Ion Transport Studies and Determination of the Cell ... - BioMedSearch
Ion Transport Studies and Determination of the Cell Wall Elastic Modulus in the Marine Alga Halicystis parvula JAMES
S. G R A V E S and J O H N
GUTKNECHT
From the Department of Physiology and Pharmacology, Duke University Medical Center, Durham, North Carolina 27710 and the Duke University Marine Laboratory, Beaufort, North Carolina 28516. Dr. Graves' present address is the Ontario Cancer Institute, Toronto, Ontario, Canada.
A ~ S x 1~A C X Using cultured cells o f the marine alga, Halicystisparvula, we measu r e d the concentrations o f 11 inorganic ions in the vacuolar sap and the electrical potential difference (PD) between the vacuole and the external solution. In normal cells u n d e r steady-state conditions a comparison o f the electrochemical equilibrium (Nernst) potential for each ion with the PD o f - 8 ~ mV (inside negative) indicates that Na + and K + are actively t r a n s p o r t e d out of the vacuole whereas all anions are p u m p e d into the cell. Although the [K +] in the vacuole is only 9 raM, the cytoplasmic [K +] is about 420 mM, which suggests that the outwardly directed p u m p is at the tonoplast. Using large Halicystis cells we perfused the vacuole with an artificial seawater and conducted a short-circuit analysis of ion transport. T h e short-circuit current (SCC) o f 299 peq • cm-2.s -~ is not significantly different from the net influx o f CI-. T h e r e is a small, but statistically significant net efflux o f K + ( 0.10 P < 0.025
- cm -~ " s-all
313-+42 (13) 299-+41 (9) 268-+24 (6)
* Data expressed as mean -+ SE (number of cells). ~: Direction indicated by sign; outward (+), inward (-). § The SCC measured during flux experiments for each ion. I1Converted to these units by: (/xA cm-Z)/(F x 10t). 1.12. F r o m this a n a l y s i s we c o n c l u d e t h a t C l - is actively t r a n s p o r t e d i n t o t h e v a c u o l e a n d t h a t t h e SCC is a g o o d m e a s u r e o f t h e r a t e o f active t r a n s p o r t o f C I - .
Turgor Pressure and Cell Wall Elastic Modulus T h e o s m o l a l i t y o f t h e v a c u o l a r s a p was m e a s u r e d in two e x p e r i m e n t s with a t o t a l o f five s a p s a m p l e s . T h e o s m o l a l i t y d i f f e r e n c e b e t w e e n t h e s a p a n d t h e A S W in w h i c h t h e cells w e r e p r e t r e a t e d was 23 ± 6 m o s m o l / k g . T h e o s m o t i c p r e s s u r e d i f f e r e n c e (AII in a t m ) was e s t i m a t e d by t h e v a n ' t H o f f e q u a t i o n (see 27): All =R
"T.
AC,
(6)
w h e r e AC is t h e o s m o l a l c o n c e n t r a t i o n d i f f e r e n c e . T h u s , t h e m e a n o s m o t i c p r e s s u r e d i f f e r e n c e is 0.54 a t m . A s s u m i n g t h a t t h e n e t flow o f w a t e r is e s s e n t i a l l y z e r o , this o s m o t i c p r e s s u r e d i f f e r e n c e is e q u a l to t h e h y d r o s t a t i c p r e s s u r e d i f f e r ence or turgor pressure. Turgor pressures for most vacuolated marine algae r a n g e f r o m 1.5 to 13 a t m (see 24). T h e v a l u e f o r H . parvula is t h e r e f o r e u n u s u a l l y low b u t is, n e v e r t h e l e s s , in a g r e e m e n t with t h e e a r l i e r v a l u e s o f 0 . 2 5 - 0 . 5 a t m r e p o r t e d f o r Halicystis (1, 20).
JAMES S. GRAVESAND JOHN GUTKNECHT Ion Transport in Halicystis
591
700
6OO
,
500
~," E ~
400
o
E "
5OO
g
(.-)
•
IOC
i~o z6o
360
46o
~6o s6o
7~o
Short- Circuit Current (peq.crn~ "= )
FIGURE 6. Relationship between unidirectional CI- influx and SCC in perfused cells. Each point corresponds to the mean value of Cl- influx and SCC in a single cell under standard conditions. The line is a least squares regression with a slope of 1.12, r = 0.98 (N = 9). When the t u r g o r pressure o f a cell was r e d u c e d (e.g., u p o n r e m o v i n g a microelectrode) we observed a conspicuous decrease in the cell volume, which suggested an unusually low d e g r e e o f wall rigidity. Dainty et al. (6) d e m o n s t r a t e d that the elastic m o d u l u s o f a cell wall is an i m p o r t a n t p a r a m e t e r in the osmotic relations o f plant cells. T h e r e f o r e , in o r d e r to estimate the elastic m o d u l u s (~ in atm) for the cell wall we d e t e r m i n e d the c h a n g e in cell volume as a function o f internally applied pressure (Fig. 7). T h e slope o f the nearly linear curve in Fig. 7 is about 1.7 atm -1. F r o m Eq. 4 (see Methods section), we f o u n d that the value o f (the reciprocal o f this slope) was 0.6 atm. D I S C U S S I O N
Steady-State Ionic Distribution We have m e a s u r e d the concentrations o f all inorganic ions in the vacuolar sap o f H. parvula. A s s u m i n g that the cells were in a steady state, we calculated the equilibrium potential o f each ion a n d c o m p a r e d it to the m e a s u r e d PD ( - 8 2 mV) to d e t e r m i n e the existence and direction o f active transport. This analysis indicates that Na + a n d K + and all anions are actively t r a n s p o r t e d . T h e electrochemical analysis for vacuolar K + (Table I) and the observed net efflux o f K + u n d e r short-circuit conditions both indicate that this ion is p u m p e d out o f the vacuole. T h e concentration o f K + in the cytoplasm is about 45 times that in the vacuolar sap (Table II). Without knowing the potential difference across each m e m b r a n e , the location o f the o u t w a r d K + p u m p c a n n o t be assigned
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THE JOURNAL OF GENERAL PHYSIOLOGY " VOLUME 6 7 - 1 9 7 6
-T o.s ~
0.5
~
o~
e-- o.3 o) ~
0.2
o.i
0i
o
6., d.2 o'.3 Pressure Difference (otm)
FIGURE 7. Increase in cell volume as a function of internally applied hydrostatic pressure; Vt is the volume at zero pressure. V~is the final volume. Points and bars represent the mean +- SE for four to five cells. with certainty. However, a passive distribution o f K + across the tonoplast would require an unusual combination o f m e m b r a n e potentials (e.g., - 170 mV at the plasmalemma and +90 mV at the tonoplast). T h u s , it seems m o r e likely that the outward K + p u m p is located at the tonoplast. T h e most c o m m o n mechanism for maintaining a high [K +] in the cytoplasm in both plant and animal cells is an inward p u m p at the plasmalemma, which is often associated with o u t w a r d Na ÷ transport. Such an inward K + p u m p at the plasmalemma o f H. parvula is not ruled out by the evidence presented. Electrochemical evidence indicates that Na + is also p u m p e d out o f the vacuole (Table I). Since the [K +] in the cytoplasm is probably more than 300 mM, for osmotic stability the [Na +] in the cytoplasm must be no greater than about onehalf the [Na +] in the vacuolar sap. Without knowing the electrical potential across the tonoplast an inward Na + p u m p cannot be assigned to that m e m b r a n e with certainty. Nevertheless, an inward Na + p u m p at the tonoplast in addition to an o u t w a r d Na + p u m p at the plasmalemma with a similar p u m p i n g rate could explain the lack o f a net Na + efflux u n d e r short-circuit conditions. In such a system the passive Na + fluxes could be m u c h smaller than the active Na + fluxes and would not then contribute significantly to the flux ratio u n d e r any experimental conditions. T h e r e is g o o d evidence for an inward Na + p u m p at the tonoplast in Nitella (40) and in Valonia (16). It t h e r e f o r e is possible that Na + and K + are t r a n s p o r t e d in opposite directions at each m e m b r a n e o f H. parvula. We have presented evidence that all inorganic anions are actively t r a n s p o r t e d into the cell (Table I). In addition, the large net influx o f CI- u n d e r short-circuit conditions (Table III) unequivocally indicates active C1- transport. Only those algae with electropositive vacuoles, namely Valonia (16) and Chaetomorpha (8).
JAMES S. GRAVES AND JOHN GUTKNECHT
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593
a p p e a r to lack C1- p u m p s . With r e g a r d to the metabolized anions, NO3-, SO4 =, a n d p h o s p h a t e , t h e r e are only a few studies in vacuolated algae which deal with the t r a n s p o r t o f these ions. Jacques a n d O s t e r h o u t (21) f o u n d that NO3- was c o n c e n t r a t e d in the sap ofValonia macryophysa by 2,000-fold a n d in the sap o f H . osterhoutii by 500-fold o v e r the concentration in natural seawater, which is less than 10 /xM. T h e a c c u m u l a t i o n o f p h o s p h a t e a n d SO4 = has been s h o w n in several species o f m a r i n e algae (23, 24, 35). Likewise, the active inward t r a n s p o r t o f p h o s p h a t e (33, 39) a n d o f SO4 = (34) has been d e m o n s t r a t e d in f r e s h w a t e r algae. With no estimation o f the cytoplasmic concentrations o f these anions, it is impossible to assign the locations o f their p u m p s . H o w e v e r , in the f r e s h w a t e r algae (30) a n d in Acetabularia (36) the C1- p u m p is located at the p l a s m a l e m m a . It also is likely that the p u m p s for the metabolized anions reside at the plasmal e m m a because a high c o n c e n t r a t i o n in the cytoplasm would be metabolically a d v a n t a g e o u s . G o o d electrochemical data for the f r e s h w a t e r alga, Hydrodictyon, indicate that the p h o s p h a t e p u m p is i n d e e d at the p l a s m a l e m m a in this cell (33).
The Short-Circuit Current In cells o f H . parvula p e r f u s e d with ASW the m e a n SCC is 299 peq • cm -2 • s -1 (ca. 29 /xA • cm-2). T h e net CI- influx o f 290 peq • cm -2 • s -Z is not statistically d i f f e r e n t f r o m the m e a n SCC, a n d the unidirectional C1- influx is p r o p o r t i o n a l to the SCC o v e r the r a n g e o f variability (Fig. 6). T h e r e is no statistically significant net Na + flux, a n d the net K + efflux is trivial c o m p a r e d to the net C1flux (Table I I I ) . T h e r e f o r e , virtually all o f the SCC is g e n e r a t e d by the active t r a n s p o r t o f C1- into the cell. In H. ovalis the m e a n SCC c o m p u t e d f r o m the data o f Blount a n d L e v e d a h l (3) is identical to the value d e t e r m i n e d for H. parvula. H o w e v e r , in H. ovalis a b o u t 39% o f the SCC is d u e to a net Na + efflux, a n d the r e m a i n d e r is d u e to net C1influx. T h u s , the fact that the SCC for both species is identical is p r o b a b l y fortuitous. T h e SCC has b e e n m e a s u r e d in only two o t h e r plant cells. In Valonia ventricosa G u t k n e c h t (17) f o u n d an SCC o f a b o u t 100 peq • cm -z • s -1. This SCC is nearly accounted for by the large net influx o f K + a n d a smaller net influx o f Na +. In Nitella clavata the SCC is only a b o u t 18 peq • cm -~ • s -1, a n d the ionic origin is u n k n o w n (42). T h u s , in Halicystis the SCC is a p p r e c i a b l y g r e a t e r t h a n the values r e p o r t e d for o t h e r plant cells a n d is m o r e like the SCC's g e n e r a t e d by s o m e animal epithelia (see 25). H. parvula (and H. osterhoutii) m a y be uniquely suited to a short-circuit c u r r e n t analysis o f ion t r a n s p o r t . I n o t h e r plant a n d animal p r e p a r a t i o n s used f o r this p u r p o s e the solutions b a t h i n g the two sides o f the " m e m b r a n e " are n o r m a l l y quite d i f f e r e n t in ionic composition, a n d at least o n e o f the solutions m u s t be substantially altered in o r d e r to attain symmetrical or short-circuit conditions. H o w e v e r , except for p H , the vacuolar sap o f H. parvula is similar in ionic composition to the external seawater. T h u s , with H. parvula the short-circuit c u r r e n t analysis o f ion t r a n s p o r t m a y be m o r e relevant to the intact cell t h a n with any system previously used.
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Osmotic Relations and Elastic Properties of the Cell Wall T h e existence o f a t u r g o r p r e s s u r e in walled plant cells a p p e a r s to be universal. Unlike animal cells, most plant ceils have an external cell wall capable o f withstanding appreciable hydrostatic pressure. T u r g o r p r e s s u r e a p p e a r s to play a critical role in m a i n t a i n i n g n o r m a l cell growth a n d m u s t t h e r e f o r e be r e g a r d e d as a physiologically i m p o r t a n t p a r a m e t e r (15). T h e t u r g o r pressure o f H . parvula is a b o u t 0.5 a t m , which is in a g r e e m e n t with previous d e t e r m i n a t i o n s for Halicystis (1, 20). This p r e s s u r e is lower t h a n those in most o t h e r walled algae, which usually r a n g e f r o m 1.5 to 13 atm. T h e cell wall o f H. parvula has an unusually low elastic m o d u l u s (i.e., low d e g r e e o f rigidity). T a b l e IV shows the volumetric elastic moduli (E) for the cell walls o f H. parvula a n d three o t h e r algae which were d e t e r m i n e d at similar pressures. It is a p p a r e n t that the • for H. parvula is at least two o r d e r s o f m a g n i t u d e lower than that o f any o t h e r alga. This low elastic m o d u l u s is consistent with the observation by Jacques (20) that the cell v o l u m e o f H. osterhoutii d e c r e a s e d by an appreciable a m o u n t w h e n it was i m p a l e d with a glass pipet. TABLE
IV
VOLUMETRIC ELASTIC MODULI OF T H E CELL WALLS OF SEVERAL ALGAE DETERMINED AT LOW PRESSURES Alga
Nitellaflexilis Valonia ventricosa Valonia utricularis Halicystisparvula
Habitat
Freshwater Marine Marine Marine
Elastic m o d u ] u s
Pressure range of measurement
atm
arm
67 182 90 0.6
0-1.0 0-1.0 0-0.5 0-0.4
Reference
(22) (46) (49) Present study
Z i m m e r m a n n a n d Steudle (49, 50) recently d e m o n s t r a t e d that the elastic moduli o f V. utricularis a n d N.flexilis are d e p e n d e n t on the t u r g o r p r e s s u r e w h e n the d e t e r m i n a t i o n is m a d e at pressures below 2 a t m . F u r t h e r m o r e , these investigators f o u n d • was also p r o p o r t i o n a l to the total cell volume (50). For V. utricularis • r a n g e d f r o m 125 to 300 atm at pressures (i.e., 3-6 atm) at which the value o f E is i n d e p e n d e n t o f p r e s s u r e . T h e analogous r a n g e o f • for N.flexilis was 100-200 a t m . Also, Dainty et al. (6) have estimated the p r e s s u r e - i n d e p e n d e n t • for one size r a n g e o f Chara corallina to be a r o u n d 700 a t m . Because the applied pressures used in this study were quite small, it is likely that the e d e t e r m i n e d for H. parvula falls within the p r e s s u r e - d e p e n d e n t region. T h e highly extensible wall o f H. parvula often leads to b r e a k a g e o f the seals a r o u n d the pipets at pressures g r e a t e r than 0.5 atm, a n d t h e r e f o r e it may be difficult or impossible to accurately m e a s u r e the p r e s s u r e - i n d e p e n d e n t e by this technique. H o w e v e r , the value o f E which exists at pressures n e a r the n o r m a l t u r g o r pressure (i.e., 0.5 a t m for H. parvula) has the greatest physiological significance, and the • o f 0.6 atm d e t e r m i n e d at pressures u p to 0.4 a t m is relevant in this r e g a r d . Also, we did not a t t e m p t to d e t e r m i n e the cell v o l u m e to e relationship for H. parvula. All o f the d e t e r m i n a t i o n s in this work were m a d e on cells between 35 and 60 m m 3 in
JAMES S. GRAVESAND JOHN GUTKNECHT Ion Transport in Halicystis
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v o l u m e , so t h e v a l u e o f 0.6 a t m is r e l e v a n t o n l y to cells in this size r a n g e . T h e m o l e c u l a r i n t e r a c t i o n s i n v o l v e d in d e t e r m i n i n g t h e elastic p r o p e r t i e s o f cell walls a r e n o t well u n d e r s t o o d . H o w e v e r , it is n o t e w o r t h y t h a t t h e c h e m i c a l c o m p o s i t i o n o f t h e walls o f Halicystis a n d a few close r e l a t i v e s is also u n u s u a l . I n a d d i t i o n to t h e c o m m o n s t r u c t u r a l c o m p o n e n t , c e l l u l o s e , Halicystis walls c o n t a i n p o l y x y l a n as t h e m a j o r s t r u c t u r a l p o l y s a c c h a r i d e (10). U n l i k e c e l l u l o s e , t h e x y l a n c h a i n s a r e h e l i c a l l y c o i l e d (11). T h e r e is u n d o u b t e d l y a r e l a t i o n b e t w e e n m o l e c u l a r s t r u c t u r e a n d e x t e n s i b i l i t y , b u t t h a t r e l a t i o n s h i p r e m a i n s to be d e t e r m i n e d . Send reprint requests to Dr. Gutknecht, Duke Marine Laboratory, Beaufort, N. C. 28516. We thank Mr. M. Bradley for providing technical assistance and Ms. M. Bisson for critically reading the manuscript. Dr. D. Hastings designed and assembled the apparatus used for vacuolar perfusion experiments. The first author (J. S. G.) was supported by a predoctoral traineeship from N. I. H. This work was supported by U.S.P.H.S. Grant HL12157.
Receivedfor publication 31 July 1975. REFERENCES 1. BLINKS, L. R. 1951. Physiology and Biochemistry of the Algae. In Manual o f Phycology. G. M. Smith, editor. Cronica Botanica Co., Waltham, Mass. 263-290. 2. BLINKS, L. R., a n d A. G. JACQUES. 1930. T h e cell sap o f Halicystis. J. Gen. Physiol. 13:733-737. 3. BLOUNT, R. W., a n d B. H. LEVEDAHL. 1960. Active sodium and chloride t r a n s p o r t in the single celled marine alga, Halicystis ovalis. Acta Physiol. Scand. 49:1-9. 4. BROOKS, S. C. 1930. Composition of the cell sap ofHalicystis ovalis. (Lyng.) Areschoug. Proc. Soc. Exp. Biol. Med. 27:409-412. 5. CHAPMAN,V.J. 1962. T h e Algae. MacMillan and Co. Ltd., London. 6. DAINTY, J., H. VINTERS, and M. T. TYREE. 1974. A study o f transcellular osmosis and the kinetics o f swelling and shrinking in cells o f Chara corallina. In Membrane T r a n s p o r t in Plants. U. Z i m m e r m a n n and J. Dainty, editors. Springer-Verlag, New York. 59-63. 7. EPPLEY, R. W., and L. R. BUNKS. 1957. Cell space and a p p a r e n t free space in the red alga, Porphyra perforata. Plant Physiol. 32:63-64. 8. FINDLAV, G. P., A. B. HOPE, M. G. PITMAN, E. A. SMITtt, and N. A. WALKEIt. 1971. Ionic relations o f marine algae. I I I . Chaetomorpha: M e m b r a n e electrical p r o p e r ties and chloride fluxes. Aust. J. Biol. Sci. 24:731-745. 9. FISKE, C. H., and Y. SUBBAROW. 1925. T h e colorimetric determination o f phosphor u s . J . Biol. Chem. 66:375-400. 10. FREI, E., and R. D. PRESTON. 196!. Variants in the structural polysaccharides o f algal cell walls. Nature (Lond.). 192:939-943. 11. FREI, E., and R. D. PRESTON. 1964. Non-cellulosic structural polysaccharides in algal cell walls. I. Xylan in siphoneous green algae. Proc. R. Soc. Lond. B Biol. Sci. 160:293-313. 12. GACE, P. W., and R. S. E~SeNBEaO. 1969. Capacitance o f the surface and transverse tubular m e m b r a n e of frog sartorius muscle fibers.J. Gen. Physiol. 53:265-278. 13. GxNz~uRg, B. Z., and J. H o g g . 1967. What does a short-circuit current measure in biological systems?J. Theor. Biol. 14:316-322.
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14. GRAVES, J. S. 1974. Ion transport and electrical properties of the marine alga, Halicystis parvula. Ph.D. Dissertation. Duke University, Durham, N. C. 15. GREEN, P. B., R. O. ERICKSON,a n d J . BUGGY. 1971. Metabolic and physical control of cell elongation rate. Plant Physiol. 47:423-430. 16. GUTKNECHT, J. 1966. Sodium, potassium and chloride transport and m e m b r a n e potentials in Valonia ventricosa. Biol. Bull. (Woods Hole). 150:331-344. 17. GUTKNECHT,J. 1967. Ion fluxes and short-circuit current in internally perfused cells of Valonia ventricosa . J. Gen. Physiol. riO:1821-1834. 18. GUTKNECHT,J. and J. DAINTY. 1968. Ionic relations of marine algae. Oceanogr. Mar. Biol. Annu. Rev. 6:163-200. 19. HANES, C. S., and F. A. ISCHERWOOD. 1949. Separation of the phosphoric esters on the filter paper chromatogram. Nature (Lond.). 164:1107-1112. 20. JACQUES, A. G. 1939. T h e kinetics of penetration. XIX. Entrance of electrolytes and of water into impaled Halicystis. J. Gen. Physiol. 22:757-773. 21. JACQUES, A. G., and W. J. V. OSTERHOUT. 1938. The accumulation of electrolytes. XI. Accumulation of nitrate by Valonia and Halicystis. J. Gen. Physiol. 21:767-773. 22. KAMIYA,N., M. TAWAWA,and T. TAKATA. 1963. The relation of turgor pressure to cell volume in Nitella with special reference to mechanical properties of the cell wall. Protoplasma. 57:501-521. 23. KESSELER, H. 1964. Zellsaftgewinnung, AFS and Vakuolenkonzentration der osmotisch wichtigsten mineralischen Bestandteile einiger Helg61ander Meeresalgen. Helg61. Wiss. Meeresunters. 11:258-269. 24. KESSELER, H. 1965. T u r g o r , osmotisches potential u n d ionale zusammensetzung des Zellsaftes einiger Meeresalgen verschiedener verbreitungsbebiete. In Proceedings of the Fifth Marine Biological Symposium, Botanica Gothoburgensia. 3:103-111. 25. KEVNES, R. D. 1969. From frog skin to sheep r u m e n : A survey of transport of salts and water across multicellular structures. Q. Rev. Biophys. 2:177-281. 26. KORNMANN, P. 1938. Zur entwicklungsgeschichte von Derbesia u n d Halicystis. Planta (Berl.). 28:464-470. 27. KOTYK, A., and K. JANACEK. 1970. Cell Membrane T r a n s p o r t . Plenum Press, New York. 28. LARKUM, A. W. D. 1968. Ionic relations of chloroplasts in vivo. Nature (Lond.). 218:447-449. 29. LowE, R. H., and H. J. EVANS. 1964. Preparation and some properties of a soluble nitrate reductase from Rhizobium japonicum. Biochim. Biophys. Acta. 85:377-389. 30. MACROBBIE,E. A. C. 1970. T h e active transport of ions in plant cells. Q. Rev. Biophys. 3:251-294. 31. PAGE,J. z. 1970. Existence of a Derbesia phase in the life history of Halicystis osterhoutii Blinks and Blinks.J. Phycol. 6:375-380. 32. PHILIe,J. R. 1958. T h e osmotic cell, solute diffusibility and the plant water economy. Plant Physiol. $$:264-275. 33. RAVEN,J. A. 1974. Phosphate transport in Hydrodictyon africanum. New Phytol. 75:421432. 34. ROBINSON,J. B. 1969. Sulfate influx in characean cells. I. General characteristics.J. Exp. Bot. 20:201-211. 35. SAnnLER, H. D. W. 1970. T h e ionic relations ofAcetabularia mediterranea. J. Exp. Bot. 21:345-359.
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36. SADDLER,H. D. W. 1970. T h e m e m b r a n e potential ofAcetabularia mediterranea.J. Gen. Physiol. 55:802-821. 37. SANUI, S., and N. PACE. 1966. Application of atomic absorption in the study o f Na, K, Mg and Ca binding by cellular membranes. Appl. Spectrosc. 20:135-141. 38. SEARS, J. R., and R. T. WILCE. 1970. Reproduction and systematics o f the marine alga Derbesia (Chlorophyceae) in New E n g l a n d . J . Phycol. 6:381-392. 39. SMITH, F. A. 1966. Active p h o s p h a t e uptake by Nitella translucens. Biochim. Biophys. Acta. 126:94-99. 40. SPAr~SWICK,R. M., and E. J. WILLIAMS. 1964. Electrical potentials and Na, K, and C1 concentrations in the vacuole and cytoplasm o f Nitella translucens. J. Exp. Bot. 15: 193-200.
41. SPANSWICK,R. M., and E. J. WXLLIAr~S.1965. Ca fluxes and m e m b r a n e potentials in NiteUa translucens. J. Exp. Bot. 16:463-473. 42. STRUNK, T . H. 1971. Correlation between metabolic p a r a m e t e r s of t r a n s p o r t and vacuolar perfusion results in Nitella clavata. J. Exp. Bot. 22:863-874. 43. TEORELL, T. 1949. Membrane electrophoresis in relation to bioelectric polarization effects. Arch. Sci. Physiol. 3:205-219. 44. USSZNG, H. H. 1949. T h e distinction by means of tracers between active t r a n s p o r t and diffusion. Acta Physiol. Scand. 19:43-56. 45. USSING, H. H., and K. ZERAHN. 1950. Active transport of sodium as the source of electric c u r r e n t in the short-circuited isolated frog skin. Acta Physiol. Scand. 23:110127. 46. VILLEGAS, L. 1967. Changes in volume and turgor pressure in Valonia cells. Biochim. Biophys. Acta. 136:590-593. 47. WHEELER, A. E. and J. Z. PAGr. 1974. T h e ultrastructure o f Derbesia tenuissima. I. Organization o f the gametophyte protoplast, gametangium, and gametangial p o r e . J . Phycol. 10:336--352. 48. ZIEGLER,J. R. and J. M. KINGSSURV. 1964. Cultural studies on the marine green alge Halicystis parvula-Derbesia tenuissima. I. Normal and abnormal sexual and asexual reproduction. Phycologia. 4:105-116. 49. ZI~traE~MAYN, U., and E. STEUDLE. 1974. T h e p r e s s u r e - d e p e n d e n c e of the hydraulic conductivity, the membrane resistance and the m e m b r a n e potential d u r i n g t u r g o r pressure regulation in Valonia utricularis. J. Membr. Biol. 16:331-352. 50. ZXMt~ERMANN, U., and E. STEUDLE. 1974. Hydraulic conductivity and volumetric elastic modulus in giant algal cells: pressure and volume d e p e n d e n c e . In Membrane T r a n s p o r t in Plants. U. Z i m m e r m a n n and J. Dainty, editors. SpringerVerlag, New York. 64-71.
S. G R A V E S and J O H N
GUTKNECHT
From the Department of Physiology and Pharmacology, Duke University Medical Center, Durham, North Carolina 27710 and the Duke University Marine Laboratory, Beaufort, North Carolina 28516. Dr. Graves' present address is the Ontario Cancer Institute, Toronto, Ontario, Canada.
A ~ S x 1~A C X Using cultured cells o f the marine alga, Halicystisparvula, we measu r e d the concentrations o f 11 inorganic ions in the vacuolar sap and the electrical potential difference (PD) between the vacuole and the external solution. In normal cells u n d e r steady-state conditions a comparison o f the electrochemical equilibrium (Nernst) potential for each ion with the PD o f - 8 ~ mV (inside negative) indicates that Na + and K + are actively t r a n s p o r t e d out of the vacuole whereas all anions are p u m p e d into the cell. Although the [K +] in the vacuole is only 9 raM, the cytoplasmic [K +] is about 420 mM, which suggests that the outwardly directed p u m p is at the tonoplast. Using large Halicystis cells we perfused the vacuole with an artificial seawater and conducted a short-circuit analysis of ion transport. T h e short-circuit current (SCC) o f 299 peq • cm-2.s -~ is not significantly different from the net influx o f CI-. T h e r e is a small, but statistically significant net efflux o f K + ( 0.10 P < 0.025
- cm -~ " s-all
313-+42 (13) 299-+41 (9) 268-+24 (6)
* Data expressed as mean -+ SE (number of cells). ~: Direction indicated by sign; outward (+), inward (-). § The SCC measured during flux experiments for each ion. I1Converted to these units by: (/xA cm-Z)/(F x 10t). 1.12. F r o m this a n a l y s i s we c o n c l u d e t h a t C l - is actively t r a n s p o r t e d i n t o t h e v a c u o l e a n d t h a t t h e SCC is a g o o d m e a s u r e o f t h e r a t e o f active t r a n s p o r t o f C I - .
Turgor Pressure and Cell Wall Elastic Modulus T h e o s m o l a l i t y o f t h e v a c u o l a r s a p was m e a s u r e d in two e x p e r i m e n t s with a t o t a l o f five s a p s a m p l e s . T h e o s m o l a l i t y d i f f e r e n c e b e t w e e n t h e s a p a n d t h e A S W in w h i c h t h e cells w e r e p r e t r e a t e d was 23 ± 6 m o s m o l / k g . T h e o s m o t i c p r e s s u r e d i f f e r e n c e (AII in a t m ) was e s t i m a t e d by t h e v a n ' t H o f f e q u a t i o n (see 27): All =R
"T.
AC,
(6)
w h e r e AC is t h e o s m o l a l c o n c e n t r a t i o n d i f f e r e n c e . T h u s , t h e m e a n o s m o t i c p r e s s u r e d i f f e r e n c e is 0.54 a t m . A s s u m i n g t h a t t h e n e t flow o f w a t e r is e s s e n t i a l l y z e r o , this o s m o t i c p r e s s u r e d i f f e r e n c e is e q u a l to t h e h y d r o s t a t i c p r e s s u r e d i f f e r ence or turgor pressure. Turgor pressures for most vacuolated marine algae r a n g e f r o m 1.5 to 13 a t m (see 24). T h e v a l u e f o r H . parvula is t h e r e f o r e u n u s u a l l y low b u t is, n e v e r t h e l e s s , in a g r e e m e n t with t h e e a r l i e r v a l u e s o f 0 . 2 5 - 0 . 5 a t m r e p o r t e d f o r Halicystis (1, 20).
JAMES S. GRAVESAND JOHN GUTKNECHT Ion Transport in Halicystis
591
700
6OO
,
500
~," E ~
400
o
E "
5OO
g
(.-)
•
IOC
i~o z6o
360
46o
~6o s6o
7~o
Short- Circuit Current (peq.crn~ "= )
FIGURE 6. Relationship between unidirectional CI- influx and SCC in perfused cells. Each point corresponds to the mean value of Cl- influx and SCC in a single cell under standard conditions. The line is a least squares regression with a slope of 1.12, r = 0.98 (N = 9). When the t u r g o r pressure o f a cell was r e d u c e d (e.g., u p o n r e m o v i n g a microelectrode) we observed a conspicuous decrease in the cell volume, which suggested an unusually low d e g r e e o f wall rigidity. Dainty et al. (6) d e m o n s t r a t e d that the elastic m o d u l u s o f a cell wall is an i m p o r t a n t p a r a m e t e r in the osmotic relations o f plant cells. T h e r e f o r e , in o r d e r to estimate the elastic m o d u l u s (~ in atm) for the cell wall we d e t e r m i n e d the c h a n g e in cell volume as a function o f internally applied pressure (Fig. 7). T h e slope o f the nearly linear curve in Fig. 7 is about 1.7 atm -1. F r o m Eq. 4 (see Methods section), we f o u n d that the value o f (the reciprocal o f this slope) was 0.6 atm. D I S C U S S I O N
Steady-State Ionic Distribution We have m e a s u r e d the concentrations o f all inorganic ions in the vacuolar sap o f H. parvula. A s s u m i n g that the cells were in a steady state, we calculated the equilibrium potential o f each ion a n d c o m p a r e d it to the m e a s u r e d PD ( - 8 2 mV) to d e t e r m i n e the existence and direction o f active transport. This analysis indicates that Na + a n d K + and all anions are actively t r a n s p o r t e d . T h e electrochemical analysis for vacuolar K + (Table I) and the observed net efflux o f K + u n d e r short-circuit conditions both indicate that this ion is p u m p e d out o f the vacuole. T h e concentration o f K + in the cytoplasm is about 45 times that in the vacuolar sap (Table II). Without knowing the potential difference across each m e m b r a n e , the location o f the o u t w a r d K + p u m p c a n n o t be assigned
592
THE JOURNAL OF GENERAL PHYSIOLOGY " VOLUME 6 7 - 1 9 7 6
-T o.s ~
0.5
~
o~
e-- o.3 o) ~
0.2
o.i
0i
o
6., d.2 o'.3 Pressure Difference (otm)
FIGURE 7. Increase in cell volume as a function of internally applied hydrostatic pressure; Vt is the volume at zero pressure. V~is the final volume. Points and bars represent the mean +- SE for four to five cells. with certainty. However, a passive distribution o f K + across the tonoplast would require an unusual combination o f m e m b r a n e potentials (e.g., - 170 mV at the plasmalemma and +90 mV at the tonoplast). T h u s , it seems m o r e likely that the outward K + p u m p is located at the tonoplast. T h e most c o m m o n mechanism for maintaining a high [K +] in the cytoplasm in both plant and animal cells is an inward p u m p at the plasmalemma, which is often associated with o u t w a r d Na ÷ transport. Such an inward K + p u m p at the plasmalemma o f H. parvula is not ruled out by the evidence presented. Electrochemical evidence indicates that Na + is also p u m p e d out o f the vacuole (Table I). Since the [K +] in the cytoplasm is probably more than 300 mM, for osmotic stability the [Na +] in the cytoplasm must be no greater than about onehalf the [Na +] in the vacuolar sap. Without knowing the electrical potential across the tonoplast an inward Na + p u m p cannot be assigned to that m e m b r a n e with certainty. Nevertheless, an inward Na + p u m p at the tonoplast in addition to an o u t w a r d Na + p u m p at the plasmalemma with a similar p u m p i n g rate could explain the lack o f a net Na + efflux u n d e r short-circuit conditions. In such a system the passive Na + fluxes could be m u c h smaller than the active Na + fluxes and would not then contribute significantly to the flux ratio u n d e r any experimental conditions. T h e r e is g o o d evidence for an inward Na + p u m p at the tonoplast in Nitella (40) and in Valonia (16). It t h e r e f o r e is possible that Na + and K + are t r a n s p o r t e d in opposite directions at each m e m b r a n e o f H. parvula. We have presented evidence that all inorganic anions are actively t r a n s p o r t e d into the cell (Table I). In addition, the large net influx o f CI- u n d e r short-circuit conditions (Table III) unequivocally indicates active C1- transport. Only those algae with electropositive vacuoles, namely Valonia (16) and Chaetomorpha (8).
JAMES S. GRAVES AND JOHN GUTKNECHT
Ion Transport in Halicystis
593
a p p e a r to lack C1- p u m p s . With r e g a r d to the metabolized anions, NO3-, SO4 =, a n d p h o s p h a t e , t h e r e are only a few studies in vacuolated algae which deal with the t r a n s p o r t o f these ions. Jacques a n d O s t e r h o u t (21) f o u n d that NO3- was c o n c e n t r a t e d in the sap ofValonia macryophysa by 2,000-fold a n d in the sap o f H . osterhoutii by 500-fold o v e r the concentration in natural seawater, which is less than 10 /xM. T h e a c c u m u l a t i o n o f p h o s p h a t e a n d SO4 = has been s h o w n in several species o f m a r i n e algae (23, 24, 35). Likewise, the active inward t r a n s p o r t o f p h o s p h a t e (33, 39) a n d o f SO4 = (34) has been d e m o n s t r a t e d in f r e s h w a t e r algae. With no estimation o f the cytoplasmic concentrations o f these anions, it is impossible to assign the locations o f their p u m p s . H o w e v e r , in the f r e s h w a t e r algae (30) a n d in Acetabularia (36) the C1- p u m p is located at the p l a s m a l e m m a . It also is likely that the p u m p s for the metabolized anions reside at the plasmal e m m a because a high c o n c e n t r a t i o n in the cytoplasm would be metabolically a d v a n t a g e o u s . G o o d electrochemical data for the f r e s h w a t e r alga, Hydrodictyon, indicate that the p h o s p h a t e p u m p is i n d e e d at the p l a s m a l e m m a in this cell (33).
The Short-Circuit Current In cells o f H . parvula p e r f u s e d with ASW the m e a n SCC is 299 peq • cm -2 • s -1 (ca. 29 /xA • cm-2). T h e net CI- influx o f 290 peq • cm -2 • s -Z is not statistically d i f f e r e n t f r o m the m e a n SCC, a n d the unidirectional C1- influx is p r o p o r t i o n a l to the SCC o v e r the r a n g e o f variability (Fig. 6). T h e r e is no statistically significant net Na + flux, a n d the net K + efflux is trivial c o m p a r e d to the net C1flux (Table I I I ) . T h e r e f o r e , virtually all o f the SCC is g e n e r a t e d by the active t r a n s p o r t o f C1- into the cell. In H. ovalis the m e a n SCC c o m p u t e d f r o m the data o f Blount a n d L e v e d a h l (3) is identical to the value d e t e r m i n e d for H. parvula. H o w e v e r , in H. ovalis a b o u t 39% o f the SCC is d u e to a net Na + efflux, a n d the r e m a i n d e r is d u e to net C1influx. T h u s , the fact that the SCC for both species is identical is p r o b a b l y fortuitous. T h e SCC has b e e n m e a s u r e d in only two o t h e r plant cells. In Valonia ventricosa G u t k n e c h t (17) f o u n d an SCC o f a b o u t 100 peq • cm -z • s -1. This SCC is nearly accounted for by the large net influx o f K + a n d a smaller net influx o f Na +. In Nitella clavata the SCC is only a b o u t 18 peq • cm -~ • s -1, a n d the ionic origin is u n k n o w n (42). T h u s , in Halicystis the SCC is a p p r e c i a b l y g r e a t e r t h a n the values r e p o r t e d for o t h e r plant cells a n d is m o r e like the SCC's g e n e r a t e d by s o m e animal epithelia (see 25). H. parvula (and H. osterhoutii) m a y be uniquely suited to a short-circuit c u r r e n t analysis o f ion t r a n s p o r t . I n o t h e r plant a n d animal p r e p a r a t i o n s used f o r this p u r p o s e the solutions b a t h i n g the two sides o f the " m e m b r a n e " are n o r m a l l y quite d i f f e r e n t in ionic composition, a n d at least o n e o f the solutions m u s t be substantially altered in o r d e r to attain symmetrical or short-circuit conditions. H o w e v e r , except for p H , the vacuolar sap o f H. parvula is similar in ionic composition to the external seawater. T h u s , with H. parvula the short-circuit c u r r e n t analysis o f ion t r a n s p o r t m a y be m o r e relevant to the intact cell t h a n with any system previously used.
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Osmotic Relations and Elastic Properties of the Cell Wall T h e existence o f a t u r g o r p r e s s u r e in walled plant cells a p p e a r s to be universal. Unlike animal cells, most plant ceils have an external cell wall capable o f withstanding appreciable hydrostatic pressure. T u r g o r p r e s s u r e a p p e a r s to play a critical role in m a i n t a i n i n g n o r m a l cell growth a n d m u s t t h e r e f o r e be r e g a r d e d as a physiologically i m p o r t a n t p a r a m e t e r (15). T h e t u r g o r pressure o f H . parvula is a b o u t 0.5 a t m , which is in a g r e e m e n t with previous d e t e r m i n a t i o n s for Halicystis (1, 20). This p r e s s u r e is lower t h a n those in most o t h e r walled algae, which usually r a n g e f r o m 1.5 to 13 atm. T h e cell wall o f H. parvula has an unusually low elastic m o d u l u s (i.e., low d e g r e e o f rigidity). T a b l e IV shows the volumetric elastic moduli (E) for the cell walls o f H. parvula a n d three o t h e r algae which were d e t e r m i n e d at similar pressures. It is a p p a r e n t that the • for H. parvula is at least two o r d e r s o f m a g n i t u d e lower than that o f any o t h e r alga. This low elastic m o d u l u s is consistent with the observation by Jacques (20) that the cell v o l u m e o f H. osterhoutii d e c r e a s e d by an appreciable a m o u n t w h e n it was i m p a l e d with a glass pipet. TABLE
IV
VOLUMETRIC ELASTIC MODULI OF T H E CELL WALLS OF SEVERAL ALGAE DETERMINED AT LOW PRESSURES Alga
Nitellaflexilis Valonia ventricosa Valonia utricularis Halicystisparvula
Habitat
Freshwater Marine Marine Marine
Elastic m o d u ] u s
Pressure range of measurement
atm
arm
67 182 90 0.6
0-1.0 0-1.0 0-0.5 0-0.4
Reference
(22) (46) (49) Present study
Z i m m e r m a n n a n d Steudle (49, 50) recently d e m o n s t r a t e d that the elastic moduli o f V. utricularis a n d N.flexilis are d e p e n d e n t on the t u r g o r p r e s s u r e w h e n the d e t e r m i n a t i o n is m a d e at pressures below 2 a t m . F u r t h e r m o r e , these investigators f o u n d • was also p r o p o r t i o n a l to the total cell volume (50). For V. utricularis • r a n g e d f r o m 125 to 300 atm at pressures (i.e., 3-6 atm) at which the value o f E is i n d e p e n d e n t o f p r e s s u r e . T h e analogous r a n g e o f • for N.flexilis was 100-200 a t m . Also, Dainty et al. (6) have estimated the p r e s s u r e - i n d e p e n d e n t • for one size r a n g e o f Chara corallina to be a r o u n d 700 a t m . Because the applied pressures used in this study were quite small, it is likely that the e d e t e r m i n e d for H. parvula falls within the p r e s s u r e - d e p e n d e n t region. T h e highly extensible wall o f H. parvula often leads to b r e a k a g e o f the seals a r o u n d the pipets at pressures g r e a t e r than 0.5 atm, a n d t h e r e f o r e it may be difficult or impossible to accurately m e a s u r e the p r e s s u r e - i n d e p e n d e n t e by this technique. H o w e v e r , the value o f E which exists at pressures n e a r the n o r m a l t u r g o r pressure (i.e., 0.5 a t m for H. parvula) has the greatest physiological significance, and the • o f 0.6 atm d e t e r m i n e d at pressures u p to 0.4 a t m is relevant in this r e g a r d . Also, we did not a t t e m p t to d e t e r m i n e the cell v o l u m e to e relationship for H. parvula. All o f the d e t e r m i n a t i o n s in this work were m a d e on cells between 35 and 60 m m 3 in
JAMES S. GRAVESAND JOHN GUTKNECHT Ion Transport in Halicystis
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v o l u m e , so t h e v a l u e o f 0.6 a t m is r e l e v a n t o n l y to cells in this size r a n g e . T h e m o l e c u l a r i n t e r a c t i o n s i n v o l v e d in d e t e r m i n i n g t h e elastic p r o p e r t i e s o f cell walls a r e n o t well u n d e r s t o o d . H o w e v e r , it is n o t e w o r t h y t h a t t h e c h e m i c a l c o m p o s i t i o n o f t h e walls o f Halicystis a n d a few close r e l a t i v e s is also u n u s u a l . I n a d d i t i o n to t h e c o m m o n s t r u c t u r a l c o m p o n e n t , c e l l u l o s e , Halicystis walls c o n t a i n p o l y x y l a n as t h e m a j o r s t r u c t u r a l p o l y s a c c h a r i d e (10). U n l i k e c e l l u l o s e , t h e x y l a n c h a i n s a r e h e l i c a l l y c o i l e d (11). T h e r e is u n d o u b t e d l y a r e l a t i o n b e t w e e n m o l e c u l a r s t r u c t u r e a n d e x t e n s i b i l i t y , b u t t h a t r e l a t i o n s h i p r e m a i n s to be d e t e r m i n e d . Send reprint requests to Dr. Gutknecht, Duke Marine Laboratory, Beaufort, N. C. 28516. We thank Mr. M. Bradley for providing technical assistance and Ms. M. Bisson for critically reading the manuscript. Dr. D. Hastings designed and assembled the apparatus used for vacuolar perfusion experiments. The first author (J. S. G.) was supported by a predoctoral traineeship from N. I. H. This work was supported by U.S.P.H.S. Grant HL12157.
Receivedfor publication 31 July 1975. REFERENCES 1. BLINKS, L. R. 1951. Physiology and Biochemistry of the Algae. In Manual o f Phycology. G. M. Smith, editor. Cronica Botanica Co., Waltham, Mass. 263-290. 2. BLINKS, L. R., a n d A. G. JACQUES. 1930. T h e cell sap o f Halicystis. J. Gen. Physiol. 13:733-737. 3. BLOUNT, R. W., a n d B. H. LEVEDAHL. 1960. Active sodium and chloride t r a n s p o r t in the single celled marine alga, Halicystis ovalis. Acta Physiol. Scand. 49:1-9. 4. BROOKS, S. C. 1930. Composition of the cell sap ofHalicystis ovalis. (Lyng.) Areschoug. Proc. Soc. Exp. Biol. Med. 27:409-412. 5. CHAPMAN,V.J. 1962. T h e Algae. MacMillan and Co. Ltd., London. 6. DAINTY, J., H. VINTERS, and M. T. TYREE. 1974. A study o f transcellular osmosis and the kinetics o f swelling and shrinking in cells o f Chara corallina. In Membrane T r a n s p o r t in Plants. U. Z i m m e r m a n n and J. Dainty, editors. Springer-Verlag, New York. 59-63. 7. EPPLEY, R. W., and L. R. BUNKS. 1957. Cell space and a p p a r e n t free space in the red alga, Porphyra perforata. Plant Physiol. 32:63-64. 8. FINDLAV, G. P., A. B. HOPE, M. G. PITMAN, E. A. SMITtt, and N. A. WALKEIt. 1971. Ionic relations o f marine algae. I I I . Chaetomorpha: M e m b r a n e electrical p r o p e r ties and chloride fluxes. Aust. J. Biol. Sci. 24:731-745. 9. FISKE, C. H., and Y. SUBBAROW. 1925. T h e colorimetric determination o f phosphor u s . J . Biol. Chem. 66:375-400. 10. FREI, E., and R. D. PRESTON. 196!. Variants in the structural polysaccharides o f algal cell walls. Nature (Lond.). 192:939-943. 11. FREI, E., and R. D. PRESTON. 1964. Non-cellulosic structural polysaccharides in algal cell walls. I. Xylan in siphoneous green algae. Proc. R. Soc. Lond. B Biol. Sci. 160:293-313. 12. GACE, P. W., and R. S. E~SeNBEaO. 1969. Capacitance o f the surface and transverse tubular m e m b r a n e of frog sartorius muscle fibers.J. Gen. Physiol. 53:265-278. 13. GxNz~uRg, B. Z., and J. H o g g . 1967. What does a short-circuit current measure in biological systems?J. Theor. Biol. 14:316-322.
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