Treatment with reagents

Ascidians, Ascidia sydneiensis samea, were collected at Otsuchi Marine Research Center, Ocean Research Institute, the University of Tokyo, Otsuchi, Iwate Prefecture and at Asamushi Marine Biological Station, Tohoku University, Asamushi, Aomori Prefecture, Japan. The ascidians were maintained in an aquarium that contained circulating natural seawater at 20°C. For the experiment, they were immersed individually in 50 ml of filtered seawater with or without several kinds of reagents for 18 to 20 hr at 20°C. The reagents tested were chloride salts (10 mM NaCl, 10 mM or 50 mM KCl, 10 mM LiCl, 5 mM CaCl₂, 10 mM NH₄Cl), ammonium sulfate (5 mM (NH₄)₂SO₄), a proton conductor (1 μM SF6847 (3,5-di-t-butyl-4-hydroxybenzilidenemalononitrile)), ionophores (5 μg/ml nigericin plus 50 mM KCl, 5 μg/ml monensin, 5 μM valinomycin plus 10 mM KCl), macrolide antibiotics (2 μM bafilomycin A₁, 10 μg/ml TJ24373, 10 μg/ml sporeamycin), inhibitors of mitochondrial ATP synthetase (F- ATPase) (5 μM oligomycin, 1 mM NaN₃), an uncoupler (1 mM 2,4- dinitrophenol), and inhibitors of P-type ATPases (1 mM ouabain, 1 mM Na₃VO₄). After the treatment, the tunic was removed and the coelomic fluid was drawn by cardiac puncture into 2 ml of ice cold artificial seawater (ASW) containing 460 mM NaCl, 9 mM KCl, 33 mM Na₂SO₄, 6 mM NaHCO₃ and 5 mM HEPES (N-2-hydroxyethylpiperazine-N′-2-ethanesulfonic acid), pH 7.0, to avoid clotting.

Cell count

An aliquot of 100 μl of coelomic fluid containing coelomic cells suspended in ASW was used to count the number of each type of coelomic cell with a hemocytometer.

Measurement of vanadium content

The vanadium content in the coelomic cells was measured by flameless atomic absorption spectrophotometry. The coelomic fluid was centrifuged at 400 × g for 10 min at 4°C. The resultant pellet was suspended in 10 ml of ASW containing 200 mM sucrose and 20 mM MOPS (3-(N-Morpholino)propanesulfonic acid) -Tris (2-Amino-2- hydroxymethyl-1,3-propanediol) at pH 8.0. Then, the suspension was centrifuged at 300 × g for 10 min at 4°C to remove giant cells having no vanadium. The cells obtained were resuspended in ASW and centrifuged twice at 400 × g for 10 min at 4°C. The pellet was resuspended in 500 μl of ASW, and 100 μl of the suspended solution was used to count the cell number as described above. The remaining cell suspension was diluted appropriately with 0.1 N HNO₃ (super special grade; Wako Pure Chemical Indust. Ltd., Japan), and 10 μl of this solution was loaded onto the flameless atomic absorption spectrophotometer (Seiko Instruments Inc., Nagano, Japan). The absorption line was 318.4 nm.

Measurement of the concentration of ATP

ATP concentrations were measured utilizing bioluminescence as described (Strehler and Totter, 1954). The coelomic fluid was adjusted to pH 7.0 with Tris, and then boiled immediately for 5 min. Next the samples (40 μl) were mixed with 50 μl of 200 mM HEPES-NaOH (pH 7.75), 5 μl of 200 mM MgSO₄, 5 μl of 1 mg/ml luciferase, and 100 μl of 1 mM D-luciferin. After fifteen seconds, photoluminescence was measured for 1 min by an Aloka BLR-101C bioluminescence reader (Aloka CO., LTD., Tokyo, Japan). ATP concentrations were expressed as nmols/mg protein.

Protein assay

Protein concentrations were measured by the method of Bradford (Bradford, 1976) using a Bio-Rad protein assay kit (Nippon Bio-Rad Laboratories, Inc., Tokyo, Japan), with bovine serum albumin as the standard.

Statistical analysis

Data were statistically analyzed by Student's t-test.

Immunohistochemical staining

The specimens were fixed with 100% methanol for 20 min and then 100% ethanol for 20 min at −20°C. Then, the specimens were embedded in a polyester wax and sliced with a microtome at a thickness of 6 μm. After removal of the polyester waxes with 100% ethanol, specimens were treated with the monoclonal antibody S4D5 which interacts specifically with vanadocytes (Uyama et al., 1991). After extensive washing, the immunoreactivity was visualized with a Histofine SAB-PO (M) immunohistochemical staining kit (Nichirei Inc., Tokyo, Japan) according to the manufacture's instructions.

Dissipation of a transmembrane proton gradient (Δ pH)

After treatment with 10 mM NH₄Cl for 20 hr, the size of the population of vanadocytes in coelomic fluid was observed to increase to about three times that of control. No such increase in any of the other cell types occurred (Fig. 1), confirming the results described previously (Hayashi et al., 1996). Treatment with chloride salts (10 mM NaCl, 10 mM or 50 mM KCl, 10 mM LiCl and 5 mM CaCl₂) had no effect. Thus, it is apparent that NH₄⁺ is responsible for the increase in the number of vanadocytes. The fact that treatment with 5 mM (NH₄)₂SO₄ also resulted in an increase in the population of vanadocytes (Fig. 1) supports this.

Fig. 1

Changes in the number of vanadocytes. The ascidian, Ascidia sydneiensis samea, was immersed in seawater with or without reagents for 20 hr, and the number of vanadocytes counted. The number per one ml of coelomic fluid was calculated. Data represent the average of three trials ± S.E. * indicates statistically significant difference from the control (P < 0.01). The number of vanadocytes was increased to about 3 to 5 times that of control by treatment with NH₄Cl, ionophores or the inhibitors of V- and F-ATPases.

NH₄⁺ accumulated in ascidian tissues was observed to abolish the pH gradient in vacuoles of vanadocytes, as described later. Therefore, neutralization of the acidic compartment might cause the increase in the number of vanadocytes. The next experiment was designed to examine whether or not the dissipation of intracellular Δ pH causes an increase in the population of vanadocytes. After treatment with 1 μM SF6847, a kind of proton conductor, the number of vanadocytes increased about four times that of control (Fig. 1). Nigericin and monensin are known to translocate H⁺ into K⁺, and H⁺ into Na⁺, respectively, across the membrane systems (Pressman, 1976). Treatment with 5 μM nigericin plus 50 mM KCl caused an increase in cell number, as did 5 μM monensin (Fig. 1). Treatment with 50 mM KCl alone had no effect. These results clearly indicated that dissipation of intracellular Δ pH awakes an increase in the number of vanadocytes.

A macrolide antibiotic, bafilomycin A₁, a specific inhibitor of V-ATPase (Bowman et al., 1988), increased the number of vanadocytes as reported previously (Hayashi et al., 1996) (Fig. 1). TJ24373 and sporeamycin, macrolide antibiotics but not inhibitors of V-ATPase, had no effect. It is, therefore, apparent that specific inhibition of V-ATPase by bafilomycin A₁ causes an increase in cell number.

Inhibition of ATP synthesis

Ion-pumping ATPases are classified into three groups, V-, F- and P-types (Pedersen and Carafoli, 1987). To examine whether F- and P-ATPases are involved in the increase in vanadocyte numbers, ascidians were treated with some inhibitors of these ATPases. Treatments with 1 mM ouabain and 1 mM Na₃VO₄, specific inhibitors of P-ATPase (Pedersen and Carafoli, 1987), caused no increase in cell number but treatments with 5 μM oligomycin and 1 mM NaN₃, known inhibitors of F-ATPase (Futai and Kanazawa, 1980), increased the number of vanadocytes (Fig. 1).

The main functions of V-ATPase and F-ATPase are to expend and to synthesize ATP, respectively, differing from the function of P-ATPase which is to form a phospho-enzyme intermediate. These results, therefore, suggest that inhibition of ATP synthesis might be involved in the increase in the number of vanadocytes. To examine this possibility, ascidians were treated with uncoupler or potassium ionophore, 2,4- dinitrophenol and valinomycin, known to inhibit ATP synthesis without inhibition of F-ATPases. Consequently, treatment with 1 mM 2,4-dinitrophenol and 5 μM valinomycin plus 10 mM KCl resulted in an increase in the number of vanadocytes (Fig. 1).

The results obtained by a series of the above experiments suggest that a decrease in ATP levels causes the increase in the number of vanadocytes. As shown in Fig. 2, ATP levels in coelomic fluid decreased by treatment with valinomycin, NaN₃ or 2,4-dinitrophenol but not with monensin or bafilomycin A₁.

Fig. 2

Levels of ATP in the coelomic fluid. Levels of ATP in the coelomic fluid were determined after treatment with or without reagents. Treatment with valinomycin, NaN₃ or 2,4-dinitrophenol decreased significantly ATP levels but treatment with monensin or bafilomycin A₁ had no such effect. * indicates statistically significant difference from the control (P < 0.01).

Vanadium contents in vanadocytes

No significant differences were observed in levels of vanadium contained in vanadocytes between, before, and after treatments, as shown in Fig. 3. The vanadium content per vanadocyte was estimated to be 5 to 7 pg.

Fig. 3

Contents of vanadium in vanadocyte. The vanadium content in vanadocytes was determined after treatment with or without reagents. No significant differences were observed between control and treated groups. The vanadium content per vanadocyte was calculated. Data represents the average of three trials ± S.E.

Source of vanadocytes

To identify the source of the vanadocytes that react to increase in their number in coelomic fluid by treatment with the above reagents, several tissues, the branchial sac, the peribranchial epithelium and the connective tissues around the alimentary canal, were stained immunohistologically with a monoclonal antibody, S4D5, specific to vanadocytes. In the non-treated animals, numerous vanadocytes were found in the connective tissues around the alimentary canal, as reported previously (Kaneko et al., 1995), but few vanadocytes were observed in other tissues. After treatment with 10 mM NH₄Cl or 2 μM bafilomycin A₁, however, vanadocytes were observed to decrease in number in the same tissues (Fig. 4). This result revealed clearly that those vanadocytes reacted with the reagents were reserved in the connective tissue around the digestive alimentary canal.

Fig. 4

Immunological detection of vanadocytes in the connective tissues around the alimentary canal. Immunohistological staining with a monoclonal antibody, S4D5, specific to vanadocytes revealed clearly that the number of vanadocytes embedded in the connective tissues decreased after treatment with 10 mM NH₄Cl or 2 μM bafilomycin A₁. No such phenomenon was observed in the other tissues examined. Therefore, the connective tissues around the alimentary canal are the source of these vanadocytes. In photograph A, a sample without treatment, a lot of immunoreactive cells are present. However, few immunoreactive cells appear in photograph B, a sample treated with 10 mM NH₄Cl. Arrowhead indicates the immunoreactive cells colored with brown color, ac, alimentary canal. Scale bar indicates 50 μm.