Journal of Molecular and Cellular Cardioloa (1979) 11, 585490
Dissociation of Cell Volume Regulation and Sodium-Potassium Exchange Pump Activity in Dog Myocardium in vitro MICHAEL
B. PINE, OSCAR H. L. BING, RONALD WEINTRAUB WALTER H. ABELMANN Departments of Medicine and Surgery and the Thorndike Laboratory,
Harvard
Medical School and Beth Israel Hospital, (Received 28 Jub
AND
Boston, Massachusetts 022 15, U.S.A.
1978, accepted in revisedform 6 .November 1978)
M. B. PINE, 0. H. L. BING, R. WEINTFUUJB AND W. H. ABELMANN. Dissociation of Cell Volume Regulation and Sodiun-Potassium Exchange Pump Activity in Dog Myocardium in vitro. Journal qf Molecular andCellular Cardiology (1979) 11, 585-590. The relation between myocardial cell potassium and water contents after hypoxia at 0°C (cold shock) in KrebsRingers-Phosphate solution was studied using 252 myocardial slices from six dogs. Losses of potassium were marked and similar after immediate cold shock, cold shock after 1 h equilibration in oxygenated media at 25°C and immediate cold shock plus 1 msr ouabain. However, myocardial water and total monovalent cations were only slightly increased by cold shock after equilibration compared to the marked increases found after immediate cold shock with and without ouabain (P < 0.01). When edematous slices were warmed to 25°C in oxygenated media, myocardial potassium increased towards control values in the absence of ouabain (P < 0.01) and decreased further when ouabain was present (P < O.Ol), but in both cases myocardial water and total monovalent cations decreased towards control values (P i 0.01). This dissociation of Na-K exchange pump activity and myocardial cell volume regulation can be only partially explained using the classic “pump-leak” theory of cell volume regulation. KEY WORDS: Cell volume regulation; pump; Ouabain.
Metabolic
blockade, Sodium-potassium
exchange
1. Introduction Cell swelling is prominent after metabolic blockade of in vitro preparations of renal cortex, diaphragm, liver and brain [4, 6, 91. This increase in water content is accompanied by an increase in monovalent cations and chloride, and has been attributed to inhibition of an energy-dependent sodium-potassium exchange pump by metabolic blockade [6]. H owever, specific blockade of this sodium-potassium Supported in part by Grants HL 10539, HL 5909, and HL 20720 from the National Heart, Lung and Blood Institute, National Institutes of Health, Grant RR-01032 from the General Clinical Research Centres Program of the Division of Research Resources, National Institutes of Health, and a grant from the Milton Fund. Data organization and analysis were performed on the PROPHET system, a national computer resource sponsored by the Chemical/Biological Information Handling Program, National Institutes of Health. Dr Bing is the recipient of a Career Development Award from the National Institutes of Health. 0 1979 Academic Press Inc. Limited (London) 0022-2828~79/060585+06$02.00/0
586
M. B. PINE
ET AL.
exchange pump by oubain has failed to produce swelling despite marked losses of potassium [5, 12, 131, suggesting that inhibition of this pump is not, in itself, sufficient to invariably result in tissue or cell swelling. In 1960, Tosteson and Hoffman [16] described and tested a model of cell volume regulation in which water content was related to sodium-potassium exchange pump activity and cell membrane permeability to monovalent cations. According to this “pump-leak” hypothesis of cell volume regulation, metabolic blockade may result in cell swelling by combining pump inhibition with an increase in membrane permeability to sodium not associated with exposure of tissues to ouabain. The present study examined the effect of metabolic inhibition secondary to hypoxia at 0°C (cold shock) on the relation between sodium-potassium exchange pump activity and cell volume regulation in slices of canine myocardium. The effect of continued pump inhibition by ouabain on removal of cold shock induced swelling when myocardium was oxygenated at 25°C was also studied. 2. Materials
and Methods
Six adult dogs were anesthetized with chloralose and urethane, and their chests were opened. Hearts were rapidly removed and submerged in Krebs-RingersPhosphate solution (KRP) which was bubbled with oxygen and maintained at 0 to 2°C. Sections of left ventricular myocardium approximately 0.5 mm thick were cut by a free-hand technique and divided into seven groups: (A) control sections submerged in oxygenated KRP at 25°C for 2 h; (B) control sections submerged in oxygenated KRP at 25°C for 3 h; (C) sections submerged in KRP which was bubbled with nitrogen at 0 to 2°C (cold shock) for 2 h; (D) sections which equilibrated in oxygenated KRP at 25°C for 1 h before being subjected to 2 h of cold shock; (E) sections subjected to 2 h of cold shock followed by 1 h of recovery in oxygenated KRP at 25°C; (F) sections subjected to 2 h of cold shock in the presence of 1 ~-IM ouabain followed by 1 h of recovery in oxygenated KRP which contained 1 mu ouabain at 25°C. Preliminary experiments revealed similar reductions in tissue potassium in dog myocardial sections incubated for 3 h in oxygenated KRP which contained either 1 or 10 mu ouabain at 25°C (K+ = 108 f 5 mEq/kg in 1 mu ouabain and 99 f 5 n-&q/kg in 10 mu ouabain) . Therefore, 1 mM ouabain was used in the present studies to maximally inhibit the ouabain sensitive sodiumpotassium exchange pump. The medium used in these experiments contained the following ions in millimoles per litre: Na+ 151.56, K+ 4.81, Gas+ 1.29, Mgs+ 1.20, Sods- 1.20, phosphate 15.63 and Cl- 127.7. Twelve tissues in each group were exposed to [sH]inulin (New England Nuclear, Boston, Massachusetts) during the last 2 h of each experiment. At the end of each experiment, tissues were taken from the bath. Excess water was removed by placing each section between two pieces of bibulous paper and applying uniform pressure for 3 s with a Petri dish cover. Tissues were weighed on
ROLE
OF NA-K
EXCHANGE
PUMP
IN CELL
VOLUME
CONTROL
587
a Mettler H54 balance, dried for 48 h at 6O”C, and then reweighed. (Additional drying for 24 h at 105°C has been shown to result in no additional reduction in weight.) Tissues exposed to [sH]inulin (two slices per heart for each group, tl = 12 per group) were rehydrated with 0.1 ml water and dissolved in 1 ml Protosol@ (New England Nuclear, Boston, Massachusetts). Ten milliliters of Econofluor TM (New England Nuclear, Boston, Massachusetts) were added and sH was counted in a Tricarb Liquid Scintillation Counter (Packard Instrument Company, Downers Grove, Illinois) using standards containing 0.05 and 0.1 ml of bathing solution. The remaining tissues (four slices per heart for each group, n = 24 per group) were dissolved in concentrated nitric acid. Tissue sodium and potassium were measured using a flame photometer (Instrumentation Laboratory, Watertown, Massachusetts) with an internal lithium standard. Extracellular water was calculated from [sH]inulin data by assuming complete equilibration of interstitium and bathing medium [IO]. Data were analyzed using analysis of variance and Duncan’s test for multiple comparisons [I]. Values are expressed as mean f S.E.M.
3. Results
Results are presented in Table 1. Since extracellular water was similar after all interventions, total tissue potassium, sodium and water accurately reflect intracellular values. There was a marked fall in myocardial potassium following immediate cold shock, cold shock after equilibration and immediate cold shock in the presence of
TABLE
1. Myocardial monovalent cations and water Potassium @q/g*
Control
2h
Monovalent
Tissue
cations dWg*
water ml/g*
Interstitial water ml/g*
251 f 38
620 *33
3.38 & 0.32
1.43 f 0.25
240 y30
634 yi5
3.39 rsd.25
l.42N:b.24
138 f 37t
650 + 44:
3.61 f 0.35?$
1.35 & 0.24
125 f 30-f
833 f 77t
4.56 & 0.38t
1.44 5 0.19
113 >7+
837 y63+ 692 f 507:
4.54 3.357 3.58 f 0.26t;
l.53N:0.24 1.55 -& 0.33
704 :45+$
3.85 & 0.24tf
P
3h
Equilibration, then cold shock Immediate cold shock Immediate cold shock and recovery
with ouabain P without ouabain with ouabain
P without ouabain
202 *tt: < 0.01 65 f 15fS
< 0.01 1.4gN;,s*o.25
* Mean f S.D. per gram dry tissue weight; t compared to controls, P < 0.01; $ compared to immediate cold shock, P < 0.01.
588
M. B. PINE
ET AL.
ouabain (P
Dissociation of Cell Volume Regulation and Sodium-Potassium Exchange Pump Activity in Dog Myocardium in vitro MICHAEL
B. PINE, OSCAR H. L. BING, RONALD WEINTRAUB WALTER H. ABELMANN Departments of Medicine and Surgery and the Thorndike Laboratory,
Harvard
Medical School and Beth Israel Hospital, (Received 28 Jub
AND
Boston, Massachusetts 022 15, U.S.A.
1978, accepted in revisedform 6 .November 1978)
M. B. PINE, 0. H. L. BING, R. WEINTFUUJB AND W. H. ABELMANN. Dissociation of Cell Volume Regulation and Sodiun-Potassium Exchange Pump Activity in Dog Myocardium in vitro. Journal qf Molecular andCellular Cardiology (1979) 11, 585-590. The relation between myocardial cell potassium and water contents after hypoxia at 0°C (cold shock) in KrebsRingers-Phosphate solution was studied using 252 myocardial slices from six dogs. Losses of potassium were marked and similar after immediate cold shock, cold shock after 1 h equilibration in oxygenated media at 25°C and immediate cold shock plus 1 msr ouabain. However, myocardial water and total monovalent cations were only slightly increased by cold shock after equilibration compared to the marked increases found after immediate cold shock with and without ouabain (P < 0.01). When edematous slices were warmed to 25°C in oxygenated media, myocardial potassium increased towards control values in the absence of ouabain (P < 0.01) and decreased further when ouabain was present (P < O.Ol), but in both cases myocardial water and total monovalent cations decreased towards control values (P i 0.01). This dissociation of Na-K exchange pump activity and myocardial cell volume regulation can be only partially explained using the classic “pump-leak” theory of cell volume regulation. KEY WORDS: Cell volume regulation; pump; Ouabain.
Metabolic
blockade, Sodium-potassium
exchange
1. Introduction Cell swelling is prominent after metabolic blockade of in vitro preparations of renal cortex, diaphragm, liver and brain [4, 6, 91. This increase in water content is accompanied by an increase in monovalent cations and chloride, and has been attributed to inhibition of an energy-dependent sodium-potassium exchange pump by metabolic blockade [6]. H owever, specific blockade of this sodium-potassium Supported in part by Grants HL 10539, HL 5909, and HL 20720 from the National Heart, Lung and Blood Institute, National Institutes of Health, Grant RR-01032 from the General Clinical Research Centres Program of the Division of Research Resources, National Institutes of Health, and a grant from the Milton Fund. Data organization and analysis were performed on the PROPHET system, a national computer resource sponsored by the Chemical/Biological Information Handling Program, National Institutes of Health. Dr Bing is the recipient of a Career Development Award from the National Institutes of Health. 0 1979 Academic Press Inc. Limited (London) 0022-2828~79/060585+06$02.00/0
586
M. B. PINE
ET AL.
exchange pump by oubain has failed to produce swelling despite marked losses of potassium [5, 12, 131, suggesting that inhibition of this pump is not, in itself, sufficient to invariably result in tissue or cell swelling. In 1960, Tosteson and Hoffman [16] described and tested a model of cell volume regulation in which water content was related to sodium-potassium exchange pump activity and cell membrane permeability to monovalent cations. According to this “pump-leak” hypothesis of cell volume regulation, metabolic blockade may result in cell swelling by combining pump inhibition with an increase in membrane permeability to sodium not associated with exposure of tissues to ouabain. The present study examined the effect of metabolic inhibition secondary to hypoxia at 0°C (cold shock) on the relation between sodium-potassium exchange pump activity and cell volume regulation in slices of canine myocardium. The effect of continued pump inhibition by ouabain on removal of cold shock induced swelling when myocardium was oxygenated at 25°C was also studied. 2. Materials
and Methods
Six adult dogs were anesthetized with chloralose and urethane, and their chests were opened. Hearts were rapidly removed and submerged in Krebs-RingersPhosphate solution (KRP) which was bubbled with oxygen and maintained at 0 to 2°C. Sections of left ventricular myocardium approximately 0.5 mm thick were cut by a free-hand technique and divided into seven groups: (A) control sections submerged in oxygenated KRP at 25°C for 2 h; (B) control sections submerged in oxygenated KRP at 25°C for 3 h; (C) sections submerged in KRP which was bubbled with nitrogen at 0 to 2°C (cold shock) for 2 h; (D) sections which equilibrated in oxygenated KRP at 25°C for 1 h before being subjected to 2 h of cold shock; (E) sections subjected to 2 h of cold shock followed by 1 h of recovery in oxygenated KRP at 25°C; (F) sections subjected to 2 h of cold shock in the presence of 1 ~-IM ouabain followed by 1 h of recovery in oxygenated KRP which contained 1 mu ouabain at 25°C. Preliminary experiments revealed similar reductions in tissue potassium in dog myocardial sections incubated for 3 h in oxygenated KRP which contained either 1 or 10 mu ouabain at 25°C (K+ = 108 f 5 mEq/kg in 1 mu ouabain and 99 f 5 n-&q/kg in 10 mu ouabain) . Therefore, 1 mM ouabain was used in the present studies to maximally inhibit the ouabain sensitive sodiumpotassium exchange pump. The medium used in these experiments contained the following ions in millimoles per litre: Na+ 151.56, K+ 4.81, Gas+ 1.29, Mgs+ 1.20, Sods- 1.20, phosphate 15.63 and Cl- 127.7. Twelve tissues in each group were exposed to [sH]inulin (New England Nuclear, Boston, Massachusetts) during the last 2 h of each experiment. At the end of each experiment, tissues were taken from the bath. Excess water was removed by placing each section between two pieces of bibulous paper and applying uniform pressure for 3 s with a Petri dish cover. Tissues were weighed on
ROLE
OF NA-K
EXCHANGE
PUMP
IN CELL
VOLUME
CONTROL
587
a Mettler H54 balance, dried for 48 h at 6O”C, and then reweighed. (Additional drying for 24 h at 105°C has been shown to result in no additional reduction in weight.) Tissues exposed to [sH]inulin (two slices per heart for each group, tl = 12 per group) were rehydrated with 0.1 ml water and dissolved in 1 ml Protosol@ (New England Nuclear, Boston, Massachusetts). Ten milliliters of Econofluor TM (New England Nuclear, Boston, Massachusetts) were added and sH was counted in a Tricarb Liquid Scintillation Counter (Packard Instrument Company, Downers Grove, Illinois) using standards containing 0.05 and 0.1 ml of bathing solution. The remaining tissues (four slices per heart for each group, n = 24 per group) were dissolved in concentrated nitric acid. Tissue sodium and potassium were measured using a flame photometer (Instrumentation Laboratory, Watertown, Massachusetts) with an internal lithium standard. Extracellular water was calculated from [sH]inulin data by assuming complete equilibration of interstitium and bathing medium [IO]. Data were analyzed using analysis of variance and Duncan’s test for multiple comparisons [I]. Values are expressed as mean f S.E.M.
3. Results
Results are presented in Table 1. Since extracellular water was similar after all interventions, total tissue potassium, sodium and water accurately reflect intracellular values. There was a marked fall in myocardial potassium following immediate cold shock, cold shock after equilibration and immediate cold shock in the presence of
TABLE
1. Myocardial monovalent cations and water Potassium @q/g*
Control
2h
Monovalent
Tissue
cations dWg*
water ml/g*
Interstitial water ml/g*
251 f 38
620 *33
3.38 & 0.32
1.43 f 0.25
240 y30
634 yi5
3.39 rsd.25
l.42N:b.24
138 f 37t
650 + 44:
3.61 f 0.35?$
1.35 & 0.24
125 f 30-f
833 f 77t
4.56 & 0.38t
1.44 5 0.19
113 >7+
837 y63+ 692 f 507:
4.54 3.357 3.58 f 0.26t;
l.53N:0.24 1.55 -& 0.33
704 :45+$
3.85 & 0.24tf
P
3h
Equilibration, then cold shock Immediate cold shock Immediate cold shock and recovery
with ouabain P without ouabain with ouabain
P without ouabain
202 *tt: < 0.01 65 f 15fS
< 0.01 1.4gN;,s*o.25
* Mean f S.D. per gram dry tissue weight; t compared to controls, P < 0.01; $ compared to immediate cold shock, P < 0.01.
588
M. B. PINE
ET AL.
ouabain (P
