Vanadium-containing tunicate blood cells are not highly acidic

384

Biochimica et Biophysica Acta, 720 (1982) 384-389 Elsevier Biomedical Press

BBA i 1046

VANADIUM-CONTAINING TUNICATE BLOOD CELLS ARE NOT HIGHLY ACIDIC A M Y L. D I N G L E Y a, K E N N E T H K U S T I N a IAN G. M A C A R A b, G U Y C. M C L E O D c and M A R Y F. ROBERTS d

" Department of Chemistry, Brandeis University, Waltham, MA 02254; b Department of Biochemistry and Molecular Biology, Harvard University, 16 Divinity Avenue, Cambridge, MA 02138; " New England Aquarium, Central Wharf, Boston, MA 02110 and d Department of Chemistry, Massachusetts Institute of Technology, Cambridge, MA 02139 (U.S.A.) (Received November 19th, 1981) (Revised manuscript received March 12th, 1982)

Key words: Vanadium; Methylamine; Transmembrane equilibration; lntracellular pH; (Tunicate blood cell)

The intracellular pH of intact blood cells of the tunicate Ascidia nigra was measured by transmembrane equilibration of [t4C]methylamine. The pH of unfractionated blood cells is 7.39-+0.10. The pH of vanadocytes, determined in a fractionation study, is 7.2. Previously used methods, in which pH values less than 3.0 are inferred from cell lysis or vital staining experiments, are shown to be unsuitable for intracellular pH determination due to the chemical composition of these vanadium-containing cells.

Introduction Vanadocytes are tunicate blood cells unusual for their ability to concentrate metal ions in reduced oxidation states. The concentration of vanadium(Ill) within the vacuoles of Ascidia nigra vanadocytes approaches 0.15 M, and these vacuoles occupy 90% of the intracellular space. In this paper we present evidence refuting claims that vanadocytes of ascidians possess highly acidic vacuoles. In addition, we review the studies in which intracellular pH values less than 3.0 are reported, and conclude that the methods used give spurious results when applied to vanadocytes. One early approach used to determine intracellular pH was simply to lyse cells in distilled water and measure the resulting change in pH of the solution. Application of the technique by several workers to vanadocytes produced a dramatic fall in p H [1-7]. Since the sulfate content of the plasma is approximately half that of seawater, a difference ascribed to accumulation of sulfate by the vanadocytes [1,6-10], it was inferred that the cells are 1 to 2 N in sulfuric acid. However, measurements of the sulfate content

of the cells have given widely diverging results [1,6,7], and it cannot be concluded that the low amount of sulfate in the plasma is the result of accumulation by the blood cells. More importantly, the experimenters did not correct their pH determinations for the considerable amount of hydrogen ion produced by cell components upon lysis. These sources of H ÷ include the hydrolysis of intracellular V(III) and V(IV) [11] and the hydrolysis of tunichrome [12]. The result of this approach is that the calculated pH is much lower than that of intact cells. A second approach used to measure intracellular pH is vital staining:diffusion of pH indicators into live cells followed by visual observation of intracellular coloration. When methylene blue, methyl red and methyl orange were added to whole vanadocyte cell suspensions, intracellular color changes corresponding to low pH were observed [4,7,13]. However, the method is subject to error, since methylene blue, methyl red and methyl orange act as irreversible redox indicators [14-21]. As vanadocytes are rich in Fe(II), V(III) and V(IV), the cell interior is very probably a highly reducing

385 environment. The likelihood that the intracellular redox potential and not the pH was measured was not eliminated. Moreover, no evidence was presented to show that the indicators were free in the intracellular fluid, and not bound to the cell membrane, which could produce a color change. Lastly, the cells of some species, such as A. nigra are intensely colored, making visual determinations of indicator color changes difficult. We now present evidence that contradicts the conclusions drawn from these earlier experiments. The ratio of intra- to extracellular concentration of amine, as measured by the use of radioisotopically-labelled methylamine, is used to measure transmembrane pH gradients [22-24] of living, intact blood cells of A. nigra.

Experimental procedures

Materials. The following compounds were used without further purification: N-2-hydroxyethylpiperizine-N'-2-ethanesulfonic acid (Hepes) and Ficoll from the Sigma Chemical Co., St. Louis, MO. and aqueous counting scintillant from Amersham, Arlington Heights, IL. The radioisotopes used were purchased as [laC]methylamine hydrogen chloride, 3H20 , 2H20 and [14C]polydextran from New England Nuclear Corp., Boston, MA. All other chemicals were obtained from Fisher Scientific Co., Pittsburgh, PA. Methods. [14C]Methylamine, [laC]polydextran and 3H20 activities were measured to ---1% precision with a Beckman LS-100 liquid scintillation system. Cell counts were made using a LevyHausser hemocytometer. Phosphorus nuclear magnetic resonance (NMR) spectra were obtained at 4°C with a Bruker 270 spectrometer operating at 109.3 MHz, using a 10 mm probe. A. nigra were obtained from Key Biscayne, FL, and maintained in salt water aquaria as described previously [25]. Methylamine distribution experiments. Suspensions of unfractionated blood cells in 50 mM Hepes/500 mM NaC1 buffer at varying pH values were prepared as described previously [26]. To prepare suspensions of vanadocytes, whole blood was layered on top of a solution of 20% Ficoll in Hepes/NaCl buffer. After centrifugation at 12000 rpm for 10 min., the vanadocytes are at the bot-

tom of the centrifuge tube, while other cells are suspended at the plasma-Ficoll interface. Each cell suspension was divided into two parts and an aliquot of 3H20 added to each. To one suspension an aliquot of [laC]methylamine was added and to the other, [14C]polydextran. The suspensions were incubated in a shaking water bath for 5 min, then centrifuged in a Beckman microfuge for 1 min. Counting was done on 200 gl aliquots of the supernatants. Part of the pellet, obtained by cutting through the centrifuge tube below the pelletsupernatant interface, was dissolved in perchloric acid or 10 N NaOH and an aliquot counted. All experiments were performed at least three times and averaged results are given. Calculation of ApH. The method of amine equilibration to determine transmembrane pH gradients has been used successfully with chromaffin granules [22], bacteria [27], lysosomes [28], chloroplasts [23,29,30] and chromatophores [31]. The technique is made possible by the fact that the neutral form of the amine, A, may freely penetrate the membrane, while the protonated form, AH ÷ , cannot [29,32]. Hence, at equilibrium, [A]in= [A]out , where the subscripts 'in' and 'out' differentiate between intra- and extracellular total concentration of neutral amine. (Concentrations in activity per volume.) From this observation, and knowledge of the distribution of amine between the intra- and extracellular space, we may derive the ApH, which is the log[H+]iJ[H+]out, as follows. K a = [A][H + ] / [ A H +]

[amine]i=/[amine]out = ([ALo + [An + ]~n)/([A]oot + [A n + ]out) = ( K a + [H+ ]in)/(Ka + [H+ ]o~t) If K a ~ [ H + ]in, [ H+ ]out, then [amine]in/[amine]o~, -------[H + ]in/[H + ]out The effect of the pK a of the amine on the accuracy of the calculated ApH has been discussed [30]. Since the pK~ value of methylamine is 10, and the pH of the cell interior is presumed to be acidic or near neutral, it is valid to use the equilibrium

386

distribution of the amine to determine the ApH. The concentration of extracellular amine is found from the ratio of the activities of 3H20 and [14C]methylamirte in the supernatant, 3H2Osup and [Z4C]methylamines,p. To calculate the intracellular amine concentration, [lnC]methylaminep~, the concentration of [14C]methylamine in the excluded volume must be subtracted from the total activity in the pellet. The excluded and intracellular volumes are calculated by the difference between the volume indicated by the activity of [14C]polydextran, V~, which does not penetrate the cell, and the total volume of the pellet, Vp¢~, as determined by the activity of 3H20. The intracellular volume determined in this manner is in good agreement with the intracellular volume obtained from cell counts and the calculated volume per cell. Thus ApH is calculated from [amineli n = (['4C]MeNH2oo, -- ( [ I a C ] M e N H 2 , . ~ ) (Vex)) / (Vpe, - Vex) [amine]out = [14C ] M e N H 2'u~/ 3 H20,~p

log([amine]in/[amine]o,,) ~ A oH.

Phosphorus NMR experiments. Unfractionated blood cells were suspended in buffer containing 2H20; the volume of the cells was 30% of the total volume. Inorganic phosphate was added to this

suspension at room temperature to a concentration of 1 mM. Since phosphate equilibrates across the cell membrane under these conditions [22], the intracellular phosphate concentration was approx. 1 mM. In a single pulse experiment, 2090 transients were collected: pulse width = 25 /~s, repetition rate = 0.5 s, sweep width = 16 KHz. A spinecho experiment was conducted to minimize broad components of the spectra; data was collected for 1 h with ~-= 60 ms between 90 ° and 180 ° pulses. Results

The results of equilibration of [14C]methylamine between unfractionated blood cells and the external buffer are summarized in Table I. To prove that the distribution was sensitive to changes in the transmembrane pH gradient, the pH of the buffer was varied from 7.40 to 8.01 in these experiments. It was found that the distribution of [)4C]methylamine changed with changing extracellular pH; however, the same intracellular pH was obtained regardless of the extracellular pH. From the data presented in Table I we calculate an intracellular pH of 7.39, S.D.= 8.0.10 -2. To demonstrate further that the distribution of amine is a measure of titratable intracellular protons, experiments were carried out with large amounts of methylamine. Under these conditions, the high concentrations of intracellular methylamine should affect the intracellular pH. [14C]Methylamine was added to a cell suspension

TABLE I METHYLAMINE EQUILIBRIUM DISTRIBUTION IN TUNICATE BLOOD CELLS ApH

Extracellular pH

Activity of MeNH 2 (cpm)/volume (ml) Conc. of MeNH 2 in out (pM)

blood cells

8.01 7.75 7.66 7.50 7.40 8.01

11 13 14 13 11 100

22652/0.110 34492/0.419 52290/0.522 33228/0.210 1278/0.0675 26048/0.312

13652/0.340 33299/0.731 38771/0.627 35264/0.360 6722/0.382 45207/0.651

0.710 0.257 0.210 0.207 0.032 0.080

7.30 7.49 7.45 7.29 7.37 7.93

Vanadocytes

7.80

12

10430/0.109

9569/0.389

0.590

7.2

Intracellular

pH

Unfractionated

387 to a final concentration of 100 /tM. From the resulting distribution, we calculate an intracellular pH of 7.93, which is more basic than that indicated when low concentrations of methylamine are used. To test whether the pH of the vanadocytes is different from that of the other A. nigra blood cell types, vanadocytes were separated from whole blood by centrifugation through 20% Ficoll. As has been reported elsewhere, this procedure enriches the amount of vanadocytes from 60% to, in our case, 88% of all cells [33]. The results of equilibration of [~4C]methylamine showed that vanadocytes are only slightly more acidic than unfractionated blood cells. The intracellular pH of vanadocytes is 7.2, which is approx. 0.2 pH units lower than the pH calculated for all blood cells, but which is within two standard deviations of that value (Table I). A single, broad (1.8 ppm) 31p resonance was detected. The chemical shift of the peak corresponds to phosphate at the pH of the buffer of the cell suspension. Discussion

Experiments have been carried out to determine the intracellular p H of A. nigra vanadocytes by measurement of the equilibrium distribution of [14C]methylamine. The distribution depends on the difference in pH between the cell interior and the extracellular medium; as the extracellular pH was varied, the amine distribution changed and the same intracellular pH was calculated. From these measured distributions, an intracellular pH of 7.39 for unfractionated blood cells and 7.2 for vanadocytes was calculated. If the amine were simply binding to the cell membrane, or to intracellular vanadium, the equilibrium distribution of amine would always be the same, regardless of external pH. When a large amount of methylamine was used, such that a significant concentration of amine is taken up by the cells, the intracellular pH was raised, since protons are being consumed in the reaction M e N H 2 + H + = M e N H ~ . It was found that incubation in 100 # M [14C]methylamine raises the intracellular pH by 0.54 units to 7.93, when the buffer pH was 8.01. This result indicates that the concentration of methylamine

used to determine the native p H does not significantly change that pH; a 10-fold increase in the concentration of amine (from 10 to 100/~M) only produced a 3.5-fold increase in H + concentration (from 7.39 to 7.93). The experimental protocol assumes that the methylamine distributes freely into the vacuoles of the blood cells. This assumption is supported by the observation that the vacuolar membranes appear to be normal bilayers [34], and that of the many organelles studied, none appear to be impervious to the amines [22,23,27-31]. Moreover, since the vacuoles occupy 90% of the total volume of the vanadocyte cells, if the amine did not enter the vacuoles, the distribution would imply a cytoplasmic pH of 6.4, which is unusually low. The intracellular pH of cells can be determined by 31p-NMR, through the chemical shift of intracellular phosphate, which is p H dependent [3537]. When tunicate blood cells were incubated with phosphate, no intracellular phosphate signal could be observed, even though a significant concentration of [alP]phosphate is taken up by the cells. Lack of an observable 3Lp-NMR resonance is attributable to the complexing of phosphate by intracellular, paramagnetic V(III) a n d / o r V(IV), which broaden the phosphorous resonance, so that it cannot be detected. This observation is further evidence that intracellular vanadium atoms are not fully surrounded by a chelating structure. A highly acidic environment within the vanadocyte was first postulated by M. Henze in 1912 [2]. He found that 11.60 ml of 0.10N N a O H were needed to neutralize 60 ml of a solution containing lysed Phallusia marnmillata blood cells. He also found that the addition of a solution of BaCI 2 to the lysed cells produced a heavy, white precipitate. The precipitate was assumed to be BaSO 4, from which the concentration of intracellular sulfate was calculated to be 90 m g / 1 0 0 ml blood. From the results of these experiments, Henze concluded that the blood cells were 1 N in sulfuric acid [1]. D.A. Webb later reworked this data, based on new cell counts, deriving an intracellular sulfuric acid concentration of 1.83 N [10]. These experiments were repeated in other laboratories, and similar results obtained [5-7]. The conclusions drawn from these experiments can be refuted by considering the effects of the

388

composition and chemistry of these cells on the methods used. Vanadocytes from many species contain high concentrations of V(III) and V(IV) which will be rapidly oxidized and hydrolyzed at the alkaline pH of the buffer, producing up to 7 mol H + / m o l V(III) and 5 mol H + / m o l V(IV) [11]. Tunichrome, an unstable organic compound present at approximately the same concentration as vanadium in the vanadocytes of A. nigra, also hydrolyzes above pH 3.5, producing 13 mol H + / m o l tunichrome [9]. Thus, at low pH of the blood cell lysates is ascribable to hydrolytic reactions of the cell contents. This conclusion is supported by the fact that lysis of blood cells of the tunicate Podoclavella molluccensis, which contain less than 4% the amount of vanadium found in the cells of A. nigra, does not produce an acidic solution [38]. Furthermore, it has been shown that Ba 2+ will form a white precipitate when added to vanadate over a wide range of pH values [39]. Barium precipitation can in fact be used for quantitative analysis of vanadate when the solutions are heated. The precipitate will form at room temperature if the vanadate is in the monomeric form. Thus, the white precipitate formed when Ba 2+ is added to lysed cells is probably a mixture of barium sulfate and barium vanadate, causing the intracellular concentration of sulfate to be overestimated. When methods other than precipitation by Ba 2+ were used, only 8 mg sulfate/100 ml blood was found, which is approximately the same concentration as is found in the plasma [7]. The fact that the sulfate content of the plasma is only 50-60% that of seawater has been confirmed by several workers [1,7-9]. This feature is unremarkable, in that similar animals also have plasma ion compositions which are different from seawater, and it cannot be used as evidence for a high concentration of sulfate within the blood cells [40]. More importantly, it has been shown that the anion transport system in vanadocytes will not accept sulfate as a substrate [26]. Later workers diffused methylene blue, methyl red and methyl orange into the blood cells, observing color changes indicative of pH values less than 3.2 [1,4,7,9,41]. However, all three of these indicators have been used as irreversible redox indicators in reactions involving Fe(II) or organic-reducing agents [14-21]. The color changes observed in the

cells may be indicative of a redox reaction, not the intraceltular pH. Also, the natural coloring of the vanadocytes of many species is very intense, and it has been admitted that the color changes are hard to judge [41]. Thus, the use of indicators is not a reliable way to determine intracellular pH in this system. The idea that the intracellular or intravacuolar environment of the vanadocyte is highly acidic was upheld for many years, primarily to account for the presence in the cells of V(III), since the aquo complex of vanadium in this oxidation state is known to be stable only below pH 3.0 [11,42 !. However, it has not been established that intracellular V(III) is present as the aquo complex, only that it is not bound to a protein or other macromolecules [29,43-45]. The vanadium inner coordination shell is most consistent with an all oxygen environment [44]. It is possible that V(III) is complexed by small ligands which keep it stable at a higher pH. The observation that the tunic of A. nigra is covered with an acidic secretion is not in conflict with our results [4,46]. Lysing of vanadocytes in the tunic would be expected to produce a low pH in or on the surface, due to the above mentioned hydrolysis of V(III), V(IV) and tunichrome.

Acknowledgements We are grateful to the Francis Bitter National Magnet Laboratory for providing the instruments used in the N M R experiments. This work was funded by National Science Foundation grant PCM-7824782.

References 1 Henze, M. (1911) Hoppe-Seyler's Z. Physiol. Chem. 72, 494- 501 2 Henze, M. (1912) Hoppe-Seyler's Z. Physiol. Chem. 79, 215-228 3 Henze, M. (1913) Hoppe-Seyler's Z. Physiol. Chem. 86, 340-345 4 Hecht, S. (1918) Am. J. Physiol. 45(3), 157-187 5 Boeri, E. (1952) Arch. Bioehim. Biophys. 37, 449-456 6 Boeri, E. and Ehrenberg, A. (1954) Arch. Biochim. Biophys. 50, 404-416 7 Endean, R. (1954) Aust. J. Mar. Freshwater Res. 6, 35-59 8 Robertson, J.D. (1949) J. Exp. Biol. 26, 182-200 9 Robertson, J.D. (1953) J. Exp. Biol. 30, 277-296

389 l0 Webb, D.A. (1939) J. Exp. Biol. 16, 499-523 II Baes, C.F., Jr. and Mesmer, R.E. (1976) The Hydrolysis of Cations, pp. 197-209, John Wiley and Sons, Inc., New York 12 Macara, I.G., McLeod, G.C. and Kustin, K. (1979) Biochem. J. 181,457-465 13 Endean, R. (1960) Quart. J. Microscop. Sci. 100, 177-197 14 Rathsberg, H. 0928) Ber. 61, 1663-1665 15 Furman, N.H. and Wallace, J.H. (1930) J. Am. Chem. Soc. 52, 1443-1447 16 Furman, N.H. and Wallace, J.H. (1930) J. Am. Chem. Soc. 52, 2347-2352 17 Bishop, E. (ed.) (1972) Indicators, pp. 79-83, 185-186 and 668-670, Pergamon Press, Braunschweig 18 Cherkesov, A.I. (1960) Zhur. Anal. Khim. 15(6), 651-655 19 Jellinek, K. and Kresteff, W. (1924) Z. anorg, allgem. Chem. 137, 333-348 20 Jellinek, K. and Kiihn, W. (1924) Z. Anorg. Allg. Chem. 138, 109-34 21 Willard, H.H. and Young, P. (1928) J. Am. Chem. Soc. 50, 1322-1334 22 Johnson, R.G. and Scarpa, A. (1976) J. Gen. Physiol. 68, 601-631 23 Rottenberg, H., Grunwald, T. and Avron, M. (1972) J. Biochem. 25, 54-63 24 Padan, E. and Rottenberg, H. (1973) Eur. J. Biochem. 40, 431-437 25 Kustin, K., Levine, S., McLeod, G.C. and Curby, W.A. (1976) Biol. Bull. 150, 426-441 26 Dingley, A.L., Kustin, K., Macara, I.G. and McLeod, G.C. Biochim. Biophys. Acta 649, 493-502 27 Kashet, E.R. and Wilson, T.H. (1973) Proc. Natl. Acad. Sci. U.S.A. 70, 2866-2869 28 Goldman, R. and Rottenberg, H. (1973) FEBS Lett. 33, 233-238

29 Rottenberg, H., Grunwald, T. and Avron, M. (1971) FEBS Lett. 13, 41-44 30 Schuldiner, S., Padan, E., Rottenberg, H. and Avron, M. (1972) Eur. J. Biochem. 25, 64-70 31 Schuldiner, S., Padan, E., Rottenberg, H., Gromet-Elhanan, Z. and Avron, M. (1974) FEBS Lett. 49, 174-177 32 Packer, L. and Crofts, A.R. (1967) Curt. Top. Bioenerg. 2, 23-64 33 Carlson, R.M.K. (1977) Ph.D. Dissertation, Stanford University 34 Gansler, H., Pfleger, K., Seifen, E. and Biehg, H.-J. (1963) Experientia 19, 232-234 35 Uregurbil, K., Schulman, R.G. and Brown, T.R. (1978) in Biological Applications of Magnetic Resonance, pp. 537589, Academic Press, New York 36 Salhany, J.M., Yamane, T., Shulman, R.G. and Ozawa, S. (1975) Proc. Natl. Acad. Sci. U.S.A. 72, 4966-4970 37 Tauber, A.I. and Roberts, M.F. (1981) FEBS Lett. 129, 105-108 38 Hawkins, C.J., Parry, D.L. and Pierce, C. (1980) Biol. Bull. 159, 669-680 39 Morette, A. (1950) Bull. Soc. Chim. (France) 17, 526-532 40 Webb, D.A. (1956) Pubbl. Staz. Zool. (Napoli) 28, 273-288 41 Endean, R. (1960) Quart. J. Microscop. Sci. 101(2), 177-197 42 Pourbaix, M., Deltombe, E. and De Zoubov, N. (1966) Atlas of Electrochemical Equilibria in Aqueous Solutions, pp. 91, 92, 234-245, Pergamon Press, Paris 43 Carlson, R.M.K. (1975) Proc. Natl. Acad. Sci. U.S.A. 72, 2217-2221 44 Tullius, T.D., Gillum, W.O., Carlson, R.M.K. and Hodgson, K.O. (1980) J. Am. Chem. Soc. 102, 5670-5676 45 Macara, I.G., McLeod, G.C. and Kustin, K. (1978) Comp. Biochem. Physiol. (1979)62A, 821-826 46 Stoecker, D. (1978) Biol. Bull 155, 615-626