Osmoregulation and Contractile Vacuole Function

We have made a major advance in determining how changes in the external osmotic environment affect several osmotic parameteres in the living Paramecium. Using the rate of fluid expulsion (R_CVC) through the contractile vacuole complex (CVC) as the assay, we found that short term exposure to a strongly hypotonic solution resulted in the cell's ability to quickly adjust to subsequent changes in external osmolarity. Short-term exposure to less hypotonic solutions resulted in the cell requiring longer times to adjust to these external changes. Exposure to isotonic or hypertonic solutions resulted in much longer times required for adjustment to subsequent changes. In all cases the cell is able to control its rate of fluid expulson (R_CVC) with the result that it keeps its cytosolic osmolarity hypertonic to its environment. Even when cells are kept in a strongly hypertonic solution, which would appear to eliminate the necessity for further contractile vacuole (CV) activity, it was found that after a period of 12h the CV activity would commence again. Whenever the external solution changed the cytosolic osmolarity always adjusted to again become hypertonic to the external solution. However, the cytosolic osmolarity did not increase linearly in relation to the external osmolarity but it rose in steps, increasing rapidly as the cytosolic osmolarity came close to being isotonic to the external osmolarity and then leveling off at a new higher plateau where it remained until the external osmolarity again reached a level close to isotonicity. Stepwise increases of cytosolic osmolarity occurred when the external osmolarity rose from 64 mosm l^-1 to 84 mosm l^-1 and again between 144 mosm l^-1 and 164 mosm l^-1. This argues that the cell needs to reestablish water segregation activity even after it has been exposed to an isotonic or hypertonic environment and that the CVC has other functions besides water balance such as the elimination of metabolic waste and/or excess ions. This work was published by Stock et al., (2001).

Figure 1. The cytosolic osmolarity in cells of P. multimicronucleatum adapted to eleven different osmolarities for 12h. Cytosolic osmolarity increases step-wise. Taken from Figure 5 in Stock et al., 2001.		Figure 2. The relationship of the K+ (A) or the Cl- (B) activity (y-axis) to the external solutions of different osmolarities (x-axis). The P. multimicronucleatum cells had been adapted for 18 hours to various sorbitol-adjusted extracellular osmolarities. Open circles show the ionic activities in the cytosol, closed circles show the ionic activities in the CV fluid. Each point and its vertical line is mean±s.d. Ion activities in both the cytosol and CV increase step-wise. Taken from Figure 2 in Stock et al., 2002a.

From the beginning of our studies in 1995 on the contractile vacuole it has always been our intention to determine how this organelle is able to sequester fluid from the cytosol and thereby regulate the cells cytosolic osmolarity. To do this we needed to know how water could be moved into the CV, if this was via osmosis along the water's concentration gradient into a hypertonic CV solution or by some active water accumulation system in which the CV content could remain hypotonic as had been reported earlier for amoeba. We have now been able to show that the CV of Paramecium is in fact hypertonic to the cytosol and that the major ions that are accumulated in the CV fluid are K⁺ and Cl^- (Stock et al., 2002a). In fact K⁺ is always kept at a level ~2.4x higher in the CV than it is in the cytosol. This was found to be true under several experimental conditions where changes in the external osmolarity and/or external ion species resulted in concomitant changes in the osmolarity of the cytosol and, in turn, the CV.

To measure the ion species in the in vivo CV as well as the in vivo cytosol our lab perfected the in situ ion-selective microelectrode technique to be used on intracellular organelles of living Paramecium. Measurements of K⁺ and Cl^- activities in the cytosol (Stock et al., 2002a) showed that increases in the K⁺ and Cl^- activities followed the same stepwise increase as did the cytosolic osmolarity as the external osmolarity was increased (Stock et al., 2001). We conclude from our experiments that i) the K⁺ and Cl^- activities of the cytosol increase with increasing external osmolarity in the same stepwise fashion as we reported for the cytosolic osmolarity, even though the external K⁺ and Cl^- concentrations do not change and ii) the K⁺ activity in the CV is maintained ~2.4x higher than that in the cytosol. The rate of water segregation (R_CVC) was not found to be directly related to the increase in the K⁺ activity in the CV.

Using ion-selective microelectrodes inserted first into the cytosol and then into the contractile vacuole (CV) of a living cell we determined and compared the concentrations of K⁺, Na⁺, Ca²⁺, and Cl^- activities in these two separate compartments. Cells were initially bathed in standard saline solution consisting of (in mmol l^-1) 2.0 KCl, 0.25 CaCl₂ and 1.0 MOPS-KOH buffer (pH 7.0) that had an osmolarity, as determined by the freezing-point depression technique, of 4 mosmol l^-1(Stock et al., 2002a). The external osmolarity was adjusted by the addition of sorbitol, a non-permeant sugar, to 24, 64, 104, 124 or 164 mosmol l^-1. Batches of cells were then adapted to each osmolarity for 18 h and ion activities under each condition were determined. The following observations and conclusions were obtained (Stock et al., 2002a)

1. At 24 mosmol l^-1 the cytosol contained (in mmol l^-1) 22.6 K⁺, 3.92 Na⁺, 27.3 Cl^-, and only a trace of Ca²⁺ (below the accurate measuring ability of the microelectrode technique). On the other hand, under the same 24 mosmol l^-1 conditions, the CV contained 56.0 K⁺, 4.67 Na⁺, 66.5 Cl^-, and 0.23 Ca²⁺, values that were significantly higher than the cyosolic concentrations for the corresponding ions. Thus the CV fluid is in fact hypertonic to the cytosol and water can flow into the CV by osmosis down its concentration gradient.
2. In cells that were adapted for 18 h to one of several higher external osmolarities, the activities of K⁺ and Cl^- in the CV also increased but, as was found to be true of the overall cytosolic osmolarity (Stock et al., 2001), these increases were stepwise, occurring only between 64 and 104 and between 124 and 164 mosmol l^-1, i.e. when the external osmolarity approaches the cytosolic osmolarity. Thus the cell seems to adjust its CV osmolarity only when the external osmolarity approaches the cytosolic osmolarity.
3. Another major observation was that the two ions present in highest concentration in both the cytosol and in the CV were K⁺ and Cl^- and that the ratio for K⁺ concentrations between these two compartments under a number of external osmolarities remains close to 2.4, i.e. the concentration in the CV is always around 2.4x higher than the cytosol. The ratio for Cl^- varied from 1.9 to 2.5. These ratios were the same, except for Cl^- in Ca²⁺-containing solutions, even when the external cationic species was changed, as when Ca²⁺ was substituted for K⁺ or when the organic ion choline was substituted for K⁺. We conclude that the plasma membrane determines the ionic composition of the cellÌs cytosol and it is the job of the CVC membrane to keep the K⁺ concentration of the CV nearly 2.4x the cytosolic concentration.
4. When cells in the standard saline medium were adapted 18 h to an external osmolarity of 124 mosmol l^-1, whether in K⁺-containing, Ca²⁺-containing or choline-containing medium, K⁺ and Cl^- activities generally rose significantly in the cytosol compared to their concentrations in 24 mosmol l^-1 but the K⁺ and Cl^- ratios between the cytosol and CV still remained between 2.0 and 2.5.
5. Fluid segregation rates of the CVC in cells adapted to K⁺-containing solutions were much higher than in those cells adapted to choline- or Ca²⁺-containing medium. This was true for external osmolarities of both 24 and 124 mosmol l^-1 although the rates were always much lower in 124 mosmol l^-1 adapted cells. Under the conditions tested the most water per unit of time expelled from a CVC occurred in cells adapted to high K⁺-containing solutions at 24 mosmol l^-1 of external osmolarity.
6. The drug Furosemide, an inhibitor of K⁺ and Cl^- transport across membranes, reduced both K⁺ and Cl^- activities by approximately 60% but did not alter the 2.0 to 2.5 ionic ratios between the cytosol and CVC. This effect resulted in a reduced water expulsion rate that, in turn, resulted in an initial swelling of the cell by 8 to 10% over the first 15 to 20 minutes of exposure.
7. One other observation was that the CVC can accumulate Ca²⁺ to a remarkable extent, much higher than 2.4x. Like other cells the Ca²⁺ concentration in the cytosol of Paramecium is maintained at a very low concentration (~10^-7 M) but this concentration can rise to 15 to 30 mmol l^-1 in the CVC in cells adapted to high Ca²⁺-containing medium, a rise of at least 43,000x over the cytosolic concentration.

The above method has now been extended to see how the CV fluid content is altered when cells are exposed to different external ions, combination of ions and to different concentrations of the same ions at two different external osmolarities (Stock et al., 2002b). Cells were exposed to 2 mM K⁺, 2 mM Na⁺, 1 mM K⁺ + 1 mM Na⁺ or 2 mM choline, all containing 1.25 mM Ca²⁺. The external osmolarities were adjusted to either 24 or 124 mosmol l^-1 by adding sorbitol. Results are summarized in Table 1 taken from Table V in Stock et al., 2002b.

Table I. Overview of the ionic activities in the contractile vacuole, the overrall cytosolic osmolarity, the estimated osmotic gradient across the contractile vacuole membrane, the rate of fluid segregation and the membrane potential of the contractile vacuole complex in P. multimicronucleatum cells adapted to either mosmol l-1 or mosmol l-1 solutions containing either K+ plus Na+, Na+ alone, K+ alone or choline as the monovalent cation(s).aionCV: Cl-, Na+, K+ and Ca2+ activities in the contractile vacuole; aionsCV: the sum of all ion activities measured in the CV; Osmc: the overall cytosolic osmolarity; RCVC: the rate of fluid segregation; VCVC: the CVC membrane potential. The values shown are taken from Stock et al., 2002b. (*) estimated by subtracting Osmc from aionsCV.

1. This set of experiments again confirms that the CV fluid is always kept hypertonic to the cytosol,
2. The water segregation rate for a given external osmolarity is positively (but not linearly) correlated with the estimated osmotic gradient across the contractile vacuole membrane. Thus, excess water enters the CV by osmosis and is eliminated from the cell when the CV membrane fuses with the plasma membrane.
3. The chloride ion serves as the counterbalancing anion for the total K⁺, Na⁺ and Ca²⁺ cation concentrations in the CV fluid.
4. Na⁺ can be used as an osmolyte but accumulates to its highest concentration if both K⁺ and Na⁺ are present in the external medium.
5. A combination of Na⁺ and K⁺ results in the highest CV osmotic gradient across the CV membrane and, in addition, the highest fluid segregation rates but does not alter the cytosolic osmolarity.
6. Both the plasma membrane and the CVC membranes play roles in fluid segregation.
7. The presence of Na⁺ moderated (reduced) K⁺ uptake but dramatically increased Ca²⁺ activity in the CV fluid. Thus, under certain conditions, we confirm that excess Ca²⁺ might play a role as CV electrolyte and so be eliminated through the CVC.
8. The pH of the cytosol is 7.0 and that of the CV is 6.4, which is only mildly acidic.

Microelectrodes were also used to measure the in situ CV potentials and membrane input resistance at the same time that video images of the CVC were recorded (Gr¯nlien et al., 2002). Cells were in standard saline solution and, as before, the osmolarity of each bathing solution was adjusted to different levels by adding various concentrations of sorbitol. In separate studies cells subjected to the same protocols were fixed, permeabilized and labeled with the DS-1 monoclonal antibody for indirect immunofluorescence studies. DS-1 is specific for V-ATPases that reside on the decorated tubules. In some cases the cells were compressed to the extent that functioning of the CVC was altered and could be resolved into a series of steps. From this study the following conclusions were reached:

1. The CV membrane potential of P. multimicronucleatum during the filling phase of the CV maintains a steady level of 77-80 mV but during each rounding phase the potential precipitously drops to 11-13 mV. Recovery occurs only at the start of the next filling phase and recovery is rapid. Input resistance is highest, approximately 160 M, during the rounding phase and falls to 60 M during fluid filling. Thus conductance of the entire CVC membrane is higher than the CV membrane alone. This confirmed the work of Tominaga et al., (1998) that the radial arms that bare the V-ATPases become detached from the CV during the filling phase.
2. Mechanically compressed cells demonstrate that the electrical membrane potential rises or decreases in stepwise fashion. An individual step represents the reattachment or detachment of one arm with or from the CV, respectively (Figure 5 from Gr¯nlien et al., 2002).

   Figure 3. (A) A schematic representation of the contractile vacuole (CV) complex in Paramecium multimicronucleatum to show the pathways of the electric currents generated by V-ATPases in the radial arms. CV, contractile vacuole; RA1-RAN, the radial arms 1-N, where N is the total number of radial arms; iH+, the current generated in a single radial arm; iRA, the passive current across the radial arm membrane caused by iH+; iCV, the passive current across the CV membrane caused by iH+ of those radial arms attached to the CV. (B) An electric circuit equivalent to A. rCV, the input resistance of the CV; rRA, the input resistance of a radial arm; SW, a switch corresponding to the attachment of the radial arm to (on) or detachment of the RA from (off) the CV; eCV, CV potential. (C) Simulated stepwise changes in the eCV based on the equivalent circuit (B) as a total of eight radial arms attach to the CV one by one. Numbers to the left signify potential steps as the radial arms are attached to the CV one by one.

3. When cells were adapted 18 h to increases in external osmolarity between 4 and 124 mosmol l^-1 the CV membrane potential was unaffected but there was a major affect on the rate of fluid segregation, R_CVC, which decreased as the external osmolarity went from 4 to 64 mosmol l^-1 and then plateaued again between 64 and 124 mosmol l^-1.
4. Immunofluorescence of CVCs indicated that the arms and their decorated tubules appeared morphologically normal in cells that had been adapted for 18 h to osmolarities of 4, 64 and 124 mosmol l^-1 even though the R_CVC decreased to a low level at 64 and 124 mosmol l^-1.
5. Returning a 124 mosmol l^-1 adapted cell to a hypotonic environment of 4 mosmol l^-1 had no affect on the membrane potential but after only 30 min the rate of fluid segregation (R_CVC) had gone from 20 to103 fl s^-1, a five-fold increase.
6. Cells 18-h adapted to 4 mosmol l^-1 then moved to 124 mosmol l^-1 for 30 min lose their CVC decorated tubule labeling. During this time the membrane potential presumably drops significantly and R_CVC will also drop to nearly 0. Returning the cell to 4 mosmol l^-1 after the 30 min pulse restores the membrane potential back to its filling phase level within 60 min and R_CVC returns to 115 fl s^-1 in 60 min. Fluorescence images show that decorated tubules are completely dispersed in cells exposed to 124 mosmol l^-1 for 30 min. Returning the cell to 4 mosmol l^-1 resulted in the recovery of the CVC and its decorated tubules within 60 min.
7. Cells adapted to increases in external osmolarity for 18 h did not show altered membrane electric potential nor did the morphological appearance of their decorated tubules change. Thus the alterations observed after 30 min of hypertonic stress in the decorated tubule morphology and the decreased CV membrane potential are restored in 18 h adapted cells. These parameters actually return to normal after only 60 min.

We believe that the engine that drives the CV water elimination process in the CV makes use of at least two forces, the proton translocating V-ATPases which are highly concentrated on the decorated spongiome membrane of the CVC (Fok et al., 1995, 2002; Gr¯nlien et al., 2002) and a tension-developing force located in the smooth spongiome (Tominaga et al., 1998b; Tani et al., 2000, 2001). The V-ATPase pumps establish a proton gradient that contributes to a positive electric potential across the CVC membrane of +60 to 80 mV (Tominaga et al., 1998a; Gr¯nlien et al., 2002) yet these pumps do not produce a strongly acid CV, the pH of the in situ CV is only mildly acidic (Stock et al., 2002b). Our current hypothesis is that protons in the CVC are probably exchanged for other cations which establish the final osmotic gradient in the CV. Thus water will flow into the CV down its concentration gradient. We must now use patch-clamp techniques to look for proton/cation exchangers in the CVC membranes to account for the accumulation of K⁺, Na⁺ and/or Ca²⁺ in the CV fluid. Cl^- channels and K⁺/Cl^- cotransporters will also be sought. The second engine produces an increased membrane tension in the smooth spongiome membrane that drives the CVC membrane dynamics including the CV Ìs ability to fuse with the plasma membrane, to release radial arms from the CV and to determine the timing of the CV rounding/relaxing cycle.

A fourth paper published in 2002 (Fok et al., 2002) reported the sequencing of the V-ATPase B subunit gene from Paramecium multimicronucleatum. This gene codes for a 56.8 kDa protein that has a 75% sequence identity, except for short N- and C-terminal regions, with the B subunits of other widely separated organisms such as yeast and man. Double monoclonal antibody labeling with the SS-1 (to the smooth spongiome) and DS-1 (probably to the E subunit of the V-ATPase) confirms that the CVC development begins by pore formation followed by smooth and decorated spongiome development almost at the same time. New CVC development is also demonstrated to be synchronized with nuclear division. Finally we were able to show by the quick-freeze deep-etch technique that much of the decorated spongiome becomes vesiculated and separates from the smooth spongiome during CVC reorganization that always accompanies new CVC formation. During vesiculation the V-ATPase-containing 20 x 30 nm "pegs" on the decorated tubules appear to disperse into 6 10-nm units which resemble the size of V-ATPases in other organisms. Thus V-ATPases seem to form compact complexes of 6 subunits in the CVC membrane.

This work has been and continues to be supported by the National Science Foundation, USA.

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