³H-Ouabain Binding Autoradiography in the Abdominal Nerve Cord of the Hawk Moth, Manduca Sexta

The localization and function of Na⁺,K⁺-adenosine triphosphatase (Na⁺,K⁺-ATPase) in neural tissue has received increasing attention in recent years (Stahl & Broderson, 1976a), due in part to the proposal that the enzyme in glial cells may have a special role in regulating the cation content of the neural extracellular space (Hertz, 1965). Efforts at localizing the Na⁺,K⁺-ATPase have thus far centred around vertebrate central nervous tissue (Schwartz, Ernst, Siegel & Agranoff, 1981; Stahl & Broderson, 1976b; Wood et al. 1977). With the growing understanding of ionic regulation in the central nervous system of insects derived mainly from the work. Treherne and colleagues (cf. Treherne & Schofield, 1979), it is of interest to localize the enzyme in this nervous tissue. We have chosen the abdominal nerve cord of the hawk moth, Manduca sexta, for this study. The general morphology of the nerve cord is summarized in Fig. 1.

Physiological studies (Pichón, Satelle & Lane, 1972) have shown that in order to maintain normal electrical activity, the nerve cord extracellular space requires low K⁺ and high Na⁺ concentrations. These ion concentrations differ from those of the haemolymph which are approximately 25 mm for K⁺ and 25 mm for Na⁺. Ion flux studies in the cockroach abdominal nerve cord (Treherne, 1961, 1966) suggest that the perineurium is responsible for these gradients and that Na⁺-K⁺ pumps may be involved in the ion transport mechanism.

Previous studies from our laboratory (Rubin, Clark & Stahl, 1980, 1981) demonstrated significant levels of the Na⁺,K⁺-ATPase in both the nerve cord and brain of sexta. It exhibited similar biochemical characteristics to Na⁺,K⁺-ATPases from OTher neural preparations with respect to its dependence on Na⁺, pH, Mg²⁺ and ATP. Some differences were noted in the interaction of K⁺ and cardioactive steroids with the insect enzyme; however, these differences were of a quantitative rather than qualitative nature. These similarities suggested that ³H-ouabain autoradiography (Shaver & Stirling, 1978; Stirling, 1972; Stirling & Lee, 1980; DiBona&Mills, 1979) could be used in M. sexta to investigate the distribution of the Na⁺-K⁺ pump at the light microscope level of resolution.

We first examined the characteristics of ³H-ouabain binding to intact abdominal nerve cords in order to establish the optimal conditions for autoradiography of ³H-ouabain. A nonspecific linear component was measured at ouabain concentrations greater than 10 μM. Thus, to ensure specific binding, only very low ouabain concentrations (< 3·6 μM) were used in the autoradiographic experiments. In these experiments, a low level of binding was observed in the perineurium and a high level of binding to the neural components, the highest activity being over the neuropil.

Pharate Manduca sexta, reared from the eggs and raised on artificial diet (Truman, 1972), were kindly supplied from the laboratory of Dr James W. Truman, Department of Zoology, University of Washington, Seattle.

The abdominal ventral nerve cords were obtained by opening the abdomen and clearing away the adhering fatty tissue while being irrigated by a stream of cool tap water. The cord was then removed by carefully pulling from the anterior end with forceps. The isolated tissue was immediately immersed in ice-cold incubation buffer containing 125mm-NaCl, 5mm-MgC1₂, 0·5% bovine serum albumin (BSA), and 12·5 mm-imidazole, pH 7·3. The BSA was included to insure stability of the enzyme during the long incubation period (Rubin et al. 1981). The entire dissection procedure took approximately 1 min to complete. For the liquid scintillation experiments the cords were cut in half with dissecting scissors, each half containing two ganglia and the intervening connective. Preliminary autoradiographic experiments indicated that the perineurium prevented ouabain and inulin from penetrating the nerve cord. Several methods of disrupting this diffusion barrier were tried. Treatment with collagenase, trypsin or hyaluronidase, or low-Ca²⁺ media did not improve penetration significantly. However, immersion in a solution of 3M-urea at room temperature greatly improved penetration (see Results). A similar treatment was used by Treherne, Schofield & Lane (1973) and Pichón & Treherne (1974) in the cock-roach to make the nerve cord permeable to Na⁺. In the present study the urea treatment produced some distortion of cellular morphology, probably due to osmotic stress, but the major cellular components were recognizable (for example, see Fig. 8). After the urea treatment, the cords were washed twice in many volumes of urea-free ice-cold buffer for several minutes before commencing the incubation.

For quantitation of ouabain binding to intact M. sexta nerve cords, the isolated tissue was transferred to incubation buffer at 37 °C containing an appropriate amount of ³H-ouabain (New England Nuclear, Boston, Massachussetts). For most of the experiments, 10 μ Ci/ml ³H-ouabain was used, corresponding to a concentration of 0·7 /IM. At appropriate times the binding reactions were stopped by quickly immersing the cords into incubation buffer without ouabain at 0°C. The unbound ouabain was then removed by washing in several volumes of cold incubation buffer or 10 mm-imidazole, pH 7·3, for 30 min. The imidazole wash was used to allow protein determinations to be done on the tissue without the interference of BSA. For the determination of the concentration dependence of ouabain binding, solutions of varying specific activities were used. The cords were incubated in groups of two half-cords per vial in 400 μl of test solution. The amount of extracellular ouabain was measured by including 0·32M-¹⁴C-sucrose (1·5 μCi/ml; New England Nuclear, Boston, Massachussetts) in the incubation media as an extracellular space marker. The binding reactions were started by transferring the test solutions to a 37 °C water bath. After 90 min the reaction was stopped by returning the vials to the 0°C bath. Each cord was then washed three times, for 10 min each time in 0·5 ml of 10 mm-imidazole, pH 7·3, to remove unbound label. For all radiochemical experiments the washed tissue was digested overnight in 1ml of 0·5 mm-NaOH/0·9 % sodium dodecyl sulphate (SDS) at room temperature with gentle agitation. For protein determinations, the tissue digest was then sonicated for 15 s using a Biosonic 11A probe sonicator at a setting of 60. The scintillation counting fluid was either Tritosol or Aquasol (New England Nuclear). Quenching was monitored in the double label experiment by the channels ratio method.

In one experiment the effect of the urea treatment on the total amount of ouabain binding was measured at equilibrium with a saturating dose (10 μM) of ³H-ouabain (10 μCi/ml). Nerve cords (20) were dissected out as described above and split into two groups. One group received the urea treatment while the other acted as the control. Each group was then homogenized in 1·0 ml of the incubation buffer and the homogenate was added in a 1: 10 dilution to 0°C incubation buffer containing a final concentration of 0·45% BSA and 1·35 mm-ADP (Rubin et al. 1981). The binding reaction was run at 37 °C. After 90 min the reaction solutions were vacuum-filtered on 0·45 μm nitrocellulose filters (Schleicher & Schuell, Keene, New Hampshire) and the unbound label was washed out with 15 ml of 10mm-imidazole, pH7·3, at 0°C as previously described (Rubin et al. 1981). The filters were then dried and the radio-activity counted. Nonspecific binding was determined using controls in which the labelled ouabain was diluted to 10⁻³M with unlabelled ouabain.

The amount of protein per half-cord was determined by a modification of the method of Lowry, Rosebrough, Farr & Randall (1951) using BSA as a standard.

The procedure for autoradiography was carried out largely as described by Stirling 1972). Nerve cords were incubated in the presence of 10 μCi/ml (0·7 μM) or 50 μCi/ ml (3·5 μM) ³H-ouabain. BSA (5 %) was included in the incubation media to provide a matrix which survives freeze drying and permits identification of radioactivity in the extracellular space in these experiments. After the incubation, the tissue was washed three times, 10 min each time, in 0°C incubation buffer, laid out on small strips of aluminium foil, and immediately frozen by dashing the strips into liquid propane (−175 °C). The frozen tissue was then dried under vacuum at 0−001 Torr. The dried tissue was osmicated in the vapour phase for 20 h at room temperature. It was infiltrated under vacuum with Spurr’s low-viscosity embedding media (Polysciences, Inc., Warrington, Pennsylvania) and orientated in flat embedding moulds. The plastic was cured overnight at 60 °C and 1–2 μm sections were cut over water on a Porter-Blum MT-2 ultramicrotome. The sections were picked up with a wire loop, placed on cleaned microscope slides and coated with nuclear track emulsion (NTB-2, Eastman Kodak, Rochester, New York). After exposure (8–113 days) the autoradiographs were developed in Kodak D-19, stained for 20–45 min in Richardson’s stain (Richardson, Jarrett & Finke, 1960), dehydrated, and cover-slipped. Latent image fading and chemography (Rogers, 1973) were absent.

Treatment of the nerve cords with 3 M-urea at room temperature was found to be a prerequisite to measuring ouabain binding in intact M. sexta nerve cords. Fig. 2 shows that at 0·7 μM-³H-ouabain, ouabain binding increased 3·5-fold during the find 15 min of exposure to the urea, and decreased after that point. This decrease was probably due to enzyme denaturation. The autoradiographic experiments described below convinced us that the increase in binding was due to an opening of the perineurial barrier thus allowing the ouabain free access to Na⁺,K⁺-ATPase sites on the underlying axons and glia. We decided that 10 min was the shortest possible exposure time to render most of the underlying Na⁺,K⁺-ATPase sites accessible to ouabain.

The specificity of ouabain uptake in the urea-treated nerve cord was examined by measuring the effect of a high concentration of K⁺, a specific inhibitor of ouabain binding to the Na-K pump. Fig. 3 shows that an increase in extracellular K⁺ from 0 to 50 mm decreased the rate of ³H-ouabain binding and the final or equilibrium binding level by approximately 35 %.

In order to localize ouabain binding using autoradiography, it is highly advantageous to remove the unbound label as this increases the signal to noise ratio. In many species the rate of ouabain dissociation is slow and highly temperature dependent. Thus, washing at 0°C in incubation buffer does not disturb bound ouabain. This question was addressed in the urea-treated nerve cord by measuring the dissociation kinetics of ouabain at 0°C. The data of Fig. 4 show that a large fraction (35 %) of the ouabain initially taken up at 37 °C is essentially irreversibly bound at 0°C. At 37 °C the half-time for dissociation is about 10 min, considerably shorter than the half-time found in the rabbit (Shaver & Stirling, 1978). These results show that if extracellular ouabain is to be washed out, it is essential that the temperature be kept near 0°C.

For quantitative considerations it is important to know the total number of specific binding sites in the tissue as well as the level of nonspecific ouabain-binding at different ouabain concentrations. Thus the concentration dependence of ouabain binding to the M. sexta nerve cord was determined. Extracellular ouabain was measured with ^l4C-sucrose. It amounted to 8·2 ± 0·8% (S.E., N= 18) of the total ouabain remaining after 30 min of washing. As shown in Fig. 5, there is a high degree of unsaturable binding in the urea-treated preparation above 10 μM-ouabain, even when the sucrose space is taken into account. This component of the binding curve could be due to cellular uptake or to a nonspecific partitioning of ouabain with cellular components which may be present in the extracellular space after urea treatment. At ouabain concentrations below 10/XM, however, the binding is clearly saturable. When the linear component is subtracted, the remaining curve shows that the total number of specific ouabain-binding sites in the tissue is 17·8 pmol/mg protein and that these are half-occupied (K_D) at 1·8 μM ouabain.

In order to assess the effect of the urea treatment on the total number of specific binding sites, we determined the degree of binding at a saturating dose (10/IM) of ouabain in homogenates of nerve cords with and without urea treatment. The homogenates of the urea-treated cords bound a total of 11·62 ± 0·80 (S.E., N = 3) pmol/mg protein whereas the untreated cords bound 12·26 ± 0·61 (S.E., N=3) pmol/mg protein. Thus, while the total amount of ouabain binding is slightly decreased in homogenates as compared to intact tissues, the urea treatment does not appreciably affect the number of sites.

Fig. 6A shows a longitudinal section through an untreated abdominal ganglion that has been incubated for 1 h with 3·6 μM-³H-ouabain and frozen immediately after the incubation, omitting the washing step. Clearly, the ouabain is largely excluded from the inner elements of the ganglion by the perineurial cell layer. A similar situation exists in the connectives (Fig. 6B). These results are shown quantitatively in Table 1 as a decrease in the relative grain density between incubation media and tissue. However, with urea treatment, ouabain penetrates the innermost regions of the ganglia and connectives as shown in Fig. 7A, B.

Fig. 8A shows ouabain binding in a urea-treated, abdominal ganglion which had been washed free of extracellular ouabain. The majority of ouabain-binding sites in the ganglion are in the neuropil. The cell body layer and perineurium also contain ouabain-binding sites above background levels, though not to the density of the neuropil. Fig. 8B shows a connective incubated under the same conditions. Heavy binding is seen in the central areas of the connective where the axons and their associated glia are located. Again, no strong binding component is seen in the area of the perineurium.

The specificity of ouabain binding in the ganglia and connectives is examined in Fig. 9A, B. In these tissues the K⁺ concentration was raised to 50HIM, which was shown above (Fig. 2) to decrease ouabain-binding at 0·7 μM-ouabain by 35 %. It is clear in these autoradiographs that the grain densities are considerably reduced, both in the ganglia and the connectives. No appreciable binding is seen in the cell body or perineurial areas. Quantitative analysis of the relative grain densities (Table 2) shows, in agreement with the radiochemical measurements, a reduction of 59 % in the ganglia and 27 % in the connectives.

It has been suggested in the cockroach that there is a flux of sodium into and out of the nerve cord that is in part regulated by Na⁺,K⁺-pumps of perineurial membranes (Treherne, 1961, 1966; Treherne & Schofield, 1979). We found evidence for these pump sites in the perineurium which had not been urea-treated and which had been exposed for autoradiography for 3–4 months. Fig. 10 shows such a washed ganglion exposed for 113 days. Some ouabain has gained access to the inner elements of the ganglion and binding is seen in the neuropil and cell body layers. Most notable, however, is the high grain density over the perineurium. Due to resolution limitations at the light microscope level it is not clear if the binding is to the haemolymph side of the perineurium. However, the limited access to the neural side and the greater area of the membranes facing the haemolymph side (Lane, 1972) suggest that most of the binding is to the latter. We could not demonstrate ouabain binding in the perineurium of the connective (autoradiograph not shown).

A question of considerable importance to the interpretation of these autoradiographs is how much of the unbound ouabain can be washed out, especially in urea-treated tissues where cellular retention may be enhanced. This question was examined by increasing the concentration of unlabelled ouabain to 1 HIM while the concentration of ³H-ouabain was kept at 50 μCi/ml. With a large excess of ouabain most of the binding sites should not be labelled and most of the radioactivity should be removed by washing. Fig. 11A, B shows a ganglion and connective, respectively, that have been incubated for 1 h with excess unlabelled ouabain and frozen immediately. Fig. 12A, B shows a similar experiment, but a subsequent washing period of 30min has been included. The great majority of ³H-ouabain was washed out; that remaining was presumably taken up into cells or bound nonspecifically.

The radiochemical measurements of ouabain-binding in this tissue clearly indicate the presence of specific binding sites, even after urea treatment. Potassium ions well-known specific inhibitor of ouabain binding to the Na⁺,K⁺-ATPase in many systems including the M. sexta brain (Rubin et al. 1981; Schwartz, Lindenmayer & Allen, 1975), also inhibits binding in the urea-treated abdominal nerve cord. In addition, the binding curve saturates at the relatively low concentration of 10 μM-³H-ouabain, corresponding to about 17·8 pmol ouabain-binding sites/mg protein. This is lower than the value of 85 pmol/mg protein previously measured in M. sexta brain homogenates (RubineZ al. 1981), a discrepancy possibly due to the presence of a large amount of protein-containing extraneural connective tissue comprising the dorsal mass in the intact connective. It is similar to the value of 44 pmol/mg protein previously reported in the frog retina (Stirling & Lee, 1980). Despite the fact that Whittam & Chipperfield (1973) showed an increase of two-to four-fold in total ouabain-binding by treating ox brain membranes with urea, we could see no effect in the M. sexta system. This was not surprising, however, since we used an intact as opposed to a partially purified preparation and since our urea treatment was somewhat different. The apparent dissociation constant (K_D) of 1·8 μM in the nerve cord is higher than the value of 0·1–0·2 μM measured in M. sexta brain homogenates. The reason for this is unclear although it is possible that the Na⁺,K⁺-ATPase in the urea-treated nerve cord preparation has a different structural integrity from that in the homogenate. This could affect the binding characteristics.

The finding that the moth nerve cord is impermeable to ouabain confirms the result of Pichón & Treherne (1974) using strophanthidin, a related cardioactive steroid inhibitor of the Na⁺,K⁺-ATPase. They showed that this compound has no effect on axonal function in the cockroach connective until the perineurial barrier is disrupted. Morphological work of Lane (1972) and McLaughlin (1974) on M. sexta showed that the perineurial tight junctions effectively exclude horseradish peroxidase and lanthanum from the inner elements of the cord. Our data (Figs 2, 6) indicate that the tight junctions are also impermeable to ouabain, a property also observed in epithelia (Shaver & Stirling, 1978; Stirling, 1972). Physiologically, the tight junctions probably act in M. sexta to prevent back-diffusion of extracellular Na⁺, which is present in high concentrations in the central nervous system to support neuronal activity, into the low-Na⁺ haemolymph. Indeed, Pichón et al. (1972) demonstrated clearly that axons in the intact M. sexta connective retained their ability to conduct action potentials for extended periods in Na⁺-free media. On the other hand, in desheathed connectives, where the perineurium has been physically disrupted, axonal activity was totally dependent on the presence of Na⁺ in the bathing medium. This suggests that the high extra-axonal Na⁺ was unable, in the intact preparation, to diffuse into the bathing medium. These tight junctions may also serve to protect the nervous system from depolarization by the high concentration of K⁺ (approximately 25 mw) present in the haemolymph.

Our finding of ouabain-binding sites in the ganglionic perineurium raises the question of the role of the perineurial Na⁺-K⁺ pump in maintaining the Na⁺ and K⁺ gradients between neural extracellular space and haemolymph inM. sexta. Treherne (1961, 1966) showed that ouabain, when applied to the outward side of the cockroach perineurium, inhibited Na⁺ efflux from the nerve cord. However, Pichón & Treherne (1974) could not demonstrate an effect of externally applied strophanthidin on the ktraneuronal potentials measured across the cockroach perineurium. Ethacrynic acid, on the other hand, had a marked effect. Thus they felt that an ethacrynic acid sensitive component in the outer perineurial membrane may have been more directly involved in the ion transport system rather than conventional Na⁺,K⁺-pumps from the same area. Since the perineurial ouabain-binding sites evident in Fig. 10 are probably predominantly associated with the outer-facing membrane (though we cannot exclude the possibility that some ouabain leaked in during the incubation and gained access to binding sites on the inner-facing membrane) it is possible that as in the cockroach, they are not directly involved in the transperineurial transport system. Recognizing that the requirements of the transport system may be quite different in these two species, there is not yet sufficient data in M. sexta to warrant firm conclusions on this point. In vertebrate sodium secreting epithelia basolateral Na⁺,K⁺ pumps have been directly tied to the secretory mechanism (DiBona & Mills, 1979; Ernst & Mills, 1977; Silva et.al. 1977). However, f or such a system to exist in M. sex ta a substantial negative transperineurial potential (neural extracellular space to haemolymph) would have to be present to allow for the transcellular concentrative movement of Na⁺. Apparently such a potential does not exist in M. sexta. Another possibility is that these pumps could function to concentrate Na⁺ in the lateral extracellular spaces just peripheral to the tight junctions to a level considerably higher than that found between the bulk haemolymph and the ECS. This would allow Na⁺ to move passively down a concentration gradient into the neural extracellular space. Schofield & Treheme (1975) have given evidence for a third possibility in the cock-roach, namely that Na⁺ is pumped from the perineurial cells and underlying electrically coupled glial cells into the neural extracellular space. This would require that pumps be localized on the neural side of the perineurium. We do not have sufficient data concerning this point since it is evident that the urea treatment destroys the perineurial binding sites shown in Fig. 10. It should be noted that we cannot identify ouabain-binding sites in the connective perineurium. This may be due to the fact that this structure is noticeably thinner than its counterpart in the ganglion, perhaps reflecting a greater degree of membrane amplification in the ganglion. This factor is commonly associated with a greater degree of ouabain binding (cf. Ernst & Mills, 1977).

This study indicates that the majority of ouabain-binding sites in the ganglia seem to be located in the synaptic areas (neuropil). There is also binding in the cell body layer, though it is hard to determine the relative levels since these structures seem most susceptible to osmotic damage by the urea-treatment. While the 30 s urea treatment that Pichón & Treherne (1974) used to make the cockroach nerve cord permeable to monovalent cations may have been more satisfactory in preserving cellular morphology, in our preparation, unfortunately, it did not make the perineurium adequately permeable to ouabain. The high numbers of ouabain-binding sites in the neuropil observed after urea treatment are not surprising in view of similar findings in the frog retina (Stirling & Lee, 1980) and sacculus (personal communication, J. A. Burnham). Most likely the small nerve fibres in this region need a high pumping capacity to maintain ion gradients (Siegel, Stahl & Swanson, 1981). We cannot, however, distinguish the relative glial and neuronal contributions to neuropillar ouabain binding because that is beyond the resolution of the technique. This would be an important point since a high level of ouabain binding in glia would support the tea that neuropillar glia respond to increases in extracellular K⁺ by transporting it actively into the glial cytoplasm.

Our demonstration of high levels of ouabain binding in the connectives is also not surprising in view of the earlier results of Pichon & Treherne (1974). They showed in the cockroach connective that the axonal resting potential was quite sensitive to strophanthidin, a cardioactive steroid with similar effects to ouabain on the Na⁺,K⁺-ATPase. Again, however, we could not distinguish between glial and neuronal binding.

In summary, we have demonstrated the existence of specific ouabain-binding sites in the intact abdominal nerve cord of the hawk moth, M. sexta. These sites are located predominantly in the axonal and neuropillar areas. However, there is a binding component in the ganglionic perineurium which may be involved in the transperineurial transport of Na⁺ from the haemolymph into the neural extracellular space.

We are indebted to Dr Arthur F. Clark for helpful discussions and to Margery Domenowske for her skillful illustration of the M. sexta central nervous system (Fig. 1). This work was supported by the Veterans Administration and by NIH Grants EY 02393 and AM 13182.