Research Article 2339 Osmoregulation in Paramecium: in situ ion gradients permit water to cascade through the cytosol to the contractile vacuole

Christian Stock1,*, Heidi K. Grønlien2, Richard D. Allen1 and Yutaka Naitoh1 1Pacific Biomedical Research Center, Snyder Hall 306, University of Hawaii at Manoa, 2538 The Mall, Honolulu, HI 96822, USA 2Department of Biology, University of Oslo, PO Box 1051, Blindern, N-0316 Oslo, Norway *Author for correspondence (e-mail: [email protected])

Accepted 15 March 2002 Journal of Cell Science 115, 2339-2348 (2002) © The Company of Biologists Ltd

Summary In vivo K+, Na+, Ca2+ and Cl– activities in the cytosol caused concomitant decreases in the cytosolic K+ and Cl– and the contractile vacuole fluid of Paramecium activities that were accompanied by a decrease in the water multimicronucleatum were determined in cells adapted to a segregation activity of the contractile vacuole complex. number of external osmolarities and ionic conditions by This implies that the cytosolic K+ and Cl– are actively co- using ion-selective microelectrodes. It was found that: (1) imported across the plasma membrane. Thus, the osmotic under standardized saline conditions K+ and Cl– were the gradients across both the plasma membrane and the major osmolytes in both the cytosol and the contractile membrane of the contractile vacuole complex ensure a vacuole fluid; and (2) the osmolarity of the contractile controlled cascade of water flow through the cell that can vacuole fluid, determined from K+ and Cl– activities only, provide for osmoregulation as well as the possible extrusion was always more than 1.5 times higher than that of the of metabolic waste by the contractile vacuole complex. cytosol. These findings indicate that excess cytosolic water crosses the contractile vacuole complex membrane Key words: Contractile vacuole complex, Ion-selective osmotically. Substitution of choline or Ca2+ for K+ in the microelectrode, K+/Cl– activity, Osmoregulation, Paramecium external solution or the external application of furosemide multimicronucleatum

Introduction and in the in vivo contractile vacuole (CV) fluid, as well as Osmoregulation in Paramecium multimicronucleatum is based their changes in response to changes in the external osmotic on an intricate interplay between the fluid segregation activity and ionic environments. Therefore, conventional liquid ion- of the contractile vacuole complex (CVC), the regulatory exchanger ion-selective microelectrodes for K+, Na+, Ca2+ and mechanisms that control the cytosolic osmolarity and the water Cl– (Ammann, 1986) were employed to measure the in vivo permeability of the plasma membrane (Stock et al., 2001). By activities of these ions in both the cytosol and the CV fluid in accumulating and expelling the excess cytosolic water that Paramecium cells under standardized conditions, and in cells enters the cell osmotically from the exterior, the CVC keeps adapted to various osmolarities and ionic conditions. This is the cytosolic osmolarity constant independently of the the first time that ion concentrations of the CV of any cell or osmolarity of the external solution as long as the external organism have been measured directly in an in vivo CV. solution remains hypotonic to the cytosol. However, in cells We found that K+ and Cl– are the major osmolytes in both that are long-term adapted to external osmolarities equal to or the in vivo cytosol and the in vivo CV fluid and that the activity higher than the cytosolic osmolarity, the cytosolic osmolarity of these ions as well as the overall fluid osmolarities remain will be shifted to a higher level, allowing the cells to continue higher in the CV than in the cytosol. We therefore propose that to acquire water at these higher external osmolarities, and thus K+ and Cl– transporters are present in both the CVC membrane for the CVC systems to resume cycling and to maintain their and the plasma membrane, and that the control of these fluid segregation activity. transporters is involved in regulating the fluid segregation Not yet understood are: (1) the mechanisms by which activity of the CVC as well as regulating cytosolic osmolarity. cytosolic water is conveyed to the CVC lumen through the CVC membrane, (2) the mechanisms by which the fluid Materials and Methods segregation activity of the CVC responds to a change in the Cells amount of water that enters the cytosol osmotically from the Cells of Paramecium multimicronucleatum (syngen 2) (Allen and Fok, external solution and (3) the mechanisms by which the 1988) were grown in axenic culture medium at 24°C (Fok and Allen, cytosolic osmolarity increases in response to a hypertonic 1979) and harvested at the late logarithmic phase. The cell density increase in the external osmolarity. was 4-6×106 cells l–1. Cell cultures of 12 ml each were centrifuged at To understand these mechanisms, it is of vital importance to 80 g for 25 seconds so that the cells formed a loose pellet. The cells know the ion species and concentrations in the in vivo cytosol were suspended in an adaptation solution (see below) and centrifuged 2340 Journal of Cell Science 115 (11) again into a loose pellet. This washing procedure was repeated twice and the cells were finally suspended in 5 ml of this solution. The cells were kept in this adaptation solution for more than 18 hours prior to experimentation. All experiments were performed at room temperature (25-27°C).

Adaptation solutions We chose to use standard saline as our standard as it has been commonly used by physiologists to study Paramecium (Kamada, 1931; Naitoh and Eckert, 1968). The first set of adaptation solutions (set A) consisted of five solutions with different osmolarities: 24, 64, 104, 124 and 164 mosmol l–1. The osmolarity was adjusted by adding different amounts of sorbitol. Besides the different sorbitol concentrations, the solutions contained (in mmol l–1): 2.0 KCl, 0.25 CaCl2 and 1.0 MOPS-KOH buffer (pH 7.0). The osmolarities of these solutions were measured by using a freezing point depression osmometer (Micro-Osmometer Model 3 MO plus, Advanced Instruments, Norwood MA). The second set (set B) of adaptation solutions consisted of two solutions each with a different osmolarity (24 and 124 mosmol l–1, adjusted by sorbitol). These solutions contained 2.0 mmol l–1 choline chloride instead of the 2.0 mmol l–1 KCl. Other ionic components were the same as those in the solutions of the first set. However, to keep the solution free of any inorganic monovalent cation, no MOPS buffer was added. In order to exclude any effects caused by the absence of the MOPS buffer we examined the CVC activity, RCVC, in buffer-free solutions of set A. RCVC of cells adapted to a MOPS-free 24 mosmol l–1 solution of set A was 65.1±17.4 fl s–1 (n=14) and did not differ from that in cells adapted to MOPS-containing 24 mosmol l–1 solution (72.8±13.1 fl s–1; n=9; Table 3). Thus, the effect of removing the MOPS buffer from these solutions was negligible. Fig. 1. Calibration and application of ion-selective electrodes. (A) K+-selective The third set (set C) of adaptation solutions consisted of – two solutions each with a different osmolarity (24 and electrode. (B) Cl selective electrode. (a) Representative traces of electrical 124 mosmol l–1, adjusted by sorbitol). These solutions potentials from each ion-selective electrode in each of three different –1 –1 calibration solutions of known ionic activities (labeled below each segment of contained 1.25 mmol l CaCl2 instead of 2.0 mmol l KCl. Other ionic components were the same as those in the the trace). (b) Representative traces of electrical potentials from each ion- solutions of the second set (B). selective electrode inserted in situ first into the cytosol and then into the CV. Ionic activities in the cytosol and the CV fluid were estimated from the calibration plots shown in c. (c) Plots of the electrical potentials estimated Ion-selective microelectrodes from the traces in (a) against their respective corresponding ion activities in the Double-barrel borosilicate glass capillaries with filaments calibration solutions. (C) Photographs showing the CV and the double-barreled (1.5 mm in outer and 0.84 mm in inner diameter; World ion-selective electrode (stars) in the cytosol (left) and the CV (right, broken Precision Instruments, Sarasota, FL) were pulled using a line). horizontal micropipette puller (Model P-97, Sutter Instrument Company, Novato, CA) to obtain double-barrel microcapillary pipettes. acetate to measure cells adapted to external osmolarity ranges of 24- Their overall outer tip diameters varied between 3 and 4.5 µm. 64, 104-124 and 164 mosmol l–1, respectively, as the cytosolic The inside glass wall of one barrel of a two-barreled micropipette osmolarities of cells adapted to those external osmolarities were was silanized (Deitmer and Munsch, 1995). A small amount (<1 µl) approximately 70, 170 and 250 mosmol l–1, respectively (Stock et al., of 5% tributylchlorosilane (Fluka Chemical, Milwaukee, WI) in 2001). A silver chloride-coated silver wire (0.25 mm thick) was put carbon tetrachloride (Mallinckrodt Chemical Works, St Louis, MO) into each barrel to conduct the electrical potential difference between was introduced into the tip of the barrel. The pipette was then baked the two barrels to an operational amplifier (INA-114, Burr-Brown, on a hot plate for 5 minutes at 460-500°C. After cooling, the tip of Tucson, AZ) for recording. the measuring electrode was filled with a small amount of liquid K+, The ion sensitivity and its linearity were tested for each electrode Cl–, Na+ or Ca2+ exchange resin (World Precision Instruments, before and after each measurement using one of three different sets Sarasota, FL). The backing solutions for the resin-containing barrels of KCl [Fig. 1A (part c), B (part c)], NaCl or CaCl2 calibration –1 + were either 100 mmol l KCl or NaCl or CaCl2 for either the K - solutions of known ionic activity and a calibration curve was drawn and Cl– or the Na+- or the Ca2+-selective electrodes, respectively. showing the relationship between the electrical potential and the ionic The non-silanized barrel of the microcapillary pipette was filled activity. The ionic activity range of the calibration solutions was with lithium acetate solution and served as the reference electrode. To chosen to include the actual ionic activity to be measured. In order to minimize the tip potential of this barrel, the ionic strength of the imitate the cytosolic ionic activities, the ionic strengths of these lithium acetate was adjusted to approximate that of the cytosol. Based solutions were also adjusted as needed to 70, 170 or 250 mmol l–1 by on the assumption that the cytosolic osmolarity is dependent mostly adding lithium acetate. Partial or complete substitution of NaCl for on monovalent electrolytes, we used 35, 85 and 125 mmol l–1 lithium the lithium acetate in the calibration solution did not make a difference Basis of osmoregulation in Paramecium 2341 in the calibration curve. Readings of the K+ activities obtained with under a coverslip to a thickness of ~26 µm using latex beads with an Na+ present in the calibration solutions hardly differed from those average diameter of 25.7 µm as spacers between the two coverslips. obtained from solutions without Na+: –0.34±0.53 mmol l–1 (n=5) and The cells were left in this condition for approximately 15 minutes –1.6±2.19 mmol l–1 (n=5) in solutions containing 5 and 30 mmol l–1 before the adaptation solution was replaced by a 1 mmol l–1 (final K+, respectively. concentration) furosemide-containing 24 mosmol l–1 adaptation The data obtained for cytosol and CV fluids were analyzed only solution or by a 0.1% (v/v) DMSO-containing adaptation solution, when the ion sensitivity of the electrode as well as its linearity were which served as the control. Images of compressed cells, viewed from the same before and after the measurement. below, were video recorded. The area of the coverslip that is covered by the cell is proportional to the cell volume. It was measured on replayed video images using NIH Image 1.62. The change in cell Determination of ion concentrations in the cytosol and the CV volume was expressed in percent change in pixel numbers that were A minute amount of adaptation solution containing an adapted cell covered by the cell. This was converted into cell area. The edge of the was introduced into a droplet of mineral oil on a coverslip. The cell cell not touching a coverslip was ignored, as it was assumed to be was immobilized by removing excess adaptation solution through a essentially the same before, during and after the volume change. The suction pipette. The tip of an ion-selective microelectrode was then significance of all data was tested using the Mann-Whitney U-test inserted into either the cytosol or into the CV (Fig. 1C) where it was (P<0.05). Values are presented as means±s.e.m. kept for several seconds to obtain a stable potential difference corresponding to the ionic activity of the compartment [Fig. 1A (part b), B (part b)]. The ionic activity was obtained from the calibration Results curve [Fig. 1A (part c), B (part c)]. When the double-barreled Ion activities in the cytosol and the CV fluid under electrode was inserted into the CV, the CV continued to accumulate standardized conditions fluid normally but fluid discharge was blocked. Table 1 shows K+, Na+, Ca2+ and Cl– activities in both the in vivo cytosol and the in vivo CV fluid of P. Determination of the fluid segregation activity by the CVC, multimicronucleatum under standardized saline conditions, (in R –1 CVC mmol l ) 2.0 KCl, 0.25 CaCl2, 20 sorbitol and 1.0 Mops-KOH The experimental procedure for this experiment was identical to that buffer (pH 7.0). Paramecium cells were adapted to this solution previously described (Stock et al., 2001). An experimental chamber for 18 hours. The most abundant ions were K+ and Cl–, in both was filled with a 0.02% poly-L-lysine solution. Cells suspended in an the cytosol and the CV fluid, while Na+, carried over from adaptation solution were introduced into the chamber at one end, earlier culture conditions, was present in only low amounts. while the poly-L-lysine solution was removed from the chamber at This was true even in adaptation solutions containing the other end by absorption with filter paper. If necessary, a solution –1 –1 + exchange was performed by this same means. Cells that adhered to 2.0 mmol l NaCl instead of 2.0 mmol l KCl (Na activity –1 the chamber were used for experimentation. was 3.3±0.9 mmol l (n=10) in the cytosol and 4.8±1.2 mmol Images of the CVs of adhered cells obtained using Nomarski l–1 (n=5) in the CV). We, therefore, focused on K+ and Cl– in microscope optics (Leitz ×63 objective, Leica Mikroskop. u. Sys. the present paper and will provide detailed information about GmbH, Wetzlar, Germany) were video-recorded (ERG-6300, Na+ activities in a subsequent study. As expected, cytosolic Panasonic Industrial, Secaucus, NJ) through a CCD camera (CCD-72, Ca2+ levels were below the sensitivity of the Ca2+-selective DAGE MTI, Michigan City, IN) together with the signals of a video electrode, while, in the CV fluid, Ca2+ was present in only trace timer (FOR. A. Japan). On replayed images of the CV, the period of amounts. The K+ and Cl– activities in the CV fluid were 2.5 to time between two successive fluid discharges and the maximum 2.4 times higher, respectively, than their activities in the cytosol diameter of the CV immediately before the start of fluid discharge (Table 2). were measured. The rate of fluid expulsion by the CVC, RCVC, was calculated by dividing the maximum volume of the CV immediately before fluid discharge (calculated from the diameter of the CV based + – on the assumption that the rounded CV is spherical) by the time that K and Cl activities in cells adapted to increasing had elapsed since the last fluid discharge. Only one CV in each cell osmolarities was measured and its RCVC evaluated. K+ and Cl– activities were determined in both the cytosol and To measure the effects of furosemide (Sigma, St Louis, MO) on the the CV fluid of P. multimicronucleatum cells that had been fluid segregation activity, the 24 mosmol l–1 adaptation solution adapted for 18 hours to 24, 64, 104, 124 or 164 mosmol l–1 containing 2 mmol l–1 K+ was carefully replaced by 1 mmol l–1 furosemide (final concentration) dissolved in 0.1% DMSO (v/v) in the same 24 mosmol l–1 adaption solution. Table 1. Ionic activities in the cytosol and the contractile vacuole (CV) under standardized conditions (standard Determination of the cytosolic osmolarity saline, 24 mosmol l–1) The cytosolic osmolarities of cells adapted for 18 hours to 24 or Ionic activity –1 124 mosmol l solutions of sets A, B and C were determined (mmol l–1) Cytosol CV according to the method previously described (Stock et al., 2001). The K+ 22.6±7.7 (7) 56.0±2.8 (5) method was essentially the same as that employed by Stoner and Na+ 3.92±1.7 (9) 4.67±1.0 (7) Dunham (Stoner and Dunham, 1970) except that we used Congo Red Ca2+ * 0.23±0.13 (5) 14 and a spectrophotometer instead of radioactive C-inulin and a Cl– 27.3±5.9 (9) 66.5±8.3 (6) scintillation counter for our determinations. *Below the innate limit of sensitivity of the Ca2+-selective microelectrode. Each value is a mean±s.d. Numbers in parentheses are numbers of Determination of changes in cell volume measurements of different cells (cytosol) or contractile vacuoles, one CV Cells in 24 mosmol l–1 adaptation solution (set A) were compressed from each cell. 2342 Journal of Cell Science 115 (11)

Table 2. K+ and Cl– activity ratios between the CV fluid and the cytosol Adaptation solution/experimental condition + 2+ External osmolarity K containing Choline containing Ca containing Furosemide DMSO (mosmol l–1)K+ Cl– K+ Cl– K+ Cl– K+ Cl– K+ Cl– 24 2.5 2.4 2.4 2.1 2.3 5.0 2.4 2.4 2.5 2.5 64 2.4 2.5 104 2.1 1.9 124 2.3 2.0 2.5 2.0 2.4 2.0 164 2.1 2.3 solutions all containing the same ionic compositions. In n=5) was significantly higher than that of cells adapted to addition, K+ and Cl– activities were also determined in cells 124 mosmol l–1 (P=0.027). adapted to 24 or 124 mosmol l–1 solutions where an equimolar Fig. 2B (closed circles) shows that the Cl– activities in the –1 –1 choline chloride or CaCl2 concentration was substituted for the CV fluid of cells adapted to 24 mosmol l (66.5±8.3 mmol l ; 2 mmol l–1 KCl. Hereafter, these solutions will be called n=6) or 64 mosmol l–1 (69.7±8.6 mmol l–1; n=5) did not differ choline- or Ca2+-containing solutions. significantly (P=0.55). The Cl– activities in cells adapted to 104 mosmol l–1 (134.6±14.8 mmol l–1; n=7) or 124 mosmol l–1 (131.4±10.4 mmol l–1; n=7) did not differ either (P=0.66). Cells adapted to K+-containing solutions However, the Cl– activity in the CV fluid of cells adapted to Cytosol 104 mosmol l–1 was significantly higher than that of cells As shown in Fig. 2A (open circles), the cytosolic K+ activities adapted to 64 mosmol l–1 (P=2.9×10–6), and the Cl– activity in of cells adapted to external osmolarities of 24 or 64 mosmol l–1 the CV fluid of cells adapted to 164 mosmol l–1 were 22.6±7.7 mmol l–1 (mean±s.d., n=7) or (193.6±21.8 mmol l–1; n=5) was significantly higher than that 21.2±5.8 mmol l–1 (n=11), respectively. They did not differ of cells adapted to 124 mosmol l–1 (P=0.0016). significantly (P=0.69; t-test). The K+ activities in cells adapted The K+ and Cl– activities in the CV fluid were always to 104 mosmol l–1 (62.1±13.7 mmol l–1; n=8) or approximately 2.3 (2.1-2.5) and 2.2 (1.9-2.5) times, 124 mosmol l–1 (60.3±18.9 mmol l–1; n=8) were nearly the respectively, more than those in the cytosol (Table 2). The same (P=0.5). However, the K+ activity in cells adapted to activity ratios were calculated by dividing the value for an ionic 104 mosmol l–1 was significantly higher than that in cells activity in the CV fluid by the corresponding value for the ionic adapted to 64 mosmol l–1 (P=2.7×10–5), and the K+ activity in activity of the same ion in the cytosol. cells adapted to 164 mosmol l–1 (83.9±16.7 mmol l–1; n=9) was significantly higher than that in cells adapted to 124 mosmol l–1 (P=0.017). Cells adapted to choline-containing solutions Fig. 2B (open circles) shows that the cytosolic Cl– activities The K+ and Cl– activities in both the cytosol and the CV fluid of cells adapted to 24 or 64 mosmol l–1 were 27.3±5.9 mmol l–1 were much lower in the choline-containing medium than in the (n=9) or 28.0±2.5 mmol l–1 (n=8), respectively. They did not K+-containing medium (compare Fig. 3Ai,ii with Fig. 3Ci,ii). differ significantly (P=0.76). The Cl– activities in cells adapted The K+ activities in the cytosol were 5.6±1.2 mmol l–1 (n=6) to 104 mosmol l–1 (69.2±4.8 mmol l–1; n=7) or 124 mosmol l–1 or 20.1±1.5 mmol l–1 (n=6), while those in the CV fluid were (66.7±5.5 mmol l–1; n=7) did not differ either (P=0.38). However, the Cl– activity in cells adapted to 104 mosmol l–1 was significantly higher than that in A B cells adapted to 64 mosmol l–1 (P=6.3×10–9), and the Cl– activity in cells adapted to 164 mosmol l–1 (100.2±21.5 mmol l–1; n=5) was significantly higher than that in cells adapted to 124 mosmol l–1 (P=0.02).

CV fluid As shown in Fig. 2A (closed circles), the K+ activities in the CV fluid of cells adapted to 24 mosmol l–1 (56.0±2.8 mmol l–1; n=5) or 64 mosmol l–1 (50.3±8.2 mmol l–1; n=5) did not differ significantly (P=0.2). The K+ activities in cells adapted to 104 (132.3±2.9 mmol l–1; n=5) or 124 mosmol l–1 External osmolarity (mosmol l-1) –1 (140.6±17.8 mmol l ; n=5) were also nearly the same + – + Fig. 2. The relationship of the K (A) or the Cl (B) activity (y-axis) to the (P=0.36). The K activity in the CV fluid of cells external solutions of different osmolarities (x-axis). The Paramecium –1 adapted to 104 mosmol l , however, was significantly multimicronucleatum cells had been adapted for 18 hours to various sorbitol- higher than that of cells adapted to 64 mosmol l–1 adjusted extracellular osmolarities. Open circles show the ionic activities in the (P=4.8×10–6), and the K+ activity in the CV fluid of cytosol, closed circles show the ionic activities in the CV fluid. Each point and cells adapted to 164 mosmol l–1 (176±28.8 mmol l–1; its vertical line is mean±s.d. Basis of osmoregulation in Paramecium 2343

+ Ai Aii adapted to K -containing solutions (compare Fig. 3Bii with Fig. 3Cii). The K+ activities in the cytosol were 8.4±1.6 mmol l–1 (n=9) or 8.3±1.5 mmol l–1 (n=9), while those in the CV fluid were 18.9±3.9 mmol l–1 (n=8) or 20.0±4.3 mmol l–1 (n=5) in cells adapted to 24 or 124 mosmol l–1, respectively. The Cl– activities in the cytosol were 17.1±4.6 mmol l–1 (n=6) or 60±4.2 mmol l–1 )

Bi 1 Bii - –1 l

(n=5), while those in the CV fluid were 86.0±14.1 mmol l l (n=6) or 117.3±8.6 mmol l–1 (n=5) in cells adapted to 24 or 124 mosmol l–1, respectively. The activity ratios between the CV fluid and the cytosol were 2.3 and 2.4 for K+ –

activity (mmo and 5.0 and 2.0 for Cl in cells adapted to 24 and - –1 Cl 124 mosmol l , respectively (Table 2). Ci Cii Ca2+ activities in the CV fluid Because of their innate limit of sensitivity, Ca2+-selective microelectrodes could not be used to detect Ca2+ in the cytosol. However, the Ca2+ activity in the CV fluid could be detected. As shown in Fig. 4, in K+-containing solutions the Ca2+ activities in the CV fluid were 0.23±0.13 mmol l–1 (n=5) and 0.7±0.3 mmol l–1 (n=5) in cells adapted to 24 Fig. 3. K+ (Ai, Bi, Ci) and Cl– activities (Aii, Bii, Cii) in the cytosol and 124 mosmol l–1. These concentrations were slightly (white bars) and in the CV fluid (black bars) of P. multimicronucleatum 2+ higher in cells adapted to solutions containing choline cells adapted to choline-containing solutions (Ai, Aii), to Ca - instead of K+, i.e. 0.7±0.4 mmol l–1 (n=5) in 24 mosmol l–1- containing solutions (Bi, Bii) and to K+-containing standard solutions adapted cells and 1.2±0.5 mmol l–1 (n=5) in that served as controls (Ci, Cii). The osmolarity of the adaptation –1 2+ solution was either 24 mosmol l–1 (left pair of bars) or 124 mosmol l–1 124 mosmol l -adapted cells. However, the Ca activities (right pair of bars). Vertical lines represent s.d. The number of were remarkably higher in the CV fluid of cells adapted measurements varies between 5 and 9; see text for details. to Ca2+-containing solutions. The activities were 15.4±5 mmol l–1 (n=5) and 29.7±8.3 mmol l–1 (n=6) in cells adapted to 24 and 124 mosmol l–1, respectively. 13.6±1.8 mmol l–1 (n=6) or 49.7±6.0 mmol l–1 (n=6) in cells adapted to 24 or 124 mosmol l–1, respectively. The Cl– activities in the cytosol were 11.5±1.1 mmol l–1 (n=6) or Effects of K+-deficiency in the external solution on the 33.1±4.4 mmol l–1 (n=7), while those in the CV fluid were rate of fluid segregation by a CVC (RCVC) and the 24.0±2.2 mmol l–1 (n=7) or 66.0±9.5 mmol l–1 (n=5) in cells cytosolic osmolarity adapted to 24 or 124 mosmol l–1, respectively. The activity As shown in Table 3, the substitution of equimolar choline or 2+ –1 + ratios between the CV fluid and the cytosol were 2.4 and 2.5 Ca for 2 mmol l K caused a marked decrease in RCVC for K+ and 2.1 and 2.0 for Cl– in cells adapted to 24 and accompanied by a decrease in the cytosolic osmolarity. –1 + 124 mosmol l , respectively (Table 2). When choline was substituted for K , RCVC was reduced by more than 50% in cells adapted to 24 mosmol l–1 and by more than 70% in cells adapted to 124 mosmol l–1, while the cytosolic Cells adapted to Ca2+-containing solutions osmolarities were reduced by 50% and by approximately 30%, + 2+ + The K activities in both the cytosol and the CV fluid of cells respectively. When Ca was used as substitute for K , RCVC was adapted to Ca2+-containing solutions (Fig. 3Bi) were much decreased by more than 40% in cells adapted to 24 mosmol l–1 lower than those of cells adapted to K+-containing solutions and by more than 60% in cells adapted to 124 mosmol l–1, while (Fig. 3Ci), whereas the Cl– activities of cells adapted to Ca2+- the cytosolic osmolarities were decreased by more than 55% and containing solutions did not differ that much from those of cells by approximately 21%, respectively.

Table 3. The fluid segregation rate of the CVC, RCVC and the cytosolic osmolarity, Osmc, in Paramecium multimicronucleatum cells adapted to two different osmolarities, 24 and 124 mosmol l–1, in the presence of one out of three different cation species, K+, choline or Ca2+ Osmolarity (mosmol l–1) 24 124 –1 –1 –1 –1 Cation species RCVC (fl s ) Osmc (mosmol l ) RCVC (fl s ) Osmc (mosmol l ) K+ 72.8±13.1 (9) 65.9±4.3 (5) 21.0±11.0 (5) 185.1±21.0 (6) Choline 32.9±16.3 (12) 32.4±5.5 (6) 5.2±7.2 (7) 131.9±7.1 (8) Ca2+ 38.2±11.4 (14) 30.0±4.2 (5) 7.9±2.8 (6) 146.7±7.7 (7)

Each value is a mean±s.d. Numbers in parentheses are numbers of measurements of different cell cultures (Osmc) or different contractile vacuoles (RCVC), one from each cell. 2344 Journal of Cell Science 115 (11)

2+ Fig. 4. Ca activities in the 40 adapted to A CV fluid of K+ 100 P. multimicronucleatum 35 choline ) 2+ adapted to either -1 Ca 80 ) –1 30 24 mosmol l (left three -1 s bars) or 124 mosmol l–1 60 (right three bars). The 25 (fl adaptation solution 40 –1 20 CVC contained either 2 mmol l R + K (white bars) or 20 2 mmol l–1 choline 15

(checked bars) or activity in CV fluid (mmol l –1 2+ 10 0 1.25 mmol l Ca (black 2+ B bars). Each column Ca represents a mean 5 value±s.d. for five or six 0 contractile vacuoles, one 24 124 per cell. External osmolarity (mosmol l-1)

Effects of the external application of furosemide Cells adapted to a 2 mmol l–1 K+-containing 24 mosmol l–1- solution were exposed to the same adaptation solution –1 containing 1 mmol l furosemide (final concentration) initially Fig. 5. Effects of 1 mmol l–1 furosemide on the rate of fluid dissolved in DMSO (final concentration 0.1% v/v). segregation, RCVC (A) and the cell volume (B) of Furosemide has been used as an inhibitor of K+ and Cl– P. multimicronucleatum. (A) Representative results obtained from a transport (for references see Discussion). K+ and Cl– activities single CV. The data in B are mean values±s.d. (vertical lines) were determined 10-20 minutes after cell exposure to obtained from five cells. furosemide. RCVC and cell volume were continuously monitored after the application of furosemide. Cl– activities in either the cytosol or the CV fluid (compare Table 1 with Table 4). K+ activities and Cl– activities As shown in Table 4, the K+ activity in the cytosol decreased by 54% from its control value measured after only the solvent RCVC and cell volume + was applied (0.1% v/v DMSO in the K -containing Fig. 5 shows the time courses of changes in RCVC (Fig. 5A) 24 mosmol l–1 adaptation solution), while the K+ activity in the and in the cell volume (Fig. 5B) after the application of – CV fluid decreased by 57% from its control value. The Cl furosemide. RCVC decreased within 5 minutes of the activity in the cytosol decreased by 52%, while that in the CV application of furosemide from its control value of 89.7±35.4 fluid decreased by 55%. to 37.2±31.2 fl s–1 (n=5), while the cell volume increased in The ratios for the K+ and Cl– activities between the CV fluid 15 minutes by 10.1±4.4% (n=5) over its original volume. The and the cytosol did not change in the presence of furosemide. addition of solvent only, 0.1% DMSO (v/v) dissolved in the The ratio for K+, as well as that for Cl–, was approximately 2.5 K+-containing 24 mosmol l–1 adaptation solution, caused in cells that were not exposed to the drug and 2.4 in cells that neither a decrease in RCVC nor an increase in the cell volume were exposed to the drug (Table 2). The exposure of cells to (data not shown). 0.1% DMSO (v/v) only did not have any effect on the K+ and

Discussion Table 4. Effects of furosemide on the K+ and Cl– activities Ionic activities of the CV under standard conditions in the cytosol and the CV fluid of P. multimicronucleatum Under standardized conditions, we found that the in vivo CV + – Control +Furosemide fluid is hypertonic to the cytosolic fluid. K and Cl activities (0.1% DMSO) (in 0.1% DMSO) in the CV fluid are 2.4-fold higher than they are in the cytosol. K+ activity (mmol l–1) This finding is different from earlier studies that concluded, Cytosol 21.4±2.4 (7) 9.9±2.6 (7) based on micropuncture and freezing-point depression studies, CV 54.7±4.4 (5) 23.8±2.8 (4) that the CV fluids of Chaos carolinensis (Riddick, 1968) and Cl– activity (mmol l–1) Amoeba proteus (Schmidt-Nielsen and Schrauger, 1963) were Cytosol 24.6±4.7 (5) 11.7±2.9 (6) both hypotonic to the cytosol. A hypertonic CV fluid, as we CV 62.4±6.4 (5) 27.8±3.8 (5) find in Paramecium, would permit water to flow down its Measurements were performed 10-20 minutes after the application of concentration gradient from the cytosol into the CV. However, furosemide or DMSO. Each value is a mean±s.d. Numbers in parentheses are this raises the question of how so much K+ and Cl– can enter numbers of measurements of different cells (cytosol) or different contractile the CV against the uphill concentration gradients of these ions. vacuoles (CVs), one from each cell. This question is addressed below. Basis of osmoregulation in Paramecium 2345 KCl is a major osmolyte in the cytosol these three osmolarity ranges are 66, 185 and 240 mosmol l–1, The presence of an ample amount of K+ together with Cl– in respectively (Stock et al., 2001). Thus, the osmolarity of the the cytosol indicates that KCl is potentially the major osmolyte CV fluid is always hypertonic to the cytosol. This fact supports that causes the cytosolic osmolarity to be hypertonic to the the idea that cytosolic water is osmotically conveyed to the external solution. The sum of the cytosolic K+ and Cl– CVC lumen. activities accounted for 67, 66 and 70% of the total cytosolic osmolarity in cells adapted to 24-64, 104-124 and 164 mosmol l–1, respectively. These percentages were obtained A hypothesis for cellular osmoregulation in Paramecium by multiplying the K+ activity by 2 (to account for equimolar Based on our findings, we propose a hypothesis that K+ and amounts of Cl–) and dividing this product by the corresponding Cl– ions are co-transported from the external solution into the cytosolic osmolarity (66, 185 and 240 mosmol l–1, cytosol to keep the cytosol hypertonic to the external solution. respectively) (Stock et al., 2001). The resulting osmotic gradient across the plasma membrane For P. caudatum adapted to external osmolarities of less than will allow water to enter the cell osmotically. At the same time, 64 mosmol l–1 Akita (Akita, 1941) obtained cytosolic K+ the CV fluid is kept hypertonic to the cytosol by the activity of concentrations ranging from 17.2 to 28.3 mmol l–1 using a K+ and Cl– transport systems in the CVC membrane, rather titration method, Yamaguchi (Yamaguchi, 1963) obtained than by bicarbonate-transport, as was proposed earlier values ranging from 16.2 to 22.0 mmol l–1 by using flame (Tominaga et al., 1998). Excess cytosolic water, therefore, can spectrophotometry, and, more recently, Oka et al. (Oka et al., flow osmotically into the CVC lumen. The building up of 1986) obtained a value of 21 mmol l–1 by using an atomic gradients will allow water to cascade from the exterior of the absorption method. Our value of 22 mmol l–1 for P. cell into the cytosol across the plasma membrane and then multimicronucleatum cells adapted to 24-64 mosmol l–1 across the CVC membrane into the CVC. We conclude that this solutions containing K+ obtained by using K+-selective is the basis for keeping the cell volume constant under natural microelectrodes is consistent with these values. In environmental conditions. Tetrahymena pyriformis, the cytosolic K+ concentration ranges –1 from 22 to 44 mmol l , depending on the culture medium used + – (for a review, see Dunham and Kropp, 1973). Support for the hypothesis that K and Cl are the major In P. multimicronucleatum, the remaining 30-34% of the osmolytes for osmoregulation in Paramecium cytosolic osmolarity not accounted for by KCl may consist in Transporting K+ and Cl– into the cytosol from the part of other inorganic ions present in smaller amounts, such external solution is needed to maintain fluid as Na+ (Table 1), and in part by a variety of charged organic segregation activity by the CVC compounds. Free amino acids, such as glycine, alanine and The presence of K+ and Cl– in the CV fluid (Fig. 2, Fig. 3) proline, have been found to act as osmolytes in Miamiensis implies that the cytosolic K+ and Cl– will be constantly avidus (Kaneshiro et al., 1969), Tetrahymena pyriformis expelled to the exterior of the cell. To maintain a normal fluid (Stoner and Dunham, 1970) and P. calkinsi (Cronkite et al., segregation activity, K+ and Cl– should be constantly 1993; Cronkite and Pierce, 1989). resupplied to the cytosol. It is unlikely that K+ and Cl– can be reabsorbed from the CV fluid into the cytosol through the CV membrane before the fluid is expelled as (1) many + – K and Cl activities in the cytosol and in the in vivo CV measurements of the ion activities in the CV fluid were fluid increase in a stepwise fashion as the external obtained just before fluid discharge and (2) slight increases in osmolarity increases linearly K+ ionic activities were detectable when the tips of ion- As shown in Fig. 2, both K+ and Cl– activities in the selective microelectrodes were placed outside the cell adjacent cytosol of P. multimicronucleatum increase in a stepwise to the pore as the CV was discharging (data not shown). Thus, fashion as the external osmolarity is increased linearly by under standard ionic conditions the cytosolic K+ and Cl– are the addition of sorbitol. These stepwise increases reflect assumed to be constantly supplied from the external solution the similar stepwise increases reported to occur in the through the plasma membrane. This idea is supported by our overall cytosolic osmolarity when the external osmolarity findings that cells adapted to solutions deprived of K+ show a approaches or exceeds the cytosolic osmolarity (Stock et al., significant decrease in the overall cytosolic osmolarity as the 2001). cytosolic K+ and Cl– activities [Table 3, Fig. 3A (parts i,ii), B K+ and Cl– are present in the in situ CV fluid and their (parts i,ii)] decrease. At a given external osmolarity a decrease activities, which are always higher in the CV than in the in the overall cytosolic osmolarity will lead to a decrease in the cytosol, increase in the same stepwise fashion as the cytosolic osmotic gradient across the plasma membrane and, therefore, + – + K and Cl activities (Fig. 2). This result indicates that K and to a reduction of RCVC (Table 3). Cl– are transported from the cytosol into the CV where they are concentrated. Effects of furosemide on K+ and Cl– activities and on cell volume The CV fluid is always hypertonic to the cytosol Furosemide is known to inhibit the reabsorption of Na+, K+ The sum total of the K+ plus the Cl– activities in the CV fluid and Cl– in the ascending limb of the loop of Henle (Brater, is approximately 120, 264 or 365 mmol l–1 in cells adapted to 1998), the [Na+, K+, Cl–] co-transport and the [K+, Cl–] co- external osmolarities of 24-64, 104-124 or 164 mosmol l–1, transport in mammalian erythrocytes (Lauf, 1984; Canessa et respectively. The cytosolic osmolarities in cells adapted to al., 1986; Garay et al., 1988). In Paramecium, the primary 2346 Journal of Cell Science 115 (11) effect of furosemide should, therefore, be essentially the same in cells adapted to K+ containing 24 and 124 mosmol l–1 as that of eliminating K+ from the external solution. Adding solutions, respectively (Fig. 3C, Table 3). As in other cells, furosemide to the external medium resulted in a reduction of choline may be actively transported from the surrounding both the K+ activity and the Cl– activity in the cytosol (Table solution through the plasma membrane into the cytosol, + – 4). RCVC decreased as the K and Cl activities in the cytosol although a choline transporter has not yet been identified in and in the CV fluid decreased (Fig. 5A). Furosemide the plasma membrane of Paramecium. Choline transporters presumably inhibits the K+ and Cl– transport through the have been found in a variety of cell species from cells of the plasma membrane. Whether furosemide can directly reduce central nervous system (Knipper et al., 1991; Laganiere et al., the K+ and Cl– transport across the CVC membrane is not 1991) to yeast (Li et al., 1991) and even in membranes of known, but is unlikely as the K+ and Cl– activity ratios organelles such as the inner membranes of rat liver between the cytosol and the CV fluid are unaltered in cells mitochondria (Porter et al., 1992). In bacteria such as treated with furosemide (Table 2). The cell volume Escherichia coli and Bacillus subtilis, choline is taken up and temporarily increased, while RCVC decreased after the cell was converted to betaine, which is then used as osmoprotectant in subjected to furosemide (Fig. 5). This implies that the osmotic high osmolarity environments (Kempf and Bremer, 1998a; water flow into the cell across the plasma membrane exceeds Kempf and Bremer, 1998b). the water flow into the CVC. In a Paramecium cell, the total When CaCl2 was substituted for the external KCl, the membrane area of the CVC is almost the same as that of the percentage of KCl in the overall cytosolic osmolytes was 56% plasma membrane [~30×108 µm2 for the CVC membrane and 11% in cells adapted to 24 and 124 mosmol l–1, (Tominaga et al., 1998; Tominaga et al., 1999) and ~40×108 respectively (Fig. 3B, Table 3). The cytosolic Cl– activity was µm2 for the plasma membrane (Stock et al., 2001)]. The water two times and seven times larger than the cytosolic K+ activity permeability of these two membranes is also the same in cells adapted to 24 and 124 mosmol l–1, respectively. This [~0.17×10–5 µm min–1 Pa–1 for the CVC membrane (C. S. et implies that there must be counter-cations for Cl– other than al., unpublished) and 0.18×10–5 µm min–1 Pa–1 for the plasma K+ present in the cytosol. Ca2+ can not be a counter-cation for membrane (Stock et al., 2001)]. Furosemide, therefore, may Cl– in the cytosol, because of its low cytosolic activity, less cause the osmotic gradient across the CVC membrane to be than 10–7 mol l–1 (Naitoh and Kaneko, 1972; Nakaoka et al., lower than that across the plasma membrane. Upon exposure 1984; Machemer, 1989), a figure that is related to its diverse to furosemide, the sum of K+ and Cl– activities decreased from functions as second messenger and regulatory ion. Arginine 46 to 22 mmol l–1 in the cytosol and from 117 to 52 mmol l–1 and lysine as well as oligopeptides with an alkaline isoelectric in the CV fluid (Table 4). If we assume that the cytosolic point may be plausible candidates as the counter-cations for osmolytes other than K+ and Cl– (a total of 66- Cl– in a pH-neutral cytosol. 46=20 mosmol l–1 before furosemide treatment) cannot In contrast to a marked decrease in K+ activity in the CV change as quickly as the cytosolic K+ and Cl– activities, the fluid, the Cl– activity in the CV fluid decreased little or even osmotic gradient across the plasma membrane will be increased following Ca2+ substitution for the external K+ (Fig. approximately 18 mosmol l–1 (22+20-24=18), while that 3B, part ii). It increased by 23% in cells adapted to across the CVC membrane will be (10+α) mosmol l–1 (52-22- 24 mosmol l–1 and decreased by 11% in cells adapted to 20=10), where α represents the activity of osmolytes other 124 mosmol l–1 (compare Fig. 3B, part ii with Fig. 3C, part ii). than K+ and Cl– in the CV fluid. As α can be assumed to be This indicates that only a small fraction of the Cl– ions are a small fraction of the total CV osmolarity, the total osmotic acting as counter-anions for K+ ions in the CV fluid of these gradient across the CVC membrane should be smaller than cells, and that, as in the cytosol, there must be cations other that across the plasma membrane. The cell will consequently than K+ in the CV fluid. swell, as was observed (Fig. 5B). As shown in Fig. 4, the Ca2+ activity in the CV fluid markedly increased under Ca2+-containing, K+-deficient conditions. This implies that Ca2+ is actively transported from The CV/cytosol ratios for K+ activity and Cl– activity the cytosol into the CVC lumen to keep the CV fluid hypertonic remain constant independently of RCVC to the cytosol. In fact, the presence of CaCl2 in the CV fluid In cells adapted to osmolarity ranges of 24 to 164 mosmol l–1, can account for 35% and 59% of the Cl– activity in the CV the ratio for K+-activity varied in a narrow range of 2.1-2.5 and fluid of cells adapted to 24 and 124 mosmol l–1, respectively. that for Cl– activity varied between 1.9-2.5 (Table 2), whereas Thus, under conditions of Ca2+ stress, the CVC can play a role –1 2+ RCVC decreased from approximately 100 to 10 fl s (Stock et in the extrusion of excess cytosolic Ca that would be al., 2001). The ratio for the K+ activity (~2.3) and that for the deleterious to many Ca2+-dependent intracellular signaling Cl– activity (~2.2) are nearly equal. This may indicate that systems. Ca2+ can enter the cytosol from the external solution much of the K+ and Cl– are, indeed, co-transported by a single via stimulus-activated Ca2+ channels in the plasma membrane transporter in the CVC membrane. and can also be released from proven intracellular Ca2+ storage sites such as alveoli (for a review, see Plattner and Klauke, 2001). Moniakis et al. (Moniakis et al., 1999) report a Ca2+- KCl is not a major osmolyte in the cytosol under ATPase in Dictyostelium discoideum that is localized to the K+-deficient conditions membranes of the CV in this cell. The gene expression for this The percentage of KCl in the overall cytosolic osmolytes Ca2+-ATPase is upregulated when cells are grown in Ca2+-rich reached only 35% and 31% in cells adapted to choline medium. 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