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
Dirk Weihrauch1,*,
Andreas Ziegler2,
Dietrich Siebers3 and
David W. Towle4
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
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Fig. 1. Active ammonia excretion across the isolated perfused gill of the shore
crab Carcinus maenas. At the beginning of all experiments, the
internal perfusate and the external bath contained symmetrical concentrations
of 100 µmol l-1 NH4Cl. (A) Omission of Tris-HCl
buffer in the saline. Rate of ammonia loss from the internal perfusate, rate
of ammonia addition to the external bath and the calculated rate of metabolic
ammonia release into the external bath are displayed (N=5). (B)
Disappearance of total ammonia from the internal perfusion medium was measured
as the rate of net active branchial ammonia excretion over an experimental
period of 3 h (N=5). Data represent means + S.E.M.
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Fig. 2. Effects of inhibitors on active ammonia excretion and transepithelial
potential difference (PDte) across isolated perfused gills
of the shore crab Carcinus maenas. The rate of disappearance of total
ammonia from the internal perfusion medium was measured with symmetrical
NH4Cl concentrations (100 µmol l-1) in the external
and internal baths (A,B,D) or with 200 µmol l-1 NH4Cl
in the internal bath and none initially in the external bath (C). (A)
Symmetrical application of bafilomycin A1 (Baf.) (1 µmol
l-1) followed by basolateral addition of ouabain (Ouab.) (5 mmol
l-1) (N=4). (B) Basolateral addition of colchicine
(Colch.) (0.2 mmol l-1) (N=6). (C) Basolateral application
of colchicine (0.2 mmol l-1) with an initial outwardly directed
NH4+ gradient of 200 µmol l-1
(N=5). (D) Basolateral addition of cytochalasin D (Cytoch.) (5
µmol l-1) (N=5). Data represent means + S.E.M.
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Fig. 3. Effects of microtubule inhibitors on the rate of active ammonia excretion
across isolated perfused gills of the shore crab Carcinus maenas
measured under initial conditions of symmetrical concentrations of
NH4Cl (100 µmol l-1) in the external and internal
baths. (A) Basolateral application of taxol (10 µmol l-1)
(N=6). (B) Basolateral application of thiabendazole (Thia.) (0.2 mmol
l-1) (N=6). Data represent means + S.E.M.
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Fig. 4. Time course of the inward negative uncorrected short-circuit current
(Itot) measured over the split gill half-lamella of
Carcinus maenas during application (bars) of basolateral colchicine
(0.2 mmol l-1) and cytochalasin D (5 µmol l-1). The
amplitudes of the current deflections, which are due to voltage pulses of 1
mV, are inversely proportional to the resistance between the tips of the
voltage electrodes.
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Fig. 6. Electron micrographs of the apical region of posterior gill epithelial
cells of the shore crab Carcinus maenas. AM, apical membrane; Bl,
basal lamina; BM, basolateral membrane; CP, clathrin-coated pit; Cu, cuticle;
Go, Golgi apparatus; M, mitochondria; Mt, microtubules; rER, rough endoplasmic
reticulum; sCu, subcuticular space; V, vesicle. Scale bars, 1 µm (A); 0.5
µm (B,C).
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Fig. 7. Partial amino acid sequence of vesicle-associated membrane protein (VAMP)
identified by PCR in gills of the shore crab Carcinus maenas aligned
with VAMP sequences from fruit fly Drosophila melanogaster (GenBank
Accession No. AAF47529), nematode Caenorhabditis elegans (GenBank
Accession No. AAB61234), seahare Aplysia californica (GenBank
Accession No. U00997), sea urchin Strongylocentrotus purpuratus
(GenBank Accession No. AAB67799) and African frog Xenopus laevis
(GenBank Accession No. P47193).
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Fig. 8. Proposed hypothetical model of active ammonia excretion across the gills of
the shore crab Carcinus maenas. According to this model,
NH4+ is pumped across the basolateral membrane by the
Na+/K+-ATPase (1) or traverses the membrane via
Cs+-sensitive channels (2). Dissociation of cytosolic
NH4+ to H+ and NH3 is accompanied
by diffusion of NH3 into vesicles acidified by a V-type
H+-ATPase (3). The ammonia-loaded vesicles (4) are then moved
via microtubules (5) to the apical membrane, where they fuse with the
external membrane, releasing NH4+ into the subcuticular
space. The NH4+ is then believed to diffuse across the
cuticle via amiloridesensitive structures (6). The possibility of an
additional ammonium transporter, probably in the basolateral membrane (7),
cannot be discounted. Rates of paracellular ammonia diffusion (8) and
non-ionic transcellular diffusion of NH3 (9) are considered to be
low at physiologically meaningful transepithelial ammonia gradients.
Pi, inorganic phosphate.
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© The Company of Biologists Ltd 2002
Imax (maximum short-circuit current) and
Kami (inhibition constant for amiloride) were obtained
from plots for the individual experiments. (C) Mean Gcut
plotted against the concentration of amiloride (in mol l-1) in the
external saline. (D) The Gcut data are shown in a
Hanes-Woolf plot. The mean values of