THE EFFECT OF LOW-CALCIUM SEA WATER AND ACTINOMYCIN-D ON THE SODIUM METABOLISM OF FUNDULUS KANSAE*

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1 J. Exp. Biol. (1974). 60, Printed in Great Britain THE EFFECT OF LOW-CALCIUM SEA WATER AND ACTINOMYCIN-D ON THE SODIUM METABOLISM OF FUNDULUS KANSAE* BY WARREN R. FLEMING, JAMES NICHOLSf AND W. T. W. POTTS Division of Biological Sciences, University of Missouri, Columbia, Mo Department of Biological Sciences, University of Lancaster, LAi 4 7 0, England (Received 4 July 1973) INTRODUCTION In a previous paper (Potts & Fleming, 1971) it was reported that transfer of the euryhaline cyprinodont, Fundulus kansae, from normal to calcium-free sea water evoked a sharp rise in sodium turnover, of about 130 %. An examination of the nature of this response was complicated by the fact that survival was poor in such an environment. This difficulty has been overcome by the observation that a similar response - at least in respect to sodium metabolism - can be demonstrated in a lowcalcium in which the animals survive indefinitely. The effect of such an environment on the kinetics of sodium efflux and on some aspects of gill metabolism has been investigated. MATERIALS AND METHODS The animals were collected from Boones Lick, Missouri, and held in the laboratory at 20 C. In most cases the animals were adapted to normal for at least 3 weeks prior to transfer into the low-calcium saline. In one experiment, in which actinomycin-d was used, the animals were moved into dilute (80 %) from fresh water, held there for 3 days, and then transferred on to the low-calcium environment. The low-calcium was prepared from a special commercial preparation (Instant Ocean) from which calcium (as CaCl 2.2H 2 O) had been withheld. This preparation did not provide a calcium-free saline, and analysis of solutions containing a normal content of sodium (467 m-equiv/1) showed a calcium content of nearly 1 m-equiv/1. Preliminary experiments showed that the animals had difficulty in surviving a direct transfer from fresh water to full-strength low-calcium ; they would, however, survive transfer into 80 % low-calcium. This solution, which contained 380 /^equiv Na + and o-8 /<equiv Ca 2+ /ml was used in the experiments described here. Gill RNA was isolated as the cetyltrimethyl ammonium salt after extraction and fractionation according to the method of Caldwell and Henderson (1970), and the total RNA in each fraction was estimated by the method of I-San Lin and Schjeide (1969). Usually, the gills from four animals were pooled for a single extraction. Supported by N.S.F. Grant GB t Present Address: Biology Department, State College of Arkansas, Conway, Arkansas EXB 60

2 268 WARREN R. FLEMING, JAMES NICHOLS AND W. T. W. POTTS Animals injected with 32 P were first adapted to 80 %, and were lightly anaesthetized with MS 222 and weighed prior to injection. The animals were given 2 /tc 32 P/g, allowed to recover from the anaesthetic for 30 min, and then transferred back to 80 % or to the low-calcium saline. Twelve hours later the animals were killed and the gill tissue was removed. The radiophosphorus present in RNA was counted by the usual liquid scintillation method after suspension in Aquasol (New England Nuclear). In order to follow the kinetics of sodium efflux, small animals weighing between 0-7 and o-8 g were injected with 0-2 fic 22 Na and, after a 30-minute equilibration period, placed in shell-vials containing 2 ml of saline and counted in a thalliumactivated sodium iodide well-crystal scintillation counter. Each fish was counted for 90 s and then transferred to a beaker containing 600 ml of the test saline - a volume of solution great enough to prevent any significant recycling of the isotope. Each animal was counted at 2-hour intervals for the first 8 hours, and at 4-hour intervals thereafter. 22 Na efflux followed the equation, C t = Ae~ k i t + Be~ k " t where k x and k 2 are the rate constants for sodium efflux from compartments A and B respectively and C t is the activity remaining after time t. The rate constants and compartment sizes were evaluated by graphical analysis after plotting the logarithm of the percentage of the radioisotope remaining against time. The methods used for analysis of whole-body sodium and potassium and for shortterm sodium efflux studies had been described elsewhere (Potts & Fleming, 1970, 1971). RESULTS The effect of a transfer from 80 % to the low-calcium on the kinetics of sodium efflux is reported in Table 1. Even in saline containing a normal amount of calcium the slow compartment is very small. Transfer into low-calcium saline accentuates this pattern; the second rate constant (k 2 ) decreases as does the size of the second compartment. In these experiments 22 Na efflux studies were started 12 h after transfer into low-calcium saline and continued for a total of 26 h. Similar experiments carried out after 9 days in low-calcium saline showed essentially similar kinetics. Other animals were transferred to low-calcium at the same time as those reported in Table 1, and these fish were killed for ion analysis at the end of the efflux experiment. As shown in Table 2, the experimental fish showed significantly lower plasma sodium levels and a less convincing drop in total body sodium levels than the controls left in 80 % calcium. Fish held in the low-calcium saline for 9 days had identical plasma and whole-body sodium levels to those in Table 2. The effect of a 12-hour exposure to low-calcium saline on radiophosphorus incorporation into gill RNA is shown in Table 3. A significant increase over the control levels is seen in both the ribosomal and srna fractions. Since exposure to low-calcium saline stimulates gill RNA metabolism, it seemed useful to examine the effect of actinomycin-d on sodium efflux and on whole-body electrolytes. Table 4 shows the response seen when animals that had been adapted to 80 % for 8 weeks were injected with 1 /ig/gm actinomycin-d and then transferred into low-calcium saline. 22 Na efflux was measured over a 2-hour period starting 20 h after transfer, and the

3 Effect of low-calcium and actinomycin-t) 269 Table 1. Changes in compartment size and rate constants of 22 Na efflux after transfer of low-calcium. Fish 1-5 are experimentals, 6-10, controls Fish A B I S o-9s ' o O-2I O-O O-II 017 O-I2 o-ii O-O cos Table 2. The effect of transfer to low-calcium on plasma Na + levels and on whole-body sodium and potassium concentrations Plasma Na +, /t-equiv/ml Total-body Na+, /t-equiv/g Total-body K+, /t-equiv/g Controls Experimentals P is7"8±i ±3-68 6S'74±3'Oi ±2-65 SS-42±i-2i Table 3. The effect of a transfer from 80% to low-calcium sea water on the incorporation of radiophosphorus into gill RNA RNA fraction Soluble tissue nucleotides Specific activity (cpm P 32 //*g RNA) Normal sea water Low-calcium 12,114 Ribosomal RNA Normal Low-calcium s-rna Normal Low-calcium 18-2

4 270 WARREN R. FLEMING, JAMES NICHOLS AND W. T. W. POTTS Table 4. The effect of actinomycin-d on sodium efflux and whole-body sodium and potassium concentrations of long-term sea-water adapted animals Total-body Na+ Total-body K+ Group n k efflux P (/tequiv/g) (/* equiv/g) SW-controls SW-act-D L-Ca 2+ controls L-Ca 2+ -act-d S 5 S 5 O-28 ±O'O4 O-27±O-O O-3I+O n.s ± Table 5. The effect of actinomycin-d on sodium efflux of short-term sea-water adapted animals k x efflux &! efflux Group n 1st period 2nd period SW-controls SW-act-D L-Ca s+ controls L-Ca a+ -act-d o-ioolo-oi Table 6. Whole-body sodium and potassium levels of short-term sea-water adapted animals after treatment with actinomycin-u andjor transfer to low-calcium Whole-body Na+ Whole-body K+ Group n (/tequiv/g) (/«equiv/g) SW controls SW-act-D L-Ca 2+ controls L-Ca 2+ act-d animals were then killed for measurement of their whole-body sodium and potassium. As shown in Table 4, the antibiotic did block the increase in sodium efflux seen in the control group. Actinomycin-D did not affect the efflux constants of those animals held in a normal calcium environment within the time of the experiment. The failure of the antibiotic to affect the sodium balance of the normal group may reflect nothing more than the stability of the gill RNA templates involved in regulating sodium efflux. With this possibility in mind, the experiment was repeated using animals that had been transferred from fresh water to 80 % 72 h prior to the time of injection. In thesefish the sodium effluxes had not yet reached the normal levels. The sodium effluxes were measured from the 22nd to 24th hours post-transfer, and from the 44th to 46th hours post-transfer. At the end of the experiment all the animals were killed and analysed for the whole-body sodium and potassium. The results are reported in Tables 5 and 6. An examination of the data presented in Table 5 shows that under the above conditions transfer into low-calcium saline again caused a substantial rise in sodium efflux which was largely blocked by the antibiotic. Further, actinomycin-d caused a significant decrease in the rate of efflux from the animals held in normal 80 % sea water. The effect of the antibiotic was more pronounced during the second efflux period. The antibiotic also affected the whole-body sodium and potassium concentrations of the animals transferred into low-calcium (Table 6) by elevating

5 Effect of low-calcium and actinomycin-d 271 sodium and reducing potassium. The low-calcium control group once again showed slightly lower whole-body sodium levels (P = < o-i > 0-05). Unexpectedly the whole-body electrolyte concentration of the group receiving actinomycin-d in normal saline was only slightly affected, although the sodium efflux rate was reduced to about 40 % of the control rate. DISCUSSION Previous studies from this laboratory have shown that a 3-day exposure to calciumfree markedly stimulated the sodium metabolism of Fundulus kansae, increasing sodium turnover from 24% to 53%/h. It was concluded that while the sodium influx was partly dependent on the external calcium concentration it must also depend upon a number of other extrinsic and intrinsic factors. A similar stimulation of the sodium fluxes has been seen in the European eel (Anguilla anguilla) after a 15-hour exposure to calcium-free (Bornancin, Cuthbert & Maetz, 1972). Such exposure caused a two-fold increase in sodium influx of animals held in saline, and a four-fold increase in sodium efflux after transfer to fresh water. Since both these fluxes were considered to be passive, and were readily reversible by the addition of calcium, it seemed probable that calcium had a generalized effect on sodium permeability. But other evidence presented by the Villefranche laboratory suggests that calcium may also play a role in sodium excretion per se. The high sodium efflux seen in potassium-enriched fresh water was sharply reduced after 15 h in calcium-free. Since the difference between the sodium efflux in potassium-enriched fresh water and the efflux in normal fresh water (following transfer from ) was considered to be active (Maetz, 1969); and since this component was in fact stimulated above control levels after addition of calcium, it seemed not unreasonable to suppose that calcium played some role in regulating both the active and passive components of sodium fluxes. On the other hand other evidence shows that the difference between the sodium efflux in normal and in potassium-enriched fresh water is due to differences in the potential across the gill membrane in the two solutions, which is a consequence of the differential permeability of the gill to sodium and potassium ions (Potts & Eddy, 1973). Further, since the continued stability of the potential difference requires the presence of environmental calcium (Potts & Eddy, 1973) it seems probable that the rapid restoration of the flux difference noted by Bornancin et al. (1972) after the addition of calcium, can be ascribed to the effect of calcium on the potential, rather than on cellular processes per se. However, the studies reported here suggest that in F. kansae, calcium does affect both the active and passive components of the sodium flux and further suggests that a proper balance of the passive and homeostatic mechanisms may be controlled not only by the presence of environmental calcium, but by additional factors such as a sodium/calcium ratio. Thus it was noted previously (Potts & Fleming, 1971) that while sodium turnover rose sharply in calcium-free, survival was poor. A similar effect on sodium metabolism was noted here after exposure to low-calcium saline, but survival was excellent. The animals became hyperexcitable after several days in the test saline, and showed little enthusiasm for the calcium-free diet provided (EDTA-washed brine-shrimp flakes), but in no case were the animals in any obvious difficulty even after 9 days exposure.

6 272 WARREN R. FLEMING, JAMES NICHOLS AND W. T. W. POTTS The rapid incorporation of radiophosphorus into gill RNA, after transfer to lowcalcium, suggests that the normal environmental calcium levels could serve either to stabilize or to inhibit the synthesis of the metabolic machinery required for sodium excretion, as well as the pathway(s) involved in sodium influx. Thus, both long-term and short-term adapted animals have lower sodium levels shortly after transfer to low-calcium saline (Tables 2 and 6), although the sodium influx must be increased in the low-calcium saline. This is a transient response, however, for no differences were noted in animals held in the test solution for 9 days. Since the response was consistent, we are led to suspect that while both components of sodium flux are stimulated after transfer, efflux slightly exceeds influx initially. The notion that low environmental calcium levels may serve to stimulate the metabolic machinery involved in sodium metabolism finds some support from the actinomycin-d experiments. In contrast to the eel (Maetz et al. 1969; Motais, 1970), the killifish is relatively insensitive to the antibiotic, especially animals that have been held in for some time. Usually a dose level twice that generally used by the Villefranche laboratory will not cause a significant drop in sodium turnover until 4 or 5 days after the injection, and that response rarely lasts more than a day. In contrast to the eel, the killifish usually survives such treatment in. Part of the difference may be due to a greater stability of the RNA templates concerned with sodium metabolism in the killifish, or it may be that the antibiotic is more effective in blocking RNA metabolism in the case of the eel. Preliminary data shows that the dose level of actinomycin-d used here does depress - but does not completely block - radiophosphorus incorporation into gill RNA. Further, the ratio of uridine/cytosine incorporated into RNA is not affected - while transfer into low-calcium affects the ratio markedly (Nichols & Fleming, unpublished data). While the antibiotic may show only a slow and transient effect on the sodium metabolism of fully adapted sea-water animals, it is highly effective in blocking the stimulation of sodium efflux seen after transfer to low-calcium saline (Table 4). Further, since the antibiotic does not affect the sodium metabolism of fishes fully adapted to sea water (Table 4) we may suppose that transfer into low-calcium serves to stimulate gill sodium metabolism specifically. However, when animals that have had only a short-term exposure to are challenged by actinomycin-d, the effects are more pronounced. These animals have not had time to become fully sea-water adapted, nor to develop a full complement of the cellular machinery used in such environments. In this case the antibiotic affected sodium efflux regardless of the environmental calcium concentration, i.e. a reduction to roughly 40 and 33 % of the control levels in normal and low-calcium saline respectively. In the former case the reduction in sodium efflux produced little change in the total electrolyte pattern; in the latter case, whole-body sodium rose sharply and whole-body potassium level fell. It seems probable, then, that transfer into lowcalcium does cause some instability of the pre-existing metabolic machinery, as well as a stimulation of the synthetic pathways that lead ultimately to a high level of balanced sodium fluxes. The nature of the response to the antibiotic after transfer to low-calcium saline apparently depends upon the amount of metabolic machinery susceptible to attack. It also suggests that the presence or level of environmental calcium may be reflected in several ways, i.e. as a regulator of permeability to mono-

7 Effect of low-calcium and actinomycin-d 273 valent ions and to water, as a stabilizer of the metabolic process involved in regulating sodium fluxes, and as an inhibitor of the synthetic processes that determine the rate of balanced sodium fluxes. SUMMARY 1. Transfer of Fundulus kansae from 80 % to a low-calcium water containing 0-4 mm/1 Ca 2+ caused a sharp rise in sodium efflux and a change in the kinetic pattern of efflux. 2. A transient drop in whole-body sodium levels occurred within 1-2 days after transfer. Both sodium and potassium levels were normal after 9 days exposure to low-calcium saline. 3. Transfer into low-calcium increased the rate of incorporation of radiophosphorus into gill RNA. 4. Actinomycin-D blocked the stimulation of sodium turnover after transfer into low-calcium. It did not affect the whole-body sodium or potassium levels of long-term sea-water adapted animals. 5. Actinomycin-D reduced the sodium efflux of short-term sea-water adapted animals regardless of the environmental calcium concentration. The antibiotic also upset the balance of sodium fluxes in those animals held in low-calcium. 6. It is suggested that in addition to the generalized effect of calcium on permeability to monovalent ions and water, calcium serves to inhibit some of the synthetic processes involved in regulating sodium metabolism, and also serves to stabilize the metabolic machinery already present. REFERENCES BORNANCIN, M., CUTHBERT, A. W. & MAETZ, J. (1972). The effects of calcium on branchial sodium fluxes in the adapted eel, Anguilla anguilla, L. J. Physiol. 222, CALDWELL, I. C. & HENDERSON, J. F. (1970). Isolation of nucleotides, nucleic acids and protein from single tissue samples by a phenol technique. Anal. Biochem. 34, I-SAN LIN, R. & SCHJEIDE, O. A. (1969). Micro estimating of RNA by the cupric ion catalyzed orcinol reaction. Anal. Biochem. 27, MAETZ, J. (1969). Sea water teleosts: Evidence for a sodium-potassium exchange in the branchial sodium-excreting pump. Science 166, MAETZ, J., NIBELLE, J., BORNANCIN, M. & MOTAIS, R. (1969). Action sur osmoregulation de l'anguille de divers antibiotiques inhibiteurs de la synthese des proteins ou du renouvellement cellulaire. Comp. Biochem. Physiol. 30, MOTAIS, R. (1970). Effect of actinomycin-d on the branchial Na-K dependent ATP-ase activity in relation to sodium balance of the eel. Comp. Biochem. Physiol. 34, POTTS, W. T. W. & FLEMING, W. R. (1970). The effects of prolactin and divalent ions on the permeability to water of Fundulus kansae. J. exp. Biol. 53, POTTS, W. T. W. & FLEMING, W. R. (1971). The effect of environmental calcium and ovine prolactin on sodium balance in Fundulus kansae, J. exp. Biol. 54, POTTS, W. T. W. & EDDY, F. B. (1973). Gill potentials and sodium fluxes in the flounder {Platichthys flesus) (in Press).

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