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Role of Na-K-atpase in the renal reabsorption of sodium in the elasmobranch, squalus acanthias
Con@. Biochem. Physiol., 1973, Vol. 44A, pp. 417 to 422. Pe~gamon Press. Printed in Great Britain
ROLE OF Na-K-ATPASE IN THE RENAL REABSORPTION OF SODIUM IN THE ELASMOBRANCH, SQUALUS ACANTHIAS” JOHN P. HAYSLETT,t LEE M. JAMPOL, MARK EPSTEIN, H. VICTOR MURDAUGH
JOHN N. FORREST, and JACK D. MYERS
Yale University School of Medicine, New Haven, Connecticut 06510; and the University of Pittsburgh School of Medicine, Pittsburgh, Pennsylvania 15213 (Received 21 April 1972)
Abstract-l.
The relative importance of Na-K-activated ATPase in active bulk transport of Na+ in the elasmobranch kidney was examined. Fractional Na+ excretion was examined before and after the administration of ouabain, associated with over a 60 per cent reduction in enzyme activity, as well as following furosemide and ethacrynic acid. 2. Marked inhibition of Na-K-ATPase with ouabain did not reduce Naf reabsorption, in contrast to furosemide and ethacrynic acid which increased fractional excretion from 0.22 to O-69. 3. These data suggest that Na-K-ATPase does not participate in bulk transport of Na+ in the elasmobranch kidney, where the conservation of Na+ is of little importance in osmoregulation. INTRODUCTION
SODIUM-POTASSIUM-ACTIVATED adenosine triphosphatase (Na-K-ATPase) has been demonstrated to play a key role in the active reciprocal transfer of sodium and potassium across the plasma membrane of individual cells (Skou, 1965) and in the bulk transport of sodium across epithelial membranes, as in the kidney (Katz & Epstein, 1967), the gill (Epstein et al., 1967) and the avian salt gland (Bonting et al., 1964). The specific activity of Na-K-ATPase in the gill of the eel (Anguilla vostrata) correlates directly with sodium extrusion across the gill and is probably of major importance in this animal as a mechanism allowing adaptation to external environments of different salinity (Jampol & Epstein, 1970). In the mammalian kidney under conditions of enhanced sodium reabsorption per g of tissue there is a parallel increase in Na-K-ATPase (Katz & Epstein, 1967). Inhibition of renal Na-K-ATPase with cardiac glycosides, in contrast, is associated with a marked decrease in the tubular reabsorption of sodium (Martinez-Maldonado et al., 1970; Torretti et al., 1972). There is also evidence to indicate that the enzyme participates in the regulation of the ionic composition of renal tubular cells (Maude, 1969). * These experiments were performed at the Mount Desert Island Biological Laboratory, Salisbury Cove, Maine, U.S.A. t Established Investigator of the American Heart Association. 417
418
JOHN P. HAYSLETT et al.
In the present experiments the relationship of the specific activity of Na-KATPase to renal sodium reabsorption in a marine elasmobranch, Squahs ucunthius, was studied since in this species the renal conservation of sodium is of lesser importance than in the mammal. MATERIALS
AND METHODS
All experiments were performed on unanesthetized fish (Spalus acanthias) maintained at 13°C in running sea water. In the clearance studies, injection and withdrawal of blood was accomplished through an indwelling polyethylene catheter (PE 50) inserted through the caudal artery into the ventral aorta. Urine was collected in a rubber balloon attached to a catheter secured to the urinary papilla. The specific activity of Na-K-ATPase and MgATPase in whole homogenates of renal tissue was determined by methods previously described (Jampol & Epstein, 1970). The measurements of sodium and potassium were performed on a flame photometer with an internal standard and of inulin on an Autoanalyzer (Fuhr et al., 1955). In nine normal fish the specific activity of Na-K-ATPase in kidney homogenate was 7.30 + 0.34 (mean + S.E.) and of Mg-ATPase was 17.29 + 3.53 /Imoles of inorganic phosphate per mg protein per hr. One hr following the injection of ouabain into the aorta, in a dose which varied from 100 to 250 pg/kg, the specific activity of Na-K-ATPase was reduced an average of 61.6 ?z25 per cent to 2.81 f O-18, while the Mg-ATPase level was unchanged at 17.29 f 3.5. A systemic dose of ouabain exceeding approximately 75 pg/kg resulted in death of the animal within 4 hr. In four fish measurements of GFR, urine volume (V), sodium and potassium excretion were made before and during 4 hr following the intra-aortic injection of ouabain (GStrophanthin, Eli Lilly & Co.), in an average dose of 57 pg/kg. The specific activity of Na-K-ATPase and Mg-ATPase was measured at the termination of the experiment in each experimental fish and compared to control fish studied on the same day.
RESULTS As shown in Table 1 the level of Na-K-ATPase was reduced an average of 61.8 + 10.7 per cent in experimental fish, while the specific activity of Mg-ATPase was unchanged, similar to the results obtained after administering larger doses of ouabain. The action of ouabain was associated with a slight increase in GFR, which rose from 1.26 + 0.20 to 1.89 _+0.16 mg/kg per hr and in urine volume which increased from O-52& 0.10 to 064 + 0.05 ml/kg per hr. Both the fractional and absolute excretion of sodium fell, however, despite the marked inhibition of Na-K-ATPase, while fractional excretion of potassium rose. In order to determine whether reduction of renal tubular sodium reabsorption could be demonstrated in this fish under other conditions, an additional group of twelve fish was studied before and after the administration of furosemide and ethacrynic acid. In these experiments both agents were found to cause a marked reduction in the tubular reabsorption of sodium and a marked rise in the fractional excretion of sodium. As shown in Table 2, during the 4 hr following the injection of ethacrynic acid (4-10 mg/kg) or furosemide (10-15 mg/kg) the GFR was not altered while urine volume increased threefold and the fractional excretion of sodium rose from
P value
SE.
lZ2
6.99
2.80 8.16 5.89 11.11
K
566.0 * 97.2
309.8 600.6 849.6 504.0
XV @equiv/hr)
O&
0.64
0.55 0.67 0.79 0.53
V
0:4
0.28
0.25 0.40 0.29 0.19
Na
1:9
8.33
4.48 9.00 6.74 13.09
K
Fractional excretion
Experimental
490.9 f 66.7
271.2 591.8 612.1 498.4
LEVELS
0:3
7.49
7.72 644 8.74 7.05
2f33
17.05
10.30 18.73 23.15 16.00
Na-KMgATPase ATPase (Pi pM/mg protein per hr)
Control
AND Na-K-ATPase
Q&V &equiv/hr)
ON RENAL FUNCTION
< 0.0025 N.S. < 0.05 < 0.01 N.S.
0:6
l-89
1.70 1.48 2.20 2.20
GFR
-
~---EFFECT OF OUABAIN
* Values for GFR and V are expressed as ml/kg per hr. Paired t-test was used in comparison of control and experimental data in individual experiments.
0:s
o:o
1.26 f 0.20
Mean
0.32 0.45 0.52 0.28
0.39
0.29 0.58 0.82 0.39
0.93 0.82 1.80 1.50
13 19 23 25
Na
0.52
V*
Experi- GFR* ment
Fractional excretion
Control
TABLE
0:9
2.93
2.33 1.02 3.64 4.75
1:s
19.16
18.10 20.29 24.24 14.00
Na-KMgATPase ATPase (Pi pM/mg protein per hr)
Experimental
8
k
420
P. HAYSLETT
JOHN
et&.
approximately 0.22 to 069 (P< 0.001). Although both agents inhibit the specific activity of Na-K-ATPase when administered in &uo in large amounts, there is in general a lack of correlation between this effect and their action to cause a natriuresis (Hook & Williamson, 1969). I n a recent report by Schmidt & Dubach (1970), the administration of furosemide in a dose of 100 mg/kg failed to reduce the enzyme level in the proximal tubule of the rat despite micropuncture studies (Brenner et al., 1969) demonstrating an effect on this segment following furosemide in a smaller dose. It seems unlikely, therefore, that the natriuretic effect of ethacrynic acid and furosemide in the dose employed in these experiments was dependent upon a reduction in Na-K-ATPase. In support of this, 4 hr following the administration of 40 mg of furosemide to two fish, Na-K-ATPase levels averaged 7.21 Pi pM/mg protein per hr, similar to control values. TABLED--EFFECT
OF ETHACRYNIC
ACID AND
FUROSEMIDE
Control
Experiment Ethacrynic acid (3 fish) Furosemide (9 fish)
GFRt
Vt
ON
RENAL
FUNCTION*
Experimental Fractional excretion, Na
GFR
V
Fractional excretion, Na
1.84 k 0.39 0.36 i 0.07 0.21 + 0.02 2.08 f 0.17 1.32 + 0.11 0.69 f 0.02 246 + 0.33 O-55k 0.05 0.24 f 0.01 2.25 + 0.22 1.54 + 0.09 0.70 f. 0.03
+ All values are given as means + SE. t Values for GFR and V are expressed as ml/kg per hr. DISCUSSION Previous studies have shown that inhibition of Na-K-ATPase in the mammalian kidney with cardiac glycosides to levels approximately two-thirds of normal, as in the present experiments, is associated with a marked natriuresis (Torretti et al., 1972). In experiments performed in non-diuretic dogs fractional sodium excretion increased from less than 1 to as much as 20-35 per cent. The reduction in sodium reabsorption was caused by inhibition of the specific activity of the enzyme since the amount of (sH)-ouabain bound to the kidney in r.&o was closely correlated with inhibition of Na-K-ATPase estimated in the same kidney as measured in vitro. There was also a correlation between the reduction of enzyme activity and inhibition of tubular reabsorption of sodium. Although this evidence suggests an important role for Na-K-ATPase in bulk transport of sodium across the tubular epithelium of the renal tubule, other mechanisms are undoubtedly important since even after enzyme inhibition greater than 90 per cent, 20 per cent or more of filtered sodium was still reabsorbed (Torretti et aZ., 1972). Proverbio et al. (1970) h ave provided evidence that active transport of sodium by the kidney occurs by at least two different pumps or by different
Na- K-ATPASE IN RENALREABSORPTION OF SODIUMLNELASMOBRANCH
421
components of the same pump. In their model Pump B derives its energy from Na-K-ATPase, is sensitive to ouabain and is an exchange mechanism whereby intracellular Na+ is interchanged with extracellular K+. Pump A, in contrast, is an electrogenic pump sensitive to ethacrynic acid, and extrudes Naf accompanied by Cl-. When viewed together with the clearance studies involving administration of cardiac glycosides these data suggest that bulk Na+ reabsorption by the mammalian kidney is in part dependent on Na-K-ATPase and in part on another mechanism(s). The relative importance of these mechanisms may, therefore, vary among epithelial membranes and among species. The failure of ouabain to reduce tubular reabsorption of sodium in the shark might be explained if Na-K-ATPase were present in the kidney in greater excess than in the dog kidney. Under these circumstances greater inhibition of enzyme activity might have been necessary in order to alter bulk sodium movement. Another more likely explanation for the lack of correlation between enzyme inhibition and sodium reabsorption in the shark relates to the role of Na-K-ATPase in the kidney under different physiological conditions. In contrast to the mammalian kidney, where renal reabsorption of sodium plays an important role in osmoregulation, the kidney of the marine elasmobranch has a lesser role. High concentrations of urea and trimethylamine in the body fluids of the sea water elasmobranch prevent the osmotic losses of water. Sodium enters the body, however, by inward diffusion through the gills and in food. There is little need therefore for active reabsorption by the renal tubules to conserve sodium. Under these circumstances the primary role of Na-K-ATPase in the kidney may be in the maintenance of intracellular concentrations of potassium and sodium, while the bulk movement of sodium across the tubular epithelial barrier is controlled primarily by another pump mechanism which is insensitive to ouabain but is inhibited by furosemide and ethacrynic acid. Acknowledgements-This work was supported by U.S.P.H.S. grants TIAM 5015, HE 13647-OlAl, HE 00834-22, 5501-RR05416-10, HE 5905-01 and the American Heart Association. REFERENCES BONTINC S. L., CARAVAGGIO L. L., CANADYM. R. & HAWKINSN. M. (1964) Studies on sodium-potassium-activated adenosine triphosphatase-XI. The salt glandof the herring gull. Archs Biochem. Biophys. 106, 49-56. BRENNERB. M., KEIMOWITZR. I., WRIGHT F. S. & BERLINERR. W. (1969) An inhibitory effect of furosemide on sodium reabsorption by the proximal tubule of the rat nephron. J. clin. Invest. 48, 290-300. EPSTEIN F. H., KATZ A. I. & PICKFORDG. E. (1967) Sodium-and-potassium-activated adenosine triphosphatase of gills: role in adaptation of teleosts to salt water. Science 156, 1245-1247. FUHR J., KACZMARCZYK J. & KRUTTGENG. (1955) Eine einfache colorimetrische Methode zur Inulinbestimmung fiir Nieren-Clearance-Untersuchungen bei Stoffwechselgesunden und Diabetikern. Klin. Wochenschr. 33, 729-730. HOOKJ. B. & WILLIAMSONH. E. (1969) Lack of correlation between natriuretic activity and inhibition of renal Na-K-activated ATPase. Proc. Sot. exp. Biol. Med. 120, 358-360.
422
JOHN P. HAYSLETTet al.
JAMPOL L. M. & EPSTEIN F. H. (1970) Sodium-potassium-activated adenosine triphosphatase and osmotic regulation by fishes. Am. J. Physiol. 218, 607-611. KATZ A. I. & EPSTEIN F. H. (1967) The role of sodium-potassium-activated adenosine triphosphatase. J. clin. Invest. 46, 1999-2011. MARTINEZ-MALDONADO M., EKNOYANG., ALLEN J. C., SUKI W. H. & SCHWARTZ A. (1970) Urine dilution and concentration after digoxin infusion into the renal artery of dogs. Proc. Sot. exp. Biol. Med. 134, 855-860. MAUDED. L. (1969) Effects of K and ouabain on fluid transport and cell Na in proximal tubule in vitro. Am. J. Physiol. 216, 1199-1206. PROVERBIOF., ROBINSONJ. W. L. & WHITTEMBURYG. (1970) Sensitivities of (Na+-K+)ATPase and Naf extrusion mechanisms to ouabain and ethacrynic acid in the cortex of the guinea pig kidney. Biochem. Biophys. Acta 211, 327-336. SCHMIDT U. & DUBACH U. C. (1970) The behavior of Na+K+-activated adenosine triphosphatase in various structures of the rat nephron after furosemide application. Nephron 7, 447-458. SKOUJ. C. (1965) Enzymatic basis for active transport of Na+ and K+ across cell membrane. Physiol. Rew. 45, 596-617. TORRETTI J., HENDLER E., WEINSTEIN E., LONGNECKERR. E. & EPSTEIN F. H. (1972) Functional significance of Na-K-ATPase in the kidney. Effects of ouabain inhibition. Am. J. Physiol. 222, 1398-1705. Key Word Index-Na-K-ATPase; acunthius; furosemide; ethacrynic acid.
renal reabsorption;
sodium
transport;
Squalus
ROLE OF Na-K-ATPASE IN THE RENAL REABSORPTION OF SODIUM IN THE ELASMOBRANCH, SQUALUS ACANTHIAS” JOHN P. HAYSLETT,t LEE M. JAMPOL, MARK EPSTEIN, H. VICTOR MURDAUGH
JOHN N. FORREST, and JACK D. MYERS
Yale University School of Medicine, New Haven, Connecticut 06510; and the University of Pittsburgh School of Medicine, Pittsburgh, Pennsylvania 15213 (Received 21 April 1972)
Abstract-l.
The relative importance of Na-K-activated ATPase in active bulk transport of Na+ in the elasmobranch kidney was examined. Fractional Na+ excretion was examined before and after the administration of ouabain, associated with over a 60 per cent reduction in enzyme activity, as well as following furosemide and ethacrynic acid. 2. Marked inhibition of Na-K-ATPase with ouabain did not reduce Naf reabsorption, in contrast to furosemide and ethacrynic acid which increased fractional excretion from 0.22 to O-69. 3. These data suggest that Na-K-ATPase does not participate in bulk transport of Na+ in the elasmobranch kidney, where the conservation of Na+ is of little importance in osmoregulation. INTRODUCTION
SODIUM-POTASSIUM-ACTIVATED adenosine triphosphatase (Na-K-ATPase) has been demonstrated to play a key role in the active reciprocal transfer of sodium and potassium across the plasma membrane of individual cells (Skou, 1965) and in the bulk transport of sodium across epithelial membranes, as in the kidney (Katz & Epstein, 1967), the gill (Epstein et al., 1967) and the avian salt gland (Bonting et al., 1964). The specific activity of Na-K-ATPase in the gill of the eel (Anguilla vostrata) correlates directly with sodium extrusion across the gill and is probably of major importance in this animal as a mechanism allowing adaptation to external environments of different salinity (Jampol & Epstein, 1970). In the mammalian kidney under conditions of enhanced sodium reabsorption per g of tissue there is a parallel increase in Na-K-ATPase (Katz & Epstein, 1967). Inhibition of renal Na-K-ATPase with cardiac glycosides, in contrast, is associated with a marked decrease in the tubular reabsorption of sodium (Martinez-Maldonado et al., 1970; Torretti et al., 1972). There is also evidence to indicate that the enzyme participates in the regulation of the ionic composition of renal tubular cells (Maude, 1969). * These experiments were performed at the Mount Desert Island Biological Laboratory, Salisbury Cove, Maine, U.S.A. t Established Investigator of the American Heart Association. 417
418
JOHN P. HAYSLETT et al.
In the present experiments the relationship of the specific activity of Na-KATPase to renal sodium reabsorption in a marine elasmobranch, Squahs ucunthius, was studied since in this species the renal conservation of sodium is of lesser importance than in the mammal. MATERIALS
AND METHODS
All experiments were performed on unanesthetized fish (Spalus acanthias) maintained at 13°C in running sea water. In the clearance studies, injection and withdrawal of blood was accomplished through an indwelling polyethylene catheter (PE 50) inserted through the caudal artery into the ventral aorta. Urine was collected in a rubber balloon attached to a catheter secured to the urinary papilla. The specific activity of Na-K-ATPase and MgATPase in whole homogenates of renal tissue was determined by methods previously described (Jampol & Epstein, 1970). The measurements of sodium and potassium were performed on a flame photometer with an internal standard and of inulin on an Autoanalyzer (Fuhr et al., 1955). In nine normal fish the specific activity of Na-K-ATPase in kidney homogenate was 7.30 + 0.34 (mean + S.E.) and of Mg-ATPase was 17.29 + 3.53 /Imoles of inorganic phosphate per mg protein per hr. One hr following the injection of ouabain into the aorta, in a dose which varied from 100 to 250 pg/kg, the specific activity of Na-K-ATPase was reduced an average of 61.6 ?z25 per cent to 2.81 f O-18, while the Mg-ATPase level was unchanged at 17.29 f 3.5. A systemic dose of ouabain exceeding approximately 75 pg/kg resulted in death of the animal within 4 hr. In four fish measurements of GFR, urine volume (V), sodium and potassium excretion were made before and during 4 hr following the intra-aortic injection of ouabain (GStrophanthin, Eli Lilly & Co.), in an average dose of 57 pg/kg. The specific activity of Na-K-ATPase and Mg-ATPase was measured at the termination of the experiment in each experimental fish and compared to control fish studied on the same day.
RESULTS As shown in Table 1 the level of Na-K-ATPase was reduced an average of 61.8 + 10.7 per cent in experimental fish, while the specific activity of Mg-ATPase was unchanged, similar to the results obtained after administering larger doses of ouabain. The action of ouabain was associated with a slight increase in GFR, which rose from 1.26 + 0.20 to 1.89 _+0.16 mg/kg per hr and in urine volume which increased from O-52& 0.10 to 064 + 0.05 ml/kg per hr. Both the fractional and absolute excretion of sodium fell, however, despite the marked inhibition of Na-K-ATPase, while fractional excretion of potassium rose. In order to determine whether reduction of renal tubular sodium reabsorption could be demonstrated in this fish under other conditions, an additional group of twelve fish was studied before and after the administration of furosemide and ethacrynic acid. In these experiments both agents were found to cause a marked reduction in the tubular reabsorption of sodium and a marked rise in the fractional excretion of sodium. As shown in Table 2, during the 4 hr following the injection of ethacrynic acid (4-10 mg/kg) or furosemide (10-15 mg/kg) the GFR was not altered while urine volume increased threefold and the fractional excretion of sodium rose from
P value
SE.
lZ2
6.99
2.80 8.16 5.89 11.11
K
566.0 * 97.2
309.8 600.6 849.6 504.0
XV @equiv/hr)
O&
0.64
0.55 0.67 0.79 0.53
V
0:4
0.28
0.25 0.40 0.29 0.19
Na
1:9
8.33
4.48 9.00 6.74 13.09
K
Fractional excretion
Experimental
490.9 f 66.7
271.2 591.8 612.1 498.4
LEVELS
0:3
7.49
7.72 644 8.74 7.05
2f33
17.05
10.30 18.73 23.15 16.00
Na-KMgATPase ATPase (Pi pM/mg protein per hr)
Control
AND Na-K-ATPase
Q&V &equiv/hr)
ON RENAL FUNCTION
< 0.0025 N.S. < 0.05 < 0.01 N.S.
0:6
l-89
1.70 1.48 2.20 2.20
GFR
-
~---EFFECT OF OUABAIN
* Values for GFR and V are expressed as ml/kg per hr. Paired t-test was used in comparison of control and experimental data in individual experiments.
0:s
o:o
1.26 f 0.20
Mean
0.32 0.45 0.52 0.28
0.39
0.29 0.58 0.82 0.39
0.93 0.82 1.80 1.50
13 19 23 25
Na
0.52
V*
Experi- GFR* ment
Fractional excretion
Control
TABLE
0:9
2.93
2.33 1.02 3.64 4.75
1:s
19.16
18.10 20.29 24.24 14.00
Na-KMgATPase ATPase (Pi pM/mg protein per hr)
Experimental
8
k
420
P. HAYSLETT
JOHN
et&.
approximately 0.22 to 069 (P< 0.001). Although both agents inhibit the specific activity of Na-K-ATPase when administered in &uo in large amounts, there is in general a lack of correlation between this effect and their action to cause a natriuresis (Hook & Williamson, 1969). I n a recent report by Schmidt & Dubach (1970), the administration of furosemide in a dose of 100 mg/kg failed to reduce the enzyme level in the proximal tubule of the rat despite micropuncture studies (Brenner et al., 1969) demonstrating an effect on this segment following furosemide in a smaller dose. It seems unlikely, therefore, that the natriuretic effect of ethacrynic acid and furosemide in the dose employed in these experiments was dependent upon a reduction in Na-K-ATPase. In support of this, 4 hr following the administration of 40 mg of furosemide to two fish, Na-K-ATPase levels averaged 7.21 Pi pM/mg protein per hr, similar to control values. TABLED--EFFECT
OF ETHACRYNIC
ACID AND
FUROSEMIDE
Control
Experiment Ethacrynic acid (3 fish) Furosemide (9 fish)
GFRt
Vt
ON
RENAL
FUNCTION*
Experimental Fractional excretion, Na
GFR
V
Fractional excretion, Na
1.84 k 0.39 0.36 i 0.07 0.21 + 0.02 2.08 f 0.17 1.32 + 0.11 0.69 f 0.02 246 + 0.33 O-55k 0.05 0.24 f 0.01 2.25 + 0.22 1.54 + 0.09 0.70 f. 0.03
+ All values are given as means + SE. t Values for GFR and V are expressed as ml/kg per hr. DISCUSSION Previous studies have shown that inhibition of Na-K-ATPase in the mammalian kidney with cardiac glycosides to levels approximately two-thirds of normal, as in the present experiments, is associated with a marked natriuresis (Torretti et al., 1972). In experiments performed in non-diuretic dogs fractional sodium excretion increased from less than 1 to as much as 20-35 per cent. The reduction in sodium reabsorption was caused by inhibition of the specific activity of the enzyme since the amount of (sH)-ouabain bound to the kidney in r.&o was closely correlated with inhibition of Na-K-ATPase estimated in the same kidney as measured in vitro. There was also a correlation between the reduction of enzyme activity and inhibition of tubular reabsorption of sodium. Although this evidence suggests an important role for Na-K-ATPase in bulk transport of sodium across the tubular epithelium of the renal tubule, other mechanisms are undoubtedly important since even after enzyme inhibition greater than 90 per cent, 20 per cent or more of filtered sodium was still reabsorbed (Torretti et aZ., 1972). Proverbio et al. (1970) h ave provided evidence that active transport of sodium by the kidney occurs by at least two different pumps or by different
Na- K-ATPASE IN RENALREABSORPTION OF SODIUMLNELASMOBRANCH
421
components of the same pump. In their model Pump B derives its energy from Na-K-ATPase, is sensitive to ouabain and is an exchange mechanism whereby intracellular Na+ is interchanged with extracellular K+. Pump A, in contrast, is an electrogenic pump sensitive to ethacrynic acid, and extrudes Naf accompanied by Cl-. When viewed together with the clearance studies involving administration of cardiac glycosides these data suggest that bulk Na+ reabsorption by the mammalian kidney is in part dependent on Na-K-ATPase and in part on another mechanism(s). The relative importance of these mechanisms may, therefore, vary among epithelial membranes and among species. The failure of ouabain to reduce tubular reabsorption of sodium in the shark might be explained if Na-K-ATPase were present in the kidney in greater excess than in the dog kidney. Under these circumstances greater inhibition of enzyme activity might have been necessary in order to alter bulk sodium movement. Another more likely explanation for the lack of correlation between enzyme inhibition and sodium reabsorption in the shark relates to the role of Na-K-ATPase in the kidney under different physiological conditions. In contrast to the mammalian kidney, where renal reabsorption of sodium plays an important role in osmoregulation, the kidney of the marine elasmobranch has a lesser role. High concentrations of urea and trimethylamine in the body fluids of the sea water elasmobranch prevent the osmotic losses of water. Sodium enters the body, however, by inward diffusion through the gills and in food. There is little need therefore for active reabsorption by the renal tubules to conserve sodium. Under these circumstances the primary role of Na-K-ATPase in the kidney may be in the maintenance of intracellular concentrations of potassium and sodium, while the bulk movement of sodium across the tubular epithelial barrier is controlled primarily by another pump mechanism which is insensitive to ouabain but is inhibited by furosemide and ethacrynic acid. Acknowledgements-This work was supported by U.S.P.H.S. grants TIAM 5015, HE 13647-OlAl, HE 00834-22, 5501-RR05416-10, HE 5905-01 and the American Heart Association. REFERENCES BONTINC S. L., CARAVAGGIO L. L., CANADYM. R. & HAWKINSN. M. (1964) Studies on sodium-potassium-activated adenosine triphosphatase-XI. The salt glandof the herring gull. Archs Biochem. Biophys. 106, 49-56. BRENNERB. M., KEIMOWITZR. I., WRIGHT F. S. & BERLINERR. W. (1969) An inhibitory effect of furosemide on sodium reabsorption by the proximal tubule of the rat nephron. J. clin. Invest. 48, 290-300. EPSTEIN F. H., KATZ A. I. & PICKFORDG. E. (1967) Sodium-and-potassium-activated adenosine triphosphatase of gills: role in adaptation of teleosts to salt water. Science 156, 1245-1247. FUHR J., KACZMARCZYK J. & KRUTTGENG. (1955) Eine einfache colorimetrische Methode zur Inulinbestimmung fiir Nieren-Clearance-Untersuchungen bei Stoffwechselgesunden und Diabetikern. Klin. Wochenschr. 33, 729-730. HOOKJ. B. & WILLIAMSONH. E. (1969) Lack of correlation between natriuretic activity and inhibition of renal Na-K-activated ATPase. Proc. Sot. exp. Biol. Med. 120, 358-360.
422
JOHN P. HAYSLETTet al.
JAMPOL L. M. & EPSTEIN F. H. (1970) Sodium-potassium-activated adenosine triphosphatase and osmotic regulation by fishes. Am. J. Physiol. 218, 607-611. KATZ A. I. & EPSTEIN F. H. (1967) The role of sodium-potassium-activated adenosine triphosphatase. J. clin. Invest. 46, 1999-2011. MARTINEZ-MALDONADO M., EKNOYANG., ALLEN J. C., SUKI W. H. & SCHWARTZ A. (1970) Urine dilution and concentration after digoxin infusion into the renal artery of dogs. Proc. Sot. exp. Biol. Med. 134, 855-860. MAUDED. L. (1969) Effects of K and ouabain on fluid transport and cell Na in proximal tubule in vitro. Am. J. Physiol. 216, 1199-1206. PROVERBIOF., ROBINSONJ. W. L. & WHITTEMBURYG. (1970) Sensitivities of (Na+-K+)ATPase and Naf extrusion mechanisms to ouabain and ethacrynic acid in the cortex of the guinea pig kidney. Biochem. Biophys. Acta 211, 327-336. SCHMIDT U. & DUBACH U. C. (1970) The behavior of Na+K+-activated adenosine triphosphatase in various structures of the rat nephron after furosemide application. Nephron 7, 447-458. SKOUJ. C. (1965) Enzymatic basis for active transport of Na+ and K+ across cell membrane. Physiol. Rew. 45, 596-617. TORRETTI J., HENDLER E., WEINSTEIN E., LONGNECKERR. E. & EPSTEIN F. H. (1972) Functional significance of Na-K-ATPase in the kidney. Effects of ouabain inhibition. Am. J. Physiol. 222, 1398-1705. Key Word Index-Na-K-ATPase; acunthius; furosemide; ethacrynic acid.
renal reabsorption;
sodium
transport;
Squalus