Using a comparative approach, the mechanisms involved in maintenance of the transmembrane K+ activity gradients in the larval body-wall muscles of two insects, Phormia terraenovae (Diptera) and Spodoptera exigua (Lepidoptera), have been investigated. Double-barrelled K+-selective microelectrodes were used to obtain simultaneous measurements of intracellular K+ activity and membrane potential, whilst ordinary microelectrodes were used to monitor input resistance. By application of a variety of general metabolic blockers, the K+ gradients in both P. terraenovae and S. exigua muscle were found to be maintained, at least in part, by a metabolic component. Differences in sensitivity to dinitrophenol of the two insects suggested that the ATP-dependence of maintenance of the K+ gradient was significantly higher in P. terraenovae than in S. exigua. Vanadate sensitivity suggested that both insects possess P-type ATPases. The K+ activity gradient in P. terraenovae muscles was also found to be ouabain-sensitive, indicating the involvement of a Na+/K+-ATPase. In contrast, the K+ gradient in S. exigua muscles proved to be totally insensitive to ouabain but sensitive to amiloride.

Application of the H+/K+-ATPase-specific inhibitor SCH 28080 suggested the presence of an H+/K+ pump similar to the mammalian gastric H+/K+-ATPase in the lepidopteran muscles. P. terraenovae muscles, however, were found to be totally insensitive to this inhibitor. Using the anion (Cl-)-dependent transport inhibitors bumetanide and SITS (4-acetamide-4-isothiocyanostilbene-2,2-disulphonic acid), P. terraenovae muscles were shown not to possess a Cl--dependent K+ transport mechanism. In contrast, a bumetanide-sensitive K+/Cl- cotransporter was likely to be involved in maintenance of the K+ gradient in S. exigua muscle. An additional SITS-sensitive Cl-/HCO3- exchanger could also have some indirect involvement in K+ maintenance through regulation of the inward Cl- gradient. The results are integrated in two ionic models, one for each insect, which could account for the bulk of K+ transport in the body-wall muscles of these insects.

Throughout the insect orders, the extracellular ion levels and transmembrane ion concentration gradients have been shown to vary greatly (see Djamgoz, 1987, for a review). These differences in extracellular ion levels are of profound importance to insect muscles since, unlike the central nervous system, there is no significant ‘blood–brain barrier’ surrounding these cells (Treherne and Pichon, 1972; Treherne and Schofield, 1981; Treherne, 1985).

Earlier studies on dipteran muscle have shown the magnitudes and directions of the prevailing ion gradients to be ‘conventional’ (Gupta et al. 1980; Dawson and Djamgoz, 1988). Thus, the K+ gradient is outwardly directed, the equilibrium potential for K+, EK, being considerably more negative than resting membrane potential, Em. The Na+ gradient is inwardly directed, the Na+ equilibrium potential, ENa, being more positive than Em, and Cl- is in passive equilibrium across the membrane (Jan and Jan, 1976; Djamgoz and Dawson, 1988). Furthermore, studies of resting membrane electrogenesis in dipteran muscle have shown that Em can be fully accounted for in terms of the partial permeabilities of Na+ and K+ (Jan and Jan, 1976; Djamgoz and Dawson, 1988). Preliminary studies in larval and prepupal dipterans (Djamgoz, 1986; Henon and Ikeda, 1981) also suggested that a ouabain-sensitive metabolic mechanism, presumably a Na+/K+-ATPase, was present in skeletal muscle.

In contrast, the ion gradients in lepidopteran muscle are ‘unconventional’ (Djamgoz, 1986, 1987; Djamgoz and Dawson, 1989). Thus, although the K+ gradient is outwardly directed, EK is generally found to be more positive than Em. An extremely weak, sometimes reversed, Na+ gradient exists, ENa also being more positive than Em. The Cl- gradient is inwardly directed, resulting in the Cl- equilibrium potential, ECl, being more negative than Em. In the study of Dawson et al. (1989) on resting membrane electrogenesis in Chilo partellus muscle, 85 % of the Em could be accounted for by the K+, Na+ and Cl- permeabilities. The remaining 15 % was proposed to be maintained by an electrogenic metabolic pump, although the possible involvement of other mechanisms, e.g. H+ and Ca2+ gradients, was not dismissed. Preliminary evidence suggested that a ouabain-sensitive mechanism was not present in lepidopteran muscle (Rheuben, 1972; Dawson et al. 1989) and this would be consistent with the prevailing ionic gradients, which are not conducive to operation of a ‘classical’ Na+/K+-ATPase (Djamgoz, 1986).

Thus, to date, our understanding of the nature and function of metabolic ion pumps/exchangers in insect muscle is limited. Since K+ is the principal ion involved in resting membrane electrogenesis in both Diptera and Lepidoptera, we have used a comparative approach to investigate the transport mechanisms involved in maintenance of the K+ activity gradients in the ventral body-wall muscle fibres of the larvae of two insects, Phormia terraenovae (Diptera) and Spodoptera exigua (Lepidoptera). Double-barrelled K+-selective microelectrodes (KSMs) were used to obtain simultaneous measurements of membrane potential (Em) and intracellular K+ activity (aKi), whilst ordinary microelectrodes (OMs) were used to monitor input resistance (Ri), following application of several general metabolic blockers and ion transport inhibitors to the muscles of these two insects. A comparison of the possible ATP-dependence of maintenance of the K+ gradients in the two insects was obtained by the use of general metabolic blockers. By using specific inhibitors of P-type ATPases and Na+-dependent transport, it was possible to elucidate the nature of the ATP-dependent K+ transporters (pumps) in each insect. In addition, the possible role of anion (Cl-)-dependent transport mechanisms in K+ activity gradient maintenance was also assessed by the use of the inhibitors bumetanide and SITS. The results suggested models which could account for the bulk of K+ transport in the two insects.

The preparations

Experiments were carried out on final-instar larvae of the dipteran Phormia terrraenovae (Robineau-Desvoidy) (reared at Imperial College, Silwood Park) and the lepidopteran Spodoptera exigua (Hubner) (supplied by Zeneca, Jealott’s Hill). All preparations were dissected dorso-ventrally under saline. P. terraenovae preparations were bathed in Rice’s saline (Finlayson and Osborne, 1970) containing (in mmol l-1): NaCl, 173; KCl, 13.5; MgCl2, 1.0; NaHCO3, 1.2; NaH2PO4, 0.8; CaCl2, 0.9; glucose, 55.5; pH 7.0. S. exigua preparations were bathed in modified Weevers saline (Weevers, 1966) containing (in mmol l-1): NaCl, 12; KCl, 30.0; MgCl2, 18.0; NaHCO3, 1.5; NaH2PO4, 1.5; CaCl2.2H2O, 2.0; sucrose, 152; pH 6.8. Preparations were allowed to equilibrate at room temperature (18–20 °C) for 30 min prior to microelectrode impalements.

K+-selective microelectrodes

Double-barrelled K+-selective microelectrodes (KSMs) were constructed and used as described previously (Djamgoz and Dawson, 1986; Fitzgerald and Djamgoz, 1995). Lengths of borosilicate glass tubing (1.0 and 1.5 mm o.d.) were glued together with Araldite (Ciba-Geigy), twisted through 180 ° in the middle in a vertical electrode puller (Narashige) and then pulled. The inside of the larger (active) barrel was silanised by exposing the opening to dimethyldichlorosilane vapour (Sigma). The tip of this barrel was filled with a column (approximately 2 mm in length) of a K+-selective, low-impedance valinomycin-based neutral ion carrier (Fluka Chemicals) (Ammann et al. 1987). The active barrel was backfilled with 0.5 mmol l-1 KCl and the filamented ‘reference barrel’ was filled with 5 mmol l-1 LiCl. KSMs were calibrated in a series of saline solutions of varying K+ activity (using equimolar Na+ as the substitute). K+ activity refers to the effective concentration representing the freely mobile (i.e. ‘active’) K+ which is available to contribute to the generation of EK. Calibration curves had slopes of 50–58 mV for a tenfold change in K+ activity. K+ activity measurements were accepted only if the calibrations of the KSM before and after the experiment were within ±5 mV of each other at every point.

Ordinary microelectrodes

Ordinary microelectrodes were pulled from borosilicate glass capillaries (1 mm o.d., 0.6 mm i.d.) on a vertical puller. They were filled with 5 mmol l-1 LiCl and had tip resistances in the range 6–10 MΩ. The microelectrodes were connected to a microprobe system (WPI M701) with an inbuilt bridge circuit for current injection which was used for measurement of cell input resistance.

Inhibitor solutions

The following general metabolic blockers were used: dinitrophenol (DNP), potassium cyanide (KCN), sodium azide and rotenone. In addition, the P-type ATPase inhibitor vanadate, the Na+/K+-ATPase-specific inhibitor ouabain, the Na+-dependent transport inhibitor amiloride, and SCH 28080, an H+/K+-ATPase-specific inhibitor, were used. The anion (Cl-)-dependent transport inhibitors bumetanide and SITS were also used. Micromolar concentrations of bumetanide inhibit Na+/K+/Cl- cotransport, whilst at millimolar concentrations there is evidence that K+/Cl- cotransport is inhibited by this drug (Lauf, 1985; Bernhardt et al. 1988; Geck and Heinz, 1986). 4-Acetamide-4-isothiocyanostilbene-2,2-disulphonic acid (SITS) is an inhibitor of a number of Cl- transport mechanisms such as Cl-/HCO3- exchange (Thomas, 1984; Jennings, 1992), Na+-dependent Cl-/HCO3- exchange (Thomas, 1977; Boron and Russell, 1983) and Na+/HCO3- cotransport (Boron and Boulpaep, 1983; Deitmer and Szatkowski, 1990). Unless otherwise stated, all drugs were used at a concentration of 1 mmol l-1, dissolved in saline. Both SCH 28080 and bumetanide were dissolved in 1 % (v/v) ethanol saline solution. At 1 %, ethanol was found to have no effect on any of the parameters measured (data not shown). With the exception of SCH 28080, which was a gift from Schering Pharmaceuticals USA, all drugs were obtained from Sigma.

Inhibitor sensitivity tests

For each inhibitor, sensitivity tests were conducted to ensure that the addition of a particular inhibitor to the saline did not cause any interference with the response of the KSMs to K+ activity (aK) (Fitzgerald and Djamgoz, 1995). KSMs were first calibrated in ‘normal’ saline solutions and then in solutions containing the inhibitor. The concentration of inhibitor (interferent) was kept constant (the same as that used in experiments) throughout the series of calibration solutions. Between three and five electrodes were calibrated per inhibitor to obtain an average curve.

With the exception of DNP, none of the inhibitors used had a significant effect on the response of the KSMs to K+ activity. DNP, however, behaved as a ‘classical’ interferent, adding significantly to the K+ activity recorded by a KSM in solutions of low aK but having no effect on the K+ activity recorded in solutions of high (greater than 97 mmol l-1) aK. Correction of the recorded values of intracellular K+ activity (aKi) for DNP interference was achieved by the method outlined in Fitzgerald and Djamgoz (1995). These corrected values of aKi are referred to throughout the paper.

Evaluation of inhibitor effects

We have evaluated whether active K+ transport was inhibited by a given inhibitor as follows. As outlined in the Introduction, previous studies have shown that Em in dipteran muscle is maintained principally by a strong outward K+ gradient, which generates an EK considerably more negative than Em. An inward Na+ gradient generates an ENa more positive than Em, whilst Cl- is in passive equilibrium and thus makes no contribution to Em (Jan and Jan, 1976; Dawson and Djamgoz, 1988; Djamgoz and Dawson, 1988). Importantly, in all these studies, the partial permeabilities of K+ and Na+ were shown to account fully for resting membrane electrogenesis in dipteran body-wall muscle. Thus, any active K+ transporter present is unlikely to contribute any additional electrogenic component to the Em, but rather seems likely to be responsible for maintenance of the Na+ and K+ gradients. From this, it follows that inhibition of an active K+ transport mechanism will reduce aKi and hence the driving force for K+ efflux. As EK then depolarises, so Em will also depolarise. However, as Em depolarises, it is also possible that voltage-dependent K+ and/or Na+ channels, known to occur in insect muscle, will in turn open and exert their effects on Em (Salkoff and Wyman, 1983; Miller and Usherwood, 1990). In the following summary of inhibitor effects, we have therefore assumed that, if a significant decrease in aKi was due to inhibition of an active K+ transporter, the EK-Em difference would change in the same direction as the corresponding Em. However, if there was a change in membrane conductance, EK-Em could change differently depending on the relative changes in Na+ and K+ conductances, GNa and GK. Such changes in conductance could also be reflected as a change in Ri.

As with Phormia terraenovae, it has been assumed that inhibition of active K+ transport in Spodoptera exigua muscle will cause a significant reduction of aKi coupled with EK-Em remaining constant or changing in the same direction. Where EK-Em changes differently and/or Ri changes, we have assumed that ionic conductance has also changed. Interpretation of these changes in EK-Em and/or Ri is based on the findings of other studies which have shown that lepidopteran muscles have ‘unconventional’ transmembrane ion gradients. Thus, although an inward K+ gradient persists, EK tends, in general, to be more positive than the corresponding Em. The Na+ gradient is extremely weak, sometimes even reversed, with ENa being slightly more positive than Em. The Cl-gradient is inwardly directed, with ECl consistently found to be more negative than Em (Djamgoz and Dawson, 1989).

Experimental procedure Time-course experiments

KSMs were used to monitor the time courses of the changes in aKi and Em (and calculated EK) for up to 60 min during application of a given inhibitor. Initial recordings of Em and aKi were made from several cells bathed in normal saline over an initial period of 10 min, designated time T0. The bathing solution was then changed for a saline + inhibitor solution, and further recordings from individual cells were made repeatedly by impaling as many cells as possible for a period of up to 55–70 min. This latter period was subdivided into approximately 10 min intervals (designated time T10 to T60). In this way, 7–10 cells on average could be sampled during each time interval, in a given preparation. In control experiments, the same procedure was followed, except that, after T0, the solution was changed to normal saline, rather than saline + inhibitor. The total number of preparations was between three and eight per treatment per species. Values of EK were calculated from the Nernst equation. Data were pooled from the total number of preparations in a given treatment. Values of Em, aKi and EK-Em are presented in Tables 1 and 2 as means ± standard error of the mean (S.E.M.). All experiments were carried out at room temperature (18–20 °C).

Table 1.

Effects of general metabolic blockers and ion transport inhibitors on Em, aKi, EK-Em and Ri in Phormia terraenovae muscle

Effects of general metabolic blockers and ion transport inhibitors on Em, aKi, EK-Em and Ri in Phormia terraenovae muscle
Effects of general metabolic blockers and ion transport inhibitors on Em, aKi, EK-Em and Ri in Phormia terraenovae muscle
Table 2.

Effects of general metabolic blockers and ion transport inhibitors on Em, aKi, EK-Em and Ri in Spodoptera exigua muscle

Effects of general metabolic blockers and ion transport inhibitors on Em, aKi, EK-Em and Ri in Spodoptera exigua muscle
Effects of general metabolic blockers and ion transport inhibitors on Em, aKi, EK-Em and Ri in Spodoptera exigua muscle

Input resistance measurements

In order to determine whether any change in membrane conductance occurred following application of inhibitors, ordinary microelectrodes (OMs) and a bridge balance amplifier were used to record input resistance (Ri) from individual muscle cells in both insects. Initial recordings of Em and Ri were made from between seven and nine cells over a period of 10 min (time T0) in preparations bathed in normal saline. The bathing medium was then changed for a saline + inhibitor solution, and the preparation was left for a further 60 min, after which additional recordings of Em and Ri were made for approximately 10 min. The timing of this latter set of measurements was designated T60. Thus, values of both Em and Ri were obtained from cells both before (T0) and after (T60) application of an inhibitor. In control treatments, the solution change after T0 was to normal saline rather than to saline + inhibitor. Between four and seven preparations were used per treatment per insect. Consequently, between 19 and 51 cells were sampled in each time interval (T0 and T60). Importantly, the Em data obtained using OMs were not significantly different from the data obtained using KSMs (data not shown). Data were only accepted if there had been no change in the resistance of either the OM or the cell as a result of the impalement of the cell. Data are presented in Tables 1 and 2 as means ± S.E.M.

Since all the drugs used in this study would ultimately result in poisoning and cell death, we have assumed that changes in Ri due to uncoupling effects will be inherent. We have therefore neglected uncoupling in our subsequent discussion of inhibitor effects. Experiments were conducted at room temperature (18–20 °C).

Statistics

Student’s t-tests (using 95 % confidence limits) were carried out on values of aKi, Em, the EK-Em difference and Ri obtained at time T0versus T60. These essential quantitative data are presented in Tables 1 and 2 as means ± S.E.M., with the results of Student’s t-tests given as follows; *P<0.05, **P<0.01, ***P<0.001.

Effects of inhibitors on Phormia terraenovae muscles

Traces from a typical experiment showing recordings of aKi and Em in Phormia terraenovae muscles, and the effect of an inhibitor (1 mmol l-1 ouabain), are shown in Fig. 1. The upper trace shows the membrane potential (Em), whilst the lower trace shows the recording of intracellular K+ activity (aKi). Quantitative data for both species are summarised in Tables 1 and 2. The percentage changes in aKi and Em between T0 and T60 [(T60-T0)/T0] are presented below as ΔaKi and ΔEm, respectively. It should be noted that, where Em depolarised, Em has been presented as a positive value. Negative values of Em denote a hyperpolarisation of Em.

Fig. 1.

Chart recording from a typical experiment showing recordings of membrane potential (Em) and intracellular K+ activity (aKi) in Phormia terraenovae muscles, under control conditions (T0) and following application of 1 mmol l-1 ouabain for 30 min (T30) and 60 min (T60). The upper trace shows Em and the lower trace shows aKi. The moments of impalement and withdrawal of the electrode from the cell are indicated by the downward-and upward-pointing arrowheads, respectively.

Fig. 1.

Chart recording from a typical experiment showing recordings of membrane potential (Em) and intracellular K+ activity (aKi) in Phormia terraenovae muscles, under control conditions (T0) and following application of 1 mmol l-1 ouabain for 30 min (T30) and 60 min (T60). The upper trace shows Em and the lower trace shows aKi. The moments of impalement and withdrawal of the electrode from the cell are indicated by the downward-and upward-pointing arrowheads, respectively.

General metabolic blockers

Of the four metabolic blockers tested, only azide was ineffective on aKi (Table 1; see Fig. 2). DNP, rotenone and KCN all reduced aKi and caused depolarisation of Em. The protonophore DNP, which uncouples phosphorylation by dissipating the mitochondrial proton gradient, had by far the most marked effects on both aKiaKi -68 %, Fig. 3A) and EmEm 54 %). The EK-Em difference closely followed the changes in EK, which ultimately depolarised to a greater extent than Em. If aKi had decreased as the result of a selective increase in GK, a hyperpolarisation of Em would have been expected. To explain the lack of change in Ri, therefore, it follows that an equal and opposite increase in the conductance of some other ion, presumably Na+, must also have occurred. Consequently, the observed decrease in aKi was probably due largely to inhibition of an ATP-dependent K+ transporter, although possible involvement of ion channel(s) could not be ruled out.

Fig. 2.

Percentage reduction of intracellular K+ activity (aKi) following application of general metabolic blockers (solid bars), P-type ATPase/Na+-dependent transport inhibitors (hatched bars) and anion (Cl-)-dependent transport inhibitors (open bars) to Phormia terraenovae muscles for 60 min. The general blockers used were dinitrophenol (DNP), rotenone (ROT), potassium cyanide (KCN) and sodium azide (AZI). The P-type ATPase/Na+-dependent transport inhibitors used were ouabain (OUA), vanadate (VAN), SCH 28080 (SCH) and amiloride (AMI). The anion (Cl-)-dependent transport inhibitors used were bumetanide (BUM) and SITS. Error bars indicate S.E.M. DNP caused a 68 % inhibition over 60 min, and a 76 % inhibition between T10 and T60 (crosshatched bar), suggesting that a strongly ATP-dependent mechanism (ion pump) was involved in regulation of the K+ gradient. The marked inhibition caused by ouabain (61 %) and vanadate (51 %) indicated that a P-type Na+/K+-ATPase is the principal mechanism involved in maintenance of the K+ gradient in these muscles. The lack of significant inhibition by bumetanide and SITS showed that no Cl--dependent mechanism is involved. CON, control. Values of N are given in Table 1.

Fig. 2.

Percentage reduction of intracellular K+ activity (aKi) following application of general metabolic blockers (solid bars), P-type ATPase/Na+-dependent transport inhibitors (hatched bars) and anion (Cl-)-dependent transport inhibitors (open bars) to Phormia terraenovae muscles for 60 min. The general blockers used were dinitrophenol (DNP), rotenone (ROT), potassium cyanide (KCN) and sodium azide (AZI). The P-type ATPase/Na+-dependent transport inhibitors used were ouabain (OUA), vanadate (VAN), SCH 28080 (SCH) and amiloride (AMI). The anion (Cl-)-dependent transport inhibitors used were bumetanide (BUM) and SITS. Error bars indicate S.E.M. DNP caused a 68 % inhibition over 60 min, and a 76 % inhibition between T10 and T60 (crosshatched bar), suggesting that a strongly ATP-dependent mechanism (ion pump) was involved in regulation of the K+ gradient. The marked inhibition caused by ouabain (61 %) and vanadate (51 %) indicated that a P-type Na+/K+-ATPase is the principal mechanism involved in maintenance of the K+ gradient in these muscles. The lack of significant inhibition by bumetanide and SITS showed that no Cl--dependent mechanism is involved. CON, control. Values of N are given in Table 1.

Fig. 3.

Time courses showing the effects of (A) the general metabolic blocker dinitrophenol, (B) the Na+/K+-ATPase-specific inhibitor ouabain and (C) the P-type ATPase inhibitor vanadate on the intracellular K+ activity (aKi) in Phormia terraenovae muscles. Values are means ± S.E.M.; values of N are given in Table 1. The inhibitors were added at T0.

Fig. 3.

Time courses showing the effects of (A) the general metabolic blocker dinitrophenol, (B) the Na+/K+-ATPase-specific inhibitor ouabain and (C) the P-type ATPase inhibitor vanadate on the intracellular K+ activity (aKi) in Phormia terraenovae muscles. Values are means ± S.E.M.; values of N are given in Table 1. The inhibitors were added at T0.

Rotenone and KCN, both well-known inhibitors of the mitochondrial electron transport chain, were considerably less effective in reducing aKi (Fig. 2). In the case of rotenone, EK-Em increased significantly since Em depolarised to a greater extent than EKaKi -19 %, ΔEm 32 %). This implied either that GK had decreased or that there had been an increase in conductance of some other ion(s) with an opposing equilibrium potential (e.g. GNa). The observed significant decrease in Ri would suggest that the latter had occurred. On the basis of these assumptions, no more than 5 mV (the depolarisation of EK) of the observed depolarisation of Em could be due directly to inhibition of an ATP-dependent K+ pump. KCN was slightly less effective than rotenone in reducing aKiaKi -12 %). Nonetheless, EK-Em remained constant, suggesting that the effects were largely due to inhibition of a metabolically driven K+ transporter. The significant drop in Ri could possibly be explained by increases in voltage-dependent conductance as Em depolarised (ΔEm 17 %). Somewhat surprisingly, azide, which acts in exactly the same way as KCN, had no significant effect on aKiaKi -10 %) but depolarised EmEm 28 %), whilst the EK-Em difference increased owing to the greater depolarisation of Em over EK. This suggested that Em may have depolarised as a result of an increase in GNa and/or a decrease in GK. Such equal and opposite conductance changes would explain the lack of effect on Ri. The reasons for the apparent lack of effect of azide on ATP-dependent K+ transport, however, remain unclear (see Fitzgerald, 1992).

P-type ATPase/Na+-dependent transport inhibitors

Of this group of inhibitors, only ouabain and vanadate caused significant reductions of aKi (Fig. 2). Ouabain was the more potent in terms both of reducing aKiaKi -61 %) and depolarising EmEm 35 %). The EK-Em difference decreased significantly over 60 min, with EK depolarising to a greater extent than Em. In addition, Ri also fell significantly. Together, these results suggested that the drop in aKi occurred largely as a result of inhibition of a Na+/K+-ATPase, in accordance with the almost absolute specificity of ouabain for this pump (e.g. Glynn, 1985; Akera, 1981). Similar to the effect of DNP in these muscles (Fig. 3A), the time course for aKi (Fig. 3B) showed a slight increase in aKi over the first 10 min before a rapid drop in aKi during the remainder of the experimental period. Earlier studies have also shown that low concentrations of ouabain initially stimulate the Na+/K+ pump (see Bonting, 1970; Lee and Dagostino, 1982).

Vanadate was also highly effective in reducing aKiaKi -51 %, Figs 2, 3C) and depolarising EmEm 33 %). In this case, however, EK-Em remained constant throughout the experimental period and Ri was unaffected, suggesting that aKi decreased through direct inhibition of a K+ transporter. Since vanadate is an inhibitor of P-type ATPases (Pederson and Carafoli, 1987), this result supports the suggestion that ouabain inhibited a Na+/K+ pump in these muscles, the Na+/K+-ATPase being an example of a P-type pump. Additional conductance changes may therefore have occurred in addition to voltage-dependent effects to result in no net change in Ri.

Amiloride had no effect on aKi (Fig. 2) but did cause depolarisation of EmEm 11 %). In addition, Ri was found to decrease significantly, which implied that there may have been a change in GNa rather than GK. Whilst one possible action of amiloride on these muscles could be inhibition of a Na+/H+ exchanger, a major target of this drug (Kleyman and Cragoe, 1988), it should be noted that Na+/H+ exchange is generally found to be inactive in resting cells. Perhaps more likely, therefore, is the possibility that the high concentration of amiloride used may have exerted ‘non-specific’ effects on a number of Na+-dependent transport processes, leading to a net increase in conductance (decrease in Ri) and depolarisation of Em.

When the H+/K+-ATPase-specific inhibitor SCH 28080 was applied to Phormia terraenovae muscles, no significant change was observed in aKiaKi -9 %, Fig. 2), although Em hyperpolarised significantly (ΔEm -8 %) after 60 min. Nonetheless, our results provided no evidence to suggest that SCH 28080 inhibited a K+ pump in Phormia terraenovae muscles.

Anion (Cl-)-dependent transport inhibitors

Consistent with earlier studies on dipteran muscle which have shown that Cl- is passively distributed across the membrane with EClEm (Jan and Jan, 1976; Dawson and Djamgoz, 1988), neither bumetanide (1 mmol l-1) nor SITS (1 mmol l-1) was found to exert any significant effects on the parameters recorded in Phormia terraenovae muscles (Fig. 2; Table 1). These results clearly demonstrated the complete lack of effect of Cl--dependent transport inhibitors against aKi (and therefore the K+ gradient) and Em.

Effects of inhibitors on Spodoptera exigua

General metabolic blockers

With the exception of DNP, the general blockers tested in Spodoptera exigua all caused significant reduction of aKi, and they all caused significant depolarisation of Em (Table 2; Fig. 4). Rotenone was found to be the most potent inhibitor (ΔaKi -38 %, ΔEm 28 %). Since EK-Em remained constant throughout treatment, aK presumably decreased as a result of inhibition of an ATP-dependent K transport mechanism rather than because of an increase in GK.

Fig. 4.

Percentage reduction of aKi in Spodoptera exigua muscle following application of the general metabolic blockers (solid bars) rotenone (ROT), cyanide (KCN), azide (AZI) and dinitrophenol (DNP), and the P-type ATPase/Na+-dependent transport inhibitors (hatched bars) ouabain (OUA), vanadate (VAN), SCH 28080 (SCH) and amiloride (AMI). The anion (Cl-)-dependent transport inhibitors used were bumetanide at two concentrations (BUM) and SITS (open bars). With the exception of DNP, all the general metabolic blockers caused significant inhibition of aKi over 60 min, although only the effects of rotenone and KCN could be attributed directly to inhibition of a K+ pump. The marked decrease in aKi observed in the presence of vanadate (35 %) and SCH 28080 (38 %) suggested that a P-type H+/K+ ATPase is involved in maintenance of the K+ gradient in the muscles of this insect. DNP caused an initial increase in aKi between T0 and T10 min followed by a steady decrease. However, the essential result is unaffected even when comparing the change in aKi between T0 and T60 (solid bar) versus the change between T10 and T60 (crosshatched bar). Bumetanide (1 mmol l-1) caused a significant reduction of aKi (36 %, P<0.001). SITS also caused a significant inhibition of 23 % (P<0.05). CON, control. Error bars indicate S.E.M.; values of N are given in Table 2.

Fig. 4.

Percentage reduction of aKi in Spodoptera exigua muscle following application of the general metabolic blockers (solid bars) rotenone (ROT), cyanide (KCN), azide (AZI) and dinitrophenol (DNP), and the P-type ATPase/Na+-dependent transport inhibitors (hatched bars) ouabain (OUA), vanadate (VAN), SCH 28080 (SCH) and amiloride (AMI). The anion (Cl-)-dependent transport inhibitors used were bumetanide at two concentrations (BUM) and SITS (open bars). With the exception of DNP, all the general metabolic blockers caused significant inhibition of aKi over 60 min, although only the effects of rotenone and KCN could be attributed directly to inhibition of a K+ pump. The marked decrease in aKi observed in the presence of vanadate (35 %) and SCH 28080 (38 %) suggested that a P-type H+/K+ ATPase is involved in maintenance of the K+ gradient in the muscles of this insect. DNP caused an initial increase in aKi between T0 and T10 min followed by a steady decrease. However, the essential result is unaffected even when comparing the change in aKi between T0 and T60 (solid bar) versus the change between T10 and T60 (crosshatched bar). Bumetanide (1 mmol l-1) caused a significant reduction of aKi (36 %, P<0.001). SITS also caused a significant inhibition of 23 % (P<0.05). CON, control. Error bars indicate S.E.M.; values of N are given in Table 2.

KCN also reduced aKiaKi -32 %, Fig. 4) with concurrent depolarisation of EmEm 26 %). During this time, the EK-Em difference and Ri exhibited no significant changes. Thus, KCN also apparently reduced aKi and depolarised Em by direct inhibition of cell metabolism and hence by inhibiting an ATP-dependent K+ transport mechanism.

In the presence of azide, the effects on aKiaKi -32 %) and EmEm 16 %) were accompanied by a significant decrease in Ri. If GK had increased selectively, EK-Em might have been expected to decrease as Em depolarised (EK being more positive than Em). However, since the opposite was observed, the main conductance increase must have been due to other ion(s), e.g. GNa. Nonetheless, aKi apparently decreased as a result of inhibition of a metabolic K+ pump.

DNP, however, appeared to have a very different effect on aKi from the other general blockers (Fig. 5A). A marked increase in aKi was observed during the first 10 min, followed by a steady decline over the remaining period. Even after 60 min, aKi had not regained its initial value (ΔaKi 15 %). Meanwhile, Em depolarised steadily throughout DNP application (ΔEm 21 %). Although Em depolarised by 2 mV during the first 10 min, contrary to the 5 mV hyperpolarisation of Em predicted for this increase in aKi, the difference between the real and predicted change was not significant. It is possible, therefore, that the increase in aKi at T10 was real and not due to an artefact. The effects on Em and EK from T10 onwards were reminiscent of pump inhibition, i.e. EK and Em changing in the same direction. It is possible, therefore, that DNP caused initial stimulation of a K+ pump, followed by inhibition. As outlined below, such an effect could be due to the action of DNP as a protonophore.

Fig. 5.

Time courses showing the effects of (A) the general metabolic blocker dinitrophenol, (B) the Na+/K+-ATPase-specific inhibitor ouabain and (C) the P-type ATPase inhibitor vanadate on the intracellular K+ activity (aKi) in Spodoptera exigua muscles. Values are means ± S.E.M.; values of N are given in Table 2. Inhibitors were added at time T0.

Fig. 5.

Time courses showing the effects of (A) the general metabolic blocker dinitrophenol, (B) the Na+/K+-ATPase-specific inhibitor ouabain and (C) the P-type ATPase inhibitor vanadate on the intracellular K+ activity (aKi) in Spodoptera exigua muscles. Values are means ± S.E.M.; values of N are given in Table 2. Inhibitors were added at time T0.

P-type ATPase/Na+-dependent transport inhibitors

Treatment with three of the four inhibitors caused significant reductions of aKi with concurrent depolarisation of Em (Fig. 4). However, in this case the exception was ouabain, which had no significant effect on either aKi or Em in these muscles. The EK-Em difference and Ri also remained constant. Thus, ouabain was found to be totally ineffective as an inhibitor of K+ transport in this insect (Fig. 5B).

Vanadate, in contrast, caused a highly significant drop in aKiaKi -35 %) and depolarisation of EmEm 26 %) (Figs 4, 5C). As in Phormia terraenovae (Table 1), the time courses for both aKi and Em were very similar, and the EK-Em difference consequently maintained a steady level throughout the experimental period. Hence, the decrease in aKi was presumably due to inhibition of a K+ pump. Ri, however, fell significantly, suggesting that vanadate may also have affected membrane conductance (possibly voltage-dependent).

Similarly, the application of the H+/K+-ATPase-specific inhibitor SCH 28080 also caused a significant decrease in aKiaKi -38 %) and depolarisation of EmEm 21 %). These effects were comparable to those of ouabain on aKi in Phormia terraenovae muscle. Ri and the EK-Em difference also decreased significantly, suggesting that there had been a net increase in membrane conductance. However, since the change in EK-Em was only very slight (3 mV), it seems most likely that the majority of the fall in aKi was due to inhibition of a K+ pump. The decrease in Ri might then result from a voltage-dependent increase in conductance involving GK and possibly some other ion(s) (Salkoff and Wyman, 1983; Miller and Usherwood, 1990; Ramaswami and Tanouye, 1989).

Amiloride-treatment of Spodoptera exigua muscles also had a pronounced effect on both aKiaKi -31 %, Fig. 4) and EmEm 17 %). EK-Em remained constant throughout the experimental period and there was no change in Ri. Thus, amiloride was also presumed to reduce aKi by inhibition of active K+ transport.

Anion (Cl-)-dependent transport inhibitors

In lepidopteran muscles, unlike dipteran muscles, there is evidence that Cl- makes a significant contribution to Em, there being a strong inward Cl- gradient associated with these cells (Dawson et al. 1989). Thus, a Cl--dependent mechanism utilising Cl- influx could possibly be involved in maintenance of aKi in Spodoptera exigua muscles. Of the anion-dependent transport inhibitor ‘treatments’ tested on these muscles, two (1 mmol l-1 bumetanide and SITS), caused significant changes in aKi after 60 min.

Treatment of Spodoptera exigua muscles with a low concentration of bumetanide (1 μmol l-1) was ineffective in reducing aKiaKi -13 %; Fig. 4). Neither Ri nor EmEm 4 %) changed, although the absolute difference EK-Em did change significantly, suggesting that there may have been some effect on membrane conductance.

In contrast, the application of 1 mmol l-1 bumetanide caused a substantial decrease in aKiaKi -36 %; Fig. 4) and depolarisation of EmEm 24 %). Although Ri decreased significantly, suggesting that membrane conductance had increased, EK-Em did not change after 60 min. This latter result implied that no significant change had occurred in GK. Presumably, therefore, 1 mmol l-1 bumetanide exerted its effects on the K+ gradient via inhibition of a Cl--dependent K+ transporter. The decrease in Ri might then be explained as follows: (1) an increase in some voltage-dependent conductance resulting from depolarisation of Em; and (2) an increase in GCl could also have been involved, although there would also need to be an increase in conductance of some other ion(s), e.g. Na+, H+, in order for Em to depolarise.

Treatment with 1 mmol l-1 SITS also caused a significant decrease in aKiaKi -23 %). Meanwhile, Ri decreased significantly, as did the absolute difference EK-Em. Both results would suggest that membrane conductance, possibly involving GK, had increased, although there was no significant change in EmEm 2 %).

The maintenance of transmembrane ion gradients can be achieved by means of two broad classes of mechanisms as follows. (1) Primary active transport, in which a primary energy source, such as energy derived from hydrolysis of adenosine triphosphate (ATP), is used to move ions against their electrochemical gradients. These mechanisms are generally referred to as metabolic ion pumps, e.g. the Na+/K+-ATPase. (2) Secondary active transport, in which the movement of one ion against its electrochemical gradient is coupled to the movement of another ion down its electrochemical gradient, such that the total free energy is negative (e.g. Na+/Ca2+ exchanger, Na+/K+/Cl- cotransporter). Either type of mechanism can be ‘electrogenic’ (i.e. transfer a net quantity of electrical charge) and may directly generate a portion of the resting membrane potential Em in a given cell.

In the present study, we have investigated the mechanisms involved in maintenance of the K+ activity gradient in the body-wall muscles of two insects with very different transmembrane ionic gradients. Using a comparative approach, we have elucidated the principal active transport mechanisms involved in maintenance of the K+ activity gradient in the muscles of both insects.

Maintenance of the K+ activity gradient in Phormia terraenovae

Relatively few data are available for the role of metabolic ion pumps in dipteran muscle. However, preliminary studies in the body-wall muscles of larval and prepupal Calliphora erythrocephala (Djamgoz, 1986) showed that application of either DNP or ouabain (1 mmol l-1) caused a gradual depolarisation (by 20–60 %) of Em over a period of up to 3 h. In the same study, cooling the preparation from 18 to 5 °C caused Em to depolarise by 10 mV in 1 min. Henon and Ikeda (1981) obtained essentially the same result in Drosophila melanogaster flight muscles, where addition of 1 mmol l-1 ouabain caused a peak depolarisation of 19 mV after 10–15 min. Studies in dictyopteran and orthopteran muscle, which have similar transmembrane ion gradients, have also shown that application of metabolic blockers, including ouabain, causes gradual depolarisation of Em over 1–3 h (Wareham et al. 1974; Leech, 1986; Djamgoz, 1986). Thus, although no data exist for the effects of metabolic blockers on aKi, these preliminary studies suggested that a DNP-and ouabain-sensitive K+ pump, presumably a Na+/K+-ATPase, could be present in dipteran muscle.

In Phormia terraenovae muscle, the application of the general metabolic blockers DNP, KCN and rotenone caused a significant reduction of aKi and a depolarisation of Em (Table 1), which could be attributed to inhibition of an ATP-dependent mechanism. Of these drugs, DNP was by far the most potent, causing a 68 % reduction in aKi over 60 min, suggesting the presence of a strongly ATP-dependent K+ pump in these muscles. Similar effects, which could also be attributed to inhibition of a K+ pump, were observed following application of the P-type ATPase inhibitor vanadate (ΔaKi -51 %) and the Na+/K+-ATPase-specific inhibitor ouabain (ΔaKi -61 %). In agreement with earlier studies, the sensitivity of aKi to these specific inhibitors suggested that a Na+/K+-ATPase is responsible for the bulk of ATP-dependent K+ transport in these muscles. Furthermore, this conclusion is consistent with studies of resting membrane electrogenesis in dipteran muscles, which have shown that Em can be fully accounted for by the partial permeabilities of K+ and Na+, the K+ and Na+ gradients presumably being maintained by the Na+/K+-ATPase. In addition, the characterisation and expression of a ouabain-sensitive α-subunit from Drosophila melanogaster flight and tubular muscles (Lebovitz et al. 1989) has provided direct evidence for the presence of a Na+/K+-ATPase in dipteran muscle.

Consistent with the lack of a Cl- gradient in dipteran muscle, no evidence for the involvement of a Cl--dependent K+ transport mechanism in K+ maintenance was obtained using bumetanide and SITS. We conclude, therefore, that a vanadate-and ouabain-sensitive Na+/K+-ATPase is the principal mechanism involved in maintenance of the K+ gradient in Phormia terraenovae muscle.

Maintenance of the K+ activity gradient in Spodoptera exigua

In contrast to the situation in Diptera, previous studies have provided some evidence to suggest that a metabolic mechanism, possibly an ion pump, may contribute directly to Em in lepidopteran muscle. As mentioned previously, Dawson et al. (1989) found that resting membrane electrogenesis in Chilo partellus skeletal muscle could not be fully accounted for by the partial permeabilities of K+, Na+ and Cl-, and some 15 % of Em remained unaccounted for. In other studies, it has also been shown that application of DNP to lepidopteran skeletal muscle induces a depolarisation of Em, albeit apparently without a change in intracellular [K+] ([K+]i) (Rheuben, 1972; Piek et al. 1973). Unlike dipteran muscle, for Lepidoptera, Rheuben (1972) noted that ouabain was ineffective on Em of the subalar muscles of adult Antherea polyphemus, while Dawson et al. (1989) also found that ouabain applied for up to 5 min had no effect on the Em of Chilo partellus muscle and that Em was constant throughout the [K+]o series applied (1–42 mmol l-1). Thus, in contrast to previous suggestions by Akera (1971), Akera et al. (1974) and Baker and Willis (1970), the apparent insensitivity of lepidopteran muscle to ouabain may not be due to the relatively high concentrations of external K+ present in the haemolymph (23–54 mmol l-1; Djamgoz, 1986). A ouabain-insensitive metabolic pump could therefore be involved in maintenance of Em in Lepidoptera.

General metabolic blockers

In comparison with Phormia terraenovae, the application of the general metabolic blockers rotenone, KCN and azide to Spodoptera exigua muscles caused considerably less reduction of aKiaKi −32 % to −38 %) which could be attributed directly to inhibition of a metabolic K+ pump. Importantly, DNP, the most potent inhibitor in P. terraenovae, was largely ineffective in S. exigua. This effect could be due to the action of DNP as a protonophore. Intracellular acidification following DNP application has been reported in a number of cells, including the Malpighian tubules of the ant Formica polyctena (Dijkstra et al. 1994; Leyssens et al. 1993; McLaughlin and Dilger, 1980). Briefly, neutral DNP molecules entering a cell return to the bath side as DNP and DNP-, leaving a proton inside the cell. Since the cell has a limited volume, the uptake of protons can alter intracellular pH, depending on the buffering capacity of the cell (McLaughlin and Dilger, 1980). DNP could therefore have caused an increase in intracellular H+ activity (aHi), which would necessitate the active extrusion of protons in order to maintain the pH balance of the cell. We have suggested that an H+/K+-ATPase is active in these muscles (see below) and an initial increase in [H+]i might stimulate this pump to remove H+ from the cell and, in so doing, cause aKi to increase.

Although we have shown previously that DNP ‘interferes’ with K+ activity recordings, this is unlikely to affect intracellular measurements (Fitzgerald and Djamgoz, 1995). Nevertheless, DNP-induced poisoning of the cells could initially generate an unknown ‘intracellular interferent’. Despite this possibility, however, the essential result is unaffected even when comparing the change in aKi between T10 and T60 min (see Fig. 4), i.e. DNP has little overall effect on aKi in S. exigua compared with P. terraenovae (see Fig. 3). Nonetheless, our results would suggest that a rotenone-, KCN-and azide-sensitive ATP-dependent K+ pump could contribute up to 38 % of K+ transport in S. exigua muscles.

P-type ATPase/Na+-dependent transport inhibitors

Unlike the situation in Phormia terraenovae, ouabain was found to be totally ineffective as an inhibitor of K+ transport in Spodoptera exigua. However, the vanadate-sensitivity of aKi did suggest the presence of a P-type ATPase in the muscle cells of this insect (Macara, 1980; Pederson and Carafoli, 1987). Of the P-type ATPases, only two, namely the Na+/K+-ATPase and the H+/K+-ATPase, are involved in K+ transport (Pederson and Carafoli, 1987; Sachs and Munson, 1991). Whilst some Na+/K+-ATPase isoforms are often referred to as being ‘ouabain-insensitive’ or ‘ouabain-resistant’, they are generally inhibited by ‘high’ concentrations (1 mmol l-1) of ouabain (Sweadner, 1989; Fambrough, 1988; Proverbio et al. 1991; Canessa et al. 1992). Ouabain-insensitivity, therefore, suggested the possible involvement of an H+/K+ pump in maintenance of aKi in S. exigua muscle. This was supported by the application of the H+/K+-ATPase-specific inhibitor SCH 28080, which also caused a significant decrease in aKi. SCH 28080 is well established as a specific inhibitor of the gastric H+/K+-ATPase and has been shown to act primarily by competitive binding to the K+ binding site on the extracellular surface of the pump (Wallmark et al. 1987; Scott et al. 1987; Keeling et al. 1988). Hence, a SCH-28080-sensitive H+/K+ pump similar to the mammalian enzyme is likely to be involved in maintenance of aKi in S. exigua muscle.

Interestingly, amiloride also caused a marked reduction in aKi which was also apparently due to inhibition of active K+ transport. Since amiloride is primarily a Na+ transport inhibitor (Benos, 1982; Kleyman and Cragoe, 1988), it is possible that Na+ and K+ transport are linked in S. exigua muscles. As previously suggested, however, the involvement of a Na+/K+-ATPase in S. exigua would be unlikely. Although amiloride-sensitive but ouabain-resistant K+ pumping has been induced in some mammalian cell lines transfected with a ouabain-resistance gene, ouaR(English et al. 1985; Epstein and Lechene, 1988; Schulz and Cantley, 1988), there have been no reports of similar effects in non-transfected cells. Alternatively, amiloride may have acted indirectly on the K+ gradient through inhibition of Na+/H+ exchange, thus causing a change in intracellular pH (pHi). This may, in turn, have affected a pHi-dependent mechanism such as the H+/K+-ATPase. Notably, English and Cantley (1984) found that [Na+]i in a cell line from Manduca sexta was vanadate-sensitive and ouabain-insensitive. It was suggested by English and Cantley (1984) that a Na+/H+ exchanger may be present in these cells. This hypothesis, however, is also questionable, assuming a weak Na+ gradient to be present in S. exigua muscle cells as found in the muscle cells of other Lepidoptera (Djamgoz and Dawson, 1989). Although it has been suggested that the amiloride-sensitive Na+/H+ exchanger can act in ‘reverse’, apparently accepting K+ as a substitute for Na+ (Cala, 1986), there is not an overwhelming body of evidence to support such a mechanism. Finally, amiloride may directly inhibit other K+ transport mechanisms (e.g. H+/K+-ATPase, K+/Cl- exchange) also found in these muscles (see below). Considering the high concentration of this drug applied, such non-specific effects may well have occurred (Kleyman and Cragoe, 1988).

The vanadate-(ΔaKi -35 %) and SCH-28080-sensitivity (ΔaKi -38 %) of aKi therefore strongly suggests that a K+ pump, not unlike the mammalian gastric H+/K+-ATPase (e.g. Ganser and Forte, 1973; Berglindh et al. 1980; Sachs et al. 1976; Rabon and Reuben, 1990), could be involved in maintenance of aKi and hence the K+ gradient in S. exigua muscle. Support for this conclusion is given by English and Cantley (1984), who also provided some evidence to suggest the presence of a ouabain-insensitive but vanadate-sensitive mechanism, presumed to be an H+/K+-ATPase, in a lepidopteran embryonic cell line (CHE) from Manduca sexta. Interestingly, Woodruff et al. (1992) have shown that an H+ gradient can make a direct contribution to the Em in ovarian follicles of the moth Hyalophora cecropia, which like lepidopteran muscles are also bathed directly in the haemolymph. In this case, the fractional contribution of H+ to Em was found to be as much as 24 %. It is important to note that active K+ transport is also well documented in insect, particularly lepidopteran, epithelia (e.g. Harvey and Nedergaard, 1964; Harvey et al. 1983; Harvey, 1992; Dow, 1984). A V-type H+-ATPase coupled to H+/K+ exchange is believed to be responsible for this mechanism of epithelial K+ transport (Wieczorek et al. 1991; Wieczorek, 1992; Moffett and Koch, 1992; Dow, 1992; Lepier et al. 1994). Although the V-type-ATPase-specific inhibitor bafilomycin A1 has not been used in the present study, the fact that this K+ transport mechanism has only ever been reported in insect epithelia would suggest that a similar mechanism is unlikely to operate in S. exigua muscle. Indeed, it was recently reported that the active K+ secretion in Malpighian tubules of F. polyctena was unaffected by application of SCH 28080 (Leyssens et al. 1994). Furthermore, the observation that SCH 28080 reduced aKi by exactly the same percentage as rotenone (38 %) would imply that an H+/K+ pump is the principal ATP-dependent mechanism involved in K+ transport in this insect.

Assuming, then, that an H+/K+-ATPase is present, it would follow that the membrane potential (Em) in Spodoptera exigua muscle must be pH-dependent. In several studies on insect muscle, changes in extracellular pH (pHe) and other external influences which may be expected to influence pHi have been found to affect the Em of lepidopteran muscle (see Djamgoz, 1987, and references therein). Rheuben (1972) showed that the Em in Antherea polyphemus was highly pH-sensitive, Em hyperpolarising and Ri decreasing when pHe was raised and the opposite occurring when pHe was lowered. Such effects can be explained by changes in chloride conductance (GCl) which, in insects, exhibits a marked pH-dependence (see Djamgoz, 1987). However, pH effects on other mechanisms not previously investigated, such as H+ conductance (GH) or inhibition of the H+/K+-ATPase, may also affect Em in Lepidoptera.

In its normal mode of operation, the gastric H+/K+-ATPase is electroneutral (Rabon et al. 1982). A similar H+/K+ pump in Lepidoptera might not, therefore, be the electrogenic mechanism proposed by Dawson et al. (1989) to be directly responsible for maintaining a part of the Em in Chilo partellus muscle. Nevertheless, it is possible that the H+ gradient, as maintained by the H+/K+ pump, could be the remaining ionic component needed to account fully for Em, according to the model of Dawson et al. (1989). Further experiments to monitor the effects of pHe on Em and aKi, however, are required before confirmation of the role of an H+ gradient in maintenance of Em. Nonetheless, it seems likely that an H+/K+ pump is responsible for ATP-dependent K+ transport in Spodoptera exigua.

Anion (Cl-)-dependent transport inhibitors

In lepidopteran muscles, unlike dipteran muscles, there is evidence that Cl- makes a significant contribution to Em, there being a strong inward Cl- gradient associated with these cells (Dawson et al. 1989). Thus, a Cl--dependent mechanism utilising Cl- influx could be involved in maintenance of aKi in Spodoptera exigua muscles. In vertebrates, high concentrations of bumetanide (1 mmol l-1) have been proposed to inhibit K+/Cl- cotransport whilst at low concentrations (1 μmol l-1), Na+/K+/Cl- cotransport is inhibited (Ellory et al. 1982; Lauf, 1984, 1985; Bernhardt et al. 1988). Whilst 1 μmol l-1 bumetanide was found to be ineffective on K+ transport in Spodoptera exigua muscle, 1 mmol l-1 bumetanide caused a significant reduction of aKi (36 %) which could be attributed to active K+ transport. Although in the past there has been some conjecture as to whether the Na+/K+/Cl- and K+/Cl- cotransporters are separate mechanisms (e.g. Lauf, 1985; Garay et al. 1986; Hall et al. 1989; Geck and Heinz, 1986), the observation that the low (1 μmol l-1) concentration of bumetanide was ineffective would suggest that the mechanism could be similar to the K+/Cl- cotransporter found in mammals (e.g. Bernhardt et al. 1988). In turn, this could agree further with the lack of a substantial inward Na+ gradient, as found in the muscles of most other Lepidoptera (Dawson et al. 1989). By analogy with the well-documented effects of bumetanide in mammals combined with the available data for its effects in insects (e.g. Schwiening and Thomas, 1992; Leyssens et al. 1994), the results of the present study and the prevailing ionic gradients in lepidopteran muscle support the conclusion that a K+/Cl- cotransport mechanism with little or no involvement of Na+ is possible. All the anion-dependent cotransporters so far studied have been found to be electroneutral (e.g. Bernhardt et al. 1988; Shetlar et al. 1990). Hence, the proposed Cl--dependent K+ cotransporter could be involved in maintenance of Em through regulation of the transmembrane K+ and Cl- gradients in these muscles.

Finally, treatment with 1 mmol l-1 SITS also caused a significant reduction in aKi (23 %) in S. exigua muscle which could be explained by the following. (1) Any direct effect of SITS on the proposed bumetanide-sensitive Cl--dependent K+ cotransporter would seem unlikely since, at least in mammals, the disulphonic stilbenes are ineffective against Na+/K+/Cl- or K+/Cl- cotransporters (Haas et al. 1982; Hoffmann, 1982; Knauf and Rothstein, 1971). (2) The disulphonic stilbenes SITS and DIDS (4,4′-diisothiocyanatostilbene-2,2′-disulphonic acid) have also been found to block Cl- channels in some epithelia (e.g. Hanrahan et al. 1985). Any effect of SITS on GCl, however, also seems unlikely to have occurred since there was no significant change in Em. A decrease in GCl would be expected to cause some depolarisation of Em. (3) Since, at least in vertebrates, none of the anion exchange/cotransport mechanisms which are inhibited by the disulphonic stilbenes transports K+, SITS is most likely to have an indirect effect on aKi. Involvement of either the Na+/HCO3- cotransporter or Na+-dependent Cl-/HCO3- exchanger seems unlikely to have occurred, bearing in mind the lack of an inward Na+ gradient in these muscles (Dawson et al. 1989). Alternatively, it is possible that SITS may have inhibited a Cl-/HCO3- exchanger and, by disrupting the Cl- gradient in this way, may indirectly have inhibited the proposed bumetanide-sensitive K+/Cl- cotransporter, resulting in a reduction of aKi. This mechanism is electroneutral (Thomas, 1984), and indeed no significant change in Em was observed after application of SITS for 60 min. Under certain conditions, e.g. reduced pHi, Cl-/HCO3- exchange in vertebrates can be found operating in reverse, with Cl- efflux coupled to HCO3- influx (Jennings, 1992). In S. exigua muscle, then, either the HCO3- gradient would need to be steeper than the Cl- gradient or, as has been suggested by Thomas (1984), some energy must be provided to extrude Cl- against its electrochemical gradient. Nonetheless, inhibition by SITS of such a Cl--extruding mechanism would increase the intracellular Cl- activity, aCli, and therefore reduce Cl- influx which, in turn, would reduce aKi, assuming that K+ and Cl- are indeed coupled.

Comparison of the mechanisms involved in maintenance of the K+ activity gradient in Phormia terraenovae and Spodoptera exigua muscles

A marked difference in the effects of the general metabolic blockers was observed in the two insects. DNP was far more potent in P. terraenovae than in S. exigua, and even rotenone, the most potent inhibitor in S. exigua, caused only some 50 % of the effect of DNP in P. terraenovae. This difference in potency of the general blockers, in particular DNP, between the two insects would suggest a greater dependence on ATP for maintenance of the K+ activity gradient in P. terraenovae compared with S. exigua.

Vanadate-sensitive, i.e. P-type, ATPases were found to be involved in maintenance of the K+ gradients in both insects. In P. terraenovae, there appears to be a ouabain-sensitive Na+/K+ pump. This is consistent with the prevailing ion gradients and agrees with previous physiological and molecular studies of dipteran muscle (Djamgoz and Dawson, 1988; Henon and Ikeda, 1981; Djamgoz, 1986; Lebovitz et al. 1989). Furthermore, this mechanism is likely to account for the bulk of ATP-dependent K+ transport in this insect, since the aKi values for ATP-dependence generally, and Na+/K+-ATPase specifically, were very similar (68 % and 61 %, respectively). Since the Cl--dependent transport inhibitors bumetanide and SITS were totally ineffective, in agreement with Cl- being in passive equilibrium across dipteran muscle membranes, the Na+/K+-ATPase would appear to be the principal mechanism maintaining the K+ gradient in this insect (Fig. 6A).

Fig. 6.

(A) Proposed scheme for maintenance of the K+ gradient in Phormia terraenovae muscle. In dipteran muscles, strong inward Na+ and outward K+ gradients prevail, whilst Cl- is in passive equilibrium as shown on the left (the relative strengths of these gradients are indicated by the thickness of the lines, i.e. the thicker the line, the steeper the gradient). In accordance with the predominant gradients, a ouabain-and vanadate-sensitive Na+/K+-ATPase is found to account for the bulk of the ATP-dependent transport (ΔaKi -61 %) in this insect (shown on the right). (B) Proposed scheme for maintenance of the K+ gradient in Spodoptera exigua muscle. In lepidopteran muscle, outward K+ and inward Cl- gradients prevail, whilst Na+ forms a very weak, almost non-existent gradient (shown on the left). On the right, the proposed K+ transport mechanisms are illustrated. A vanadate-and SCH-28080-sensitive H+/K+-ATPase was found to account for the bulk of ATP-dependent K+ transport. Consistent with there being a strong inward Cl- gradient, a bumetanide-sensitive K+/Cl- cotransporter was also found to be involved in K+ transport. The possibility, however, that some Na+ influx is also associated with this cotransport cannot be ruled out (indicated by the dotted line). Together, these two mechanisms could account for a large part (approximately 70 %) of K+ transport in this insect. An additional SITS-sensitive Cl-/HCO3- exchanger could indirectly affect K+ transport through regulation of the Cl- gradient and hence K+/Cl- cotransport. It is possible that these different mechanisms operate in synchrony, as indicated by the dotted routes.

Fig. 6.

(A) Proposed scheme for maintenance of the K+ gradient in Phormia terraenovae muscle. In dipteran muscles, strong inward Na+ and outward K+ gradients prevail, whilst Cl- is in passive equilibrium as shown on the left (the relative strengths of these gradients are indicated by the thickness of the lines, i.e. the thicker the line, the steeper the gradient). In accordance with the predominant gradients, a ouabain-and vanadate-sensitive Na+/K+-ATPase is found to account for the bulk of the ATP-dependent transport (ΔaKi -61 %) in this insect (shown on the right). (B) Proposed scheme for maintenance of the K+ gradient in Spodoptera exigua muscle. In lepidopteran muscle, outward K+ and inward Cl- gradients prevail, whilst Na+ forms a very weak, almost non-existent gradient (shown on the left). On the right, the proposed K+ transport mechanisms are illustrated. A vanadate-and SCH-28080-sensitive H+/K+-ATPase was found to account for the bulk of ATP-dependent K+ transport. Consistent with there being a strong inward Cl- gradient, a bumetanide-sensitive K+/Cl- cotransporter was also found to be involved in K+ transport. The possibility, however, that some Na+ influx is also associated with this cotransport cannot be ruled out (indicated by the dotted line). Together, these two mechanisms could account for a large part (approximately 70 %) of K+ transport in this insect. An additional SITS-sensitive Cl-/HCO3- exchanger could indirectly affect K+ transport through regulation of the Cl- gradient and hence K+/Cl- cotransport. It is possible that these different mechanisms operate in synchrony, as indicated by the dotted routes.

In contrast, a ouabain-insensitive but vanadate-and SCH-28080-sensitive pump, probably an H+/K+-ATPase, appears to be present in S. exigua. This is consistent with there being a strong K+ gradient but a very weak Na+ gradient in lepidopteran muscle (Djamgoz and Dawson, 1989), and would further agree with preliminary studies in other lepidopteran tissues which suggested that the H+ gradient may be involved in K+ transport (English and Cantley, 1984; Woodruff et al. 1992). This mechanism could account for approximately 40 % of the total K+ transport in this insect, the aKi values for both ATP-dependence generally, and H+/K+-ATPase specifically, being the same. However, consistent with the presence of a strong inward Cl- gradient, a bumetanide-sensitive K+/Cl- cotransporter appeared to be responsible for a further 36 % of K+ transport. In turn, a SITS-sensitive exchanger, possibly a Cl-/HCO3- exchanger, could also affect K+ transport indirectly, perhaps by regulating the inward Cl- gradient and hence K+/Cl- cotransport. Together, it would be possible for these mechanisms to operate in concert as shown in Fig. 6B. Accordingly, active extrusion of Cl- from the cell by Cl-/HCO3- exchange would maintain the inward Cl- gradient and, hence, K+ transport via K+/Cl- cotransport; incoming HCO3- could be removed from the cell as CO2 and H2O.

This study was supported by a SERC-CASE studentship to E.M.F. (Zeneca Agrochemicals as collaborating body).

Akera
,
T.
(
1971
).
Quantitative aspects of the interaction between ouabain and (Na++K+)-activated ATPase in vitro
.
Biochim. biophys. Acta
249
,
53
62
.
Akera
,
T.
(
1981
).
Effects of cardiac glycosides on Na+,K+-ATPase
. In
Handbook of Experimental Pharmacology
, vol.
56/I
(ed.
K.
Greef
), pp.
287
336
. Berlin: Springer.
Akera
,
T.
,
Brady
,
T. M.
,
So
,
R. H. M.
,
Tobin
,
T.
and
Baskin
,
S. I.
(
1974
).
Factors and agents that influence cardiac glycosides Na+–K+ATPase interaction
.
Ann. N. Y. Acad. Sci
.
242
,
617
634
.
Ammann
,
D.
,
Chao
,
P.
and
Simon
,
W.
(
1987
).
Valinomycin-based K+selective micro-electrodes with low electrical membrane resistance
.
Neurosci. Lett.
74
,
221
226
.
Baker
,
P. F.
and
Willis
,
J. S.
(
1970
).
Potassium ions and the binding of cardiac glycosides to mammalian cells
.
Nature
226
,
521
523
.
Benos
,
D.
(
1982
).
Amiloride: a molecular probe of sodium transport in tissues and cells
.
Am. J. Physiol.
242
,
C131
C145
.
Berglindh
,
T.
,
Dibona
,
D. R.
,
Pace
,
C. S.
and
Sachs
,
G.
(
1980
).
ATP dependence of H+secretion
.
J. Cell Biol.
85
,
392
401
.
Bernhardt
,
I.
,
Hall
,
A. C.
and
Ellory
,
J. C.
(
1988
).
Transport pathways for monovalent cations through erythrocyte membranes
.
Studia biophys.
126
,
5
21
.
Bonting
,
S. L.
(
1970
).
Sodium–potassium activated adenosinetriphosphatase and cation transport
. In
Membranes and Ion Transport
, vol.
1
(ed.
E. E.
Bittar
), pp.
257
363
.
London
:
John Wiley and Sons Ltd
.
Boron
,
W. F.
and
Boulpaep
,
E. L.
(
1983
).
Intracellular pH regulation in the renal proximal tubule of the salamander: Basolateral HCO3- transport
.
J. gen. Physiol.
81
,
53
94
.
Boron
,
W. F.
and
Russell
,
J. M.
(
1983
).
Stoichiometry and ion dependencies of the intracellular-pH-regulating mechanism in giant squid axons
.
J. gen. Physiol
.
81
,
373
399
.
Cala
,
P. M.
(
1986
).
Volume-sensitive alkali metal–H+transport in Amphiuma red blood cells
.
Curr. Topics Membr. Transport
26
,
79
99
.
Canessa
,
C. M.
,
Horisberger
,
J.-D.
,
Louvard
,
D.
and
Rossier
,
B. C.
(
1992
).
Mutation of a cysteine in the first transmembrane segment of Na,K-ATPase alpha subunit confers ouabain resistance
.
EMBO J.
11
,
1681
1687
.
Dawson
,
J.
and
Djamgoz
,
M. B. A.
(
1988
).
Quantitative analysis of resting membrane electrogenesis in insect (Diptera) skeletal muscle. I. Intracellular K+, Na+and Cl-activities, measured using liquid ion-exchanger and neutral ion-carrier microelectrodes
.
J. exp. Biol.
136
,
417
432
.
Dawson
,
J.
,
Djamgoz
,
M. B. A.
,
Hardie
,
J.
and
Irving
,
S. N.
(
1989
).
Components of resting membrane electrogenesis in lepidopteran skeletal muscle
.
J. Insect Physiol
.
35
,
659
666
.
Deitmer
,
J. W.
and
Szatkowski
,
M.
(
1990
).
Membrane potential dependence of intracellular pH regulation by identified glial cells in the leech nervous system
.
J. Physiol., Lond.
421
,
617
631
.
Dijkstra
,
S.
,
Lohrmann
,
E.
,
Van Kerkhove
,
E.
,
Steels
,
P.
and
Greger
,
R.
(
1994
).
Effects of dinitrophenol on active-transport processes and cell membranes in the Malpighian tubule of Formica
.
Pflügers Arch.
428
,
150
156
.
Djamgoz
,
M. B. A.
(
1986
).
Electrophysiological aspects of metabolic pumping in insect muscle
.
Comp. Biochem. Physiol.
84A
,
207
215
.
Djamgoz
,
M. B. A.
(
1987
).
Insect muscle: intracellular ion concentrations and mechanisms of resting potential generation
.
J. Insect Physiol.
33
,
287
314
.
Djamgoz
,
M. B. A.
and
Dawson
,
C. J.
(
1986
).
Procedures for manufacturing double-barrelled ion-sensitive microelectrodes employing liquid sensors
.
J. biochem. biophys. Meth.
13
,
9
21
.
Djamgoz
,
M. B. A.
and
Dawson
,
J.
(
1988
).
Quantitative analysis of resting membrane electrogenesis in insect (Diptera) skeletal muscle. II. Testing of a model involving contributions from potassium and sodium ions and the anomalous effect of reducing extracellular sodium
.
J. exp. Biol.
136
,
433
449
.
Djamgoz
,
M. B. A.
and
Dawson
,
J.
(
1989
).
Ion-sensitive microelectrode measurements of intracellular K+, Na+, Cl-activities in lepidopteran skeletal muscle
.
J. Insect Physiol.
35
,
165
173
.
Dow
,
J. A. T.
(
1984
).
Extremely high pH in biological systems: a model for carbonate transport
.
Am. J. Physiol
.
246
,
R633
R635
.
Dow
,
J. A. T.
(
1992
).
pH gradients in lepidopteran midgut
.
J. exp. Biol.
172
,
355
375
.
Ellory
,
J. C.
,
Dunham
,
P. B.
,
Logue
,
P. J.
and
Stewart
,
G. W.
(
1982
).
Anion-dependent cation transport in erythrocytes
.
Phil. Trans. R. Soc. Lond. B
299
,
483
495
.
English
,
L. H.
and
Cantley
,
L. C.
(
1984
).
Characterisation of monovalent ion transport systems in an insect cell line (Manduca sexta embryonic cell line CHE)
.
J. Cell Physiol.
121
,
125
132
.
English
,
L. H.
,
Epstein
,
J.
,
Cantley
,
L.
,
Housman
,
D.
and
Levenson
,
R.
(
1985
).
Expression of an ouabain resistance gene in transfected cells. Ouabain treatment induces a K+ transport system
.
J. biol. Chem.
260
,
1114
1119
.
Epstein
,
J. A.
and
Lechene
,
C.
(
1988
).
Ouabain-resistant, amiloridesensitive Na+–K+pumping activity and morphological changes are inducible
.
Am. J. Physiol.
254C
,
847
855
.
Fambrough
,
D. M.
(
1988
).
The sodium pump becomes a family
.
Trends Neurosci.
11
,
325
328
.
Finlayson
,
L. H.
and
Osborne
,
M. P.
(
1970
).
Electrical activity of neurohaemal tissue in the stick insect, Carausius morosus
.
J. Insect Physiol.
16
,
791
800
.
Fitzgerald
,
E. M.
(
1992
).
Maintenance of the K+activity gradient in insect muscle. PhD thesis
.
University of London
.
Fitzgerald
,
E. M.
and
Djamgoz
,
M. B. A.
(
1995
).
Sensitivity of valinomycin-based K+-selective micro-electrodes to inhibitors of K+transport
.
J. Neurosci. Meth.
59
,
273
277
.
Ganser
,
A. L.
and
Forte
,
J. G.
(
1973
).
K+-stimulated ATPase in purified microsomes of bullfrog oxyntic cells
.
Biochim. biophys. Acta
307
,
169
180
.
Garay
,
R. P.
,
Hannaert
,
P. A.
,
Nazaret
,
C.
and
Cragoe
,
E. J.
, Jr
(
1986
).
The significance of the relative effects of loop diuretics and anti-brain edema agents on the Na+,K+,Cl-cotransport system and the Cl-/NaCO3-anion exchanger
.
Naunyn-Schmied. Arch. Pharmac
.
334
,
202
209
.
Geck
,
P.
and
Heinz
,
E.
(
1986
).
The Na–K–2Cl cotransport system
.
J. Membr. Biol.
91
,
91
105
.
Glynn
,
I. M.
(
1985
).
The Na+,K+-transporting adenosine triphosphatase
. In
The Enzymes of Biological Membranes, 2nd edn
, vol.
3
(ed.
A. N.
Martonosi
), pp.
35
114
.
New York
:
Plenum Press
.
Gupta
,
B. L.
,
Wall
,
B. J.
,
Oschman
,
J. L.
and
Hall
,
T. A.
(
1980
).
Direct microprobe evidence of local concentration gradients and recycling of electrolytes during fluid absorption in the rectal papillae of Calliphora
.
J. exp. Biol.
88
,
21
47
.
Haas
,
M.
,
Schmidt
, III,
W. F.
and
Mcmanus
,
T. J.
(
1982
).
Catecholamine-stimulated ion transport in duck red blood cells. Gradient effects in electrically neutral (Na+K+2Cl) co-transport
.
J. gen. Physiol.
80
,
125
147
.
Hall
,
K.
,
Perez
,
G.
,
Anderson
,
D.
,
Sachs
,
G.
,
Hersey
,
S.
and
Kaplan
,
J.
(
1989
).
An unsuspected membrane spanning peptide in gastric H,K-ATPase
.
FASEB J
.
3
,
A873
.
Hanrahan
,
J. W.
,
Alles
,
W. P.
and
Lewis
,
S. A.
(
1985
).
Single anion-selective channels in basolateral membrane of a mammalian tight epithelium
.
Proc. natn. Acad. Sci. U.S.A
.
82
,
7791
7795
.
Harvey
,
W. R.
(
1992
).
Physiology of V-ATPases
.
J. exp. Biol.
172
,
1
17
.
Harvey
,
W. R.
,
Cioffi
,
M.
,
Dow
,
J. A. T.
and
Wolfersberger
,
M. G.
(
1983
).
Potassium ion transport ATPase in insect epithelia
.
J. exp. Biol.
106
,
91
117
.
Harvey
,
W. R.
and
Nedergaard
,
S.
(
1964
).
Sodium independent active transport of potassium in the isolated midgut of the Cecropia silkworm
.
Proc. natn. Acad. Sci. U.S.A.
51
,
757
765
.
Henon
,
B. K.
and
Ikeda
,
K.
(
1981
).
Changes in membrane properties of the Drosophila dorsal longitudinal flight muscle induced by sodium pump inhibitors
.
J. exp. Biol.
90
,
175
183
.
Hoffmann
,
E. K.
(
1982
).
Anion-exchange and anion cation cotransport systems in mammalian-cells
.
Phil. Trans. R. Soc. Lond. B
299
,
519
535
.
Jan
,
L. Y.
and
Jan
,
Y. N.
(
1976
).
Properties of the larval neuromuscular junction in Drosophila melanogaster
.
J. Physiol., Lond.
262
,
189
214
.
Jennings
,
M. L.
(
1992
).
Cellular anion transport
. In
The Kidney: Physiology and Pathophysiology, 2nd edn
(ed.
D. W.
Seldin
and
G.
Giebisch
), pp.
113
145
.
New York
:
Raven Press
.
Keeling
,
D. J.
,
Laing
,
S. M.
and
Senn-Bilfinger
,
J.
(
1988
).
SCH 28080 is a lumenally acting, K+-site inhibitor of the gastric (H++K+)-ATPase
.
Biochem. Pharmac
.
37
,
123
130
.
Kleyman
,
T. R.
and
Cragoe
,
E. J.
, Jr
(
1988
).
Amiloride and its analogs as tools in the study of ion transport
.
J. Membr. Biol.
105
,
1
21
.
Knauf
,
P. A.
and
Rothstein
,
A.
(
1971
).
Chemical modification of membranes. II. Permeation paths for sulphydril reagents
.
J. gen. Physiol.
58
,
190
210
.
Lauf
,
P. K.
(
1984
).
Thiol-dependent passive K+Cl--transport in sheep red blood cells. VI. Functional-heterogeneity and immunological identity with volume-stimulated K+(Rb+) fluxes
.
J. Membr. Biol.
82
,
167
178
.
Lauf
,
P. K.
(
1985
).
K+:Cl-cotransport: sulfhydrides, divalent cations and the mechanism of volume activation in a red cell
.
J. Membr. Biol.
88
,
1
13
.
Lebovitz
,
R. M.
,
Takeyasu
,
K.
and
Fambrough
,
D. M.
(
1989
).
Molecular characterisation and expression of the (Na++K+)-ATPase alpha-subunit in Drosophila melanogaster
.
EMBO J.
8
,
193
202
.
Lee
,
C. O.
and
Dagostino
,
M.
(
1982
).
Effects of strophanthidin on intracellular Na+ion activity and twitch tension of constantly driven canine Purkinje fibers
.
Biophys. J.
40
,
185
198
.
Leech
,
C. A.
(
1986
).
Resting potential and potassium-selective electrode measurements in locust skeletal muscles
.
J. exp. Biol
.
122
,
439
442
.
Lepier
,
A.
,
Azuma
,
M.
,
Harvey
,
W. R.
and
Wieczorek
,
H.
(
1994
).
K+/H+antiport in the tobacco hornworm midgut: the K+-transporting component of the K+pump
.
J. exp. Biol.
196
,
361
373
.
Leyssens
,
A.
,
Djikstra
,
S.
,
Van Kerkhove
,
E.
and
Steels
,
P.
(
1994
).
Mechanisms of K+uptake across the Malpighian tubules of Formica polyctena: the effect of ions and inhibitors
.
J. exp. Biol.
195
,
123
145
.
Leyssens
,
A.
,
Zhang
,
S.-L.
,
Van Kerkhove
,
E.
and
Steels
,
P.
(
1993
).
Both dinitrophenol and Ba2+reduce KCl and fluid secretion in Malpighian tubules of Formica polyctena: the role of the apical H+and K+concentration gradient
.
J. Insect Physiol.
39
,
1061
1073
.
Macara
,
I. G.
(
1980
).
Vanadium – an element in search of a role
.
Trends biochem. Sci.
50
,
92
95
.
Mclaughlin
,
S. G. A.
and
Dilger
,
J. P.
(
1980
).
Transport of protons across membranes by weak acids
.
Physiol. Rev.
60
,
825
863
.
Miller
,
B. A.
and
Usherwood
,
P. N. R.
(
1990
).
Characteristics of five types of K+channel in cultured locust muscle
.
J. exp. Biol.
154
,
45
65
.
Moffett
,
D. F.
and
Koch
,
A. R.
(
1992
).
Driving forces and pathways for H+and K+transport in insect midgut goblet cells
.
J. exp. Biol.
172
,
403
415
.
Pederson
,
P. L.
and
Carafoli
,
E.
(
1987
).
Ion motive ATPases. II. Energy coupling and work output
.
Trends biochem. Sci.
12
,
186
189
.
Piek
,
T.
,
Njio
,
K. D.
and
Mantel
,
P.
(
1973
).
Effects of anions and dinitrophenol on resting membrane potentials of insect muscle fibres
.
J. Insect Physiol
.
19
,
2373
2392
.
Proverbio
,
F.
,
Marin
,
R.
and
Proverbio
,
T.
(
1991
).
The ouabaininsensitive sodium pump
.
Comp. Biochem. Physiol.
99A
,
279
283
.
Rabon
,
E. C.
,
Mcfall
,
T. L.
and
Sachs
,
G.
(
1982
).
The gastric H,K ATPase stoichiometry
.
J. biol. Chem.
257
,
6296
6299
.
Rabon
,
E. C.
and
Reuben
,
M. A.
(
1990
).
The mechanism and structure of the gastric H,K-ATPase
.
A. Rev. Physiol.
52
,
321
344
.
Ramaswami
,
M.
and
Tanouye
,
M.
(
1989
).
Two sodium-channel genes in Drosophila: implications for channel diversity
.
Proc. natn. Acad. Sci. U.S.A
.
86
,
2079
2082
.
Rheuben
,
M. B.
(
1972
).
The resting potential of moth muscle fibre
.
J. Physiol., Lond.
225
,
529
554
.
Sachs
,
G.
,
Chang
,
G. G.
,
Rabon
,
E.
,
Schackman
,
R.
,
Lewin
,
M.
and
Saccomani
,
G.
(
1976
).
A nonelectrogenic H+ pump in plasma membranes of hog stomach
.
J. biol. Chem.
251
,
7690
7698
.
Sachs
,
G.
and
Munson
,
K.
(
1991
).
Mammalian phosphorylating ionmotive ATPases
.
Curr. Opin. Cell Biol.
3
,
685
694
.
Salkoff
,
L.
and
Wyman
,
R.
(
1983
).
Ion channels in Drosophila muscle
.
Trends Neurosci
.
8
,
128
133
Schulz
,
J. T.
and
Cantley
,
L. C.
(
1988
).
CV-1 cell recipients of the mouse ouabain-resistance gene express a ouabain-insensitive Na,K-ATPase after growth in cardioactive steroids
.
J. biol. Chem.
263
,
624
632
.
Schwiening
,
C. J.
and
Thomas
,
R. C.
(
1992
).
Mechanism of pHi regulation by locust neurones in isolated ganglia: a microelectrode study
.
J. Physiol., Lond.
447
,
693
709
.
Scott
,
C. K.
,
Sundell
,
E.
and
Castrovilly
,
L.
(
1987
).
Studies on the mechanism of action of the gastric microsomal (H+K)-ATPase inhibitors SCH 32651 and SCH 28080
.
Biochem. Pharmac
.
36
,
97
104
.
Shetlar
,
R. E.
,
SchöLerman
,
B.
,
Morrison
,
A. L.
and
Kinne
,
R. K. H.
(
1990
).
Characterisation of a Na+–K+-2Cl-cotransport system in oocytes from Xenopus laevis
.
Biochim. biophys. Acta
1023
,
184
190
.
Sweadner
,
K. J.
(
1989
).
Isozymes of the Na+/K+-ATPase
.
Biochim. biophys. Acta
988
,
185
220
.
Thomas
,
R. C.
(
1977
).
The role of bicarbonate, chloride and sodium ions in the regulation of intracellular pH in snail neurones
.
J. Physiol., Lond.
273
,
317
338
.
Thomas
,
R. C.
(
1984
).
Experimental displacement of intracellular pH and the mechanism of its subsequent recovery
.
J. Physiol., Lond.
354
,
3P
22P
.
Treherne
,
J. E.
(
1985
).
Blood–brain barrier
. In
Comprehensive Insect Physiology, Biochemistry and Pharmacology
, vol. 5, Nervous System: Structure and Motor Function (ed.
G. A.
Kerkut
and
L. I.
Gilbert
), pp.
118
138
. Oxford: Permagon Press.
Treherne
,
J. E.
and
Pichon
,
Y.
(
1972
).
The insect blood–brain barrier
.
Adv. Insect Physiol.
9
,
257
313
.
Treherne
,
J. E.
and
Schofield
,
P. K.
(
1981
).
Mechanisms of ionic homeostasis in the central nervous system of an insect
.
J. exp. Biol.
95
,
61
73
.
Wallmark
,
B.
,
Briving
,
C.
,
Fryklund
,
J.
,
Munson
,
K.
,
Jackson
,
R.
,
Mendlein
,
J.
,
Rabon
,
E.
and
Sachs
,
G.
(
1987
).
Inhibition of gastric H+, K+-ATPase and acid secretion by SCH 28080, a substituted Pyridyl(1, 2a) imidazole
.
J. biol. Chem
.
262
,
2077
2084
.
Wareham
,
A. C.
,
Duncan
,
C. J.
and
Bowler
,
K.
(
1974
).
The resting potential of cockroach muscle membrane
.
Comp. Biochem. Physiol
.
48A
,
765
797
.
Weevers
,
R. DE G.
(
1966
).
A lepidopteran saline: effects of inorganic cation concentrations on sensory, reflex and motor responses in a herbivorous insect
.
J. exp. Biol.
44
,
163
175
.
Wieczorek
,
H.
(
1992
).
The insect V-ATPase, a plasma membrane proton pump energising secondary active transport: molecular analysis of electrogenic K+ transport in the tobacco hornworm midgut
.
J. exp. Biol
.
172
,
335
343
.
Wieczorek
,
H.
,
Putzenlechner
,
M.
,
Zeiske
,
W.
and
Klein
,
U.
(
1991
).
A vacuolar-type proton pump energises K+/H+antiport in an animal plasma membrane
.
J. biol. Chem.
266
,
15340
15347
.
Woodruff
,
R. I.
,
Munz
,
A.
and
Telfer
,
W. H.
(
1992
).
Steady-state potentials in ovarian follicles of a moth, Hyalophora cecropia
.
J. Insect Physiol.
38
,
49
60
.