Unidirectional ion movements in the hindgut of larval Sarcophaga bullata (Diptera: Sarcophagidae)

The isolated hindgut of larval Sarcophaga bullata is capable of secreting K⁺, Na+, NH₄+ and Cl⁻ into the gut lumen, under the appropriate conditions (Prusch, 1972). The hindgut of Sarcophaga also maintains a transepithelial potential difference (TEP) such that the lumen of the gut is normally negative with respect to the haemolymph (Prusch, 1974). Since the TEP is maintained in the presence of identical ionic solutions on either side of the gut, the origin of the potential is most likely due to the electrogenic transport of some ion(s). The secretion of K⁺ from the haemolymph into the midgut lumen of Hyalophora cecropia results in the establishment of a TEP such that the lumen in this case is positive with respect to the haemolymph (Harvey, Haskell & Nedergaard, 1968).

On the basis of net ion flux and TEP measurements in the isolated hindgut of S. bullata, it was determined that K+, NH₄+ and Cl⁻ were actively secreted. Addition of small amounts of NH₄⁺ to the external medium resulted in a large decrease in K⁺ secretion and a concomitant large increase in NH₄+ secretion. Cl⁻ secretion was maintained in both the presence and absence of external NH₄⁺. These results were used to develop a model of the hindgut system in which Cl⁻ was being secreted into the hindgut lumen constantly and independently of cation movements (Prusch, 1974). In addition, a cation pump was thought to be present in the hindgut epithelium in which K⁺ and NH₄⁺ competed for the same transport site. In order to determine if this was the case, unidirectional ion fluxes across the larval, isolated hindgut were measured with the appropriate radioisotopes. The results of this study indicate that K⁺ and NH₄+ are secreted independently of each other and that anion and cation movements are not entirely independent.

The hindgut of third instar larvae of Sarcophaga bullata (Parker) was removed as described previously (Prusch, 1971) and mounted in a perfusion chamber as is shown in Fig. 1. The perfusion chamber consisted of a plastic box, which would hold 20 ml of the desired medium. The isolated gut was tied with silk thread to 30-gauge syringe needles mounted at opposite ends of the chamber. The isolated gut was perfused with a Sage syringe pump at approximately 7 μl/min. The external medium was constantly stirred with a magnet stirrer and aerated. The solutions used in these experiments were as described previously (Prusch, 1974).

Unidirectional isotope influx was measured by adding the appropriate radio-isotope (⁴²K, ²⁴Na, or ³⁶C1; obtained from New England Nuclear Corp.) to the external medium. For a given experiment, approximately 100 μ Ci of the isotope was added to the external medium. The perfusate was collected at specified time intervals in metal planchettes, dried and counted in a Nuclear Chicago gas flow counter.

Unidirectional isotope efflux was measured by perfusing the isolated gut with medium which contained the desired radioisotope (approximately 50 μ Ci/ml). Aliquots of the external medium (100 μl) were taken at 5 min intervals, placed in metal planchettes, dried and counted. The volume of the medium in the chamber was maintained constant by replacing each 100 μl aliquot withdrawn from the chamber by an equal volume of fresh medium.

The influx of K⁺ from the outside control medium into the isolated gut lumen is 1·05 (±0·11) ×10⁻⁸ moles/cm².min (9) (mean + s.e.m., followed by the number of determinations in parentheses). A representative ⁴²K influx is shown in Fig. 2, in which the linear portion of the influx curve intercepts the abscissa at approximately 25 min. This lag time in K⁺ influx is independent of the concentration of K⁺ in the external medium and the associated changes in (see below). The lag time for labelled K⁺ entry into the isolated midgut of H. cecropia was found to be only 2–4 min (Harvey & Zerahn, 1969). Zerahn (1973) found that the lag time was influenced by the thickness of the gut tissue. These authors concluded that the lag time was caused by diffusion delays and the time to achieve uniform specific activity in the gut cells. The lag time in Sarcophaga could also be influenced by the cuticular lining of the hindgut.

It was noted previously (Prusch, 1972) that CN in the external medium decreases K⁺ secretion by the isolated hindgut. Addition of CN to the external medium (5×10⁻⁴ M) in these experiments decreased K⁺ influx from 1·05 × 10⁻⁸ to 0·53 × 10⁻⁸ moles/cm². min (Fig. 3). Presence of CN in the external medium has a negligible effect on K⁺ efflux from the gut lumen (see below).

Substitution of SO₄²⁻ for Cl⁻ in the outside control medium depolarizes the transepithelial potential across the isolated hindgut from —15 to +33 mV (Prusch, 1974). Under these same conditions (Fig. 4) the unidirectional K⁺ influx decreases from 1·05 × 10⁻⁸ to 1·89 × 10⁻⁸ moles/cm².min. Addition of Cl⁻ to the previous Cl⁻-free control medium increases K⁺ influx to near control levels.

The effect of changes in external K⁺ concentration on is shown in Fig. 5. initially increases with increasing external K⁺ and then levels off, demonstrating saturation kinetics. Saturation of apparently occurs between 6 and 10 mm-K⁺ in the external medium. One half saturation occurs at about 3 mm-K+.

The isolated hindgut of S. bullata is apparently capable of secreting K+, Na+, NH₄+ and Cl⁻ into the gut lumen. Addition of small amounts of NH₄+ (1 mm) to the control medium bathing the isolated hindgut brings about a decrease in K⁺ secretion and a concomitant increase in NH₄⁺ secretion. It was suggested that a cation pumping mechanism was present in the hindgut epithelium which would move K⁺ in the absence of external NH₄⁺, but when NH₄⁺ was present the pump mechanism would preferentially switch to NH₄+. That is, there is a competition between NH₄⁺ and K⁺ for the same cation pump site with NH₄⁺ having a much higher affinity for the site (Prusch, 1974). In order to investigate this possibility further, the effects of NH₄⁺ on were investigated. Addition of NH₄⁺ to the external medium, either during or at the start of the experiment, had no appreciable effect on ⁠, indicating no competition between NH₄⁺ and K+.

The efflux of from the isolated hindgut lumen is 3·46 (±1·09) × 10⁻⁹ moles/cm². min (7). Addition of 1 mm-NH₄⁺ to the external control medium increases from 3·46 ×10⁻⁹ to 1·49 ×10⁻⁸ moles/cm².min (Fig. 6). The effect of NH₄⁺ on the net K⁺ flux across the isolated hindgut is not then a decrease in due to an NH₄⁺/K⁺ competition but to an increase in ⁠.Substitution of SO₄²⁻ for Cl⁻ in the external medium increases from 3·46 to 5·01 × 10⁻⁹ moles/ cm². min.

Unidirectional Na+ influx into the isolated hindgut lumen from the control medium is 1·61 (± 0·09) × 10⁻⁸ moles/cm².min (6). Addition of 1 mm-NH₄⁺ to the outside medium slightly decreases to 1·15 × 10⁻⁸ moles/cm².min. as a function of changes in the Na⁺ concentration of the external control medium. In this case does not demonstrate saturation kinetics, but is roughly a linear function of the external Na+ concentration, indicating that the influx of Na+ into the hindgut lumen is not carrier-mediated but moves by diffusion in response to the Na⁺ concentration gradient.

The efflux of Na⁺ from the isolated hindgut lumen is 1·91 (± 0·58) × 10⁻⁹ moles/ cm². min (5). Addition of 1 mm-NH₄⁺ to the external control medium increases to 2·83 ×10⁻⁹ moles/cm².min.

Unidirectional Cl⁻ influx into the isolated hindgut lumen from the control medium is 2·26 (±0·54) × 10⁻⁸ moles/cm².min (7). If K⁺ is eliminated from the external medium, is reduced to 5·92 × 10⁻⁹ moles/cm².min. This depression of in K+-free control medium is reversible and increases close to control levels upon restoration of external K+. Addition of r mm NH₄⁺ to the external medium results in a marked increase in (Fig. 8), compared to the control influx, from 2 ·26 to 4 ·42 × 10⁻⁸ moles/cm². min. as a function of changes in the external Cl⁻ concentration is shown in Fig. 9. As is the case with ⁠, Cl⁻ influx demonstrates saturation kinetics, increasing with increasing external Cl⁻ up to 10 mm, when only slight further increases are noted. The external Cl⁻ concentration at one-half saturation is approximately 5 mm.

Unidirectional Cl⁻ efflux out of the isolated hindgut lumen is 2 ·96 (± 1 ·49) × 10⁻⁹ moles/cm².min (6). Addition of NH₄⁺ to the external medium (1 mm) did not appreciably change although a slight increase (to 3 ·11 × 10⁻⁹ moles/ cm².min) was noted.

The experimental and calculated flux ratios for K+, Na+, and Cl⁻ under control conditions are summarized in Table 1. Under these conditions, the transepithelial potential difference across the isolated hindgut is 15 mV, lumen negative in respect to the outside medium. The calculated flux ratio f₁/ f₀, for univalent cations is 1 ·81. The experimental flux ratio for Na+, 1 ·56, is relatively close to the calculated ratio and indicates that the movement of Na⁺ in this situation is essentially passive. The K⁺ flux ratio was found to be 3 ·03, which is almost twice the calculated ratio and indicates that the movement of K⁺ is influenced by other than passive forces. The same can be applied to the Cl⁻ flux ratio where the experimental ratio of 7 ·64 is an order of magnitude higher than the calculated ratio of 0 ·55.

Addition of NH₄⁺ or absence of Cl⁻ in the external control medium alters the flux ratios of K+. In Cl⁻-free medium, is 1 ·89 × 10⁻⁹ and is 5 ·01 × 10⁻⁹. The experimental flux ratio for K⁺ is therefore 0 ·38. Under these conditions the TEP is +33 mV (Prusch, 1974) and the calculated ratio is consequently 0 ·27. When Cl⁻ is present in the external medium the experimental and calculated flux ratios differ considerably, but when Cl⁻ is removed the ratios become similar. A very nearly identical situation is seen when NH₄⁺ is added to the external control medium. In this case the TEP is +12 mV and the experimental and calculated K⁺ flux ratios are virtually the same (0 ·71 and 0 ·62 respectively).

The distribution of Na⁺ across the isolated hindgut of S. bullata is passive, according to unidirectional flux and transepithelial potential measurements. The calculated flux ratio for Na+ is 1 ·81, whereas the measured flux ratio was 1 ·56 (Table 1). The closeness of the experimental and calculated flux ratios indicates that Na⁺ distribution in this system is a passive process (Ussing, 1949). Further evidence for the passive distribution of Na⁺ in the hindgut comes from measurements of as a function of changes in external Na+ concentration (Fig. 7). Instead of saturating, increases in a linear manner with increased external Na⁺; this indicates fairly conclusively that Na+ movement across the hindgut is not carrier-mediated.

The increase with time of Na⁺ in the isolated hindgut sac preparation (Prusch, 1972) comes about essentially because of the potential difference across the gut. In the control medium, the TEP is 15 mV (lumen negative) and Na+ is accumulated in the hindgut. When the TEP is changed, J^Na+ in either direction changes according to the polarity of the potential. For example, when NH₄⁺ is added to the external medium the potential changes from —15 to +33 mV (Prusch, 1974). As would be expected from a passive Na+ distribution across the hindgut, this results in a decreased and an increased ⁠.

The distribution of Na+ in other systems may be either active or passive. The isolated midgut of H. cecropia transports K⁺ independently of Na+ (Harvey & Nedergaard, 1964), but under the proper experimental conditions, i.e. when Mg²⁺ and Ca²⁺ are removed from the medium, the midgut of H. cecropia will transport Na⁺ (Zerahn, 1971). In cockroach midgut, Na+ transport is linked to K⁺ transport (O’Riordan, 1969).

K+ distribution across isolated Sarcophaga hindgut is at least partially active. The calculated K⁺ flux ratio is 1 ·81, whereas the experimental flux ratio is 3 ·03. A carrier-mediated transport system for K⁺ is also indicated by the saturation of with increases in external K⁺ (Fig. 5). Furthermore, addition of CN (5 × 10⁻⁴ M) to the external medium results in an approximately 50% decrease in unidirectional K⁺ influx (Fig. 3). All of these observations support the conclusion that K⁺ is actively secreted into the hindgut lumen of Sarcophaga.

Although the movement of [K+ into the hindgut is at least partially an active process, J^K+ is not entirely independent of anion movements. Substitution of SO₄²⁻ for Cl⁻ in the external control medium changes the TEP from —15 to +33 mV (Prusch, 1974) and brings about a concomitant decrease in and increase in ⁠.This is what would be expected from a passive cation distribution when the TEP depolarizes, reverses polarity and becomes positive.

The flux ratio analyses under the same conditions indicate that the flux of K⁺ in this condition is not entirely passive. The calculated flux ratio for K⁺ movement in Cl^_-free medium is 0 ·22, whereas the experimental ratio is 0 ·38. Consequently, one component of J^K+ is passive and is influenced by the potential across the gut, and another component is definitely active.

The transepithelial transport of K⁺ in insects has been known for some time and has been demonstrated in a variety of systems. Active K⁺ transport across Malpighian tubules is responsible for the production of an isosmotic primary excretory fluid (Ramsay, 1953). In H. cecropia midgut, K⁺ is actively moved from the haemolymph into the gut lumen (Harvey & Nedergaard, 1964; Harvey et al. 1968) in order to compensate for excess K⁺ in the diet. The secretion of K⁺ by Sarcophaga hindgut may serve a similar role, i.e. excretion of excess dietary K⁺. Active K⁺ transport has also been demonstrated in Calliphora salivary glands (Oschman & Berridge, 1970).

The movement of Cl⁻ into the hindgut lumen of Sarcophaga is an active process. Evidence in support of this conclusion comes from the flux ratio analysis of unidirectional Cl⁻ fluxes and the demonstration of saturation kinetics for ⁠.The calculated Cl⁻ flux ratio is 0 ·55, whereas the experimental ratio is 7 ·64. As is the case for K⁺ unidirectional fluxes, Cl⁻ movements are not independent of the co-ion movement, which in this case is K+. In K+-free medium, the TEP hyperpolarizes to —63 mV and decreases from 2 ·26 to 0 ·59 × 10⁻⁸ moles/cm².min. Movements of both K⁺ and Cl⁻ in this system obviously influence the movement of the other ion, and to this extent the fluxes of K⁺ and Cl⁻ are not independent of one another.

Chloride transport has been indicated in a variety of insect epithelial systems. In Rhodnius Malpighian tubules Maddrell (1971) provides evidence for a Cl⁻ pump in the apical cell membrane. Transport of Cl⁻ is also seen in Calliphora salivary glands, where its secretion is controlled by the presence of 5-hydroxytryptamine (Berridge & Prince, 1971). Active Cl⁻ movement has also been demonstrated across the body surface and anal papillae of freshwater insect larvae (Shaw & Stobbart, 1963).

From this investigation and previous studies with this system (Prusch, 1972, 1974) it has been established that the isolated hindgut of S. bullata is capable of actively transporting the NH₄⁺ ion. Other more common mechanisms for NH₄⁺ excretion in various organisms include NH₄+-Na⁺ exchange mechanisms such as that seen across fish gills (Maetz & Garcia-Romeu, 1964) and freshwater crayfish (Shaw, 1960) or the non-ionic diffusion of NH₃ and its subsequent entrapment and excretion as NH₄⁺ in the mammalian kidney (Milne, Schribner & Crawford, 1958). Neither of these two mechanisms is involved in NH₄⁺ excretion in Sarcophaga as was demonstrated previously (Prusch, 1972). From transepithelial potential and net flux measurements, it had been suggested (Prusch, 1974) that NH₄⁺ and K⁺ competed for the same pump site in the hindgut epithelium. An NH₄+-K+ competition for the same pump site has been demonstrated in the mammalian red blood cell (Post & Jolly, 1957), but from unidirectional K⁺ flux measurements in the isolated hindgut it was shown that K⁺ and NH₄⁺ enter the hindgut independently of each other. Addition of NH₄⁺ to the external medium does not affect the unidirectional influx of K⁺ but greatly increases K⁺ efflux. The increase in K⁺ efflux, which is most likely a passive event, is probably due to the change in potential across the isolated hindgut elicited by the addition of NH₄+. The TEP in the control medium is — 15 mV, but with the addition of NH₄⁺ (1 mm) the TEP reverses polarity and becomes +12 mV.

The hindgut of Sarcophaga bullata actively transports K+, NH₄⁺ and Cl⁻ from the haemolymph into the gut lumen. The concentration of Na+ in the lumen increases simply because of the electrical potential difference across the gut. Because NH₄⁺ is transported independently of other cations in this system and because of the relatively simple structure of the gut, the isolated hindgut of S. bullata represents an ideal model system in which to investigate the active transport of the NH₄⁺ ion.