J Comp Physiol B (1998) 168: 364±376
Ó Springer-Verlag 1998
ORIGINAL PAPER
D. Weihrauch á W. Becker á U. Postel á S. Riestenpatt
D. Siebers
Active excretion of ammonia across the gills of the shore crab
Carcinus maenas and its relation to osmoregulatory ion uptake
Accepted: 11 March 1998
Abstract The mechanism of transbranchial excretion of
total ammonia of brackish-water acclimated shore crabs,
Carcinus maenas was examined using isolated, perfused
gills. Applying physiological gradients of NH4Cl (100±
200 lmol á l)1) directed from the haemolymph space to
the bath showed that the e‚ux of total ammonia consisted of two components. The saturable component
(excretion of NH‡
4 ) greatly exceeded the linear component (di€usion of NH3). When an outwardly directed
gradient (200 lmol á l)1) was applied, total ammonia in
the perfusate was reduced by more than 50% during a
single passage of saline through the gill. E‚uxes of
ammonia along the gradient were sensitive to basolateral
dinitrophenol, ouabain, and Cs+ and to apical amiloride. Acetazolamide (1 mmol á l)1 basolateral) or Cl)free conditions had no substantial e€ects on ammonia
¯ux, which was thus independent of both carbonic anhydrase mediated pH regulation and osmoregulatory
NaCl uptake. When an inwardly directed gradient
(200 lmol á l)1) was employed, in¯ux rates were about
10-fold smaller and una€ected by basolateral ouabain
(5 mmol á l)1) or dinitrophenol (0.5 mmol á l)1). Under
symmetrical conditions (100 lmol á l)1 NH4Cl on both
sides) ammonia was actively excreted against the gradient of total ammonia, which increased strongly during
the experiment and against the gradient of the partial
pressure of NH3. The active excretion rate was reduced
to 7% of controls by basolateral dinitrophenol
(0.5 mmol á l)1), to 44% by basolateral ouabain
(5 mmol á l)1), to 46% by Na+-free conditions and to
42% by basolateral Cs+ (10 mmol á l)1), indicating
+
basolateral membrane transport of NH‡
4 via the Na /
+
+
K -ATPase and K -channels and a second active,
D. Weihrauch (&) á D. Siebers á U. Postel á S. Riestenpatt
Biologische Anstalt Helgoland (Zentrale),
Notkestr. 31, D-22607 Hamburg, Germany
W.Becker
Zoologisches Institut und Museum, UniversitaÈt Hamburg,
Martin-Luther-King-Platz 3, D-20146 Hamburg, Germany
apically located, Na+ independent transport mechanism
of NH‡
4 . Anterior gills, which are less capable of active
ion uptake than posterior gills, exhibited even increased
rates of active excretion of ammonia. We conclude that,
under physiological conditions, branchial excretion of
ammonia is a directed process with a high degree of effectiveness. It even allows active extrusion against an
inwardly directed gradient, if necessary.
Key words Excretion ammonia á Ouabain á Carcinus á
crab
Abbreviations AZ Acetazolamide á CA carbonic
anhydrase á DNP 2,4-dinitrophenol á FW fresh
weight á Kt transport constant á PNH3 partial pressure of
non-ionic ammonia á nPNH3 gradients of PNH3 á PDte
transepithelial potential di€erence á TAmm total
ammonia á TRIS Tris-(hydroxymethyl)aminomethan á Vmax maximum rates of excretion
Introduction
As in the vast majority of aquatic animals, most crustaceans excrete their metabolic nitrogenous end products
largely as ammonia, regardless of whether they occupy
marine, fresh-water, or terrestrial habitats (Kormanik
and Cameron 1981; Claybrook 1983). The main site of
N-excretion in crustaceans is the gill epithelium, while
antennal and maxillary glands play a minor role (Regnault 1987). In spiny lobsters Jasus edwardsii and blue
crabs Callinectes sapidus less than 2% of total excreted
ammonia is eliminated via the urine (Binns and Peterson
1969; Cameron and Batterton 1978).
Usually the term ammonia or total ammonia (TAmm)
is used to describe the mixture of nonionic ammonia
(NH3) and ammonium ions (NH‡
4 ). In solution, both
forms of ammonia exist in a pH-dependent equilibrium.
The relationship of the concentrations of NH3 and NH‡
4
can be calculated using the Henderson-Hasselbalch
equation for ammonia:
365
pH ˆ pK ‡ log
‰NH3 Š
‰NH‡
4Š
…1†
At 20 °C and a concentration of 250 mmolál)1 NaCl, pK
amounts to 9.48 (Cameron and Heisler 1983). Using this
pK and a physiological pH of 7.8, approximately 2% of
total ammonia is present as nonionic NH3. However, the
higher lipid solubility of NH3 makes it more di€usible
through phospholipid bilayers. In crab gills the permeability ratio for NH3 and NH‡
4 indicates that NH3 is
approximately 53 times more permeable (Cameron and
Heisler 1983).
The ionic form of ammonia can also permeate biological membranes, although in di€erent ways. Due to
its hydrophilic properties NH‡
4 can pass lipid bilayers
only very slowly. The transport of NH‡
4 proceeds via
membrane-associated carrier proteins or, as reported by
Wilkie (1997) for marine teleosts, via the highly cationand ammonium-permeable shallow tight junctions
between chloride or accessory cells.
Kormanik and Cameron (1981) reported that ammonia excretion of sea water adapted blue crabs Callinectes sapidus occurs mainly by di€usion of nonionic
NH3. Other authors have obtained experimental evidence for at least partial excretion of ammonia in its
ionic form, NH‡
4 (Pressley et al. 1981; Lucu et al. 1989;
Siebers et al. 1995).
Experiments with intact crabs Cancer pagurus revealed a permeability of the gills for ionic NH‡
4 (Kormanik and Evans 1984). In other studies on Callinectes
sapidus, a correlation of ammonia excretion with Na+
absorption was found (Pressley et al. 1981). The same
result was obtained by experiments using the chinese
crab Eriocheir sinensis (PeÂqueux and Gilles 1981), the
shrimp Macrobrachium rosenbergii (Armstrong et al.
1981) and the shore crab Carcinus maenas (Lucu et al.
1989). Experimental studies employing membrane vesicles of gill epithelia (Towle and Hùlleland 1987) and
isolated, perfused gills (Lucu et al. 1989) indicated that
+
NH‡
ions in activation of
4 ions can substitute for K
+
+
the ouabain-sensitive Na /K -ATPase. In the apical
membrane the presence of an amiloride-sensitive Na+/
‡
NH‡
4 antiporter translocating NH4 from the epithelial
cell to the ambient medium in exchange for Na+ was
reported by Pressley et al. (1981) for the crab Callinectes
sapidus and by Lucu et al. (1989) and Siebers et al.
(1995) for the shore crab Carcinus maenas.
Previous studies in this laboratory have shown that
excretion of TAmm across the gill epithelium of the shore
crab proceeds at least partially via some of the transporting proteins operative in osmoregulatory NaCl uptake. The following investigations were undertaken to
obtain further information about the mode of excretion
of total ammonia in the euryhaline shore crab Carcinus
maenas.
Of both forms of ammonia comprising TAmm, NH3
has been long recognized as being the most toxic. For
example, ®sh tolerate relatively high concentrations of
NH‡
4 , but su€er severe damage from micromolar con-
centrations of NH3 (Rice and Stokes 1975). At toxic
internal levels ammonia is detrimental to the metabolism
of glutamine-glutamate (Stryer 1991), branchial gas
exchange in salmonids (Burrows 1964), oxidative metabolism (Arillo et al. 1981) and to the central nervous
system (Cooper and Plum 1987). Therefore, excretion
of ammonia must also be considered in the context of
reducing NH3-toxicity.
Materials and methods
Crabs
Shore crabs (C. maenas L.) were obtained from a ®sherman in Kiel
Bay (Baltic Sea). In the laboratory adult males of approximately 5±
7.5 cm carapace width were maintained in 200 l aquaria (ca. one
crab per 20 l). In order to avoid moulting, light periods were
reduced to 8 h per day. The water was continuously aerated and
®ltered over gravel, and the temperature was kept at 16 °C. Before
experimental use the crabs were acclimated for at least 1 month in
dilute seawater (10&). The crabs were fed 3 times a week with small
pieces of bovine heart.
Preparation and perfusion of gills
The crabs were killed by destroying the ventral ganglion using a
spike, which was pressed through the ventral side of the body wall.
The carapace was lifted and the gills were removed. The gills were
perfused according to Siebers et al. (1985) with a ¯ow rate of
0.135 ml á min)1. During perfusion, transepithelial potential differences (PDte) were monitored using a millivolt meter (type 197,
Keithley, Cleveland, USA), connected with the perfusate and the
bath solutions by means of two Ag/AgCl electrodes (type 373-S7,
Ingold, Frankfurt/Main, Germany). PDte was measured in individual gills to control the success of the preparation and the e€ects
of changing the composition of the bath or perfusion solution.
Previous experiments employing NaCl salines symmetrically in the
bath and in the haemolymph space of the gills have shown PDte of
about )5 to )8 mV (haemolymph side negative) in ion-transporting
posterior gills 7±9 and about )3 to )4 mV in the less transporting
anterior gills 4±6. When a constant PDte was established (within
approximately 30 min) the external bath and the perfusion solution
were replaced, followed by a sampling period of 30 min (controls).
In order to quantify the small concentrations of TAmm originating from gill metabolism the experimental periods were prolonged to 1 h in this experiment. Samples of 2 ml were taken from
the bath (original volume 30 ml) and from the perfusate after passage through the gill. In order to continue the experiment with the
same gill the procedure was repeated with modi®ed salines (changes
of the ionic composition or addition of inhibitors of ionic transport
or energy metabolism). Following application of a modi®ed saline
for 0.5 h ¯uxes of TAmm during the following hour were measured
again in 2 ml samples taken from the bath and the perfusate. There
was no indication of changes in the output of endogenous ammonia
with time (see Fig. 1). In the experiments on active excretion of
ammonia across the gill epithelium, concentration changes in TAmm
measured in the bath and in the perfusate were used for calculations
of ¯uxes. In the experiments on e‚ux of ammonia across the posterior gills along varying gradients, concentrations of TAmm were
measured in the bath at perfusate concentrations between 800 and
4800 lmol á l)1 and as decreases in the perfusate at perfusate concentrations between 25 and 400 lmol á l)1.
The samples were analysed for their content of TAmm on the day
of experimentation or were sealed and immediately frozen at
)70 °C with measurement of ammonia taking place within 2 days.
At the end of the experiment the gill was cut above the clamp, dried
under light pressure between two sheets of soft paper (Kleenex) and
weighed.
366
Determination of ammonia
Concentration of TAmm in magnetically stirred samples was determined using a gas sensitive NH3 electrode (Ingold, type
152 303 000) connected to a digital pH meter (Knick, type 646). In
order to transform total ammonia into NH3, 30 ll of an alkaline
solution (1.36 mol á l)1 trisodiumcitrate dihydrate, 1 mol á l)1
NaOH) were added to the 2-ml sample directly before measurement.
For the calculation of the concentration of TAmm a calibration curve
with de®ned solutions of ammonia (3 á 10)6±4 á 10)4 mol
)1
NH‡
4 á l ) prepared from an ammonia standard solution (Orion,
no. 951006) in saline was used. The electrode potentials (mV)
measured were plotted against the logarithms of the concentrations
of originally dissolved TAmm of the the calibration solution. The
calibration curves obtained were straight lines with a high degree of
accuracy, as evidenced by regression coecients of 0.999 or, in a few
cases, 0.998. The millivolt values of the samples were calculated as
TAmm-concentrations, using the lin-log equation of the calibration
line. Calibration solutions and samples were measured under the
same conditions (identical ionic strength, a sample volume of 2 ml,
temperature of 12 °C, identical stirring conditions). Fluxes (J) were
expressed in lmol á g FW)1 á h)1 and calculated according to
…Cbeg ÿ Cend † V
…2†
1000 t FW
where Cbeg is the concentration of ammonia in the sample (lmol á l)1) at the beginning of the experiment, Cend is the concentration
of ammonia in the sample at the end of the experiment (lmol á l)1),
V is the volume of the external bath or perfusate (ml), t is the
sampling period (h) and FW is the fresh weight of the gill (g).
The drift of the electrode over time was small, usually less than
5% per hour, however, the drift was linear over time. Therefore a
)1
standard sample (0.1 mmol NH‡
4 á l ) was included after every
tenth measurement. From the values measured a time-dependent
increment (positive or negative) was calculated for the correction of
every sample. The sensitivity of the electrode measurements
thus amounted to ‹1 lmol TAmm á l)1 in the concentration range
4±50 lmol á l)1 and to ca. ‹1.5 lmol TAmm á l)1 in the concentration range 50±100 lmol á l)1.
Jˆ
Calculations of NH3 partial pressures
As reviewed by Wilkie (1997) for ®sh gills, ammonia excretion
patterns follow blood-to-water NH3 partial pressure gradients
(DP NH3 † in both freshwater and marine environments. In order to
obtain information on the ammonia excretion pattern in the shore
crab, DP NH3 was calculated for all experimental conditions. Using
the pK of 9.48 (see introduction), the concentration of TAmm, and
the pH-value measured concentrations of NH3 were calculated
according to Eq. 1. Partial pressures of NH3 (PNH3 ), considering it
as a dissolved gas, were calculated according to Eq. 3:
PNH3 ˆ ‰NH3 Š=a
2.5 mmol á l)1 TRIS. In order to analyse the e€ects of TRIS added
to the bicarbonate bu€er, some speci®ed experiments were carried
out with salines bu€ered only with bicarbonate. For replacing Na+
or Cl) -ions in the salines, choline-Cl or Na-gluconate were used in
equimolar concentrations. Sodium-free salines contained 2 mmol á l)1 KHCO3 instead of NaHCO3.
Acetazolamide (AZ) and amiloride were purchased from Sigma
(St. Louis, USA), the ammonia standard (0.1 mol á l)1) was obtained from Orion Research Incorporated (Boston, USA), choline
chloride was obtained from Aldrich (Steinheim, Germany), Mg-Dgluconate and ouabain were obtained from Fluka (Buchs, Switzerland), CsCl, 2,4-dinitrophenol (DNP) and all other chemicals
were of analytical grade and purchased from Merck (Darmstadt,
Germany).
Calculations
The transport constants (Kt) and the maximum rates of excretion of
total ammonia along varying gradients of ammonia directed from
the haemolymph to the external medium (Vmax) (Fig. 2A) were
calculated by assuming the linear portion observed at higher gradients of TAmm as a di€usive component. Shifting the linear portion
to the concentration of zero (gradient-driven di€usion does not
occur when no concentration gradient is present) permits the subtraction of the di€usive, linear portion from the measured e‚uxes
of TAmm. The resulting, obviously saturable, component (Fig. 2A,
line c) was linearized (Fig. 2B) using the Hanes-Woolf-plot (LuÈthje
1990) to obtain transport constants and maximum excretion rates
of the saturable process. All values are presented as means ‹ SEM.
Di€erences between groups were tested with the paired StudentÔs
t-test. Statistical signi®cance was assumed for P < 0.05.
Results
Transbranchial potential di€erences
In order to monitor the proper physiological functioning
of isolated, perfused gills during the experiments, the
PDte between the bath and the perfusion solution was
measured. Only gills generating an initial and continuously negative PDte were employed. Variations in the
concentrations of ammonia in the bath and perfusion
solutions did not signi®cantly a€ect the PDte. Measurements of the ¯uxes of ammonia under control conditions showed that there was no correlation between the
rates of the transbranchial ¯uxes of TAmm and the PDte.
…3†;
where a is the solubility coecient and [NH3] the concentration
of NH3. For our calculations we used the value a ˆ
43.67 mmol á l)1 á torr)1 for plasma at 20 °C (Cameron and Heisler 1983). DPNH3 was calculated from the di€erences in (PNH3 †
between external and internal media.
Salines and chemicals
The haemolymph-like saline (control solution) used as perfusate
and bathing solution contained (mmol á l)1): 248 NaCl, 5 CaCl2, 5
KCl, 4 MgCl2, 2 NaHCO3, 2.5 Tris-(hydroxymethyl)-aminomethan, and had a pH of 7.8. At the beginning of each experiment pH
values of the perfusion and bathing solutions were controlled and,
if necessary, readjusted. In addition, pH was measured in the internal and external saline at the end of each experiment. In order to
keep changes in pH due to dissolution of NH4Cl small the physiological bu€ering with bicarbonate was increased by addition of
Production and release of ammonia by posterior gills
In order to measure ¯uxes of total ammonia across the
gill epithelium along varying gradients, it is necessary to
know the contributions to these ¯uxes by the production
and release of ammonia from gill metabolism. Therefore
no ammonia was added to the salines at the beginning.
Total liberation of ammonia by the gill epithelium was
5.4 ‹ 0.2 lmol á g FW)1 á h)1 (n ˆ 4) (Fig. 1). Of this,
81.5% was released to the apical and 18.5% to the
basolateral side. Since the pH of the salines remained
constant at 7.8 the DPNH3 across the epithelium remained negligibly small.
Using Na+-free salines total liberation of ammonia
was similar (6.0 ‹ 0.5 lmol á g FW)1 á h)1, n ˆ 4);
367
3 lmol á l)1 (Fig. 1). Internal concentrations that had
been decreased as a result of e‚uxes can thus only be
underestimated by this ®gure.
E‚ux of ammonia across the posterior gills
along varying gradients
Fig. 1 Production and release rates of ammonia by the gill epithelium
of Carcinus maenas in controls and under Na+-free conditions. Gills
were bathed and perfused with identical salines without ammonia.
Data represent means ‹ SEM of four observations (P < 0.01) (FW
fresh weight)
however, the portion of ammonia transported to the
basolateral side increased signi®cantly (P < 0.01) to
35.1% of the total release (Fig. 1). Another experiment
showed that the addition of 2 mmol á l)1 glucose to the
perfusion solution reduced the liberation of total ammonia signi®cantly to 68.5% (n ˆ 9; P < 0.01) of
controls, without signi®cant changes in the proportion
of e‚uxes of ammonia to the apical and basolateral side
(data not shown).
In the experiments employing a ®xed gradient of
TAmm directed from perfusate to bath (Table 1) the
contributions of the production and release of ammonia
by the gill to changes in TAmm on either side are dicult
to quantify and represent a factor of uncertainty. We
have, however, no reason to assume that the production
and release of total ammonia by the gill had been increased. In order to avoid overestimation of ¯ux rates of
ammonia, only the concentration changes detected in
the perfusate were used for ¯ux calculations. The portion of TAmm due to release of metabolic ammonia into
the perfusate is considered not to exceed approximately
Table 1 Percentage inhibition
of the excretion of total ammonia across the gill epithelium
along a gradient of 200
lmol . l)1 NH4Cl directed from
the haemolymph side to the
external bath (DNP 2,4 -dinitrophenol)
By applying varying concentrations of NH4Cl (25±4800
lmol á l)1) in the perfusion saline without NH‡
4 in the
external bath, it was possible to determine e‚ux rates of
total ammonia across the gill epithelium along the respective gradients.
The gradient-driven excretion of total ammonia was
composed of a saturable and a linear component
(Fig. 2A, a). The non-saturable, linear component (r ˆ
0.998; Fig. 2B) dominated at unphysiologically high
gradients between the haemolymph and external medium (1600±4800 lmol á l)1), the saturable component
(Fig. 2A, c) dominated at the lower physiological conditions. The saturable curve plotted in a Hanes-Woolfdiagram exhibited a regression of 0.998. A Kt of 301
lmol á l)1 NH‡
and Vmax of 104 lmol NH‡
4
4 á g
)1
)1
FW á h were calculated from this plot (Fig. 2B).
Measurements of the PDte indicate that the ion transport processes in the gills were not damaged by unphysiologically high concentrations of ammonia (data
not shown).
At the beginning of the experiment the pH of the
perfusion solutions and in the bath was kept constant at
7.8. After each experiment pH of the bath and the perfusion solution was measured. This allowed us to calculate PNH3 in both saline solutions before and after the
experiment and the respective DPNH3 , which are the
driving force for di€usive translocation of NH3. During
the experiments (0.5 h) the pH of the perfusate changed
slightly by 0.08 units. At low concentrations pH increased from 7.80 at the beginning to ca. 7.84 (Fig. 3a).
Along with increasing TAmm pH decreased steadily to
reach ®nal values of 7.76. In the bath ± obviously due to
the bu€ered saline and the comparatively high volume of
30 ml ± the pH remained constant at 7.80 ‹ 0.02 at all
concentrations applied. Measured excretion rates of total ammonia were plotted against calculated DPNH3 at
Manipulation
Concentration of )1
inhibitor (mmol . l )
Reduction of
ammonia e‚ux (%)
Number of
observations (n)
Acetazolamideb
-Cl)a,b
-Cl)a,b/ouabainb
DNPb
Ouabainb
Ouabainb/Cs+b
Cs+a
Amiloridea
1
±
±/5
0.5
5
5/10
10
0.1
18
24
51
55
52
73
12
55
6
8
8
6
7
7
4
12
a
b
manipulation on the apical side of the gill epithelium
manipulation on the basolateral side of the gill epithelium
368
Fig. 2A E‚ux rates for total ammonia (TAmm:NH3/NH‡
4 ) across
isolated, perfused gills along various gradients directed from the
haemolymph into the external bath (a excretion of ammonia: data
represent means ‹ SEM of at least six observations; b linear
component; c saturable component). B Hanes-Woolf-plot of data
obtained from the saturable component of mean-e‚uxes to calculate
the transport constant Kt and the maximum release rate Vmax
the beginning and at the end of the experiment (Fig. 3b).
A ¯ux of NH3 driven by DPNH3 should be linear over
time. This linearity was, however, only observed at
higher DPNH3 ; indicating that at lower DPNH3 another
mode of excretion other than di€usion of NH3 must be
operating.
Flux of ammonia across the posterior gills
along a ®xed gradient: e€ects of dinitrophenol,
ouabain, Cs+, amiloride, acetazolamide
and substitution of Cl)
Based on the concentrations of ammonia measured in
the haemolymph of C. maenas (147 ‹ 37 lmol á l)1,
1 day after feeding, n ˆ 21), the posterior gills immersed in control solutions were perfused with the same
saline but containing in addition 200 lmol á l)1 NH4Cl.
Measuring TAmm of the perfusion medium and the
perfusate after passage through the gill showed that
Fig. 3A,B E‚ux rates for TAmm across isolated, perfused gills along
various gradients directed from the haemolymph into the external
bath as shown in Fig 2. At the beginning the pH was 7.8 in the
internal and the external saline. A Changes in pH of the perfusate
after the experimental period of 0.5 h B Fluxes of total ammonia
plotted against the gradients of PNH3 before (closed circles, r of the
linear component ˆ 0.998) and after the experiment (open circles, r of
the linear component ˆ 0.986)
TAmm had been reduced from 200 lmol á l)1 to a mean
concentration of 90.9 lmol á l)1. This result shows that
the original TAmm of the perfusion solution was reduced
by more than 50% during a single passage of saline
through the gill. Using the perfusion rate of 8 ml á h)1,
the FW of the gills (between 18 and 40 mg), and the
measured reductions of TAmm in the perfusate of individual gills, an e‚ux of TAmm from the basolateral to the
apical side of the epithelium (Jb->a) of 35.1 ‹ 5.3
lmol á g FW)1 á h)1 (n ˆ 32) was calculated. Mean
e‚ux rates decreased sligthly by 4.0 ‹ 0.4 lmol á g
FW)1 á h)1 (n ˆ 3) when measurements were continued
up to 4 h under control conditions (data not shown).
Addition of 0.5 mmol á l)1 DNP, an inhibitor of oxidative phosphorylation and a H+-shunt reagent, to the
perfusion saline reduced the e‚ux of total ammonia
(Jb->a) along the gradient of 200 lmol á l)1 NH‡
4 directed from the haemolymph side into the external bath
from 62.9 ‹ 5.5 lmol á g FW)1 á h)1 (controls) to
28.2 ‹ 4.0 lmol á g FW)1 á h)1 (n ˆ 6; P < 0.01)
(Table 1). Basolateral application of 5 mmol á l)1
369
ouabain, a potent and speci®c inhibitor of Na+/K+ATPase (Skou 1965), decreased the e‚ux (Jb->a) from
26.4 ‹ 4.5 (controls) to 12.8 ‹ 2.9 lmol á g FW)1 á h)1
(n ˆ 7; P < 0.01). The e‚ux rates further decreased to
7.2 ‹ 1.9 lmol á g FW)1 á h)1 (P < 0.01) when, in
addition to ouabain, 10 mmol á l)1 of Cs+, an inhibitor
of K+-channels (Van Driessche and Zeiske 1980; Riestenpatt 1995), was added to the perfusion saline
(Table 1). Apical application of Cs+ had no substantial
e€ect on e‚ux rates [controls: 44.2 ‹ 6.4 lmol . g
FW)1 á h)1, Cs+apical: 39.1 ‹ 5.9 lmol á g FW)1 á h)1;
(n ˆ 4; P < 0.05)]. Addition of 0.1 mmol á l)1 amiloride, a potent inhibitor of Na+/H+ antiporters in
crustacean epithelia (Shetlar and Towle 1989; Ahearn
et al. 1990; Ahearn 1996), resulted in a reduction of ef¯ux rates of TAmm from 48.9 ‹ 1.9 lmol á g FW)1 á h)1
(controls) to 21.6 ‹ 1.4 lmol á g FW)1 á h)1 (n ˆ 12;
P < 0.01) (Table 1).
High activities of carbonic anhydrase (CA) were
found in the gill tissue of C. maenas (BoÈttcher and
Siebers 1993). In order to investigate the e€ects of AZ, a
potent inhibitor of CA (Maren 1977), 1 mmol á l)1 AZ
was added to the perfusion saline. Compared to controls
Jb->a of total ammonia along the gradient of 200
lmol á l)1 NH‡
4 directed from the perfusion solution to
the bath was slightly reduced from 35.8 ‹ 4.3 to 29.5 ‹
3.9 l mol á g FW)1 á h)1 (n ˆ 6; P < 0.05) (Table 1).
Previous investigations have shown that active osmoand ionoregulatory ion absorption across the posterior
gills of the shore crab strictly proceeds in a coupled
mode of Na+ and Cl) (Onken and Siebers 1992). The
question as to whether or not ammonia excretion is directly coupled to active ion uptake was tested by symmetrical substitution of Cl) by gluconate. Using this
saline no active ion uptake occurs (Riestenpatt et al.
1996). When Cl) ions in the perfusion and the bathing
solution were replaced by gluconate, e‚ux rates of
ammonia decreased slightly from 28.8 ‹ 3.6 to
21.8 ‹ 3.1 lmol á g FW)1 á h)1 (n ˆ 8; P < 0.01).
Additional basolateral application of 5 mmol á l)1
ouabain to the perfusion solution further reduced the
e‚ux rates to 14.2 ‹ 1.8 lmol á g FW)1 á h)1 (P <
0.01) (Table 1).
In¯ux of ammonia across the posterior gills
In order to investigate gradient-driven ¯ux rates of
ammonia from the external bath to the haemolymph
space, a gradient of NH4Cl (200 lmol á l)1) from the
bath to the perfusion solution was applied, which resulted in an in¯ux of ammonia (Ja->b) of 3.7 ‹ 0.3
lmol á g FW)1 á h)1 (n ˆ 9). Compared with the previously measured e‚ux rates (Jb->a ˆ 39.3 ‹ 6.5
lmol á g FW)1 á h)1) in¯ux rates of ammonia were approximately 10-fold smaller than e‚uxes (Fig. 4). Additional basolateral application of 5 mmol á l)1 ouabain
had no signi®cant e€ect on the in¯ux of ammonia (in¯uxouabain 3.5 ‹ 0.5 lmol á g FW)1 á h)1; n ˆ 9). The
Fig. 4 Rates of in¯ux and e‚ux for total ammonia across)1 isolated,
perfused gills of C. maenas along a gradient of 200 lmol . l NH4Cl.
)1
The white bar represents)1 e‚uxes (haemolymph side: 200 lmol . l
.
NH4Cl; bath: 0 lmol l NH4Cl); the black bar represents in¯uxes
(haemolymph side: no NH4Cl; bath: 200 lmol . l)1 NH4Cl). Data
represent means ‹ SEM of nine observations (P < 0.01)
same negative e€ect was found after basolateral addition
of 0.5 mmol á l)1 DNP (in¯uxcontrols: 4.2 ‹ 2.0 lmol á g
FW)1 á h)1; in¯uxDNP: 4.3 ‹ 2.1 lmol á g FW)1 á h)1 ;
n ˆ 3) (data not shown).
Active excretion of ammonia across the gill
epithelium: e€ects of DNP, ouabain, Cs+
and of substitution of Na+, and active e‚ux
of ammonia across anterior and posterior gills
In previous experiments ¯ux of ammonia was measured
using concentration gradients of ammonia between the
haemolymph and the external bath. Under these conditions, gradient-driven ¯ux of NH3 and NH‡
4 and active net movement of NH‡
mediated
by
energy
4
dependent processes cannot be distinguished. In a series
of experiments 100 lmol á l)1 of NH‡
4 were added to
both the perfusion saline and the external bath. Measurements of the concentration of TAmm in the perfusion
solutions and the external bath show that ammonia is
actively excreted across the gill epithelium. Since the
perfusate passes the vessels of the gill only once, the
reduction of TAmm in the perfusate after passage
through the gill is a direct measure of the proportion of
ammonia (%) excreted from the perfusate across the gill
into the bath. Under these symmetrical conditions,
66.7% of TAmm in the perfusate was eliminated during
a single passage and an active net e‚ux of
9.2 ‹ 0.8 lmol á g FW)1 á h)1 (n ˆ 7) was calculated.
At the beginning of the experiment TAmm in the bath
and the perfusate was 100 lmol á l)1 without a DPNH3 .
After 0.5 h of experiment TAmm in the bath had increased to 115.3 ‹ 3.9 lmol . l)1. TAmm in the perfusate
was reduced from 100 to 33.3 ‹ 5.5 lmol á l)1. Increases in the bath and decreases in the perfusates were
calculated for a standard gill of 30 mg FW. In this experiment an additional release of ammonia
(6.1 ‹ 1.6 lmol á g FW)1 á h)1) generated from the gill
370
metabolism and excreted mainly into the bath can be
calculated. During the experiment PNH3 changed from
46.9 to 53.9 ltorr in the bath and from 46.9 to 15.7 ltorr
in the perfusate. DPNH3 therefore changed from zero to
38.2 ltorr and was directed into the perfusate. This result shows that the gill has excreted ammonia against a
large gradient of TAmm increasing from zero to
82 lmol . l)1 during the experiment and an opposing
DPNH3 increasing from zero to 38.2 ltorr.
In contrast to partial inhibition by DNP of ammonia
excretion along an outwardly directed gradient (see
above), an almost complete reduction of active net e‚ux
across the gill epithelium was observed using symmetrical
concentrations of ammonia (100 lmol á l)1 NH‡
4 ) in the
bath and in the perfusion solution. E‚uxes (Jactive, b->a)
under control conditions amounted to 15.4 ‹ 2.5
lmol á g FW)1 á h)1 and were reversibly blocked to
1.2 ‹ 0.5 lmol á g FW)1 á h)1 (n ˆ 5; P < 0.001) following basolateral addition of 0.5 mmol á l)1 DNP
(Fig. 5a).
Results on the e‚ux of ammonia along an outwardly
directed gradient (see above) showed that the Na+/K+ATPase plays an important role in the excretion of
ammonia across the posterior gills of C. maenas. Fol-
lowing the addition of 5 mmol á l)1 ouabain to the perfusion solution the active net e‚ux in the symmetrical
presence of 100 lmol á l)1 NH‡
4 was reduced from
9.2 ‹ 0.8 lmol á g FW)1 á h)1 (controls) to 3.7 ‹ 0.3
(n ˆ 7; P < 0.001) (Fig. 5b). When we applied Na+free salines prepared by isoionic replacement of Na+ by
choline, active net e‚ux of NH‡
4 decreased from
10.9 ‹ 2.3 lmol á g FW)1 á h)1 (controls) to 4.7 ‹ 0.6
lmol á g FW)1 á h)1 (n ˆ 3; P < 0.05) (Fig. 5c).
Basolateral application of 10 mmol á l)1 CsCl inhibited
active net e‚ux (Jactive, b->a) of ammonia from
12.5 ‹ 1.8 (controls) to 5.0 ‹ 0.6 lmol á g FW)1 á h)1
(n ˆ 5; P < 0.01). Additional enrichment of the perfusion solution with 5 mmol á l)1 ouabain increased the
inhibitory e€ect of Cs+. Net e‚uxes of ammonia ®nally
decreased to 1.8 ‹ 0.8 lmol á g FW)1 á h)1 (n ˆ 5;
P < 0.001) (Fig. 5d).
In order to obtain information about the presence of
active excretion of ammonia in the di€erent gills of the
shore crab, gills 4±9 were symmetrically exposed to 100
lmol á l)1 NH4Cl. As shown in Fig. 6, anterior gills 4±5
are also capable of active excretion of ammonia. The
excretion rates were even larger than those observed in
anterior gills 7±9.
Fig. 5a±d E€ects of di€erent inhibitors and of omission of sodium on
active excretion of ammonia across the gill epithelium of C. maenas.
At the beginning of all experiments
the perfusate and the external bath
)1
contained 100
lmol . l NH4Cl. a Basolateral application of
)1
n ˆ 5, P < 0.001); b baso0.5 mmol . l 2,4-dinitrophenol (DNP;
)1
lateral application of 5 mmol . l ouabain (n ˆ 7, P < 0.001); c
(n ˆ 3, P < 0.05); d basolateral
symmetrical Na+-free conditions
)1
)1
application of 10 mmol . l Cs+ and Cs+ + 5 mmol . l ouabain
(n ˆ 5, P < 0.01). Data represent means ‹ SEM (i basolateral, e
apical)
Fig. 6 Active excretion of ammonia across anterior gills 4±6 and
posterior gills 7±9 and transepithelial potential di€erences. At the
beginning of all experiments
the perfusate and the external bath
)1
contained 100 lmol . l NH4Cl. Data represent means ‹ SEM
(n ˆ 5 for all gills except gill 6 and 9 where n ˆ 4)
371
Active excretion of ammonia across posterior gills:
utilization of salines bu€ered without TRIS
Using salines bu€ered with 2 mmol . l)1 bicarbonate
alone (omitting TRIS) ¯ux rates of ammonia and
changes of PNH3 were measured under symmetrical application of 100 lmol á l)1 ammonia at the beginning of
the experiment. After the 0.5 h experiment TAmm in the
bath had increased from 100 to 113.4 ‹ 2.3 lmol . l)1.
TAmm in the perfusate was reduced from 100 to
55.8 ‹ 7.6 lmol . l)1(n ˆ 6) (Fig. 7A). Increases in the
bath and decreases in the perfusates were calculated for
a standard gill of 30 mg FW. Taking into consideration
the reduction of ammonia in the perfusate a net excretion of ammonia from the haemolymph space into the
bath of 13.2 ‹ 2.3 lmol á g FW)1 á h)1 (n ˆ 6) was
calculated. Measurements of ammonia in the bath
showed an additional increase of ammonia that must
have resulted from the N-metabolism of the gill itself
and amounted to 13.7 ‹ 3.1 lmol á g FW)1 á h)1
Fig. 7A,B Active excretion of ammonia across posterior)1 gills 7 and 8
using symmetrical salines bu€ered only by 2 mmol . l bicarbonate
[omitting Tris-(hydroxymethyl)-aminomethan]. At the beginning of all
experiments
the perfusate and the external bath contained 100
)1
lmol . l NH4Cl (n ˆ 6). Data represent means ‹ SEM. A Concentrations of TAmm in the bath and in the perfusate before and after
an experimental period of 0.5 h. B Flux rates of TAmm indicating
active net e‚uxes from the perfusate into the bath (Gill release
calculated proportion of TAmm resulting from the N-metabolism of
the ionocytes and liberated mainly into the bath)
(Fig. 7B). During the experiment PNH3 changed from
46.9 to 53.2 ‹ 0.4 ltorr in the bath and from 46.9 to
21.6 ‹ 2.7 ltorr in the perfusate. DPNH3 therefore
changed from 0 to 31.6 ‹ 7.0 ltorr and was directed
into the perfusate. This result shows that the gill has
excreted ammonia against a large concentration gradient
of TAmm increasing from 0 to 57.6 lmol . l)1 during the
experiment, and an opposing DPNH3 that increased from
0 to 31.6 ltorr.
Discussion
E‚ux of ammonia across the posterior gills
along varying gradients
Release of TAmm along varying gradients directed from
the haemolymph to the external medium was composed
of a saturable and a non-saturable component. The saturable component indicates that transport of the ionic
form of ammonia (NH‡
4 ) proceeds via a ®nite number of
transporting proteins. The non-saturable component
most probably shows simple di€usion of non-ionic NH3,
dependent on the partial pressure gradient. Simple diffusion of NH3 under physiologically meaningful concentration gradients of TAmm comprised only a small
portion of total N-e‚ux, amounting to approximately
16% at a concentration gradient of TAmm of 100
lmol á l)1, and 18% at a gradient of 200 lmol á l)1.
Under
physiological
concentration
gradients
branchial N-excretion in Carcinus maenas is thus considered to proceed mostly as NH‡
4 . These ®ndings are in
line with the results of the in¯ux experiments (Fig. 4). In
contrast, Kormanik and Cameron (1981) showed that
branchial N-excretion in sea water-adapted blue crabs
Callinectes sapidus occurs mainly as non-ionic NH3. In
Carcinus maenas excretion of NH3 exceeded the rates of
the saturable component only at unphysiologically high
concentration gradients of TAmm (larger than ca.
1.5 mmol á l)1) between haemolymph and ambient medium. The results di€er from the ®ndings by Lucu et al.
(1989) who reported only saturable e‚ux of TAmm
across isolated, posterior gills of the shore crab without
any linear component. The tendency of slight increases
in the pH of the perfusate from 7.80 to ca. 7.84 at low
perfusate concentrations, followed by a steady decrease
to ®nal values of ca. 7.76 along with increasing perfusate
concentrations, can be explained by considering the
NH3/NH‡
4 equilibrium of TAmm:
‡
NH‡
4 ‡ H2 O , NH3 ‡ H3 O
…4†
At low perfusate concentrations of TAmm the preferable
outward transport of NH‡
4 may result in alkalinization
of the perfusate, while at higher perfusate concentrations
the high di€usion rate of NH3 may result in acidi®cation. The changes in pH due to transport of ammonia
probably became visible because of limitations in the
capacity of pH regulation by the gill (Siebers et al. 1994).
372
E‚uxes of ammonia across posterior gills
along a ®xed gradient
Application of a near-physiological 200 lmol á l)1 gradient of NH4Cl directed from the haemolymph to the
external side of the gill resulted in e‚ux rates indicative
of a high degree of e€ectiveness in removing ammonia
from the haemolymph: TAmm in the perfusate was reduced by more than 50% during a single passage
through the gill. Involvement of Na+/K+-ATPase is
obvious from the ®nding that ¯uxes of ammonia along
the 200 lmol á l)1 gradient was reduced by 52% following basolateral addition of ouabain (Fig. 8). The
e‚ux rates further decreased to reach a total reduction
of 73% when, in addition to ouabain, 10 mmol á l)1 Cs+
was added to the basolateral side (Table 1). The results
suggest that apart from Na+/K+-ATPase, Cs+-sensitive K+-channels located in the basolateral membrane
(Riestenpatt et al. 1996), which do not discriminate between K+ and NH‡
4 , also play a role in the translocation
of NH‡
4 from the haemolymph across the basolateral
membrane into the epithelial cell (Fig. 8). Application of
Cs+ in the external medium reduced e‚ux rates for
ammonia by only 12%, a result implying that apical
Cs+-sensitive K+-channels are not involved in N-excretion. Ouabain sensitivity of ammonia ¯uxes across
the gills has also been shown in the chinese crab Eriocheir sinensis (PeÂqueux and Gilles 1981) and in the shore
crab Carcinus maenas (Lucu et al. 1989).
In comparison to controls, addition of 0.5 mmol á l)1
DNP to the perfusion saline reduced the e‚ux of TAmm
along its gradient by 55%, implying that the process
depends on provision of energy. The ®nding that the
addition of both ouabain and DNP resulted in an incomplete reduction of ammonium e‚ux by only approximately 55% suggests that the remaining 35% of
e‚ux (an assumed maximum ®gure when considering
ca. 20% passive nonionic di€usion) is driven by an
ammonium gradient across the apical membrane, generated from NH‡
4 translocation into the epithelial cell
via basolateral K+-channels. We assume that the gradient across the apical membrane drives the ¯ux out o€
the epithelial cell via transporting structures, which have
gone unidenti®ed until now. Apical amiloride (0.1 mmol
. )1
l ) reduced the e‚ux of ammonia along its gradient by
55%, a result allowing the assumption that amiloridesensitive cation antiporters are responsible for the exit of
ammonia across the apical side of the epithelial cell
(Fig. 8). Following the addition of 0.3 mmol . l)1
amiloride to the ambient medium, excretion rates of
ammonia of intact blue crabs Callinectes sapidus were
inhibited by 63% in specimens adapted to a salinity of
17& and by 67% in specimens adapted to 35& sea
water (Pressley et al. 1981). Also, isolated non-perfused
gills of the blue crab excreted 45% (acclimated to 17&
salinity) and 40% (acclimated to 35& salinity) less ammonia after the addition of amiloride. Based on the
correlation between amiloride sensitivity of Na+-in¯ux
and ammonia excretion in the blue crab, the authors
proposed the presence of a Na+/NH‡
4 -exchanger in the
apical membrane.
With respect to published inhibition constants for
amiloride of 200 lmol . l)1 of the K+/H+-antiporter in
the apical membrane of the midgut of the tobacco
hornworm Manduca sexta (Wieczorek et al. 1991) and of
280 lmol . l)1 of the 2Na+/H+-antiporter in the apical
membrane of branchial epithelial cells of the shore crab
(Shetlar and Towle 1989), we propose that the incomplete amiloride-induced reduction of the e‚uxes of
ammonia observed in our experiments resulted from the
relatively low doses of the drug administered. However,
one cannot exclude the possibility that the release of
ammonia that could not be inhibited by amiloride may
have proceeded via amiloride-insensitive apical cationtranslocating structures. An amiloride-sensitive cation
pore of low selectivity for monovalent cations including
NH‡
4 was identi®ed by S. Riestenpatt (personal communication) in the isolated branchial cuticle of the shore
crab. The possibility that this pore is a potential translocation site for NH‡
4 cannot be excluded.
Relation between nitrogen excretion
and osmoregulatory ion transport, excretion
of respiratory CO2 and pH regulation
of the haemolymph in posterior gills
Fig. 8 Preliminary model of ammonia excretion across the gill
epithelium of the shore crab C. maenas (1 ouabain sensitive Na+/
K+-ATPase, 2 Cs+)sensitive K+ channel, 3 routes of NH3 di€usion,
4 unknown active translocation mechanism of NH‡
4 , 5 amiloridsensitive cation-transporting structure of the cuticle)
Replacement of Cl) ions by gluconate in the perfusion
and the bathing solution resulted in slight decreases of
e‚ux rates of ammonia of 24% (Table 1). Since active
translocation of NaCl depends strictly on the presence of
373
Na+ and Cl) ions in the perfusate and the bathing solution (Onken and Siebers 1992), this result implies that
only a small portion of the excretion of ammonia along
its gradient is coupled to the process of ion absorption.
Additional basolateral application of ouabain to the
perfusion solution reduced the e‚ux by 51% (Table 1), a
®gure already known from application of ouabain alone.
The data obtained from application of acetazolamide
and omission of Cl)indicate that excretion of ammonia is
not necessarily coupled to active osmoregulatory ion
absorption in spite of the the fact that it utilizes some of
the proteins (basolateral Na+/K+-ATPase, basolateral
K+-channel, apical amiloride sensitive cation-transporting structures; see Table 1) that play a role in this
process. In spite of the well established role of CA in CO2
excretion (BoÈttcher et al. 1995) and pH regulation (Siebers et al. 1994), this enzyme seems not to be involved in
active ion uptake across the posterior gills of the shore
crab. Only small or no e€ects of AZ were observed on
PDte (Siebers et al. 1994) and on the in¯ux of sodium or
chloride (BoÈttcher et al. 1991) in isolated perfused gills,
and on the short circuit current measured in isolated gill
half lamellae (Onken and Siebers 1992). In contrast to
the ®ndings in the shore crab, Henry and Cameron
(1982) considered the extensive salinity modi®cations of
speci®c CA activity in posterior gills of hyperregulating
blue crabs Callinectes sapidus, and deduced that one of
the potential roles of the enzyme is osmoregulatory (see
also Henry 1988). In the chinese crab Eriocheir sinensis
AZ is a strong inhibitor of active ion transport (Onken et
al. 1995). In this crab, active Na+-independent Cl)-uptake is driven by an apical V-type H+-pump and proceeds via apical Cl)/HCO3)-exchange (apical H+-pump
and anion exchange in concerted action with cellular
carbonic anhydrase) and basolateral Cl)channels (Onken et al. 1995). In the shore crab the coupled in¯ux of
sodium and chloride is energized by only one pump, the
Na+/K+-ATPase, and the in¯ux of these ions across the
apical membrane is considered to proceed via apical
Na+/K+/2Cl)- cotransport (Riestenpatt et al. 1996).
Basolateral application of the comparatively high concentration of AZ (1 mmol . l)1) reduced the e‚ux of
ammonia along a 200 lmol á l)1 gradient by only 18%
(Table 1). Considering that e‚ux along the gradient was
reduced by approximately 10% per hour under control
conditions (see results) this ®nding shows that the majority of TAmm excretion does not depend on the carbonic anhydrase mediated processes of excretion of
respiratory CO2 (BoÈttcher et al. 1995) or pH regulation
of the haemolymph (Siebers et al. 1994). In these experiments on isolated perfused gills the e€ects of AZ
(0.1 mmol . l)1) on pH regulation and excretion of CO2
were observed within approximately 10 min following its
application, implying that 30 min preincubation of perfused gills with 1 mmol á l)1 AZ (this paper) was sucient for drug equilibration.
In¯ux of ammonia
If the ¯ux of ammonia exclusively proceeded as nonionic
NH3-di€usion, it can be anticipated that in¯ux and e‚ux
will equal each other when the gradients of TAmm and
PNH3 directed from internal to external medium, and vice
versa, are identical. However, compared with the e‚uxes
of ammonia along a 200 lmol á l)1 gradient directed
from the basolateral to the apical side of the epithelium
(Jb->a ˆ 39.3 ‹ 6.5 lmol á g FW)1 á h)1), the in¯uxes
proceeding along an equal gradient in the opposite direction (Ja->b ˆ 3.7 ‹ 0.3 lmol á g FW)1 á h)1)
amounted to only approximately 10% of e‚uxes
(Fig. 4). Due to the insensitivity of in¯ux to ouabain and
DNP this 10% is considered as nonionic di€usion of
NH3, which does not proceed via ouabain-sensitive Na+/
K+-ATPase nor on DNP-sensitive provision of energy.
In addition, the results of the in¯ux experiment show that
¯ux of ammonia across the gills of the shore crab is
highly directed. It has been shown by Cameron (1986) for
intact specimens of the crab Callinectes sapidus that rapid
net in¯ux of ammonia occurs at high external concentrations of TAmm (1 mmol á l)1). Since our in¯ux experiments were conducted on isolated gills at comparatively
low external concentrations we cannot exclude the possibility that at high external concentrations of TAmm high
net in¯uxes may also occur.
Production and release of ammonia
by the posterior gills
The posterior gills of Carcinus maenas are multifunctional organs responsible for acid-base regulation (Siebers et al. 1994; BoÈttcher et al. 1995), N-excretion
(Lucu et al. 1989; Siebers et al. 1995) and osmoregulatory ion uptake (Siebers et al. 1985; Riestenpatt et al.
1996). These processes require energy produced by cell
metabolism. In order to quantify the production and
release of ammonia by the posterior gills no ammonia
was added to any of the salines at the beginning of the
experiments. Of the TAmm liberated by the gill epithelium (Fig. 1) 81.5% was transported to the apical and
18.5% to the basolateral side. Application of Na+-free
salines increased the liberation of ammonia to the
basolateral side to 35.1%, probably as a result of deactivation of Na+/K+-ATPase. Following Na+-omission, approximately two-thirds of total liberation of
ammonia still proceeded to the apical side. In the absence of Na+ this high amount of ammonia excreted to
the external medium cannot proceed via an apical Na+/
NH‡
4 exchanger, which was considered to operate in
this process by Lucu et al. (1989). Addition of
2 mmol á l)1 glucose to the perfusion solution reduced
the liberation of total ammonia signi®cantly to 68.5%
of controls without signi®cant changes in the pattern of
e‚ux of ammonia in the apical and basolateral direction. This result indicates that even in the presence of
sucient glucose approximately two-thirds of metabo-
374
lism is still accounted for by amino acids or other Ncontaining molecules.
Active excretion of ammonia across the gill epithelium
In contrast to experiments dealing with gradient-driven
¯uxes of ammonia, 100 lmol á l)1 of NH4Cl were added
to both the perfusion saline and the external bath to
characterize the potential presence of active mechanisms
of net e‚ux (Jactive, b->a) of ammonia across the gill
epithelium. Even under these symmetrical conditions,
69.3% of TAmm originally contained in the perfusate was
eliminated during a single passage through the gill, a
result showing active net excretion of NH‡
4 . The gills of
the shore crab seem to counteract the in¯ux of toxic
ammonia with active excretion when the external concentrations are elevated up to or higher than internal
levels. As shown for Callinectes sapidus this capacity
may be limited when external concentrations rise to
higher levels of approximately 1 mmol á l)1 (Cameron
1986). It is presently not known whether the capability
of active excretion of ammonia observed in Carcinus
maenas is also present in other crustacean species.
Dependence on metabolic energy is shown by the
e€ects of DNP, which after basolateral addition nearly
reduced the process completely (by 93%; Fig. 5). Dependence of active excretion of ammonia on Na+/K+ATPase is obvious from sensitivity to ouabain inhibiting
it by 56% with regard to controls (Figs. 5, 8). Under
Na+-free conditions active extrusion of ammonia was
reduced to 54% of controls. It is worth mentioning that
blocking of the access to energy by DNP resulted in a
nearly complete reduction of this process, but inhibition
of Na+/K+-ATPase and omission of Na+ decreased
active extrusion by one-half. This ®nding allows the
assumption that besides Na+/K+-ATPase a second active process, which is independent of the presence of
Na+ ions, is involved in active excretion of ammonia.
This process is not known at present (Fig. 8).
Involvement of basolateral K+-channels (Fig. 8) in
this mechanism is evident from its sensitivity to Cs+ administered on the haemolymph side of the epithelium,
which inhibited active ammonia extrusion by 58%. Additional application of ouabain reduced the active process
by 86%, a ®nding showing that the entrance of NH‡
4 from
the haemolymph space into the epithelial cell almost entirely proceeds via Na+/K+-ATPase and K+-channels.
With respect to basolateral entrance into the epithelial
cell, excretion of ammonia along its gradient equals active
extrusion. We therefore consider the second active component to be located on the apical side (Fig. 8).
Active e‚ux of ammonia across anterior
and posterior gills
Of the nine gills located on each side of the cephalothorax of the shore crab the posterior gills are assumed
to play the dominant role in active ion uptake (Siebers et
al. 1982; Siebers et al. 1987; see also Towle 1981 for
other crustaceans) and carbonic anhydrase-dependent
pH regulation (BoÈttcher and Siebers 1993; BoÈttcher et
al. 1995). In anterior gills the capability of active ion
uptake is markedly reduced. This is also obvious from
the transepithelial potential di€erences shown in Fig. 6.
Interestingly, this pattern was not found when considering active excretion of ammonia. The capacity for
active excretion of ammonia was even more pronounced
in the anterior gills 4±6 (gills 1±3 were too small for
perfusion). This observation is consistent with the assumption that active excretion of ammonia across the
gills of the shore crab does not depend on the coupled
active transport of NaCl, though it utilizes some of the
membrane proteins (Na+/K+-ATPase and basolateral
K+-channel) operating in it. With respect to large sodium in¯ux rates (approximately 800 lmol á g
FW)1 á h)1) (Siebers et al. 1987), active excretion rates
of ammonia are small (10±20 lmol á g FW)1 á h)1).
Thus it can be assumed that the lower activities of Na+/
K+-ATPase in anterior gills (Siebers et al. 1987) are also
sucient for the translocation of ammonia from the
haemolymph space into the interior of the ionocyte.
Active excretion of ammonia across posterior gills:
utilization of salines bu€ered without TRIS
It may be argued that the di€usion of ammonia gas
across the gill depends on the protonation of NH3 to
NH‡
4 in order to maintain the small but signi®cant PNH3
gradient. In a saline more heavily bu€ered by
2.5 mmol á l)1 TRIS those protons may be less available,
so NH3 may build up in the external boundary layer and
potentially retard di€usion. In order to analyse the
transport pattern under more physiological conditions,
rates of active excretion of ammonia across the posterior
gills were additionally measured in the absence of TRIS,
using only bicarbonate bu€ering (Fig. 7). Under these
conditions active excretion rates of ammonia and
changes in PNH3 in the bath and the perfusion solution
were nearly similar to the ®ndings measured during bicarbonate/TRIS bu€ering. So, even under more physiological conditions ammonia was actively excreted
against a gradient of TAmm and PNH3 .
Conclusions
The N-excretory pattern detected in the gills of the shore
crab is a highly ¯exible system allowing excretion of
ammonia over a wide range of concentration gradients
of TAmm, which may vary with respect to internal and
environmental levels. N-excretion under physiological
conditions mainly consisted of carrier-mediated e‚ux of
NH‡
4 . These e‚ux mechanisms utilize some of the
transport proteins playing a role in active osmoregulatory ion uptake (Fig. 8). Because of the toxicity of NH3,
375
N-excretion must proceed independently of other physiological processes. However, even at physiologically
relevant low concentration gradients of TAmm a small
portion of TAmm was liberated as non-ionized NH3. This
portion steadily increased along with an increasing
concentration gradient that could reach very high levels
(5 mmol . l)1 and probably higher, see Fig. 2). Under
physiological conditions excretion of ammonia across
the gills is a directed process with a high degree of effectiveness. It even allows active extrusion against an
inwardly directed gradient, if necessary.
Acknowledgements This article is based on a doctoral study loy
Dirk Weibranch in the Faculty of Biology, University of Hamburg.
The ®nancial support of this work by the Deutsche Forschungs
Gemeinschaft (Si 295/2-3) is gratefully acknowledged.
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Communicated by G. Heldmaier