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The Effect of Low Calcium Sea Water and Actinomycin D on the Sodium Metabolism of Fundulus Kansae


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J. Exp. Biol. (1974). 60, 267-273 2 6 7 Printed in Great Britain





Division of Biological Sciences, University of Missouri, Columbia, Mo. 65201. Department of Biological Sciences, University of Lancaster,

LAi 4 7 0 , England

(Received 4 July 1973)


In a previous paper (Potts & Fleming, 1971) it was reported that transfer of the euryhaline cyprinodont, Fundulus kansae, from normal sea water 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 low-calcium sea water 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.


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 sea water 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 %) sea water from fresh water, held there for 3 days, and then transferred on to the low-calcium environment.

The low-calcium sea water was prepared from a special commercial preparation (Instant Ocean) from which calcium (as CaCl2.2H2O) had been withheld. This pre-paration did not provide a calcium-free saline, and analysis of solutions containing a normal sea water 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 sea water; they would, however, survive transfer into 80 % low-calcium sea water. This solution, which contained 380 /^equiv Na+ and o-8 /<equiv Ca2+/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 23798.

t Present Address: Biology Department, State College of Arkansas, Conway, Arkansas 72032.



Animals injected with 32P were first adapted to 80 % sea water, and were lightly anaesthetized with MS 222 and weighed prior to injection. The animals were given 2 /tc32P/g, allowed to recover from the anaesthetic for 30 min, and then transferred back to 80 % sea water 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 22Na and, after a 30-minute equilibration period, placed in shell-vials containing 2 ml of saline and counted in a thallium-activated 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. 22Na efflux followed the equation, C

t = Ae~kit + Be~k"t where kx and k2 are the rate

constants for sodium efflux from compartments A and B respectively and Ct is the

activity remaining after time t. The rate constants and compartment sizes were evalu-ated by graphical analysis after plotting the logarithm of the percentage of the radio-isotope remaining against time.

The methods used for analysis of whole-body sodium and potassium and for short-term sodium efflux studies had been described elsewhere (Potts & Fleming, 1970,



The effect of a transfer from 80 % sea water to the low-calcium sea water 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 (k2) decreases as does the size

of the second compartment. In these experiments 22Na 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 sea water 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 sea water. 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 in-corporation 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 % sea water for 8 weeks were injected with 1 /ig/gm actinomycin-D and then transferred into low-calcium saline. 22


Effect of low-calcium sea water and actinomycin-T)


Table 1. Changes in compartment size and rate constants of 22Na efflux after transfer of low-calcium sea water. Fish 1-5 are experimentals, 6-10, controls

Fish A B

I 2 3 4 S 6 7 8 9 1 0

0 9 7 0 9 2 0-94

0 9 4 o-9S 0-89

0 8 3 0 8 8 0 8 9


0 4 8



0 3 9 o-37 0-19 O-2I 0 1 7 0-28 0-19

O-O3 0 0 8 0-06 0-06 0 0 5 O-II 0 1 7 O-I2 o - i i 0-24

0-02 0 0 4 O-O2 0-04 0-04 0-07 0-06 0-08 0 0 8

c o s

Table 2. The effect of transfer to low-calcium sea water on plasma Na+ levels and on whole-body sodium and potassium concentrations

Controls Experimentals


Plasma Na+, /t-equiv/ml iS7"8±i-93 Total-body Na+, /t-equiv/g 75-4° ±3-68 6S'74±3'Oi Total-body K+, /t-equiv/g 54-92 ±2-65 SS-42±i-2i


0 0 5

Table 3. The effect of a transfer from 8 0 % sea water to low-calcium sea water on the incorporation of radiophosphorus into gill RNA

RNA fraction Soluble tissue nucleotides

Specific activity (cpm P32//*g RNA)



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-Ca2+ controls


S 5 S 5

O-28 ±O'O4 O-27±O-O2 0-4810-03 O-3I+O-02

— 63-3914-1

n.s. 71-01+2-2 53-4612-2 — 63-7514-4 51-53 ±2-9 0-05 69-9011-9 57-1212-1

Table 5. The effect of actinomycin-D on sodium efflux of short-term sea-water adapted animals

kx efflux &! efflux

Group n 1st period 2nd period

SW-controls 5 0-192 + 0-02 0-21210-03 SW-act-D 5 0-13610-02 0-08410-01

L-Cas+controls 5 0-278 + 0-01 0-32410-03

L-Caa+-act-D 5 0-16810-02 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 sea water

Whole-body Na+ Whole-body K+ Group n (/tequiv/g) (/«equiv/g)

SW controls 5 68-1211-32 45-20+1-53 SW-act-D 5 72-61+4-32 44-03 + 5-68

L-Ca2+controls 5 62-16 + 2-74 50-12 + 4-85

L-Ca2+act-D 5 84-89 + 5-56 36-14 + 2-88

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 sea water 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 % sea water 72 h prior to the time of injection. In these fish 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.


Effect of low-calcium sea water 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.


Previous studies from this laboratory have shown that a 3-day exposure to calcium-free sea water 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 sea water (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 sea water. Since the difference between the sodium efflux in potassium-enriched fresh water and the efflux in normal fresh water (following transfer from sea water) 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 un-reasonable 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.



The rapid incorporation of radiophosphorus into gill RNA, after transfer to

low-calcium sea water, suggests that the normal environmental low-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 meta-bolic machinery involved in sodium metabolism finds some support from the actino-mycin-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 sea water 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 sea water. Part of the differ-ence 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 - radio-phosphorus incorporation into gill RNA. Further, the ratio of uridine/cytosine incorporated into RNA is not affected - while transfer into low-calcium sea water affects the ratio markedly (Nichols & Fleming, unpublished data).

While the antibiotic may show only a slow and transient effect on the sodium meta-bolism of fully adapted sea-water animals, it is highly effective in blocking the stimula-tion 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 sea water serves to stimulate gill sodium metabolism specifically.


mono-Effect of low-calcium sea water 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.


1. Transfer of Fundulus kansae from 80 % sea water to a low-calcium water con-taining 0-4 mm/1 Ca2+ 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 sea water increased the rate of incorporation of radio-phosphorus into gill RNA.

4. Actinomycin-D blocked the stimulation of sodium turnover after transfer into low-calcium sea water. 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 sea water.

6. It is suggested that in addition to the generalized effect of calcium on perme-ability 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.


BORNANCIN, M., CUTHBERT, A. W. & MAETZ, J. (1972). The effects of calcium on branchial sodium fluxes in the sea water adapted eel, Anguilla anguilla, L. J. Physiol. 222, 487-96.

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, 303—11.

I-SAN LIN, R. & SCHJEIDE, O. A. (1969). Micro estimating of RNA by the cupric ion catalyzed orcinol reaction. Anal. Biochem. 27, 473-83.

MAETZ, J. (1969). Sea water teleosts: Evidence for a sodium-potassium exchange in the branchial sodium-excreting pump. Science 166, 613-15.

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, 1125-51.

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, 497-501.

POTTS, W. T. W. & FLEMING, W. R. (1970). The effects of prolactin and divalent ions on the perme-ability to water of Fundulus kansae. J. exp. Biol. 53, 317-27.

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, 63-75.

POTTS, W. T. W. & EDDY, F. B. (1973). Gill potentials and sodium fluxes in the flounder {Platichthys



Table 3. The effect of a transfer from 80% sea water to low-calcium seawater on the incorporation of radiophosphorus into gill RNA


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