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Uptake of vanadium by the ascidian Ascidia ceratodes
Comp. Biochem. Physiol. Vol. 99A,No. I/2, pp. 151-158, 1991 Printed in Great Britain
0300-9629/91 $3.00+ 0.00 PergamonPress plc
UPTAKE OF VANADIUM BY THE ASCIDIAN ASCIDIA CERA TODES DANIEL H. ANDERSON,JOHN R. BERG and JAMESH. SWINEHART Department of Chemistry, University of California, Davis, CA 95616, U.S.A. (Received 16 August 1990) Abstraet--l. The incorporation of vanadium from seawater into the plasma and blood cells of the phlebobranch ascidian Ascidia ceratodes has been investigated using the radioisotope 4sV. 2. There are two paths by which vanadium enters the blood cells. One path, I, involves direct uptake from the plasma by the blood cells. 3. The other path, II, results in the long-term incorporation in the blood cells. 4. Possible mechanisms for II are: (a) the slow loss from remote sites into the plasma and then direct incorporation into blood cells or (b) incorporation during blood-cell production and development. 5. Studies of the exchange of 4sV between labelled plasma and unlabelled blood cells show that plasma and plasma simulants with in vitro added vanadium (V) are not good models for the in vivo uptake of vanadium by blood cells from plasma.
INTRODUCTION It has been known for some time that many ascidians have the unique property of accumulating vanadium into their blood cells from seawater (Henze, 1911; Webb, 1939). The ability to accumulate vanadium and the major oxidation state of the vanadium found in vanadium-containing species have been used as a basis for assigning phylogenetic position (Swinehart et al., 1974; Hawkins et al., 1983). Vanadium in seawater is present as vanadium (V), V(V), while that in ascidians is predominantly V(III) and/or V(IV), with V(V) being found at a relatively low concentration in the plasma. Species in the suborder Phlebobranchia have predominantly V(III); in the suborder Aplousobranchia V(IV) and V(III); and in the Stolidobranchia only much lower levels of vanadium, the oxidation state of which is not known (Michibata et aL, 1986). All ascidians examined contain some iron, as well as other transition metals (for review see Biggs and Swinehart, 1976). Most work not involving vanadium has concentrated on the iron content and speciation in the Stolidobranchia. Endean (1955) observed high levels of iron(II) in the blood cells of the stolidobranch Pyura stolonifera. The presence and oxidation state of iron in the blood cells of P. stolonifera (Hawkins et al., 1980) and other species of the Stolidobranchia (Agudelo et al., 1983) have been confirmed. In general, the levels of iron in the stolidobranch ascidians are several orders of magnitude less than that of vanadium in phlebobranch and aplousobranch ascidians. The paths by which vanadium is removed from seawater and enters the blood cells of ascidians are not known. It is established that V(V), generally monitored by the radiotracer ~V, is removed from seawater by a first-order process which does not saturate as the concentrations of V(V) is increased (Goldberg et aL, 1951; Kustin and McLeod, 1977). There is evidence that vanadium accumulates in the branchial sac, mucus associated with the branchial
sac, and the gastrointestinal tract (Bielig et al., 1961a,b; Goldberg et al., 1951) and these areas have been cited as regions of vanadium uptake. The purpose of this work is to define the paths by which vanadium is transferred from seawater to blood cells. Vanadium is monitored as ~ V by examining its loss from seawater and its appearance in the plasma and blood cells as a function of time up to 56 days. Autoradiographic measurements of the blood cells after various incubation times aid in defining the order in which vanadium appears in different types of blood cells. The phlebobranch Ascidia ceratodes (Huntsman), which contains primarily V(III) in its blood cells, is the object of this study. MATERIALS AND METHODS 4sV incorporation experiments Carrier free 48V, as the V(IV) chloride in 1.0 N HC1, was obtained from Amersham Corp. These stock solutions initially contained activities ranging from 0.21 to 1.59 mCi/ ml at concentrations of 0.3-1.0/~g of V/ml. Sufficient ~V was added to filtered seawater obtained from the Bodega Marine Laboratory, University of California, Davis to yield the desired initial activity. The concentration of vanadium in seawater is about 10-~ M. The added ~V represented no more than 0.1% of that value. The seawater was cooled to 12°C (ambient seawater temperature) and aerated for 24 hr to allow for the oxidation of V(IV) to V(V), the oxidation state of vanadium in seawater, and the complete equilibration of V'(V) with the constituents of seawater. In some experiments the seawater was sampled and counted upon addition of the 48V and after 24 hr, prior to addition of the animals. No difference in activity was observed. In a number of series of experiments carried out at different times, 30- to 40-g specimens of A. ceratodes collected in Bodega Harbor, CA and maintained in aquaria were added to each portion of ~V-containing seawater. One or two animals were added to 21 of seawater in 81 cylindrical aquaria, and at any time up to three aquaria were in use. In all experiments the seawater was maintained at 12°Cwith aeration during the course of the experiment, and animals
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DANIEL H . ANDERSON et al.
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Some mixtures were shaken for 20 min, prior to treatment as above. The same general techniques were used to determine the effect of the in vitro addition of V(V) and 48V to plasma and seawater, and 48V to plasma on the distribution of 48V between plasma and cells• In these experiments the 48V, initially present as V(IV), was neutralized and oxidized to V(V) before addition to the plasma or seawater.
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Fig. 1. Ratio of 4SV activity (cpm/mV) in seawater, plasma, and blood cells during uptake by A. ceratodes to that of the initial 48V activity in seawater versus incubation time. Seawater • (two experiments), plasma C) (two experiments), and blood cells • (three experiments). The labels SEAWATER x 100, and PLASMA x 10 mean that the ratios were multiplied by the indicated number prior to graphing. One animal per (3 or • . occupied the same positions in the vessels throughout the experiment. If an animal did not appear to be healthy, as manifested by siphon closure and general appearance, it was discarded. At intervals over periods of up to 56 days the seawater was sampled, and animals were removed and killed to collect their blood• A shallow incision was made through the heart and blood was allowed to drain into a clean dry test tube. Four to five millilitres of blood per animal were obtained• One-millilitre portions of the blood were centrifuged and the plasma decanted from the packed cells. The 48V activity of the cells, plasma and seawater were determined with a Beckman Model 310 gamma ray counter. The activity was corrected for the ambient background levels, and for the 16.1 day half-life of 4sV. In some experiments the animal was dissected and the activities of selected organs were measured• These data combined with the 48V activities of the plasma and blood cells were used to ascertain a minimum percentage of the amount of the activity removed from seawater which was contained in the animal• A blood volume of 10 ml for a 30- to 40-g specimen was used in all calculations. This is 10% more than the maximum volume of blood that can be removed. 48V pulse experiments were carried out by placing five animals in 3 1 of 48V-containing seawater for one day. After this incubation period, three animals were removed from the 48V-containing seawater and placed in a tank containing 3 1 of unlabelled seawater and two animals remained in the original tank. The 48V-labelled seawater in the animals transferred to the unlabeUed tank was not expressed to avoid injury to the animals. The activity of the seawater in both tanks was measured at various times, and 1 and 9 days after the transfer took place the 48V activities of the blood ceils and plasma from one animal from each tank were measured.
48V exchange between plasma and cells Samples of whole blood containing 48V were obtained at various incubation times from animals placed in labelled seawater as described in the previous section. The plasma and cells were separated. Two types of experiments were carried out. One experiment involved the mixing of plasma from l ml of 48V-frce blood with 48V-containing blood cells from 1 ml of 48V-containing blood• The second experiment involved the mixing of 48V-containing plasma from I ml of 48V-containing blood with 48V-free blood cells from 1 ml of blood• The mixtures were gently shaken in an ice bath for 3rain, centrifuged, decanted and the 48V activities of the blood cells and plasma were measured.
Samples of blood from animals incubated with 48Vcontaining seawater for times up to 56 days were fixed with formaldehyde. Two to four drops of 37% formaldehyde were added to 1-ml portions of whole blood and the blood was kept in an ice bath for 20-30 min. Smears of the blood were made on microscope slides and allowed to air dry. Kodak NTB-3 nuclear track emulsion was diluted in a darkroom with an equal volume of water, warmed to 40°C and used to coat the slides by dipping. The emulsion was allowed to air dry, the slides were stored in a light tight box for between 3 and 30 days and developed at 17°C with a Kodak Dektol developer-water mixture (1:2) for 2rain, distilled water for 0.5 rain, Kodak fixer 3 rain and rinse 20 rain. The slides were examined with a microscope under bright field illumination with no stain. Photomicrographs were made with a Zeiss Photoscope II with automatic exposure control and Kodak Ectachrome EPY film. Autoradiography of cell smears made from blood to which 48V was added in vitro was also done. 48V(V) was added to unlabelled whole blood to yield activities similar to those of animals kept in 48V-containing seawater for I-2 days. The blood was kept in an ice bath for 30 min, after which it was fixed and treated as previously described.
RESULTS In vivo plasma and cell incorporation o f 4sV from
seawater Figure 1 shows typical levels o f 48V in seawater, b l o o d cells a n d p l a s m a over a period o f 56 days d u r i n g u p t a k e o f the radioisotope by A. ceratodes. T h e 4sV level at any time is represented by the ratio of the activity in a particular m e d i u m to the activity initially present in seawater. T h e d a t a show t h a t in the plasma a m a x i m u m is reached in approximately 1-2 days a n d in the b l o o d cells in approximately 20 days• The seawater activity initially decreases rapidly, b u t the decrease slows a n d some activity is always f o u n d in the seawater. The results o f a pulse-labelling experiment are s h o w n in Fig. 2. T h e 4sV activities o f the plasmas f r o m animals in the two e n v i r o n m e n t s at 1 a n d 9 days after transfer were the same to within the experimental error f o u n d w h e n o t h e r animals exposed to the same conditions were sampled. After 1 day the activities o f the b l o o d cells f r o m the different environm e n t s were the same (within the n o r m a l variation), b u t at 9 days the activity o f the b l o o d cells from animals in 48V-containing seawater was approximately 4 times t h a t o f cells f r o m animals placed in seawater c o n t a i n i n g n o 48V. Additionally, the 4sV activity in the unlabelled seawater reached a plateau of 140 c p m / m l the first day after the three animals were transferred a n d t h e n slowly declined. T h e level of activity at the plateau, 140 cpm/ml, was considerably higher t h a n the activity o f 32 c p m / m l expected f r o m the dilution of 48V-containing seawater in the branchial sacs o f the ascidians.
Uptake of V by Ascidia ceratodes
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Table 2. In vitro studies: percentage of aV activity exchanged from labelled to unlabelled media 4SV labelled plasma to '~V labelled cells to Time unlabelled cells unlabelled plasma (days) Expt C Expt D Expt C Expt D 1 9.2 32.6 1.5 17.2 23.6 2.5 19.8 11.8 3 13.7 19.8 8 8.1 3.5 11 15.1 2.7 15 7.6 2.0 21 16.0 18.2 3.3 1.4 29 24.5 3.6 30 19.4 2.5 43 17.8 3.6 50 6.6 14.8 56 12.7 2.2
3-
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TIME (days) Fig. 2. Pulse labelling experiment measuring 48V activity (cpm/ml) for five animals incubated in 4sV for 1 day. (a) Three animals placed in unlabelled seawater, plasma • and blood • cells measured 1 and 9 days after transfer. 0a) Two animals remain in ~V seawater, plasma <> and blood cells [] measured at I and 9 days after transfer.
animals dissected after 1, 2 and 3 days o f incubation show the percentage o f the activity removed from seawater that is associated with the digestive tract, the branchial sac and the blood is 6 7 + 5%. These sources represent less than 50% of the total body weight of the animal. The external surface of the test contained a small amount of activity compared to that of the branchial sac and digestive tract.
Exchange o f 48V between cells, blood cells and plasma Table 1 contains typical data showing the a V activity imbalance between the 48V removed from seawater and that found in the blood plasma and cells at various times. The data in experiment A come from a variety o f incubation studies and contain values for the 4sV activity removed from seawater, found in the plasma and cells, and that accounted for in the plasma and cells recorded as a percentage o f the total removed from seawater. At earlier times a smaller percentage of the activity removed from seawater is accounted for in the blood compared to later times. A second experiment, B, generated data on the 48V activities of the plasma and cells over a 10-day incubation period. The data show that the sum o f the activities of the plasma and blood cells is approximately constant during the first 6 days and then the sum increases rapidly due to the rapidly increasing level in the blood cells. The data from three
Table 2 contains data from two sets o f experiments, C and D, showing the distribution after the exchange of 48V between labelled plasma and unlabelled blood cells, and unlabelled plasma and labelled blood cells in which the labellecl blood cells or plasma were taken from animals incubated with ' s V for periods up to 56 days. The plasma and blood cells were mixed for 3 min, a period in which equilibrium was established, separated and the activity of the blood cells and plasma measured. Extending the mixing time to 20 min did not change the amount o f activity exchanged in any experiment. The data show there is no dependence of animal incubation time on the a m o u n t of activity taken up from the labelled plasma by the unlabelled cells. An average o f 18% of the activity available is adsorbed and taken up by the cells. Except for the 50-day time in experiment D, the data do show a clear incubation time dependence in
Table 1.4SVuptake: seawater loss, activity in plasma and cells, and percentage accounted for in plasma and cells Experiment A Experiment B Seawater loss* Seawater loss* Time Plasma$ Cellst Plasmat Cellst (days) (clam x 10-6) %5 (cpm x 10-6) 1 1.66 0.20 0.012 13 6.3 0.70 0.01 2.7 0.15 0.012 6 4.4 1.6 0.42 4 2 7.4 0.65 0.03 2.5 20.6 1.8 0.36 10 3 4.0 0.58 0.13 18 7.9 0.50 0.10 4 8.2 0.30 0.25 6 8.6 0.05 0.70 8 5.9 0.09 2.4 43 8.6 0.03 1.6 10 8.7 0.03 3.0 15 10.2 0.67 4.7 48 21 6.8 0.45 5.9 94 30 5.6 0.22 3.8 71 *Loss from seawater (2 I, initial activity 2000-4000 cpm/ml) per animal, tActivity found per animal (10 ml blood volumeper animal assumed). :[:Percentageloss from seawater accounted for in the blood plasma and cells. CBPA 99/I/2--K
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Table 3. '~V in vitro studies: percentage exchange of 48V from plasma and seawater to unlabelled cells Number of samples 2 5 2 3 2*
Experimental Conditions Time for uptake Added VO](min) (raM) 10 3-15 3-10 3-15 8-15
0 0 0 0.5 0.5
'~V uptake by cells (%) 76_+6 74 _+6 75 _+9 69 _+3 25 _+2
*Seawater, other samples plasma.
the amount of activity released by the labelled cells to the unlabelled plasma. At the shortest time (1 day) 33% of the activity is released, while at the longest time (56 days) only 2% of the activity is released. Table 3 contains data on the distribution of 4sV after exchange between unlabelled blood cells and labelled plasma, plasma containing V(V), and seawater. In these experiments the 48V was added directly to the medium for comparison with the experiments recorded in Table 2 in which the labelled medium was generated by the natural incorporation of the radioisotope. The data show that when the 48V level is the same in both types of labelled plasma, the cells take up more of the radioisotope from the directly labelled plasma and seawater than from the naturally labelled plasma. Within experimental air the presence of V(V) in the directly labelled medium does not affect the uptake of 4sV by the blood cells. Autoradiography of 4SV incorporation into blood cells
Figure 3a shows a fixed blood-cell smear from an animal which contains no 4sV and Fig. 3b shows a smear from an animal incubated with 4sV for 17 days, about the time the 4sv level reaches a maximum in the blood cells (see Fig. 1). Clearly the production of silver granules is more pronounced in some types of cells from the animal incubated with 4sV than from the one not exposed to ~sv. However, some cells from the animal not incubated with 4sV do produce silver granules. It is difficult to determine the specific cell type labelled with silver granules, but the labelled cells are not morula (vanadocytes) or pigment cells. The labelled cells appear to be amoebocytes and/or compartment cells. These types of cells exhibit labelling similar to that in Fig. 3b for incubation times between 8 and 56 days, the longest incubation time. The number of cells and difference in the degree of labelling between cells of 48V incubated and non-incubated animals increases with increasing incubation time. At 56 days some of the morula cells appear to be labelled with silver granules. In vitro experiments in which 48V was added to whole blood for 0.5 hr at levels attain in 1-2 days in the plasma of animals incubated with 4sV showed labelling in the same cell types as observed in in vivo experiments. Many more cells were labelled in this experiment than in a 1-2-days in vivo incubation experiment. DISCUSSION
Possible mechanisms for vanadium incorporation by blood cells
The data presented here suggest that at least two pathways exist for the incorporation of
vanadium into the blood cells of A. ceratodes. One pathway, termed I, involves the rapid uptake of vanadium directly into the plasma and subsequent direct uptake by blood cells. Another pathway II, is consistent with the slow incorporation of vanadium into the blood cells. Several pieces of evidence are consistent with the hypothesis that at least two mechanisms are available for the incorporation of vanadium into the blood cells of A. ceratodes. (1) The data in Fig. 1 on the disappearance of 48V from seawater and its appearance in plasma and blood cells show that the incorporation of 4sV into the plasma reaches a maximum after 1-2 days, then rapidly declines and after 6 days the change in activity is small. The maximum level of 48V in the blood cells is achieved after 20 days. (2) Data in Table 1 on the imbalance between the 4sV removed from seawater and that found in the plasma and cells show that the amount of activity lost from the seawater which is accounted for in the blood cells and plasma increases with increasing time. Additional data show that 65% of the activity lost from seawater at incubation times of 1-3 days can be accounted for in the branchial sac, digestive tract and blood, and since these tissues and the blood represent less than 50% of the total body weight of the animal, a greater percentage must be present in the whole animal. (3) The individual 48V activities of the plasma and blood cells in Table 1 show that for times up to 6 days, but not longer, the total activity in the blood is approximately constant and as the activity of the plasma decreases the activity of the blood cells increases. (4) The data in Fig. 2, showing the results of a pulse experiment in which five animals were incubated for 1 day in 48V-containing seawater and three were transferred to unlabelled seawater while two remained in labelled seawater, indicate that part of the 4sV incorporated into blood cells at short times (1 day after transfer) comes from 48V contained in the plasma, but a major portion of the activity found in the blood cells at long times (9 days after transfer) must come from somewhere other than the plasma. These combined data suggest two uptake processes are operative. The first, I, requires up to 6 days and involves a direct seawater-plasmablood cell transfer, and the second, II, incorporates vanadium unaccounted for in the plasma at short times and the process required about 20 days for the vanadium to fully appear in the blood cells. The data in Fig. 1 and Table 1 are consistent with the results of other workers. Goldberg et al. (1951) studied the removal of 48V from seawater by A. ceratodes. The approximate times for disappearance of 48V were about the same. A study by Bielig and co-workers (1961a) of in vivo uptake of 48V by Phallusia mamillata, a phlebobranch ascidian in the same family as A. ceratodes, demonstrates similar removal from seawater; after 2 days only 2% of the activity removed from the seawater is accounted for
Uptake of V by Ascidia ceratodes
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Fig. 3. Blood cell smears from animals with: (a) no 4sV incubation and (b) incubation with 4SV-labelled seawater for 17 days. Photographic emulsion exposed for 30 days in both cases. Light grey cells are morula and pigment cells, clear cells are compartment or amoebocyte cells. in the blood, and of the activity in the blood 75% is in the plasma with 25% in the blood cells. A similar study (Bielig et al., 1961b) using Ciona intestinalis, a vanadium-containing aplousobranch ascidian not closely related to A. ceratodes, shows a similar uptake from seawater, and
plasma/blood cell ratios of 4sV activities of 82:8 at 6 hr and 5:95 at 5 days. A mechanistic analysis has been made by Kustin and McLeod (1977). A regulated uptake involving chelation or ion exchange at the assimilation site followed by transport was proposed.
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Characteristics o f paths I and H
The scheme below outlines the general characteristics of paths I and II. I
V(V)seawater ----~-V(V)plasma----~Vcells---~V'cells
other location II Table 2 contains data related to the direct incorporation and potential equilibration of 48V from plasma into blood cells by path I. If in vivo generated 48V-labelled plasma from two series of experiments is mixed with unlabelled blood cells for 3 rain, 12 + 5 and 17 + 5% of the 48V originally in the plasma associates with the blood cells and this quantity is independent of the in vivo incubation time of the plasma (1 to 56 days) and mixing times up to 20 rain, the maximum mixing time used. One interpretation of these data is that an equilibrium exists between the plasma and blood cells involving V(V). If such an equilibrium occurs, the ratio of 48V activity between plasma and blood cells should ideally be a constant. In reality, there will be some variation in the ratio due to slight changes in the V(V) levels in the plasma (Anderson and Swinehart, in press) and individual and seasonal variations in the total cell count and distribution of blood cell types (Biggs and Swinehart, 1979). Although some variation occurs in the ratio, it can be concluded that the ratio of V(V) bound to blood cells to that in the plasma is about 15/85 or 0.18 + 0.10 if all of the data are considered together. These data do not distinguish as to which types of cells take up vanadium and it is possible that the V(V) in path I is in equilibrium with a cell type found at low and/or variable levels in the blood. A study of the blood of A. ceratodes has shown that there is a substantial seasonal and individual variation in the distribution of blood cell types, in particular, compartment cells and amoebocytes (Biggs and Swinehart, 1979). Autoradiographic studies of blood cells from short-time in vivo incubation experiments do not show excessive labelling in blood cells due to the low levels of 48V incorporation, but 48V incorporation from in vitro labelled plasma into compartment and amoebocyte cells does occur. These data do not necessarily mean that compartment and/or amoebocyte cells take up vanadium by path I, but long-term incorporation of 48V in in vivo experiments does occur in these types of cells, Fig. 3a and b, and there is some indication that labelling of morula cells occurs at the longest incubation times. There is a substantial difference in the equilibration properties of in vivo and in vitro 48V-labelled plasma and unlabelled blood cells. The data in Table 3 are for experiments in which 48V is added directly to blood plasma or seawater followed by mixing with unlabelled blood cells. In these experiments the distribution of 48V from in vitro labelled plasma is much different than in those described in Table 2. If 48V is added directly to plasma at the activity found after
1-2 days in in vivo incubation experiments, a much greater percentage of the vanadium associates with the blood cells, 70-80% vs 15 + 5% for incubation of in vivo eV-labelled plasma with unlabelled cells. Thus, it can be concluded that in vitro 48V-labelled plasma preparations used to measure the uptake of V(V) by blood cells are not good models for the natural uptake process. The V-51 nuclear magnetic resonance (NMR) signal of V(V) in plasma from A. ceratodes is + 3 ppm from that of comparable concentrations of V(V) in seawater (Anderson and Swinehart, in press). A V-51 NMR signal similar to that in seawater is obtained for V(V) in 0.1 M HEPES buffer at pH 7.5, but not for V(V) bound to transferrin (Butler et al., 1987). Therefore, in vivo incorporated V(V) in the plasma of A. ceratodes is not present in a transferrinlike environment. The difference in the equilibration of V(V) between plasma and blood cells when the e V tracer is introduced by in vivo uptake and the equilibration of V(V) when ¢V is added to seawater suggests that V(V) in plasma is not equivalent to V(V) in seawater. If blood cells labelled with 4gV from in vivo incubation studies are mixed with unlabelled plasma (Table 2), the percentage of the activity originally in the blood cells transferred to the plasma decreased with increasing incubation time of the blood cells. In a typical set of experiments the percentages of the initial activity in the blood cells transferred to the plasma are: 1 day, 33%; 3 days, 20% and 8-20 days, 3 + 1%. The larger values in the 1-3-day range are not due to the high levels of 4gV activity in the plasma during this time period. Therefore, it can be concluded that the small amount of activity in the blood cells during the l-3-day measurements is in part easily lost to the plasma, while the larger amount of activity incorporated in the 8-56day time period is only slightly available for exchange into the plasma. Thus, there is a slow, irreversible incorporation of V(V) into the blood cells even at short incubation times, but there is always some V(V) associated with the blood cells that is available for equilibration with the plasma. The percentage of 48V available for this equilibration, which may involve desorption, decreases with increasing time. A question arises as to how the V(V) enters the blood cells. A portion of the V(V) incorporated enters the blood cells directly from the plasma, path I. This path may have some of the characteristics of the direct uptake process studied by Kustin and coworkers (Dingley et al., 1981) in which V(V) and 48V-containing buffers were mixed with unlabelled blood cells. The characteristics found for this process were rapid uptake of 48V with half-lives of less than a minute, a large ratio for 48V in blood cells to that in plasma, and V(IV) formation and subsequent disappearance as measured by the appearance of an electron spin resonance (ESR) signal. The measurement of V(IV) ESR signals in blood cells taken directly from A. ceratodes (Frank et al., 1986) may be a manifestation of this path. However, as seen from data previously presented, uptake of V(V), as measured by 48V, from artificial mixtures representing plasma into blood cells can have very different
Uptake of V by Ascidia ceratodes characteristics from that using plasma containing in vivo incorporated V(V). The long-term path, II, involves the transfer of V(V), measured as 48V, to some site or sites in the animal, followed by long-term incorporation into the blood cells. Our data show that the branchial sac, the digestive tract, and associated tissues contain at least 50% of the activity removed during 1-3-day incubation with 48V. Goldberg et al. (19 51) monitored the uptake of 4aV by A. ceratodes and C. intestinalis by following the disappearance of the isotope from seawater and the incorporation of 4sV into various types of tissue using autoradiographic techniques. The approximate time for the disappearance of 4aV from seawater recorded in their work is in agreement with the data in Fig. 1. Their autoradiographic studies of A. ceratodes at a 15-day incubation time showed no 4sV in muscles of the mantle (part of the branchial sac), very little in the mantle and test, some in the branchial sac, and high levels in the mucus, gut wall and ovary. This is similar to our findings t'or dissected branchial sacs and digestive tracts. It is suggested that the 48V detected in the branchial sac comes in part from blood found in blood vessels of this tissue. In vivo uptake of 48V by the closely related ascidian Phallusia mamillata (Bielig et al., 1961a) occurs only at the branchial sac, while uptake by C. intestinalis, more distantly related to A. ceratodes, is found at the branchial sac and gastrointestinal tract (Bielig et al., 1961b) and gut wall (Goldberg et al., 1951). Therefore, a great deal of the 48V is incorporated in other tissue and does not enter blood cells directly by path I. However, a great deal of this 4sV does ultimately enter the blood cells, but just how it enters is not clear. Several possibilities exist. One alternative is that of path IIa in which 4sV enters the blood cells by re-entering the plasma by a circuitous route and then is taken up directly into the blood cells, a long-term path I. Another alternative, IIb is uptake through the production and development of blood cells. Three pieces of circumstantial evidence suggest this pathway. (1) The time taken for the maximum 4sV level to be reached in the blood cells, 20 days, corresponds to the time measured for cell development in Styela clara (Ermak, 1975). (2) In vivo experiments in which A. ceratodes is incubated with 4sV for varying lengths of time and autoradiographic studies of the resulting blood cells show incorporation of 48V in compartment and/or amoebocyte cells but not morula (vanadocyte) or pigment cells, although at the longest incubation time, 56 days, some morula cells appear to be labelled. In discussions of the development of blood cells it has been suggested, but highly debated, that compartment and amoebocyte cells are precursors to morula cells (Goodbody, 1974; Wright, 1981). (3) Much of the 4sV taken up from seawater at long times is localized in the gut (Goldberg et al., 1951) where haematopoietic tissue is found in many ascidians (Wright, 1981). While these pieces of evidence are not conclusive and do not eliminate reincorporation into plasma and then directly into blood cells, they are suggestive of path II incorporation of vanadium through the natural development of blood cells. The general conclusions reached from this work are:
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(1) There are two paths by which vanadium is incorporated into the blood cells of the phlebobranch A. ceratodes, a predominantly V(III)-containing ascidian. (2) One path, I, involves a rapid equilibration of V(V) between the plasma and blood cells, followed by a slow irreversible incorporation into the blood cells. (3) Another path, II, involves the accumulation of vanadium in a tissue and a slow incorporation into the blood cells by a mechanism, which may involve (a) the slow loss from remote sites into the plasma and direct incorporation into blood cells or (b) the production and development of blood cells. (4) Vanadium (V) added by in vitro methods to plasma or plasma simulants is not a good model for in vivo V(V)-labelled plasma-blood cell interactions. Acknowledgements--This work was supported by the Committee on Research, University of California, Davis, in the form of Faculty Research grants and Intercampus Travel grants to the Bodega Marine Laboratory of the University of California. REFERENCES
Agudelo M. I., Kustin K., McLeod G. C., Robinson W. E. and Wang R. T. (1983) Iron accumulation in tunicate blood cells. I. Distribution and oxidation state of iron in blood of Boltenia ovifera, Styela clara, and Molgula manhattensis. Biol. Bull. 165, 100-109. Anderson D. H. and Swinehart J. H. The distribution of vanadium and sulfur in the blood cells and the nature of vanadium in the plasma of the ascidian Ascidia ceratodes. Comp. Biochem. Physiol. (in press). Bielig H. J., Jost E., Pfleger K,, Rummel W, and Seifent E. (1961a) Aufnahme und Verteilung yon Vanadin bei der Tunicate Phallusia mamillata Cuvier. Hoppe-Seyler's Z. Physiol. Chem. 325, 122-131. Bielig H. J., Plteger K., Rummel W. and Seifen E. (1961b) Aufnahme und Verteilung von Vanadin bei der Tunicate Ciona intestinals, L. Hoppe-Seyler's Z. Physiol. Chem. 326, 240-258. Biggs W. R. and Swinehart J. H. (1976) Vanadium in selected biological systems. In Metals Ions in Biological Systems (Edited by Siegel H.), pp. 141-195. M. Dekker Inc., New York. Biggs W. R. and Swinehart J. H. (1979) Studies of the blood of Ascidia ceratodes. Experientia 35, 1047-1048. Butler A., Danzitz M. J. and Eckert H. (1987) V-51 NMR as a probe of metal ion binding in metalloproteins. J. Am. Chem. Soc. 109, 1864-1865. Dingley A. L., Kustin K., Macara I. G. and McLeod G. C. (1981) Accumulation of vanadium by tunicate blood cells occurs via a specific anion transport system. Biochim. Biophys. Acta 649, 493-502. Endean R. (1955) Studies of the blood and test of some Australian ascidians. I. The blood of Pyura stolonifera. Austrl. J. Mar. Freshwater Res. 6, 35-59. Ermak T. H. (1975) An autoradiographic demonstration of blood cell renewal in Styela claava (Urochordata: Ascidiacea). Experientia 31, 837-839. Frank P., Carlson R. M. K. and Hodgson K. O. (1986) Vanadyl ion epr as a noninvasive probe of pH in intact vanadocytes from Ascidia ceratodes. Inorg. Chem. 25, 470-478. Goldberg E. D., McBlair W. and Taylor K, M. (1951) The uptake of vanadium by tunicates. Biol. Bull. 191, 84-94. Goodbody I. (1974) The physiology of ascidians. In Advances in Marine Biology (Edited by Russel F. S. and Yonge M.), Vol. 12, pp. 1-149. Academic Press, New York.
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Hawkins C. J., Kott P., Parry D. L. and Swinehart J. H. (1983) Vanadium content and oxidation state as related to ascidian phylogeny. Comp. Biochem. Physiol. 76B, 555-558. Hawkins C. J., Merefield P. M., Parry D. L., Biggs W. R. and' Swinehart J. H. (1980) Comparative study of the blood plasma of the ascidians Pyura stolonifera and Ascidia ceratodes. Biol. Bull. 159, 656-668. Henze M. (1911) Die Vanadiumverbindung der Blutkorperchen. Hoppe-Seyler's Z. Physiol. Chem. 72, 494-50 I. Kustin K. and McLeod G. C. (1977) Interactions between metal ions and living organisms in sea water. In Inorganic Biochemistry II, pp. 1-34. Springer, Berlin.
Michibata H., Terada T., Anada N., Yamakawa K. and Numakunai T. (1986) The accumulation and distribution of vanadium, iron, and manganese in some solitary ascidians. Biol. Bull. 171, 672-681. Swinehart J. H., Biggs W. R., Halko D. J. and Schroeder N. C. (1974) The vanadium and selected metal content of some ascidians. Biol. Bull. 146, 302-312. Webb D. A. (1939) Studies on the ultimate composition of biological material. J, Esp. Biol. 16, 499-523. Wright R. K. (1981) Urochordates. In Invertebrate Blood Cells, Arthropods to Urochordates, Invertebrates and Vertebrates Compared (Edited by Ratcliffe N. A. and Rowley A. F.), Vol. 2, pp. 565-626. Academic Press, London.
0300-9629/91 $3.00+ 0.00 PergamonPress plc
UPTAKE OF VANADIUM BY THE ASCIDIAN ASCIDIA CERA TODES DANIEL H. ANDERSON,JOHN R. BERG and JAMESH. SWINEHART Department of Chemistry, University of California, Davis, CA 95616, U.S.A. (Received 16 August 1990) Abstraet--l. The incorporation of vanadium from seawater into the plasma and blood cells of the phlebobranch ascidian Ascidia ceratodes has been investigated using the radioisotope 4sV. 2. There are two paths by which vanadium enters the blood cells. One path, I, involves direct uptake from the plasma by the blood cells. 3. The other path, II, results in the long-term incorporation in the blood cells. 4. Possible mechanisms for II are: (a) the slow loss from remote sites into the plasma and then direct incorporation into blood cells or (b) incorporation during blood-cell production and development. 5. Studies of the exchange of 4sV between labelled plasma and unlabelled blood cells show that plasma and plasma simulants with in vitro added vanadium (V) are not good models for the in vivo uptake of vanadium by blood cells from plasma.
INTRODUCTION It has been known for some time that many ascidians have the unique property of accumulating vanadium into their blood cells from seawater (Henze, 1911; Webb, 1939). The ability to accumulate vanadium and the major oxidation state of the vanadium found in vanadium-containing species have been used as a basis for assigning phylogenetic position (Swinehart et al., 1974; Hawkins et al., 1983). Vanadium in seawater is present as vanadium (V), V(V), while that in ascidians is predominantly V(III) and/or V(IV), with V(V) being found at a relatively low concentration in the plasma. Species in the suborder Phlebobranchia have predominantly V(III); in the suborder Aplousobranchia V(IV) and V(III); and in the Stolidobranchia only much lower levels of vanadium, the oxidation state of which is not known (Michibata et aL, 1986). All ascidians examined contain some iron, as well as other transition metals (for review see Biggs and Swinehart, 1976). Most work not involving vanadium has concentrated on the iron content and speciation in the Stolidobranchia. Endean (1955) observed high levels of iron(II) in the blood cells of the stolidobranch Pyura stolonifera. The presence and oxidation state of iron in the blood cells of P. stolonifera (Hawkins et al., 1980) and other species of the Stolidobranchia (Agudelo et al., 1983) have been confirmed. In general, the levels of iron in the stolidobranch ascidians are several orders of magnitude less than that of vanadium in phlebobranch and aplousobranch ascidians. The paths by which vanadium is removed from seawater and enters the blood cells of ascidians are not known. It is established that V(V), generally monitored by the radiotracer ~V, is removed from seawater by a first-order process which does not saturate as the concentrations of V(V) is increased (Goldberg et aL, 1951; Kustin and McLeod, 1977). There is evidence that vanadium accumulates in the branchial sac, mucus associated with the branchial
sac, and the gastrointestinal tract (Bielig et al., 1961a,b; Goldberg et al., 1951) and these areas have been cited as regions of vanadium uptake. The purpose of this work is to define the paths by which vanadium is transferred from seawater to blood cells. Vanadium is monitored as ~ V by examining its loss from seawater and its appearance in the plasma and blood cells as a function of time up to 56 days. Autoradiographic measurements of the blood cells after various incubation times aid in defining the order in which vanadium appears in different types of blood cells. The phlebobranch Ascidia ceratodes (Huntsman), which contains primarily V(III) in its blood cells, is the object of this study. MATERIALS AND METHODS 4sV incorporation experiments Carrier free 48V, as the V(IV) chloride in 1.0 N HC1, was obtained from Amersham Corp. These stock solutions initially contained activities ranging from 0.21 to 1.59 mCi/ ml at concentrations of 0.3-1.0/~g of V/ml. Sufficient ~V was added to filtered seawater obtained from the Bodega Marine Laboratory, University of California, Davis to yield the desired initial activity. The concentration of vanadium in seawater is about 10-~ M. The added ~V represented no more than 0.1% of that value. The seawater was cooled to 12°C (ambient seawater temperature) and aerated for 24 hr to allow for the oxidation of V(IV) to V(V), the oxidation state of vanadium in seawater, and the complete equilibration of V'(V) with the constituents of seawater. In some experiments the seawater was sampled and counted upon addition of the 48V and after 24 hr, prior to addition of the animals. No difference in activity was observed. In a number of series of experiments carried out at different times, 30- to 40-g specimens of A. ceratodes collected in Bodega Harbor, CA and maintained in aquaria were added to each portion of ~V-containing seawater. One or two animals were added to 21 of seawater in 81 cylindrical aquaria, and at any time up to three aquaria were in use. In all experiments the seawater was maintained at 12°Cwith aeration during the course of the experiment, and animals
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DANIEL H . ANDERSON et al.
152
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Some mixtures were shaken for 20 min, prior to treatment as above. The same general techniques were used to determine the effect of the in vitro addition of V(V) and 48V to plasma and seawater, and 48V to plasma on the distribution of 48V between plasma and cells• In these experiments the 48V, initially present as V(IV), was neutralized and oxidized to V(V) before addition to the plasma or seawater.
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Fig. 1. Ratio of 4SV activity (cpm/mV) in seawater, plasma, and blood cells during uptake by A. ceratodes to that of the initial 48V activity in seawater versus incubation time. Seawater • (two experiments), plasma C) (two experiments), and blood cells • (three experiments). The labels SEAWATER x 100, and PLASMA x 10 mean that the ratios were multiplied by the indicated number prior to graphing. One animal per (3 or • . occupied the same positions in the vessels throughout the experiment. If an animal did not appear to be healthy, as manifested by siphon closure and general appearance, it was discarded. At intervals over periods of up to 56 days the seawater was sampled, and animals were removed and killed to collect their blood• A shallow incision was made through the heart and blood was allowed to drain into a clean dry test tube. Four to five millilitres of blood per animal were obtained• One-millilitre portions of the blood were centrifuged and the plasma decanted from the packed cells. The 48V activity of the cells, plasma and seawater were determined with a Beckman Model 310 gamma ray counter. The activity was corrected for the ambient background levels, and for the 16.1 day half-life of 4sV. In some experiments the animal was dissected and the activities of selected organs were measured• These data combined with the 48V activities of the plasma and blood cells were used to ascertain a minimum percentage of the amount of the activity removed from seawater which was contained in the animal• A blood volume of 10 ml for a 30- to 40-g specimen was used in all calculations. This is 10% more than the maximum volume of blood that can be removed. 48V pulse experiments were carried out by placing five animals in 3 1 of 48V-containing seawater for one day. After this incubation period, three animals were removed from the 48V-containing seawater and placed in a tank containing 3 1 of unlabelled seawater and two animals remained in the original tank. The 48V-labelled seawater in the animals transferred to the unlabeUed tank was not expressed to avoid injury to the animals. The activity of the seawater in both tanks was measured at various times, and 1 and 9 days after the transfer took place the 48V activities of the blood ceils and plasma from one animal from each tank were measured.
48V exchange between plasma and cells Samples of whole blood containing 48V were obtained at various incubation times from animals placed in labelled seawater as described in the previous section. The plasma and cells were separated. Two types of experiments were carried out. One experiment involved the mixing of plasma from l ml of 48V-frce blood with 48V-containing blood cells from 1 ml of 48V-containing blood• The second experiment involved the mixing of 48V-containing plasma from I ml of 48V-containing blood with 48V-free blood cells from 1 ml of blood• The mixtures were gently shaken in an ice bath for 3rain, centrifuged, decanted and the 48V activities of the blood cells and plasma were measured.
Samples of blood from animals incubated with 48Vcontaining seawater for times up to 56 days were fixed with formaldehyde. Two to four drops of 37% formaldehyde were added to 1-ml portions of whole blood and the blood was kept in an ice bath for 20-30 min. Smears of the blood were made on microscope slides and allowed to air dry. Kodak NTB-3 nuclear track emulsion was diluted in a darkroom with an equal volume of water, warmed to 40°C and used to coat the slides by dipping. The emulsion was allowed to air dry, the slides were stored in a light tight box for between 3 and 30 days and developed at 17°C with a Kodak Dektol developer-water mixture (1:2) for 2rain, distilled water for 0.5 rain, Kodak fixer 3 rain and rinse 20 rain. The slides were examined with a microscope under bright field illumination with no stain. Photomicrographs were made with a Zeiss Photoscope II with automatic exposure control and Kodak Ectachrome EPY film. Autoradiography of cell smears made from blood to which 48V was added in vitro was also done. 48V(V) was added to unlabelled whole blood to yield activities similar to those of animals kept in 48V-containing seawater for I-2 days. The blood was kept in an ice bath for 30 min, after which it was fixed and treated as previously described.
RESULTS In vivo plasma and cell incorporation o f 4sV from
seawater Figure 1 shows typical levels o f 48V in seawater, b l o o d cells a n d p l a s m a over a period o f 56 days d u r i n g u p t a k e o f the radioisotope by A. ceratodes. T h e 4sV level at any time is represented by the ratio of the activity in a particular m e d i u m to the activity initially present in seawater. T h e d a t a show t h a t in the plasma a m a x i m u m is reached in approximately 1-2 days a n d in the b l o o d cells in approximately 20 days• The seawater activity initially decreases rapidly, b u t the decrease slows a n d some activity is always f o u n d in the seawater. The results o f a pulse-labelling experiment are s h o w n in Fig. 2. T h e 4sV activities o f the plasmas f r o m animals in the two e n v i r o n m e n t s at 1 a n d 9 days after transfer were the same to within the experimental error f o u n d w h e n o t h e r animals exposed to the same conditions were sampled. After 1 day the activities o f the b l o o d cells f r o m the different environm e n t s were the same (within the n o r m a l variation), b u t at 9 days the activity o f the b l o o d cells from animals in 48V-containing seawater was approximately 4 times t h a t o f cells f r o m animals placed in seawater c o n t a i n i n g n o 48V. Additionally, the 4sV activity in the unlabelled seawater reached a plateau of 140 c p m / m l the first day after the three animals were transferred a n d t h e n slowly declined. T h e level of activity at the plateau, 140 cpm/ml, was considerably higher t h a n the activity o f 32 c p m / m l expected f r o m the dilution of 48V-containing seawater in the branchial sacs o f the ascidians.
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Table 2. In vitro studies: percentage of aV activity exchanged from labelled to unlabelled media 4SV labelled plasma to '~V labelled cells to Time unlabelled cells unlabelled plasma (days) Expt C Expt D Expt C Expt D 1 9.2 32.6 1.5 17.2 23.6 2.5 19.8 11.8 3 13.7 19.8 8 8.1 3.5 11 15.1 2.7 15 7.6 2.0 21 16.0 18.2 3.3 1.4 29 24.5 3.6 30 19.4 2.5 43 17.8 3.6 50 6.6 14.8 56 12.7 2.2
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TIME (days) Fig. 2. Pulse labelling experiment measuring 48V activity (cpm/ml) for five animals incubated in 4sV for 1 day. (a) Three animals placed in unlabelled seawater, plasma • and blood • cells measured 1 and 9 days after transfer. 0a) Two animals remain in ~V seawater, plasma <> and blood cells [] measured at I and 9 days after transfer.
animals dissected after 1, 2 and 3 days o f incubation show the percentage o f the activity removed from seawater that is associated with the digestive tract, the branchial sac and the blood is 6 7 + 5%. These sources represent less than 50% of the total body weight of the animal. The external surface of the test contained a small amount of activity compared to that of the branchial sac and digestive tract.
Exchange o f 48V between cells, blood cells and plasma Table 1 contains typical data showing the a V activity imbalance between the 48V removed from seawater and that found in the blood plasma and cells at various times. The data in experiment A come from a variety o f incubation studies and contain values for the 4sV activity removed from seawater, found in the plasma and cells, and that accounted for in the plasma and cells recorded as a percentage o f the total removed from seawater. At earlier times a smaller percentage of the activity removed from seawater is accounted for in the blood compared to later times. A second experiment, B, generated data on the 48V activities of the plasma and cells over a 10-day incubation period. The data show that the sum o f the activities of the plasma and blood cells is approximately constant during the first 6 days and then the sum increases rapidly due to the rapidly increasing level in the blood cells. The data from three
Table 2 contains data from two sets o f experiments, C and D, showing the distribution after the exchange of 48V between labelled plasma and unlabelled blood cells, and unlabelled plasma and labelled blood cells in which the labellecl blood cells or plasma were taken from animals incubated with ' s V for periods up to 56 days. The plasma and blood cells were mixed for 3 min, a period in which equilibrium was established, separated and the activity of the blood cells and plasma measured. Extending the mixing time to 20 min did not change the amount o f activity exchanged in any experiment. The data show there is no dependence of animal incubation time on the a m o u n t of activity taken up from the labelled plasma by the unlabelled cells. An average o f 18% of the activity available is adsorbed and taken up by the cells. Except for the 50-day time in experiment D, the data do show a clear incubation time dependence in
Table 1.4SVuptake: seawater loss, activity in plasma and cells, and percentage accounted for in plasma and cells Experiment A Experiment B Seawater loss* Seawater loss* Time Plasma$ Cellst Plasmat Cellst (days) (clam x 10-6) %5 (cpm x 10-6) 1 1.66 0.20 0.012 13 6.3 0.70 0.01 2.7 0.15 0.012 6 4.4 1.6 0.42 4 2 7.4 0.65 0.03 2.5 20.6 1.8 0.36 10 3 4.0 0.58 0.13 18 7.9 0.50 0.10 4 8.2 0.30 0.25 6 8.6 0.05 0.70 8 5.9 0.09 2.4 43 8.6 0.03 1.6 10 8.7 0.03 3.0 15 10.2 0.67 4.7 48 21 6.8 0.45 5.9 94 30 5.6 0.22 3.8 71 *Loss from seawater (2 I, initial activity 2000-4000 cpm/ml) per animal, tActivity found per animal (10 ml blood volumeper animal assumed). :[:Percentageloss from seawater accounted for in the blood plasma and cells. CBPA 99/I/2--K
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Table 3. '~V in vitro studies: percentage exchange of 48V from plasma and seawater to unlabelled cells Number of samples 2 5 2 3 2*
Experimental Conditions Time for uptake Added VO](min) (raM) 10 3-15 3-10 3-15 8-15
0 0 0 0.5 0.5
'~V uptake by cells (%) 76_+6 74 _+6 75 _+9 69 _+3 25 _+2
*Seawater, other samples plasma.
the amount of activity released by the labelled cells to the unlabelled plasma. At the shortest time (1 day) 33% of the activity is released, while at the longest time (56 days) only 2% of the activity is released. Table 3 contains data on the distribution of 4sV after exchange between unlabelled blood cells and labelled plasma, plasma containing V(V), and seawater. In these experiments the 48V was added directly to the medium for comparison with the experiments recorded in Table 2 in which the labelled medium was generated by the natural incorporation of the radioisotope. The data show that when the 48V level is the same in both types of labelled plasma, the cells take up more of the radioisotope from the directly labelled plasma and seawater than from the naturally labelled plasma. Within experimental air the presence of V(V) in the directly labelled medium does not affect the uptake of 4sV by the blood cells. Autoradiography of 4SV incorporation into blood cells
Figure 3a shows a fixed blood-cell smear from an animal which contains no 4sV and Fig. 3b shows a smear from an animal incubated with 4sV for 17 days, about the time the 4sv level reaches a maximum in the blood cells (see Fig. 1). Clearly the production of silver granules is more pronounced in some types of cells from the animal incubated with 4sV than from the one not exposed to ~sv. However, some cells from the animal not incubated with 4sV do produce silver granules. It is difficult to determine the specific cell type labelled with silver granules, but the labelled cells are not morula (vanadocytes) or pigment cells. The labelled cells appear to be amoebocytes and/or compartment cells. These types of cells exhibit labelling similar to that in Fig. 3b for incubation times between 8 and 56 days, the longest incubation time. The number of cells and difference in the degree of labelling between cells of 48V incubated and non-incubated animals increases with increasing incubation time. At 56 days some of the morula cells appear to be labelled with silver granules. In vitro experiments in which 48V was added to whole blood for 0.5 hr at levels attain in 1-2 days in the plasma of animals incubated with 4sV showed labelling in the same cell types as observed in in vivo experiments. Many more cells were labelled in this experiment than in a 1-2-days in vivo incubation experiment. DISCUSSION
Possible mechanisms for vanadium incorporation by blood cells
The data presented here suggest that at least two pathways exist for the incorporation of
vanadium into the blood cells of A. ceratodes. One pathway, termed I, involves the rapid uptake of vanadium directly into the plasma and subsequent direct uptake by blood cells. Another pathway II, is consistent with the slow incorporation of vanadium into the blood cells. Several pieces of evidence are consistent with the hypothesis that at least two mechanisms are available for the incorporation of vanadium into the blood cells of A. ceratodes. (1) The data in Fig. 1 on the disappearance of 48V from seawater and its appearance in plasma and blood cells show that the incorporation of 4sV into the plasma reaches a maximum after 1-2 days, then rapidly declines and after 6 days the change in activity is small. The maximum level of 48V in the blood cells is achieved after 20 days. (2) Data in Table 1 on the imbalance between the 4sV removed from seawater and that found in the plasma and cells show that the amount of activity lost from the seawater which is accounted for in the blood cells and plasma increases with increasing time. Additional data show that 65% of the activity lost from seawater at incubation times of 1-3 days can be accounted for in the branchial sac, digestive tract and blood, and since these tissues and the blood represent less than 50% of the total body weight of the animal, a greater percentage must be present in the whole animal. (3) The individual 48V activities of the plasma and blood cells in Table 1 show that for times up to 6 days, but not longer, the total activity in the blood is approximately constant and as the activity of the plasma decreases the activity of the blood cells increases. (4) The data in Fig. 2, showing the results of a pulse experiment in which five animals were incubated for 1 day in 48V-containing seawater and three were transferred to unlabelled seawater while two remained in labelled seawater, indicate that part of the 4sV incorporated into blood cells at short times (1 day after transfer) comes from 48V contained in the plasma, but a major portion of the activity found in the blood cells at long times (9 days after transfer) must come from somewhere other than the plasma. These combined data suggest two uptake processes are operative. The first, I, requires up to 6 days and involves a direct seawater-plasmablood cell transfer, and the second, II, incorporates vanadium unaccounted for in the plasma at short times and the process required about 20 days for the vanadium to fully appear in the blood cells. The data in Fig. 1 and Table 1 are consistent with the results of other workers. Goldberg et al. (1951) studied the removal of 48V from seawater by A. ceratodes. The approximate times for disappearance of 48V were about the same. A study by Bielig and co-workers (1961a) of in vivo uptake of 48V by Phallusia mamillata, a phlebobranch ascidian in the same family as A. ceratodes, demonstrates similar removal from seawater; after 2 days only 2% of the activity removed from the seawater is accounted for
Uptake of V by Ascidia ceratodes
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Fig. 3. Blood cell smears from animals with: (a) no 4sV incubation and (b) incubation with 4SV-labelled seawater for 17 days. Photographic emulsion exposed for 30 days in both cases. Light grey cells are morula and pigment cells, clear cells are compartment or amoebocyte cells. in the blood, and of the activity in the blood 75% is in the plasma with 25% in the blood cells. A similar study (Bielig et al., 1961b) using Ciona intestinalis, a vanadium-containing aplousobranch ascidian not closely related to A. ceratodes, shows a similar uptake from seawater, and
plasma/blood cell ratios of 4sV activities of 82:8 at 6 hr and 5:95 at 5 days. A mechanistic analysis has been made by Kustin and McLeod (1977). A regulated uptake involving chelation or ion exchange at the assimilation site followed by transport was proposed.
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Characteristics o f paths I and H
The scheme below outlines the general characteristics of paths I and II. I
V(V)seawater ----~-V(V)plasma----~Vcells---~V'cells
other location II Table 2 contains data related to the direct incorporation and potential equilibration of 48V from plasma into blood cells by path I. If in vivo generated 48V-labelled plasma from two series of experiments is mixed with unlabelled blood cells for 3 rain, 12 + 5 and 17 + 5% of the 48V originally in the plasma associates with the blood cells and this quantity is independent of the in vivo incubation time of the plasma (1 to 56 days) and mixing times up to 20 rain, the maximum mixing time used. One interpretation of these data is that an equilibrium exists between the plasma and blood cells involving V(V). If such an equilibrium occurs, the ratio of 48V activity between plasma and blood cells should ideally be a constant. In reality, there will be some variation in the ratio due to slight changes in the V(V) levels in the plasma (Anderson and Swinehart, in press) and individual and seasonal variations in the total cell count and distribution of blood cell types (Biggs and Swinehart, 1979). Although some variation occurs in the ratio, it can be concluded that the ratio of V(V) bound to blood cells to that in the plasma is about 15/85 or 0.18 + 0.10 if all of the data are considered together. These data do not distinguish as to which types of cells take up vanadium and it is possible that the V(V) in path I is in equilibrium with a cell type found at low and/or variable levels in the blood. A study of the blood of A. ceratodes has shown that there is a substantial seasonal and individual variation in the distribution of blood cell types, in particular, compartment cells and amoebocytes (Biggs and Swinehart, 1979). Autoradiographic studies of blood cells from short-time in vivo incubation experiments do not show excessive labelling in blood cells due to the low levels of 48V incorporation, but 48V incorporation from in vitro labelled plasma into compartment and amoebocyte cells does occur. These data do not necessarily mean that compartment and/or amoebocyte cells take up vanadium by path I, but long-term incorporation of 48V in in vivo experiments does occur in these types of cells, Fig. 3a and b, and there is some indication that labelling of morula cells occurs at the longest incubation times. There is a substantial difference in the equilibration properties of in vivo and in vitro 48V-labelled plasma and unlabelled blood cells. The data in Table 3 are for experiments in which 48V is added directly to blood plasma or seawater followed by mixing with unlabelled blood cells. In these experiments the distribution of 48V from in vitro labelled plasma is much different than in those described in Table 2. If 48V is added directly to plasma at the activity found after
1-2 days in in vivo incubation experiments, a much greater percentage of the vanadium associates with the blood cells, 70-80% vs 15 + 5% for incubation of in vivo eV-labelled plasma with unlabelled cells. Thus, it can be concluded that in vitro 48V-labelled plasma preparations used to measure the uptake of V(V) by blood cells are not good models for the natural uptake process. The V-51 nuclear magnetic resonance (NMR) signal of V(V) in plasma from A. ceratodes is + 3 ppm from that of comparable concentrations of V(V) in seawater (Anderson and Swinehart, in press). A V-51 NMR signal similar to that in seawater is obtained for V(V) in 0.1 M HEPES buffer at pH 7.5, but not for V(V) bound to transferrin (Butler et al., 1987). Therefore, in vivo incorporated V(V) in the plasma of A. ceratodes is not present in a transferrinlike environment. The difference in the equilibration of V(V) between plasma and blood cells when the e V tracer is introduced by in vivo uptake and the equilibration of V(V) when ¢V is added to seawater suggests that V(V) in plasma is not equivalent to V(V) in seawater. If blood cells labelled with 4gV from in vivo incubation studies are mixed with unlabelled plasma (Table 2), the percentage of the activity originally in the blood cells transferred to the plasma decreased with increasing incubation time of the blood cells. In a typical set of experiments the percentages of the initial activity in the blood cells transferred to the plasma are: 1 day, 33%; 3 days, 20% and 8-20 days, 3 + 1%. The larger values in the 1-3-day range are not due to the high levels of 4gV activity in the plasma during this time period. Therefore, it can be concluded that the small amount of activity in the blood cells during the l-3-day measurements is in part easily lost to the plasma, while the larger amount of activity incorporated in the 8-56day time period is only slightly available for exchange into the plasma. Thus, there is a slow, irreversible incorporation of V(V) into the blood cells even at short incubation times, but there is always some V(V) associated with the blood cells that is available for equilibration with the plasma. The percentage of 48V available for this equilibration, which may involve desorption, decreases with increasing time. A question arises as to how the V(V) enters the blood cells. A portion of the V(V) incorporated enters the blood cells directly from the plasma, path I. This path may have some of the characteristics of the direct uptake process studied by Kustin and coworkers (Dingley et al., 1981) in which V(V) and 48V-containing buffers were mixed with unlabelled blood cells. The characteristics found for this process were rapid uptake of 48V with half-lives of less than a minute, a large ratio for 48V in blood cells to that in plasma, and V(IV) formation and subsequent disappearance as measured by the appearance of an electron spin resonance (ESR) signal. The measurement of V(IV) ESR signals in blood cells taken directly from A. ceratodes (Frank et al., 1986) may be a manifestation of this path. However, as seen from data previously presented, uptake of V(V), as measured by 48V, from artificial mixtures representing plasma into blood cells can have very different
Uptake of V by Ascidia ceratodes characteristics from that using plasma containing in vivo incorporated V(V). The long-term path, II, involves the transfer of V(V), measured as 48V, to some site or sites in the animal, followed by long-term incorporation into the blood cells. Our data show that the branchial sac, the digestive tract, and associated tissues contain at least 50% of the activity removed during 1-3-day incubation with 48V. Goldberg et al. (19 51) monitored the uptake of 4aV by A. ceratodes and C. intestinalis by following the disappearance of the isotope from seawater and the incorporation of 4sV into various types of tissue using autoradiographic techniques. The approximate time for the disappearance of 4aV from seawater recorded in their work is in agreement with the data in Fig. 1. Their autoradiographic studies of A. ceratodes at a 15-day incubation time showed no 4sV in muscles of the mantle (part of the branchial sac), very little in the mantle and test, some in the branchial sac, and high levels in the mucus, gut wall and ovary. This is similar to our findings t'or dissected branchial sacs and digestive tracts. It is suggested that the 48V detected in the branchial sac comes in part from blood found in blood vessels of this tissue. In vivo uptake of 48V by the closely related ascidian Phallusia mamillata (Bielig et al., 1961a) occurs only at the branchial sac, while uptake by C. intestinalis, more distantly related to A. ceratodes, is found at the branchial sac and gastrointestinal tract (Bielig et al., 1961b) and gut wall (Goldberg et al., 1951). Therefore, a great deal of the 48V is incorporated in other tissue and does not enter blood cells directly by path I. However, a great deal of this 4sV does ultimately enter the blood cells, but just how it enters is not clear. Several possibilities exist. One alternative is that of path IIa in which 4sV enters the blood cells by re-entering the plasma by a circuitous route and then is taken up directly into the blood cells, a long-term path I. Another alternative, IIb is uptake through the production and development of blood cells. Three pieces of circumstantial evidence suggest this pathway. (1) The time taken for the maximum 4sV level to be reached in the blood cells, 20 days, corresponds to the time measured for cell development in Styela clara (Ermak, 1975). (2) In vivo experiments in which A. ceratodes is incubated with 4sV for varying lengths of time and autoradiographic studies of the resulting blood cells show incorporation of 48V in compartment and/or amoebocyte cells but not morula (vanadocyte) or pigment cells, although at the longest incubation time, 56 days, some morula cells appear to be labelled. In discussions of the development of blood cells it has been suggested, but highly debated, that compartment and amoebocyte cells are precursors to morula cells (Goodbody, 1974; Wright, 1981). (3) Much of the 4sV taken up from seawater at long times is localized in the gut (Goldberg et al., 1951) where haematopoietic tissue is found in many ascidians (Wright, 1981). While these pieces of evidence are not conclusive and do not eliminate reincorporation into plasma and then directly into blood cells, they are suggestive of path II incorporation of vanadium through the natural development of blood cells. The general conclusions reached from this work are:
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(1) There are two paths by which vanadium is incorporated into the blood cells of the phlebobranch A. ceratodes, a predominantly V(III)-containing ascidian. (2) One path, I, involves a rapid equilibration of V(V) between the plasma and blood cells, followed by a slow irreversible incorporation into the blood cells. (3) Another path, II, involves the accumulation of vanadium in a tissue and a slow incorporation into the blood cells by a mechanism, which may involve (a) the slow loss from remote sites into the plasma and direct incorporation into blood cells or (b) the production and development of blood cells. (4) Vanadium (V) added by in vitro methods to plasma or plasma simulants is not a good model for in vivo V(V)-labelled plasma-blood cell interactions. Acknowledgements--This work was supported by the Committee on Research, University of California, Davis, in the form of Faculty Research grants and Intercampus Travel grants to the Bodega Marine Laboratory of the University of California. REFERENCES
Agudelo M. I., Kustin K., McLeod G. C., Robinson W. E. and Wang R. T. (1983) Iron accumulation in tunicate blood cells. I. Distribution and oxidation state of iron in blood of Boltenia ovifera, Styela clara, and Molgula manhattensis. Biol. Bull. 165, 100-109. Anderson D. H. and Swinehart J. H. The distribution of vanadium and sulfur in the blood cells and the nature of vanadium in the plasma of the ascidian Ascidia ceratodes. Comp. Biochem. Physiol. (in press). Bielig H. J., Jost E., Pfleger K,, Rummel W, and Seifent E. (1961a) Aufnahme und Verteilung yon Vanadin bei der Tunicate Phallusia mamillata Cuvier. Hoppe-Seyler's Z. Physiol. Chem. 325, 122-131. Bielig H. J., Plteger K., Rummel W. and Seifen E. (1961b) Aufnahme und Verteilung von Vanadin bei der Tunicate Ciona intestinals, L. Hoppe-Seyler's Z. Physiol. Chem. 326, 240-258. Biggs W. R. and Swinehart J. H. (1976) Vanadium in selected biological systems. In Metals Ions in Biological Systems (Edited by Siegel H.), pp. 141-195. M. Dekker Inc., New York. Biggs W. R. and Swinehart J. H. (1979) Studies of the blood of Ascidia ceratodes. Experientia 35, 1047-1048. Butler A., Danzitz M. J. and Eckert H. (1987) V-51 NMR as a probe of metal ion binding in metalloproteins. J. Am. Chem. Soc. 109, 1864-1865. Dingley A. L., Kustin K., Macara I. G. and McLeod G. C. (1981) Accumulation of vanadium by tunicate blood cells occurs via a specific anion transport system. Biochim. Biophys. Acta 649, 493-502. Endean R. (1955) Studies of the blood and test of some Australian ascidians. I. The blood of Pyura stolonifera. Austrl. J. Mar. Freshwater Res. 6, 35-59. Ermak T. H. (1975) An autoradiographic demonstration of blood cell renewal in Styela claava (Urochordata: Ascidiacea). Experientia 31, 837-839. Frank P., Carlson R. M. K. and Hodgson K. O. (1986) Vanadyl ion epr as a noninvasive probe of pH in intact vanadocytes from Ascidia ceratodes. Inorg. Chem. 25, 470-478. Goldberg E. D., McBlair W. and Taylor K, M. (1951) The uptake of vanadium by tunicates. Biol. Bull. 191, 84-94. Goodbody I. (1974) The physiology of ascidians. In Advances in Marine Biology (Edited by Russel F. S. and Yonge M.), Vol. 12, pp. 1-149. Academic Press, New York.
158
DANIELH. ANDERSONet al.
Hawkins C. J., Kott P., Parry D. L. and Swinehart J. H. (1983) Vanadium content and oxidation state as related to ascidian phylogeny. Comp. Biochem. Physiol. 76B, 555-558. Hawkins C. J., Merefield P. M., Parry D. L., Biggs W. R. and' Swinehart J. H. (1980) Comparative study of the blood plasma of the ascidians Pyura stolonifera and Ascidia ceratodes. Biol. Bull. 159, 656-668. Henze M. (1911) Die Vanadiumverbindung der Blutkorperchen. Hoppe-Seyler's Z. Physiol. Chem. 72, 494-50 I. Kustin K. and McLeod G. C. (1977) Interactions between metal ions and living organisms in sea water. In Inorganic Biochemistry II, pp. 1-34. Springer, Berlin.
Michibata H., Terada T., Anada N., Yamakawa K. and Numakunai T. (1986) The accumulation and distribution of vanadium, iron, and manganese in some solitary ascidians. Biol. Bull. 171, 672-681. Swinehart J. H., Biggs W. R., Halko D. J. and Schroeder N. C. (1974) The vanadium and selected metal content of some ascidians. Biol. Bull. 146, 302-312. Webb D. A. (1939) Studies on the ultimate composition of biological material. J, Esp. Biol. 16, 499-523. Wright R. K. (1981) Urochordates. In Invertebrate Blood Cells, Arthropods to Urochordates, Invertebrates and Vertebrates Compared (Edited by Ratcliffe N. A. and Rowley A. F.), Vol. 2, pp. 565-626. Academic Press, London.