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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 Weihrauch^1,*, 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

View larger version (15K): [in a new window]   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.

View larger version (16K): [in a new window]   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.

View larger version (15K): [in a new window]   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.

View larger version (14K): [in a new window]   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.

View larger version (23K): [in a new window]   Fig. 5. Dose-dependence of cuticular short-circuit current (Isc) and transcuticular conductance (Gcut) inhibition by amiloride. A clamp voltage of 10 mV with reference to the apical side was maintained to force transcuticular NH4+ fluxes. In four experiments on different isolated cuticles, amiloride was added at increasing concentrations to the external solution. (A) Mean Isc plotted against the concentration of amiloride (in mol l-1) in the external saline. (B) The Isc data are shown in a Hanes-Woolf plot. The mean values of 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 Gmax (maximum transcuticular conductance) and KAmi were obtained from plots for the individual experiments. Values are means ± S.E.M.

View larger version (155K): [in a new window]   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).

View larger version (29K): [in a new window]   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).

View larger version (40K): [in a new window]   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.