Download Membrane lipids and gill Na+/K+-ATPase in eel

2385
The Journal of Experimental Biology 202, 2385–2392 (1999)
Printed in Great Britain © The Company of Biologists Limited 1999
JEB2189
LIPID RESTRUCTURING DOES NOT CONTRIBUTE TO ELEVATED ACTIVITIES OF
Na+/K+-ATPase IN BASOLATERAL MEMBRANES FROM THE GILL OF SEAWATERACCLIMATED EEL (ANGUILLA ROSTRATA)
ELIZABETH L. CROCKETT*
Department of Biological Sciences, Ohio University, Athens, OH 45701, USA and Mount Desert Island Biological
Laboratory, Salisbury Cove, ME 04672, USA
*e-mail: [email protected]
Accepted 25 May; published on WWW 9 August 1999
Summary
present in gill basolateral membranes, makes up more than
In teleost fishes, increases in gill
activity
60 % of the total phospholipid content in both freshwateraccompanying the transition from fresh water to sea water
and seawater-acclimated animals. The contents of other
may be attributed to changes in either the numbers of
phospholipids and major fatty acyl chains are also similar
enzyme molecules present or to turnover number (kcat).
+
+
for the two acclimation groups. Cholesterol/phospholipid
The sensitivity of Na /K -ATPase to its chemical/physical
molar ratios are 0.28 for freshwater and 0.29 for seawater
environment in the membrane makes it plausible that
animals. The similarity between lipid compositions in
modulation of enzyme activity may be driven, in part, by
membranes from freshwater- and seawater-acclimated eels
changes in membrane properties. In the current study, I
indicates that lipid restructuring is not a mechanism for
test the hypothesis that lipid compositional changes
+
+
modulation of gill Na+/K+-ATPase activity in Anguilla
(restructuring) contribute to the modulation of gill Na /K rostrata, at least during the acclimation time course used in
ATPase activity. An enriched preparation of basolateral
the present study.
membranes was prepared from the gills of freshwater- and
seawater-acclimated American eel (Anguilla rostrata).
Phospholipid class distribution, fatty acyl chain
Key words: Na+/K+-ATPase, gill, salinity adaptation, plasma
compositions and cholesterol contents were determined.
membrane, eel, Anguilla rostrata, Na+ pump.
Phosphatidylcholine, the most abundant phospholipid
Na+/K+-ATPase
Introduction
Many aquatic organisms exist in fluctuating salinities or
spend portions of their life cycle in a mixture of marine,
freshwater or estuarine habitats. Despite wide differences
in ionic compositions and concentrations of these
environments, teleost fishes maintain a relatively constant
internal milieu (Evans, 1979). The gill tissue of teleosts is a
key interface between an organism and its environment. In
addition to its roles in gas exchange, in the elimination of
nitrogenous waste and in acid–base balance, the gill plays
a central part in ion- and osmoregulatory processes
(McDonald et al., 1991).
Variation in environmental salinity invokes a suite of
responses in teleosts. These responses involve several different
levels of biological organization such as (1) rates of drinking
(Hirano, 1974; Karnaky, 1980), (2) cell morphology and
composition in the gill (Virabhadrachari, 1961; Karnaky et al.,
1976a; Kültz et al., 1995) and (3) the activities of enzymes
associated with osmoregulatory processes such as Na+/K+ATPase (Epstein et al., 1967; Sargent and Thomson, 1974).
Modulation of the activity of Na+/K+-ATPase in gill is
arguably among the most widespread of these responses. It is
well documented that Na+/K+-ATPase activities from the gills
of euryhaline organisms are positively correlated with salinity
(e.g. Epstein et al., 1967; Sargent and Thomson, 1974;
Karnaky et al., 1976a,b; Towle et al., 1977).
Modification of Na+/K+-ATPase activity may involve either
changes in gene expression (affecting Na+ pump numbers or
subunit isoform expression) or various post-translational
adjustments that directly affect catalytic rate (Ewart and Klip,
1995; Lee et al., 1998). Levels of Na+/K+-ATPase α-mRNA
rise in the gill of the European eel (Anguilla anguilla) upon
transfer from fresh water to sea water (Luke et al., 1993; Cutler
et al., 1995). Similarly, a 3 day exposure to 25 ‰ sea water
results in an increase in both Na+/K+-ATPase activities and
mRNA levels for the α-subunit of the enzyme in brown trout
gill, yet the sixfold elevation in enzyme activity after 50 days
is not matched by mRNA levels (Madsen et al., 1995). While
an increase in activity of Na+/K+-ATPase during exposure to
elevated salinities has been shown to parallel the number of
Na+ pumps (measured by ouabain binding) in the gill of
2386 E. L. CROCKETT
Fundulus heteroclitus (Karnaky et al., 1976b), this is not
always the case. In this same species, a 30 min acute exposure
to 30 ‰ sea water results in an almost twofold increase in
pump activity in the gill without a corresponding rise in
ouabain binding (Towle et al., 1977). Post-translational
modifications affecting turnover number (kcat) may include
reversible phosphorylation, changes in the subcellular
distribution of the enzyme and lipid modulation of enzyme
activity (Towle, 1981; Ewart and Klip, 1995). As suggested by
Towle (1981), modulation of membrane lipids may be
responsible for the rapid rise (after 30 min) of Na+/K+-ATPase
activity in the gill of F. heteroclitus exposed to high salinity.
Compositional changes in membrane lipids (restructuring)
may, in fact, contribute to variation in Na+/K+-ATPase activity
at some point during the animal’s adjustment to varying
environmental salinities. Lipid compositional changes have
been noted in tissues from fishes acclimated to different
salinities (Daikoku et al., 1982; Leray et al., 1984).
Furthermore, many membrane-associated proteins are
influenced by the chemical and/or physical properties of the
membrane (Deuticke and Haest, 1987; Vemuri and Philipson,
1989). Na+/K+-ATPase is among the integral membrane
proteins that appear to be particularly sensitive to variation in
membrane physical properties (Grisham and Barnett, 1973;
Kimelberg and Papahadjopoulos, 1974; Gibbs and Somero,
1990). This sensitivity may reflect the large number of
membrane-spanning domains associated with the catalytic (α)
subunit of the protein. Lingrel and Kuntzweiler (1994) have
used a ‘working’ model of 10 membrane-spanning regions
accounting for 30 % of the protein directly embedded in the
lipid matrix of the bilayer.
While phospholipids are required for Na+/K+-ATPase
activity (Hokin and Hexum, 1972; Stahl, 1973), it is debatable
whether a particular phospholipid class is necessary for
maximal activity (Hokin and Hexum, 1972; Stahl, 1973;
Harris, 1985). In addition, the neutral lipid cholesterol may
also play a role in modulation of enzyme activity. In
reconstitution studies using Na+/K+-ATPase from bovine
kidney, maximal enzyme activities occur at physiological
(intermediate) levels of cholesterol; a decrease in activity is
observed at both low and high levels of membrane cholesterol
(Yeagle et al., 1988). Reduced Na+/K+-ATPase activities
parallel decreases in membrane fluidity in trout red cell
membranes supplemented with cholesterol compared with
native membranes (Raynard and Cossins, 1991). Even within
the physiological range of cholesterol levels in basolateral
membranes (BLMs) from trout intestinal epithelia, Na+/K+ATPase activity is highest in membranes with the lowest levels
of this lipid (Crockett and Hazel, 1997). The fluidity of the
bilayer and other physical properties of the membrane seem to
be particularly important factors influencing Na+/K+-ATPase
activity (Harris, 1985; Yeagle et al., 1988; Gibbs and Somero,
1990).
The purpose of the present study is to test the hypothesis that
lipid restructuring plays a part in the modulation of Na+/K+ATPase activity in the gill of salinity-acclimated euryhaline
teleosts. Na+/K+-ATPase activities and lipid compositions
(phospholipid class, fatty acyl composition and cholesterol
content) were determined in basolateral membranes prepared
from the gill of the American eel (Anguilla rostrata)
acclimated to either fresh water or sea water.
Materials and methods
Animals
Yellow-phase American eel (Anguilla rostrata LeSueur)
were collected in fresh water (Penobscot River, Maine, USA)
by commercial fishermen in June 1997. Eels (100–450 g) were
acclimated to either fresh water or sea water (32 ‰) for a
minimum of 2 weeks before use. A gradual increase in salinity
(5 ‰ per day) was made before reaching the final salinity in
seawater aquaria. Polyvinylchloride tubes were placed in each
aquarium for animals to hide in, thus reducing activity levels
and stress (Egginton, 1986a). The temperatures in the
freshwater aquaria were matched to those of sea water (13 °C)
using chilling units. Eels were not fed during the acclimation
period.
Membrane preparations
Eels were anaesthetized in 0.1 % (w/v) MS-222 dissolved in
either fresh water or sea water, depending on the acclimation
history of the animal, and neutralized with NaHCO3. Eel gills
1 NaCl through
were perfused with 10 ml of 175 mmol l−
the ventricle until the gill blanched. Whole gill baskets were
removed, and individual arches were cut out, rinsed in
homogenization medium (see below) and blotted dry. Gill
epithelia were scraped on an ice-cold glass stage. Scrapings
were pooled (2–3.5 g wet mass), typically from as many as 10
1
fish (eight on average), and homogenized in 300 mmol l−
−
1
sucrose, 25 mmol l
Hepes (pH 7.6 at 25 °C) using a
motor-driven homogenizer (Biohomogenizer, Biospec
Products, Inc.) for 15 s, three times. Homogenates were
transferred to, and further homogenized (one pass) in, a 7 ml
Ten-Broeck (TB) ground-glass homogenizer.
The starting point for the plasma membrane preparation was
from Klaren et al. (1993), although most centrifugation steps
and conditions were developed empirically to achieve an
enriched basolateral membrane preparation (Fig. 1).
Homogenates were centrifuged for 10 min in a fixed-angle
rotor (Sorvall SS-34) at 1400 gmax. The supernatant (S1) was
set aside, and the pellet (P1) was resuspended (three passes) in
1 ml of homogenization medium in 7 ml TB homogenizers. The
resuspended pellet was then brought to 20 ml with
homogenization medium and centrifuged again at 1,400 gmax
(Sorvall SS-34), this time for 20 min. The supernatant from this
second centrifugation step (S2) was combined with S1. The
combined supernatants (S1+S2) were then centrifuged in a
fixed-angle rotor (Beckman 70 Ti) at 93 000 gmax for 90 min.
The pellet (P3) from this step was resuspended in 0.5 ml of
homogenization medium using three passes in a 2 ml TB
ground-glass homogenizer. This suspension was then layered
on 18 % Percoll (in Hepes-buffered sucrose solution; final
Membrane lipids and gill Na+/K+-ATPase in eel 2387
Homogenate
1400 gmax (10 min)
S1
P1
1400 gmax (20 min)
S2
P2
93 000 gmax (90 min)
P3
S3
Percoll, 37 000 gmax (25 min)
Membrane
band
165 000 gmax (90 min)
P4
Fig. 1. Preparation of gill plasma membranes enriched in basolateral
membranes using differential and density-gradient centrifugation. P,
pellet, S, supernatant.
1) and centrifuged at
sucrose concentration 300 mmol l−
37 000 gmax for 25 min in a Beckman 70 Ti rotor to selfgenerate a gradient. The plasma membrane band was recovered
from the low-density region of the gradient using a 66 %
sucrose solution (Hepes-buffered) pumped to the bottom of
tube. Plasma membranes were recovered by resuspension in
1 NaCl in 25 mmol l−
1
rinsing medium (175 mmol l−
Hepes, pH 7.6 at 25 °C) and a final centrifugation step
(165 000 gmax for 90 min in Beckman 70 Ti rotor). The final
plasma membrane pellet (P4) was dislodged from the Percoll
pellet by gentle agitation and resuspended in several drops of
1 Hepes (pH 7.6) using a 2 ml TB homogenizer
25 mmol l−
(10 passes). Final fractions and samples of homogenates were
stored frozen at −80 °C until assayed for organelle marker
activity (see below). Final fractions for lipid analyses were
stored under a nitrogen atmosphere and frozen at −80 °C.
Organelle marker enzymes were assayed at 25 °C
(controlled with a refrigerated recirculating bath) in
homogenates and final plasma membrane fractions (P4) to
determine the extent of enrichment of basolateral membranes
and to ensure that enrichment factors were comparable for each
acclimation group (freshwater and seawater). Na+/K+-ATPase
was used as a marker for basolateral membranes and assayed
according to Barnett (1970). γ-Glutamyltranspeptidase was
also used as a plasma membrane marker (Tate and Meister,
1985). Contamination by intracellular membranes was
monitored by assaying both succinate cytochrome c reductase,
a mitochondrial marker (King, 1967), and NADPH
cytochrome c reductase, an enzyme associated with the
endoplasmic reticulum (Masters et al., 1967).
Preparation of lipid extracts and analyses of phospholipid
class/fatty acyl chain compositions
Lipids were extracted from plasma membrane fractions
(P4) following the method of Bligh and Dyer (1959).
Butylated hydroxytoluene (0.01 %) was used in the extraction
procedure to prevent autoxidation. Phospholipid class
composition for plasma membranes prepared from
freshwater- and seawater-acclimated animals was determined
using Iatroscan TLC-FID (Mark 5, Bioscan Inc.). The
procedure used was similar to that described by Hazel (1985).
Chromarods (SIII) were developed overnight prior to use and
burned twice immediately before spotting to remove any
contamination from the rods. Rods were spotted with 1 µl of
standard or sample (10–15 µg standard or sample per
microlitre CHCl3) using a blunt-tipped 2 µl syringe (Hamilton
7102). Rods were dried at 60 °C for 5 min before
development. Two solvent systems were used for stepwise
separation of neutral and polar lipid classes. Each solvent
system was allowed to develop completely. The first solvent
system (hexane/diethyl ether/formic acid, 80:20:2;
development time 34 min) was used to determine whether
neutral lipids other than cholesterol (which was analyzed by
a different method, see below) could be detected. No
significant peak other than that for cholesterol was observed.
The second development (to resolve phospholipids) included
a polar solvent system (CHCl3/MeOH/H2O, 80:35:3;
development time 55 min). Double development with the
second solvent system was used to improve the resolution of
phospholipid classes. Between and after all developments,
rods were dried for 5 min at 60 °C. Rods were scanned (30 s
1
per rod) with hydrogen at a flow rate of 180 ml min−
−
1
and with air at a flow rate of 2 l min
. Standard curves
for the phospholipids were generated using four replicates for
each of six concentrations. Repeated attempts to separate
phosphatidylethanolamine (PE) and phosphatidylinositol
(PI) using a variety of different solvent systems were
unsuccessful, so PE and PI are reported as a combination of
the two phospholipid classes.
Fatty acyl composition analysis was performed by Avanti
Polar Lipids, Inc (Alabaster, AL, USA). Briefly, lipid extracts
were hydrolyzed and methylated with 1 % sulphuric acid in
methanol at 70 °C. After hydrolysis and methylation, hexane
was added, and samples were vortexed and centrifuged. The
upper phase containing the fatty acid methyl esters (FAMEs)
was removed. Samples (2 µl) were injected into a Hewlett
Packard 5890 gas chromatograph with flame ionization
detection (GC/FID). A J and W column (DB225, 30 m) was
used (isothermal at 220 °C, helium as the carrier gas). Samples
were analyzed against a FAME calibration standard and
control. Peaks were included in the analysis (1) if they
constituted more than 1 % of total FAMEs, or (2) if less than
1 %, they were identifiable.
2388 E. L. CROCKETT
previous reports, Na+/K+-ATPase activities are elevated more
than twofold in seawater-acclimated fish compared with their
freshwater counterparts. In contrast, differences in the activity
of the other enzyme associated with the plasma membrane, γglutamyltranspeptidase (γ-GT), are not statistically significant
between acclimation groups.
Cholesterol, total phospholipid and protein determinations
The cholesterol contents of membrane fractions were
analyzed using a fluorometric/enzymatic assay as originally
developed and described by Crockett and Hazel (1995). The
time courses of the enzymatic reaction (cholesterol oxidation)
were examined initially to determine the incubation time
necessary for the enzyme (cholesterol oxidase) to react with all
membrane cholesterol. A 3 h incubation was determined to be
the optimal incubation time for gill plasma membranes. Total
phospholipid was quantified as acid-hydrolyzable phosphate
(Rouser et al., 1970). The amount of membrane protein present
was measured using the bicinchoninic acid method (Smith et
al., 1985).
Membrane enrichment
Membranes (P4 fractions) prepared from both acclimation
groups have eightfold higher protein-specific activities
of Na+/K+-ATPase compared with activities in crude
homogenates (Table 1). This result has two implications. First,
there is significant enrichment of basolateral membranes in the
final membrane fraction and, second, the degree of enrichment
is similar for membranes prepared from both freshwater- and
seawater-acclimated animals. Enrichment factors for γ-GT
are similar to those for Na+/K+-ATPase. The mitochondrial
enzyme (succinate cytochrome c reductase) and the
endoplasmic reticulum marker enzyme (NADPH cytochrome
c reductase) are enriched threefold or less in the plasma
membrane (P4) fraction, indicating some contamination by
intracellular membranes. Of the total activity of each of the two
plasma membrane markers (Na+/K+-ATPase and γ-GT) in the
crude homogenate, 13 % is recovered in the P4 fraction, while
only 4 % of the enzyme activity of intracellular markers is
recovered. Importantly, intracellular membranes make up
similar portions of the P4 fractions for both freshwater and
seawater treatments.
Statistics
Results from freshwater and seawater acclimations were
analyzed statistically using Student’s t-test. The level of
significance was set at P<0.05.
Chemicals
Phospholipid class standards were from Avanti Polar Lipids,
Inc. (Alabaster, AL, USA) and included saturated fatty acyl
chains in the number 1 position on the glycerol backbone and
unsaturated fatty acyl chains in the number 2 position on
the glycerol backbone. FAME standards were from Nu-Chek
Prep, Inc. (Elysian, MN, USA). Pyruvate kinase-lactate
dehydrogenase (PK-LDH) used in assays for Na+/K+-ATPase
was from Boehringer-Mannheim (Indianapolis, IN, USA).
Bicinchoninic acid (BCA) protein assay reagent was purchased
from Pierce Chemical Co. (Rockford, IL, USA). All other
biochemicals were from Sigma Chemical Co. (St Louis, MO,
USA). Solvents used were HPLC-grade or better.
Phospholipid and cholesterol compositions
Phospholipid
class
compositions
are
virtually
indistinguishable for gill plasma membranes from freshwaterand seawater-acclimated eels (Fig. 2). Phosphatidylcholine
(PC) is by far the most abundant phospholipid, making up 61 %
of the total phospholipid content of the membrane, while
phosphatidylethanolamine (PE) and phosphatidylinositol (PI)
combined represent 23 % of the phospholipid component of the
membrane. Sphingomyelin (SM) constitutes 12 % and
phosphatidylserine (PS) 4 % of the phospholipid matrix.
Results
Enzymes activities from gill homogenates
A comparison of enzyme activities in the gill homogenates
from freshwater- and seawater-acclimated eels reveals several
trends (Table 1). In keeping with the pattern observed in
Table 1. Marker enzyme activities in homogenates and enrichment factors in final fractions (P4)
Freshwater-acclimated
Marker
Na+/K+-ATPase
γ-GT
S-CCR
N-CCR
Homogenate activity
(µU mg−1
protein)
P4
Enrichment
(fold)
39±1.9
15±0.9
5.3±0.8
3.4±0.3
8.0±1.0
8.3±0.7
3.1±0.5
2.9±0.1
Seawater-acclimated
Homogenate activity
(µU mg−1
protein)
88±3.7***
12±0.6
7.2±0.05
3.8±0.2
P4
Enrichment
(fold)
8.4±0.6
9±0.6
2.5±0.4
2.5±0.2
Values are means ± S.E.M. (N=3).
***P<0.001 for freshwater/seawater comparison.
Enrichment factors are calculated as protein specific activity of P4 (membrane) versus protein specific activity of homogenate.
P4, fourth pellet (final membrane fraction); µU, microunit (nmol product formed min−1); γ-GT, γ-glutamyltranspeptidase; S-CCR, succinate
cytochrome c reductase; N-CCR, NADPH cytochrome c reductase.
Membrane lipids and gill Na+/K+-ATPase in eel 2389
FW
SW
Percentage composition
60
Table 3. Cholesterol contents in gill plasma membranes from
freshwater- and seawater-acclimated eels
45
Cholesterol/protein
(µg µg−1)
Cholesterol/phospholipid
(µmol µmol−1)
30
15
0
PE (and PI)
PC
SM
PS
Fig. 2. Percentage compositions of phospholipid class in basolateral
membranes from gills of freshwater (FW)- and seawater (SW)acclimated eel. Values are presented as means + S.E.M. (N=3). No
statistically significant differences were found between freshwater
and seawater eels. PE, phosphatidylethanolamine; PC,
phosphatidylcholine; PI, phosphatidylinositol; SM, sphingomyelin;
PS, phosphatidylserine.
Fatty acyl chain compositions for membranes prepared from
freshwater- and seawater-acclimated animals are also similar
between acclimation groups (Table 2). An exception to this
trend is in the fatty acid 20:2 and an unidentified fatty acid with
a retention time relative to 18:0 (RRT) of 3.14. This latter fatty
acid is probably a polyunsaturate. Both 20:2 and RRT 3.14
Table 2. Percentage composition of acyl chains in gill plasma
membranes from freshwater- and seawater-acclimated eels
Fatty acid
Freshwater-acclimated
14:0
RRT 0.7
16:0
16:1
RRT 0.9
18:0
18:1
RRT 1.05
18:2
18:3
20:0
20:1
20:2
20:3
20:4
RRT 1.95
RRT 2.79
RRT 3.14
22:6
24:1
0.93±0.06
1.93±0.33
13.0±1.12
3.59±0.14
1.93±0.53
9.45±0.45
12.1±0.73
3.40±0.58
1.17±0.37
0.43±0.07
0.48±0.02
0.67±0.06
0.36±0.04
0.34±0.02
15.5±0.96
2.90±0.11
2.55±0.25
2.18±0.13
9.60±0.75
0.52±0.23
Seawater-acclimated
1.07±0.11
1.55±0.07
14.8±0.85
4.07±0.24
1.56±0.04
10.6±0.63
12.0±1.21
4.87±1.46
1.54±0.03
0.45±0.04
0.50±0.04
0.64±0.08
0.68±0.04**
0.37±0.01
16.5±0.68
3.32±0.29
3.12±0.13
2.96±0.16**
10.33±0.89
0.37±0.15
Values are means ± S.E.M. (N=3).
Unidentified fatty acids, representing at least 1 % of the total, are
identified by retention times (RRT) relative to 18:0.
Freshwater/seawater comparisons are not significantly different
for most fatty acids; ** P<0.02.
Freshwateracclimated
Seawateracclimated
0.160±0.003
0.155±0.006
0.280±0.004
0.292±0.007
Values are means ± S.E.M. (N=3).
Mass ratios were calculated for cholesterol/protein, whereas molar
ratios were calculated for cholesterol/phospholipid.
Freshwater/seawater comparisons are not significantly different
for either ratio.
levels are elevated in the seawater-acclimated animals. Three
unsaturated fatty acids make up nearly 40 % of the total fatty
acyl chain composition of membranes from each acclimation
group. These include oleic acid (18:1), which makes up 12 %,
and two polyunsaturated fatty acids (PUFAs), arachidonic acid
(20:4), contributing 16–17 %, and docosahexaenoic acid
(22:6), contributing 10 % of the total fatty acid complement of
the gill membranes. The two most predominant saturated fatty
acids, palmitic (16:0) and stearic (18:0) acids, make up
13–15 % and 9–10 % of the fatty acid composition,
respectively. Unsaturation indices (the mean number of double
bonds per fatty acyl chain) are 2.1±0.35 for membranes from
both acclimation groups.
Cholesterol contents, normalized to either membraneassociated protein or phospholipid, are not significantly
different for the two acclimation groups (Table 3), indicating
that cholesterol levels are comparable in the gill plasma
membranes of freshwater- and seawater-acclimated eels.
Discussion
Characterization of P4 membranes
A mixture of cell types is present in the gill epithelium of
teleost fishes, with the majority of Na+/K+-ATPase occurring
in the basolateral membranes of chloride cells (Sargent et al.,
1975; Karnaky et al., 1976b; Perry, 1997). It is, therefore, most
meaningful to compare lipid compositions in basolateral
membranes that originate largely from this cell type to
determine whether lipid restructuring contributes to the
modulation of Na+/K+-ATPase activity. While the membranes
present in P4 (the final membrane fraction) are likely to
represent a mixture of cell types, a significant portion of the
membranous material in P4 is basolateral membranes of
chloride cell origin. The rationale for this is outlined below.
The eightfold enrichment of Na+/K+-ATPase in the final
membrane fraction (P4) indicates that the membranes in the P4
fraction are primarily basolateral surfaces. This conclusion is
based largely on the fact that the Na+/K+-ATPase is localized
to basolateral membranes in gill epithelia of teleosts (e.g.
Karnaky et al., 1976b). While the precise localization of a
second plasma membrane marker, γ-glutamyltranspeptidase (γ-
2390 E. L. CROCKETT
GT), in teleost gill is unknown, it is quite plausible that
localization of γ-GT in teleost gill may be basolateral given (1)
that the enzyme has virtually the same enrichment factor as
Na+/K+-ATPase (Table 1) and (2) that γ-GT, in addition to
its apical distribution, is also associated with basolateral
membranes in other epithelia such as renal proximal tubules
(Spater et al., 1982).
A comparison can be made between protein-specific
Na+/K+-ATPase activities in the final membrane fraction (P4)
obtained in the present study with activities in a chloride cell
preparation (‘zone 3’) reported previously (Sargent et al.,
1975). (Zone 3 in the earlier study was prepared from the gill
epithelium of a related species, the European eel Anguilla
anguilla, using density-gradient centrifugation.) Relative
distributions of epithelial cell types were estimated in zone 3.
While zone 3 contained only 45 % chloride cells by cell
number, it contained 90 % chloride cells by volume. Because
of the larger volume occupied by chloride cells in this
preparation, the majority of membranous material is likely to
be of chloride cell origin. Preparations fortified with chloride
cell membranes should have relatively high protein-specific
activities of Na+/K+-ATPase. A comparison of the proteinspecific activities of Na+/K+-ATPase in P4 (present study;
assayed at 25 °C) and zone 3 (Sargent et al., 1975; assayed at
20 °C) reveals the following: activities from freshwateracclimated
animals
are
1 mg−
1 protein for P4 and
0.310 µmol product min−
1 mg−
1 protein for zone 3,
0.082 µmol product min−
while activities from seawater-acclimated animals are
1 mg−
1 protein for P4 and
0.73 µmol product min−
−
1
−
1 protein for zone 3.
0.65 µmol product min
mg
Because protein-specific enzyme activities in P4 are higher
than or comparable with those in zone 3, these data indicate
that P4 contains a large proportion of basolateral membranes
from chloride cells.
Lipid restructuring in salinity-acclimated and -acclimatized
animals
To the author’s knowledge this is the first report detailing
the lipid composition of enriched plasma membranes from the
gills of salinity-acclimated teleost fishes. While previous
studies have characterized gill lipids from freshwater- and
seawater-acclimated (or acclimatized) animals, they have done
so using whole-gill lipid extracts in which a mixture of plasma
and intracellular membrane lipids is present.
While the present data reveal the expected rise (upon
seawater-acclimation) in Na+/K+-ATPase activity, this
increase in activity is not accompanied by any major changes
in phospholipid or neutral lipid compositions. The absence of
a significant difference in compositions of fatty acyl chains in
gill basolateral membranes from freshwater- and seawateracclimated eels is in keeping with the similarity of total gill
fatty acids in the European eel (Anguilla anguilla) acclimated
for 3 months to either fresh water or sea water (Thomson et
al., 1977). In addition, gill Na+/K+-ATPase activities are higher
in 1 °C-acclimated Atlantic cod (Gadus morhua) than in 8 °C-
acclimated animals, yet lipid class and fatty acid compositions
are the same after a 17 day acclimation period (Staurnes et al.,
1994).
The absence of lipid compositional differences noted above
is in contrast, however, with the results of other studies that
have used either salinity-acclimated or -acclimatized (fieldcollected) animals. Differences in phospholipid and fatty acyl
chain compositions of various tissues (including gill) are found
in salinity-acclimated guppies (Daikoku et al., 1982).
Variations in fatty acyl chain compositions of the gill have also
been noted in the European eel (Thomson et al., 1977) and
brown trout (Pagliarani et al., 1991) after the animals have
been acclimatized to either fresh water or sea water (eel) or
fresh water and brackish water (trout). These differences in
fatty acid composition of salinity-acclimatized fishes may
largely reflect dietary changes.
Despite the results presented in the present study, the
possibility cannot be ruled out that lipid restructuring (when it
does occur and regardless of its bases) plays some role in altering
the catalytic character of Na+/K+-ATPase during salinity
adaptation. Variation in discontinuity temperatures in Arrhenius
plots of Na+/K+-ATPase correspond with differences in the
relative abundances of certain polyunsaturated fatty acids in
salinity-acclimatized teleosts (Thomson et al., 1977). Thomson
et al. (1977) reported striking differences in the relative amounts
of 20:4 and 22:6 in the gills from freshwater- and seawateracclimatized Anguilla anguilla. Gill lipids from freshwateracclimatized animals consist of 22 % of 20:4 and only 8 % of
22:6, while seawater animals are particularly rich in 22:6 (19 %)
with relatively low amounts of 20:4 (9 %) (Thomson et al., 1977).
These data suggest that at least some of the catalytic
discrepancies between the enzyme in freshwater and seawater
animals may be determined by lipid remodelling.
A role for lipid restructuring during salinity change may be of
heightened importance in apical membranes compared with
basolateral domains of the gill epithelium. As pointed out by
Hazel and Williams (1990), membranes that face varying ionic
environments (such as the apical membranes of the gill) may
need to undergo compositional changes to ameliorate changes in
chemical and physical properties of the membranes that could
otherwise occur. Changes in the lipid compositions of apical
membranes have been reported for intestinal brush border from
rainbow trout after transfer to sea water (Leray et al., 1984). After
only 1 day of seawater acclimation, the brush-border membranes
have twofold or greater levels of 22:6 in PC and PE (PC
representing approximately 45 % and PE approximately 22 % of
the phospholipid class composition of the membrane) than their
freshwater counterparts (Leray et al., 1984). Lipid restructuring
in this case is not induced by dietary differences. Significant
differences are also detected in the relative levels of the saturated
fatty acid 18:0, levels of which decline in the brush-border
membranes upon seawater acclimation (Leray et al., 1984).
Organisms whose environments span freshwater, marine or
brackish environments undergo either (1) a profound
metamorphosis (e.g. diadromous fishes such as the American
eel) affecting the behaviour, morphology, physiology and
Membrane lipids and gill Na+/K+-ATPase in eel 2391
biochemistry of the animal, or (2) adjustments in the absence
of metamorphosis (e.g. many intertidal or estuarine
organisms). The catadromous adult eel undergoes a ‘silvering’
process that transforms a yellow-phase eel into a silver-phase
individual. This metamorphosis entails marked changes in both
the locomotory musculature (Egginton, 1986a,b) and the
ultrastructural attributes of the gill epithelium (Fontaine et al.,
1995). Silvering occurs prior to the downstream migration of
the eel through the estuary and is presumably hormonally
driven (Fontaine et al., 1995). The time required to migrate
through the estuary (i.e. to make the transition between
freshwater and seawater environments) is relatively short (4
days; Barbin et al., 1998) and, therefore, the animal has
probably acquired most, if not all, of the necessary changes
during metamorphosis for life as a marine teleost.
Some of the responses in a yellow-phase eel acclimated to
sea water would be expected to be similar to those changes that
occur during silvering in the eel. Elevated activities of gill
Na+/K+-ATPase, compared with the freshwater condition,
appear to be a requisite for life in sea water. Whether different
mechanisms are responsible for the increase in enzyme activity
in animals acclimated to sea water compared with those
undergoing metamorphosis, or even for organisms living in an
estuary and experiencing daily salinity fluctuations as a result
of tidal cycling, is not known. Seawater-acclimated fishes show
changes in Na+/K+-ATPase subunit expression concomitant
with altered activities of gill Na+/K+-ATPase (Luke et al.,
1993; Cutler et al., 1995; Lee et al., 1998). While altering the
rates of subunit gene expression may also be a mechanism for
altering the catalytic activity of Na+/K+-ATPase during
metamorphosis, it is still plausible that lipid restructuring may
also contribute because dramatic changes in fatty acid
composition in other tissues have been reported in
metamorphic trout (Sheridan et al., 1985).
In summary, the data presented here demonstrate that lipid
restructuring in the basolateral membranes of the gill does not
contribute to increases in Na+/K+-ATPase activity during
seawater-acclimation of yellow-phase eels. This does not,
however, exclude the possibility that lipid remodelling may
provide a mechanism for influencing enzyme activity in
salinity-acclimatized, metamorphic and/or intertidal animals.
The underlying mechanism(s) for modulation of Na+/K+ATPase activity during these different scenarios remain(s) to
be elucidated.
Many thanks to Dr Barbara Kent, Gary Gorczyca and the rest
of the staff at the Mount Desert Island Biological Laboratory. I
especially thank Dr R. Patrick Hassett for all his help with the
acclimations and Dr Jeffrey Hazel for comments on the
manuscript. This research was supported by NSF IBN 9628952.
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