Central Role Of the Apical Membrane H⁺-Atpase in Electrogenesis and Epithelial Transport in Malpighian Tubules

In the past, our laboratory has investigated the mechanisms of transepithelial electrolyte and fluid secretion in Malpighian tubules of the yellow fever mosquito Aedes aegypti by focusing on the mechanism and regulation of (i) Na⁺ and K⁺ entry across the basolateral membrane of principal cells, and (ii) Cl⁻ transport through the shunt pathway (Beyenbach, 1995). Greatly aided by the availability of bafilomycin, we have now begun to explore the role of the apical membrane of principal cells in transepithelial electrolyte secretion. This membrane is thought to be the site of a vacuolar H⁺-ATPase, which in other insect epithelia is believed to energize secondary active electrolyte transport across membranes and whole epithelia (Harvey and Wieczorek, 1997; Wieczorek et al., 1999).

In the present study, we have found a bafilomycin-sensitive electrogenic pump, presumably a V-type H⁺-ATPase, in the apical membrane of principal cells. The pump polarizes not only the apical membrane but also the basolateral membrane by virtue of electrical coupling through the shunt pathway. Electrical coupling allows the electrical driving forces at the basolateral and apical membranes to rise and fall in parallel so that cation entry across the basolateral membrane matches cation extrusion across the apical membrane.

Mosquitoes of the genus Aedes aegypti L. (NIH strain) were reared as described by Pannabecker et al. (1993). Adults were kept under a 12 h:12 h light:dark regimen and offered 3 % sucrose ad libitum. Only adult female mosquitoes between 3 and 6 days old were used for experiments. Malpighian tubules were dissected from cold-anaesthetized mosquitoes as described previously (Pannabecker et al., 1993).

Mosquito Ringer’s solution contained in (mmol l⁻¹): NaCl 150, KCl 3.4, CaCl₂ 1.7, MgCl₂ 0.6, NaHCO₃ 1.8, Hepes 25, and glucose 5. The pH was adjusted to 7.0 with NaOH. Bafilomycin A₁ was purchased from K. Altendorf (Osnabrück, Germany), dissolved in 100 % dimethyl sulfoxide (DMSO, Sigma) as 100 and 10 mmol l⁻¹ stock solutions, and stored at —20 °C until use. At the concentration of bafilomycin A₁ normally used (10⁻⁵ mol l⁻¹), the DMSO concentration was 0.01 % or 0.1 %, which had no effect on transepithelial fluid secretion in isolated Malpighian tubules.

Rates of transepithelial fluid secretion were measured in isolated Malpighian tubules by the method of Ramsay adapted by our laboratory (Plawner et al., 1991). Six consecutive measurements of cumulative volume over time (30 min) yielded the fluid secretion rate, first in the absence (control) and then in the presence of bafilomycin A₁. Thus, each tubule served as its own control. The time course of the effect of bafilomycin A₁ on fluid secretion was evaluated by measuring secreted volume every 2 min after addition of the antibiotic to the peritubular bath.

Isolated Malpighian tubules (0.3–0.6 mm long) were cannulated and perfused in vitro using methods described previously (Beyenbach and Frömter, 1985). The transepithelial voltage (V_t) was measured continuously via Ag/AgCl electrodes and 4 % Ringer/agar bridges positioned in the tubule lumen and the peritubular bath (ground). The basolateral membrane voltage of principal cells (V_bl) was measured using a conventional microelectrode filled with 850 mmol l⁻¹ potassium acetate and 150 mmol l⁻¹ potassium chloride. The apical membrane voltage of the principal cell (V_a) was calculated as the difference between V_t and V_bl. The transepithelial resistance, R_t, and the fractional resistance of the apical membrane, fR_a, were measured by cable analysis (Helman, 1972), except that we used a double-barrelled perfusion pipette to separate current injection into the tubule lumen through one barrel (100–500 nA, 300 ms) from the measurement of transepithelial voltage through the other (Beyenbach and Frömter, 1985).

Each perfused tubule served as its own control. The flow rate of Ringer’s solution through the peritubular bath was 0.5 ml min⁻¹ at steady state and increased briefly to 4 ml min⁻¹ when the peritubular Ringer’s solution was changed to include bafilomycin A₁. The tubule lumen was perfused with Ringer’s solution through the voltage barrel of a double-barrelled perfusion pipette at rates between 1 and 5 nl min⁻¹. An exchange pipette lodged in the voltage barrel served to change the luminal perfusion fluid rapidly.

Electrophysiological data are interpreted on the basis of the Ussing–Windhager model (1964) of epithelial ion transport (Fig. 1). The circuit model uses a minimum number of components to account for the transepithelial voltage V_t and resistance R_t: the transcellular resistance R_c, the shunt resistance R_sh and the electromotive force of the transcellular active transport pathway E_c. The requirement for energy input (ATP) confines the active transport pathway (consisting of E_c and R_c) to epithelial cells which, in the case of Aedes aegypti Malpighian tubules, are the principal cells. The shunt pathway is located outside principal cells (Fig. 1). It may be the paracellular pathway through septate junctions (Pannabecker et al., 1993) or a pathway through stellate cells (O’Donnell et al., 1998). The cations Na⁺ and K⁺ are secreted from the haemolymph to the tubule lumen via the active transport pathway, and Cl⁻ is secreted via the shunt pathway (Beyenbach, 1995).

The statistical significance of the data was evaluated with Student’s t-test for paired data (each tubule serving as its own control) and for unpaired data (comparisons of sample means with unequal variances). Values are presented as means ±S.E.M.

The inhibitory effects of bafilomycin A₁ (10⁻⁵ mol l⁻¹) on rates of transepithelial fluid secretion and on virtual short-circuit current (vI_sc) developed gradually (Fig. 2). The mean rate of transepithelial fluid secretion was 0.62±0.05 nl min⁻¹ in 13 Malpighian tubules, each serving as its own control. After adding bafilomycin A₁ to the peritubular Ringer, rates of fluid secretion began to decline and reached 60 % inhibition 40 min later. Complete (100 %) inhibition of transepithelial fluid secretion was not observed in the presence of 10⁻⁵ mol l⁻¹ bafilomycin A₁, but it was observed in some tubules at a concentration of 5×10⁻⁵ mol l⁻¹.

Inhibition of vI_sc roughly paralleled the inhibition of fluid secretion (Fig. 2). The virtual short-circuit current, a measure of active transport, was 6.44 μA cm⁻¹ under control conditions in nine Malpighian tubules (Table 1). Upon addition of bafilomycin A₁ (10⁻⁵ mol l⁻¹) to the peritubular Ringer, vI_sc started a progressive decline that reached 70 % inhibition 30 min later (Fig. 2). Short exposures to peritubular bafilomycin A₁ (5–10 min) were reversible; longer exposures (15 min and longer) were not (data not shown). ‘Complete inhibition’ of vI_sc was observed only after the sudden collapse of the transepithelial voltage and resistance that presumably reflects the toxic effects of bafilomycin A₁ (see Fig. 5).

Dose–response curves for bafilomycin A₁ were assessed between 10 and 40 min of exposure to the antibiotic, or shortly before voltages and resistances collapsed to values close to zero (see Fig. 5). Fig. 3 shows that peritubular bafilomycin A₁ had little or no effect on transepithelial fluid secretion and vI_sc at concentrations below 4×10⁻⁷ mol l⁻¹. Inhibitory effects became significant (P<0.05) at a concentration of 10⁻⁶ mol l⁻¹ and reached 50 % inhibition at a concentration of approximately 5×10⁻⁶ mol l⁻¹ (Fig. 3). The correlation coefficient for the effects on fluid secretion (r=0.97) was higher than that for the effects on virtual short-circuit current (r=0.84).

In vitro microperfusion of Malpighian tubules allows solution changes in the tubule lumen. The act of perfusing the lumen can be confirmed by visual inspection of fluid rising in the pipette holding the distal end of the tubule. Application of bafilomycin A₁ to the tubule lumen had no effect on transepithelial voltage and resistance (Fig. 4A). In contrast, application of bafilomycin A₁ from the peritubular side depolarized the transepithelial voltage while increasing transepithelial resistance (Fig. 4B). Similar results were obtained with three additional Malpighian tubules (not shown).

The effect of another inhibitor, dinitrophenol (DNP), on transport across Malpighian tubules showed similar polarity. Dinitrophenol added to peritubular Ringer is known to depolarize transepithelial voltage and to increase transepithelial resistance (Pannabecker et al., 1992). In the present study, we extended these findings to show that DNP, like bafilomycin A₁, had no effect when presented from the tubule lumen (Fig. 4C), but was effective when added to the peritubular bath (Fig. 4D).

Details of the inhibitory effects of bafilomycin A₁ on the electrophysiological correlates of transepithelial transport were studied in nine perfused Malpighian tubules (Table 1). Fig. 5 illustrates a representative experiment in which, under control conditions, the transepithelial voltage was 61.2 mV (lumen-positive), the apical membrane voltage was 142.8 mV (lumen-positive) and the basolateral membrane voltage was 81.6 mV (cell-negative). After the addition of bafilomycin A₁ to the peritubular bath, all voltages began to decline towards zero as the transepithelial resistance increased (Fig. 5). The virtual short-circuit current decreased in parallel, indicating the progressive inhibition of transcellular active transport. After exposure to bafilomycin A₁ for approximately 35 min, all voltages suddenly collapsed to zero in parallel with a drop in the transepithelial resistance. Accordingly, the effects of bafilomycin A₁ can be divided into two phases: (i) inhibitory effects that increase gradually over the course of 30 min or less, followed by (ii) an irreversible loss of all voltages and the transepithelial resistance.

The initial inhibitory effects of bafilomycin A₁ on transcellular transport (fall of vI_sc) are paralleled by an increase in the fractional resistance of the apical membrane and a decrease in the electrical diameter of the tubule (Fig. 5). These effects signal a shift of the transepithelial resistance towards the apical membrane, the site of the H⁺-ATPase. The irreversible collapse of voltage and resistance after 35 min of bafilomycin A₁ treatment probably reflects toxic effects. Bafilomycin A₁ inhibits not only the H⁺-ATPase in the apical membrane, but also in endosomes, vacuoles and other cell organelles. Inhibition of these H⁺-ATPases could contribute to the sudden collapse of voltages and resistances.

Observations made in all nine Malpighian tubules are summarized in Table 1, which shows data collected in the absence (control) and in the presence of bafilomycin A₁ shortly before voltages and resistance collapsed. Bafilomycin A₁ had significant effects on every electrophysiological indicator of transepithelial transport (Table 1). Virtual short-circuit current and all voltages (membrane and transepithelial) dropped to values less than 40 % of control values, while transepithelial resistance increased by 34 %. Measurements of fractional resistance indicated that, under control conditions, the apical membrane is the site of 27 % of the transcellular resistance, leaving 73 % to the basolateral membrane of principal cells (Table 1). In the presence of bafilomycin A₁, this distribution shifts, with 57 % of the transecellular resistance relocating at the apical membrane, leaving 43 % to the basolateral membrane. The shift of resistance to the apical membrane is confirmed in measurement of the core resistance, which increases by 48 % in the presence of bafilomycin A₁, indicating a decrease in the electrical diameter of the tubule (Table 1).

Dose-dependent or time-dependent effects of stimulators or inhibitors of transport can be used according to the protocol suggested by Yonath and Civan (1971) to estimate epithelial shunt conductance and the electromotive force of the active transport pathway. Such a plot of vI_scversus transepithelial conductance (as a function of time in bafilomycin A₁) is shown in Fig. 6, yielding a shunt conductance of 20.2 μS cm⁻¹ resistance 49.6 kΩ cm) and 87.8 mV as the electromotive force (E_c) of the active transport pathway. Table 2 summarizes similar Yonath–Civan estimates taken in all 14 tubule experiments. This summary yields a mean shunt resistance of 20.7 kΩ cm, a mean transcellular resistance of 17.6 kΩ cm and (shunt a mean electromotive force of the active transport pathway of 117.3 mV (Table 2; Fig. 7).

In vitro microperfusion is the method of choice for measuring electrophysiological correlates of ion transport across Malpighian tubules. The ratio of the transepithelial voltage to the transepithelial resistance yields the virtual short-circuit current, which is a measure of the rate of active transport of Na⁺ and K⁺ into the tubule lumen (equation 4). It is a virtual current because it is calculated from open-circuit measurements of transepithelial voltage and resistance rather than from real measurements of current at the transepithelial voltage-clamp of 0 mV. The virtual short-circuit current is an estimate of the maximum rate of active transport that would be possible if transepithelial electrolyte movement were not hindered by the resistance Cl⁻ encounters during transport through the shunt (Fig. 1; equation 4). The great advantage of measuring virtual short-circuit current is that effects on the active transport pathway can be observed while the experiment is in progress with greater resolution than that afforded by measurements of ion flux.

Vacuolar-type H⁺-ATPases are proton pumps that generate large voltages across the membranes in which they are found. The molecular constituents of the pump are a cytoplasmic catalytic V₁ complex that utilizes ATP and a transmembranous V_o complex that functions as a proton channel (Merzendorfer et al., 1997). The V₁ and V_o complexes may associate and dissociate, which is thought to be under genetic regulation at the transcriptional and post-transcriptional levels (Wieczorek et al., 1999).

Bafilomycin A₁ inhibits H⁺-ATPases by blocking the proton channel of the pump (Crider et al., 1994; Gill et al., 1998; Peng et al., 1999). In the present study, inhibition of the virtual short-circuit current was observed when bafilomycin A₁ was presented from the peritubular side but not when it was present on the luminal side (Fig. 4). Hence, bafilomycin A₁ does not block the proton channel from its opening into the tubule lumen, but gains access to its blocking site from the cytoplasm, after permeation of the basolateral membrane (Fig. 4).

The bafilomycin A₁ inhibition of both short circuit-current and transepithelial fluid secretion illustrates the central role of proton conduction through the H⁺-ATPase in powering salt and water transport across Malpighian tubules (Fig. 2). The short-circuit current reflects energy-dependent transport of Na⁺ and K⁺ through principal cells (Fig. 1). Na⁺ and K⁺ enter principal cells via channel- and carrier-mediated pathways (Sawyer and Beyenbach, 1985; Hegarty et al., 1991, 1992). Extrusion of Na⁺ and K⁺ from the cell is thought to be energized by the H⁺-ATPase located in the apical membrane, i.e. the luminal brush border (Fig. 7). According to the model advanced by Harvey, Wieczorek and coworkers, the H⁺-ATPase mediates the primary active transport step of translocating protons into the extracellular space, the tubule lumen in the case of Aedes aegypti Malpighian tubules (Harvey and Wieczorek, 1997; Wieczorek et al., 1991; Wieczorek, 1992; Lepier et al., 1994). The H⁺ electrochemical potential thus generated across the apical membrane powers secondary active transport of Na⁺ and K⁺ into the lumen via H⁺/Na⁺ and H⁺/K⁺ antiport with stoichiometries that have yet to be determined for Aedes aegypti Malpighian tubules. Interference in the active transport of protons, by depriving the H⁺-ATPase of ATP (treatment with dinitrophenol) or by blocking the proton channel of the H⁺-ATPase with bafilomycin A₁, inhibits (i) current generation at the apical membrane, (ii) secondary active transport of Na⁺ and K⁺ across the apical membrane, and (iii) intraepithelial current and, hence Cl⁻ transport, through the shunt (Fig. 1). The result is inhibition of transepithelial electrolyte and water transport.

The bafilomycin A₁ concentration that inhibits 50 % of the short-circuit current and transepithelial fluid secretion (IC₅₀), is 5 μmol l⁻¹, which is high in comparison with the nanomolar IC₅₀ concentrations measured in other cells (Kohn et al., 1993; Ohta et al., 1998). However, the IC₅₀ measured in the present study is based on bafilomycin A₁ concentrations in the extracellular medium and not on intracellular concentrations, where the antibiotic is active. Measuring a similar high IC₅₀ in Malpighian tubules of ants, Dijkstra et al. (1994) concluded that the H⁺-ATPase is only weakly bafilomycin-sensitive, which may also be true for Aedes aegypti Malpighian tubules. The need for high extracellular concentrations of bafilomycin A₁ in Malpighian tubules may also reflect poor permeation through plasma membranes, as suggested by the inability of bafilomycin A₁ to affect the virtual short-circuit current when the antibiotic is presented from the tubule lumen (Fig. 4). The unusual permeability properties of the brush-border apical membrane of Aedes aegypti Malpighian tubules are further documented by the failure of DNP to exert effects from the extracellular environment of the tubule lumen (Fig. 4). A low permeability of the apical membrane to bafilomycin was also noted in Malpighian tubules of the ant Formica polyctena (Weltens et al., 1992).

The inhibition of electrolyte and water transport is immediate and reversible when DNP is used to block ATP synthesis (Pannabecker et al., 1992). The inhibition by bafilomycin A₁ is slow, gradual and later irreversible (Figs 2, 5). The difference in time course could be related to differences in membrane permeability for bafilomycin A₁ and DNP, the latter being notably more permeable across the basolateral membrane. Significantly, the inhibition of the virtual short-circuit current by both DNP and bafilomycin A₁ is accompanied by a significant increase in transepithelial resistance that includes the increase in resistance of the apical membrane where the H⁺-ATPase is located (Fig. 5; Table 1). Blocking the proton channel of the H⁺-ATPase is expected to increase apical membrane resistance, which is reflected in an increase of apical membrane fractional resistance, fR_a (Table 1). Depriving the H⁺-ATPase of ATP should not, in itself, alter the conductance of the associated pathway. Nevertheless, the fact that similar inhibition is caused by agents differing widely in their mechanisms of action, channel block by bafilomycin A₁ and inhibition of ATP synthesis by DNP, documents the central role of the apical membrane V-ATPase in transepithelial electrolyte and fluid secretion in Malpighian tubules.

In a previous study, we used dinitrophenol (DNP) to inhibit transcellular transport and to increase transcellular resistance to such a degree that measurements of the transepithelial resistance approach the resistance of the shunt (Pannabecker et al., 1992). This strategy yielded a shunt resistance of 16.8 kΩ cm (Fig. 7). In the present study, we used bafilomycin A₁ and applied the method of Yonath and Civan (1971) to estimate a shunt resistance of 20.7 kΩ cm, which is not significantly different (P=0.41) from that measured with DNP (Pannabecker et al., 1992). In contrast, the estimate of the transcellular resistance by the method of Yonath and Civan was significantly (P=0.05) lower (17.6 kΩ cm) than R_c estimated using DNP (44.0 kΩ cm), suggesting that DNP blocks electroconductive pathways in addition to those blocked by bafilomycin (Fig. 7). The Yonath–Civan estimate of the electromotive force (117.3 mV) of the active transport pathway (E_c) was also significantly lower (P=0.04) than E_c estimated using the DNP method (190.0 mV) (Fig. 7). Again, DNP and bafilomycin may affect targets they do not share, hence the different estimates of R_c and E_c. However, in spite of these differences, the effects of DNP and bafilomycin on the active transport pathway are large enough to yield similar estimates of the shunt resistance (Fig. 7).

Dinitrophenol reduces transepithelial voltage and voltages across basolateral and apical membranes of principal cells to values close to or indistinguishable from zero (Pannabecker et al., 1992). The tubule does not appear to suffer adverse effects in spite of the abolition of active transport and all voltages. For one reason, the effects of DNP on voltage and resistance are fully reversible upon washout (Pannabecker et al., 1992, 1993). For another reason, receptor-mediated mechanisms, such as that of leucokinin-VIII, remain intact in the presence of DNP (Pannabecker et al., 1993).

The nullification of apical and basolateral membrane voltages in the presence of DNP without a trace of ionic diffusion potentials has been an enigma to us since we first made this observation (Pannabecker et al., 1992) because it implies the absence of Na⁺ and K⁺ diffusion potentials across the basolateral membrane of principal cells after DNP treatment, despite their unquestionable presence under control conditions (Sawyer and Beyenbach, 1985). Although the effects of bafilomycin A₁ are not as quick, they are qualitatively similar to those of DNP (Fig. 4). The similarities allude to mechanisms of electrogenesis that in H⁺-ATPase-supported systems are markedly different from those in Na⁺/K⁺-ATPase-supported systems. In cells supported largely by the Na⁺/K⁺-ATPase, the pump generates electrochemical potential differences for Na⁺ and K⁺ across plasma membranes that are subsequently used to produce membrane voltages (via K⁺ channels) and to power carriers (electrogenic or neutral) such as Na⁺/H⁺ exchange, Na⁺/glucose cotransport, Na⁺/Ca²⁺ exchange, etc. In cells supported largely by H⁺-ATPases, the pump generates electrochemical potential differences for H⁺ that can subsequently be used to power H⁺/Na⁺ exchange and other H⁺-based carriers (Wieczorek, 1992; Bertram and Wessing, 1994). More importantly, voltage is generated by the H⁺-ATPase itself in the process of transporting H⁺ alone. The H⁺-ATPase is therefore electrogenic, generating large voltages in those membranes where they are located.

Wieczorek et al. (1991) have long proposed that V-ATPases energize the membranes in which they are located. The present study extends this proposition, by way of electrical coupling, to membranes not containing V-ATPases. In the case of the Malpighian tubules of Aedes aegypti, the apical membrane is electrically coupled to the basolateral membrane via a shunt pathway that has a resistance similar to that of the transcellular pathway (Fig. 7). The situation is similar in the Malpighian tubules of ants, in which current generated by the pump in the apical membrane flows through the shunt pathway and returns to the apical membrane via the basolateral membrane to complete the intraepithelial current loop (Weltens et al., 1992).

The transepithelial voltage is therefore the product of current times shunt resistance and, hence, is lumen-positive. The basolateral membrane voltage is the product of the current and the basolateral membrane resistance plus or minus any electromotive force associated with that membrane. In Aedes aegypti Malpighian tubules, the principal determinant of cell-negative voltages across the basolateral membrane is the product of current and basolateral membrane resistance, with the current coming from the apical membrane through the shunt (Pannabecker et al., 1992; Beyenbach, 1995). Electrical coupling of apical and basolateral membranes explains the parallel changes in V_a, V_bl and V_t as they approach or reach zero in the presence of bafilomycin A₁ or DNP (Fig. 5). Moreover, V_t and V_bl are always less than V_a, which they track (Fig. 5), revealing the apical membrane H⁺-ATPase as the source of intraepithelial current that is central to transepithelial transport and to electrogenesis at both membranes of principal cells.

The authors thank the National Science Foundation for enabling this study (IBN 9604394).