Volume Regulation by Flounder Red Blood Cells in Anisotonic Media PETER M. CALA From the Mount Desert Island Biological Laboratory, Salisbury Cove, Maine 04672, and the Department of Physiology and Pharmacology, Duke University Medical Center, Durham, North Carolina 27710. Dr. Cala's present address is the Department of Human Physiology, University of California School of Medicine, Davis, California 95616.
A B S T R A C X T h e nucleated high K, low Na red blood cells of the winter flounder demonstrated a volume regulatory response subsequent to osmotic swelling or shrinkage. During volume regulation the net water flow was secondary to net inorganic cation flux. Volume regulation after osmotic swelling is referred to as regulatory volume decrease (RVD) and was characterized by net K and water loss. Since the electrochemical gradient for K is directed out of the cell there is no need to invoke active processes to explain RVD. When osmotically shrunken, the flound e r erythrocyte demonstrated a regulatory volume increase (RVI) back toward control cell volume. T h e water movements characteristic of RVI were a consequence o f net cellular NaCI and KCI uptake with Na accounting for 75% of the increase in intraceilular cation content. Since the Na electrochemical gradient is directed into the cell, net Na uptake was the result o f Na flux via dissipative pathways. T h e addition o f 10 -4 M ouabain to suspensions of flounder erythrocytes was without effect u p o n net water movements d u r i n g volume regulation. T h e presence o f ouabain did however lead to a decreased ratio of intraceilular K:Na. Analysis o f net Na and K fluxes in the presence and absence o f ouabain led to the conclusion that Na and K fluxes via both conservative and dissipative pathways are increased in response to osmotic swelling or shrinkage. In addition, the Na and K flux rate through both p u m p and leak pathways decreased in a parallel fashion as cell volume was regulated. Taken as a whole, the Na and K movements t h r o u g h the flounder erythrocyte m e m b r a n e demonstrated a functional d e p e n d e n c e d u r i n g volume regulation. INTRODUCTION
T h e p h e n o m e n o n o f c e l l u l a r v o l u m e r e g u l a t i o n is c h a r a c t e r i z e d by t h e a d j u s t m e n t o f cell v o l u m e b a c k t o w a r d o r i g i n a l , s t e a d y - s t a t e v o l u m e a f t e r o s m o t i c p e r t u r b a t i o n . T h e v o l u m e r e g u l a t o r y r e s p o n s e h a s b e e n d e s c r i b e d by a n u m b e r o f w o r k e r s s t u d y i n g a v a r i e t y o f d i f f e r e n t v e r t e b r a t e cell t y p e s (1-10). V o l u m e r e g u l a t i o n by v e r t e b r a t e cells is t h e r e s u l t o f n e t i n o r g a n i c c a t i o n f l u x a n d o s m o t i c a l l y o b l i g e d w a t e r flow. O n e o f t h e e a r l y i n v e s t i g a t i o n s was p e r f o r m e d by F u g e l l i u s i n g r e d b l o o d cells o f t h e E u r o p e a n f l o u n d e r Pleuronectesflesus (2). T h i s a u t h o r d e m o n s t r a t e d t h a t w h i l e v o l u m e r e g u l a t i o n d i d o c c u r in r e s p o n s e to o s m o t i c s w e l l i n g , o n l y o n e - e i g h t h o f t h e f l u i d t r a n s p o r t e d c o u l d b e l i n k e d to c h a n g e s in t h e c e l l u l a r c o n t e n t o f n i n h y d r i n - p o s i t i v e s u b s t a n c e s . T h e v o l u m e THE JOURNAL OF GENERAL PHYSIOLOGY ' VOLUME 69,
1977 ' p a g e s 5 3 7 - 5 5 2
537
538
THE JOURNAL
OF GENERAL PHYSIOLOGY
" VOLUME
69 ' 1977
regulatory response of duck erythrocytes was studied by Kregenow (4, 5). These cells were shown to rely almost solely upon net inorganic ion fluxes as a means of effecting net water flow during volume regulation. When swollen, the duck red cell decreases volume as a result of net KCI and water loss. In response to osmotic shrinkage the duck erythrocyte increases volume by gaining NaCI, KC1, and osmotically obliged water. Net K uptake accounts for 75% of the total cation gained by the duck erythrocyte during regulatory volume increase (RVI). Volume regulation by shrunken duck red cells was observed only u n d e r conditions o f elevated external K. T h e volume regulatory response of mouse leukemic cells (L5178Y) was studied by Roti Roti and Rothstein (10). T h e cells were observed to demonstrate both regulatory volume decrease (RVD) and RVI. As was the case with the duck erythrocyte, RVD was characterized by net loss of KCI and osmotically obliged water. When osmotically shrunken by transfer from control (325 mosM) to hypertonic media the cells did not demonstrate a volume regulatory response. If, however, after RVD in hypotonic medium (150 mosM) the cells were osmotically shrunk by resuspension in control (325 mosM) medium the cell volume was regulated back toward control values as a result of net KC1 and osmotically obliged water uptake. Upon the basis of an analysis of net and unidirectional fluxes Roti Roti and Rothstein concluded that the ion fluxes responsible for water flow after osmotic swelling are the result of increased membrane permeability to K. After osmotic shrinkage the cells are thought to regulate their volume as a result of K uptake mediated by the pump. In contrast, unidirectional flux studies pe r f or m e d upon human red cells (8) suggest a net Na and K loss after swelling and net uptake of both Na and K after shrinkage. Since the unidirectional flux data predict net contragradient Na and K fluxes during RVD and RVI, relbectively, it would seem that volume regulation by human erythrocytes is d ependent upon metabolic energy (8). T h e red blood cells of the winter flounder (Pseudopleuronectesamericanus) were chosen for the present study. This study is intended to illustrate the dynamic changes in the Na and K transport pathways during cell volume regulation. Data will be presented demonstrating that, as in the systems described above, the red blood cells of the winter flounder regulate volume by adjusting intracellular Na and/or K content. T h e data will show that after osmotic swelling the flounder red cell loses K, presumably anion, and osmotically obliged water. Since these cells are of high K (130 mM/liter cell water), low Na (24 mM/liter cell water) type, the net K loss associated with RVD is passive. When flounder red cells are osmotically shrunk, they recover volume as a result of net Na, K, C1, and water uptake. Na is the major cation accumulated, accounting for 75% of the total cation gained. Since Na moves into the cell down its electrochemical gradient during RVI, the net ion flux leading to net water flow appears to be passive. T h e net gain in intracellular K during RVI is inexplicable by passive means and probably represents cation rather than volume regulation. This paper will present data demonstrating that the volume regulatory response of flounder red blood cells is a direct result of changes in permeability to Na and K. T h e behavior o f the Na-K pum p during the volume regulatory
CALA VolumeRegulation by Red Blood Cells in Anisotonic Media
539
r e s p o n s e a n d its c o n t r i b u t i o n s will be d i s c u s s e d . I n a d d i t i o n , e v i d e n c e will be p r e s e n t e d which suggests that d u r i n g the volume regulatory response, transm e m b r a n e N a a n d K m o v e m e n t s a r e , at least f u n c t i o n a l l y , l i n k e d . MATERIALS
AND
METHODS
General Protocol Blood was drawn from adult winter flounder which had been captured and maintained in seawater at 10°C for no longer than 1 wk. Blood was taken from the caudal vein into a heparinized syringe and immediately centrifuged, and the plasma and buffy coat were removed by aspiration. T h e cells were then washed four times in 30 vol of solution A (Table I). Since the analysis of flounder plasma for total osmolarity gave a mean value of 340 mosM with a range of 150 mosM, it was considered necessary to preincubate cells overnight in solution A to assure that a steady state with respect to cell volume and ion content was reached before experimental treatment. All experiments were carried out at TABLE
I
EXPERIMENTAL MEDIA Solution
NaC1 KCI CaCI~ MgCle Dextrose Imidazole penicillin (U) Streptomycin sulfate pH
A
B
mM
raM
170 3 0.75 1 5 3 10e/liter 0.25 g/liter 7.95
100 3 0.75 1 5 3 108/liter 0.25 g/liter 7.95
All ringers were gassed with air saturated with H20 at 5°C before use. pH 7.95 and 10°C. After preincubation the cell suspension was split into experimental and control groups. T h e suspensions were then centrifuged, the supernate was removed by aspiration, and the resultant cell pellets were resuspended in known volumes of the desired solutions (Tables I and II). Samples were taken at known intervals by removal of 250-/zl or 500-/zl portions, which were transferred to preweighed siliconized Pyrex culture tubes (6 x 60 ram). T h e samples were centrifuged for 3 min at 20,000 g in a Clay Adams autocrit centrifuge (model 0571, head CT-2915, Clay Adams, Div. of Becton, Dickinson & Co., Parsippany, N. J.). T h e resultant cell pellets were immediately separated from the supernate by aspiration and both were stored for analysis. Before analytical treatment, the cell pellets were dried to constant weight (24-48 h) in the culture tubes at 80°C. The dried pellets were then extracted in 250 p,1 of 15 mM LiNO3 (internal standard for flame photometry).
Chemical Analysis for H20, Na, K, and Cl All cells taken for analysis of Na, K, C1, or H20 were suspended at a hematocrit of 8% in media containing tracer quantities of [14C]polyethylene glycol([14C]PEG) mol wt 4,000, which served as an extracellular space marker. This marker was chosen as a result of studies by Schmidt-Nielsen et al., (11). While there was some indication of marker
540
THE JOURNAL
or
GENERAL
PHYSIOLOGY
" VOLUME
69
• 1977
metabolism when inulin was used, [14C]PEG appeared to function as a good marker of extracellular space. T h e extracellular marker was used in all experiments since standard corrections lead to errors because of r a n d o m variability as well as an inverse relationship between cell volume and extracellularly trapped fluid. T h e relatively high hematocrit was chosen in o r d e r to provide a sufficient n u m b e r of cells for chemical analysis. It should also be noted that 14C associated with PEG was the only isotope present. A 40-gl sample of the cell extract was taken for liquid scintillation counting o f [t4C]PEG as was a 10-gl sample o f the experimental bathing medium. 14C cpm/g,1 o f extracetlular fluid were d e t e r m i n e d and used to correct for the extracellular contribution to cell water and ion content. T h e ~4C associated with PEG was counted in a Packard Tri-Carb Liquid Scintillation Counter model 3002 (Packard Instrument Co., Inc., Downers Grove, Ill.). T h e scintillation cocktail consisted o f 800 ml T o l u e n e , 200 ml ethanol, 0.3 g 1,4-b/s[2-(5phenyloxazolyl)]benzene (POP), 7 g 2,5-diphenyloxazole (PPO), and 5 g carbosil. From the same pellet extract used for scintillation counting, a 100-/zl portion was removed for chemical determination o f Na and K. T h e analysis was p e r f o r m e d with an InstrumentaTABLE
II
EXPERIMENTAL PROTOCOL 1, Withdraw blood from the caudal vein into a heparinized syringe. 2, Wash cells four times in 30 vol each wash, with solution A at 10°C. 3, Preincubate cells overnight in solution A at a hematocrit of
A B S T R A C X T h e nucleated high K, low Na red blood cells of the winter flounder demonstrated a volume regulatory response subsequent to osmotic swelling or shrinkage. During volume regulation the net water flow was secondary to net inorganic cation flux. Volume regulation after osmotic swelling is referred to as regulatory volume decrease (RVD) and was characterized by net K and water loss. Since the electrochemical gradient for K is directed out of the cell there is no need to invoke active processes to explain RVD. When osmotically shrunken, the flound e r erythrocyte demonstrated a regulatory volume increase (RVI) back toward control cell volume. T h e water movements characteristic of RVI were a consequence o f net cellular NaCI and KCI uptake with Na accounting for 75% of the increase in intraceilular cation content. Since the Na electrochemical gradient is directed into the cell, net Na uptake was the result o f Na flux via dissipative pathways. T h e addition o f 10 -4 M ouabain to suspensions of flounder erythrocytes was without effect u p o n net water movements d u r i n g volume regulation. T h e presence o f ouabain did however lead to a decreased ratio of intraceilular K:Na. Analysis o f net Na and K fluxes in the presence and absence o f ouabain led to the conclusion that Na and K fluxes via both conservative and dissipative pathways are increased in response to osmotic swelling or shrinkage. In addition, the Na and K flux rate through both p u m p and leak pathways decreased in a parallel fashion as cell volume was regulated. Taken as a whole, the Na and K movements t h r o u g h the flounder erythrocyte m e m b r a n e demonstrated a functional d e p e n d e n c e d u r i n g volume regulation. INTRODUCTION
T h e p h e n o m e n o n o f c e l l u l a r v o l u m e r e g u l a t i o n is c h a r a c t e r i z e d by t h e a d j u s t m e n t o f cell v o l u m e b a c k t o w a r d o r i g i n a l , s t e a d y - s t a t e v o l u m e a f t e r o s m o t i c p e r t u r b a t i o n . T h e v o l u m e r e g u l a t o r y r e s p o n s e h a s b e e n d e s c r i b e d by a n u m b e r o f w o r k e r s s t u d y i n g a v a r i e t y o f d i f f e r e n t v e r t e b r a t e cell t y p e s (1-10). V o l u m e r e g u l a t i o n by v e r t e b r a t e cells is t h e r e s u l t o f n e t i n o r g a n i c c a t i o n f l u x a n d o s m o t i c a l l y o b l i g e d w a t e r flow. O n e o f t h e e a r l y i n v e s t i g a t i o n s was p e r f o r m e d by F u g e l l i u s i n g r e d b l o o d cells o f t h e E u r o p e a n f l o u n d e r Pleuronectesflesus (2). T h i s a u t h o r d e m o n s t r a t e d t h a t w h i l e v o l u m e r e g u l a t i o n d i d o c c u r in r e s p o n s e to o s m o t i c s w e l l i n g , o n l y o n e - e i g h t h o f t h e f l u i d t r a n s p o r t e d c o u l d b e l i n k e d to c h a n g e s in t h e c e l l u l a r c o n t e n t o f n i n h y d r i n - p o s i t i v e s u b s t a n c e s . T h e v o l u m e THE JOURNAL OF GENERAL PHYSIOLOGY ' VOLUME 69,
1977 ' p a g e s 5 3 7 - 5 5 2
537
538
THE JOURNAL
OF GENERAL PHYSIOLOGY
" VOLUME
69 ' 1977
regulatory response of duck erythrocytes was studied by Kregenow (4, 5). These cells were shown to rely almost solely upon net inorganic ion fluxes as a means of effecting net water flow during volume regulation. When swollen, the duck red cell decreases volume as a result of net KCI and water loss. In response to osmotic shrinkage the duck erythrocyte increases volume by gaining NaCI, KC1, and osmotically obliged water. Net K uptake accounts for 75% of the total cation gained by the duck erythrocyte during regulatory volume increase (RVI). Volume regulation by shrunken duck red cells was observed only u n d e r conditions o f elevated external K. T h e volume regulatory response of mouse leukemic cells (L5178Y) was studied by Roti Roti and Rothstein (10). T h e cells were observed to demonstrate both regulatory volume decrease (RVD) and RVI. As was the case with the duck erythrocyte, RVD was characterized by net loss of KCI and osmotically obliged water. When osmotically shrunken by transfer from control (325 mosM) to hypertonic media the cells did not demonstrate a volume regulatory response. If, however, after RVD in hypotonic medium (150 mosM) the cells were osmotically shrunk by resuspension in control (325 mosM) medium the cell volume was regulated back toward control values as a result of net KC1 and osmotically obliged water uptake. Upon the basis of an analysis of net and unidirectional fluxes Roti Roti and Rothstein concluded that the ion fluxes responsible for water flow after osmotic swelling are the result of increased membrane permeability to K. After osmotic shrinkage the cells are thought to regulate their volume as a result of K uptake mediated by the pump. In contrast, unidirectional flux studies pe r f or m e d upon human red cells (8) suggest a net Na and K loss after swelling and net uptake of both Na and K after shrinkage. Since the unidirectional flux data predict net contragradient Na and K fluxes during RVD and RVI, relbectively, it would seem that volume regulation by human erythrocytes is d ependent upon metabolic energy (8). T h e red blood cells of the winter flounder (Pseudopleuronectesamericanus) were chosen for the present study. This study is intended to illustrate the dynamic changes in the Na and K transport pathways during cell volume regulation. Data will be presented demonstrating that, as in the systems described above, the red blood cells of the winter flounder regulate volume by adjusting intracellular Na and/or K content. T h e data will show that after osmotic swelling the flounder red cell loses K, presumably anion, and osmotically obliged water. Since these cells are of high K (130 mM/liter cell water), low Na (24 mM/liter cell water) type, the net K loss associated with RVD is passive. When flounder red cells are osmotically shrunk, they recover volume as a result of net Na, K, C1, and water uptake. Na is the major cation accumulated, accounting for 75% of the total cation gained. Since Na moves into the cell down its electrochemical gradient during RVI, the net ion flux leading to net water flow appears to be passive. T h e net gain in intracellular K during RVI is inexplicable by passive means and probably represents cation rather than volume regulation. This paper will present data demonstrating that the volume regulatory response of flounder red blood cells is a direct result of changes in permeability to Na and K. T h e behavior o f the Na-K pum p during the volume regulatory
CALA VolumeRegulation by Red Blood Cells in Anisotonic Media
539
r e s p o n s e a n d its c o n t r i b u t i o n s will be d i s c u s s e d . I n a d d i t i o n , e v i d e n c e will be p r e s e n t e d which suggests that d u r i n g the volume regulatory response, transm e m b r a n e N a a n d K m o v e m e n t s a r e , at least f u n c t i o n a l l y , l i n k e d . MATERIALS
AND
METHODS
General Protocol Blood was drawn from adult winter flounder which had been captured and maintained in seawater at 10°C for no longer than 1 wk. Blood was taken from the caudal vein into a heparinized syringe and immediately centrifuged, and the plasma and buffy coat were removed by aspiration. T h e cells were then washed four times in 30 vol of solution A (Table I). Since the analysis of flounder plasma for total osmolarity gave a mean value of 340 mosM with a range of 150 mosM, it was considered necessary to preincubate cells overnight in solution A to assure that a steady state with respect to cell volume and ion content was reached before experimental treatment. All experiments were carried out at TABLE
I
EXPERIMENTAL MEDIA Solution
NaC1 KCI CaCI~ MgCle Dextrose Imidazole penicillin (U) Streptomycin sulfate pH
A
B
mM
raM
170 3 0.75 1 5 3 10e/liter 0.25 g/liter 7.95
100 3 0.75 1 5 3 108/liter 0.25 g/liter 7.95
All ringers were gassed with air saturated with H20 at 5°C before use. pH 7.95 and 10°C. After preincubation the cell suspension was split into experimental and control groups. T h e suspensions were then centrifuged, the supernate was removed by aspiration, and the resultant cell pellets were resuspended in known volumes of the desired solutions (Tables I and II). Samples were taken at known intervals by removal of 250-/zl or 500-/zl portions, which were transferred to preweighed siliconized Pyrex culture tubes (6 x 60 ram). T h e samples were centrifuged for 3 min at 20,000 g in a Clay Adams autocrit centrifuge (model 0571, head CT-2915, Clay Adams, Div. of Becton, Dickinson & Co., Parsippany, N. J.). T h e resultant cell pellets were immediately separated from the supernate by aspiration and both were stored for analysis. Before analytical treatment, the cell pellets were dried to constant weight (24-48 h) in the culture tubes at 80°C. The dried pellets were then extracted in 250 p,1 of 15 mM LiNO3 (internal standard for flame photometry).
Chemical Analysis for H20, Na, K, and Cl All cells taken for analysis of Na, K, C1, or H20 were suspended at a hematocrit of 8% in media containing tracer quantities of [14C]polyethylene glycol([14C]PEG) mol wt 4,000, which served as an extracellular space marker. This marker was chosen as a result of studies by Schmidt-Nielsen et al., (11). While there was some indication of marker
540
THE JOURNAL
or
GENERAL
PHYSIOLOGY
" VOLUME
69
• 1977
metabolism when inulin was used, [14C]PEG appeared to function as a good marker of extracellular space. T h e extracellular marker was used in all experiments since standard corrections lead to errors because of r a n d o m variability as well as an inverse relationship between cell volume and extracellularly trapped fluid. T h e relatively high hematocrit was chosen in o r d e r to provide a sufficient n u m b e r of cells for chemical analysis. It should also be noted that 14C associated with PEG was the only isotope present. A 40-gl sample of the cell extract was taken for liquid scintillation counting o f [t4C]PEG as was a 10-gl sample o f the experimental bathing medium. 14C cpm/g,1 o f extracetlular fluid were d e t e r m i n e d and used to correct for the extracellular contribution to cell water and ion content. T h e ~4C associated with PEG was counted in a Packard Tri-Carb Liquid Scintillation Counter model 3002 (Packard Instrument Co., Inc., Downers Grove, Ill.). T h e scintillation cocktail consisted o f 800 ml T o l u e n e , 200 ml ethanol, 0.3 g 1,4-b/s[2-(5phenyloxazolyl)]benzene (POP), 7 g 2,5-diphenyloxazole (PPO), and 5 g carbosil. From the same pellet extract used for scintillation counting, a 100-/zl portion was removed for chemical determination o f Na and K. T h e analysis was p e r f o r m e d with an InstrumentaTABLE
II
EXPERIMENTAL PROTOCOL 1, Withdraw blood from the caudal vein into a heparinized syringe. 2, Wash cells four times in 30 vol each wash, with solution A at 10°C. 3, Preincubate cells overnight in solution A at a hematocrit of
