Active ammonia excretion across the gills of the green shore crab Carcinus maenas: participation of Na+/K+-ATPase, V-type H+-ATPase and functional microtubules
1 Lake Forest College, Lake Forest, IL 60045, USA
2 Universität Ulm, Germany
3 Alfred-Wegener-Institut, Bremerhaven, Germany
4 Mount Desert Island Biological Laboratory, Salsbury Cove, ME 04672,
USA
* Author for correspondence at present address: University of Illinois at Chicago, Department of Physiology and Biophysics, 835 S. Wolcott Avenue, Chicago, IL 60612-7342, USA (e-mail: Weihrauchblues{at}gmx.net)
Accepted 6 June 2002
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Summary |
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Key words: Ammonia excretion, amiloride, crab, Carcinus maenas, colchicine, cuticle, microtubule, V-type H+-ATPase
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Introduction |
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Cellular uptake or excretion of non-ionic NH3 is generally
thought to occur by diffusion across the lipid bilayer of cellular membranes,
although permeability to NH3 is much lower than to CO2
(Knepper et al., 1989).
Indeed, some plasma membranes of animal cells are relatively impermeable to
NH3 (Burckhardt and
Frömter, 1992
; Garvin et
al., 1987
). The entry of charged ammonium ions into animal cells
may be mediated by several transport proteins:
NH4+-permeable K+ channels
(Latorre and Miller, 1983
),
the Na+/K+/2Cl- cotransporter
(Kinne et al., 1986
), the
Na+/H+ exchanger
(Kinsella and Aronson, 1981
)
or a recently described ammonium transporter related to the rhesus protein
(Marini et al., 2000
). In
addition, abundant evidence suggests that NH4+ may be
transported actively, utilizing the ubiquitous
Na+/K+-dependent ATPase
(Skou, 1960
;
Towle and Hølleland,
1987
; Wall,
1997
).
Cellular excretion of NH4+ may be mediated by an
apical Na+/NH4+ exchanger, as suggested for
mammalian renal proximal tubules (Hamm and
Simon, 1990) and the gills of marine teleosts
(Randall et al., 1999
). In
freshwater teleost gills (Wilson et al.,
1994
) and mammalian inner medullary collecting ducts
(Knepper et al., 1989
),
acidification exterior to the outer apical membrane may induce passive
diffusion of non-ionic ammonia by diffusion trapping. Experiments with intact
blue crabs (Callinectes sapidus) suggest that ammonia is excreted
across the gills by diffusion of non-ionic NH3 in animals
acclimated to sea water (Kormanik and
Cameron, 1981
) but by Na+/NH4+
exchange in animals acclimated to low salinities
(Pressley et al., 1981
). Very
little ammonia is excreted in the urine in this species
(Cameron and Batterton,
1978
).
The mechanism by which ammonia crosses the epithelial layer of the
excreting tissue is not generally considered, it being assumed that
NH3 and NH4+ diffuse through the cytoplasm in
a free state. However, under such conditions, the toxic effects of ammonia
could be felt by multiple intracellular targets. In this study, we present the
experimental basis for a novel mechanism of active ammonia excretion across a
moderately tight epithelium, the gills of the euryhaline shore crab
Carcinus maenas. We have previously demonstrated that isolated
perfused gills from this animal are capable of transporting ammonia against a
concentration gradient under physiologically meaningful conditions (Weihrauch
et al., 1998,
1999
). Active ammonia
excretion across these gills was inhibited by apical amiloride, basolateral
Cs+ or basolateral ouabain, implicating the participation of an
apical Na+/NH4+ exchanger, basolateral
NH4+-permeable K+ channels and basolateral
Na+/NH4+-dependent ATPase, respectively. We
show here that an intracellular V-type H+-ATPase and an intact
microtubule system are also required for active excretion of ammonia in this
system, leading to an experimental model that includes acidification and
ammonia-loading of intracellular vesicles, transport of vesicles along
microtubules and exocytosis. Ultrastructural and molecular evidence support
the model.
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Materials and methods |
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Measurement of active ammonia flux across perfused gills
Gills were prepared and perfused according to previously described methods
(Siebers et al., 1985;
Weihrauch et al., 1998
) at a
flow rate of 0.135 ml min-1. Transepithelial potential differences
(PDte) were monitored using a millivolt meter (Keithley,
type 197) to evaluate the quality of the preparation. Only gills generating an
initial and continuously negative PDte (-7.1±1.0
mV, N=32; mean ± S.E.M.) were employed.
The morphology of the phyllobranchiate gills provides sufficient surface
area, roughly 247 cm2 g-1 fresh gill mass
(Riestenpatt, 1995), to
measure transepithelial ammonia fluxes directly. Excretion rates and ammonia
concentrations were measured according to methods described in detail in an
earlier publication (Weihrauch et al.,
1998
). Briefly, unless mentioned otherwise, gills were perfused
and bathed in saline (Sstandard) initially containing symmetrical
amounts of ammonia (100 µmol l-1 NH4+) at
identical pH values. Consequently, all measured net fluxes must be based on
active transport mechanisms. The external medium was stirred constantly during
each treatment. The concentration of ammonia selected was within the range of
hemolymph ammonia levels measured in C. maenas
(Durand and Regnault, 1998
;
Weihrauch et al., 1999
). When
a constant PDte had been established (within approximately
30 min), the external bath and the perfusion solution were replaced and
measurements were made for 30 min (controls). To continue the experiment with
the same gill, the procedure was repeated with modified salines. Following
application of a modified saline for 30 min, fluxes of total ammonia over the
following 30 min were measured again. 2 ml samples were taken from the bath
and internal perfusate after each experimental step.
Total ammonia concentrations (TAmm) were determined
with a gas-sensitive NH3 electrode (Ingold, type 152303000). The
sensitivity of the electrode measurements was approximately ±1.5
µmol l-1 in the TAmm concentration range
50-100 µmol l-1. To calculate net excretion, only the ammonia
removed from the internal perfusion saline was considered to avoid including
metabolically produced ammonia (Weihrauch
et al., 1998). An alternative approach would be to measure the
appearance of total ammonia in the external bath. However, as we show below,
such a measurement would lead to an overestimation of transport rates as a
result of ammonia production by the gill itself.
Measurement of gill resistance and calculation of transepithelial
conductance
After removing the gills, single gill lamellae were isolated and split into
their two halves (Schwarz and Graszynski,
1989). In this way, a single epithelial layer covered by an apical
cuticle was obtained. Isolated cuticle was prepared by carefully removing the
epithelial cells using a smooth rounded metal wire. Split gill lamellae or
isolated cuticle were mounted in a modified Ussing chamber, allowing area
(0.02 cm2)-specific short-circuit currents
(Isc) and transepithelial resistances
(Rte) to be measured. The chamber compartments were
continuously superfused with saline at a rate of 0.5 ml min-1 by
means of a peristaltic pump.
To measure PDte, Ag/AgCl electrodes were connected
via agar bridges (3% agar in 3 mmol l-1 KCl) to the
chamber compartments; the separation distance of the preparation was less than
0.1 cm. A second pair of Ag/AgCl electrodes, connected through agar bridges,
served as current electrodes to short-circuit the PDte
with an automatic clamping device (VCC 600, Physiologic Instruments, San
Diego, USA). Isc and Rte were
calculated according to Riestenpatt et al.
(1996). The transepithelial
and transcuticular conductances were calculated as
Gte=1/Rte and
Gcut=1/Rcut, respectively, where
Rcut is the transcuticular resistance.
To assess the effects of NH4Cl solutions on cuticular
Isc and Rcut, the isolated cuticle was
superfused (0.5 ml min-1) on both sides with a non-physiological
ammonia-containing saline (SNH4Cl) consisting of 248
mmol l-1 NH4Cl and 2.5 mmol l-1 Tris adjusted
to pH 7.8 with HCl. A clamp voltage of 10 mV with reference to the apical side
was maintained to force transcuticular ion fluxes. Resistances for
Sstandard and SNH4Cl were 9.0±0.1
cm2 (N=6) and 7.1±0.4
cm-2
(N=5) (means ± S.E.M.), respectively.
Statistical analyses
All data presented in this study were corrected by the resistances of the
salines employed. All results are presented as means ± S.E.M.
Differences between groups were tested using one-way analysis of variance
(ANOVA) and the NewmanKeuls multiple-comparison test. Statistical
significance was assumed for P<0.05.
Salines and chemicals
The ionic composition of the salines (Sstandard) used to bathe
and perfuse isolated gills and to measure transepithelial
resistance/conductance in gill half-lamellae contained (mmol l-1)
248 NaCl, 5 CaCl2, 5 KCl, 4 MgCl2, 2 NaHCO3,
2.5 Tris and 0.1 NH4Cl. Immediately before use, 2 mmol
l-1 glucose was added to the basolateral salines in all
experiments, and the pH of all salines was adjusted to 7.8 (HCl). Amiloride,
bafilomycin A1, colchicine, cytochalasin D, ouabain and taxol were
purchased from Sigma (St Louis, USA). The ammonia standard (0.1 mol
l-1) was obtained from Orion Research Incorporated (Boston, USA).
CsCl and all other salts were of analytical grade and were purchased from
Merck (Darmstadt, Germany). Thiabendazole was kindly provided by Dr R.
Gräf (Munich).
Electron microscopy
Gills from crabs acclimated to 10 salinity were shockfrozen in a
high-pressure freezer (Wohlwend Engineering GMBH, Sennwald, Switzerland)
(Studer et al., 1989
),
followed by freeze-substitution in 1% OSO4 in acetone
and embedding in Epon. Ultrathin sections were stained with uranyl acetate and
lead citrate and viewed with a Philips electron microscope at 80 kV.
Molecular identification of vesicle-associated membrane protein
cDNA
Total RNA was extracted from gill tissue under RNAse-free conditions
(Chomczynski and Sacchi, 1987).
Reverse transcription of poly(A) mRNA was initiated with oligo-dT primer and
Superscript II reverse transcriptase (Invitrogen). Amplification of a putative
vesicle-associated membrane protein (VAMP) cDNA sequence was achieved
via polymerase chain reaction (PCR) using degenerate primers based on
published VAMP sequences (DiAntonio et al.,
1993
; Mandic and Lowe,
1999
). The forward primer had the following composition:
5'-CARCARACNCARGCNCARGTNGA-3'; the reverse primer had the
following composition: 5'-ATDATCATCATYTTNARRTTYT-3', designed to
produce a 198-nucleotide PCR product. Following separation on agarose gels,
the PCR product was extracted from gel slices (Qiagen QiaQuick) and sequenced
at the Marine DNA Sequencing Center of Mount Desert Island Biological
Laboratory. The partial sequence was submitted to a BLAST search of GenBank
(Altschul et al., 1997
) and
analyzed for open reading frame using DNASIS. The partial VAMP cDNA sequence
from C. maenas gill was submitted to GenBank (Accession number
AY035549).
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Results |
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In the ensuing perfusion experiments, all solutions were buffered with 2.5 mmol l-1 Tris to ensure pH stability under all experimental conditions. Measuring active ammonia excretion over time, the initial net ammonia efflux was 14.7±2.5 µmol g-1 fresh mass h-1 (N=5) and was thus within the range measured under Tris-free conditions. Over a period of 3h, the excretion rate decreased slightly but non-significantly by 4.7 µmol g-1 fresh mass h-1 (N=5) (Fig. 1B). Control rates of active ammonia excretion with symmetrical ammonia concentrations varied between experiments, ranging from approximately 12 to 26 µmol g-1 fresh mass h-1, perhaps reflecting variability within natural populations.
Symmetrical addition of 1 µmol l-1 bafilomycin A1,
a specific inhibitor of V-type H+-ATPase
(Bowman et al., 1988), lowered
the transbranchial active net ammonia efflux by 66% from 18.3±2.6
µmol g-1 fresh mass h-1 (control) to 6.3±1.7
µmol g-1 fresh mass h-1 (N=7;
P<0.001). The concentration of 1 µmol l-1
bafilomycin A1 employed is known to produce maximal inhibition of
crustacean gill V-type H+- ATPase without affecting the activity of
Na+/K+-ATPase or F1F0-ATPase
(Putzenlechner, 1994
). After
washout of the inhibitor, a minor but non-significant recovery of the efflux
rate was detected (data not shown). A similar experiment was performed to
verify whether the inhibitory effect of bafilomycin A1 could be
augmented by additional application of ouabain, a specific inhibitor of the
Na+/K+-ATPase. The initial control efflux (19±3.8
µmol g-1 fresh mass h-1) was reduced by application
of 1 µmol l-1 bafilomycin A1 to 6.3±2.1
µmol g-1 fresh mass h-1 (N=4;
P<0.001) and was further reduced to 0.9±1.4 µmol
g-1 fresh mass h-1 by subsequent basolateral application
of 5 mmol l-1 ouabain (N=4; P<0.05)
(Fig. 2A). A partial but
statistically insignificant recovery was measured after washout. It should be
noted that the Ki for ouabain in C. maenas
(Ki=2.9x10-4 mol l-1) and
other crustaceans is more than two orders of magnitude higher than the
Ki in mammals (Postel
et al., 1998
; Towle,
1984
).
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In the next series of experiments, the effects of inhibitors of the
cytoskeleton on active ammonia excretion were investigated. Basolateral
application of 0.2 mmol l-1 colchicine, an inhibitor of the
microtubule system (Wilson and Farrell,
1986), led to almost complete inhibition (to 2.3±1.3
µmol g-1 fresh mass h-1) of the initial control
efflux of 26.2±3.9 µmol g-1 fresh mass h-1
(N=6; P<0.001) (Fig.
2B). Following washout, the efflux recovered significantly to
15.0±5.4 µmol g-1 fresh mass h-1. After
establishing an outwardly directed ammonia gradient by adding 200 µmol
l-1 NH4+ to the perfusing saline and none to
the external bath, colchicine still reduced the initial efflux rate
(37.1±3.9 µmol g-1 fresh mass h-1) by 74% of
the control value to 10.4±4.3 µmol g-1 fresh mass
h-1 (N=5; P<0.001). After washout, efflux
recovered to 72% of the control value (Fig.
2C).
In contrast to drugs affecting the microtubule system, no significant
effects on active ammonia excretion were observed when 5 µmol
l-1 cytochalasin D, a specific inhibitor of actin filaments
(MacLean-Fletcher and Pollard,
1980), was added to the perfusion saline (N=5)
(Fig. 2D). The slight and
non-significant decrease in the excretion rate from 20.1±2.8 to
16.5±2.7 µmol g-1 fresh mass h-1
(P>0.05) resembled the control rates over the experimental period
(Fig. 1B)
(Weihrauch et al., 1998
).
In all the above experiments, PDte was monitored to
detect changes in the electrophysiological variables of the gill
(Fig. 2). With the exception of
the addition of ouabain, which resulted in a reversible decrease in
PDte of 55% (Fig.
2A) due to a disruption of transepithelial Na+
transport (Siebers et al.,
1985), application of the various inhibitors had no significant
effects on PDte, indicating that Na+ transport
pathways were not affected by the remaining treatments.
For two other blockers of the microtubule system, microtubule
hyper-stabilizing taxol (Nogales et al.,
1995) and microtubule destabilizing thiabendazole
(Davidse and Flach, 1978
), a
strong inhibitory effect on active ammonia excretion was observed
(Fig. 3). Whereas basolateral
application of 10 µmol l-1 taxol led to a decrease in the
control rate (12.4±3.2 µmol g-1 fresh mass
h-1) of 77% to 2.1±1.0 µmol g-1 fresh mass
h-1 (N=6; P<0.001), basolateral addition of
0.2 mmol l-1 thiabendazole altered the efflux rate of
11.5±2.5 µmol g-1 fresh mass h-1 to an
apparent influx of ammonia (2.7±1.8 µmol g-1 fresh mass
h-1) (N=6; P<0.001), probably as a result of
metabolic ammonia production and release across the basolateral membrane. A
statistically significant recovery of ammonia efflux was observed after
washout of thiabendazole but not of taxol.
|
To evaluate changes in the electrical variables of the gill epithelium in
response to application of the cytoskeleton inhibitors colchicine and
cytochalasin D, the highly sensitive transepithelial short-circuit current
(Isc) and transepithelial resistance
(Rte) were measured employing the preparation of the split
gill lamella mounted in an Ussing-type chamber. Basolateral application of
either 0.2 mmol l-1 colchicine or 5 µmol l-1
cytochalasin D had no significant effect on Isc
(colchicine, 318.8±51.6 µA cm-2; control,
320.2±52.9 µA cm-2, N=5; cytochalasin D,
353.4±61.5 µA cm-2; control, 358.8±64.9 µA
cm-2, N=4) (Fig.
4). Control values of Rte measured in parallel
were not altered following the addition of colchicine (24.1±1.7
cm2, N=5) or cytochalasin D (28.3±1.7
cm2, N=4) (data not shown).
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Previous studies have shown that amiloride, a blocker of epithelial
Na+ channels and the Na+/H+ exchanger
(Kleyman and Cragoe, 1988),
has a strong inhibitory effect on ammonia excretion by isolated crab gills
when applied to the apical (cuticle) side of the epithelium
(Lucu et al., 1989
;
Weihrauch et al., 1998
). To
investigate whether the amiloride-induced reduction in the rate of ammonia
excretion may be based on a possible effect on the electrophysiological
properties of isolated cuticle (Lignon,
1987
) in addition to any effect on the epithelial cells
themselves, NH4+-dependent Isc and
Gcut of the isolated gill cuticle of C. maenas
were measured in a micro Ussing chamber. As expected in a cell-free system, a
transcuticle potential difference (PDcut) of 0 mV was
measured (N=4). Following the imposition of a clamp voltage of 10 mV,
a negative Isc of -5800±1368 µA cm-2
and a Gcut of 683.0±165.2 mS cm-2 were
measured. The detected current is probably the result of
NH4+ effluxes, since cuticular permeability in C.
maenas has been described to be 100- to 1000-fold smaller for monovalent
anions than for monovalent cations
(Lignon, 1987
). After apical
application of various amiloride concentrations (0.001-1 mmol l-1),
dose-dependent inhibition was observed for both Isc and
Gcut (Fig.
5). Linear regression in a Hanes-Woolf plot revealed simple
Michaelis-Menten kinetics for Isc and
Gcut. For the NH4+-dependent
Isc, Kami was 19.1
µmoll-1 and
Imax was -4638 µA
cm-2. Kami for
NH4+-dependent Gcut was 20.4
µmoll-1 and
Gmax was 549.8 mS
cm-2 (Fig. 5).
Symmetrical application of 1 µmoll-1 bafilomycin A1
had no significant effect on cuticular NH4+-dependent
Isc (control, -4294±206 µA cm-2;
bafilomycin, -3984±217 µA cm-2) and
Gcut (control, 454±45 mS cm-2;
bafilomycin, 400±80 mS cm-2) (N=3).
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Because our inhibitor studies indicated important roles for the V-type
H+-ATPase and microtubules in active ammonia excretion, we sought
ultrastructural and molecular evidence that would help to support or refute
such possibilities. A previous study showed that the B-subunit of the V-type
H+-ATPase was distributed throughout the cytoplasm of gill
epithelial cells in C. maenas rather than being located specifically
in the apical membrane, suggesting that the H+-ATPase may be
associated with cytoplasmic vesicles
(Weihrauch et al., 2001b) as
well as with the apical membrane. In the present study, transmission electron
microscopy of sections obtained from posterior gills of C. maenas
acclimated to 100 µmoll-1 external ammonia revealed an
apparently dynamic system of membrane vesicles and Golgi bodies associated
with the apical region of the epithelium
(Fig. 6A). We also observed a
well-developed microtubule assemblage associated with the apical membrane
(Fig. 6B,C). In several
sections, we were able to detect apparent interactions between membrane
vesicles and the microtubules.
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To ascertain whether any of the known exocytotic mechanisms are expressed
in C. maenas gill, we attempted to identify one of the expected
components, vesicle-associated membrane protein (VAMP, also called
synaptobrevin) (Trimble et al.,
1988). Using PCR with degenerate primers based on published VAMP
sequences, we identified a VAMP-related sequence in a cDNA mixture prepared
from C. maenas gill. Translation of the 132-nucleotide fragment
revealed an amino acid sequence that is highly homologous to VAMP sequences
determined for other invertebrate and vertebrate species
(Fig. 7).
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Discussion |
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Active ammonia transport across biological membranes could be mediated by a
proton pump via two different mechanisms: (i) the proton gradient
generated by the pump drives a parallel
H+/NH4+ exchanger, or (ii) the proton pump
generates a partial pressure gradient for NH3 over the membrane,
inducing transmembrane diffusion of gaseous ammonia. The latter has been
suggested for ammonia transport across the apical membrane in the gills of
freshwater trout (Wilson et al.,
1994) since in this tissue the H+-ATPase is localized
predominantly in the apical membrane
(Sullivan et al., 1995
). In
addition, buffering the external medium with Hepes dramatically inhibited
ammonia excretion across fish gill because, under this condition, the partial
pressure gradient for NH3 diffusion across the apical membrane was
abolished.
However, in gill epithelial cells of C. maenas, the V-type
H+-ATPase is distributed throughout the cytoplasm and only faintly
detectable in the apical region (Weihrauch
et al., 2001b). In the present study, no significant changes in pH
in the apical medium were detected during excretion nor was the active ammonia
excretion rate altered when a buffer (2.5 mmoll-1 Tris-HCl) was
applied in the apical saline (Fig.
1) (Weihrauch et al.,
1998
). These results indicate that in C. maenas
acidification of the outer apical membrane is not responsible for driving a
putative H+/NH4+ exchanger nor does it
generate a partial pressure gradient for diffusion of non-ionic NH3
across the apical membrane. However, we cannot discount the possibility of
unstirred layers between the gill lamellae and thus cannot exclude the
possibility of pH gradients immediately external to the cuticle. Indeed, we
suspect that the subcuticular space, between the cuticle and the apical
membrane, represents a classic unstirred layer. Diffusion of NH3
coupled with transport of H+ to produce NH4+
could theoretically occur in these unstirred layers to which buffer might not
penetrate.
However, we suggest that the proton pump of crab gills, rather than being
restricted to apical membranes, may be inserted into the membranes of
cytoplasmic vesicles, generating an inwardly directed partial pressure
gradient for NH3 and leading to the accumulation of
NH4+ within the vesicles. It has been shown that
radioactively labeled methylamine diffuses into acidified vesicles, where it
is protonated into its membrane-impermeable ionic form methylammonium
(Riejngoud and Tager, 1973).
We suggest that a similar mechanism functions in crab gills, where cytoplasmic
NH3 diffuses into vesicles acidified by the V-type
H+-ATPase and is trapped in the vesicles as
NH4+.
The almost complete inhibition of both active and gradient-driven net
ammonia excretion by microtubule inhibitors (colchicine, thiabendazole and
taxol) indicates a microtubule-dependent ammonia excretion mechanism. In
contrast, blocking the actin filaments with cytochalasin D, which causes a
small increase in exocytotic and endocytotic movements in frog nephron
epithelia (Verrey et al.,
1995), had no significant effect on active ammonia excretion
across the isolated gill. We suggest that NH4+-loaded
vesicles are transported along the microtubule network to the apical membrane,
where ammonia is released by membrane fusion and exocytosis. This suggestion
is supported by the abundance of Golgi bodies and vesicles in gill epithelial
cells (Fig. 6A), by the
presence of bundles of microtubules oriented towards the apical membrane and
by the endo/exocytotic activities represented by apparent clathrin-coated pits
(Fig. 6B,C).
Following application of bafilomycin A1, colchicine, thiabendazole, taxol and cytochalasin D, PDte remained unchanged, indicating that changes in ammonia excretion during treatment with these inhibitors were not caused by alterations to the osmoregulatory NaCl uptake machinery or by damage to the integrity of the preparation. The unaltered Isc and Gte across the split half-lamella during exposure to colchicine also supported our conclusion that a functional microtubule network is necessary for the process of active ammonia excretion but that its inhibition does not affect the electrical variables of the gill epithelium, at least over the short term (Fig. 2).
The possibility of an apical amiloride-sensitive
Na+/NH4+ exchange across the apical membrane
prompted our examination of the role of the cuticle. Amiloride has been shown
to have an inhibitory effect on ammonia transport in renal proximal tubules
(Knepper et al., 1989),
colonic crypt cells (Ramirez et al.,
1999
), teleost gills (Randall
et al., 1999
) and crustacean gills
(Lucu et al., 1989
;
Weihrauch et al., 1998
). In
contrast to vertebrate tissues, crustacean gills are covered with an
ion-selective cuticle. Although the conductance of the isolated gill cuticle
of C. maenas has been shown to be approximately 10 times higher than
that of the combined epithelium plus cuticle
(Riestenpatt, 1995
), ion
selectivity is demonstrable, with the permeability for monovalent anions
(Cl-, HCO3-) being 100-1000 times lower than
that for Na+ (Lignon,
1987
).
The present study employing the isolated cuticle showed that
NH4+-dependent Isc and
Gcut were inhibited by amiloride in a dose-dependent
manner. At an amiloride concentration of 100µmoll-1, a
concentration commonly used in investigating Na+ and
NH4+ fluxes across the gill epithelia of C.
maenas (Lucu et al.,
1989; Lucu and Siebers,
1986
; Onken and Siebers,
1992
; Weihrauch et al.,
1998
), more than 70% of the cuticular
NH4+-dependent Isc and
Gcut were blocked. In a recent electrophysiological study
investigating the effects of amiloride on Na+ influx across split
gill lamellae and isolated cuticle of C. maenas, it was shown that
apical amiloride inhibits both the Na+-dependent transepithelial
Isc and Gte and also the
transcuticular Isc and Gcut with
similar values for Kami
(Onken and Riestenpatt, 2002
).
These authors suggested that the effects of amiloride were due to an
interaction between the diuretic and the outer cuticle and not with
transporters in the apical cell membrane or paracellular junctions. They
concluded, however, that amiloride may interact directly with cellular
transporters in the gills of other crab species
(Onken and Riestenpatt,
2002
).
The values of Kami for the cuticular
NH4+-dependent Isc and
Gcut (approximately 20 µmoll-1) obtained in
our study are approximately 20-fold higher than the values calculated for the
Na+-dependent Isc and Gcut
(approximately 1 µmoll-1) employing an identical experimental
design. Comparison of the NH4+-dependent
Gcut (683±165 mS cm-2) and the
Na+-dependent Gcut (583±71 mS
cm-2) (Riestenpatt,
1995) showed a higher conductance of the cuticle for
NH4+ ions than for Na+ ions, confirming
earlier measurements (Lignon,
1987
). We only can speculate that because of the smaller hydrated
ionic size of NH4+ (approximately 0.38nm versus
approximately 0.56 nm for Na+), blockage of a cation-permeable
structure in the cuticle by amiloride is less efficient. However, from these
experiments, the presence of a Na+/NH4+
exchanger in the apical membrane itself cannot be excluded.
On the basis of previous observations and insights gained from the present
study, we have constructed a hypothetical model for transbranchial ammonia
excretion in C. maenas functioning at physiological ammonia
concentrations. In this model (Fig.
8), we suggest that hemolymph ammonia enters the epithelial cell
across the basolateral membrane via
NH4+-permeable Cs+-sensitive channels and
also via the Na+/K+-ATPase in exchange for
Na+ (Lucu et al.,
1989; Towle and
Hølleland, 1987
). The nature of the
NH4+-permeable channel is unknown, but it may be related
to a recently described rhesus-like protein that appears to mediate transfer
of NH4+ across cell membranes
(Marini et al., 2000
). Using
reverse transcription and PCR, we have recently identified such a rhesus-like
protein in the gills of C. maenas
(Weihrauch et al., 2001a
).
|
The pool of ammonia imported from the hemolymph and produced by gill
metabolism occurs within the cytoplasm in a pH-dependent equilibrium between
NH4+ and NH3 (pKAmmonia=9.48)
(Cameron and Heisler, 1983). In
our working model, we suggest that non-ionic NH3 diffuses along its
partial pressure gradient into intracellular vesicles acidified by a proton
pump. Because of the low pH within the vesicles, NH3 would be
converted into its membrane-impermeable ionic form NH4+
and therefore trapped in this compartment. Our microtubule inhibitor studies
suggest that the NH4+-loaded vesicles may be transported
along the microtubule network to the apical membrane, where
NH4+ would be released by exocytosis into the
subcuticular space. Our demonstration of a vesicle-associated membrane protein
(VAMP) sequence in cDNA prepared from C. maenas gill RNA
(Fig. 7) shows that at least
one component of the exocytotic machinery is expressed in this tissue,
providing circumstantial evidence for branchial exocytotic activity.
From the subcuticular space, NH4+ would diffuse along
a concentration gradient via amiloride-sensitive structures across
the cuticle into the external medium of the gill chamber. At physiologically
meaningful outwardly directed ammonia gradients (50-400
µmoll-1), transepithelial diffusion of ammonia is considered to
be low, comprising 12-21% of the total efflux
(Weihrauch et al., 1998).
In the present report, the proposed mechanism of exocytotic ammonia
excretion is supported only by indirect evidence. To investigate its validity
more thoroughly, more direct experiments are necessary. Feasible future
approaches include measurements of the capacitance as an indicator of the
exocytotic activity of the apical membrane under control and high-ammonia
conditions (Zeiske et al.,
1998), the use of radioactively labeled
methylamine/methyl-ammonium as a traceable competitive inhibitor for ammonia
movements (Talor et al., 1987
)
and the use of laser confocal microscopy combined with video-image analysis to
trace intracellular pH-labeled compartments
(Miller et al., 1994
).
The possibility of an exocytotic ammonia excretion mechanism should be
considered since, in this situation, toxic ammonia is trapped in vesicles
within the cell rather than diffusing through the entire cytoplasm, where it
could cause major damage. In aquatic animals facing an inwardly directed
ammonia gradient in the natural environment
(Weihrauch et al., 1999), the
active component of the mechanism would provide protection for the gill
epithelial cells and, indeed, for the entire organism against passive
NH4+ influxes. Microtubule-mediated transport of
ammonia-loaded vesicles and exocytosis at the apical membrane would permit a
potent ammonia detoxification mechanism in such organisms without compromising
ionic permeability. Whether such a mechanism applies broadly across species is
not known. However, some aquatic species, including the South American rainbow
crab Chasmagnathus granulatus
(Rebelo et al., 1999
), the
prawn Nephrops norvegicus
(Schmitt and Uglow, 1997
) and
three fish species in the family Batrachoididae
(Wang and Walsh, 2000
),
tolerate high environmental ammonia levels. Among the adaptive mechanisms in
these species may be an active ammonia excretion process similar to that
described here.
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Acknowledgments |
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References |
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