AMER. ZOOL., 22:709-721 (1982)
Kinetic and Thermodynamic Aspects of Sodium-Coupled
Amino Acid Transport by Marine Invertebrates 1
ROBERT L. PRESTON
Department of Biology, Illinois State University,
Normal, Illinois 61761
AND
BRUCE R. STEVENS
Department of Physiology, School of Medicine,
University of California, Los Angeles,
Los Angeles, California 90024
SYNOPSIS. Marine invertebrates absorb amino acids directly across their external body
surfaces. This absorption process occurs via carrier-mediated transport systems which,
recent evidence suggests, may be sodium-dependent. Steady-state amino acid gradients
are maintained at levels exceeding 103-106 times that of the external environment. Examination of the standing gradients of total free amino acids, Na, and K in seven invertebrates suggests that an Na/amino acid cotransport model, or an Na/K/amino acid cotransport model can account for amino acid gradients of this magnitude. However, the
Na : amino acid coupling coefficients must be 2 or 3 depending on factors such as membrane potential and the intracellular Na activities. Evidence from studies of L-alanine
transport in the integument of the polychaete Glycera dibranchiata is presented showing
that, for this case, the Na : alanine coupling coefficient is 3. It is concluded that the models
presented are plausible and readily testable explanations for the observed amino acid
gradients in marine invertebrates.
phens (1978) demonstrated that uptake of
Soft-bodied marine invertebrates absorb FAA can supply 37% of the oxidative refree amino acids (FAA) and other organic quirements of the mussel Mytilus califormolecules such as hexoses directly across nianus at an environmental concentration
their external body surface. The absorbed of 1 fiM. In some species the dissolved orsubstances can supply, in part, the nutri- ganic matter's contribution to the nutritional needs of the organisms. This hy- tional needs of the organisms is much lowpothesis was proposed at the turn of the er (<10%). However, it has been argued
century by Putter (1909). However, it that in nutritionally marginal environfoundered for lack of definitive evidence. ments this level may still be significant for
Technical advances in chromatography, survival.
This ability to absorb organic molecules
chemical analysis, and radioisotope tracers
permitted Stephens and others during the is nearly ubiquitous in marine invertelast 20 years to establish unequivocally the brates. Stephens (1981) and West el al.
validity of this proposal. For example, Ste- (1977) report that uptake has been obphens and co-workers (1978) have found served in 18 classes in 13 phyla and in at
that FAA uptake by the sand dollar Den- least 80 different species. The exceptions
draster excentricus can supply 100% of the appear to be arthropods which have thick
animal's energy needs at external amino impermeable chitinous exoskeletons. This
acid concentrations (>35 /xM) likely to oc- absorptive ability is also lacking in freshcur in their environment. Wright and Ste- water invertebrates and in both freshwater
or marine vertebrates (with the exception
of hagfish, which can absorb amino acids
1
From the Symposium on The Role of Uptake of Or- directly from the medium). The increasing
ganic Solutes in Nutrition of Marine Organisms presented
interest in this field is evident from the onat the Annual Meeting of the American Society of
Zoologists, 27-30 December 1980, at Seattle, Wash- going series of reviews on this topic (Stephens, 1967, 1972,1981;Jorgensen, 1976;
ington.
INTRODUCTION
709
710
R. L. PRESTON AND B. R. STEVENS
Sepers, 1977; Stewart, 1979; Schlichter, ents by a variant of the same hypothesis.
1980; Wright and Stephens, 1982).
The purpose of this article is to review
From the cellular point of view, the sol- briefly the cellular aspects of amino acid
ute absorptive abilities of marine inverte- transport in marine invertebrates with embrates are intriguing. The surface epithelia phasis on kinetics of uptake, thermodycontain cells which have microvilli exposed namics of accumulation, and the applicato the external medium (e.g., Goreau and bility of the Na-coupled mechanisms to
Philpott, 1965; Gupta et al., 1966; Lloyd, invertebrates. An example, the transport
1969; Menton and Eisen, 1970; Chien et of L-alanine by the body wall of the polyal., 1972). By analogy with vertebrate in- chaete Glycera dibranchiata will be detestinal and renal epithelia, the function of scribed. This will be followed by a discusthose microvillous epithelia appears to be sion of the energetics of Na-coupled amino
nutrient absorption. The absorption pro- acid transport in marine invertebrates.
cess itself occurs primarily via carrier-mediated transport systems. This process is
DISCUSSION
also dependent on the sodium concentration in the external medium. Invertebrate Kinetics of uptake
tissues differ from mammalian tissues,
The presence of carrier-mediated FAA
however, in that they seem to have consid- transport has been inferred from numererably higher affinities for amino acid ous studies conducted on marine invertetransport, but generally lower permeabili- brate tissues (reviewed by Stewart, 1979;
ties. Most animal cells maintain relatively see also Jorgensen, 1976; Stephens, 1981).
higher concentrations of amino acids in- The primary criterion has been the saturtracellularly compared with extracellular ability of the transport system with increasfluids. These standing gradients appear to ing external substrate concentration, which
be regulated by transport processes. In could be quantitatively described by Mimammalian tissues the standing gradient chaelis-Menten kinetics. The rate of upwhich can be maintained is typically on the take (usually measured using radioisotoorder of 5—20 fold for the total pool. That pically labeled substrate) is initially linear,
is, the internal concentration of free amino then levels off at higher concentrations of
acid may be 20 mM while the blood plasma substrate, reaching a maximum velocity
and interstitial fluids may contain only 1 (Jmax)- A calculated half-saturation conmM free amino acid. In marine inverte- stant (K,) can be used to approximate the
brates, on the other hand, these gradients apparent affinity of the amino acid for the
are usually one order of magnitude higher. transport carrier. The initial influx (Jj) is
Standing gradients typically range from 20 given by the hyperbolic relation:
to 1,000 fold compared with the blood or
_ JmaJS]
coelomic fluid of an organism. Compared
(1)
J» = K, + [S]
with sea water levels of amino acids these
concentration gradients may be yet higher
by one or two orders of magnitude; in some where [S] = external substrate concentracases exceeding gradients of 10H (see later tion, and J max and K, are defined as above.
discussion). With few exceptions, these The lower the concentration at which the
spectacular concentrative abilities have yet carrier becomes half-saturated, the higher
to be analysed in detail. Based on numer- is the apparent affinity of the carrier for
ous reports of preliminary kinetic analyses, the substrate. (A rigorous definition of afit is reasonable to assume that concentra- finity requires evaluation of the dissociative amino acid transport can be coupled tion constant of the amino acid from the
to the Xa electrochemical gradient. This carrier which is rarely evaluated directly.)
mechanism operates in many mammalian In addition, other criteria defining the
tissues. Recent evidence (Stevens and Pres- presence of transport systems have been
ton. 1980a, b, c) suggests that it is possible occasionally used such as substrate anato explain the observed amino acid gradi- logue competition, the presence of exchange diffusion, counterflow and trans-
TRANSPORT BY MARINE INVERTEBRATES
concentration effects. (For a discussion of
criteria for carrier-mediated systems, see
Stein, 1967, 1981; Wilbrand, 1975.) Often
an additional diffusion component has
been observed. This implies the presence
of a significant cellular or paracellular
pathway for solute diffusion. But in some
cases, it can also be explained as arising
from systematic error due to the presence
of substrate trapped in the extracellular
spaces (ECS) or the inappropriate use of
ECS markers (Wright and Stephens, 1977).
Table 1 presents a summary of apparent
kinetic constants for amino acid uptake
from a variety of marine invertebrates. The
qualification "apparent" must be added
since a diverse number of techniques have
been used to obtain these data. This table
only presents data for surface epithelia (ostensibly) or gill tissues, deleting data on invertebrate intestine, etc. Other cautions
must also be added. The Kt values reported in Table 1 should be regarded as maximum estimates due to the possibility of
the unforseen presence of unstirred layers
(USL) existing near the epithelial surface.
The overestimation of K, caused by USL
has been formally discussed by Winne
(1973), and experimentally demonstrated
by Wright and Stephens (1978) in Mytilus
in vitro gill segments. For example, Wright
and Stephens (1978) showed that the K,
for glycine uptake varied from 35 \xM to
1.3 fjiM depending on the degree of vigorous stirring of the incubation medium.
An additional caveat is that many of these
measurements did not use ESC markers.
When short incubation times are required
to measure initial velocities, this may produce a particularly significant correction in
the data. In spite of these problems, comparison of the K, values in Table 1 leads
to some interesting conclusions. The apparent K,'s range from 0.17 fiM for
L-phenylalanine uptake in the pogono-
711
ties. Although there are some areas of
overlap, the Kt's for mammalian systems
are usually in the 0.1-10 mM range. Only
in a few exceptional cases, such as glutamate transport by dog red blood cells, are
the K,'s in the low micromolar range (Preston et al., 1982). Although J max units have
not been standardized, it is apparent (from
reviewing the mammalian literature) that
substrate permeabilities are generally lower in marine invertebrates than in mammalian tissues.
The K, values are also of interest because it is assumed that these values should
be in the same range as the environmental
concentrations to which the system is exposed (Atkinson, 1969; Crowly, 1975). For
example, the L-alanine K, for Glycera dibranchiata is 86 /JLM, and the expected environmental range is 10-200 fjuM (Stephens, 1975; Crowe et al., 1977). The K,
of glycine uptake in Mytilus californianus is
1-5 JJLM, and the environmental range is
0.2-2 /JLM (North, 1975).
Amino acid gradients
Marine invertebrates maintain high
standing concentration gradients of intracellular amino acids compared with coelomic fluid (e.g., Clark, 1968; Preston,
1970). When compared to external sea
water these gradients are yet higher by one
to four orders of magnitude. Table 2 presents a list of the total FAA pools in a representative number of marine organisms.
In addition, the standing total FAA concentration gradients for the entire pool
compared at two external FAA concentrations have been calculated. The concentrations chosen, 0.2 /JLM and 200 /JLM, are intended to bracket the most probable range
of external FAA to which most organisms
are likely to be exposed (North, 1975; Stephens, 1975; Crowe et al., 1977; Stephens
etal., 1978; Henrichs and Farrington, 1979;
phoran, Siboglinum fiordicum to 2,300 /JLM Jorgensen and Kristensen 1980a. The calfor L-aspartate in the polychaete, Glycera culated gradients range from 103:1 to 106:1
dibranchiata. Most values fall in the 10-200 in Asterias rubens, to 2.5 x 103:1 to 2.5 X
\iM range. Comparison of these values with 106:1 in Nereis succinea. Individual FAA
those for amino acid transport in mam- concentrations in sea water are variable demalian tissues supports the conclusion that, pending on habitat (compare, e.g., Henrichs
in general, the transport systems in marine and Farrington, 1979; Rosenfeld, 1979; Jorinvertebrates have higher substrate affini- gensen and Kristensen, 1980a, b). This
712
R. L. PRESTON AND B. R. STEVENS
TABLE 1. Transport kinetic constants in marine invertebrates.
Organism
Substrate
Reference
Jm,x"
A. ESTABLISHED Na-DEPENDENT SYSTEMS
Annelids
Polychaetes
Bankia gouldii
8.2
Glycine
Glycera dibranchiata
86
Alanine
67.2b
0.23c
23
Glycera dibranchiata
Alanine
86
0.02c
12.5
Nereis limnicola
Nereis succinea
Glycine
Glycine
37
113
1.23
1.60
—
Stewart and Dean, 1980
Stevens and Preston, 1980a,
b,c
Stevens and Preston, 1980a,
b, c
Stephens, 1964
Stephens, 1964
20
30
0.69
0.45
0.02
15
15
15
Siebers and Bulnheim, 1977
Siebers and Bulnheim, 1977
Siebers and Ehlers, 1978
Stewart and Bamford, 1975
Wright and Stephens, 1977
Wright and Stephens, 1977
McCrea, 1976
Ahearn and Townsley, 1975
DeBurgh et al., 1977; DeBurgh, 1978
12.5
Oligochaetes
Enchytraeus albidus
Enchytraeus albidus
Enchytraeus albidus
Glycine
Valine
Asparagine
5-10
Molluscs
Mya arenaria
Mytilus californianus
Mytilus californianus
Modiolus modiolus
Alanine
Glycine
Cycloleucine
Glycine
95
34
63
22
31.2"
16.8"
10
16
16
—
4.6
4.5
Echinoderms
Chiridota rigida
AIB
5.9
9.0
24
Paracentratus lividus
Alanine
8.4
1.62"
10
37
113
61
1.13
1.60
2.30
—
—
15
20
0.36
15
Stephens, 1964
Stephens, 1964
Jorgensen and Kristensen,
1980a
Jorgensen, 1979
37
3-6
2.0
0.02
15
—
Siebers and Bulnheim, 1977
Siebers and Bulnheim, 1977
Glycine
31
2.3
22
Anderson and Bedford,
Phenylalanine
12
16.8"
10
Bamford and Campbell,
1976
7-9
1.6"
12
Shick, 1975
2,080
0.96
0.16
0.16
0.20
0.16
1.06
21
21
21
21
21
21
21
21
21
Chien
Chien
Chien
Chien
Chien
Chien
Chien
Chien
Chien
B. SALINITY-DEPENDEN T SYSTEMS
Annelids
Polychaetes
Nereis limnicola
Glycine
Nereis succinea
Glycine
Nereis succinea
Alanine
Nereis virens
Valine
Oligochaetes
Enchytraeus albidus
Enchytraeus albidus
Molluscs
Rangia cuneata
Glycine
Glutamate
1 j
Mytilus edulis
1 O
Cnidaria
Aurelia aurila
Glycine
C. OTHER SYSTEMS
Annelids
Polychaetes
Glycera dibranchiata
Glycera dibranchiata
Glycera dibranchiata
Glycera dibranchiata
Glycera dibranchiata
Glycera dibranchiata
Glycera dibranchiata
Glycera dibranchiata
Glycera dibranchiata
Alanine
Arginine
Aspartate
Glycine
Valine
Proline
Glutamate
Phenvlalanine
Taurine
217
2,300
252
1,200
368
393
201
630
—
0.08'-
et ai, 1972
et al., 1972
et ai, 1972
et ai, 1972
et ai, 1972
et ai, 1972
et ai, 1972
et ai, 1972
et al.. 1972
713
TRANSPORT BY MARINE INVERTEBRATES
TABLE 1. Continued.
Organism
Substrate
K,
Jmax"
Temp
(°C)
Reference
Jorgensen and Kristensen,
1980a
Jorgensen and Kristensen,
Nereis diversicolor
Alanine
30
0.74
15
Nereis diversicolor
Serine
87
1.46
15
Nereis diversicolor
Glutamate
22
0.24
15
Nereis succinea
Serine
84
2.76
15
Nereis succinea
Glutamate
24
0.27
15
Nereis virens
Alanine
68
1.63
15
Nereis virens
Serine
72
1.54
15
Nereis virens
Glutamate
32
0.32
15
Nereis virens
Clymenella torquata
Clymenella torquata
Clymenella torquata
Clymenella torquata
Glutamate
Glycine
Valine
Phenyalanine
Lysine
71
200
270
50
120
0.16
10.0
4.40
3.70
3.60
15
—
—
—
—
Jorgensen and Kristensen,
1980a
Jorgensen and Kristensen,
1980a
Jorgensen and Kristensen,
1980a
Jorgensen and Kristensen,
1980a
Taylor, 1969
Stephens, 1962a, b, c, 1963
Stephens, 1962a, b, c, 1963
Stephens, 1962a, b, c, 1963
Stephens, 1962a, b, c, 1963
A1B
17
50
0.47
0.15
15
15
Siebers and Bulnheim, 1977
Siebers and Bulnheim, 1977
Alanine
Lysine
Cycloleucine
Cycloleucine
Glycine
Alanine
24
40
19
21
3
94
2.52"
2.04"
47.0"
107"
9"
31.7"
10
10
—
—
10
Bamford and McCrea, 1975
Bamford and McCrea, 1975
Crowe etal, 1977
Crowe etal., 1977
Wright and Stephens, 1978
Stewart, 1978a
Leucine
49
18.7"
10
Stewart, 1978a
Methionine
74
24.2"
10
Stewart, 1978a
Phenylalanine
74
18.9"
10
Stewart, 1978a
Serine
121
19.7"
10
Stewart, 1978a
Glycine
126
28.6"
10
Stewart, 1978a
Lysine
40
10
Stewart, 1978a
10
Stewart, 19786
i youu
Jorgensen and Kristensen,
1980a
Jorgensen and Kristensen,
lyoUa
Oligochaetes
Enchytraeus albidus
Enchytraeus albidus
Molluscs
Cerastoderma edule
Cerastoderma edule
Modiolus demissus
Modiolus demissus
Modiolus demissus
Mya arenaria (isolated
gills)
Mya arenaria (isolated
gills)
Mya arenaria (isolated
gills)
Mya arenaria (isolated
gills)
Mya arenaria (isolated
gills)
Mya arenaria (isolated
gills)
Mya arenaria (isolated
gills)
Mya arenaria (isolated
gills)
Mya arenaria (isolated
gills)
Mytilus califomianus
Mytilus califomianus
Mytilus edulis
Alanine
AIB
Aspartate
Glycine
Cycloleucine
Alanine
122
5.52"
12.9"
23
9
0.70"
10
Stewart, 19786
3
250
1.7
12.7
0.35 c
1.2
23
22
14
Wright and Stephens, 1978
Wright el al, 1975
Jorgensen, 1976
—
Stephens etal., 1978
Echinoderms
215C
Glycine
74
Siboglinum ekmani
Alanine
2.7
0.18
6
Siboglinum ekmani
Glycine
1.7
0.31
6
Dendraster excentricus
Pogonophora
Southward and Southward,
1980
Southward and Southward,
1980
714
R. L. PRESTON AND B. R. STEVENS
TABLE 1. Continued.
Organism
Substrate
Temp
K,
Jmax"
0.64
0.08
6
Phenylalanine
1.5
0.15
6
Siboglinum fiordicum
Phenylalanine
0.17
0.61
6
Siboglinum atlanticum
Mixed amino
acids
10
Siboglinum ekmani
Glutamate
Siboglinum ekmani
Reference
TO
0.05-0.13
—
Southward
1980
Southward
1980
Southward
1970
Southward
1970
and Southward,
and Southward,
and Southward,
and Southward,
Sipunculids
Golfingia gouldii
Glycine
100
0.1
—
Virkar, 1963
Leucine
Lysine
1.9
2.6
—
—
—
Bajorat, 1979
Bajorat, 1979
Cnidaria
Anemonia sulcata
Anemonia sulcata
a
Units of /xmol/g/hr, except: b /xmol/g dry weight/hr; c /nmol/cmVhr; all units of K, are /xM. In parts A. and
B. the medium [Na] was usually that of sea water (420—480 mVW). Reported diffusional constants are
KD = 7.5 x 10"e liter/hr/cm2 for alanine in Glycera (Stevens and Preston, 1980a, b), 0.025 nmol/g/min for A1B
in Chiridota (Ahearn and Townsley, 1975), and 0.1 mmol/g/hr for glutamate in Enchytraeus (Siebers and
Bulnheim, 1977). All amino acids are the L-stereoisomer.
could lead to variable calculated FAA FAA and primary amines in sea water usgradients. The data of Table 2 were ob- ing radioisotope, fluorometry, and chrotained by the simplifying assumption that matography techniques. At an external
individual FAA concentrations are propor- concentration of 86 /xM, Glycera body wall
tional in sea water and the intracellular maintains a [14C]L-alanine steady state grapool. The broad gradient range (0.2-200 dient of 343:1 (Stevens and Preston, 1980c).
fj.M) was chosen to encompass the inherent Other polychaetes can remove fluoresvariability of internal and environmental camine-positive material down to 3 /JLM
FAA composition. These assumptions thus (Capitella capitata), and 5-20 /JLM (Nereis
allow relative comparisons of FAA gradi- diversicolor) (Jorgensen, 1976). Mytilus
ents obtainable by the organisms listed in californianus removes glycine down to 300
nM (Wright and Stephens, 1978), and
Table 2.
Additional recent evidence suggesting Echinaster removes mixed amino acids down
maintenance of very high gradients has to 500 nM (Ferguson, 1980a, b). Manahan
been provided by experimental measure- et al. (1982) have shown that Mytilus edulis
ments of uptake and steady state levels of can remove amino acids down to 38 nM.
TABLE 2. Amino acid, Xa, and K gradients in marine invertebrates.
Obtainable gradients
Measured gradients
Organism
Arenicola marinaa'h
Glycera dibranchiata"
Nereis succineaA
Mytilus edulis''
Sepia officinalis'
Eledone cirrhosa1
Asterias rubens"'h
Tissue
Muscle
Body wall
Muscle
Adductor muscle
Mantle muscle
Mantle muscle
Podial tissue
[A],
INaUNal,
|K],:[KL
449
280
495
238
483
326
221
385:57.8
440:166
479:48.1
490:79
492:30.8
438:33.3
433:134
88.3:9.7
190:9
234:8.9
152:12.5
189:10.5
167:9.3
103:9.2
[A],:= 0.2 iiM
[A], = 200 ILM
X 10 3
2.2 x 10"
2.2
1.4
2.5
1.2
2.4
1.6
1.1
1.4
2.5
1.2
2.4
1.6
1.1
AH unidentified concentrations are m.Vf. The values for the obtainable gradients when [A], = 0.2 fiM or
200 fxM are xl0 3 and x l()6 respectively. References: a Shumway and Davenport, 1977; " Xeseterov and Skulski,
lW.i: ' Stevens and Preston. 19H<)«. b. ,: '' Freel ,1 al.. 1973; ' Potts. 1958; ' Robertson, 196.U u Gilli-s. 197.");
h
Bin von, 1978.
715
TRANSPORT BY MARINE INVERTEBRATES
Compared to measured intracellular FAA
pools of 0.2-0.5 M (see, e.g., Awapara,
1962), the calculated concentration gradients are maintained on the order of 106:1.
Although these are exceptionally high
gradients, there is a plausible transport
mechanism which could produce them.
From a theoretical viewpoint, it appears an
Na/FAA cotransport mechanism could
drive accumulation to these levels under
certain conditions. For the purposes of discussion here we will not consider the contribution of intracellular metabolism to
FAA pool maintenance (e.g., Campbell and
Bishop, 1970), and we will assume that FAA
concentrations in whole body wall or muscle are identical with concentrations in the
epithelial cells. To test the limits of the
Na cotransport model, we wish to make the
most conservative assumption—that a
transport carrier system can account entirely for maintenance of large steady state
FAA gradients.
Low [K]
+
-
C _
High [Na]
Low [AA]
J)
/ ^
AA
J
High
AA
-
G<]
L o w [Na]
High [AA]
Na
ATP - — ! ^
nNa
J~
»nNa
ADP + Pi»
'
FIG. 1. General model for Na-coupled amino acid
cotransport. The accumulation of intracellular amino
acid is driven by the coupling of amino acid influx to
the simultaneous influx of Na on the same transport
carrier. The energetically "downhill" movement of
Na permits "uphill" accumulation of amino acid. The
cotransport system may also be driven in part by the
membrane potential if the carrier or carrier-substrate
complex is charged. The Na and K gradients are
maintained by the Na/K pump.
cotransport has been the observation of reduction in FAA uptake when the Na in the
medium is replaced by choline, Li, or K.
Table 1 summarizes the data on Na dependency for a number of marine inverGeneral Na cotransport model
tebrates tested this way. Considering the
It has been generally concluded that Na- mass of uptake data available, it is surpriscoupled transport is the major mechanism ing that relatively few experiments of this
for accumulative nonelectrolyte transport sort have been conducted. A number of
in eukaryotes (Schultz and Curran, 1970; studies have also shown salinity-dependent
Crane, 1977). Most of the detailed studies FAA uptake in marine invertebrates (Taon Na/FAA cotransport have been con- ble 1). Only a few studies have measured
ducted on mammalian systems, especially the quantitative effects of Na on transport
kidney (Mircheff et ai, 1981), intestine kinetics and steady state levels of cellular
(Preston et ai, 1974; Stevens et ai, 1982), FAA pools (Stevens, 1977; Wright and Steand Ehrlich ascites cells {e.g., Christensen, phens, 1977; Stevens and Preston, 1980a,
1979). Figure 1 presents the general fea- b, c). To illustrate the applicability of the
tures of this model. Most cells contain high Na cotransport hypothesis to marine inlevels of K and FAA compared with the vertebrate tissue it will be useful to considexternal medium. Intracellular Na is usu- er a specific example, the transport of
ally lower than the external medium. The L-alanine by Glycera body wall.
driving force for FAA accumulation is derived from coupling the energetically uphill Alanine transport by Glycera:
flow of amino acid to the downhill flow of An Na cotransport model
Na via a carrier which simultaneously binds
The transport of alanine by the body wall
both moieties. The Na gradient is main- of Glycera dibranchiata has been studied by
tained by an Na/K pump fuelled by ATP. us (Stevens and Preston, 1980a, b, c). The
This Na/K pump has been found in vir- transport kinetics, Na dependency, and
tually all eukaryotic cells with few excep- steady state accumulations of L-alanine
tions (Parker, 1977). If the carrier or the were measured. Each of these topics will
carrier/substrate complex is charged, the be discussed in turn.
membrane potential may also contribute to
Initial influx measurements were obthe driving force for FAA accumulation.
tained using modified influx chambers
The most popular criterion for Na/FAA originally designed for mammalian uptake
716
C
R. L. PRESTON AND B. R. STEVENS
determined that two carrier systems existed by which alanine uptake was obligatorily coupled to Na. One system dominates
at high [Na] and one dominates at low [Na].
The crossover point is at 190 mM Na.
From these data it was possible to formulate a mathematical model describing
the effects of varying external Na and alanine concentration on alanine influx. This
model is described by equation 2, and a
schematic interpretation of the model is
presented in Figure 3:
5 -
0.01
0.025
0.05
[alanine]
FIG. 2. Na-dependency of L-alanine transport by the
integument of Glycera dibranchiata. The initial influx
of L-alanine (Jaianine) in the presence (•) and absence
(O) of Na in sea water as a function of medium
L-alanine concentration is composed of two components: A saturable component (
) and a diffusional component (
). In the absence of Na the flux
is entirely diffusional.
J"[Na]3[AlaJ
+ ,K'[Ala] + Kt[Na]3
V
+ [Na]3[Ala]
J"[Na]3[Ala]
3
2K'Kt + 2K'[Ala] + Kt[Na]
+ [Na]3[Ala]
(2)
+ KD[Ala]
where Ji = total alanine influx; ,,2J" =
maximal influx constants for systems 1 and
2 (J" = 2.30 x 10"7 and 2J" = 1.80 x 10~8
mol/hr/cm2); Kt = alanine Michaelis constant (8.63 x 10"5 M alanine); 1>2K' =
transport constants for Na effects on
alanine maximal flux for systems 1 and
2 dK' = 8.52 x 10"2 and 2K' = 4.00 X 10~5
[meq/liter]3); KD = apparent diffusion constant for alanine (7.48 X 10~6liter/hr/cm2).
Basically this model suggests that 3 Na
must bind to the carrier before alanine is
translocated from the sea water to the cytoplasmic side of the integument epithelial
cell membrane (see Fig. 3). At the cytoplasmic side, the carrier releases the 3 Na
and alanine. It then "returns" to the outside for another cycle. The relevant assumptions of this formulation are discussed elsewhere (Stevens and Preston,
1980ft).
J.= I ,K'Kt
measurements. The chambers allowed only
the external surface of the Glycera in vitro
body wall to be exposed to vigorously
stirred artificial sea water. The sea water
(12°C) contained the substrate [14C]L-alanine and the ECS marker [3H]inulin. Na
concentrations were varied using choline
chloride substituted for NaCl. The initial
rate of transport of L-alanine as a function
of alanine concentration in the presence
and absence of Na is shown in Figure 2.
In the presence of Na, uptake was saturable with an apparent added linear component at high substrate concentrations. Jn
the absence of Na, only the linear component remained. Alanine influx was competitively inhibited by phenylalanine, methionine, and valine in the presence of Na,
but no competition was observed in the absence of Na. These facts suggested that the
linear component was passive diffusion.
Varying [Na] affected only the J max of the
carrier-mediated alanine uptake. Using An explanation for observed large
graphical techniques, it was possible to show steady state FAA gradients
that 3 Na were transported for each alaIn addition to kinetic modeling of memnine molecule. This ratio (Na transport- brane events, the coupling coefficient is
ed : alanine transported), known as the useful in evaluating the free energy availcoupling coefficient (CC), can in principle able from the Na electrochemical gradient
be measured directly by simultaneous up- for amino acid uptake and accumulation.
take of [14C]alanine and 22Na. However, the In a plausible Na/FAA cotransport model,
background Na flux was high in Glycera, adequate conjugate forces are required to
and in practice this was not possible. It was sustain the FAA gradient. The energy may
TRANSPORT BY MARINE INVERTEBRATES
Na 3
FIG. 3. Model for Na-coupled L-alanine transport
by Glycera integument. The carrier (X) binds 3 Na
and 1 alanine molecules (random order). The quinary complex (X-Na3-Ala) and the carrier (X) are capable of translocation (P^ P2). The translocation rate
constants (P3, P4) for the other complexes (X-Na3,
X-Ala), are apparently low. Dissociation constants K,,
K2, K3 and K4 characterize each step. On the cytoplasmic side the alanine and 3 Na are released and
the carrier recycles to the external membrane surface. There is also a diffusional flux of alanine with
rate constant (KD) which may be transcellular or paracellular. For details of the assumptions in this model
see Stevens and Preston (1980i).
be obtained by exploiting existing gradients of ions (e.g., Na and K electrochemical
gradients generated by a membrane-bound
Na/K-APTase). This relationship has been
formally expressed by Jacquez and Schafer
(1969) for the case where FAA uptake is
coupled to the Na gradient, the K gradient, the membrane potential, or various
combinations of these factors:
GT =
[Na],
[Na],
- (m - n)FV
RTln
[K]
(3)
where GT = free energy available for
transport of amino acid A; R = gas constant; T = absolute
temperature;
F = Faraday constant; V = membrane potential; the subscripts represent internal (i)
and external (e) concentrations; the other
constants k, m, n are the number of molecules of A, Na, and/or K (respectively)
717
transported during each transport event.
In the case of mammalian intestine (Schultz
and Curran, 1970; Stevens et al., 1982) K
is not directly involved (i.e., n = 0). Other
tissues appear to be directly dependent on
K for FAA uptake and accumulation (Jacquez and Schafer, 1969; Murer et al., 1980;
Sacktor and Schneider, 1980). We have
previously shown experimentally and theoretically (Stevens and Preston, 19806, c)
that, independent of K, large FAA gradients (>343:1) in Glycera can be maintained
by coupling directly to the Na concentration gradient plus the resting membrane
potential of - 7 5 mV (a typical potential
for marine worm epithelia—Bilbaut, 1980).
That is, m = 3, n = 0, and k = 1. As mentioned above, FAA gradients up to 106:l
can be generated in various organisms.
However, there are no reports of invertebrate epithelia that have been rigorously
tested for direct coupling of K and/or
membrane potential to explain observed
Na-dependent FAA transport and steady
state gradients. By employing published
values for Na gradients, K gradients,
and typical marine invertebrate membrane
potentials (—40 to —80 mV—Ashley and
Ridgway, 1970; Bilbaut, 1980), we have
evaluated, for this paper, the feasibility of
the above model (equation 3, Fig. 3) for
FAA transport and accumulation in the
seven invertebrates listed in Table 2. The
cytoplasmic activities of Na (activity coefficient — 0.4) and K (activity coefficient =1)
were used instead of concentrations to
more accurately reflect the "true" ion gradients (see Lev and Armstrong, 1975; Walker
and Brown, 1977). By inserting into equation 3 the observed Na and K standing
gradients (see Table 2) and the typical
membrane potential range (see above), we
find that each of the organisms listed in
Table 2 can maintain lower FAA gradients
(103:l) when the CC = 2 or more, and
can maintain higher FAA gradients (106:l)
when the CC = 3. It should be noted that
the FAA gradients can be maintained in
all these organisms entirely by application
of the Glycera-type mechanism, but that
the additional coupling to K reduces the
dependence on membrane potential and/
or allows FAA accumulation several orders
718
R. L. PRESTON AND B. R. STEVENS
of magnitude in excess of 106:l. Therefore, although this model has been confirmed only in Glycera, the model may apply to nearly all marine invertebrates. This
should be readily testable by simply measuring standing FAA, Na, and K gradients
(and appropriate ionic activity coefficients),
the coupling coefficients and membrane
potential.
Systems with CC > 1 have been observed for transport of glycine in shrimp
midgut (CC = 2, Ahearn, 1976), glutamate in crab nerve (CC = 2, Baker and
Potashner, 1971), and succinate in rabbit
kidney (CC = 3, Wright et al., 1981). It is
reasonable to argue that cells of marine
invertebrates which generate large standing FAA gradients possess the Glyceratype mechanism, or a variation which includes direct coupling to K.
CONCLUSION
From the data presented here, it is clear
that Na cotransport offers a plausible explanation for the characteristics of amino
acid transport and accumulation observed
in marine invertebrates. The co-transport
hypothesis is worthy of considerably more
effort to prove or disprove its applicability. Recent advances in the technology
of ion-selective microelectrodes, high performance liquid chromatography of FAA,
and the standard radioactive tracer techniques should allow direct measurements
of the appropriate parameters discussed
above. Kinetic analysis and modeling are
standardly applied to mammalian systems.
It is hoped that these approaches will be
applied with vigor to marine invertebrate
epithelia in the near future.
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