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).