A M . ZOOLOGIST 10-365-376 (1970).
The Study of NaCl Transport in Aquatic Animals
LEONARD B. KIRSCHNER
Department of Zoology, Washington State University,
Pullman, Washington 99163
SYNOPSIS. The first part o£ the paper is devoted to the methods used for studying
transfer of ions between intact aquatic animals and their environments. The use of
flux-force relationships is described, and some alternative criteria for delimiting active
transport are examined.
The second part discusses recent research on hyper- and hypotonic regulators.
In the first paper of this symposium, Dr.
Koch developed criteria for passive and
active transfer of material across permeable interfaces. The next two papers discussed, among other things, applications of
these principles to single cells and isolated
transport epithelia such as the frog's skin.
The purpose here is to show that the same
analytical approach can be applied to intact animals. We will begin by describing
some methods used for obtaining values for
the forces acting across permeable parts of
the body surface, and then will show how
the appropriate fluxes can be estimated. A
brief survey of recent research on aquatic
animals will illustrate the kinds of information that have become available as a
result of such investigations.
FLUX-FORCE RELATIONSHIPS AND ACTIVE
TRANSPORT
The Electrochemical Potential Difference
One force acting on a substance is the
gradient of its chemical potential. For
most useful solutions of the diffusion equation (i.e. the flux-ratio or Goldman equations discussed by Dr. Koch) values are
needed for concentrations on both sides of
a transport site, usually in the body fluid
and external bathing medium. With the
armament of microsampling and analytical
procedures available this rarely presents a
problem.
The work in my laboratory has been supported
by grants from the National Science Foundation
(GB-811), the National Institute of General Medical Sciences (GM-04254), and the State of Washington Initiative Measure 171.
If we are examining movement of ions it
is also necessary to measure any potential
difference across the body surface. Electrical measurements are made too infrequently, but they have been reported in
several animals and appear to develop
across transport epithelia such as skin or
gills. We will call them transepithelial potentials (TEP's). Some recent research on
electrogenesis will be described later, and
it suffices here to note that they are variable, not merely among different kinds of
animals but even within a species. That is,
the magnitude and polarity may depend
on environmental conditions (concentrations, temperature, etc.) and on the state
of the animal. This is illustrated by the
range of values in Table 1. Obviously, it is
dangerous to rely on published values unless the conditions under which they were
obtained are reproduced.
Fortunately, the TEP is reasonably simple to measure in most cases. A saline
bridge (often 3M KC1 in agar) can be
inserted through the body wall in such a
way that electrical leakage is negligible. A
second bridge is in contact with the external solution, and each is connected
through a calomel electrode to a potentiometer (Fig. 1). In our work the animals
are lightly anesthetized (0.25% urethane or
0.01% tricaine methane sulfonate), a
procedure which appears to affect neither
ionic fluxes nor TEP's (unpublished
studies on frogs and salamanders).
Undirectional Measurements of Flux
365
Tracer experiments, which provide data
36G
LEONARD B. KTRSCHNER
TABLE 1. TEP's of fresh-water animals in dilute media
Species
Salmo gairdneri*
Bnna esadenla
Rana pipiens
Astacus pallipes
Ambystoma tigrinum
Anguilla anguilla
Blennius pholis
Palaemonetes antennarius
Aedes aegypti
•Nfn
-L^ <*OUt
TEP
(mM)
(mVolts)
0.9
3
1
2.0
1.2
<1
45
0.5
2.9
5
85
13
5
15
—18
2
—33
9
Reference
Keistetter (unpublished)
Barker-Jorgensen, et al. (1954)
Brown, 1962
Bryan, 1960
Dietz, et al. (1967)
Maetz and Companini (1966)
House (1963)
Parry and Potts (1965)
Stobbart (1965)
* The external medium contained 0.5 mM CaCl2. In the absence of Ca2* the TEP was about
—20mV.
for estimating unidirectional ionic fluxes,
can be designed in many ways. If a pair of
radio-active isotopes is available {e.g.,
- N a and -4Na, •<!«C1 and 38C1) they can be
used simultaneously, one to measure
influx and the other efflux. This is a powerful technique, especially when the two
fluxes are not equal; that is, when there is
net uptake or loss of the ion. However, it
has never been employed in whole-animal
experiments, and under certain conditions
the same information can be obtained with
only one isotope. The latter may be
added to the external bathing solution or
can be injected into the animal; examples
of both will be shown below. In principle,
samples for analysis can be taken either
from the animal's blood or from the external bath, but in practice bath samples are
usually used because they are more easily
obtained.
The theoretical basis of isotopic fluxmeasurements has been described by several investigators (Robertson, 1957, Solo-
FIG. 1. Measurement of the transepithelial potential difference in an aquatic animal.
mon, 1960, Sheppard, 1962). A recent
study of NaCl movement in teleosts also has
a useful section on tracer methods (Motais, 1967). In most of the research on
intact aquatic animals, two simplifying assumptions are usually made, and it sometimes appears that the investigator does
not appreciate them. One of these is that
the animal is in a steady state (influx and
efflux are equal); the other is that the
animal and external medium comprise a
simple two-compartment system. The first
is sometimes correct, but often is not, for
experimental animals are frequently in
positive or negative salt balance. The second is probably never true. Animals are
simply not well-mixed bags of the element
studied. At the very least they comprise
two large fluid compartments, muscle and
extracellular fluids, each with its pool of
the element and communicating with the
other through a system of cell membranes
whose permeability is very different from
that of the skin or gills. Thus, an animal
and its environment consist of at least
three compartments in series, and this can
complicate tracer kinetics. The papers by
Motais and Solomon present good discussions of the kinds of multicompartment
analysis that are sometimes necessary. Fortunately, the two-compartment analysis often suffices.
The treatment of tracer kinetics developed in the Appendix leads to an apparently simple relationship between
tracer movement and one-way ionic flux
across the body surface of an intact animal. For example, if we add an isotope of
367
TRANSPORT OF N A C L IN AQUATIC ANIMALS
InQ* = —
20
40
Minutes
FIG. 2. Uptake of
gairdneri.
3G
C1- and net Cl- flux in S.
Na+ to the external medium, the influx of
sodium, Jin, is
dQ*
^
'
T
V
Jnet^Mn
dt
J1U =
,
(3)
•^out—'Mn
where the symbols are denned in the Appendix, and as noted there, no assumption
is made about whether or not the animal is
in a steady state. A typical experiment is
shown in Figure 2 where the gills of a
rainbow trout (Salmo gairdneri) were perfused with artificial pond water (NaCl =
0.9 111M) containing 36 C1-. Samples removed every 20 minutes permitted measurement of the total radioactivity and total CI~ in the perfusate. The derivative in
Equation (3) is simply a tangent to the
isotope curve at any time during the experiment, and the specific activities (Xln
and Xout) are measured at the same time.
The net flux, Jnet, is a tangent to the total
chloride curve also measured at that time.
Note that all of these quantities may be
changing very rapidly, but as long as an
instantaneous value for each can be estimated, Ji,, can be calculated. If the experiment is short and Xln remains much smaller than X,,ut, the terms containing Xin can
be neglected, and it is not necessary to get
a blood sample. If, in addition, J net is small
so that Qout is nearly constant, J in is given
by Equation (5)
ln
(5)
Qoat
where Qout is the total quantity of ion in
the external solution, Q*out is the total radioactivity left in the solution at time t,
and Q*out(o) is the initial quantity of isotope. This method, too, requires no blood
samples. In addition, the relationship between InQ* and t is linear, and graphical
analysis is less subjective than in the previous example.
Figure 3 shows a different set of experimental results with larval salamanders.
The isotope was injected into the animal
and its appearance in the external bath
measured. For the first six hours the animal was losing sodium, and if the experiment had ended with only the data collected during that period, the general, non
steady-state solution, Equation (3), would
have been required. Other examples of
non steady-state situations have involved
salamanders (Kirschner, et al. 1970) and
fish (Motais, 1967) adapted to one salinity
and transferred to another for measurement.
Partitioning a Unidirectional Flux
Values for the total influx and efflux of
an ion are useful, but are often not adequate for the flux-force analysis we wish to
make. This is best explained by illustration. In a fresh-water animal, virtually the
entire influx goes across the body surface,
and hence is subjected to the electrochemical potential gradient across it. But the
r
^—-r
'^"^
X
§20
CPM
si
"5
- B to
No
/ -
10
20
Hours
FIG. 3. Efflux of B NV and net Na* flux in Ambystoma tigrinum.
368
LEONARD B. KJRSCHNER
TABLE 2. Partitioning sodium influx in larval
Ambystoma tigrinum
Experiment
Procedure
la
lb
2
cloaca blocked**
control**
free-flow***
JTtotal*
out
0.7
1.8
0.88
dTrenal
out
—
—
0.57
*
J.kln*
out
0.7
—
0.31
* (ieq (10 g m ^ h i - 1 .
** Efflux of sodium was measured in animals in
which urine flow was blocked by a purse-string
suture around the anal papilla (la). The values
were compared with controls which had been
sham-stitched. The influx (not shown) was
equal to total efflux.
*** Efflux of sodium was partitioned by measuring
the flow of urine with inulin as described in the
text. Influx was equal to the total efflux.
efflux comprises two independent outflows.
Part of the loss is across the body surface,
but another fraction is in the urine. Only
the former is affected by the electrochemical gradient, and this is the efflux value
needed. It can be obtained by either of two
methods. If the excretory aperture can be
blocked, loss through the kidney is eliminated and the measured efflux equals that
across the body surface. This was done (Alvarado and Kirschner, 1963) for larval
salamanders, with results shown in the top
row of Table 2. The second row of data
shows total fluxes in control animals. Note
that blocking the excretion of urine reduces the efflux of Na+ by 61%. The relevant flux data are in column 3, and the
flux ratio across the body surface is 2.6.
With the animals in a steady-state, use of
the total fluxes would give a ratio of 1.0.
The second method is to measure renal
efflux and subtract it from the total. Flow
of urine can be measured by the rate of
inulin excretion and its concentration in a
terminal urine sample (Fig. 4). The ion
concentration in the terminal sample is
also measured and assumed to be constant
during the measurement. The product of
volume flow and ion concentration gives
the rate of excretion of the ion in the
urine. Data for salamanders are shown in
the last line of Table 2, and the renal
contribution was 63% of the total efflux, in
good agreement with the other method.
The same need to partition a total flux
arises in the case of hypotonic regulators,
such as marine fish, but here it is the
influx that must be analyzed. The situation
is complicated by a rapid "exchangediffusion" (see below), but considering
only diffusive movement and active transport, part of the total occurs through the
gill and another fraction through absorption in the gut. When flux-force analyses
are attempted it will be necessary to eliminate the latter to obtain Jln and Jout values
for the gill alone.
Identification of Active Transport
When we have measured the electrochemical potential difference and fluxes
across the body surface we can use the
flux-ratio criterion to determine whether
an ion is actively transported or is merely
moving passively. If the latter, the following relationship must hold
Jin
(6)
Jout
where the "expected" flux ratio is calculated from concentrations (Cout and Cin)
and the potential difference (E). The "observed" flux ratio is obtained from tracer
measurements of Jin and Jout. When the two
s
Slope II2XIO 5 CPM/HR
Urine [CMJ 345X10* CPM/ML
Volume Flow 0326 ML/HR
Hours
FIG. 4. Inulin excretion by Ambystoma tigrinum.
The "C-inulin was injected subcutaneously two
days before Lhe measurements were made. The
linear increase in concciuralion of inulin in the
medium suggests that its concentration in the
blood was practically constant during the experiment.
369
TRANSPORT OF N A C L IN AQUATIC ANIMALS
TABLE 3. Active transport of sodium and chloride in aquatic animals
Concentration
(mM)
Animal
Ion
out
in
c»/c,
E*
(mV)
Ambystoma
Jtana
Salmo
Astacus
Ambystoma
Sana
Salmo
Astacus
Na
Na
Na
Na
Cl
Cl
Cl
Cl
1.2
3
1.0
o
1.2
3
1.0
0.3
100
100
150
205
80
85
130
184
0.012
0.030
0.007
0.010
0.015
0.035
0.008
0.016
+ 14
+85
+ 15
+ 5
+14
+85
+ 15
—28
Jln/J out
expected found
0.007
0.001
0.004
0.008
0.026
0.93
0.034
0.021
2.6
0.6
1.4
1.1
1
1.1
1.8
1.0
Ref.**
1,2
3
4
5
1,2
3
4
6
* Sign is that of the body fluids.
** 1. Alvarado and Kirschner, 1963. 2. Dietz, et al., 1967. 3. Barkcr-Jorgenson, et al., 1954.
4. Unpublished. 5. Bryan, 1960. 6. Shaw, 19606.
estimates agree, the observed movements
are assumed to be passive. If they differ
appreciably we usually attribute the discrepancy to active transport. Table 3
shows the results of this type of analysis for
both Na+ and CI~ fluxes in four aquatic
animals. In every case the observed flux ratio for sodium is several orders higher than
that predicted by Equation (6). We conclude that Na+ is actively transported inward. For three of the animals it is clear
that Cl~ is also transported inward. In the
frog, however, the observed ratio is not appreciably different from the predicted, and
movement of Cl~ appears to be passive in
these experiments.
It is worth noting that a different approach is sometimes used. Total fluxes are
measured and if they are equal (i.e., if the
animal is in a steady state) the Nernst equation (rf. Koch's Equation 14) is used as the
criterion for passive movement. But, as
noted in the previous section, part of a
total flux may be unrelated or only indirectly related to the electrochemical gradient across the body surface, and use of
the Nernst relationship may be unwarranted.
The flux ratio is a powerful tool for
delimiting active transport but is not infallible. That is, there are cases in which the
flux ratio does not appear to conform to
the requirements of passive movement, but
which we hesitate to call active transport.
One of these, "file diffusion", has never
been noted in epithelia and will not be
discussed. A second, "entrainment", was
mentioned by Dr. Lindley and involves
movement of one substance against a gradient of its potential because it is coupled
to the downhill movement of a second substance. The energy for moving the former
derives from the electrochemical gradient
of the latter, and no metabolic input is
required. Entry of Na+ and Cl~ into the
cells of epithelia such as the skins or gills
of aquatic animals may involve such coupled transfers of ions. This will be described briefly later. There is also one situation in which the flux ratio is closer to
unity than would be the case if the fluxes
were clue to simple passive leaks with or
without a transport system. This is a special case of entrainment in which movement of an ion in one direction is obligatorily coupled with the simultaneous
movement of the same species in the other
direction, a process called "exchangediffusion" (Ussing, 1947). It should be
noted that it differs fundamentally from
active or diffusive transport both of which
can generate net transfer. Exchangediffusion cannot; in fact, without tracers
we should be ignorant of its existence.
However, it adds a component to both
unidirectional fluxes, one that is insensitive to the electrochemical gradient and to
transport inhibitors.
Exchange-diffusion
of sodium has been observed in crayfish
(Shaw, 1959) and in larval salamanders
and rainbow trout (unpublished data). A
particularly troublesome case in hypotonic
370
LEONARD B. KIRSCHNER
regulators will be described below.
Other criteria for active transport are
therefore useful, although none is free of
objection if applied alone. If a flux is coupled with energy metabolism it may well
be active, although the use of compounds
like cyanide and dinitrophenol to test for
metabolic linkage has obvious drawbacks
in intact animals. Specific transport inhibitors like the cardiac glycosides must also be
used cautiously, although those that are
effective from the outside may prove to be
powerful tools.
There is an impressive body of evidence
to show that active ion transport systems are
saturable. That is, the active flux depends
on concentration of the transported ion at
low concentrations but approaches an upper limiting value (Jmttx) at high concentrations. Such saturation kinetics have been
shown for several fresh-water forms. Na+
movement across trout gills provides a
good example (Fig. 5). This relationship
is described approximately by
J,m,x[Na+]0Ut
10
20
50
Sodium
FIG. 5. liillux ot sodium and external [Na+] in S.
gairdncri. The points are mean values for small
groups of animals. Vertical bars show ± 1 s.e. The
high value at 7.5 mM may represent a change in
permeability. It is also seen in salamanders at
about 10 mM.
not characteristic of diffusive movement
and provide a criterion for chemically
mediated transfer, including active transport.
TRENDS IN RECENT RESEARCH
Hyperlonic Regulators
K nl H-[Na+] 0Ut
and its derivation can be found in almost
any treatment of enzyme kinetics (cf. Potts
and Parry, 1964). The system is characterized by the two parameters Jmnx (the maximum pump flux) and Km which is the
concentration at which the flux is half
maximum (Jir, = i/2 Jmnx). Some values of
these parameters in fresh-water species are
given in Table 4. Their significance in
determining how dilute an environment
can be tolerated has been discussed by
Shaw (1961). But in the present context
we simply note that saturation kinetics are
It is clear that fresh-water animals actively transport both Na+ and Cl~ inwards. Moreover, Krogh (1938) showed
that the two mechanisms are independent.
If one of these animals is placed in Na2SO4,
the cation can be absorbed even though
SO.J-- cannot; and in KCI solutions,
Cl~, but not K+, is taken up. Similar
observations have been made on crayfish
(Shaw, 1960 a, b), larval salamanders
(Dietz, el al. 1967) and rainbow trout
(unpublished data), which suggests that
the mechanisms are widely distributed. In
order to preserve electrostatic neutrality in
TABLE 4. Parameters of sodium influx in fresh-water animals
Kill
Animal
Antaeus pallipes
Gammarus pulex
Salmo gairdneri
Aedes aegypti
Gammarus lacu.ilris
Sana pipifns
2
1
1
* fioq (cm )- hr" .
80
mM
Jmajr
(mM)
^eq (10 gm)- 1 hi- 1
0.2-0.3
0.15
3.6
20
3.3
]20
26
0.5
0.55
0.14
5
1.5*
Reference
Shaw (1959)
Shaw and Suteliffe (1961)
Kerstetter (unpublished)
Stobbart(1965)
Suteliffe and Shaw (1965)
Brown (1962)
371
TRANSPORT OF X A C I . IN AQUATIC; ANIMALS
such single ion transfers the skin or gills
must contain a pair of ionic exchange systems each capable of operating independently of the other. Little is known about
the nature or location of these exchange
mechanisms. For many years it has been
believed that absorption of cations involves coupling of influx of Na+ with excretion of NH 4 +, because the two were
approximately equivalent when animals
were in pond water (Krogh, 1938, Shaw,
1960a, Dietz, et al., 1967). Recent work
rules out this mechanism in at least one
case and makes it suspect generally. The
evidence can be summed up as follows. In
one species of frog, Calyptocephalella gayi,
no NH4+ is excreted across the body surface; absorption of Na+ is balanced by excretion of H+ (Garcia-Romeau, et al.,
1969). If one examines the fluxes in the
work previously cited it can be seen that
even when ammonia is excreted the relationship with sodium-uptake is rarely stoichiometric. For example, in the salamander, NH4+ efflux averaged only 70% of
Na+ influx. Recent, unpublished work on
the trout shows that NH4+ excretion in
pond water (XaCl = 1 mM) is about the
same as Na+ uptake, but when the latter
changes by as much as a factor of 10 there
is no corresponding change in the former.
Thus, obligatory coupling must be ruled
out in this animal. Some evidence suggests
that exchange in the trout, as in Calyptocephalella gayi, is with H+. More research
may show that this is a general rule; as a
corollary, the correspondence noted between Na+ influx and XH4+ efflux may be
fortuitous.
There is some evidence that uptake of
Cl- is coupled with excretion of HCO 3 - in
frogs (Krogh, 1937, Garcia-Romeau, et al.,
1969), goldfish
(Maetz and GarciaRomeau, 1964) and crayfish (Shaw,
1969b).
It is tempting, but speculative, to place
the exchange steps at the outer membrane
of the epithelial cells where they would
permit entry of Xa+ and Cl~ into the cell.
The obligatory coupling suggested is an
interesting example of entrainment in
which uphill movement of Xa+ and Clare generated by outward diffusion of H+
and HCO-j- ions generated within the
cells. A role for the en/.yme, carbonic anhydrase, was suggested by the work of Maetz
and Garcia-Romeau (1964) in goldfish
and has also been established in trout
(unpublished data).
Jl entry of Xa+ and Cl~ into the cells is
mediated by a pair of ion-exchange
mechanisms, there still remains the problem of transporting them from cells to
blood. This step has been studied only in
isolated epithelia and will not be reviewed
here. It is unlikely that research on intact
animals will contribute much to our understanding of this important process.
One interesting and important concomitant of ion transport is the generation of
transepithelial electrical potential differences (TJGP's), and studies on intact animals provide some of the data. As noted
above, when animals are in fresh water
(NaCl = 1-5 mM) there is a TEP across
the body surface. In nearly every case the
body fluids are positive to the environment.
If the external NaCl is replaced by a nonabsorbable salt (e.g., K2SO4) the TEP is
abolished or replaced by a small TEP of
the opposite polarity. Stepwise increases in
100
eo
60
/
»
40
"
1
2
a
\
/
/
\
20
0
/
V
-ZO
01
10
[No*]mM
10
100
FIG. 6. TEP and external [Na+] in Rana pipiens.
Measurements were made as described by Dietz, et
al. (1967), and the same results were obtained on
their larval salamanders. These frogs had been
salt-depleted for 10 days and the TEP's are considerably higher than those .seen in animals adapled
to pond-waier.
372
LEONARD B. KTRSCHNER
NTa+ concentration increase the TEP in potonic to the environment is a wellsteps (Fig. 6). At concentrations below 10 known characteristic of marine teleosts,
mM the TEP is indifferent to the anion, but appears among the invertebrates as
which appears to show that electrogenesis well, e.g., in the brine shrimp, Artemia.
depends primarily on transport of sodium. The problems of osmotic desiccation and
The TEP is little affected by varying salt loading have been recognized for many
[Cl~] in the absence of sodium (i.e., in years, as well as the broad outlines of
KC1 solutions) even though net uptake of their solutions. These animals drink sea
Cl- takes place (Dietz, et al., 1967). At water and absorb much of the fluid
higher concentrations the anion makes a ingested so that the total inflow of water
difference. The decrease in TEP caused by just balances osmotic and renal losses. The
Cl— may reflect an increase in permeabili- main solutes absorbed in the gut are exNa+ and Cl- through the gills,
ty of the skin to chloride. This pheno- creted,
2
+
and
SO42~ by the kidney. This
Mg
menon deserves further investigation. One
study on isolated frogs' skins showed that means that three effector systems are inabout half of the TEP develops across each volved; the gut, gills, and kidney. Research
membrane in dilute NaCl, but that the on the latter is still confined primarily to
concentration-dependent step is at the out- determining rates of formation and the
er membrane. The potential difference composition of urine under different conacross the inner membrane was relatively ditions (Hickman, 1969). However, the instable as external [Na+] varied (Biber, et testine and gills have been subjected to
al., 196f>). It would be feasible to repeat scrutiny recently, and the results show interesting differences from fresh-water forms.
this in intact animals.
When Artemia drinks, the fluid entering
In marked contrast, the only published
measurements of TEP's for fish in fresh the gut is first diluted by water entering
water (on Anguilla anguilla and Blennius from the blood although it remains hyperpholis) showed the body fluids to be nega- tonic to the latter. The direction of net
tive in dilute solutions. The values were fluid movement then reverses as water and
about —18 raV for the eel in about 0.5 mM salt are absorbed (Croghan, \9b%b). PreNaCl (Maetz and Campanini, 1966) and liminary dilution of the gut contents also
—3 mV for the brackish water blenny in occurred in isolated preparations from the
about 40 mM (House, 1963). Preliminary fish, Coitus scorpius (House and Green,
experiments with Salmo gardneri in 1 mM 1965), and Anguilla anguilla (Sharratt, et
NaCl also showed that the body fluids were al., 1964), after which there was net water
negative by about 10 mV, but that if CaCl2 absorption from the still hypertonic
was added to the external solution the po- lumen. Movement of water was apparently
larity was reversed and resembled that in coupled with transfer of salt since replacthe amphibians (unpublished data). The ing NaCl with non-penetrating solutes
study on larval salamanders (Dietz, et al., abolished net water absorption. This is an1967) also showed the external [Ca2+] other case of entrainment of one flux with
makes the body fluids more positive in di- another. These observations were conlute media. The role of Ca2+ in generating firmed in elegant experiments on the
the TEP needs further investigation as perfused gut in intact eels (Skadauge and
does: (1) the contribution of each mem- Maetz, 1967). This study on a euryhaline
brane of cells in epithelia other than frog's fish also showed that (1) transport of salt
skin, and (2) the roles of membrane per- by the gut was faster in animals adapted to
meability and the transport processes them- sea water than in those adapted to fresh
water, and (2) the osmotic permeability of
selves.
this organ was higher in sea water.
The gills of hypotonic regulators have
Hypotonic regulators
been known since the work of Keys (1931)
The ability to maintain body fluids hy- to excrete NaCl absorbed in the gut. Only
TRANSPORT OF NACL IN AQUATIC ANIMALS
recently, however, has the process been
examined in detail. Croghan's work on Artemia indicated that unidirectional ionic
fluxes were spectacularly high. In both
Crustacea (Thuet, et al., 1969, Smith,
19696; and teleosts (Motais, 1967) fluxes
are two orders of magnitude faster in euryhaline animals adapted to sea water than
when they are in fresh water. In the
flounder, Platichthys flesus, for example,
Na+ fluxes were 2.6 meq (100 g m ) - 1 ^ - 1 ,
amounting to turnover of 47% of the total
body Na+ per hour (Motais, 1967). These
rates are not peculiar to euryhaline forms;
in nine teleost species, six of them stenohaline marine and three euryhaline animals adapted to sea water, N+ fluxes were
in the range 1.2-4.8 meq (100 gm)-1!^—1.
Movement of chloride across the gill is
equally rapid. As in fresh-water animals
cationic and anionic fluxes are dissociable
(Motais, 1967, Thuet, et al, 1969).
At present the mechanisms underlying
NaCl fluxes are uncertain. Some evidence
in fish suggests that as much as 90% of the
total flux of each ion is due to exchange
diffusion (Motais, et al., 1966). Experiments with Artemia also showed that
fluxes behaved as though they were largely
due to Na+—Na+ and C1"~-C1~ exchanges
(Thuet, et al., 1969). But a more thorough
examination of the flux-force relationships
in Artemia showed that Na+ efflux was not
by exchange but represented a true leak.
That is, the gills in Artemia are extremely
permeable to Na+ (Smith, 1969a, b). On
the other hand, Cl~ movement did appear
to have a very large exchange component.
It was pointed out earlier that such a forced
exchange of identical ions is physiologically
useless since it can cause no net transfer of
ions (in contrast, forced exchange of one
ion for a different ionic species, as in freshwater animals, generates net movement of
both). It does, however, create technical difficulties. For example, when exchange diffusion is rapid compared with other routes,
the flux ratio is nearly unity and almost independent of the electrochemical gradient
(c/. Dr. Lindley's Fig. 5). This is because
most of the ions are not moving independ-
373
ently under the influence of the gradient,
but rather through a chemical mechanism
in which transfer in one direction is tightly
coupled to equivalent transfer in the other.
As a consequence the analysis of flux ratio
has never been applied to the extrusion of
NaCl across the gills of hypotonic regulators.
Tn spite of this difficulty it is possible to
make some statements of a qualitative
nature about mechanisms. We begin with
the observation that NaCl is extruded
from the animal into the more concentrated environment. The TEP has been measured in only three animals, the blenny eel
(House, 1963), the yellow eel (Maetz and
Campanini, 1966), and the brine shrimp
(Smith, 1969fl). In each, the environment
was negative to the body fluids. Thus, the
electrochemical gradient favors inward
movement of anions. Net chloride extrusion means that there must be an active
transport mechanism oriented outward.
But the situation is more equivocal for
Na+. The electrical gradient favors net cation outflux, and the values reported
(about 18 mV for the eel, 30 mV for the
blenny, and 23 mV for Artemia) are
roughly appropriate to offset the concentration difference between blood and sea
water. Extrusion could be purely passive
although present data do not rule out
transport.
Additional detail has been added, primarily in studies on the eel, Angnilla anguilla, and flounder, Platichthys flesus.
The total fluxes of Na+ and Cl~ arc independent of each other (Motais, el al.,
1966). Even in the absence of any exchange, efflux of Na+ and Cl~ is about an
order of magnitude more rapid than fluxes
in fresh-water forms, and they remain
high for some time after a euryhaline animal is transferred from sea water to fresh
water. This may reflect prolonged activity
of the active efflux mechanism in the gills
but may also be partly due to increased
renal excretion of salt. Here is another
example of the need to partition a total
flux in order to interpret data unequivocally. In any event, the net efflux is part of
374
LEONARD B. KIRSCHNER
the process of adaptation from a marine to
a fresh-water milieu and results in lowering the concentration of NaCl and osmotic
pressure in the blood.
One recent development may relate to
the biochemical mechanism of transport in
hypotonic regulators. It has been reported
that levels of an ATPase related to sodium and potassium transport (the Na/K
ATPase) are higher in the intestine and
gills of fish in sea water than in fresh water
(Epstein, et id., 1967; Kamiah and Utida,
1968). Moreover in euryhaline fish the enzyme level increases in gut and gills on
transfer from fresh to sea water and the
opposite happens in the kidney. This, of
course, parallels changes in the movement
of salt in these organs. There is no problem in interpreting changes in the kidney
and intestine, but the situation in the gill
requires more study. As noted above, we
are not even sure that Na+ is excreted
actively by the gill. Extrusion of Cl~ is
undoubtedly active, but this ion has never
been shown to play a role in ATPase activity.
Al'L'ENDIX
A simple relationship can be derived as
follows for determining both J in and J o u t
using only a single tracer. Consider two ion
pools, the body fluids of an animal and
the external environment, separated by a
permeable surface such as skin or gills. Let
the total quantity in the pools be Q in and
Q out . At the beginning of an experiment a
quantity, Q*, of radioactive ion is added to
the external medium. T h e proportion of
Q*
counts min —1
radioactive ions, (
^mole" 1 ) is called the specific activity and
denoted X ollt . A similar quantity, the specific activity of the body fluids, X in , is initially zero since the isotope is added outside,
but it increases with time as radioactive
ions cross the body surface. During the
experiment, isotope enters the animal at a
rate proportional to the total influx: i.e.,
Ji n X ollt . Efflux of isotope similarly is J out X in .
At any instant the rate of change in
amount of isotope in the medium is
dQ,*,,t
— Jln^-out—JoutX in .
dt
(I)
If the two fluxes are unequal there will be
a net movement of ion into or out of the
animal. The net flux is
net — J i n — J m i t •
\*-)
We can eliminate J out in (1) and solve for
dQ*t
dt
Jin =
(3)
'Vint—Ain
Equation (3) is completely general; no assumption is made about number of compartments, and the system need not be in a
steady state. It is also valid when isotope
is injected into the animal and its appearance in the medium is followed. We need
only be able to take samples from medium
and blood periodically to measure the radioactivity (Q*) and total ion (Q) in each.
When these are plotted for the external
solution, as in Figure 2, the derivatives,
dQ*/dt and dQ/dt (=J,,,.,), and X,,,,t are
all available. X m is determined from blood
samples.
In many situations it is possible to design experiments so that no blood sample
is required. When isotope enters the animal it mixes with a large pool (Qin) of
unlabelled material. Hence, X in is much
smaller than X out early in the experiment,
and terms containing X ln can be neglected.
Equation (1) then becomes
J,,,
dt
Q.,uf
In addition, if J,,,.t is not too large, the total
quantity of ion in the medium will not
vary much in a short experiment, and if
Q out is constant, (4) can be integrated to
give
In Q* t =
-
J.. t + C.
Qout
(5)
TRANSPORT OF NACL IN AQUATIC ANIMALS
The constant is the logarithm of Q*out
initially (i.e., at t = o). In this case a
semilogarithmic plot should be linear with
slope —Jin/Qout- An analogous equation can
be developed for disappearance of isotope
from the animal into an unlabelled medium, and in that case the medium need not
be sampled.
Equation (5) is often used in experiments with intact animals. But it is important that the boundary conditions be observed: i.e., X ln <<X 01]t and Q essentially
constant. In the experiment shown in Figure 2, Xin was only about 5% of Xout even
after 60 minutes, but Qout decreased by
about 18%. The latter change is not really
negligible and Equation (4) rather than
(5) should be used.
Having measured Jin and Jnct, Jout can be
calculated from Equation (2).
REFERENCES
Alvarado, R., and L. B. Kirschner. 1963. Osmotic
and ionic regulation in Ambysloma tigrinum.
Comp. Biochcm. Physiol. 10:55-67.
Barker-Jovgensen, C, H. Levi, and K. Zerahn. 1954.
On active uptake of sodium and chloride
ions in Anurans. Acta Physiol. Scand. 30:178-190.
Biber, T., R. A. Chez, and P. F. Curran. 19C6. Na
transport across frog skin at low external Na
concern rations. J. Gen. Physiol. 49:1161-1176.
Brown, A. C. 1962. Current and potential of frog
skin in vivo and in vitro. J. Cell. Comp. Physiol.
00:263-270.
Bryan, G. W. 1960. Sodium regulation in the
crayfish Aslacus fluviatilis. J. Exp. Biol. 37:83-99.
Croghan, P. C. 1958. Ionic fluxes in Artemia salina.
J. Exp. Biol. 35:425-436.
Croghan, P. C. 1958. The mechanism of osmotic
regulation in Artemia salina: the physiology of
the gut. J. Exp. Biol. 35:243-249.
Diet/., T., L. B. Kirschner, and D. Porter. 1967.
The roles of sodium transport and anion permeability in generating transepithelial potential
differences in larval salamanders. J. Exp. Biol.
46:85-96.
Epstein, F. II., A. I. Katz, and G. E. Pickford. 1967.
Sodium- and potassium-activated adenosine
triphosphatase of gills: Role in adaptation of
teleosts to salt water. Science 156:1245-1247.
Garcia-Romeu, F., A. Salibian, and S. PezzaniHernandez. 1969. The nature of the in vivo sodium and chloride uptake mechanisms through the
epithelium of the Chilean frog, Calyptocephalella gayi. J. Gen. Physiol. 53:816-835.
Hickman, C. P. 1969. The kidney. In W. S. Hoar
and D J. Randall, [ed.], Fish physiology, Vol. 1.
375
Academic Press, New York.
House, C. R. 1963. Osmotic regulation in the
brackish water leleost Blennius pholis. J. Exp.
Biol. 40:87-104.
House, C. R., and K. Green. 1965. Ion and water
transport in isolated intestine of the marine
teleost, Cottiis scorpius. J. Exp. Biol. 42:177-189.
Kamiah, M., and S. Utida. 1968. Changes in activity
of sodium-potassium-activated adenosine triphosphatase in gills during adaptation of the Japanese eel to sea water. Comp. Biochcm. Physiol.
26:675-685.
Keys, A. B. 1931. Chloride and water secretion and
absorption by the gills of the eel. Z. Vergl.
Physiol. 15:364-388.
Kirschner, L. B., T. Kerstetter, and D. Porter.
1970. Adaptation of larval Ambystoma tigrinum
to concentrated environments. Amer. J. Physiol.
(In press).
Krogh, A. 1937. Osmotic regulation in the frog
(liana esculenta) by active absorption of chloride ions. Skand. Arch. Physiol. 76:60-73.
Krogh, A. 1938. The active absorption o£ ions in
some freshwater animals. Z. Vergl. Physiol.
25:235-250.
Maetz, J., and G. Campanini. 1966. Potentiels transepitheliaux de la branchie d'Anguille in vivo en
eau douce et en eau de mer. J. Physiol. (Paris).
58:248.
Maetz, J., and F. Garcia-Romeu. 1964. The
mechanism oC sodium and chloride uptake by
the gills of a freshwater fish, Carassius auratus.
J. Gen. Physiol. 47:1209-1227.
Motais, R. 1967. Les mcchanismes d'^changcs ionique branchiaux chez les tel<Sost£ens. Ann. Inst.
Oceangr. 45:1-84.
Motais, R., F. Garcia-Romeu, and J. Maetz. 1966.
Exchange diffusion effect and euryhalinity in
teleosts. J. Gen. Physiol. 50:391-422.
Pany, G., and W. T. W. Potts. 1965. Sodium
balance in the freshwater prawn Palaemonetes
anlennarius. J. Exp. Biol. 42:415-421.
Potts, W. T. AV., and G. Parry. 1964. Osmotic and
ionic regulation in animals. Pergamon Press, Oxford, England.
Robertson, J. S. 1957. Theory and use of tracers in
determining transfer rates in biological systems.
Physiol. Rev. 37:133-154.
Sharrat, B. M., I. Chester-Jones, and D. Bellamy.
1964. Adaptation of the silver eel (Anguilla anguilla) to sea water and to artificial media together with observations on the role of the gut.
Comp. Biochem. Physiol. 11:19-30.
Shaw, J. 1959. The absorption of sodium ions by
the crayfish Aslacus pallipes. J. Exp. Biol.
36:126-144.
Shaw, J. 1960a. The absorption of sodium ions by
the crayfish. Astacus pallipes. J. Exp. Biol.
37:534-547.
Shaw, J. 1960b. The absorption of chloride ions by
the crayfish Astacus pallipes. J. Exp. Biol.
37:557-572.
Shaw, J. 1961. Sodium balance in Eriocheir sinensis.
376
LEONARD B. KIRSCHNER
The adaptation of the Crustacea to fresh water.
J. Exp. Biol. 38:154-162.
Shaw, J., and D. W. Sutcliffe. 1961. Studies on
sodium balance in Gammarus duebeni and G.
pulex. J. Exp. Biol. 38:1-16.
Sheppard, C. W. 1962. Basic principles of the tracer
method. John Wiley and Sons, New York.
Skadaugc, E., and J. Maeu. 1967. Etude in vivo de
l'absorption intestinale d'eau et d'electrolytes
chez /tnguilla anguilla adapte a des milieux de
salinitcs diverses. C. R. Acad. Sci. Paris
265:347-350.
Smith, P. G. 1969a. The ionic relations of Arlemia
salina, I. J. Exp. Biol. 51:727-738.
Smith, P. G. 19696. The ionic relations of Arlemia
salina, II. J. Exp. Biol. 51:739-758.
Solomon, A. K. J900. Compartmental methods of
kinetic analaysis. In C. F. Comar and F. Bionner,
[ed.], Mineral metabolism, Vol. I, Part A.
Academic Press, New York.
Stobbart, R. H. J965. The effect of some anions
and cations on the fluxes and net uplake of
sodium in the larva of Aedes aegypli. J. Exp.
Biol. 42:29-44.
Sutcliffe, D. W., and J. Shaw. 1969. The sodium
balance mechanism in ihe fresh waier amphipod
Gammarus luciutris. J. Exp. Biol. 46:519-528.
Thuet, P., R. Molais, and J. Maelz. 1968. Les
mechanismes de l'euryhalinile chez le crustace de
salines Arlemia sidina. Comp. Biochem. Physiol.
26:793-818.
Ussing, H. H. 1947. Interpreiation of ihe exchange
of radiosodium in isolated muscle. Nature
160:262-263.