AJEBAK 53 (Pt. 6) 489-498 (1975)
REGULATION OF SHEEP ERYTHROCYTE VOLUME IN ANISOTONIC MEDIA bv SIRIBHINYA BENYAJATP AND R. J. H. MORRIS (From the Department of Physiology, University of New England, Armidale, N.S.W., Australia.' (Aecepted for publication August 25, 1975.) Summary. Sheep erythrocytes of hi^li and low potas.sium types were incubated in non-haemolytic hypotonic and hypertonic media for 4-5 h at 30°. After initial swelling or shrinking, they readjusted their volume toward their initial isotonic volume. The volume regulation was associated with specific changes in cation flnxes. In the swollen cells, efflux of both sodium and potassium was increased and influx of both cations was slightly decreased; the converse was true for the shnmken eells. All four fluxes were changed in a direction that led to return to normal voluuie. The difference in the response of the two types of sheep erythrocytes to changes of extracellular fluid osmolality resided in the different activity of their catiou transport systems. It is concluded that sheep erythroeytes possess some means of regulating their volume in vitro which is linked to cation permeability. The exact nature of the physical mechanisms by which the)' accomplish tliis remains to be elucidated.
INTRODUCTION. The regulation of cell volume involves a balance between efflux and influx of electrolytes and of water. On the basis of their studies with sheep red blood cells, Tosteson and Hoffman (1960) have proposed a model wherein the cell controls its volume in the steady state by the action of sodium and potassium leaks workiiiii aud Iloflinan, 1960). Since K and Na (luxes changed in the same direction, it seems unlikely that ion movement is produced by active
495
VOLUME REGULATION OF SHEEP ERYTHROCYTES
transport. The possibility exists of aii increase iu passive membrane potential change that may alter passive catiou permeability directly {Donlon and Rothstein, 1969), but the indicated potential change is small and the chloride ratio depends also on Cl movement induced by small pH changes. The potential change seems to result from altered flux rather than to cause ion movement. TABLE 1. Chanf-es in chloride ratio* associated with the volume response o/ sheep erythrocytes i HK and LK types) in anisotonie metlla'^
Sheep HK
LK
Medium osmolality mosm/kg H ^O Mean ± S.D.
[CL]o/[CLl, Time I min
1'5 h
3 Oh
4-5 h
Isotonic (306+3-5)
1-40 ±0 20
1 38 ±0 58
1 26 ±0 39
1 49 ±0-10
Hypotonic (285 ±5-0)
1 01 ±0 05
1 25 ±0 06
1 69 ±0-54
3-46 ±0-74
Hypertonic (326±4-9)
5'43 + 0'67
2-7i ±019
1-45 ±011
1 31 ±0 20
Isotonic (306±5 4)
1 40 ±0 09
1 49 ±0 36
1 60 ±0 44
1 32 ±017
Hypotonic (286±6'0)
134 ±0 07
1-58 ±0 22
1-79 ±0-40
2 07 ±0-62
Hvpertonic (326±4'0)
3-73 ±0-59
1 34 ±012
1-07 ±0-04
±011
1-01
* The chloride concentrations used were those in plasma water and cell water. + Data were the average of 2 experiments in isotonic media and 3 experiments in anisotonic media.
The differences iu characteristics of catiou transport mechanisms between HK and LK cells may partly explain the differences in response to tonicity and volume alterations. In hypotonic media HK cells tended to restore volume fa.ster than did LK cells. This could be due to the K loss being iu the same direction as the electrochemical potential gradient for K, whereas in LK cells the concentration of cellular K is close to that in plasma. The LK cells have weakly active sodium efflux, having only one quarter the active cation pumping capacity of HK cells {Tosteson and Hoffman, 1960). There is also a difference in passive permeability between the two cell types. Whether this accounts for the faster regulatory response of LK cells in hypertonic media is not known. The possibility exists also that an ethacr)nic acid-sensitive, ouabaiii-insensitive, ATP-iudependeut pump may play a role iu the ion transport, a system shown to exist in the human red cell (Hoflman and Kregenow, 1966). In most studies of volume regulation b\ mammalian cells the regulation of volume is not interfered with appreciably by ouabain, a potent inhibitor of the active Na-K transport system (Kregenow, 1971a, b; Posnansky aud Solomon, 1972a, b; Rorive and Kleiuzciler, 1972a, b; Oellasega and Grantham, 1973; Parker, 1973).
496
SIRIBHINYA RENYA[ATI AND R. ]. H. MORRIS
Sheep erythrocytes of both potassium types appear to regulate their volume more slowly than do mouse lymphoblasts (Buekhold et ai, 1965; Roti Roti and Rothstein, 1973) and duek erythrocytes (Kregenow, 1971a, b). The restoration of volume was about half completed in 4-5 h and haemolysis prevented the couthiuation of the incubation. (The median corpuscular fragilities of these cells were 0-720S^ NaCl for HK and 0-762% for LK respecti\'ely). Parker (1973) TABLE 2. Changes in Na and K fluxes associated with the volume regulatory response of HK and LK .sheep crvthrocvtes.
Flux sample
Cumulative influx m.equiv/cells originally in 1 litre 1-5 Na* Control (306 mosmolal) Hypotonic (285 mosmolal) Hypertonic (NaCl, 326 mosmolal) Hypertonic (KCl, 319 mosmolal)
Time (h) 3 0 4 5
Net flux
m.equiv/cells originally in 1 litre 1 5
Time (h) 3 0
Calculated cumulative efflux m.equiv/cells originally in 1 litre Time (h)
4 5
15
3- 0
4-5
3-7
50
10 0
-0- 9
+ 0-4
+ 1-0
4-5
4- 7
9 0
3-5
3 4
12 7
-2- 6
-0'5
-4-1
0-9
3- 9
16-8
2-5
7 6
X6
-1- 3
+ 0-4
+ 4-1
3-8
7- 2
4-4
1-7
7- 0
9 0
-2- 1
-1-0
+ 2-4
3-8
8- 0
6-6
Control (306 mosmolal) Hypotonic ^285 mosmolal) Hypertonic (NaCI. 326 mosmolal) Hypertonic (KCl, 319 mosmolal)
0 7
12
1 5
-2- 1
-2-5
-4-7
2-7
3' 7
6-2
0-6
I- 1
14
-5- 6
-7-0
-9-6
6-2
8- 0
11-1
0-5
1 1
14
-3- 4
-2-2
-2-6
3-9
3- 3
2-6
0-7
20
2 6
-2- 1
-0-3
+ 1-18
2-X
1- 3
0-8
Control (306 mosmolal) Hypotonic (286 mosmolal) Hypertonic (NaCI, 326 mosmolal) Hypertonic (KCl, 315 mosmolal)
3-4
8- 9
II 8
-2- 3
+ 2-3
-0-01
5-7
6- 0
118
1-5
6 7
9 1
+ 1- 1
-2-1
-7-8
0-4
8- 8
16-8
4-9
9 5
12 5
-2- 2
-1-2
+ 0-3
7-0
10 7
12-2
4-9
12- 0
13 5
-4 1
- I -2 + 2-2
9-0
13- 2
11-2
0 7
0 8
0 9
+ 0- 2
-0 2
-0-8
0-4
I- 0
17
0-3
0 7
0-9
+ 0- 03
-0 6
-1-9
0-3
1 2
2-8
0-2
0 2
0 5
-0' 9
-0-5
+ 0-4
1-1
0 7
0-1
0-3
0- X
1 3
-0 2
+ 0-1
+0 9
0-5
0- 6
0-4
Control (306 mosmolal) Hypotonic (286 mosmolal) Hypertonic (NaCI. 326 mosmolal) Hypertonic (KCl. 315 mosmolal)
Sheep erythrocytes were incubated in either isotonic. hypotonic or hypertonic media as in Methods. The fluxes were calculated by the method of Kregenow (1971a). The values from measurements at the earlier time periods were added to the values from later time periods to arrive at cumulative flux values at 3 and 4-5 h. The results shown are means of 2 experiments; the coefficient of variation between the experiments was less than SJ^.
VOLUME REGULATION OF SHEEP ERYTHROCYTES
497
studied the sodium, potassium and chloride movements in dog (low potas-sium) erythrocytes .subjected tu osmotic stress. Calculation from his Table III indicated that, after incubating cells in hypertonic NaCl solution, the return of all concentrations was complete in 8-16 h. Acknowledgements. We are grateful to Professor J. V. Kvaiw of the Physiology Departinent for the use of the sheep and for hi.s interest during this work. This work was presented as partial fulfilment of the requirements for the B.Se. (Hons.) degree at the l'ni\tTSity of New England while the author (S.B.) was an Australian Colombo Plan .scholar.
REFERENCES. BRENDA,
ADAMS,
R. B.. and
GHEGG, E . C . (1965): 'Osmotie adaptation of mouse lymphoblasts.' Biochim. biophys. Acta, 102, 600. COTLOVE, E., TRANTHAM, H . V., and BOW-
MAN, R. L. (1958): 'An in.strument and method for automatic, rapid, accurate and sensitive titratiou of chloride in biologic samples.' /. Lab. clin. Med., 51, 461. OKLLASE(;A.
M., and
CIIANIHAM,
J.
J.
(1973): "Hegulation of renal tubule cell volume in hypotonic media.* Am. } . Physiol, 224, 1288. D()Ni.(iN, J. A., and ROTHSTEIN, A. (1969): Ths cation permeability of erythrocytes in low ionic strength media of various tonicities.' ]. Membrane Biol., I, 37. EVANS, J. V. (1954): 'Electrolyte concentrations in red blood cells of British breed of sheep.* Nature, 174, 931. FLOHKIN. M . (1962); 'La regulation isosuiotique intracellulaire chez les invertebres barins euryhalins,' Bull. Acad. R. Belg.. 48. 687. . K. (1967): 'Regulation of cell volume in Flounder (Plueronectus Flesus) erythroeytes accompanying a decrease in plasma osinolarity.' Comp. Bioehetn. Physiol, 22, 253. HOFFMAN, J. F., and
KRKCENOW.
F . M.
(1966): 'The characterization of new energy dependent cation transport processes iu red blcwd cells.' Ann. N.Y. Aead. Sd.. 137, .566-570.
KRE(;EN()W, F . M . (1971a): 'The response of duek erythrocytes to nonhemolytic hypotonic media.* /. ^icn. Physiol, 58, 372. KiiEGENow, F. M (1971b): 'The response of duck erythrocytes to hypertonic media.' ;. f,'cn. Physiol.. 58. 396. , J. C. (1973): 'Dog red blood cells: Adjustment of salt and water content in vitro.' ]. gen. Physiol, 62, 147. PARKER, J. C , and HOFFMAN, J. F. (1965):
'Interdependence of cation permeability, cell volume and metabolism in dog red cells.' Fedn Proc, 24, 589. POSNANSKY,
M.,
and
SOLOMON,
A,
K.
(1972a); 'Effect of cell volume on potassium tran.sport in human red cells.' Biochim. hiophys. Acta, 274, 111. POSNANSKY.
M.,
and
SOLOMON,
A.
K.
(1972b): 'Regulation of human red eell volume by linked cation fluxes.' /. Membrane Biol, 10, 259. RORIVE, C, and KLEINZELLER, A, (1972a):
'Effect of pH on the water and electrolyte content of rat diaphragm.' Arc/i.s Biochem. Biophys.. 152. 876. RoHivE, G., and KLEINZELLEH, A. (1972b): 'The effect of ATP and Ca2+ on the cell volume in isolated kidney tubules.' Biochim. biophys. Acta. 274, 226. Ron
ROTI,
L . W . , and
ROTHSTEIN,
A.
(1973): 'Adaptation of mouse leukemic cells (L 5178Y) to anisotonie media. I. Gell volume regulation.' Expl Cell Res , 79, 295.
498
SIRIHHINYA BENYAJATI AND R . J. H. MORRIS
SHAAFI, K. 1., and
HAJJAK, J. J.
(1971):
'Sodium movement in high .sodium feline red cells.' /. fien. Phy.'iiol. 57, 684. SOMOGYI. M . (1945): 'Determination of blood sugar.' ]. hiol Chem., 160, 69. TOSTESON,
TOSTESUN,
D.
C'., and
IIOFFMAN,
J.
E.
(19fiO): 'Regulatitni of cell volume by active cation transport in high and low potassiun, sheep red cells.- /. gen. ^''^''"'- ^- ^^^-
D . C , and HOFFMAN, J. E.
VIRKAH. R. A. (1966) r 'The role of free
(1958): 'Cation transport in high and low potassium sheep red cells.' /. gen. Physiol, 50, 2513.
amino acids in the adaptation to reduced salinity in the Sipunculid Golfingia Gouldii.' Comp. Biocliem. Phys-iol, 18, 617.
REGULATION OF SHEEP ERYTHROCYTE VOLUME IN ANISOTONIC MEDIA bv SIRIBHINYA BENYAJATP AND R. J. H. MORRIS (From the Department of Physiology, University of New England, Armidale, N.S.W., Australia.' (Aecepted for publication August 25, 1975.) Summary. Sheep erythrocytes of hi^li and low potas.sium types were incubated in non-haemolytic hypotonic and hypertonic media for 4-5 h at 30°. After initial swelling or shrinking, they readjusted their volume toward their initial isotonic volume. The volume regulation was associated with specific changes in cation flnxes. In the swollen cells, efflux of both sodium and potassium was increased and influx of both cations was slightly decreased; the converse was true for the shnmken eells. All four fluxes were changed in a direction that led to return to normal voluuie. The difference in the response of the two types of sheep erythrocytes to changes of extracellular fluid osmolality resided in the different activity of their catiou transport systems. It is concluded that sheep erythroeytes possess some means of regulating their volume in vitro which is linked to cation permeability. The exact nature of the physical mechanisms by which the)' accomplish tliis remains to be elucidated.
INTRODUCTION. The regulation of cell volume involves a balance between efflux and influx of electrolytes and of water. On the basis of their studies with sheep red blood cells, Tosteson and Hoffman (1960) have proposed a model wherein the cell controls its volume in the steady state by the action of sodium and potassium leaks workiiiii aud Iloflinan, 1960). Since K and Na (luxes changed in the same direction, it seems unlikely that ion movement is produced by active
495
VOLUME REGULATION OF SHEEP ERYTHROCYTES
transport. The possibility exists of aii increase iu passive membrane potential change that may alter passive catiou permeability directly {Donlon and Rothstein, 1969), but the indicated potential change is small and the chloride ratio depends also on Cl movement induced by small pH changes. The potential change seems to result from altered flux rather than to cause ion movement. TABLE 1. Chanf-es in chloride ratio* associated with the volume response o/ sheep erythrocytes i HK and LK types) in anisotonie metlla'^
Sheep HK
LK
Medium osmolality mosm/kg H ^O Mean ± S.D.
[CL]o/[CLl, Time I min
1'5 h
3 Oh
4-5 h
Isotonic (306+3-5)
1-40 ±0 20
1 38 ±0 58
1 26 ±0 39
1 49 ±0-10
Hypotonic (285 ±5-0)
1 01 ±0 05
1 25 ±0 06
1 69 ±0-54
3-46 ±0-74
Hypertonic (326±4-9)
5'43 + 0'67
2-7i ±019
1-45 ±011
1 31 ±0 20
Isotonic (306±5 4)
1 40 ±0 09
1 49 ±0 36
1 60 ±0 44
1 32 ±017
Hypotonic (286±6'0)
134 ±0 07
1-58 ±0 22
1-79 ±0-40
2 07 ±0-62
Hvpertonic (326±4'0)
3-73 ±0-59
1 34 ±012
1-07 ±0-04
±011
1-01
* The chloride concentrations used were those in plasma water and cell water. + Data were the average of 2 experiments in isotonic media and 3 experiments in anisotonic media.
The differences iu characteristics of catiou transport mechanisms between HK and LK cells may partly explain the differences in response to tonicity and volume alterations. In hypotonic media HK cells tended to restore volume fa.ster than did LK cells. This could be due to the K loss being iu the same direction as the electrochemical potential gradient for K, whereas in LK cells the concentration of cellular K is close to that in plasma. The LK cells have weakly active sodium efflux, having only one quarter the active cation pumping capacity of HK cells {Tosteson and Hoffman, 1960). There is also a difference in passive permeability between the two cell types. Whether this accounts for the faster regulatory response of LK cells in hypertonic media is not known. The possibility exists also that an ethacr)nic acid-sensitive, ouabaiii-insensitive, ATP-iudependeut pump may play a role iu the ion transport, a system shown to exist in the human red cell (Hoflman and Kregenow, 1966). In most studies of volume regulation b\ mammalian cells the regulation of volume is not interfered with appreciably by ouabain, a potent inhibitor of the active Na-K transport system (Kregenow, 1971a, b; Posnansky aud Solomon, 1972a, b; Rorive and Kleiuzciler, 1972a, b; Oellasega and Grantham, 1973; Parker, 1973).
496
SIRIBHINYA RENYA[ATI AND R. ]. H. MORRIS
Sheep erythrocytes of both potassium types appear to regulate their volume more slowly than do mouse lymphoblasts (Buekhold et ai, 1965; Roti Roti and Rothstein, 1973) and duek erythrocytes (Kregenow, 1971a, b). The restoration of volume was about half completed in 4-5 h and haemolysis prevented the couthiuation of the incubation. (The median corpuscular fragilities of these cells were 0-720S^ NaCl for HK and 0-762% for LK respecti\'ely). Parker (1973) TABLE 2. Changes in Na and K fluxes associated with the volume regulatory response of HK and LK .sheep crvthrocvtes.
Flux sample
Cumulative influx m.equiv/cells originally in 1 litre 1-5 Na* Control (306 mosmolal) Hypotonic (285 mosmolal) Hypertonic (NaCl, 326 mosmolal) Hypertonic (KCl, 319 mosmolal)
Time (h) 3 0 4 5
Net flux
m.equiv/cells originally in 1 litre 1 5
Time (h) 3 0
Calculated cumulative efflux m.equiv/cells originally in 1 litre Time (h)
4 5
15
3- 0
4-5
3-7
50
10 0
-0- 9
+ 0-4
+ 1-0
4-5
4- 7
9 0
3-5
3 4
12 7
-2- 6
-0'5
-4-1
0-9
3- 9
16-8
2-5
7 6
X6
-1- 3
+ 0-4
+ 4-1
3-8
7- 2
4-4
1-7
7- 0
9 0
-2- 1
-1-0
+ 2-4
3-8
8- 0
6-6
Control (306 mosmolal) Hypotonic ^285 mosmolal) Hypertonic (NaCI. 326 mosmolal) Hypertonic (KCl, 319 mosmolal)
0 7
12
1 5
-2- 1
-2-5
-4-7
2-7
3' 7
6-2
0-6
I- 1
14
-5- 6
-7-0
-9-6
6-2
8- 0
11-1
0-5
1 1
14
-3- 4
-2-2
-2-6
3-9
3- 3
2-6
0-7
20
2 6
-2- 1
-0-3
+ 1-18
2-X
1- 3
0-8
Control (306 mosmolal) Hypotonic (286 mosmolal) Hypertonic (NaCI, 326 mosmolal) Hypertonic (KCl, 315 mosmolal)
3-4
8- 9
II 8
-2- 3
+ 2-3
-0-01
5-7
6- 0
118
1-5
6 7
9 1
+ 1- 1
-2-1
-7-8
0-4
8- 8
16-8
4-9
9 5
12 5
-2- 2
-1-2
+ 0-3
7-0
10 7
12-2
4-9
12- 0
13 5
-4 1
- I -2 + 2-2
9-0
13- 2
11-2
0 7
0 8
0 9
+ 0- 2
-0 2
-0-8
0-4
I- 0
17
0-3
0 7
0-9
+ 0- 03
-0 6
-1-9
0-3
1 2
2-8
0-2
0 2
0 5
-0' 9
-0-5
+ 0-4
1-1
0 7
0-1
0-3
0- X
1 3
-0 2
+ 0-1
+0 9
0-5
0- 6
0-4
Control (306 mosmolal) Hypotonic (286 mosmolal) Hypertonic (NaCI. 326 mosmolal) Hypertonic (KCl. 315 mosmolal)
Sheep erythrocytes were incubated in either isotonic. hypotonic or hypertonic media as in Methods. The fluxes were calculated by the method of Kregenow (1971a). The values from measurements at the earlier time periods were added to the values from later time periods to arrive at cumulative flux values at 3 and 4-5 h. The results shown are means of 2 experiments; the coefficient of variation between the experiments was less than SJ^.
VOLUME REGULATION OF SHEEP ERYTHROCYTES
497
studied the sodium, potassium and chloride movements in dog (low potas-sium) erythrocytes .subjected tu osmotic stress. Calculation from his Table III indicated that, after incubating cells in hypertonic NaCl solution, the return of all concentrations was complete in 8-16 h. Acknowledgements. We are grateful to Professor J. V. Kvaiw of the Physiology Departinent for the use of the sheep and for hi.s interest during this work. This work was presented as partial fulfilment of the requirements for the B.Se. (Hons.) degree at the l'ni\tTSity of New England while the author (S.B.) was an Australian Colombo Plan .scholar.
REFERENCES. BRENDA,
ADAMS,
R. B.. and
GHEGG, E . C . (1965): 'Osmotie adaptation of mouse lymphoblasts.' Biochim. biophys. Acta, 102, 600. COTLOVE, E., TRANTHAM, H . V., and BOW-
MAN, R. L. (1958): 'An in.strument and method for automatic, rapid, accurate and sensitive titratiou of chloride in biologic samples.' /. Lab. clin. Med., 51, 461. OKLLASE(;A.
M., and
CIIANIHAM,
J.
J.
(1973): "Hegulation of renal tubule cell volume in hypotonic media.* Am. } . Physiol, 224, 1288. D()Ni.(iN, J. A., and ROTHSTEIN, A. (1969): Ths cation permeability of erythrocytes in low ionic strength media of various tonicities.' ]. Membrane Biol., I, 37. EVANS, J. V. (1954): 'Electrolyte concentrations in red blood cells of British breed of sheep.* Nature, 174, 931. FLOHKIN. M . (1962); 'La regulation isosuiotique intracellulaire chez les invertebres barins euryhalins,' Bull. Acad. R. Belg.. 48. 687. . K. (1967): 'Regulation of cell volume in Flounder (Plueronectus Flesus) erythroeytes accompanying a decrease in plasma osinolarity.' Comp. Bioehetn. Physiol, 22, 253. HOFFMAN, J. F., and
KRKCENOW.
F . M.
(1966): 'The characterization of new energy dependent cation transport processes iu red blcwd cells.' Ann. N.Y. Aead. Sd.. 137, .566-570.
KRE(;EN()W, F . M . (1971a): 'The response of duek erythrocytes to nonhemolytic hypotonic media.* /. ^icn. Physiol, 58, 372. KiiEGENow, F. M (1971b): 'The response of duck erythrocytes to hypertonic media.' ;. f,'cn. Physiol.. 58. 396. , J. C. (1973): 'Dog red blood cells: Adjustment of salt and water content in vitro.' ]. gen. Physiol, 62, 147. PARKER, J. C , and HOFFMAN, J. F. (1965):
'Interdependence of cation permeability, cell volume and metabolism in dog red cells.' Fedn Proc, 24, 589. POSNANSKY,
M.,
and
SOLOMON,
A,
K.
(1972a); 'Effect of cell volume on potassium tran.sport in human red cells.' Biochim. hiophys. Acta, 274, 111. POSNANSKY.
M.,
and
SOLOMON,
A.
K.
(1972b): 'Regulation of human red eell volume by linked cation fluxes.' /. Membrane Biol, 10, 259. RORIVE, C, and KLEINZELLER, A, (1972a):
'Effect of pH on the water and electrolyte content of rat diaphragm.' Arc/i.s Biochem. Biophys.. 152. 876. RoHivE, G., and KLEINZELLEH, A. (1972b): 'The effect of ATP and Ca2+ on the cell volume in isolated kidney tubules.' Biochim. biophys. Acta. 274, 226. Ron
ROTI,
L . W . , and
ROTHSTEIN,
A.
(1973): 'Adaptation of mouse leukemic cells (L 5178Y) to anisotonie media. I. Gell volume regulation.' Expl Cell Res , 79, 295.
498
SIRIHHINYA BENYAJATI AND R . J. H. MORRIS
SHAAFI, K. 1., and
HAJJAK, J. J.
(1971):
'Sodium movement in high .sodium feline red cells.' /. fien. Phy.'iiol. 57, 684. SOMOGYI. M . (1945): 'Determination of blood sugar.' ]. hiol Chem., 160, 69. TOSTESON,
TOSTESUN,
D.
C'., and
IIOFFMAN,
J.
E.
(19fiO): 'Regulatitni of cell volume by active cation transport in high and low potassiun, sheep red cells.- /. gen. ^''^''"'- ^- ^^^-
D . C , and HOFFMAN, J. E.
VIRKAH. R. A. (1966) r 'The role of free
(1958): 'Cation transport in high and low potassium sheep red cells.' /. gen. Physiol, 50, 2513.
amino acids in the adaptation to reduced salinity in the Sipunculid Golfingia Gouldii.' Comp. Biocliem. Phys-iol, 18, 617.
