The freshwater protozoan Paramecium multimicronucleatum maintains a constant cytosolic osmolarity through the exocytotic activity of its contractile vacuole complex. The contractile vacuole (CV) expels the excess cytosolic water, acquired osmotically, to the exterior of the cell at fairly regular intervals. In a single exocytotic cycle, the CV swells as the cytosolic water enters the vacuole, rounds up, and then rapidly shrinks as the fluid is expelled through the pore of the CV. The exocytotic activity of the CV complex can sometimes be slowed and distorted by microelectrode impalement. Using a CV with exocytotic activity slowed in this way makes it possible to determine more precisely the time course of changes in the exocytotic-activity-related membrane capacitance and membrane potential of the organelle. We have clearly demonstrated that the radial arms of the CV were always severed after the CV had exhibited rounding. Microelectrode impalement sometimes caused a failure of the CV pore to open after rounding up, so that the CV entered the next fluid-filling phase without expelling its fluid. The radial arms remained severed from the CV during such prolonged rounding phases and then rejoined the CV at the start of the next fluid-filling phase. The rounding of the CV corresponds to an increase in the tension of the CV membrane. This suggested that the periodic development of increased tension in the CV membrane might be the primary event leading to periodic severing of the radial arms and the opening of the pore. We then observed that the CV and its radial arms sometimes became fragmented into vesicles when the cell had been mechanically ruptured in a salt solution. Many of the resulting in vitro vesicles showed periodic rounding and slackening which occurred at different times so that they were out of phase one with the other. This indicates that the membranes of the CV and the radial arms maintain their ability to develop a periodic increase in tension even after the cell has been ruptured. We propose the hypothesis that the CV membrane (together with its associated cytoskeletal structures) possesses a mechanism by which its tension is periodically increased. Such a periodic change in membrane tension may govern the exocytotic cycle of the contractile vacuole complex by increasing the tension to a point that the radial arms sever from the CV and the pore opens. Conversely, a decrease in the tension causes closure of the pore and rejoining of the radial arms to the CV. Transformation of the CV membrane into 40 nm tubules is assumed to be responsible for the development of tension in the planar CV membrane. The causes of this periodic tubulation must now be sought.

In the freshwater protozoan Paramecium multomicronucleatum, excess cytosolic water, acquired osmotically, is segregated and expelled to the cell exterior through the activity of the contractile vacuole complex. This process keeps the cell volume constant. The electrophysiological parameters of the organelle were measured in situ using a fine-tipped microelectrode inserted into the contractile vacuole, the exocytotic vesicle of the organelle to which the segregated fluid is transported before being expelled to the exterior. The input capacitance decreased markedly immediately before fluid expulsion and regained its previous value when fluid filling resumed after fluid expulsion. This change in the capacitance proved that the contractile vacuole became disconnected from its radial arms, which project from the vacuole, before fluid expulsion occurred and then reconnected with the arms after fluid expulsion. A positive electrical potential was recorded from the contractile vacuole only when it was connected to the radial arms. This implies that the electrogenic mechanism resides exclusively in the radial arms and supports the idea that the decorated spongiomes, V-type proton-pump-covered terminal tubules of the radial arms that end blindly in the cytosol, are electrogenic. The linear current­voltage relationship of the contractile vacuole membrane also implies that few voltage-activated ion channels are present in the membrane. To explain the movement of water into the contractile vacuole complex, we favour the hypothesis that the potential generated across the decorated spongiome membrane can be used to drive counter-anions from the cytosol into the lumen of the complex. The anions could then act as an osmolite to pull cytosolic water into the lumen of the organelle.

To examine the forces needed for discharge of the fluid contents from the contractile vacuole of Paramecium multimicronucleatum, the time course of the decrease in vacuole diameter during systole (the fluid-discharging period) was compared with that of various vacuole discharge models. The observed time course did not fit that predicted by a model in which contraction of an actin­myosin network surrounding the vacuole caused discharge nor that predicted by a model in which the surface tension of the lipid bilayer of the vacuole caused discharge. Rather, it fitted that predicted by a model in which the cell's cytosolic pressure was responsible for discharge. Cytochalasin B, an effective inhibitor of actin polymerization, had no effect on the in vivo time course of systole. An injection of a monoclonal antibody raised against the proton pumps of the decorated spongiomes (now known to be the locus of fluid segregation in P. multimicronucleatum) disrupted the decorated spongiomes and reduced the rate of fluid segregation, whereas it did not alter the time course of systole. We conclude that in P. multimicronucleatum the internal pressure of the contractile vacuole is caused predominantly by the cytosolic pressure and that the fluid-segregation mechanism does not directly affect the fluid-discharge mechanism. Elimination of this cytosolic pressure by rupturing the cell revealed the presence of a novel fluid-discharge mechanism, apparently centered in the vacuole membrane. The involvement of tubulation of the vacuole membrane as the force-generating mechanism for fluid discharge in disrupted cells is discussed.

The precise relationship between the rate of contractile vacuole fluid discharge and the vacuole diameter at the start of systole was determined in cells of Paramecium multimicronucleatum subjected to various external conditions. The rate of discharge was higher when the diameter was larger. When the rate of discharge was plotted against the diameter, the points fell around a single parabolic line passing through the origin and were independent of the external conditions employed. This implies that the rate of discharge is proportional to the square of the vacuole diameter. We have previously proposed a hypothesis in which membrane tension in the contractile vacuole is altered as its planar membrane becomes tubular or as tubules become planar membrane (termed the membrane area-proportional tension hypothesis). We propose here that it is this change in membrane tension which determines the vacuole pore shape and sets the subsequent rate of fluid discharge.

1. A voltage-clamped Paramecium produced an inward membrane current upon thermal or mechanical stimulation of its anterior region, whereas it produced an outward membrane current upon similar stimulation of its posterior region. 2. Anterior thermo- and mechanoreceptor currents decreased when the membrane potential was shifted in a positive direction, showing sign reversal at a positive membrane potential, whereas posterior thermo- and mechanoreceptor currents decreased when the membrane potential was shifted in a negative direction, showing sign reversal at a membrane potential more negative than the resting potential. 3. The reversal potential for both anterior receptor currents shifted in a positive direction when external [Ca2+] was increased, whereas those for both posterior receptor currents shifted in a positive direction when external [K+] was increased. 4. External [Mg2+], [Mn2+], [Na+], [Rb+] and [TEA+] had similar effects on the thermo- and mechanoreceptor currents. 5. Thermoreceptor currents decreased whereas mechanoreceptor currents increased as the ambient temperature was raised. 6. When a mechanical stimulus was applied to the membrane where a thermoreceptor current was being produced, an algebraic summation of these receptor currents occurred. 7. It is concluded that thermoreceptor currents are dependent on ion channels different from those responsible for the mechanoreceptor currents, although the ionic pores for the channels are similar to each other in various respects. 8. A possibility that a thermoreceptor mechanism exclusively shares a Ca2+ pore in the anterior membrane, or a K+ pore in the posterior membrane, with a mechanoreceptor mechanism is discussed.