The giant mudskipper Periophthalmodon schlosseri facilitates active NH4+ excretion by increasing acid excretion and decreasing NH3 permeability in the skin
1 Department of Biological Sciences, National University of Singapore, 10
Kent Ridge Road, Singapore 117543, Republic of Singapore
2 Department of Biology and Chemistry, City University of Hong Kong, Tat
Chee Avenue, Hong Kong, China
3 Department of Zoology, University of Guelph, Guelph, Ontario, Canada NIG
2W1
4 Centro Interdisciplinar de Investigação Marinha e
Ambiental-CIIMAR, Rua do Campo Alegre 823, 4150-180 Porto, Portugal
5 Natural Sciences, National Institute of Education, Nanyang Technological
University, 1 Nanyang Walk, Singapore 637616, Republic of Singapore
* Author for correspondence (e-mail: dbsipyk{at}nus.edu.sg)
Accepted 7 November 2003
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Summary |
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Key words: ammonia, excretion, permeability, cholesterol, fatty acid, gill, H+-ATPase, lipid, membrane, membrane fluidity, mudskipper, Periophthalmodon schlosseri, phospholipid, proton pump, skin
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Introduction |
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P. schlosseri has a very high tolerance of environmental ammonia.
It can survive for more than 1 week in 100 mmol l-1
NH4Cl, an ammonia concentration that most other fish species would
not survive for a few hours (Peng et al.,
1998). It does not produce urea, or store ammonia in its body when
exposed to high concentrations of environmental ammonia. Under such
experimental conditions, the plasma ammonia concentration remains low
(Peng et al., 1998
;
Randall et al., 1999
). Randall
et al. (1999
) demonstrated
that P. schlosseri could actively excrete NH4+
even when the blood-to-water NH3 partial pressure gradient
(
PNH3) was reversed. Using a pharmacological
approach, it was shown that the branchial Na+/K+
(NH4+)-ATPase and the Na+/H+
(NH4+) exchanger were involved in the process. Wilson et
al. (2000
) confirmed the
presence of these transporters using immunolocalization techniques.
Active pumping of NH4+ is energetically more
efficient than turning ammonia into urea or glutamine
(Ip et al., 2001;
Jow et al., 1999
), because
only one mole of ATP is required for every two moles of
NH4+ eliminated (substituting K+ transport
via Na+,K+-ATPase). However, for such a system
to function effectively there must be mechanisms to prevent the back diffusion
of NH3 when the concentration of total ammonia in the environment
reaches a level that would impose an inwardly directed
PNH3. There are two possible solutions to such a
problem. Firstly, P. schlosseri may be capable of acidifying the
external medium, as suggested by Chew et al.
(2003
); the excreted
NH4+ stays in the ionized form and is prevented from
penetrating back into the body as NH3. Secondly, the skin of P.
schlosseri may have a low permeability to NH3, despite the
fact that cutaneous respiration accounts for 50% of aerial or aquatic
respiration (Clayton, 1993
), a
percentage that is greater than in fishes that respire in water. Indeed,
membranes with low NH3 permeability have been suggested to be
present in the apical membrane of the ascending limb of the loop of Henle in
the mammalian kidney (Kikeri et al.,
1989
).
To test the validity of the first possible solution, i.e. acid excretion, attempts were made to measure the pH of the water within the burrows of P. schlosseri in its natural habitat in comparison to open waters in the vicinity. We speculated that the pH of the former was lower than that of the latter. Next, in order to show that the fish is the major cause of the decrease in pH of the external medium, efforts were made to verify the capabilities of P. schlosseri to actively excrete NH4+ to, and lower the pH of, the bulk water in an artificial burrow. To analyze the environmental signals that prompted the increase in acid excretion, effects of environmental pH or ammonia on the rate of acid excretion in P. schlosseri were studied. In order to verify that acid excretion in this fish involves a V-type H+-ATPase, bafilomycin was tested as an inhibitor. Furthermore, we hypothesized that mechanisms involved in NH4+ and acid excretions in P. schlosseri occurred in close proximity to each other in the head region of the fish. Therefore, experiments were performed with a specimen positioned in an artificial chamber with the head and tail regions separated by a rubber septum just behind the opercula into two compartments, in which waters at various pH values or concentrations of NH4Cl were introduced.
To test the validity of the second possible solution, i.e. low
NH3 permeability in the skin, 15NH4Cl was
introduced into the head or tail compartments in the artificial chamber, and
the recovery of 15N-ammonia in the tail or head compartments were
analyzed. We hypothesized that the permeability of NH3 through the
surfaces of the head region was greater than that of the tail region of the
fish. In a separate experiment, dissected skin was set up across an
Ussing-type apparatus to determine the permeation of NH3 through
the skin directly. We also examined the role of skin lipids in mediating
ammonia permeability. While carriers like aquaporins can enhance the
permeability of membranes to NH3
(Wood, 1993;
Nakhoul et al., 2001
), the
lower limits of permeability are set by the lipid properties of membranes. The
lipid-water partition coefficient for NH3 is low (Evans and
Cameron, 1989; Wood, 1993
)
suggesting membrane permeability to NH3 is generally low. In
certain biological situations, however, membrane permeability to
NH3 has to be further reduced. For example, the very low
NH3 permeability of the ascending limb of the loop of Henle in the
mammalian kidney has been attributed to its membrane lipid composition
(Kikeri et al., 1989
). Several
mechanisms for reducing the permeability of membranes to NH3 are
apparent from model studies. In particular, the cholesterol and phospholipid
fatty acid contents of artificial membranes have been shown to affect the
permeability or artificial membranes to NH3 (Lande et al.,
1994
,
1995
). In the face of very
high external levels of total ammonia such as those encountered by P.
schlosseri, reductions of the permeability of skin would help reduce the
influx of NH3. Hence, we analyzed the lipid composition
(phospholipids, phospholipid fatty acids and cholesterol) of the skin of
P. schlosseri to test the hypothesis that its skin would have a lipid
composition that would render low NH3 permeability. In addition,
attempts were made to elucidate if P. schlosseri was capable of
altering the lipid composition of its skin in response to long-term ammonia
exposure.
In this report, NH3 represents unionized molecular ammonia, NH4+ represents ammonium ion, and total ammonia refers to the sum of NH3 and NH4+.
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Materials and methods |
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Preparation of SW for experiments
50%SW was aerated for at least 24 h before the addition of a biological
buffer. Trizma-Base (pKa=8.1, buffering range=7-9) was used to
buffer the pH of the 50%SW at 7.0, 7.6, 8.0 or 8.5.
2-(N-morpholino)ethanesulfonic acid (MES; pKa=6.1,
buffering range=5.5-6.7) was used to buffer the 50%SW at pH 6.0. The final
concentration of buffer used was 2 mmol l-1. After adding the
buffer, the pH of the 50%SW was adjusted to the desired value using
concentrated HCl or 2 mol l-1 NaOH. The buffered water was strongly
aerated over night and the pH adjusted accurately again the next day before
usage. The pH was determined with an Orion model 420A pH-meter (Boston, MA,
USA) and a Corning G-P Combo w/RJ Tris-electrode (Halstead, Essex, UK).
Preliminary results obtained indicated that Tris did not affect the ammonia
excretory rate in P. schlosseri.
Experiment 1: pH of water from natural and artificial burrows
Water samples were collected from six different burrows of P.
schlosseri on the mudflat and from the adjacent canal (N=6) at
Pasir Ris, Singapore, during the non-breeding season between July and
September 2001. The identity of the burrow was judged by the characteristic
fin marks left around the opening of the burrow as a result of the
mudskipper's locomotory activities on land. Sampling was made at the water
surface or 30 cm deep inside the burrow. The pH and salinity of the water
sample were determined by a hand-held pH meter and an YSI Model 33 S-C-T meter
(Yellow Spring, OH, USA), respectively.
Specimens (70-80 g, N=4) were placed individually in each
artificial burrow containing unbuffered 50%SW (pH 8.2). The artificial burrow
was constructed using a translucent plastic hose (5 cm i.d.; 260 cm length;
Togawa Super Sun-braid hose, Japan), with a plastic box containing a thin
layer of water attached to one end, and a rubber stopper at the other. The
hose (5000 ml) was bent to a U-shape and mounted on a wood board. The box
served as the `land-surface', with which the fish could have the choice of
entering into the artificial burrow or staying on `land'. The artificial
burrows were kept in a dark cabinet such that the behaviour of the fish could
be observed with minimal disturbances. A three-way stopcock valves (Connecta,
Helsingborg, Sweden) was installed at 170 cm from the opening end of the hose
to facilitate the collection of water samples. Water samples were collected at
0 h, 3 h, 6 h and 24 h during the first day of experiment. Subsequently, water
samples were taken every 24 h, and the experiment was continued for 8 days in
order to verify the steady state concentration of total ammonia build-up
inside the burrow by P. schlosseri. The pH of the ambient 50%SW was
measured using an Orion model 420A-pH meter and a Corning G-P Combo w/RJ
Tris-electrode. Ammonia concentration was determined according to Chew et al.
(2003).
Experiment 2: Effects of environmental pH or ammonia on the rate of acid excretion
Specimens were placed individually into darkened 1000 ml conical flasks and
acclimatized overnight in ordinary 50%SW. After acclimation, specimens were
pre-adapted for 1 h in 5 volumes (w/v) of 50%SW containing 2 mmol
l-1 Tris adjusted to pH 7.0 at 25°C. During pre-adaptation,
experimental and recovery periods, strong aeration (using an air stone fully
immersed in the medium) was maintained in the conical flask to drive off any
CO2 produced by the fish, thereby removing the acidification effect
resulted from the hydration of the excreted CO2. Preliminary
studies revealed that if this was not done, the rates of H+
excretion would be lower than those reported herein because excreted
CO2 also contributed to the decrease in environmental pH, which in
turn influenced the rate of H+ excretion.
After the pre-adaptation period, the ordinary 50%SW was drained off and buffered 50%SW of pH 7.0 (control condition) was added. After exactly 1 h, this buffered pH 7.0 50%SW was collected in a plastic vial for determination of the baseline rates of acid and ammonia excretion. The same volume of buffered 50%SW containing 2 mmol l-1 MES at pH 6.0 or 2 mmol l-1 Tris at pH 7.0, 7.6, 8.0 or 8.5 (experimental condition) was immediately added to the conical flask. This buffered 50%SW was again collected in a plastic vial after exactly 1 h fordetermination of the effects of pH on the rates of acid and ammonia excretion. The fish was rinsed several times with ordinary 50%SW to get rid of remnants of the test solution, and then left to recover overnight in 50%SW with strong aeration in the conical flask. Recovery studies in buffered pH 7.0 50%SW were conducted on the following day.
Preliminary results showed that strong aeration affected the pH of the buffered 50%SW during the experimental or recovery period. Therefore, a flask containing buffered 50%SW of the same pH but without fish was used as a blank for comparison. The blank was strongly aerated to the same extent as the solutions used in the experiments with fish, and the pH recorded after 1 h.
Titration was done using a burette, and water pH was measured using an
Orion model 420A pH-meter and a Corning G-P Combo w/RJ Tris-electrode. The
titrant used was 0.01 mol l-1 NaOH or 0.01 mol l-1 HCl.
The pH of the media in the presence of fish was titrated back to those of the
corresponding blank values. Preliminary results obtained revealed that the
titratable acid flux obtained using this method was similar to that obtained
by titration to pH 4.0, as prescribed in the method of McDonald and Wood
(1981).
For total ammonia analysis, water samples were acidified to pH 2.0 with 1 mol l-1 HCl to keep the ammonia present in its ionized form (NH4+). The ammonia concentrations of water samples were analyzed using a Tecator Aquatec System (Hoganas, Sweden) equipped with an Ammonium Cassette. The resolution power of the Aquatec Analyzer was tested by calibrations at three different concentrations (30, 31 and 32 mmol l-1) of standard NH4Cl solution. The calculated concentrations (N=5) from the readings obtained were 30.16±0.26, 30.96±0.34 and 32.12±0.23 mmol l-1, respectively, which were significantly different from each other.
Net acid flux (µmol h-1 g-1 fish) was obtained by summing the fluxes (µmol h-1 g-1 fish) of titratable acid and total ammonia, because a portion of the excreted acid (H+) would react with the ammonia excreted as NH3 to form NH4+.
To verify that a V-type H+-ATPase was involved, specimens (26-34 g) were exposed to pH 7.0 (N=3) or pH 9.0 (N=3) in the presence of bafilomycin A1 (Sigma Chemical Co.) for 1 h during the experimental condition (using a 500 ml conical flask). Six fish were consecutively tested in the same 120 ml volume of buffered (10 mmol l-1 Tris) medium containing 8 µmol l-1 of bafilomycin. Bafilomycin was dissolved in dimethysulphoxide (DMSO) before mixing with the 120 ml of buffered 50%SW. A control medium without bafilomycin, but containing 0.17% DMSO, was used to test whether DMSO has any effect on the rates of H+ and ammonia excretion by the fish. At the end of the 1 h period, the pH of the bafilomycin-containing medium was recorded and 0.05 ml of the medium was collected for total ammonia assay. The pH of the remaining medium was then adjusted back to the desired value (either pH 7.0 or 9.0) before putting the next fish into it.
To evaluate the effects of environmental ammonia alone on the rate of net acid flux, specimens were pre-adapted for 1 h at pH 7.0 following the acclimation period. For the control condition, the fish was exposed to buffered 50%SW at pH 7.0 or 8.0 for 1 h. For the experimental condition, the fish was exposed to buffered 50%SW at pH 7.0 or 8.0 containing 10, 20 or 30 mmol l-1 NH4Cl. The titratable acid flux and ammonia flux were determined in order to obtain the net acid flux. Again, flasks containing the test media with buffer, but without fish, served as blanks.
Experiment 3: Ammonia and H+ fluxes in the head or tail portion of specimens in two half chambers separated by a rubber septum
Specimens were anaesthetized with neutralized MS 222 (Sigma Chemical Co.,
USA) at a final concentration (w/v) of 0.125% for 10 min. The anesthetized
specimen was positioned in a special chamber with the rubber septum, creating
a partition between the head and the tail of the fish. The apparatus was
fabricated locally using transparent plastic. The volumes of the `head' and
the `tail' compartments were 100 ml and 400 ml, respectively. Preliminary
tests using KMnO4 proved that the set-up was leak-proof for a
minimum of 24 h. The fish was left to acclimatize in the chambers in ordinary
50%SW for at least 2 h. After that, the 50%SW in the compartments was replaced
with 50%SW containing Tris alone (pH 7.0 or pH 8.0) or 50%SW containing Tris
(pH 7.0 or 8.0) and 30 mmol l-1 NH4Cl. Water samples
were taken at the start of the experiment (0 h) and at 24 h. The pH and total
ammonia concentration in the water samples were determined as described
above.
To evaluate the effects of ammonia exposure on ammonia and H+ fluxes in the head and tail regions, specimens were exposed to 30 mmol l-1 NH4Cl in a plastic aquarium for 6 days, with renewal of the external medium every 2 days. They were then thoroughly rinsed with Tris-50%SW, anesthetized and placed individually in the compartment chamber to determine ammonia and H+ fluxes under various experimental conditions.
Experiment 4: The flux of 15N-ammonia through the head or tail regions of live specimens
Experiments were performed using the compartment chambers and
15NH4Cl (99.7%, Boehringer, Germany). The 50%SW in the
compartments was replaced with Tris-50%SW (pH 7.0 or pH 8.0) alone or
Tris-50%SW (pH 7.0 or pH 8.0) containing 5, 10 or 20 mmol l-1
15NH4Cl. One water sample was taken immediately at 0 h
and another after 6 h. Due to the limited amount of
15NH4Cl available at the time of the experiments,
results were obtained as average of duplicates for each experimental condition
only.
15N- and 14N-NH3 were released from
samples using 0.1 mol l-1 borate buffer (pH 10.4) and trapped on
slips of glass filter paper (Whatman CF/C) in glass vials (22 ml)according to
the method of Iwata and Deguchi
(1995).
15N-measurement was performed with a quadrupole type mass
spectrometer (QP 2000, Shimadzu, Japan). The ion source temperature was
maintained at 250°C under vacuum conditions for sample analysis. The ratio
of 14N-ammonia and 15N-ammonia in each water sample was
determined and the concentration of 15N-ammonia calculated and
expressed as % atom excess. Calculation of % atom excess was as follows
(Constantin and Schnell, 1990
):
(Ratiosample-Ratiostandard)x 100, where
Ratio=15NH3/(14NH3+15NH3).
Known concentrations of 14NH4Cl or 15NH4Cl (50-100 µg) were used to test the recovery efficiency of the method used above. Ammonia liberated on the glass filter paper was introduced directly to the mass spectrometer. The recovery was 98.9%±0.2 (N=6).
Experiment 5: Transepithelial ammonia flux across the dissected skin
A portion of the fish skin (about 4 cm2) was carefully descaled,
dissected out from a freshly killed specimen, rinsed with saline solution
(0.9% NaCl, with 2 mmol l-1 Hepes buffer; this same composition was
used throughout the experiment), and immediately mounted in an Ussing-type
apparatus. The apparatus was made of two separate pieces of Plexiglass,
between which, the skin could by mounted with two rubber O-rings. The chambers
had a total volume of 13 cm3 and a circular opening of 3.14
cm2 (2 cm diameter).
After mounting the skin, both half chambers were filled with saline
solution (pH 7.0), and the skin was allowed to acclimatize to this artificial
condition for 10 min. Subsequently, the solution was replaced, with one
half-chamber being filled with saline solution at pH 7.0 alone, and the other
half with saline solution containing 10 mmol l-1 NH4Cl
at pH 8.0. There were two gradients in this setup: (1) a tenfold
NH4Cl gradient and (2) a 1-unit pH gradient. In this way, an
ammonia concentration gradient was set up across the skin, and any
NH3 diffusing down the chemical potential gradient would be trapped
as NH4+. Mixing of the media in the two half chambers
was achieved by two streams of air currents directed at the surface of the
media through two Pasteur pipettes, which also ensured oxygenation of the
media. A sample (100 µl) was collected every 10 min for a period of 1 h,
and immediately acidified with 5 µl of 2 mol l-1 HCl. Total
ammonia content was determined later colorimetrically according to the method
of Chew et al. (2003). By
reversing the ammonia gradient across the skin, both NH3 efflux
(serosa-mucosa) and influx (mucosa-serosa) could be determined.
Since P. schlosseri is `amphibious', with the skin highly adapted
for cutaneous respiration in water and on land
(Clayton, 1993), experiments
were also performed on the skin of the frog Rana catesbiana (100-120
g), obtainable commercially from markets in Singapore, for comparison.
Naturally, the skins of these animals had different thickness (x),
which directly affects the concentration (c) gradient
(
c/
x). The thickness (µm) of the skins was
determined using a Leica TCS-SP2 RS Confocal Laser Scanning Microscope (Tokyo,
Japan) at 488 nm with a reflection model 10x objective lens. Since flux
= -P(
c/
x), where P is the permeability
constant, the NH3 flux across the skins of these animals cannot be
compared directly, but because
c was constant in these
experiments, it can be incorporated into P. Hence, in this study, permeability
constants, calculated as flux (
x), i.e. µmol
min-1 cm-2 (10-4 cm), or 10-4
µmol min-1 cm-1, were compared instead.
Experiment 6: The lipid composition of the skin
Specimens were exposed to 50%SW containing 10 mmol l-1 Tris (pH
7.0) with (ammonia exposed) or without (control) 30 mmol l-1
NH4Cl for 6 days. The external medium was renewed daily. No food
was provided throughout the experimental period. After 6 days, specimens were
killed with a strong blow to the head. The lateral skin was dissected
immediately, with special care not to sample any muscle, rinsed in saline
solution and frozen in liquid nitrogen. Samples were stored at -80°C until
analysis.
Lipids were extracted from the skin according to the method of Folch et al.
(1957). Phospholipids were
isolated from the neutral lipids by acetone precipitation using a modified
technique based on the procedure of Hoevet et al.
(1968
). The neutral lipids
(cholesterol is part of this fraction) and phospholipid fractions were dried
separately under nitrogen.
For the determination of cholesterol, the neutral lipids were redissolved in 1 ml of isopropanol per gram of skin (w/w). Thereafter, 10 ml of sample were assayed for cholesterol using a Sigma Diagnostics Kit #401 (St Louis, MO, USA).
The dried phospholipids were resuspended in 0.2 ml g-1 tissue
(w/w) of chloroform:methanol (1:1), and the phospholipids separated on
silica-coated chromatography plates (Z29297-4, Merck, Darmstadt, Germany)
using a solvent system consisting of chloroform:methanol:acetic acid:water,
100:75:7:4 (v:v:v:v). Developed plates were exposed to iodine vapour for
visualization of phospholipid spots. Six phospholipid classes were detected
and completely separated. The total amount of phospholipids (Tot PLs) in each
class was estimated by summing the different fatty acids recovered in that
class. Fatty acid composition of individual phospholipid classes was analyzed
according to the methods of Gillis and Ballantyne
(1999). Methylated fatty acids
were analyzed using a chromatography column (J & W DB-225MS, Folsom, CA,
USA; Cat. # 122-2932, dimension - 30 m length x 0.2 mm i.d. x 0.25
µm film thickness) in a gas chromatograph (HP 6890; Hewlett
Packard-Agilent, Toronto, ON, Canada) fitted with a flame ionization detector
(FID, HP 6890 series) and an automatic injector (HP 6890 series
Split/Splitless Inlet). The column oven temperature was programmed with an
initial increase from 150°C to 210°C in the first 6 min followed by
isothermal separation at 210°C for 30 min. Fatty acids were identified by
comparison of retention times of a known standard containing all fatty acids
of interest. Chain lengths shorter than C:14 were not resolved under the
present conditions, and thus are not reported.
Statistical analysis
Results are presented as means ± standard error (S.E.M.).
Differences between means were evaluated by one-way analysis of variance
(ANOVA) followed by Student-Neuman-Keul's multiple range test or two-tail
Student's t-test, where applicable. Any difference where
P<0.05 was regarded as statistically significant.
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Results |
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The ammonia concentrations in water samples collected from the surface water and 30 cm below the water surface in the burrow were 2.67±0.34 and 2.98±0.27 mmol l-1, respectively. For water collected from the adjacent canal, the ammonia concentration was 0.034±0.003 mmol l-1, which was significantly lower than those in the burrow water.
Capability of P. schlosseri to alter the pH of the medium and actively excrete ammonia into it within an artificial burrow
In the presence of a specimen of P. schlosseri, the pH of the
50%SW in the artificial burrow changed significantly from pH 8.2 to pH 7.0
within 24 h. (Fig. 1A). A
decrease in pH of about 0.60 units was observed in the burrow during the first
3 h (Fig. 1A). The ammonia
concentration in the ambient 50%SW in the artificial burrow reached 1 mmol
l-1 after 1 day (Fig.
1B). By day 6, the ambient ammonia concentration increased to 10
mmol l-1 with no further changes thereafter.
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Proton excretion in response to environmental pH or ammonia
There was an apparent influx of titratable acid to the fish when the
specimen was exposed to pH 6.0 (Fig.
2). In contrast, at pH 7.0, 7.6, 8.0 or 8.5, there were
significant increases in the titratable acid flux to the medium
(Fig. 2), increasing in the
order pH 7.0<7.6<8.0<8.5. Alkaline pH had no effect on ammonia
excretion in this mudskipper; the ammonia excretion rates at pH 8.5 or 8.0
were comparable to that at pH 7.0(Fig.
2). As a result, the net acid efflux in P. schlosseri
increased when the mudskipper was exposed to alkaline pH
(Fig. 2). Between pH 7.0 and pH
8.5, the net acid flux appeared to vary linearly with the change in the pH in
the external medium. Bafilomycin exhibited a large inhibitory effect on the
net acid flux of specimens exposed to pH 7.0 or 9.0
(Fig. 3).
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When exposed to pH 7.0 in the presence of 20 or 30 mmol l-1 NH4Cl, the net acid fluxes were significantly higher than the respective control values (Fig. 4). Increases in net acid flux were observed in specimens exposed to 10, 20 or 30 mmol l-1 NH4Cl at pH 8.0.
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Ammonia and H+ excretion in the head or tail regions of P. schlosseri
Ambient total ammonia concentration in the head compartment increased when
specimens were exposed to increasing NH3 concentrations (achieved
via increasing pH) in the body compartment
(Table 1). Preliminary
experiments showed that specimens exposed to 30 mmol l-1
NH4Cl at pH 9.0 in the tail compartment died after 6 h. The final
pH of the media in the head or tail compartments was comparable to the initial
pH values. With increasing NH3 concentration (because of the
increasing pH) in the tail compartment, ammonia excretion through the head
region still occurred despite the presence of 30 mmol l-1
NH4Cl in the head compartment
(Table 2). P.
schlosseri was able to drastically alter the pH of the medium in the head
compartment but not that in the tail compartment (Tables
1,
2).
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The total ammonia concentrations built up in the head compartment containing specimens subjected to prior exposure to 30 mmol l-1 NH4Cl at pH 7.0 for 6 days (Table 3) were lower than in those containing specimens not subjected to prior ammonia exposure (Tables 1, 2).
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Permeation of 15NH4Cl through the head or tail regions of P. schlosseri
When specimens were exposed to 15NH4Cl at pH 7.0 or
8.0 in the head compartment, 15N-ammonia enrichment (expressed as %
atom excess of 15N) was detected in the external medium in the tail
compartment after 6 h (Table
4). In contrast, when 15NH4Cl was added to
the tail compartment containing medium at pH 7.0, no 15N-ammonia
was detected in the medium in the head compartment, except with 20 mmol
l-1 NH4Cl (Table
5). However, with a pH 8.0 medium in the tail compartment, a
greater enrichment of 15N-ammonia was observed in the medium in the
head compartment (Table 5).
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NH3 flux through dissected skin in an Ussing-type apparatus
The skin of P. schlosseri (average thickness 132 µm) was
symmetrical with respect to NH3 permeability. The NH3
fluxes (N=5) from mucosa to serosa (0.0105±0.0005 mmol
min-1 cm-2) and from serosa to mucosa
(0.0096±0.0007 mmol min-1 cm-2) were comparable.
The permeability constant was calculated to be 1.33x10-4 mmol
min-1 cm-1.
The NH3 flux across the skin of R. catesbiana (average thickness 440 µm) was 0.0054±0.0011 mmol min-1 cm-2 (N=3). The permeability constant was 2.38x10-4 mmol min-1 cm-1, which was 1.8-fold that of P. schlosseri.
The lipid compositions in the skin of P. schlosseri
Six classes of phospholipids were detected from the skin of P.
schlosseri, namely phosphatidylcholine (PC), phosphatidylserine (PS),
phosphatidylinositol (PI), phosphatidylethanolamine (PE), cardiolipin (CL) and
sphingomyelin (SM). PC was the most abundant phospholipid class, comprising
almost half of the total phospholipid content in the skin
(Table 6). The next highest
were PS and PE, contributing about 15% each, followed by SM with 13%. The two
minor categories were PI and CL, with 5 and 1%, respectively. The skin of
P. schlosseri had a high cholesterol content
(Table 7). The ratio of
cholesterol:phospholipids was 6.2±0.3 (N=5).
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In these lipids, the amount of total saturated fatty acids was twice that of the total monounsaturated fatty acids, which in turn were twofold higher than that of the total polyunsaturated fatty acids (Table 7). Among all the phospholipids determined in this study, the most abundant fatty acid was the saturated fatty acid 18:0 (about 29%), followed by the monounsaturated fatty acid 18:1 (23%), and by another saturated fatty acid, 16:0 (17%) (Table 7). The major saturated fatty acids recovered were 18:0 and 16:0 (52% and 34% of the total saturated fatty acids, respectively). For monounsaturated fatty acids, 18:1 was the major fatty acid (84% of the total monounsaturated fatty acids), followed by 16:1 (12%). All others represented only 1.5% or less of this class. Among the polyunsaturated fatty acids (PUFAs), 20:4(n-6) was the main representative (37% of the total PUFAs), followed by 18:2(n-6) and 22:4(n-6) (22% and 18%, respectively). A few fatty acids were absent in this class, e.g. 20:3(n-3) and 22:2(n-6), or present only in traces (less than 1 nmol g-1), e.g. 18:3(n-3) and 20:2(n-6). Specifically, among the PUFAs, n3 was completely absent from SM and PI in the control specimens. Cardiolipin also lacked n3 PUFAs in the control fish (Table 8), despite its being an exclusively mitochondrial phospholipid.
|
Effects of ammonia exposure on lipid compositions in the skin
The cholesterol content in the skin increased significantly to
5.5±0.03 µmol g-1 after specimens were exposed to 30 mmol
l-1 NH4Cl for 6 days
(Table 7). Ammonia exposure led
to a significant decrease in SM, from 13.3% to 12.7%
(Table 6), but had no effect on
the content of other phospholipids. Exposure to 30 mmol l-1
NH4Cl for 6 days led to significant increases in the amounts of
total fatty acids in the phospholipid fraction, the total saturated fatty
acids and the total monounsaturated fatty acids, but not the PUFAs
(Table 7). With respect to the
percentages of various fatty acids within a certain class of phospholipid in
the skin of P. schlosseri, changes were detected in the PC and PI
fractions (Table 8). In PC, the
total PUFAs increased from 11.3 to 13.1%, and total n6 PUFAs from 9.4% to
10.8%. In PI, both total polyunsaturated fatty acids and n6 PUFAs decreased
(from 30.4% to 26.4%).
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Discussion |
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In the laboratory, we confirmed that P. schlosseri was capable of
lowering the pH of the water in the artificial burrow and elevating the total
ammonia concentration therein. At the beginning, the concentration of total
ammonia in the 50%SW inside the artificial burrow was close to zero, and it
can be deduced that the specimen altered the pH of the external medium solely
as a response to the alkalinity of the medium (pH 8.2). Ammonia excretion at
this stage might be achieved through NH3 diffusion and
`NH3 trapping' (Wilkie,
1997). Surprisingly, within 8 days, a single specimen of P.
schlosseri was able to establish the ambient total ammonia concentration
inside the artificial burrow to 10 mmol l-1. In nature, burrows of
P. schlosseri are constructed on high ground and subjected to tides
only 2-3 days a year (Clayton,
1993
); removal of ammonia would be difficult in the absence of
tidal inundation. The ability to excrete ammonia against a concentration
gradient allows P. schlosseri to survive in a `closed' environment
where ammonia could build up to high concentrations, especially during the
breeding seasons when eggs are developing therein.
As the ambient ammonia concentration builds up, it is imperative that the excreted NH4+ does not dissociate to NH3 and H+ because NH3 can diffuse back into the fish. If acid excretion were also responsive to environmental ammonia at neutral pH (see below), there would be a continuous excretion of acid. This would maintain a low pH (high concentrations of H+) in the boundary water layer of the branchial epithelia, preventing the excreted NH4+ from dissociating and avoiding a back flux of NH3. In essence, this is a process of `NH4+ trapping' in contrast to `NH3 trapping' at the beginning of the experiment.
Increased net acid (H+) excretion in response to environmental pH or ammonia
Instead of studying the expired water from the gills of P.
schlosseri, we monitored the decrease in pH of the bulk water, which was
made up to 10 volumes (w/v) of the fish. The decrease in pH was very large and
rapid at pH 8.5 or pH 9.0, even in the presence of 2 mmol l-1 of
Tris. Hence, large quantities of acid must be excreted to manipulate the
external pH of alkaline waters. At pH 6.0, this mudskipper was capable of
decreasing the net acid efflux, and achieving an increase in the ambient pH
through NH3 excretion. NH3 combined with H+
in the medium, leading to an apparent influx of titratable acid. At alkaline
pH, P. schlosseri responded by excreting more acid to the external
medium. This would explain why the pH of the water within its burrow was close
to neutral.
Bafilomycin, a specific inhibitor of V-ATPases
(Bowman et al., 1988) had a
large inhibitory effect on the net acid flux of specimens exposed to pH 7.0 or
9.0. V-ATPases are H+-translocating enzymes that occur in the
endomembranes of all eukaryotes and in the plasma membranes of many eukaryotes
(Merzendorfer et al., 1997
).
It is possible that the increased rate of proton excretion by the mudskipper
was due to the increased rate of fusion of the V-ATPase-containing vesicles to
the apical surfaces (Merzendorfer et al.,
1997
) of the branchial epithelium and opercular membrane, thereby
increasing the density of the V-ATPases in these regions.
More importantly, P. schlosseri was capable of increasing the rate of net acid excretion in response to the presence of 20 or 30 mmol l-1 NH4Cl in the external medium at pH 7.0 or 8.0, which served as direct evidence linking net acid excretion with defense against environmental ammonia toxicity in these fish.
Active ammonia excretion and acid excretion occurred in the head region of P. schlosseri
In agreement with previous observations made on whole fish
(Chew et al., 2003), active
NH4+ excretion (sets 1, 2 and 3 of
Table 2) through the head
region of P. schlosseri was unaffected by alkaline pH (set 3 of
Table 2). More importantly, our
results reveal that both active NH4+ excretion and
H+ excretion occurred in the head region, where the gills and the
opercular membranes are located. Theoretically, it is imperative for these two
mechanisms to be located together. The branchial and opercular surfaces have
the important functions of allowing passage for gases and other ions.
Excretion of acid to trap the actively excreted NH4+ is
likely to be more effective than modifying the fluidity of these surfaces to
change the permeability of NH3, which would also affect the
permeability of other molecules.
The exact pH of the branchial boundary water layer in P.
schlosseri is unknown. However, with such a large efflux of H+
(3 µmol min-1 g-1) into the bulk of the external
medium, it is logical to conclude that the concentration of H+ in
the boundary layer must be high. Hence, the NH4+
actively pumped out to the boundary layer was likely to remain in the ionized
form. As a result, there would be no back diffusion of NH3. Active
NH4+ excretion accompanied by increased acid excretion
allows P. schlosseri to reserve other ammonia detoxification
mechanisms (e.g. glutamine formation; Peng
et al., 1998) to function at much higher concentrations of
environmental ammonia, rendering it an extremely high ammonia tolerance (96 h
LC50 of 120 mmol l-1 NH4Cl;
Peng et al., 1998
).
The skin of P. schlosseri had a low permeability to NH3
Unlike branchial and opercular surfaces in the head region, the skin of the
tail portion of P. schlosseri apparently adopted other mechanisms to
reduce the penetration of exogenous ammonia. Judging from
Table 1, the skin of P.
schlosseri was permeable to NH3, albeit NH3
permeability could be low. In animals, membranes with low permeability to
NH3 have been suggested to be present in the ascending limb of the
loop of Henle of the mouse (Kikeri et al.,
1989), and the luminal surface of colonic crypt cells
(Singh et al., 1995
) and the
apical membranes of bladder cells (Chang et
al., 1994
) of the rabbit.
In this study, 15N was a qualitative index of NH3 entry into the specimen, and not an indicator of the actual NH3 entry rate. With 15NH4Cl added to the head compartment at pH 7.0, high levels of 15N-ammonia enrichment (69-83% atom excess) were detected in the tail compartment, despite only a very small amount of ammonia being excreted through the tail region. This reconfirms that the branchial and opercular epithelial surfaces were indeed permeable to ammonia. For specimens exposed to ammonia but at pH 8.0 in the head compartment, the level of 15N-ammonia enrichment detected in the tail compartment was slightly lower. This was unexpected because the concentration of NH3 at pH 8.0 was tenfold higher than at pH 7.0. This could be due to the activation of the acid excretion mechanism at pH 8.0 (see above), rendering the boundary water layer acidic which, in effect, reduced the influx of exogenous NH3 through the branchial surfaces.
In contrast, no 15N-ammonia was detected in the external medium
in the head compartment when 5 or 10 mmol l-1
15NH4Cl was added to the tail compartment at pH 7.0.
This means that NH3 entry into the body through the skin was low at
neutral pH, and a significant amount of NH3 entry occurred only at
20 mmol l-1 NH4Cl. These results suggest that the skin
of P. schlosseri, despite being responsible for 50% of O2
uptake in air or in water (Clayton,
1993), was much less permeable to NH3 than the
branchial epithelial surface. However, with a higher percentage of
NH3 at pH 8.0 in the tail compartment, the 15N-ammonia
recovered from the head compartment increased (from
Table 5), confirming that
NH3 could permeate the skin, albeit with relatively low
permeability.
Using an Ussing-type apparatus, it could be shown that the flux of
NH3 through the skin of P. schlosseri was low
(approximately 0.01 µmol min-1 cm-2). We could not
find equivalent results from other fishes for direct comparison, but the
permeability constant for NH3 calculated for the frog R.
catesbiana, which is also amphibious and dependent on cutaneous
respiration, was indeed higher (1.8-fold) than that of P. schlosseri.
Lohrmann and Feldman (1994)
measured the unidirectional ammonia flux through portions of distal colon,
using a 20 mmol l-1 ammonia solution (with no pH difference) to
create a gradient, and reported an ammonia flux of 2.8 µEq h-1
cm-2, or equivalent to 0.047 µmol min-1
cm-2. While the skin and the colon have different morphologies and
functions, the
80% smaller NH3 flux under a much greater
NH3 gradient (taking into consideration the pH effect) in the case
of P. schlosseri suggests that its skin has indeed a low
NH3 permeability.
In order to gauge the usefulness of the decreased NH3
permeability of the skin in reducing the load on the ammonia excretory
capacity of P. schlosseri, a theoretical calculation was performed on
a hypothetical specimen of 110 g, which had a skin surface area of 9.25
cm2. For such a specimen, the total ammonia flux in 1 h through the
skin was 0.01 µmol min-1 cm-2x9.25
cm2x60 min=5.55 µmol, when exposed to 10 mmol
l-1 NH4Cl at pH 8.0. The ammonia excretion rate in
P. schlosseri at pH 7.0 (or pH 8.0) was 0.65 µmol h-1
g-1 (Lim et al.,
2001), or 71.5 µmol h-1 for a 110 g fish. The
majority of this NH3 is excreted through the head region,
presumably through the gills. To remove the excess amount of 5.55 µmol
(entered through the skin) via the gills within the same period (1 h)
would mean an extra load of (5.55/71.5)x100=7.76% to the gills in the
head region. This is a very small percentage, and the calculated quantity is
already an overestimated value because the blood pH was not 7.0 and the plasma
ammonia concentration was not zero, which were the conditions in the
Ussing-type apparatus in vitro.
Lipid compositions of the skin of P. schlosseri suggested its low fluidity
The low NH3 permeability in the skin of P. schlosseri
was likely to be due to its low membrane fluidity. It has been shown that
NH3 permeability in artificial vesicles decreased with decreasing
lipid fluidity (Lande et al.,
1995). The fluidity of a membrane is influenced by the cholesterol
content, phospholipid composition and the fatty acid composition of the
phospholipids.
The rigid steroid ring structure of cholesterol restricts the molecular
motion available to adjacent phospholipid hydrocarbon chains, increasing
membrane orderness (Robertson and Hazel,
1999). Cellular membranes of fish undergo compensatory lipid
compositional changes during thermal acclimation
(Hazel and Williams, 1990
).
For example, gill membranes of trout kept at 5°C have a lower cholesterol
content than those kept at 20°C
(Robertson and Hazel, 1999
).
This reflects an increase in structural disorder with acclimation to lower
temperatures to compensate for the increasing order due to the lower
temperature. The cholesterol content (4.5 µmol g-1) in P.
schlosseri skin was high compared with those of tissues from other
animals (Borchman et al., 1989
;
Crockett and Hazel, 1995
;
Cuculeseu et al., 1995; Fines et al.,
2001
; Molitoris et al.,
1985
; Smith and Ploch,
1991
), indicating the low fluidity and hence low permeability of
its skin. Furthermore, ammonia exposure could lead to higher cholesterol
content in the skin of P. schlosseri (see below).
Compared to fish gills, PE, a destabilizing phospholipid
(Gillis and Ballantyne, 1999),
is present in a smaller amount in the skin of P. schlosseri
(<15%). This would also contribute to a more ordered, less permeable
membrane. PS is present in a greater amount in the skin of this mudskipper
(15-20%) than in fish gill (approximately 8%). Charged lipids such as PS
strongly perturb phospholipid head group motion, probably by changing the
pattern of hydrogen bonding between water molecules, and thus easily segregate
into distinct domains (Aloia and Mlekusch,
1988
). According to Hazel and Williams
(1990
), an increase in PE,
using PS as a substrate for its formation, is adaptive at low environmental
temperatures as it would enhance membrane fluidity. Hence, these results again
suggest that the skin of P. schlosseri has relatively low membrane
fluidity.
PC was the major phospholipid present in the skin of P.
schlosseri. It is known to stabilize the bilayer, favoring the formation
of a laminar structure (Gillis and
Ballantyne, 1999). Stabilizing the lipid bilayer is an important
adaptation in warm temperatures (Logue et
al., 2000
). The PC:PE ratio can be used as an indication of
membrane fluidity, and may be altered according to the environmental and
physiological conditions encountered by an animal
(Hazel and Williams, 1990
).
The PC:PE ratio of 3.4 in the skin of P. schlosseri was much higher
than those of tissues (intestinal mucosa or kidney membranes) of other fishes
(Acierno et al., 1996
;
Hazel and Landrey, 1988
).
Although no information on the lipid compositions of other fish skins is
available, together with the results discussed above, it can be concluded that
the skin of P. schlosseri has a high degree of stability or order,
consistent with low membrane fluidity.
When we consider the different types of fatty acids present in various
phospholipid classes as percentages of the total fatty acids in P.
schlosseri, it becomes obvious that the saturated fatty acids (52%)
predominate, followed by fatty acids with one double bond (monoenes, 28%) and
the PUFAs (20%). These percentages are remarkably different from those of the
skin of the trout (Ghioni et al.,
1997), in which monoenes predominate (48%), followed by the PUFAs
(26%), and the saturated fatty acids (22%). Unsaturated fatty acids adopt a
more expanded conformation, occupy greater areas in monomolecular films, pack
less compactly, and possess lower melting points than their saturated
homologs. Hence, a nearly ubiquitous response to cold temperature is a
reduction in the proportion of saturated fatty acids, and a corresponding
increase in the proportion of unsaturated fatty acids in the lipids of
cellular membranes (Hazel and Williams,
1990
). In general, the relatively high content of saturated fatty
acids in the skin of P. schlosseri supports the above conclusion that
its skin is less fluid, and possibly less permeable, to NH3.
Ammonia exposure affected the lipid composition of the skin of P. schlosseri and its NH3 permeability
After exposure of P. schlosseri to ammonia for 6 days, there were
detectable changes in the amount of ammonia entering through the tail region
(compare Table 3, sets 2, 3 and
4, with Table 1, sets 2 and 3
and Table 2, set 2). At the
same time, in specimens subjected to prior exposure to ammonia, there were
changes in lipid compositions in the skin, some of which could be linked to a
lower NH3 permeability. Taken together, these results suggest that
the skin in the tail region of P. schlosseri decreased its
permeability to ammonia after prolonged exposure to NH4Cl.
More importantly, the cholesterol content in the skin of P.
schlosseri increased after 6 days of exposure to exogenous ammonia. This
result suggests a definite role for cholesterol in an active defense mechanism
against environmental ammonia toxicity, because an increase in cholesterol
content would imply a decrease in the fluidity of the membrane in the skin.
Since the specimens were not fed during the experimental period, it is logical
to deduce that cholesterol synthesis must be enhanced in response to
ammonia-loading conditions, or that cholesterol was mobilized from other
tissues. Lande et al. (1995)
studied the NH3 permeability of artificial unilamellar vesicles, in
which cholesterol and SM contents, together with acyl chain saturation, were
varied to create a range of fluidities. They concluded that lower fluidity did
indeed correspond with lower NH3 permeability. This would explain
why there was an apparent decrease in the influx of NH3 through the
skin of the specimens that had been exposed to NH4Cl for 6 days, as
described above.
An ecological perspective
The tolerance of P. schlosseri to environmental ammonia is much
higher than those of other fishes, in part because of its capability to
actively excrete NH4+. For active excretion of
NH4+ to be efficacious, back diffusion of NH3
must be prevented. Our results suggest that P. schlosseri solves the
problem of back diffusion of NH3 differently through the gills
(head region) and through the skin (tail region). Acid excretion occurred in
the head region, in response to alkaline pH and/or environmental ammonia. On
the other hand, the skin has relatively low permeability to NH3,
and its lipid compositions can be altered after long-term (6 days) exposure to
environmental ammonia. P. schlosseri is the only species of
mudskipper found exclusively in the tropics. Other species of mudskippers
(e.g. Periophthalmus spp. and Boleophthalmus spp.) can be
found in both tropical and temperate regions (e.g. Japan and China). It is
possible that, by reducing the membrane fluidity of the skin to enhance
ammonia tolerance, P. schlosseri loses its ability to survive in
areas of lower temperature. In temperate regions, the temperature during
winter can drop to a level that would cause even lower membrane fluidity,
which would limit the permeability of the skin to various other molecules that
are essential to life. Therefore, efforts should be made in the future to
compare membrane fluidity and permeability to NH3 in tropical and
temperate fish species.
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Acknowledgments |
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References |
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