J. Euk. Microbiol.. 44(5), 1997 pp 503-510 0 1997 by the Society of Protozoologists

Osmotic Homeostasis in Dictyostelium Discoideum : Excretion of Amino Acids and Ingested Solutes

THEODORE L. STECK,*J LUCIUS CHIARAVIGLI0,*,2 and STEPHEN MEREDITH** *Department of Biochemistry and Molecular Biology, University of Chicago, 920 East 58th Street, Chicago, Illinois 60637, USA, and

**Department of Pathology, University Of Chicago, Chicago, Illinois 60637, USA

ABSTRACT. The response to osmotic stress in axenically cultured Dictyostelium discoideum was examined. Hypoosmotic buffers elicited two changes in the large (-50 mM) cytosolic pool of amino acids: a) the total size of the pool diminished, while b) about half of the initial pool was excreted. Hyperosmotic stress had the opposite effect. Among the predominant amino acids in the pool were glycine, alanine and proline. Putrescine, the major diamine, was neither excreted nor modulated. Recently ingested radioactive amino acids were excreted in preference to those in the cytoplasm, suggesting that the endocytic pathway might be involved in water excretion. Furthermore, hypoosmotic stress stimulated the selective excretion of small, membrane-impermeable fluorescent dyes which had been ingested into endocytic vacuoles. Caffeine inhibited the excretion of the fluorophores but not the amino acids. We conclude that the response of Dictyostelium to osmotic stress is complex and includes both modulation of the cytoplasmic amino acid pool and the excretion of amino acids and other small solutes from the endocytic pathway.

Supplementary key words. Acidification, amoeba, caffeine, contractile vacuoles, endocytic vacuoles, fluorophores, proton pumps, shock.

ECAUSE biological membranes are slightly permeable to B water, cells of a11 types behave as osmometers 110, 181. Maintaining a constant volume is therefore a universal issue in cellular homeostasis [13]. Animals minimize the impact of ex- ternal osmotic transients by maintaining a constant internal mi- lieu, while bacteria, algae, fungi and plants are protected from osmotic swelling by rigid cell walls and from osmotic shrinkage by the accumulation of cytosolic osmolytes [12, 371. Presum- ably because cell walls would impede endocytosis, many pro- tista and other simple eukaryotes must forego these mechanical barriers; instead, they excrete osmotic water from their cytosol via contractile vacuoles [32, 431.

The molecular basis for contractile vacuole function is poorly understood. In a typical protozoan, a few large vacuoles (which we refer to as bladders) fill slowly and then empty abruptly by venting under pressure through a pore in the plasma membrane [9 ] . The bladders seem to be fed by a tubulo-vesicular spon- giome which extends throughout the cytoplasm. Characteristi- cally, spongiomes have a high surface/volume ratio, favoring water uptake, while bladders minimize this ratio, presumably to limit the diffusion of water back into the cytosol from their transiently hypoosmotic interiors [32, 431.

Spongiomes are highly enriched in a proton pump, the V-H+- ATPase [ l l , 17, 23-25]. Proton gradients established by this pump could drive the antiport of cytosolic solutes into the spon- giome; these then could serve as osmolytes to draw water into the lumen of the contractile vacuole complex [17, 251. The membrane potential generated by the V-H+-ATPase could thus be the primary energy source for water transport. The contrac- tile vacuole complex would then function as a two-chambered pump: the spongiome taking up cytosolic fluid by chern-os- motic work and the bladder expelling it mechanically [43].

Besides the contractile vacuole complex, endocytic and other vacuoles have been observed to take up water when protozoa are placed in hypoosmotic media [5, 20, 401. Such vesicles could play a role in volume homeostasis [41].

Amino acids are widely used as osmolytes in cell volume

’ To whom correspondence should be addressed. Telephone: 3 12-702- 1329; Email: t-steck@uchicago.edu

ZCurrent address: 343 112 Mono Ave., Pismo Beach, CA 93449, USA.

Abbreviations: bicine, N,N-bis(2-hydroxyethyl)glycine; DIDS, 4,4’- diisothiocyanatostilbene-2,2’-disulfonate; DMSO, dimethylsulfoxide; HEPES, N-2-hydroxyethylpiperazine-N’-2-ethanesulfonic acid; MES, 4-morpholine-ethanesulfonate; NBD-CI, 7-chloro-4-nitrobenzo-2-oxa- 1,3-diazole; V-H+-ATPase, vacuolar proton pump.

regulation by prokaryotes, microbial eukaryotes and metazoans alike [4, 6, 13, 181. Whether they might serve as the antiported solute in the excretion of by contractile vacuoles or contribute to the uptake of water by endocytic vacuoles has not been ex- amined. We therefore tested for such functions in Dictyostelium discoideum, a soil amoeba which must respond at times to the osmotic stresses of both rainwater and mud.

MATERTALS AND METHODS

Buffers. The axenic strain A X - 3 of Dictyostelium discoideum was grown in a rich broth [3] with an apparent osmotic activity of approximately 200 mOsM, as determined by freezing point depression. We scaled our test buffers accordingly: low osmotic Buffer L contained 5 mM Nap, (pH 6.5) + 5 mM glucose + 1 mM MgC1, (calculated ideal osmolarity -20 mOsM); medium osmotic Buffer M contained Buffer L + 75 mM NaCl (-170 mOsM); and high osmotic Buffer H contained Buffer L + 150 mM NaCl (-320 mOsM). Buffer I, was 1 mh4 MgC1, + 5 mM glucose (-8 mOsM).

Analysis of amine release. For both growth and experimen- tal incubations, cells were kept swirling at room temperature [3]. In a typical experiment, cells were harvested in the mid- exponential phase of growth ( 5 X 106/ml), washed with Buffer M, pelleted, and resuspended at 5 X 107/ml in test buffer. Ali- quots were withdrawn at timed intervals and the cells pelleted in a microcentrifuge. To recover their free amines, the unspun input as well as the supernatant and pellet fractions were mixed with four volumes of isopropanol and allowed to sit for 10 min at room temperature, then chilled. After centrifugation of the precipitated macromolecules, the isopropanol-soluble extracts from 2 X lo5 cells were analyzed fluorometrically in duplicate for primary amines by reaction with o-phthaldialdehyde [33]. These analyses included buffer blanks and were calibrated against a glycine reference standard.

Amino acid analysis. Aliquots of isopropanol extracts were dried under vacuum; the residues were dissolved, reacted with phenylisothiocyanate, and analyzed by HPLC, as described [16]. Each set of determinations was preceded and followed by calibration with a standard set of amino acids and, when ap- propriate, putrescine. Norleucine was routinely added to isopro- pan01 extracts as a recovery standard.

That this procedure was reliable was indicated by the full recovery of [14C]alanine added to cells undergoing extraction and derivatization. We also showed that there was no loss of volatile amines under vacuum during preparation for amino acid analysis by performing o-phthaldialdehyde analyses on

503

504 J. EUK. MICROBIOL., VOL. 44, NO. 5 , SEPTEMBER-OCTOBER 1997

I I I I 1

0 50 100 150

NaC1, mM Fig. 1. Response of cells to osmotic strength. Cells were washed

with and adapted to Buffer M (see Materials and Methods). Aliquots were centrifuged and the cells suspended immediately at 5 X 107/ml in Buffer L supplemented with 0-150 mM NaCI. After 20 rnin of re- sponse, aliquots were centrifuged and unspun, supernatant and pellet fractions analyzed for amines. Open circles, total amines; triangles, cel- lular fraction; closed circles, excreted fraction.

control samples lacking derivatization before and after the evaporation step.

Excretion of ingested solute probes. Cultured cells were washed, resuspended in Buffer M and swirled for 15 min to permit them to recover from the stress of manipulation. The cells were then fed probes for a defined interval, washed twice and resuspended in media of varied osmotic strength. The time course of excretion was then determined by the analysis of un- centrifuged, supernatant and pellet fractions as described [28, 351. The excitation and emission wavelengths used in the flu- orescence assays were for FITC-dextran, 495 and 520 nm; for pyranine, 403 and 505 nm; and for pyrene tetrasulfonate, 372 and 403 nm.

RESULTS Responses to osmotic stress. As judged by the wet weight

of pellets, cells approximately doubled in aqueous volume over 10 min following transfer from a physiological buffer (-200 mOsM) to one of low osmolarity (-20 mOsM). They then re- gained their approximate initial volume over about three hours. Of interest here was the excretion of solutes during acute os- motic stresses.

Dictyostelium in Buffer M typically had an amine content of -50 ymol/109 cells (i.e. -50 mM); however, the values varied widely among experiments. The pool of amines responded acutely and in two different ways to changes in the osmolarity of the medium (Fig. 1): a) Half or more of the cellular amines were excreted from cells transferred to buffers of low osmolal- ity, whereas only a very small fraction was excreted at high osmolarity. b) Total intracellular amines increased with osmo- larity, reaching values of 80 mM or more in media of > 300 mOsM. Both of these responses appeared to be homeostatic in that they would help to maintain cell volume constant. The total amines in the system and the fraction released from the cells varied among experiments but the pattern was highly reproduc- ible.

I I I I

1 1 I I

0 10 20 30 MINUTES

Fig. 2. Time course of response of cells to hypoosmotic medium. Cells were washed from culture medium into Buffer H and allowed to adapt for 30 min. They were then pelleted and resuspended in Buffer Z at 5 X lo7 cells/ml. At intervals, aliquots were mixed with an equal volume of a stopping solution (300 mM NaCl in 20% DMSO), centr- fuged and the supernatant and pellet fractions analyzed for amines. Open circles, total amines; triangles, cellular fraction: closed circles, excreted fraction.

The release of amines in response to hypoosmotic stress seemed to be physiological and not due to injury. For example, amine excretion was inhibited at 0" C. Furthermore, the release of cell protein was not increased by incubation in hypoosmotic versus hyper-osmotic buffer. Finally, cells incubated in low os- motic buffer were fully viable (i.e. grew normally overnight) following their return to growth medium. We observed a similar excretion of amines under low osmotic challenge in the ciliated fresh-water protozoan, Tetrahymena thermophila (not shown).

The response of the cellular amine pool was followed over time after cells were transferred from hyperosmotic to hypoos- motic buffer (Fig. 2). The release of amines was initially brisk, but this process stopped after - 10 min in the experiment shown here and no later than 20 min in other experiments. It was typical for more than half of the amine pool to be excreted. In addition, total amines in the system fell by more than a third during the response period. Between the drop in total amines and the excretion of amines, the level of intracellular amines fell to about 20% of the input during the 30 min incubation in low osmotic buffer; i.e. to -10 mM.

The production and release of NH,+ also increased when cells were transferred into low osmotic buffers; however, this parameter was not pursued further.

Amino acid chromatograms (e.g. Fig. 3) were complex, featuring not only most of the common amino acids, but also the major cellular diamine, putrescine [ 191; an unknown amine (H') running just slower than histidine; the internal standard, norleucine (L); and reagent peaks, some of which are labeled Rgt. Nevertheless, the chromatographic analysis made several important points. a) The quantities of cellular amino acids ob- served accounted for the amines detected above with o-phthal- dialdehyde. The variance of total cell amine content with os- motic stress was likewise attributable to amino acids (not shown). Similarly, the excreted amines in Fig. 1 and 2 were predominantly amino acids (Fig. 3A). Thus, no other major form of amine need be postulated. b) The most abundant spe-

STECK. CHIARAVIGLIO & MEREDITH-OSMOSIS IN DICTYOSTELIUM 505

2.500

2.000

1.500

1 .ooo

0.500

3.000

2.500

2.000

1.500

1 .ooo 0.500

1.600

1.200

0.800

0.400

2.000

1.500

1 .ooo

0.500

0.000

Rgt Putrescine

Time (minutes) Fig. 3. Amino acid analysis of the response to hypoosmotic stress. Cells were transferred from Buffer M to Buffer L and incubated for 30

min. An aliquot was centrifuged, and 8 X lo6 cell equivalents of supernatant (excreted fraction, trace A), pellet (unexcreted fraction, trace B) and uncentrifuged input (trace C) were analyzed along with standards (trace D) as in Materials and Methods. Putrescine [19] was identified in other runs using an authentic standard. Single letters label the common amino acids: H’ is an unidentified amine running slightly slower than histidine; n L is the norleucine internal standard; and Rgt labels peaks from the reagent and solvents. The extinction coefficients for the amino acids vary on average by only a few percent, so that the major peaks can be directly compared.

506 J. EUK. MICROBIOL., VOL. 44, NO. 5, SEPTEMBER-OCTOBER 1997

cies of amino acids in whole cells were glycine > alanine > proline (Fig. 3C). A fourth major component (H') ran slightly slower than histidine. Since it reacted with phenylisothiocyan- ate, it is probably an amine and perhaps an amino acid deriv- ative. All four of these constituents (as well as the minor amino acid, serine) were preferentially excreted (compare tracings A and B). We also observed that all four of these species de- creased when cells were transferred to buffer of high osmolarity (not shown). c) Putrescine was not excreted following hypoos- motic stress (compare panels A, B and C). Furthermore, the putrescine concentration did not vary substantially between cells incubated in low and high osmotic buffers (not shown). These findings prompt two inferences: a) that putrescine is not used as osmotic ballast; and b) that the release of amino acids was specific and not the result of a generalized leakage of small molecules from osmotically-damaged cells. d) Several nonpolar amino acids, including tyrosine, methionine, vafine, leucine, isoleucine and phenylalanine, were detected at low levels in cells washed with Buffer M. The abundance of these minor amino acids rose several-fold when cells were incubated for 30 min in hyperosmotic buffer. Conversely, these amino acids be- came undetectable following incubation of cells for 30 min in low osmotic strength buffer. Thus, the response to osmotic stresses may include the hydrolysis and synthesis of cellular proteins.

Excretion of radiolabeled amino acids in response to os- motic stress. Dictyostelium apparently takes up amino acids principally by pinocytosis. Cells incubated with various [14C]amino acids subsequently released them when transferred to low but not high osmotic buffers. We sought to determine whether the radioactive amino acids exited from the cytosol or from the endocytic pathway. We therefore fed ['"C] alanine or ['"C] glutamate (representing major and minor Dictyostelium amino acids) for 1 h and chased for 15 min to permit its trans- port to the cytosol. Meanwhile, another aliquot of cells was incubated with the same probe for 15 min but not chased; this should have left the bulk of the probe in the lysosomes [25, 281. The release of isotope from cells transferred to high and low osmotic buffers was then followed. Little probe was re- leased from the cells in hyperosmotic buffer, while considerable release occurred in low osmotic buffer. Excretion of the radio- active amino acid probes was inhibited at 0" C.

Of particular interest was that the label taken up for only 15 min was more efficiently released than that ingested for one hour and then chased for 15 min (Fig. 4). These results suggest preferential excretion from the endocytic pathway as compared to the cytosol. This excretion was selective: less than 5% of lysosomal acid phosphatase and the ingested macromolecular marker, FITC-dextran, was released over 30 min following transfer to either high or low osmotic media.

The experiment in Fig. 4 was performed in the presence of cycloheximide, which inhibited protein synthesis by > 90%. We also showed, in control studies using isopropanol to precip- itate macromolecules, that nearly ail the isotope remained sol- uble in these experiments. Furthermore, [I4C]alanine gave re- sults similar to those for ['4C]glutamate (not shown). In other control experiments, we examined the release of ingested [14C]glucose. After feeding for an hour and chasing for an ad- ditional 15 min, only 7% of the ingested label was released during a 20 min incubation in low osmotic buffer. The release of [14C]glucose fed for only 15 min was higher (17% in 20 min) but was not influenced by the osmotic strength of the medium. We conclude that glucose does not behave as a reg- ulated osmolyte and therefore serves as a reference control for the amino acids.

Excretion of fluorescent probes from the endocytic path-

I . I . I

3000 *

w g 1000 /------. E 3 t

300: m t- 2 3 8 2000

looo 1 0 % 0 10 20

C

v\

0 7 7

J * 0 10 20

1 . 1

2000

1000

0

2000

1000

0

MINUTES

Fig. 4. Excretion of [14C]glutamate in response to osmotic stress. Cells were washed with Buffer M, pre-incubated in Buffer M containing 0.5 mg/ml cycloheximide for 15 min to block protein synthesis, then incubated in the same buffer with 1 pCi carrier-free ['4C]glutamic acid for 1 h (left panels A & B) or 15 min (right panels C & D). The cells labeled for 1 h were then washed and chased with unlabeled buffer for 15 min. Both samples were then washed twice more, all in Buffer M. The cells were then resuspended at 5 X 107/ml in Buffer L (top panels A & C ) or Buffer H (bottom panels B & D) containing cycloheximide and, at intervals, aliquots centrifuged and the supernatant (circles) and pellet (triangles) fractions analyzed for radioactivity.

way. The preferential release of newly ingested amino acids prompted us to examine other ingested probes. We found that while the release of FITC-dextran was minimal in all buffers, the excretion of three small, membrane-impermeable fluorop- hores was extensive and was stimulated in low osmotic strength medium (Fig. 5) . The release of small fluorescent probes was inhibited at 0" C (not shown). The linear time course of excre- tion over 30 min was distinct from the hyperbolic curves seen for the unlabeled and radioactive amino acids (Fig. 4). The selective excretion of small but not large ingested fluorescent probes is reminiscent of the size fractionation observed for the endocytic pathway of macrophages 123.

Effect of caffeine on excretory responses to osmotic stress. Caffeine is an inhibitor of endocytosis and perhaps of general endomembrane membrane movement in Dictyostelium [ 11. Fur- thermore, we observed that caffeine caused sustained cell swell- ing in the low osmotic buffer (not shown); this could possibly represent interference with water excretion. We therefore tested the ability of caffeine to block the observed excretory responses to low osmotic stress. In Fig. 6, two of the small fluorescent dyes, pyranine and pyrene tetrasulfonate, were fed to cells to load their lysosomes. The cells were then incubated with or without 7.5 mh4 caffeine before being shifted to buffers of low or high osmotic strength. We found again that both pyranine (Fig. 6A) and, to a lesser extent, pyrene tetrasulfonate (not shown) were promptly excreted, more by cells in low osmotic than high osmotic buffer. Caffeine inhibited the excretion of both dyes in both low osmotic and high osmotic buffer. How- ever, caffeine did not inhibit the excretion of cellular amnes (Fig. 6B) or ingested ['4C]alanine under conditions where high osmotic medium was inhibitory (not shown). These results sug- gest that the excretion of the ingested fluorophores and the ami- no acids followed different pathways (see Discussion).

STECK. CHIARAVIGLIO & MEREDITH-OSMOSIS IN DICTYOSTELIUM 507

0 300

200

100

0

600

400[,/: 200 Ly 1300

0 0 0 10 20 30 0 10 2 0 30

MINUTES MINUTES

Fig. 5. Excretion of ingested endocytic markers in response to os- motic stress. Cells were washed and incubated for 20 min in Buffer M containing FITC-dextran (mol. wt. -70,000; 1 mg/ml) (top panels A & B); pyranine (8-hydroxypyrene-l,3,6-trisulfonic acid; 0.25 mM) (middle panels C & D); and pyrene tetrasulfonate (0.5 mM) (bottom panels, E & F), all from Molecular Probes, Inc. The loaded cells were washed, placed in Buffer L (left panels) or Buffer H (right panels) and the release of probes followed over 30 min by fluorometry.

Role for an acidified intracellular compartment? In view of the high density of V-H+-ATPase proton pumps in Dictyos- telium spongiomes [17, 23-25], we sought evidence for their participation in water volume homeostasis. We found that two nonspecific inhibitors of the V-H+-ATPase, 0.1 mh4 NBD-Cl and 0.1 mM N-ethyl maleimide, caused prompt swelling of cells in the low osmotic buffer. However, neither agent blocked the excretion of amino acids (not shown). We also found that neither bafilomycin A, nor concanamycin A, both potent and specific inhibitors of V-H+-ATPases [S], caused cell swelling or affected the excretion of amino acids even at extremely high concentrations of up to 10 pg/ml (-10 pM). This may be be- cause the proton pump in Dictyostelium is not very sensitive to these agents (unpublished observations) and/or because the agents were taken up poorly into the cells.

We monitored the extracellular pH of cells transferred to un- buffered hypoosmotic and isoosmotic media (namely, 5 mh4 glucose + 1 mM MgCl, + 0 or 75 mM NaCl) and folind no difference over a hour of incubation. We therefore have no ev- idence for excretion of protons in response to osmotic stress.

In vitro proton ATPase activity. We also tested the ability of amino acids to uncouple proton electrochemical gradients generated in acidosomes, proton-tight vesicle fragments of the spongiomes of Dictyostelium [25]. To minimize perturbations, we used fresh cell homogenates in which acidosomes contain nearly all the V-H+-ATPase and in vitro acidifying potential [23, 291. V-H+-ATPase enzyme activity was determined under coupling conditions (see Fig. 5 in [29]). As shown in Fig. 7, alanine did not substitute for C1-, an electrogenic anion which discharges the membrane electrical potential. Indeed, it did not stimulate as well as MES, an untransported impermeant anion

which presumably increased the ATPase activity by raising the ionic strength non-specifically (unpublished observations). Un- coupling by alanine (measured as stimulation of V-H+-ATPase activity) was also weak in the presence of imidazole, a mem- brane-permeable buffer which dissipates the proton concentra- tion gradient across the membrane. Similar results were ob- tained with glycine (not shown). Furthermore, preparing ho- mogenates in the presence of Mg2+ to maintain putative con- nections with endocytic vacuoles [27, 30, 311 did not change the results. Thus, there was no evidence for the coupling of amino acid transport to proton gradients in vitro.

DISCUSSION The rate of swelling of Dictyostelium in low osmotic buffers

suggests a plasma membrane water permeability of -3 X cms-l (L. Mahadevan and T. L. Steck, unpubl.). While this val- ue is two orders of magnitude lower than that typical of ver- tebrate plasma membranes, it corresponds well to values for the amoeba, Chaos chaos, and for synthetic bilayers made of cho- lesterol and sphingomyelin [lo, 18, 341. While low, this finite permeability nevertheless causes protozoa like Dictyostelium to absorb water osmotically and then excrete it actively. Our re- sults bear on three interrelated aspects of this vital but poorly understood homeostatic activity.

Amino acid excretion. That Dictyostelium uses amino acids as osmotic ballast is strongly suggested by its large cytoplasmic pool of a few small, neutral amino acids. Furthermore, the size of this pool varied acutely with shifts in the osmotic strength of the medium. This response included the excretion of more than half of the pool. Amino acids make a good osmotic ballast: a) They are less likely than inorganic electrolytes to perturb protein functions at high concentrations [37]. b) They can be stored at low osmotic activity as cytosolic polymers. c) They can create a high-capacity system; that is, the cellular proteins in Dictyostelium hold the equivalent of a - 1 M solution of free amino acids. Invertebrates commonly use cellular proteins for such osmotic purposes [cf. 4, 151. However, that polymerization is, in fact, the mechanism which decreased the amino acid pool in osmotically stressed Dictyostelium seems unlikely for two reasons. First, this effect was not blocked by cycloheximide under conditions where protein synthesis was inhibited by > 90%. Secondly, storage protein(s) composed of the few pre- dominant amino acids detected here would be highly unusual; furthermore, they might not fold into stable structures. It is perhaps more likely that the amino acids in question are mod- ulated by catabolism to smaller compounds, such as NH,+ and HCO,-, which could offer the advantage of doubling the os- motic potency of the parent amino acids [14].

Following transfer to hypoosmotic buffer, the cells swelled for about 10-20 min before volume recovery began. However, the excretory response to the stress began immediately (Fig. 2). Clearly, the excretion of amino acids did not prevent the cells from swelling during this period, suggesting that the relation- ship between the acute excretion of amino acids and water elim- ination is not simple. It could be that the modulation of the amino acid pool is simply a short-term maneuver which coun- ters acute changes in the external osmotic environment. For example, amino acid excretion could serve to reduce the work which cells shifted to a hypoosmotic milieu expend to return to their normal volume.

These findings notwithstanding, amino acids might still be used to draw water out of the cytosol of swollen cells. It is possible, for example, that these osmolytes are pumped from the cytosol into the spongiomes and then delivered to the blad- ders. They might initially be excreted from the bladders as an isoosmotic solution, to remove both osmotic load and excess

508 J. EUK. MICROBIOL., VOL. 44, NO. 5, SEPTEMBER-OCTOBER 1997

w 200 u

u u1

100 E

i t t 1

W

m I+ 4

30 Q)

0 H

20 a

E 10 a

I+ 0

0 10 20 30 0 10 20 30 MINUTES

Fig. 6 . Effect of caffeine on the response to osmotic stress. Cells were washed with Buffer M, resuspended to 5 X lo7 cells/ml and allowed to adapt for 15 rnin before the introduction of pyranine and pyrene tetrasulfonate to 1 mM and 0.1 mM, respectively. After 15 min of feeding, the cells were washed into Buffer H containing or lacking 7.5 mM caffeine and incubated for 10 min at 5 X lo7 cells/ml. They were then pelleted and resuspended in Buffer Z lacking caffeine (open symbols) or containing caffeine (filled symbols) and incubated at room temperature. At intervals, aliquots were mixed with an equal volume of stopping solution (300 mM NaCl in 20% DMSO), centrifuged and the supernatants (circles) and pellet fractions (squares) analyzed for fluorescent probes (see Fig. 5 ) and amines (see Fig. 1). Panel A: pyranine; panel B, amines. The data for pyrene tetrasulfononate (not shown) paralleled those for pyranine.

n

El F: 0 ln 7+ U

4

o-6 I rl u a l 4

0.4

0.2

0.0 Fig. 7. Effect of alanine on V-H+-ATPase hydrolytic activity. Cells

were washed and homogenized at 2 X lo8 cellslml in 100 rnM sucrose containing 5 mM bicine-KOH (pH 8.5) as described [29]. The homog- enate was centrifuged at 2,000 X g for 1 rnin and aliquots of the su- pernatant fraction analyzed for V-H+-ATPase activity in 500 ( ~ 1 mixtures containing: 4 X lo6 cell equivalents of homogenate + 100 mM sucrose + 10 mM K-HEPES (pH 7.5) 2 potassium (K) or imid- azole (Im) salts of 20 mM chloride (Cl), MES or alanine (Ala) + 2.5 mM MgMES, 2 10 pM DIDS + 2 mM Na,ATP (added last). After a 10 min incubation at 30" C, the reaction was stopped with 10% trichlo- roacetic acid, the mixtures centrifuged and the supernatants analyzed for P,as described [29]. Ordinate, optical absorbance at 750 nm. Values are averages of duplicates with the DIDS-inhibited blank subtracted.

water. Subsequent to this adjustment period, the amino acids entering the bladder might be fully retrieved to the cytosol just before each contractile event. Such a feature would have the critically important benefit of forestalling the loss of valuable cell constituents over long periods of stress. To illustrate: the estimated excretion rate of 0.1 cell volumeshin of a 0.2 M isoosmotic amino acid solution would entirely exhaust the pool of free amino acids as well as the entire cell protein reservoir during an hour-long rain storm.

It thus seems reasonable to suppose that, whatever the cy- tosolic osmolyte(s), they would be reclaimed to forestall fatal depletion 1431. Osmolyte salvage might then be an essential design feature of the bladders. There is some evidence that the contractile vacuoles in other organisms contain a moderately hypoosmotic fluid [32, 431. Since contractile vacuole bladder membranes presumably have a finite water permeability, retain- ing their osmolytes until just before contraction would minimize the time during which water could be drawn back to the cytosol from the hypoosmotic fluid. This could explain the very acute period of contraction characteristic of these bladders (> 2 s; ref. [9]).

Proton pumping and water excretion. A strong case can be made for the role of proton pumping in water accumulation by the contractile vacuole complex. First, there i s a great abun- dance of V-H+-ATPase in the spongiomes of Dictyostelium [17, 23, 251, Acanthamoeba [24], and Paramecium I l l ] . The spon- giome membranes of other protozoans also show an abundance of large particles on their cytoplasmic faces [21], further evi- dence that a high density of these proton pumps is characteristic of spongiomes. Functional support for this premise is the find- ing that specific inhibitors of V-H+-ATPase promote osmotic swelling in Paramecium [l 11 and Dictyostelium [40]. In addi-

STECK. CHIARAVIGLIO & MEREDITH-OSMOSIS IN DIC7YOSTELIUM 509

tion, the accumulation in the contractile vacuole bladders of Dictyostelium of the acidotropic dye, neutral red, was observed to be blocked by mutation of a subunit of the V-H+-ATPase

The observed level of V-H+-ATPase activity in Dictyostelium [23, 291 is approximately that needed to power the excretion of the volume of water estimated to enter the cells under hypoos- motic conditions (> 0.1 cell volumes/min) (L. Mahadevan and T. L. Steck, unpubl.). This inference is based on the reasonable assumption that the proton gradient drives the antiport of cyt- solic osmolytes (possibly, amino acids) into the lumen of the contractile vacuole spongiome with a stoichiometry of two per ATP hydrolyzed {see 7, 381.

It is puzzling that the sensitive acidotropic fluorophore, ac- ridine orange, is not demonstrably accumulated in contractile vacuole complexes [e.g. 17, 251 even though isolated spongi- omes, as acidosomes, vigorously concentrate acridine orange [30, 311. This finding can be explained if the coupled antiport of cytosolic osmolytes into the spongiomes returns the lumenal protons to the cytosol. It is also possible that the membrane potential generated by the V-H+-ATPase creates an electrical gradient rather than a pH gradient. This premise is unlikely for two reasons: First, the electrical gradient estimated for contrac- tile vacuole bladders, while of the appropriate polarity, is quite small; namely, 10-20 mV [43]. Secondly, isolated spongiome (acidosome) membranes appear to have a large anion conduc- tance pathway which would dissipate any electrical gradient

Despite the robust response of cytosolic amino acids to os- motic stress and that amino acids are antiported by proton gra- dients across other organelle membranes like synaptic vesicles and plant vacuoles [38, 391, certain of our results do not favor amino acid antiport in spongiomes. First, the amino acid excre- tion response to hypoosmotic medium was normal in cells pre- treated with sulfhydryl reagents (NEM and NBD-Cl) which in- hibit the V-H+-ATPase, although cell swelling still occurred (not shown). Secondly, amino acids were not effective in dis- charging either the chemical or the electrical component of the proton gradient established in the acidosomes in homogenates supplemented with MgATP (Fig. 7). (It is conceivable that cell homogenization disrupted this capacity, perhaps removing or diluting a soluble factor.) It is also unlikely that proton symport is the process by which osmolytes are pumped from the bladder to the cytosol. For symport to function in this capacity in the absence of a significant electrical gradient, the bladder proton concentration would have to be comparable to that of the lu- menal osmolytes (presumably > 0.1 M). Not only is the V-H+- ATPase not likely to create such a strong pH gradient [22], but this exceptional acidity should be obvious upon exposing the cells to acridine orange. The putative retrieval of osmolytes from bladders thus remains a mystery.

Involvement of the endocytic pathway. If cytosolic osmo- larity is adjusted by protozoa to maintain a constant intracellular volume in the face of fluctuating external osmolarity, then their osmotically-sensitive cytoplasmic organelles must either vary in volume or adjust their own osmotic load accordingly. The lysosomes in Dictyostelium appear to use both options: in re- sponse to hypoosmotic buffer, they both swell conspicuously (e.g. [40]) and selectively excrete small ingested solutes (Fig. 5). Their rapid release suggests that the ingested probes need not traverse the entire endocytic pathway and be dumped by terminal post-lysosomal vacuoles, as is the case in unstressed cells [28]. Rather, the excreted solutes might be selectively transferred from the endocytic pathway to shuttle vesicles which then fuse with the Dlasma membrane 1361. It mav be

~421.

~291.

sion of clathrin heavy chains becomes swollen in hypoosmotic medium and fails to elaborate large lucent vacuoles [26], al- though the cells still excrete amino acids normally (OUT unpubl. observations).

The function of excretion from the endocytic pathway might not only be to minimize the osmotic load arising from digestion products in the endocytic circuit but also to draw water from the cytosol for excretion. Such a mechanism was suggested long ago for Amoeba miru [20]. In this regard, it appears that the ingested [I4C]amino acids in the endocytic compartment were excreted in preference to those in the cytosol (Fig. 4). Nevertheless, it also seems likely from the large fraction of amino acids excreted (Fig. 1, 2) that most of the pool was cytosolic. Furthermore, endocytosed amino acids were not es- sential to the osmotic response: cells starved of nutrients for an hour responded normally to a subsequent hypoosmotic chal- lenge (not shown). Perhaps the swelling of the endocytic com- partments in cells subjected to a low osmotic stress is fostered by their uptake of amino acids from the cytosol (conceivably by proton antiport), whereupon these solutes are excreted along with their ingested counterparts.

There is intriguing preliminary evidence for a specific con- nection between the endocytic pathway and spongiomes. The two organelles appear to be associated in cell homogenates. This association is reversible, dependent on Mg2+ and cytosolic proteins and does not appear to involve membrane fusion [27, 30, 311. It is therefore conceivable that digestive vacuoles make reversible membrane junctions with spongiomes (perhaps akin to gap junctions) through which digestion products directly pass without entering the cytosol. Both ingested and cytosolic amino acids (upon transfer to the endocytic pathway) could be deliv- ered to the contractile vacuole complex in this way. While we have not observed ingested membrane-impermeable fluorescent probes in either the contractile vacuole complex or in the cy- tosol (not shown), it could be that the fluorophores were too highly diluted in the high-capacity contractile vacuole water stream to be readily visible by conventional fluorescence mi- croscopy.

On the other hand, it could also be that amino acids are excreted by a different mechanism than the ingested dyes. In particular, caffeine caused cells to swell as it inhibited their excretion of ingested dyes in response to hypoosmotic stress. However, it did not alter the release of either resident or newly ingested amino acids (Fig. 6). Furthermore, the kinetics of ex- cretion of ingested dyes differed from that of amino acids, both ingested and resident (Fig. 2 versus Fig. 5).

Given such complexity, we must conclude that it is now too early to construct a satisfying and parsimonious hypothesis for the mechanisms governing water homeostasis in Dictyostelium.

ACKNOWLEDGMENTS We are grateful to Malti Lavasa, Keiki Hinami and Helene

Auer for their excellent technical assistance. We also thank Ter- ry O'Halloran for providing the clathrin deficient Dictyostelium mutant line [26] Aaron Turkewitz for cells of Tetruhymena thermophilu and Karlheinz Altendorf for bafilomycin A, and other inhibitors of V-H+-ATPase. This work was supported by grant GM47282 to TLS from the National Institutes of Health.

LITERATURE CITED 1. Aubry, L., Klein, G . & Satre, M. 1993. Endo-lysosomal acidifi-

cation in Dictyostelium discoideum amoebae. Effects of two endocytosis inhibitors: caffeine and cycloheximide. Eur. J . Cell Biol., 61:225-28.

2. Berthiaume, E. I?, Medina, C . & Swanson, J. A. 1995. Molecular size-fractionation during, endocytosis in macrophates. J. Cell Biol., 129: L~ -

relevant that a mutant of Dictyostelium deficient in the expres- 989-998.

510 J. EUK. MICROBIOL., VOL. 44, NO. 5, SEPTEMBER-OCTOBER 1997

3. Clark, R. L., Retzinger, G. S., & Steck, T. L. 1980. Novel mor- phogenesis in Ax-3, a mutant strain of the cellular slime mould Dic- tyostelium discoideum. J. Gen. Microbiol., 121:3 19-33 1.

4. Cronkite, D. L., Diekman, A. B., Lewallen, B. & Phillips, L. 1993. Aminotransferase and the production of alanine during hyperos- motic stress in Paramecium calkinsi. J. Euk. Microbiol., 40:796-800.

5. Cronkite, D. L., Neuman, J., Walker, D. & Pierce, S. K. 1991. The response of contractile and non-contractile vacuoles of Paramecium calkinsi to widely varying salinities. J. Protozool., 38:565-573.

6. Cronkite, D. L. & Pierce, S. K. 1989. Free amino acids and cell volume regulation in the euryhaline ciliate Paramecium calkinsi. J. Exp. Zool., 251:275-284.

7. Davies, J. M., Hunt, I. & Sanders, D. 1994. Vacuo1arH’-pumping ATPase variable transport coupling ratio controlled by pH. Proc. Natl. Acad. Sci., 91:8547-8551.

8. Drose, S., Bindseil, K. U., Bowman, E. J., Siebers, A., Zeeck, A. & Altendorf, K. 1993. Inhibitory effect of modified bafilomycins and concanamycins on P- and V-type adenosinetriphosphatases. Biochem- istry, 32:3902-3906.

9. Dunham, I? B. & Stoner, L. C. 1969. Indentation of the pellicle of Tetrahymena at the contractile vacuole pore before systole. J. Cell Biol., 43:184-188.

10. Finkelstein, A. 1976. Water and nonelectrolyte permeability of lipid bilayer membranes. J. Gen. Physiol., 68: 127-135.

11. Fok, A. K., Aihara, M. S., Ishida, M., Nolta, K. V. & Steck, T. L. 1995. The pegs on the decorated tubules of the contractile vacuole complex of Paramecium are proton pumps. J. Cell Science, 108:3163- 3170.

12. Gilles, R. 1979. Intracellular organic osmotic effectors. In : Gil- les, R. (ed.), Mechahisms of Osmoregulation in Animals. John Wiley & Sons, New York. Pp. 11 1-154.

13. Goldstein, L. (ed.) 1994. Cellular volume regulation-mecha- nisms and control. J. Exp. Zool., 268:77-175.

14. Halpern, M. L., Kamel, K. S., Ethier, J. H., Stinebaugh, B. J. & Jungas, R. L. 1992. Biochemistry and physiology of ammonium ex- cretion. In: Seldin, D. W. & Giebisch, G. (ed.), The Kidney: Physiology and Pathophysiology. Raven Press, Ltd., New York. Pp. 264-2679.

15. Hawkins, A. J. S & Hilbish, T. J. 1992. The costs of cell volume regulation: protein metabolism during hyperosmotic adjustment. J. Mar. Biol. Ass. UK, 72:569-578.

16. Heinrikson, R. L. & Meredith, S. C. 1984. Amino acid analysis by reverse-phase high-performance liquid chromatography: precolumn derivitization with phenylisothiocyanate. Anal. Biochem., 136:65-74.

17. Heuser, J., Zhu, Q. & Clarke, M. 1993. Proton pumps populate the contractile vacuoles of Dictyostelium amoebae. J. Cell Biol., 121: 1311-1327.

18. Hoffmann, E. K. 1977. Control of cell volume. In : Gupta, B. L., Moreton, R. B., Oschman, J. L. & Wall, B. J. (ed.), Transport of Ions and Water in Animals. Academic Press, New York. Pp. 286-332.

19. Klein, G., Cotter, D. A., Martin, J.-B. & Satre, M. A natural- abundance ‘)C-NMR study of Dictyostelium discoideum metabolism. Eur. J. Biochem., 193: 135-142.

20. Mast, S . 0. & Hopkins, D. L. 1941. Regulation of the water content of Amoeba mira and adaptation to changes in the osmotic con- centration of the surrounding medium. J . Cell Comp. Physiol., 17:31- 48.

21. McKanna, J. A. 1976. Fine structure of fluid segregation organ- elles of Paramecium contractile vacuoles. f. Ultructruct. Res., 54: 1-10.

22. Miiller, M. L., Irkens-Kiersecker, U., Runinstein, B. & Taiz, L. 1996. On the mechanism of hyperacidification in lemon. J . Bid. Chem., 271: 19 16-1924.

23. Nolta, K. V., Padh, H. & Steck, T. L. 1991. Acidosomes from

Dictyostelium: Initial biochemical characterization. J. Biol. Chem., 266: 183 18-1 8323.

24. Nolta, K. V., Padh, H. & Steck, T L. 1993. An immunocyto- chemical analysis of the vacuolar proton pump in Dictyostelium dis- coideum. J. Cell Sci., 105:849-859.

25. Nolta, K. V. & Steck, T. L. 1994. Isolation and initial charac- terization of the bipartite contractile vacuole complex from Dictyoste- lium discoideum. J . Biol. Chem., 269:2225-2233.

26. O’Halloran, T. J. & Anderson, R. G. W. 1992. Clathrin heavy chain is required for pinocytosis, the presence of large vacuoles, and development in Dictyostelium. J. Cell B i d , 118: 1371-1377.

27. Padh, H. 1995. Electromagnetic purification of endocytic vac- uoles and acidosomes from Dictyostelium. Arch. Biochem. Biophys., 316:643-648.

28. Padh, H., Ha, J., Lavasa, M. & Steck, T. L. 1993. A post-lyso- soma1 compartment in Dicryostelium discoideum. J. Biol. Chem., 268: 6742-6747.

29. Padh, H., Lavasa, M. & Steck, T. L. 1989. Proton H+-ATPase in Dictyostelium discoideum. Biochim. Biophys. Acta, 982:27 1-278.

30. Padh, H., Lavasa, M. & Steck, T. L. 1991a. Endosomes are acidified by association with discrete proton-pumping vacuoles in Dic- tyostelium. J. Biol. Chem., 266:55 14-5520.

31. Padh, H., Lavasa, M. & Steck, T. L. 1991b. Reconstitution of the association of endocytic vacuoles and acidosomes from Dictyoste- lium. J . Biol. Chem., 266: 12123-12126.

32. Patterson, D. J. 1980. Contractile vacuoles and associated struc- tures: their organization and function. Bid. Rev., 55: 1-46.

33. Peterson, G. L. 1983. Determination of total protein. Meth. En- zymol., 91:95-119.

34. Prescott, D. M. & Zeuthen, E. 1953. Water diffusion and water filtration across cell surfaces. Acta Physiol. Scand., 28:77-94.

35. Rodriguez-Paris, J . M., Nolta, K. N. & Steck, T. L. 1993. Char- acterization of lysosomes isolated from Dictyostelium discoideum by magnetic fractionation. J. Biol. Chem., 268:9110-9116.

36. Ruscetti, T., Cardelli, J. A., Niswonger, M. L. & O’Halloran, T. J. 1994. Clathrin heavy chain functions in sorting and secretion of lysosomal enzymes in Dictyostelium discoideum. J. Cell Biol., 126:343- 352.

37. Somero, G. 1986. Protons, osniolytes, and fitness of internal milieu for protein function. Am. J. Physiol., 251:R197-R213.

38. Sze, H., Ward, J. M. & Lai, S. 1992. Vacuolar H+-translocating ATPases from plants: structure, function, and isoforms. J. Bioenerg. Biomembr., 24:37 1-38 1.

39. Tabb, J. S., Kish, P. E., van Dyke, R. & Ueda, T. 1992. Gluta- mate transport into synaptic vesicles. J. Biol. Chem., 267: 15412-15418.

40. Temesvari, L. A,, Rodriguez-Paris, J. M., Bush, J. M., Zhang, L. & Cardelli, J. A. 1996. Involvement of the vacuolar proton-translocat- ing ATPase in multiple steps of the endo-lysosomal system and in the contractile vacuole system of Dictyostelium discoideum. J. Cell Science, 109: 1479-1495.

41. Van Rossum, G. D. V., Russo, M. A. & Schisselbauer, J. C. 1987. Role of cytoplasmic vesicles in volume maintenance. Curr. Topics Membr. Transport, 30:45-74.

42. Xie, Y., Coukell, M. B. & Gombos, Z. 1996. Antisense RNA inhibition of the putative vacuolar H+-ATPase proteolipid of Dictyos- relium reduces intracellular Ca2+ transport and cell viability. J. Cell Sci., 109:489-497.

43. Zeuthen, T. 1992. From contractile vacuole to leaky epithelia. Coupling between salt and water fluxes in biological membranes. Bio- chim. Biophys. Acta, 1113:229-258.

Received I I-IS-96, 3-24-97: accepted 6-2-97