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Evaluation of gravity-dependent membrane potential shift in Paramecium
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Adv. Space Res. Vol. 23, No. 12, pp. 206%2073,1999 0 1999 COSPAR. Published by Elsevier Science Ltd. All rights reserved Printed in Great Britain 0273-1177/99$20.00 + 0.00 PII: SO273-1177(99)00164-7
EVALUATION OF GRAVITY- DEPENDENT MEMBRANE POTENTIAL SHIFT IN PmfluUM S. A. Baba, Y. Mogami, and T. Otsu
Department of Bio/ogy, Ochanomim Universi& Okwh 2-Z-4 To&o 112-8610, Japan
ABSTRACT It is still debated whether or not gravity can stimulate unicellular organisms. This question may be settled by revealing changes in the membrane potential in a manner depending on the gravitational forces imposed on the cell. We estimated the gravity- dependent membrane potential shift to be about 1 mV G-l for Paramecium showing gravikinesis at l- 5 G, on the basis of measurements of gravity- induced changes in active propulsion and those of propulsive velocity in solutions, in which the membrane potential has been measured electrophysiologically. The shift in membrane potential to this extent may occur from mechanoreceptive changes in K’ or Ca” conductance by about 1% and might be at the limit of electrophysiological measurement using membrane potential- sensitive dyes. Our measurements of propulsive velocity vs membrane potential also suggested that the reported propulsive force of Paramecium measured in a solution of graded densities with the aid of a video centrifuge microscope at 350 G was 11 times as large as that for - 29 mV, i.e., the resting membrane potential at [K’10 = 1 mM and [Ca”+], = 1 mM, and, by extrapolation, that Paramecium was hyperpolarized to - 60 mV by gravity stimulation of lOO- G equivalent, the value corrected by considering the reduction of density difference between the interior and exterior of the cell in the graded density solution. The estimated shift of the membrane potential from - 29 mV to - 60 mV by lOO- G equivalent stimulation, i.e., 0.3 mV G-‘, could reach the magnitude entirely 01999 COSPAR. Published by Elsevier Science Ltd. feasible to be measured more directly.
INTRODUCTION The action of gravity on Paramecium in water on earth is about 0.1 nN estimated as difference between the gravitational and buoyant forces (Machemer, 1998). The gravity may also act to expand the membrane by a positive pressure inside the cell reaching to 0.08 Pa at the bottom when Paramecium lies with the anteroposterior axis parallel to the gravity vector (Ooya etal., 1992). Thus the action of gravity seems minute as compared with experimental pressure differences often used to stimulate mechanoreceptors, e.g., a negative pressure of about 600 Pa on Necturusproximal tubule induces an increase in the open probability of stretchactivated K’ channels by a factor of 4 (Sackin, 1989). It has been demonstrated that Paramecium increases propulsion when pointing upwards and by decreasing it when pointing downwards, i.e., it performs gravikinesis. Upward and downward swimming velocities and the sedimentation rates of freely moving paramecia were analysed under natural and experimental gravity
2065
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S. A. Baba et al.
conditions (Baba et al., 1989; Ooya et al., 1992) and from cells fixed in orientation parallel to the gravity vector applying a DC field (Machemer eta]., 1991). We show that the gravity- induced membrane potential shift during gravikinesis is about 1 mV G-’ or even smaller, which may arise from changes in either K’ or Ca” conductance by about 1%. The method to measure the gravity- dependent membrane potential shift more convincingly will also be discussed.
Fig. 1. Long- armed centrifuge used for measurements of propulsive velocities and the sedimentation rate of Paramecium in the range of 2- 5 G. Jointed freely at the edges of 2- m long arms are two baskets, one holding a Paramecium observation chamber and a CCD camera and the other (not shown here) serving as a counter- balance. The observation chamber is set to an artificial vertical position after the centrifugal acceleration has become stable at a given value.
ACTIVE PROPULSION
CHANGES INDUCED BY GRAVITY
Ooya et al. (1992) have measured changes in active propulsion by analysing swimming velocities with respect to the swimming direction at 1 through 5 G using a long- armed centrifuge as shown in Figure 1. The average of the absolute values of increase and decease in active propulsion, for cells with the anterior ends upwards and downwards, respectively, was 0.075 + 0.009 mm s-’ (mean 2 SE., N = 9 replicates of experiments, each with about 2000 cells). This value applies to Paramecium caudatum specified by a path curvature of greater than 0.85 mm-‘, which will be referred to as curved swimmers. The change in active propulsion was 0.035 -+ 0.007 mm s-’ (mean 2 SE., N = 9) for cells with a curvature less than 0.1 mm” (straight swimmers) at 1 G. The induced- changes in active propulsion increase with increasing experimental gravitational forces (Figure 2). The dependence of the induced active propulsive velocity A!$ on the gravitational acceleration Gmay be described by the equation: AYp=&(G-
T,)
where &and T,are the sensitivity and threshold for gravitational acceleration of Paramecium, respectively.
Gravity-dependent
2067
Membrane Potential Shift
The sensitivity and threshold were determined from measurements shown in Figure 2 by the method of nonlinear least squares, i.e., A’,,= 0.085 f: 0.004 mm s.’ G-’ and TG= 0.27 + 0.12 G (fit + u, degree of freedom (dof) = 3, x2 per dof = 3.9) for curved swimmers and S, = 0.049 5 0.005 mm s-’ GM’and TG= 0.37 + 0.18 G (fit + u, dof = 3, x2 per dof = 1.0) for straight swimmers (for details of the method, see Bevington and Robinson, 1992). We will refer later to these values for S, as the gravireceptive sensitivity at 1 G through 5 G. It should be noted that Hemmersbach ef al. (1996a, b) have also found by space experiments the threshold for gravitaxis of Paramecium biaurela between 0.16 G and 0.3 G, values comparable to those for TGobtained above. Machemer ef al (1991) have reported 0.038 mm s-’ (computed from medians of upward and downward swimming velocities and the sedimentation rate, each from some 1500 cells) for the average change in active propulsion for Paramecium caudatum captured in orientation by galvanotaxis. These findings suggest that Paramecium can compensate sedimentation due to difference between the gravitational and buoyant forces by 63% (curved swimmers), 29% (straight swimmers) and by 32% (galvanotactic swimmers) at 1 G, since the sedimentation rate measured from Paramecium caudatum immobilized in a solution containing Ni2’ is 0.119 -C0.032 mm sS1(mean t S.D., N = 475 cells) (Ooya ef al., 1992) and 0.117 mm s-* G-’ (slope of sedimentation against 6’) (Nagel et al., 1997). Later on we will use the value of 0.12 mm s“ for the sedimentation rate at 1 G.
0
1
2
4 Grav:ty
5
6
(G)
Fig. 2. Gravity- induced changes in active propulsion in Paramecium caudatum under natural and experimental gravitational acceleration. Values are mean ? SE. from nine replicates of experiments, each with some 2000 cells. Open symbols, cells with a path curvature of less than 0.10 mm-’ (straight swimmers); filled symbols, cells with a path curvature greater than 0.85 mm“ (curved swimmers). The lines are drawn by the method of least squares. 24 ‘C. (Replotted from Ooya et al, 1992).
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Kuroda and Kamiya (1989) have determined the propulsive force to be 7 nN for Paramecium under centrifugal acceleration at 350 G. At 300- 400 G, Paramecium caua’atum was collected in a zone nearly 1.04 g cm3 being isopycnic to the body in a solution of graded densities calibrated with density marker beads. Most cells swam upwards to a position of density calibrated to be 1.03 g cm”, moved back to the position of 1.04 g cm” and repeated this up- and- down movement as long as centrifugation continued. Thus the propulsive force can be calculated by the equation:
where v represents the volume of the cell, 0, the density of the cell, D,,, the density of the medium on the level that the cell can reach, and CIthe centrifugal acceleration at that time. Kuroda and Kamiya (1989) have estimated vas 2 x lo7 cm3, (0, - 0,) as 0.01 g cmm3and a as 350 G and thereby obtained 7 nN for E It should be noted, however, that the value of a itself is not a measure of gravity stimulation when compared with that for Paramecium discussed earlier. The gravity stimulation for Paramecium in a solution of graded density is instead to be measured by (0, - D,,,)a/(P - pO)where P is the density of the cell and p,, the density of water at 1 G, provided that the gravireceptors of Paramecium distribute on the surface membrane but not in the interior of the cell (Ooya et al., 1992; Machemer et al., 1991). If this was the case, the strength of stimulation by gravity at 350 G in the density gradient solution should be evaluated to be lOO- G equivalent by using 1.033 g cm” for p and 1.000 g cm” for PO(Ooya etar!, 1992). The difference between 0, and P may reflect adaptation of Paramecium to high density solutions where the density of the cell becomes higher than that in water (Murakami, 1998). The active propulsive force of 7 nN thus obtained under centrifugal acceleration at 350 G seems substantially larger than that in water at 1 G determined by hydrodynamics. At a Reynolds number of 5 10m3applicable to Paramecium (Machemer, 1998) the force of sedimentation& for Paramecium sedimenting with the major axis parallel to the gravity vector and that of propulsion Fp are balanced with hydrodynamic resistances proportional to the velocity of sedimentation Y, and that of propulsion VP,respectively. Hence, they are given by
K=Pw
(3)
Fp=P‘&
(4)
and
where ,u is the viscosity of the medium and D the drag coefficient for Paramecium with the major axis parallel to the flow. D is evaluated theoretically by approximating the cell body a prolate spheroid as follows: D=
47ca ln(2ulb) -0.5
where a is the major semiaxis of the spheroid and b the minor semiaxis (Happel and Brenner, 1973). Using a = 0.1 mm, b = 0.025 mm and p = 0.009 P, we obtain @ = 0.7 nN mm-’ s. Since the force of sedimentation is the difference between the gravitational and buoyant forces, C;, is also given by
4 =
(6) From Eqs. 3 and 6, and using described
Gravity-dependent
2069
Membrane Potential Shit?
values for CI,b, p, p. and c = 0.12 mm s-l, we obtain ,_W= 0.7 nN mm-’ s, which is consistent with the hydrodynamic value calculated above. Now we can evaluate the propulsive force of Parameczkm from the propulsive velocity using Eq. 4 and obtain J$ = 0.63 nN for cells swimming at 5 = 0.892 2 0.434 mm se1 (mean rt S.D., N = 44 cells) with the membrane potential of - 29 mV at 1 G (see below). This indicates that the propulsive force of Paramecium in a solution of graded densities at a gravity stimulation of lOO-G equivalent is 11 times larger than that of Paramecium at 1 G.
0.25
1.0
4.0
16.0
WI0 MW Fig. 3. Propulsive velocity of Parameciumcazdztumin solutions containing KC1at various concentrations. The velocity was determined from the distance along a helical trajectory divided by an elapsed time of that trajectory with 3- dimensional corrections based on geometrical parameters. Solutions contain 1 mM CaCl,, 1 mM Tris-HCI (pH 7.3) in addition to the indicated concentrations of KCl. 24 “C.
PROPULSIVE VELOCITY VERSUS MEMBRANE POTENTIAL The propulsive velocity of Parameciumcaudbtum(syngen 3, mating type V) swimming along a helical path in solutions containing 1 mM CaCl,, 1 mM Tris- HCl (pH 7.3) and either of 0.25, 1.0,4.0 or 16.0 mM KC1 was measured by means of video- microscopy and image analysis as shown in Figure 3 (details will be published elsewhere). The dependence of membrane potential E, on the concentration of K’ in the medium containing Ca2+at a room temperature of about 20 “C may be evaluated by a modified Goldman- Hodgkin- Katz equation (Goldman, 1943; Hodgkin and Katz, 1949; Oka etaal., 1986):
Em =%log
in mM ‘JK +lo-pJJK +I;+@
2PK[K +li +8Pca[Ca 2’li
(7)
2070
S. A. Baba et al.
20
10
5
O
.E. 3 .i
-10
5 a Q) 5 -20 L “E $
-30
-40
-50
0.25
1.0
16.0
[K+]* (rtdj” Fig. 4. Membrane potential of Paramecium cazu’atum as a function of the external potassium ion concentration. Plotted are the means of membrane potential with S.D. bars measured in solutions containing 1 mM CaCl,, 1 mM Tris-HCl (pH 7.2-7.4) in addition to the indicated concentrations of KCl; 0, Naitoh and Eckert (1968), 0, Naitoh and Eckert (1972); q, Naitoh and Eckert (1973); n , Oami (1998); 0, Nakaoka (personal communication). The curve is drawn by the method of least squares. Experiments were performed at room temperatures of 17-24 “C.
where P,and PC, are the permeability constants of K’ and Ca”, respectively, [K’],, [K’], [Ca2’10and [Ca”‘]i are concentrations of respective ions in the extra- and intracellular compartments, and D is given by: D=&[K’lO-[K
+]i)*+4(p,[~+]~+4p,[cu*+]~)(p,[K+]i+4p,[ca*+];).(8)
Assuming PK [K’], >>PC, [Ca2+]fnand putting [Ca”+],= 1 mM into Eqs. 7 and 8, we obtain the equation:
inmM
where A, =[K’]i and AZ= P&P,.
(9)
AI and A, were determined by the method of non-linear least squares,
Gravity-dependent
.^
_^
-3u
-4u
2071
Membrane Potential Shift
-20
-30
Membrane
Potential
-10
0
(mV)
Fig. 5. Propulsive velocity of Paramecium caudatzm as a function of the membrane potential. Replotted from Fig. 3 using the Goldman equation, which has been fitted to observations as shown in Fig. 4.
whereby Eq. 9 was nicely fitted to measurements by several authors (Naitoh and Eckert, 1968, 1972, 1973; Oami, 1998; Nakaoka, personal communication) of E,,,vs [K’10at [Ca”‘], = 1 mM and at room temperatures 2.0 + 0.1 (fit 2 o, dof = 24, x2 per dof = of 17-24 “C (Fig. 4), that is, [K’]i = 22.2 2 0.5 mM and PJ&= 3.6, for details of the method, see Bevington and Robinson, 1992). Using Eq. 9 with these values, the propulsive velocity can be plotted against the membrane potential as shown in Figure 5. GRAVITY-INDUCED MEMBRANE POTENTIAL SHIFT The shift of the membrane potential induced by gravity stimulation at 1 through 5 G is estimated to be 1.2 mV G-’ and 0.7 mV G-’ for curved and straight swimmers, respectively. This value results from the S, of Eq. 1 divided by the average slope, - 0.07 mm s-’ mV' ,of the propulsive velocity vs the membrane potential around - 29 mV in Figure 5. The values seem minute to be detected either electrophysiologically or optically by using membrane potential sensitive dyes. However, it must be large enough to enable Paramecium to cope with the sedimenting action of gravity by gravikinesis as well as by gravitaxis as discussed previously (Ooya etaL, 1992; Mogami and Baba, 1998). The magnitude of change in ionic conductance required for the membrane potential shift of the order of 1 mV can be estimated using the equation:
Em=
gK
&a
-EK+
-EC,
gK +&a
gK +&‘a
in mM
(10)
2072
S. A. Baba et al
where g, and g, are conductances for K’ and Ca”, respectively, and E,and E,, the equilibrium potentials for the respective ions (see Randall et al!, 1997). EK and EC0are calculated to be - 81 mV and 116 mV at a room temperature of 20 ‘C, respectively, by the Nemst equations (see Randall et al, 1997): EK = 58 log([K’],, /[K’]J
in mV
(11)
and E,, = 29 log([Ca”],
/[Ca”+]J
in mV
(12)
in which [K’10 = 1 mM, [K’], = 25 mM, [Ca”+], = 1 mM and [Ca”], = 10e7 M are used. Putting E,,, = -29 mV, EK = - 81 mV and EC, = 116 mV into Eq. 10, g, is estimated to be about 0.36 xg,. Thus, a small amount of change either in g, or gca, i.e., Ag, or Agca, will produce a change A Emin the membrane potential as given by
A&
BE,= -EK+ 1.4&
kh
-----EC, 3 .8&:*
in mM
(13)
where Ag, and Ag,, have been neglected for g, and gca, respectively, in the denominators on the right side. Eq. 13 states that a change either in g, or in g,, by 1% will produce a shift in E,,, by 0.6 mV or 0.3 mV. Therefore, gravireceptive changes in K’ or Ca” conductance by an extent of this order may explain the gravireceptive membrane potential shift as described above. The dependence of propulsive velocity on the membrane potential shown in Figure 5 also indicates, by extrapolation with a slope of - 0.12 mm s-’ mV’ to the upper- left of the curve, that Paramecium exerting the propulsive force up to 7 nN, which is equivalent to the velocity 11 times larger than that at - 29 mV as discussed earlier, may be hyperpolarized to - 60 mV. The shift of 31 mV, from - 29 mV to - 60 mV, divided by 100-G equivalent stimulation gives the sensitivity of about 0.3 mV G -r . The estimation of the shift by 100-G equivalent stimulation may be only qualitative because of limited accuracy in measuring propulsive forces in the solution of graded densities. The shift could, however, reach the magnitude entirely feasible to be measured more directly. A more quantitative analysis of propulsive forces of Paramecium with the membrane potential being monitored by a membrane potential sensitive dye in solutions of graded densities under centrifugal acceleration of up to 350 g will clarify the nature of gravireception in unicellular organisms. ACKNOWLEDGEMENTS We are grateful to Dr. Yasuo Nakaoka of Osaka University for providing his unpublished measurements. This study is carried out as a part of “Ground Research Announcement for the Space Utilization” promoted by NASDA and Japan Space Forum.
REFERENCES Baba, S. A., M. Ooya, Y. Mogami, M. Okuno, A. Izumi- Kurotani and M. Yamashita, Gravireception in Paramecium with a Note on Digital Image Analysis of Videorecords of Swimming Paramecium, BioL ScL Space, 3,285 (1989). Bevington, P. R. and D. K. Robinson, Data Reakctzbn and Error Analyds for the Physical Sciences, McGraw Hill Co., New York, NY (1992).
Gravity-dependent Membrane Potential Shift
2073
Goldman, D. E., Potential, Impedance and Rectification in Membranes, J Gen. Physiol., 27, 37- 60 (1943). Happel, J. and H. Brenner, l;ow Reyno(dF Number Hydroodynamics,2nd ed., Martinus Nijhoff Pub., Dordrecht (1973). Hemmersbach, R., R. Voormamrs and D. P. H der, Graviresponses in Parameciumbzizurehzunder Different Accelerations: Studies on the Ground and in Space, J; tip. Viol! ,199,2199-2205 (1996). Hemmersbach, R., R. Voormanns, W. Briegleb, N. Rieder and D. P. H der, InfIuence of Accelerations on the Spatial Orientation of bxoa’es and Paramecium,J; Biofechnot! ,47,271-278 (1996). Hodgkin, A. L. and B. Katz, The Effect of Sodium Ions on the Electrical Activity of the Giant Axon of the Squid, J; Phy.siol, London, 108,37-77 (1949). Kuroda, K. and N. Kamiya, Propulsive Force of Paramecium as Revealed by the Video Centrifuge Microscope, fip. CellRex., 184,268-272 (1989) Machemer, H., S. Machemer- R hnisch, R. Br ucker, and K. Takahashi, Gravikinesis in Paramecium: Theory and Isolation of a Physiological Response to the Natural Gravity Vector, J Camp. PXrysio1 A. 16&l-12 (1991). Machemer, H., Mechanisms of Gravireception and Response in Unicellular Systems, Adv. Space Res., 21, 1243-1251(1998). Mogami, Y. and S. A. Baba, Super- helix model: A Physiological Model for Gravitaxis of Paramecium&v. Space l&x, 21,1291-1300 (1998). Murakami, A., Short- term Responses of Gravitaxis to Altered Gravity in Paramecizun,A&. SpaceRex, 21, 1253-1261(1998). Nagel, U., D. Watzke, D.- Ch. Neugebauer, S. Machemer- R hnisch, R. Br ucker and H. Machemer, Analysis of Sedimentation of Immobilized Cells under Normal and Hypergravity, Mcrogravi@Sci. Z&ho& 10,41-52 (1997). Naitoh, Y. and R. Eckert, Electrical Properties of Paramecium caua’abm: Modification by Bound and Free Cations, Z Yerg P/lyssiol., 61,427-452 (1968). Naitoh, Y. and R. Eckert, A Regenerative Calcium Response in Paramecium, J Ezp. Biol., 56, 667- 681 (1972). Naitoh, Y. and R. Eckert, Sensory Mechanisms in Paramecium II Ionic Basis of the Hyperpolarizing Mechanoreceptor Potential, J fip Viol., 59,53-65 (1973). Oami, K., Ionic Mechanisms of Depolarizing and Hyperpolarizing Quinine Receptor Potentials in Paramecium caudzhm, J; Camp. Physiol. A, 182,403-409 (1998). Oka, T., Y. Nakaoka and F. Oosawa, Changes in Membrane Potential during Adaptation to External Potassium Ions in Paramecium caz&hm, J: Ezp. Biol., 126,111-117 (1986). Ooya, M., Y. Mogami, A. Izumi-Kurotani and S. A. Baba, Gravity-induced Changes in Propulsion of Paramecium cazdztum: A Possible Role of Gravireception in Protozoan Behaviour, J &Z Brbl., 163, 153-167 (1992). Randall, D., W. Berggren and K. French, &ckertAnimalPhysiology Mecha&ns andAa!bptations,4th ed., W. H. Freeman and Co., New York (1997). Sackin, H., A Stretch-activated K +Channel Sensitive to Ceil Volume. Boc Natn. Acaa! Sci. c! J: A., 86, 1731-1735 (1989).
Adv. Space Res. Vol. 23, No. 12, pp. 206%2073,1999 0 1999 COSPAR. Published by Elsevier Science Ltd. All rights reserved Printed in Great Britain 0273-1177/99$20.00 + 0.00 PII: SO273-1177(99)00164-7
EVALUATION OF GRAVITY- DEPENDENT MEMBRANE POTENTIAL SHIFT IN PmfluUM S. A. Baba, Y. Mogami, and T. Otsu
Department of Bio/ogy, Ochanomim Universi& Okwh 2-Z-4 To&o 112-8610, Japan
ABSTRACT It is still debated whether or not gravity can stimulate unicellular organisms. This question may be settled by revealing changes in the membrane potential in a manner depending on the gravitational forces imposed on the cell. We estimated the gravity- dependent membrane potential shift to be about 1 mV G-l for Paramecium showing gravikinesis at l- 5 G, on the basis of measurements of gravity- induced changes in active propulsion and those of propulsive velocity in solutions, in which the membrane potential has been measured electrophysiologically. The shift in membrane potential to this extent may occur from mechanoreceptive changes in K’ or Ca” conductance by about 1% and might be at the limit of electrophysiological measurement using membrane potential- sensitive dyes. Our measurements of propulsive velocity vs membrane potential also suggested that the reported propulsive force of Paramecium measured in a solution of graded densities with the aid of a video centrifuge microscope at 350 G was 11 times as large as that for - 29 mV, i.e., the resting membrane potential at [K’10 = 1 mM and [Ca”+], = 1 mM, and, by extrapolation, that Paramecium was hyperpolarized to - 60 mV by gravity stimulation of lOO- G equivalent, the value corrected by considering the reduction of density difference between the interior and exterior of the cell in the graded density solution. The estimated shift of the membrane potential from - 29 mV to - 60 mV by lOO- G equivalent stimulation, i.e., 0.3 mV G-‘, could reach the magnitude entirely 01999 COSPAR. Published by Elsevier Science Ltd. feasible to be measured more directly.
INTRODUCTION The action of gravity on Paramecium in water on earth is about 0.1 nN estimated as difference between the gravitational and buoyant forces (Machemer, 1998). The gravity may also act to expand the membrane by a positive pressure inside the cell reaching to 0.08 Pa at the bottom when Paramecium lies with the anteroposterior axis parallel to the gravity vector (Ooya etal., 1992). Thus the action of gravity seems minute as compared with experimental pressure differences often used to stimulate mechanoreceptors, e.g., a negative pressure of about 600 Pa on Necturusproximal tubule induces an increase in the open probability of stretchactivated K’ channels by a factor of 4 (Sackin, 1989). It has been demonstrated that Paramecium increases propulsion when pointing upwards and by decreasing it when pointing downwards, i.e., it performs gravikinesis. Upward and downward swimming velocities and the sedimentation rates of freely moving paramecia were analysed under natural and experimental gravity
2065
2066
S. A. Baba et al.
conditions (Baba et al., 1989; Ooya et al., 1992) and from cells fixed in orientation parallel to the gravity vector applying a DC field (Machemer eta]., 1991). We show that the gravity- induced membrane potential shift during gravikinesis is about 1 mV G-’ or even smaller, which may arise from changes in either K’ or Ca” conductance by about 1%. The method to measure the gravity- dependent membrane potential shift more convincingly will also be discussed.
Fig. 1. Long- armed centrifuge used for measurements of propulsive velocities and the sedimentation rate of Paramecium in the range of 2- 5 G. Jointed freely at the edges of 2- m long arms are two baskets, one holding a Paramecium observation chamber and a CCD camera and the other (not shown here) serving as a counter- balance. The observation chamber is set to an artificial vertical position after the centrifugal acceleration has become stable at a given value.
ACTIVE PROPULSION
CHANGES INDUCED BY GRAVITY
Ooya et al. (1992) have measured changes in active propulsion by analysing swimming velocities with respect to the swimming direction at 1 through 5 G using a long- armed centrifuge as shown in Figure 1. The average of the absolute values of increase and decease in active propulsion, for cells with the anterior ends upwards and downwards, respectively, was 0.075 + 0.009 mm s-’ (mean 2 SE., N = 9 replicates of experiments, each with about 2000 cells). This value applies to Paramecium caudatum specified by a path curvature of greater than 0.85 mm-‘, which will be referred to as curved swimmers. The change in active propulsion was 0.035 -+ 0.007 mm s-’ (mean 2 SE., N = 9) for cells with a curvature less than 0.1 mm” (straight swimmers) at 1 G. The induced- changes in active propulsion increase with increasing experimental gravitational forces (Figure 2). The dependence of the induced active propulsive velocity A!$ on the gravitational acceleration Gmay be described by the equation: AYp=&(G-
T,)
where &and T,are the sensitivity and threshold for gravitational acceleration of Paramecium, respectively.
Gravity-dependent
2067
Membrane Potential Shift
The sensitivity and threshold were determined from measurements shown in Figure 2 by the method of nonlinear least squares, i.e., A’,,= 0.085 f: 0.004 mm s.’ G-’ and TG= 0.27 + 0.12 G (fit + u, degree of freedom (dof) = 3, x2 per dof = 3.9) for curved swimmers and S, = 0.049 5 0.005 mm s-’ GM’and TG= 0.37 + 0.18 G (fit + u, dof = 3, x2 per dof = 1.0) for straight swimmers (for details of the method, see Bevington and Robinson, 1992). We will refer later to these values for S, as the gravireceptive sensitivity at 1 G through 5 G. It should be noted that Hemmersbach ef al. (1996a, b) have also found by space experiments the threshold for gravitaxis of Paramecium biaurela between 0.16 G and 0.3 G, values comparable to those for TGobtained above. Machemer ef al (1991) have reported 0.038 mm s-’ (computed from medians of upward and downward swimming velocities and the sedimentation rate, each from some 1500 cells) for the average change in active propulsion for Paramecium caudatum captured in orientation by galvanotaxis. These findings suggest that Paramecium can compensate sedimentation due to difference between the gravitational and buoyant forces by 63% (curved swimmers), 29% (straight swimmers) and by 32% (galvanotactic swimmers) at 1 G, since the sedimentation rate measured from Paramecium caudatum immobilized in a solution containing Ni2’ is 0.119 -C0.032 mm sS1(mean t S.D., N = 475 cells) (Ooya ef al., 1992) and 0.117 mm s-* G-’ (slope of sedimentation against 6’) (Nagel et al., 1997). Later on we will use the value of 0.12 mm s“ for the sedimentation rate at 1 G.
0
1
2
4 Grav:ty
5
6
(G)
Fig. 2. Gravity- induced changes in active propulsion in Paramecium caudatum under natural and experimental gravitational acceleration. Values are mean ? SE. from nine replicates of experiments, each with some 2000 cells. Open symbols, cells with a path curvature of less than 0.10 mm-’ (straight swimmers); filled symbols, cells with a path curvature greater than 0.85 mm“ (curved swimmers). The lines are drawn by the method of least squares. 24 ‘C. (Replotted from Ooya et al, 1992).
2068
S. A. Baba et al
Kuroda and Kamiya (1989) have determined the propulsive force to be 7 nN for Paramecium under centrifugal acceleration at 350 G. At 300- 400 G, Paramecium caua’atum was collected in a zone nearly 1.04 g cm3 being isopycnic to the body in a solution of graded densities calibrated with density marker beads. Most cells swam upwards to a position of density calibrated to be 1.03 g cm”, moved back to the position of 1.04 g cm” and repeated this up- and- down movement as long as centrifugation continued. Thus the propulsive force can be calculated by the equation:
where v represents the volume of the cell, 0, the density of the cell, D,,, the density of the medium on the level that the cell can reach, and CIthe centrifugal acceleration at that time. Kuroda and Kamiya (1989) have estimated vas 2 x lo7 cm3, (0, - 0,) as 0.01 g cmm3and a as 350 G and thereby obtained 7 nN for E It should be noted, however, that the value of a itself is not a measure of gravity stimulation when compared with that for Paramecium discussed earlier. The gravity stimulation for Paramecium in a solution of graded density is instead to be measured by (0, - D,,,)a/(P - pO)where P is the density of the cell and p,, the density of water at 1 G, provided that the gravireceptors of Paramecium distribute on the surface membrane but not in the interior of the cell (Ooya et al., 1992; Machemer et al., 1991). If this was the case, the strength of stimulation by gravity at 350 G in the density gradient solution should be evaluated to be lOO- G equivalent by using 1.033 g cm” for p and 1.000 g cm” for PO(Ooya etar!, 1992). The difference between 0, and P may reflect adaptation of Paramecium to high density solutions where the density of the cell becomes higher than that in water (Murakami, 1998). The active propulsive force of 7 nN thus obtained under centrifugal acceleration at 350 G seems substantially larger than that in water at 1 G determined by hydrodynamics. At a Reynolds number of 5 10m3applicable to Paramecium (Machemer, 1998) the force of sedimentation& for Paramecium sedimenting with the major axis parallel to the gravity vector and that of propulsion Fp are balanced with hydrodynamic resistances proportional to the velocity of sedimentation Y, and that of propulsion VP,respectively. Hence, they are given by
K=Pw
(3)
Fp=P‘&
(4)
and
where ,u is the viscosity of the medium and D the drag coefficient for Paramecium with the major axis parallel to the flow. D is evaluated theoretically by approximating the cell body a prolate spheroid as follows: D=
47ca ln(2ulb) -0.5
where a is the major semiaxis of the spheroid and b the minor semiaxis (Happel and Brenner, 1973). Using a = 0.1 mm, b = 0.025 mm and p = 0.009 P, we obtain @ = 0.7 nN mm-’ s. Since the force of sedimentation is the difference between the gravitational and buoyant forces, C;, is also given by
4 =
(6) From Eqs. 3 and 6, and using described
Gravity-dependent
2069
Membrane Potential Shit?
values for CI,b, p, p. and c = 0.12 mm s-l, we obtain ,_W= 0.7 nN mm-’ s, which is consistent with the hydrodynamic value calculated above. Now we can evaluate the propulsive force of Parameczkm from the propulsive velocity using Eq. 4 and obtain J$ = 0.63 nN for cells swimming at 5 = 0.892 2 0.434 mm se1 (mean rt S.D., N = 44 cells) with the membrane potential of - 29 mV at 1 G (see below). This indicates that the propulsive force of Paramecium in a solution of graded densities at a gravity stimulation of lOO-G equivalent is 11 times larger than that of Paramecium at 1 G.
0.25
1.0
4.0
16.0
WI0 MW Fig. 3. Propulsive velocity of Parameciumcazdztumin solutions containing KC1at various concentrations. The velocity was determined from the distance along a helical trajectory divided by an elapsed time of that trajectory with 3- dimensional corrections based on geometrical parameters. Solutions contain 1 mM CaCl,, 1 mM Tris-HCI (pH 7.3) in addition to the indicated concentrations of KCl. 24 “C.
PROPULSIVE VELOCITY VERSUS MEMBRANE POTENTIAL The propulsive velocity of Parameciumcaudbtum(syngen 3, mating type V) swimming along a helical path in solutions containing 1 mM CaCl,, 1 mM Tris- HCl (pH 7.3) and either of 0.25, 1.0,4.0 or 16.0 mM KC1 was measured by means of video- microscopy and image analysis as shown in Figure 3 (details will be published elsewhere). The dependence of membrane potential E, on the concentration of K’ in the medium containing Ca2+at a room temperature of about 20 “C may be evaluated by a modified Goldman- Hodgkin- Katz equation (Goldman, 1943; Hodgkin and Katz, 1949; Oka etaal., 1986):
Em =%log
in mM ‘JK +lo-pJJK +I;+@
2PK[K +li +8Pca[Ca 2’li
(7)
2070
S. A. Baba et al.
20
10
5
O
.E. 3 .i
-10
5 a Q) 5 -20 L “E $
-30
-40
-50
0.25
1.0
16.0
[K+]* (rtdj” Fig. 4. Membrane potential of Paramecium cazu’atum as a function of the external potassium ion concentration. Plotted are the means of membrane potential with S.D. bars measured in solutions containing 1 mM CaCl,, 1 mM Tris-HCl (pH 7.2-7.4) in addition to the indicated concentrations of KCl; 0, Naitoh and Eckert (1968), 0, Naitoh and Eckert (1972); q, Naitoh and Eckert (1973); n , Oami (1998); 0, Nakaoka (personal communication). The curve is drawn by the method of least squares. Experiments were performed at room temperatures of 17-24 “C.
where P,and PC, are the permeability constants of K’ and Ca”, respectively, [K’],, [K’], [Ca2’10and [Ca”‘]i are concentrations of respective ions in the extra- and intracellular compartments, and D is given by: D=&[K’lO-[K
+]i)*+4(p,[~+]~+4p,[cu*+]~)(p,[K+]i+4p,[ca*+];).(8)
Assuming PK [K’], >>PC, [Ca2+]fnand putting [Ca”+],= 1 mM into Eqs. 7 and 8, we obtain the equation:
inmM
where A, =[K’]i and AZ= P&P,.
(9)
AI and A, were determined by the method of non-linear least squares,
Gravity-dependent
.^
_^
-3u
-4u
2071
Membrane Potential Shift
-20
-30
Membrane
Potential
-10
0
(mV)
Fig. 5. Propulsive velocity of Paramecium caudatzm as a function of the membrane potential. Replotted from Fig. 3 using the Goldman equation, which has been fitted to observations as shown in Fig. 4.
whereby Eq. 9 was nicely fitted to measurements by several authors (Naitoh and Eckert, 1968, 1972, 1973; Oami, 1998; Nakaoka, personal communication) of E,,,vs [K’10at [Ca”‘], = 1 mM and at room temperatures 2.0 + 0.1 (fit 2 o, dof = 24, x2 per dof = of 17-24 “C (Fig. 4), that is, [K’]i = 22.2 2 0.5 mM and PJ&= 3.6, for details of the method, see Bevington and Robinson, 1992). Using Eq. 9 with these values, the propulsive velocity can be plotted against the membrane potential as shown in Figure 5. GRAVITY-INDUCED MEMBRANE POTENTIAL SHIFT The shift of the membrane potential induced by gravity stimulation at 1 through 5 G is estimated to be 1.2 mV G-’ and 0.7 mV G-’ for curved and straight swimmers, respectively. This value results from the S, of Eq. 1 divided by the average slope, - 0.07 mm s-’ mV' ,of the propulsive velocity vs the membrane potential around - 29 mV in Figure 5. The values seem minute to be detected either electrophysiologically or optically by using membrane potential sensitive dyes. However, it must be large enough to enable Paramecium to cope with the sedimenting action of gravity by gravikinesis as well as by gravitaxis as discussed previously (Ooya etaL, 1992; Mogami and Baba, 1998). The magnitude of change in ionic conductance required for the membrane potential shift of the order of 1 mV can be estimated using the equation:
Em=
gK
&a
-EK+
-EC,
gK +&a
gK +&‘a
in mM
(10)
2072
S. A. Baba et al
where g, and g, are conductances for K’ and Ca”, respectively, and E,and E,, the equilibrium potentials for the respective ions (see Randall et al!, 1997). EK and EC0are calculated to be - 81 mV and 116 mV at a room temperature of 20 ‘C, respectively, by the Nemst equations (see Randall et al, 1997): EK = 58 log([K’],, /[K’]J
in mV
(11)
and E,, = 29 log([Ca”],
/[Ca”+]J
in mV
(12)
in which [K’10 = 1 mM, [K’], = 25 mM, [Ca”+], = 1 mM and [Ca”], = 10e7 M are used. Putting E,,, = -29 mV, EK = - 81 mV and EC, = 116 mV into Eq. 10, g, is estimated to be about 0.36 xg,. Thus, a small amount of change either in g, or gca, i.e., Ag, or Agca, will produce a change A Emin the membrane potential as given by
A&
BE,= -EK+ 1.4&
kh
-----EC, 3 .8&:*
in mM
(13)
where Ag, and Ag,, have been neglected for g, and gca, respectively, in the denominators on the right side. Eq. 13 states that a change either in g, or in g,, by 1% will produce a shift in E,,, by 0.6 mV or 0.3 mV. Therefore, gravireceptive changes in K’ or Ca” conductance by an extent of this order may explain the gravireceptive membrane potential shift as described above. The dependence of propulsive velocity on the membrane potential shown in Figure 5 also indicates, by extrapolation with a slope of - 0.12 mm s-’ mV’ to the upper- left of the curve, that Paramecium exerting the propulsive force up to 7 nN, which is equivalent to the velocity 11 times larger than that at - 29 mV as discussed earlier, may be hyperpolarized to - 60 mV. The shift of 31 mV, from - 29 mV to - 60 mV, divided by 100-G equivalent stimulation gives the sensitivity of about 0.3 mV G -r . The estimation of the shift by 100-G equivalent stimulation may be only qualitative because of limited accuracy in measuring propulsive forces in the solution of graded densities. The shift could, however, reach the magnitude entirely feasible to be measured more directly. A more quantitative analysis of propulsive forces of Paramecium with the membrane potential being monitored by a membrane potential sensitive dye in solutions of graded densities under centrifugal acceleration of up to 350 g will clarify the nature of gravireception in unicellular organisms. ACKNOWLEDGEMENTS We are grateful to Dr. Yasuo Nakaoka of Osaka University for providing his unpublished measurements. This study is carried out as a part of “Ground Research Announcement for the Space Utilization” promoted by NASDA and Japan Space Forum.
REFERENCES Baba, S. A., M. Ooya, Y. Mogami, M. Okuno, A. Izumi- Kurotani and M. Yamashita, Gravireception in Paramecium with a Note on Digital Image Analysis of Videorecords of Swimming Paramecium, BioL ScL Space, 3,285 (1989). Bevington, P. R. and D. K. Robinson, Data Reakctzbn and Error Analyds for the Physical Sciences, McGraw Hill Co., New York, NY (1992).
Gravity-dependent Membrane Potential Shift
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Goldman, D. E., Potential, Impedance and Rectification in Membranes, J Gen. Physiol., 27, 37- 60 (1943). Happel, J. and H. Brenner, l;ow Reyno(dF Number Hydroodynamics,2nd ed., Martinus Nijhoff Pub., Dordrecht (1973). Hemmersbach, R., R. Voormamrs and D. P. H der, Graviresponses in Parameciumbzizurehzunder Different Accelerations: Studies on the Ground and in Space, J; tip. Viol! ,199,2199-2205 (1996). Hemmersbach, R., R. Voormanns, W. Briegleb, N. Rieder and D. P. H der, InfIuence of Accelerations on the Spatial Orientation of bxoa’es and Paramecium,J; Biofechnot! ,47,271-278 (1996). Hodgkin, A. L. and B. Katz, The Effect of Sodium Ions on the Electrical Activity of the Giant Axon of the Squid, J; Phy.siol, London, 108,37-77 (1949). Kuroda, K. and N. Kamiya, Propulsive Force of Paramecium as Revealed by the Video Centrifuge Microscope, fip. CellRex., 184,268-272 (1989) Machemer, H., S. Machemer- R hnisch, R. Br ucker, and K. Takahashi, Gravikinesis in Paramecium: Theory and Isolation of a Physiological Response to the Natural Gravity Vector, J Camp. PXrysio1 A. 16&l-12 (1991). Machemer, H., Mechanisms of Gravireception and Response in Unicellular Systems, Adv. Space Res., 21, 1243-1251(1998). Mogami, Y. and S. A. Baba, Super- helix model: A Physiological Model for Gravitaxis of Paramecium&v. Space l&x, 21,1291-1300 (1998). Murakami, A., Short- term Responses of Gravitaxis to Altered Gravity in Paramecizun,A&. SpaceRex, 21, 1253-1261(1998). Nagel, U., D. Watzke, D.- Ch. Neugebauer, S. Machemer- R hnisch, R. Br ucker and H. Machemer, Analysis of Sedimentation of Immobilized Cells under Normal and Hypergravity, Mcrogravi@Sci. Z&ho& 10,41-52 (1997). Naitoh, Y. and R. Eckert, Electrical Properties of Paramecium caua’abm: Modification by Bound and Free Cations, Z Yerg P/lyssiol., 61,427-452 (1968). Naitoh, Y. and R. Eckert, A Regenerative Calcium Response in Paramecium, J Ezp. Biol., 56, 667- 681 (1972). Naitoh, Y. and R. Eckert, Sensory Mechanisms in Paramecium II Ionic Basis of the Hyperpolarizing Mechanoreceptor Potential, J fip Viol., 59,53-65 (1973). Oami, K., Ionic Mechanisms of Depolarizing and Hyperpolarizing Quinine Receptor Potentials in Paramecium caudzhm, J; Camp. Physiol. A, 182,403-409 (1998). Oka, T., Y. Nakaoka and F. Oosawa, Changes in Membrane Potential during Adaptation to External Potassium Ions in Paramecium caz&hm, J: Ezp. Biol., 126,111-117 (1986). Ooya, M., Y. Mogami, A. Izumi-Kurotani and S. A. Baba, Gravity-induced Changes in Propulsion of Paramecium cazdztum: A Possible Role of Gravireception in Protozoan Behaviour, J &Z Brbl., 163, 153-167 (1992). Randall, D., W. Berggren and K. French, &ckertAnimalPhysiology Mecha&ns andAa!bptations,4th ed., W. H. Freeman and Co., New York (1997). Sackin, H., A Stretch-activated K +Channel Sensitive to Ceil Volume. Boc Natn. Acaa! Sci. c! J: A., 86, 1731-1735 (1989).