+]bl.

Effects of bumetanide

Fluid secretion experiments

The loop diuretic bumetanide inhibits K+/Cland Na+/K+/2Clcotransport systems (see Ellory and Hall, 1988). A concentration of 10−5 mol l−1is usually sufficient for maximal inhibition of the Na+/K+/2Clcotransporter, but much higher concentrations are generally needed to affect the K+/Clcotransporter (see Ellory and Hall, 1988; Palfrey and O’Donnell, 1992). Bumetanide was tested in 5, 51 and 113 mmol l−1[K+]bl. In 51 mmol l−1[K+]bl, 10−4 mol l−1bumetanide significantly diminished fluid secretion rate; the application of 10−3 mol l−1almost completely abolished it (Fig. 4A, Table 2B). Only at 10−4 mol l−1was the effect reversible. In the lower [K+]bl (5 mmol l−1), bumetanide reversibly inhibited fluid secretion when applied at 10−5 mol l−1. The inhibitory effect of 10−4 mol l−1bumetanide was more pronounced and the fluid secretion did not recover within the 30 min wash-out period (Fig. 4B, Table 2B). In the high-K+, Na+-free solution, in contrast, bumetanide (10−4 mol l−1) had no significant effect (Fig. 4C, Table 2B).

Fig. 4.

Effect of bumetanide on the fluid secretion rate (A) in 51 mmol l−1 [K+]bl at 10−5 (●), 10−4 (●) and 10−3 (.△) mol l−1, (B) in 5 mmol l−1 [K+]bl at 10−5 (◯) and 10−4 (●) mol l−1 and (C) in 113 mmol l−1 [K+]bl (Na+-free) at 10−4 mol l−1. Mean values ± S.E.M. *Statistically significant difference between the experimental relative fluid secretion rate and the rate in the control series at the same moment (unpaired Student’s t-test, P<0.05). Effect of 10−4 mol l−1 bumetanide (D) on Vte and (E) on Vbl and [K+]i in 51 and 5 mmol l−1 [K+]bl.

Fig. 4.

Effect of bumetanide on the fluid secretion rate (A) in 51 mmol l−1 [K+]bl at 10−5 (●), 10−4 (●) and 10−3 (.△) mol l−1, (B) in 5 mmol l−1 [K+]bl at 10−5 (◯) and 10−4 (●) mol l−1 and (C) in 113 mmol l−1 [K+]bl (Na+-free) at 10−4 mol l−1. Mean values ± S.E.M. *Statistically significant difference between the experimental relative fluid secretion rate and the rate in the control series at the same moment (unpaired Student’s t-test, P<0.05). Effect of 10−4 mol l−1 bumetanide (D) on Vte and (E) on Vbl and [K+]i in 51 and 5 mmol l−1 [K+]bl.

Electrical potential measurements

Since bumetanide (10−4 mol l−1) diminished the fluid secretion rate in 51 and 5 mmol l−1[K+]bl, electrophysiological effects were tested in these bath solutions. As is shown in Fig. 4D, in 51 mmol l−1[K+]bl, Vte remained constant after the addition of 10−4 mol l−1bumetanide; in 5 mmol l−1[K+]bl, Vte was 4 mV higher than the control value. On average (see Table 3A), neither in 5 nor 51 mmol l−1[K+]bl could a significant effect on Vte be detected. In 2 out of the 11 experiments performed in 51 mmol l−1[K+]bl, the drug was applied for more than 20 min without any clear change in potential difference being observed.

K+-selective measurements

The effect of 10−4 mol l−1bumetanide was tested on Vbl and [K+]i in 5 and 51 mmol l−1[K+]bl on the same tubule (Fig. 4E). In control conditions, an increase in [K+]bl from 5 to 51 mmol l−1caused a depolarization of Vbl and an increase in [K+]i. On introduction of the bumetanide-containing bath solution in 51 mmol l−1[K+]bl, Vbl increased slightly and [K+]i remained virtually unaffected. When [K+]bl was lowered to 5 mmol l−1in the presence of the drug, Vbl hyperpolarized and [K+]i decreased. On returning to 51 mmol l−1[K+]bl in the presence of bumetanide, Vbl depolarized again, while [K+]i approached its previous value in 51 mmol l−1[K+]bl. Thus, bumetanide did not inhibit the rise of [K+]i on increasing [K+]bl. Washing out bumetanide in 5 mmol l−1[K+]bl did not change the Vbl and [K+]i values measured in the presence of bumetanide. The small (irreversible) changes in Vbl and [K+]i occurring in this experiment during administration of bumetanide were not systematically observed in other experiments. On average, there was no statistically significant effect of 10−4 mol l−1bumetanide on Vbl or [K+]i (Table 3B). The lack of an effect on Vbl was confirmed in eight experiments performed in 51 mmol l−1[K+]bl with 57 mmol l−1Clin the solution (Clsubstituted by citrate): even after 20 min of application of 10−3 mol l−1bumetanide, a concentration which effectively inhibited fluid secretion (Verhulst et al. 1988), Vbl did not change significantly (R. Weltens, personal communication).

Combined effect of Ba2+ and bumetanide

Both Ba2+and bumetanide are able to impair fluid secretion substantially, probably by affecting different mechanisms. Neither Ba2+nor bumetanide seemed to block a change in intracellular [K+] when applied separately. It was of interest, therefore, to observe their combined action on fluid secretion and K+-selective measurements.

Fluid secretion experiments

After three periods of 10 min in control Ringer containing 51 mmol l−1[K+]bl and 143 mmol l−1Cl, 6 mmol l−1Ba2+was added during the following three 10 min periods. This caused a decrease in fluid secretion to 18±10% (N=3) of that in the previous control period. Over the next 30 min, 10−4 mol l−1bumetanide was applied in the presence of Ba2+. Fluid secretion was completely inhibited within 20 min. The effect was only poorly reversible within the wash-out period (23±12% recovery after 30 min). In a control series, 6 mmol l−1Ba2+alone was added for 60 min (N=3). This treatment also resulted in complete inhibition of fluid secretion.

Electrical potential and K+-selective measurements

In another series of experiments, the effects of a change in [K+]bl on Vbl and [K+]i were examined in the same cell of an isolated tubule, first in the presence of 6 mmol l−1Ba2+and then in the presence of Ba2+together with 10−4 mol l−1bumetanide. The change in [K+]bl from 113 to 5 mmol l−1was carried out in both directions. The results are summarized in Table 4. Surprisingly a change in [K+]i was still observed when both bumetanide and Ba2+were present in the medium, although it was statistically smaller by 6±1.5 mmol l−1(paired t-test, N=6) than in the presence of Ba2+alone (P<0.01, paired t-test).

Table 4.

Effect of 10−4 mol l−1 bumetanide on the change in Vbl and [K+]i induced by an increase in bath K+ concentration in the presence of Ba2+

Effect of 10−4 mol l−1 bumetanide on the change in Vbl and [K+]i induced by an increase in bath K+ concentration in the presence of Ba2+
Effect of 10−4 mol l−1 bumetanide on the change in Vbl and [K+]i induced by an increase in bath K+ concentration in the presence of Ba2+

In this series of experiments, the tubules were less sensitive to Ba2+. Ba2+still caused some hyperpolarization of Vbl (compare Vbl with the control values in Table 1 and in Weltens et al. 1992), but the effect of [K+]bl on Vbl was only partly abolished by Ba2+(compare the effect in Tables 1 and 4).

Basal K+channels are not the only K+entry mechanism in Malpighian tubules of Formica polyctena

In the present study, we have shown that in the presence of 6 mmol l−1Ba2+in response to a rise in bath [K+], [K+]i increased to the same extent and with a similar time course as in the absence of Ba2+(Fig. 1 and Table 1). This result and previous findings in Formica polyctena (see Introduction for references) strongly argue for the role of other K+uptake systems, besides entry through conductive channels, in maintaining fluid secretion and in adapting the cytosolic K+concentration to the surrounding K+concentration.

Ba2+itself seemed to have contradictory effects on [K+]i: in some experiments, [K+]i increased in the presence of Ba2+; in others, it decreased. As Ba2+slows down both K+entry across the basal membrane and K+exit across the apical membrane (see Leyssens et al. 1993b), a difference in balance between these two effects might explain the rise in [K+]i in some tubules and the decrease in others.

Basal primary active K+-transporting ATPases

A Na+/K+-ATPase has been found in insect Malpighian tubules, but only in a few species does its activity affect fluid secretion significantly (for a review, see Nicolson, 1993).

In Malpighian tubules of Formica polyctena fluid secretion continued at a very high rate in a high-K+(113 mmol l−1) but completely Na+-free solution (Van Kerkhove et al. 1989), and omission of Na+from a 51 mmol l−1K+solution had no effect (Table 2A). The latter findings do not support a significant role for any Na+-dependent K+uptake mechanism in fluid secretion by Malpighian tubules of Formica polyctena, at least in the presence of relatively high K+concentrations.

In low K+concentrations, omission of Na+from the bath decreased fluid secretion (Table 2A), but the Na+/K+-ATPase inhibitor ouabain was without effect in the presence of 5 mmol l−1K+on either fluid secretion (Table 2B) or Vbl in the presence or absence of Ba2+(Fig. 2A,B, Table 3A). Thus, although the insensitivity of Na+/K+-ATPase to ouabain in Formica polyctena tubules, or even excretion and sequestration of ouabain (for a review, see Vaughan and Jungreis, 1977; Anstee and Bowler, 1979), cannot be excluded, our results suggest that, even in a low-K+medium, an active Na+/K+pump is of little importance for transepithelial K+transport in Malpighian tubules of Formica polyctena. The small Na+-dependence of the fluid secretion rate in low [K+]bl can probably be explained by the presence of another Na+-dependent transport system, i.e. a Na+/K+/2Clcotransport system (see below).

Another candidate for active K+uptake, the K+/H+-ATPase, if present, does not seem to play any role in fluid secretion: no effect of its blocker Sch28080 could be detected.

Since vanadate interacts with both Na+/K+- and K+/H+-ATPases, no significant effect on fluid secretion was expected on adding vanadate to the bath solution. Surprisingly, when applied at 10−3 mol l−1, vanadate strongly inhibited the fluid secretion rate, the inhibitory effect becoming statistically significant after 20 min (Fig. 3A). In both 5 and 51 mmol l−1[K+]bl, Vte significantly depolarized to a new steady-state level within 5 min of the addition of vanadate. The observation that Vbl, [K+]i and [K+]l remained unaffected (Fig. 3D and Table 3A,B) seems to indicate that K+(Cl?) and water transport were affected to the same extent: the speed of passage of K+and water was slowed down without changing the actual K+concentrations. This, in itself, seems to exclude an effect of vanadate on a Na+/K+-ATPase, as this would have altered the K+and Na+concentrations in the cell and, consequently, Vbl. Furthermore, since the inhibitory effects of vanadate on fluid secretion rate in Na+-containing (i.e. 51 mmol l−1[K+]bl) and Na+-free (i.e. 113 mmol l−1[K+]bl) conditions were comparable (Fig. 3A,B, Table 2B), its strong inhibitory effect was probably not due to an interaction with a hypothetical Na+/K+-ATPase. These results are different from the response of Malpighian tubules of Locusta migratoria, where both vanadate and ouabain caused a slow depolarization of both Vbl and Vap (Baldrick et al. 1988).

Because in Formica polyctena there was no evidence for the presence of a K+/H+-ATPase either (see above), we had to consider other possible sites of action. A complex indirect effect of vanadate was suggested for Malpighian tubules of Drosophila melanogaster (Bertram et al. 1991). A multitude of stimulatory and inhibitory intracellular mechanisms for vanadate have been reviewed by Chasteen (1983) and Nechay et al. (1986). The effects on the fluid secretion rate and electrophysiological variables of Formica polyctena Malpighian tubules were comparable to, and followed a similar time course to, the effects of the V-type ATPase inhibitors bafilomycin A1 and N-ethylmaleimide (NEM) reported by Weltens et al. (1992) on the same preparation. In the latter study, the reduction of the Ba2+-induced hyperpolarization was attributed to an electrogenic current generated by the apical H+-ATPase and blocked by bafilomycin A1 and NEM. Unlike ouabain (in 5 mmol l−1[K+]bl), vanadate (10−3 mol l−1in 51 mmol l−1[K+]bl) did significantly reduce the Ba2+-induced hyperpolarization of Vbl (Fig. 3C, Table 3A).

Reports of a direct effect of vanadate on the apical H+pump are controversial. According to Forgac (1989), vacuolar-type H+-ATPases are insensitive up to at least 1 mmol l−1. Chatterjee et al. (1992), however, described the presence of a novel V-type H+-ATPase in osteoclast plasma membrane vesicles with a unique pharmacology and with specific isoforms of two subunits in the catalytic portion of the enzyme; the H+-ATPase activity was completely inhibited by 10−3 mol l−1vanadate. 5×10−4 mol l−1vanadate caused 50% inhibition of the ATP-dependent proton uptake of isolated chromaffin granules and yeast vacuoles (Beltrán and Nelson, 1992). This is in line with a small effect (10–15% inhibition only) of a lower concentration of vanadate (10−4 mol l−1) on the cation-stimulated ATPase activity in purified goblet cell apical membranes of Manduca sexta (Wieczorek et al. 1986), later identified as a V-type H+-ATPase (Wieczorek et al. 1989). The effect was not significant and was considered to be negligible by the authors at that time, but might represent the threshold for vanadate-sensitivity of this ATPase.

In summary, no conclusive evidence was found for the existence of basal active K+uptake mechanisms in exchange for either Na+or H+. Their role in maintaining an asymmetric ion distribution across the basal membrane and a particular rate of fluid secretion is apparently minimal. The strong inhibitory effect of vanadate on fluid secretion can probably be explained by an effect on the apical H+pump.

Coupled entry mechanisms for K+, Cl-(and Na+) in the basal membrane

The existence of coupled entry mechanisms for K+, Na+and Clhas been investigated in Malpighian tubules of other insects, but only in a low bath K+concentration (for a review, see Nicolson, 1993). In Malpighian tubules of Formica polyctena a more extensive study was carried out to find evidence for an electroneutral coupled transport of K+in different bath K+(and Na+) concentrations.

In high-K+(113 mmol l−1) Na+-free conditions, K+entry via a Na+/K+/2Clcotransporter can be excluded. K+uptake via a K+/Clcotransporter is not very likely either, as bumetanide had no effect at a concentration as high as 10−4 mol l−1(Fig. 4C, Table 2B).

In 51 mmol l−1[K+]bl, the fluid secretion rate was not affected by omitting Na+from the bath solution (Table 2A). Although bumetanide binding to a Na+/K+/2Clcotransporter should be favoured in the 51 mmol l−1 K+, 62 mmol l−1 Na+bath solution (see Haas and Forbush, 1986), no change in fluid secretion rate was observed after the addition of 10−5 mol l−1bumetanide (Fig. 4A, Table 2B). These findings do not favour K+entry via a Na+/K+/2Clcotransporter. However, the significant reduction of the fluid secretion rate after the addition of 10−4 mol l−1bumetanide suggests that a large portion of the basal K+uptake may occur via a K+/Clcotransporter (Fig. 4A, Table 2B). Since, at this concentration, no effect of bumetanide was observed in 113 mmol l−1[K+]bl, specific effects are unlikely. The observation that Brhas a stimulatory effect on fluid secretion in 51 mmol l−1[K+]bl corroborates the hypothesis of a K+/Clcotransport system (Table 2A; see Ellory and Hall, 1988). At 10−3 mol l−1, the inhibitory effect of bumetanide was much more pronounced. However, at this high concentration, interaction with other Cl-dependent transport mechanisms cannot be excluded; in whole-cell recordings of the rat lacrimal gland, it was shown that 4X10−4 mol l−1bumetanide inhibited Clchannels, for instance (Evans et al. 1986).

In a low-K+, high-Na+medium (i.e. 10 or 5 mmol l−1[K+]bl), both Na+omission and Clsubstitution by Brcaused a reversible reduction of the secretion rate (Table 2A), and bumetanide clearly inhibited fluid secretion at 10−5 mol l−1(see Fig. 4B, Table 2B). These findings argue for the presence of a Na+/K+/2Clcotransporter (Ellory and Hall, 1988). Evidence for a Na+/K+/2Clcotransporter (Maddrell, 1969; O’Donnell and Maddrell, 1984; Hegarty et al. 1991) and/or Cl-dependent K+uptake (Wessing et al. 1987; Baldrick et al. 1988) has also been found for Malpighian tubules of other species.

That bumetanide (and/or another loop diuretic, furosemide, for other species) affects electroneutral transport mechanisms is confirmed by the lack of a significant effect on electrical potential differences (Table 3A,B) (cf. Vbl in Locusta migratoria, Baldrick et al. 1988, and Onymacris, Nicolson and Isaacson, 1987; Vap in Aedes aegypti, Hegarty et al. 1991). Long-term application, however (several minutes), might change the intracellular K+, Cland/or Na+concentrations and hence Vbl (cfr. Rhodnius prolixus, O’Donnell and Maddrell, 1984; Aedes aegypti, Hegarty et al. 1991). In Malpighian tubules of Formica polyctena, [K+]i did not change much, as Vbl did not alter.

An important observation was that bumetanide did not hamper the response of [K+]i to a change in [K+]bl from 5 to 51 mmol l−1(Fig. 4E, Table 3B). This means that other pathways, probably K+channels, can ensure basal K+uptake and adapt the intracellular K+content to the surrounding K+concentration. This finding indirectly points to the existence of an inwardly directed basal electrochemical K+gradient in the latter conditions.

To summarize, in a high-K+(113 mmol l−1) Na+-free solution, coupled entry of K+and Clseems to be of little importance. In 51 mmol l−1[K+]bl, a substantial portion of K+entry can occur via a K+/Clcotransporter. In low [K+]bl, a Na+/K+/2Clcotransporter seems to become important.

Combined effects of Ba2+and bumetanide

When both Ba2+and bumetanide were present, fluid secretion was completely blocked. It was possible, however, that this was due solely to the continued presence of Ba2+. Also, the effect of [K+]bl on [K+]i was reduced, but not abolished. This could mean either that the proposed model (see below) is still incomplete and that other K+ uptake mechanisms are present in the basal membrane or that Ba2+does slow down K+ entry, but does not completely block it (see, for instance, the difference in sensitivity to Ba2+of the series of tubules in Tables 1 and 4). Dijkstra et al. (1994) found that Ba2+always increased the basal membrane resistance, but that the hyperpolarization of Vbl was smaller in the ant tubules in their study than in those investigated by Weltens et al. (1992). It would be interesting to study the Ba2+-sensitivity of single K+ channels using the patch-clamp technique. The observation that Ba2+can completely block fluid secretion if applied for long enough can be explained by its concomitant hyperpolarizing effect on the apical membrane. This will severely reduce the turnover of the electrogenic H+ pump and the build-up of the proton concentration gradient that is needed to drive K+ into the lumen (see Leyssens et al. 1993b).

Transport model for transcellular K+transport in Malpighian tubules of Formica ployctena

Several hypothetical transport mechanisms (Fig. 10 in Leyssens et al. 1992) have now been investigated. Previous results have shown that, at the apical side, K+ extrusion into the lumen is realized via the combination of an electrogenic H+ pump and an electroneutral K+/H+ antiporter (see Weltens et al. 1992; Zhang et al. 1994; Leyssens et al. 1993a). The present study shows that the pathways for basal K+ entry seem to be conductive channels and secondary active cotransporters and that their relative importance varies depending on the bath K+ (and Na+) concentration. Primary active K+ uptake systems, i.e. a Na+/K+-or K+/H+-ATPase, if present, do not seem to contribute significantly to transepithelial transport or to the maintenance of intracellular K+ (and Na+) levels. Fig. 5 shows the proposed transport model for Malpighian tubules of Formica polyctena.

Fig. 5.

Model for transcellular K+ transport in 5 or 10 (A), 51 (B) and 113 mmol l−1 (C) [K+]bl, summarizing the results in the present study. It has previously been shown (1) that K+ transport increases (larger arrows) at a higher [K+]bl (Van Kerkhove et al. 1989); (2) that the apical and basal membrane potential differences decrease (smaller + and -signs in the figure) when [K+]bl increases (Leyssens et al. 1992); (3) that [K+]l and [K+]i increase (indicated by larger symbols in the figure) when [K+]bl increases (Leyssens et al. 1993a); and (4) that an H+ pump is present in the apical membrane in parallel with a K+/H+ antiporter (Weltens et al. 1992; Zhang et al. 1994; Leyssens et al. 1993a,b).

Fig. 5.

Model for transcellular K+ transport in 5 or 10 (A), 51 (B) and 113 mmol l−1 (C) [K+]bl, summarizing the results in the present study. It has previously been shown (1) that K+ transport increases (larger arrows) at a higher [K+]bl (Van Kerkhove et al. 1989); (2) that the apical and basal membrane potential differences decrease (smaller + and -signs in the figure) when [K+]bl increases (Leyssens et al. 1992); (3) that [K+]l and [K+]i increase (indicated by larger symbols in the figure) when [K+]bl increases (Leyssens et al. 1993a); and (4) that an H+ pump is present in the apical membrane in parallel with a K+/H+ antiporter (Weltens et al. 1992; Zhang et al. 1994; Leyssens et al. 1993a,b).

In high-K+, Na+-free conditions, a coupled entry of K+ and Cl seems to be of little importance. K+ uptake probably occurs via basal K+ channels, driven by a favourable electrochemical gradient (Fig. 5C) (see also Leyssens et al. 1993a). In more physiological conditions, i.e. 51 mmol l−1 [K+]bl, when the basal electrochemical K+ gradient is smaller (see Leyssens et al. 1993a), K+ entry via channels may become less important and a K+/Cl cotransport system probably accounts for a large portion of K+ uptake (Fig. 5B). In 5 or 10 mmol l−1 [K+]bl (and high-Na+ conditions), little K+ is available for transepithelial transport; the basal electrochemical K+ gradient is even smaller than in 51 mmol l−1 [K+]bl and possibly outwardly directed (see Leyssens et al. 1993a). The fluid secretion rate is low but still present. In this condition, a Na+/K+/2Cl cotransporter may take over a large part of the basal K+ uptake (Fig. 5A).

Furthermore, the different basal transport systems for K+ entry may function at different rates depending on the basal K+ (and Na+) concentration and may thereby determine the amount of K+ available for transport across the apical membrane via the K+/H+ antiporter. The rise in the intracellular [K+] in response to an increased basal K+ concentration could occur either via K+ channels or via a coupled K+/Cl entry mechanism.

Questions remaining to be answered

In a low-K+, high-Na+ solution, the intracellular K+ content is markedly reduced (see Leyssens et al. 1993a). Consequently, intracellular K+ has to be replaced by another cation, probably Na+. A Na+ conductance could not be detected (see Leyssens et al. 1992), but the uptake of Na+ might occur via a Na+/K+/2Cl cotransporter. The luminal K+ concentration also decreased when [K+]bl was lowered, but not to the same extent as [K+]i (see Leyssens et al. 1993a). This implies that K+ is still the major cation secreted. Consequently, although the apical K+/H+ antiporter may have some affinity for Na+ when intracellular Na+ levels rise, secretion of K+ still predominates. This means that net basal K+ uptake must exceed net Na+ uptake. Additional uptake via a K+/Cl cotransporter seems unlikely; it would need a very low intracellular Cl concentration. Preliminary results show that this is not the case (unpublished observations). As no evidence was found for a Na+/K+-ATPase, we have no indication of how intracellular [Na+] is regulated in our preparation. Theoretically, in view of the high intracellular pH found in Malpighian tubules of Formica polyctena (Zhang et al. 1994), a Na+/H+ antiporter in the basal membrane, working in reverse mode and unexpectedly functioning at an alkaline pH, might extrude Na+ in exchange for H+.

The transepithelial Cl pathway awaits further investigation. In high [K+]bl (113 mmol l−1), the electroneutral K+/Cl cotransporter did not seem to be functional and no relative Cl conductance was found. This suggests that Cl transport is primarily paracellular. The lack of a relative Cl conductance in the basal membrane (Leyssens et al. 1992) points to a paracellular pathway for Cl down an electrical gradient in 113 mmol l−1 [K+]bl. In 51 and 5 mmol l−1 [K+]bl, K+/Cl and Na+/K+/2Cl cotransporters, respectively, seem to be functional and at least part of the Cl transport may occur transcellularly; hence, Cl channels would be required to ensure Cl exit across the apical membrane. In a few experiments in 5 mmol l−1 [K+]bl, a small hyperpolarization of Vap was observed when bumetanide was applied (Fig. 4D) (cf. Rhodnius prolixus, O’Donnell and Maddrell, 1984; Locusta migratoria, Baldrick et al. 1988). If a Cl conductance were present in the apical membrane, this might be explained by a drop in intracellular Cl concentration due to inhibition of basal Cl influx. The existence of a transcellular Cl pathway was also suggested by a study on symmetrically perfused Formica polyctena tubules (Dijkstra et al. 1994). Cl channels in the apical membrane have been detected by a patch-clamp study in Aedes aegypti (Wright and Beyenbach, 1987) and in Drosophila melanogaster tubules (J. Dow and B. Harvey, personal communication). Apical Cl channels (as in rectal gland; Greger et al. 1989) and/or bumetanide-sensitive basal cotransporters (as in Aedes tubules, Hegarty et al. 1991) could be a site of regulation by hormones and second messengers to accelerate fluid secretion rate even at lower bath K+ concentrations.

Preliminary results obtained from intracellular and luminal ion-selective measurements in different bath K+ concentrations have demonstrated that the calculated gradients for Cl and K+ are consistent with the transport mechanisms for K+ and Cl proposed in the model.

The authors wish to thank Mr P. Pirotte for making the electrodes and performing part of the fluid secretion measurements, Mrs J. Vanderhallen for preparing the solutions, Mr R. Van Werde for help with the electronics, Mr W. Leyssens for administrative tasks, Ms K. Ungricht for typing the tables and Mr and Mrs Withofs for artwork. This work was supported by a grant from NFWO (Nationaal Fonds voor Wetenschappelijk Onderzoek, Belgium) and by a grant from the EC (European Community: SC1-CT90-0480).

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