- Email: [email protected]
Effects of substrates and inhibitors of the tricarboxylic acid cycle on proximal tubular fluid transport in vitro
216
SHORT COMMUNICATIONS
other hand, neither does it appear to be particularly rich in glycosaminoglycans, as would have been the case if the surface zone had contained a layer of hvaluronic acid as postulated by BALAZSet al2. Further studies are in progress to determine the variation in the distribution of fixed charge density near the articular surface as a function of age. Tile principle of using cations from dilute solution to balance the fixed negative charges in structural tissue can readily be extended to localise and estimate anionic groups by such techniques as autoradiography (e.g. with 4~Ca) and electron-probe microanalysis.
Biomechanics Unit, Department of Mechanical Engineering, Imperial College, University of London, London (Great Britain)
ALICE MAROUDAS HILARY THOMAS
I A. MAROUDAS, H. MUIR AND J. V~7INGHAM, Biochim. Biophys. Acta, 177 (1969) 492. 2 l5". HELFFERICH, Ion Exchange, McGraw-Hill, New York, 1962, p. 143. 3 C. A. ANTONOPOULOS, S. GARDELL, J. A. SZIRMAI AND ]E. R. DE TYSSONSK, Biochim. Biophys. Acta, 83 (1964) I. 4 T. BITTER AND H. MUIR, Anal. Biochem., 4 (1962) 33 o. 5 M. B. MATHEXVS, Biochem. J., 96 ([965) 7Io. 6 M. SCHUBERT, Federation Proc., 25 (1966) lO47. 7 D. H. COLLINS AND T. F. MCELLIGOTT, Ann. Rheumatic Diseases, 19 (196o) 31. 8 C. V~rEISS, L. ROSENBERG AND A. J. HELFET, J . Bone Joint Surg., 5oA (4) (1968) 663. 9 E. A. BALAZS, G. D. BLOOM AND D. A. S\VANN, Federation Proc., 25 (1966) 1813.
Received April I5th, 197o Biochim. Biophys. Acta, 215 (197 o) 214-216
BBA 23588
Effects of substrates and inhibitors of the tricarboxylic acid cycle on proximal tubular fluid transport in vitro I have studied the effects of substrates and inhibitors of the tricarboxylic acid cycle on proximal tubular fluid transport using a technique of stop-flow microperfusion of single tubules in leached rat renal cortex slices. The experimental method has been described in detail elsewhere 1. Briefly, a surface cortical slice taken from a kidney of a pentobarbital-anesthetized male Sprague-Dawley rat is mounted in a micropuncture chamber. Here it is bathed in a rapidly flowing, recirculating oxygenated bicarbonate-saline solution at 37-38°. The basic medium provided (mM) 14o Na +, 5 K+, 121.6 Cl-, 20 HCOa-, 2.4 HP042-, 0.6 H2PO~-, I.o Ca2+ and 1.2 MgSO 4. Most of the media contained one or more substrates or inhibitors. These were all sodium salts of carboxylic acids and replaced an equivalent amount of NaC1, so that the Na + concentration was the s~me in all media. Before mounting, the slices were leached for 4-6 h at 2 ° in a medium identical to that subsequently used in the micropuncture chamber. The shrinking-droplet method Biochim. Biophys. Acta, 215 (197 o) 216-219
SHORT COMMUNICATIONS
217
of GERTZ2 was used to measure the rate of proximal tubular fluid transport. Since the perfusate is isosmotic (14o mM) NaC1, the rate of fluid reabsorption is directly related to the net rate of transport of sodium salts from the tubular lumen. For convenience, the results have been expressed in terms of the fluid reabsorptive halftime, t~2. 1.0 0.7 o.5
0.3 0
10 20 Time (sec)
30
40
Fig. i. Relative perfusate volume (V/Vo) plotted as a function of time in representative experim e n t s on slices leached w i t h o u t s u b s t r a t e ((2)--(2)) and with 3 mM ~-ketoglutarate ( @ - - O ) .
As in studies with fresh kidney slices 1, the relative volume of perfusing fluid remaining within the tubule, V / V o, decreased exponentially with time under all experimental conditions. Fig. i shows data from two representative experiments on leached slices. Table I summarizes data from all experiments. Half-time for the reabsorption of isosmotic saline perfusates b y proximal tubules of fresh slices bathed in media providing io mM e-ketoglutarate as substrate is about 19 sec (ref. I). The half-time in slices leached in substrate-free media is significantly longer than this, 39.6 sec. However, when the leaching and bathing media contain 3 mM pyruvate, oxaloacetate or =-ketoglutarate, or io mM succinate, t~ becomes approximately the same as in fresh slices. Apparently leaching in a substrate-free medium depletes proximal tubular cells of one or more diffusible metabolites required for normal rates of NaC1 transport, while the above substrates are able to restore and maintain adequate levels of these substances. The effectiveness of these compounds in stimulating transport in leached slices can be most easily explained by their ability to provide the two intermediates needed to initiate and maintain operation of the tricarboxylic acid cycle: oxaloacetate and acetyl-CoA. The failure of acetate to restore normal rates of transport could be due to its inability to produce the oxaloacetate required b y the cycle. I t is more difficult to understand why citrate, fumarate and malate fail to yield normal rates of transport in leached slices. If transport were dependent simply upon operation of the tricarboxylic acid cycle, these compounds should have been as effective as pyruvate and the other cycle intermediates. Possible explanations for this discrepancy are: (i) Substrates which fail to support transport m a y be unable to penetrate cellular and/or mitochondrial membranes sufficiently rapidly to supply the cycle. Consistent with this hypothesis is the fact that the minimal effective concentrations of a-ketoglutarate and succinate (greater than I and 3 mM, respectively) are considerably higher than their concentrations in fresh kidney tissue a, as would be expected if permeability barriers had to be overcome. HASLAM AND KREBS4 have found more direct evidence that the rate of penetration of substrates across kidney mitochondrial Biochim. Biophys. Acta, 215 (197 o) 216-219
218
SHORT COMMUNICATIONS
TABLE I H A L F - T I M E FOR T U B U L A R F L U I D REABSORPTION (tl/2) I N SLICES L E A C H E D W I T H VARIOUS SUBSTRATE~
AND SUBSTRATE--INHIBITOR COMBINATIONS N u m b e r of t u b u l e s perfused is indicated in parentheses.
Substrate/inhibitor
tl 6 ~ S.E.
None
39.6 _~ 4.I*
(17)
mM 0¢-ketoglutarate 2 mM ~-ketoglutarate 3 mM ~-ketoglutarate i o mM ~-ketoglutarate 3 mM ~-ketoglutarate 6 mM monofluoroacetate 3 mM ~-ketoglutarate o.5 mM inethoxyindolcarboxylic acid 3 mM a - k e t o g l u t a r a t e 3 mM m a l o n a t e 3 mM a - k e t o g l u t a r a t e 7 mM m a l o n a t e 3 mM a - k e t o g l u t a r a t e 15 mM m a l o n a t e
28.i i 1.4" 20.2 ~_ 0.6
(2i) (7)
I9.I
18.o :z I . I
(25) (19)
22.o ~ 5.1
(io)
1
3 mM succinate io mM succinate io mM succinate 6 mM monofluoroacetate io mM succinate 0.5 mM methoxyindolcarboxylic acid 3 mM f u m a r a t e 3 mM nlalate i o m M malate 3 mM oxaloacetate 3 mM oxaloacetate 6 mM monofluoroacetate 3 mM oxaloacetate 0. 5 mM m e t h o x y i n d o l c a r b o x y l i c acid 3 mM oxaloacetate 15 mM m a l o n a t e
:ix o . 8
32.2 ~ 3.4** (1 i) i8.6 ~ 0. 9
(IO)
17-6 zc 2.4
(9)
31.2 ~ 4.6** (12) 33.0 ~z 4.2* 20. 7 ~ 1. 3
44.4 -3- 5.8** ( I i ) 21.6 _~_ 2. 5
(9)
39.o ~ 3.0*
(II)
27.6 ~ 3.0 26.1 ~ 1.2"
(8) (12)
i8.o ~ 1.2
(13)
4 o. 2 ~ 3.2 * * (I i) 30.0 ~ 3.o** (14) 26. 4 ~ 2.8**
3 mM citrate
34.2 ~_ 4.8*
3 mM p y r u v a t e
20. 4 ~ 3.8
io mM acetate io mM acetate 3 mM succinate
(io) (14)
(8) (11) (8)
27.8 ~- 2.2*
(14)
18.6 i
(14)
1.2
* S u b s t r a t e e x p e r i m e n t s where t~i is significantly greater (P < o.oi) t h a n in experinlent~ with 3 mM ~-ketoglutarate. ** S u b s t r a t e - i n h i b i t o r c o m b i n a t i o n s where tg2 is significantly greater t h a n with the subs t r a t e alone.
membranes may be rate limiting for cycle oxidations. They noted that the rate of oxidation of NADH within rat kidney mitochondria by externally added oxaloacetate (or the rate of reduction of mitochondrial NAD + by malate) increased markedly when Biochim. Biophys. Acta, 2I 5 (197o) 216-219
.~FfORT COMMUNICATIONS
219
the mitochondrial membrane was rendered more permeable by detergent treatment. It is interesting in light of the differences between the transport-stimulating abilities of oxaloacetate and malate noted in the present experiments that in untreated mitochondria the apparent rate of penetration of oxaloacetate was about ioo-fold greater than that of malate. (2) The transport-stimulating substrates may enter into reactions which play a key role in proximal tubular salt transport. There has been considerable interest in the possibility that certain biochemical reactions m a y provide energy specifically for ion transportS, 6, though in the best-studied system--aldosterone-stimulated Na + transport by the toad urinary bladder--recent evidence fails to support this concepV. As far as the present results are concerned, it is difficult to see what common reactions would be stimulated by ~-ketoglutarate and succinate and by oxaloacetate and pyruvate but not by the intervening intermediates fumarate and malate. Since proximal tubules depend for their energy supply upon oxidative metabolism, we would expect interference with normal operation of the tricarboxylic acid cycle to slow fluid transport. I have tested the effects of the following inhibitors of the cycle enzymes: (i) monofluoroacetic acid, which by condensation with oxaloacetate forms fluoroeitrate, a competitive inhibitor of the aconitase reaction; (2) malonate, a competitive inhibitor of suceinate dehydrogenase; and (3) 5-methoxyindol-2-carboxylic acid, an inhibitor of the lipoic acid-dependent oxidation of pyruvate and ~-ketoglutarate 8. Each of these compounds interferes with fluid transport under certain conditions (Table I). However, fluoroacetate does not prolong t,~ in the presence of ~-ketoglutarate and methoxyindolearboxylic acid is not inhibitory in the presence of succinate. In both of these experiments a large number of energy-yielding steps remain between the added substrate and the presumed site of cycle blockade. Apparently then a rate of ATP production sufficient to maintain normal rates of fluid transport can be achieved without the participation of all the cycle oxidations.
Department of Physiology, New York Medical College, New York, N.Y. ioo29 (U.S.A.) I 2 3 4 5 6 7 8
DAVID L. MAUDE
D. L. 3,{AUDE,Am. J. Physiol., 2I 4 (1968) 1315. K. H. GERTZ,Arch. Ges. Physiol., 276 (1963) 336. C. E. FROHMAN, J. M. ORTEN AND A. H. SMITH, J. Biol. Chem., 193 (1951) 803. J. M. HASLAM AND H. A. KREBS, Biochem. J., lO 7 (1968) 659G. M. FIMOGNARI, G. A. PORTER AND I. S. EDELMAN, Biochim. Biophys. Acta, 135 (1967) 89. G. ~V. G. SHARP AND A. LEAF, J. Gen. Physiol., 51 (1968) 27IS. M. Z. FALCHUK AND G. W. G. SHARP, Biochim. Biophys. Acta, 153 (1968) 706. N. BAUMAN AND C. J. HILL, Biochemistry, 7 (1968) 1322.
Received April I3th, 197o Biochim. Biophys. Acta, 215 (197 o) 216-219
SHORT COMMUNICATIONS
other hand, neither does it appear to be particularly rich in glycosaminoglycans, as would have been the case if the surface zone had contained a layer of hvaluronic acid as postulated by BALAZSet al2. Further studies are in progress to determine the variation in the distribution of fixed charge density near the articular surface as a function of age. Tile principle of using cations from dilute solution to balance the fixed negative charges in structural tissue can readily be extended to localise and estimate anionic groups by such techniques as autoradiography (e.g. with 4~Ca) and electron-probe microanalysis.
Biomechanics Unit, Department of Mechanical Engineering, Imperial College, University of London, London (Great Britain)
ALICE MAROUDAS HILARY THOMAS
I A. MAROUDAS, H. MUIR AND J. V~7INGHAM, Biochim. Biophys. Acta, 177 (1969) 492. 2 l5". HELFFERICH, Ion Exchange, McGraw-Hill, New York, 1962, p. 143. 3 C. A. ANTONOPOULOS, S. GARDELL, J. A. SZIRMAI AND ]E. R. DE TYSSONSK, Biochim. Biophys. Acta, 83 (1964) I. 4 T. BITTER AND H. MUIR, Anal. Biochem., 4 (1962) 33 o. 5 M. B. MATHEXVS, Biochem. J., 96 ([965) 7Io. 6 M. SCHUBERT, Federation Proc., 25 (1966) lO47. 7 D. H. COLLINS AND T. F. MCELLIGOTT, Ann. Rheumatic Diseases, 19 (196o) 31. 8 C. V~rEISS, L. ROSENBERG AND A. J. HELFET, J . Bone Joint Surg., 5oA (4) (1968) 663. 9 E. A. BALAZS, G. D. BLOOM AND D. A. S\VANN, Federation Proc., 25 (1966) 1813.
Received April I5th, 197o Biochim. Biophys. Acta, 215 (197 o) 214-216
BBA 23588
Effects of substrates and inhibitors of the tricarboxylic acid cycle on proximal tubular fluid transport in vitro I have studied the effects of substrates and inhibitors of the tricarboxylic acid cycle on proximal tubular fluid transport using a technique of stop-flow microperfusion of single tubules in leached rat renal cortex slices. The experimental method has been described in detail elsewhere 1. Briefly, a surface cortical slice taken from a kidney of a pentobarbital-anesthetized male Sprague-Dawley rat is mounted in a micropuncture chamber. Here it is bathed in a rapidly flowing, recirculating oxygenated bicarbonate-saline solution at 37-38°. The basic medium provided (mM) 14o Na +, 5 K+, 121.6 Cl-, 20 HCOa-, 2.4 HP042-, 0.6 H2PO~-, I.o Ca2+ and 1.2 MgSO 4. Most of the media contained one or more substrates or inhibitors. These were all sodium salts of carboxylic acids and replaced an equivalent amount of NaC1, so that the Na + concentration was the s~me in all media. Before mounting, the slices were leached for 4-6 h at 2 ° in a medium identical to that subsequently used in the micropuncture chamber. The shrinking-droplet method Biochim. Biophys. Acta, 215 (197 o) 216-219
SHORT COMMUNICATIONS
217
of GERTZ2 was used to measure the rate of proximal tubular fluid transport. Since the perfusate is isosmotic (14o mM) NaC1, the rate of fluid reabsorption is directly related to the net rate of transport of sodium salts from the tubular lumen. For convenience, the results have been expressed in terms of the fluid reabsorptive halftime, t~2. 1.0 0.7 o.5
0.3 0
10 20 Time (sec)
30
40
Fig. i. Relative perfusate volume (V/Vo) plotted as a function of time in representative experim e n t s on slices leached w i t h o u t s u b s t r a t e ((2)--(2)) and with 3 mM ~-ketoglutarate ( @ - - O ) .
As in studies with fresh kidney slices 1, the relative volume of perfusing fluid remaining within the tubule, V / V o, decreased exponentially with time under all experimental conditions. Fig. i shows data from two representative experiments on leached slices. Table I summarizes data from all experiments. Half-time for the reabsorption of isosmotic saline perfusates b y proximal tubules of fresh slices bathed in media providing io mM e-ketoglutarate as substrate is about 19 sec (ref. I). The half-time in slices leached in substrate-free media is significantly longer than this, 39.6 sec. However, when the leaching and bathing media contain 3 mM pyruvate, oxaloacetate or =-ketoglutarate, or io mM succinate, t~ becomes approximately the same as in fresh slices. Apparently leaching in a substrate-free medium depletes proximal tubular cells of one or more diffusible metabolites required for normal rates of NaC1 transport, while the above substrates are able to restore and maintain adequate levels of these substances. The effectiveness of these compounds in stimulating transport in leached slices can be most easily explained by their ability to provide the two intermediates needed to initiate and maintain operation of the tricarboxylic acid cycle: oxaloacetate and acetyl-CoA. The failure of acetate to restore normal rates of transport could be due to its inability to produce the oxaloacetate required b y the cycle. I t is more difficult to understand why citrate, fumarate and malate fail to yield normal rates of transport in leached slices. If transport were dependent simply upon operation of the tricarboxylic acid cycle, these compounds should have been as effective as pyruvate and the other cycle intermediates. Possible explanations for this discrepancy are: (i) Substrates which fail to support transport m a y be unable to penetrate cellular and/or mitochondrial membranes sufficiently rapidly to supply the cycle. Consistent with this hypothesis is the fact that the minimal effective concentrations of a-ketoglutarate and succinate (greater than I and 3 mM, respectively) are considerably higher than their concentrations in fresh kidney tissue a, as would be expected if permeability barriers had to be overcome. HASLAM AND KREBS4 have found more direct evidence that the rate of penetration of substrates across kidney mitochondrial Biochim. Biophys. Acta, 215 (197 o) 216-219
218
SHORT COMMUNICATIONS
TABLE I H A L F - T I M E FOR T U B U L A R F L U I D REABSORPTION (tl/2) I N SLICES L E A C H E D W I T H VARIOUS SUBSTRATE~
AND SUBSTRATE--INHIBITOR COMBINATIONS N u m b e r of t u b u l e s perfused is indicated in parentheses.
Substrate/inhibitor
tl 6 ~ S.E.
None
39.6 _~ 4.I*
(17)
mM 0¢-ketoglutarate 2 mM ~-ketoglutarate 3 mM ~-ketoglutarate i o mM ~-ketoglutarate 3 mM ~-ketoglutarate 6 mM monofluoroacetate 3 mM ~-ketoglutarate o.5 mM inethoxyindolcarboxylic acid 3 mM a - k e t o g l u t a r a t e 3 mM m a l o n a t e 3 mM a - k e t o g l u t a r a t e 7 mM m a l o n a t e 3 mM a - k e t o g l u t a r a t e 15 mM m a l o n a t e
28.i i 1.4" 20.2 ~_ 0.6
(2i) (7)
I9.I
18.o :z I . I
(25) (19)
22.o ~ 5.1
(io)
1
3 mM succinate io mM succinate io mM succinate 6 mM monofluoroacetate io mM succinate 0.5 mM methoxyindolcarboxylic acid 3 mM f u m a r a t e 3 mM nlalate i o m M malate 3 mM oxaloacetate 3 mM oxaloacetate 6 mM monofluoroacetate 3 mM oxaloacetate 0. 5 mM m e t h o x y i n d o l c a r b o x y l i c acid 3 mM oxaloacetate 15 mM m a l o n a t e
:ix o . 8
32.2 ~ 3.4** (1 i) i8.6 ~ 0. 9
(IO)
17-6 zc 2.4
(9)
31.2 ~ 4.6** (12) 33.0 ~z 4.2* 20. 7 ~ 1. 3
44.4 -3- 5.8** ( I i ) 21.6 _~_ 2. 5
(9)
39.o ~ 3.0*
(II)
27.6 ~ 3.0 26.1 ~ 1.2"
(8) (12)
i8.o ~ 1.2
(13)
4 o. 2 ~ 3.2 * * (I i) 30.0 ~ 3.o** (14) 26. 4 ~ 2.8**
3 mM citrate
34.2 ~_ 4.8*
3 mM p y r u v a t e
20. 4 ~ 3.8
io mM acetate io mM acetate 3 mM succinate
(io) (14)
(8) (11) (8)
27.8 ~- 2.2*
(14)
18.6 i
(14)
1.2
* S u b s t r a t e e x p e r i m e n t s where t~i is significantly greater (P < o.oi) t h a n in experinlent~ with 3 mM ~-ketoglutarate. ** S u b s t r a t e - i n h i b i t o r c o m b i n a t i o n s where tg2 is significantly greater t h a n with the subs t r a t e alone.
membranes may be rate limiting for cycle oxidations. They noted that the rate of oxidation of NADH within rat kidney mitochondria by externally added oxaloacetate (or the rate of reduction of mitochondrial NAD + by malate) increased markedly when Biochim. Biophys. Acta, 2I 5 (197o) 216-219
.~FfORT COMMUNICATIONS
219
the mitochondrial membrane was rendered more permeable by detergent treatment. It is interesting in light of the differences between the transport-stimulating abilities of oxaloacetate and malate noted in the present experiments that in untreated mitochondria the apparent rate of penetration of oxaloacetate was about ioo-fold greater than that of malate. (2) The transport-stimulating substrates may enter into reactions which play a key role in proximal tubular salt transport. There has been considerable interest in the possibility that certain biochemical reactions m a y provide energy specifically for ion transportS, 6, though in the best-studied system--aldosterone-stimulated Na + transport by the toad urinary bladder--recent evidence fails to support this concepV. As far as the present results are concerned, it is difficult to see what common reactions would be stimulated by ~-ketoglutarate and succinate and by oxaloacetate and pyruvate but not by the intervening intermediates fumarate and malate. Since proximal tubules depend for their energy supply upon oxidative metabolism, we would expect interference with normal operation of the tricarboxylic acid cycle to slow fluid transport. I have tested the effects of the following inhibitors of the cycle enzymes: (i) monofluoroacetic acid, which by condensation with oxaloacetate forms fluoroeitrate, a competitive inhibitor of the aconitase reaction; (2) malonate, a competitive inhibitor of suceinate dehydrogenase; and (3) 5-methoxyindol-2-carboxylic acid, an inhibitor of the lipoic acid-dependent oxidation of pyruvate and ~-ketoglutarate 8. Each of these compounds interferes with fluid transport under certain conditions (Table I). However, fluoroacetate does not prolong t,~ in the presence of ~-ketoglutarate and methoxyindolearboxylic acid is not inhibitory in the presence of succinate. In both of these experiments a large number of energy-yielding steps remain between the added substrate and the presumed site of cycle blockade. Apparently then a rate of ATP production sufficient to maintain normal rates of fluid transport can be achieved without the participation of all the cycle oxidations.
Department of Physiology, New York Medical College, New York, N.Y. ioo29 (U.S.A.) I 2 3 4 5 6 7 8
DAVID L. MAUDE
D. L. 3,{AUDE,Am. J. Physiol., 2I 4 (1968) 1315. K. H. GERTZ,Arch. Ges. Physiol., 276 (1963) 336. C. E. FROHMAN, J. M. ORTEN AND A. H. SMITH, J. Biol. Chem., 193 (1951) 803. J. M. HASLAM AND H. A. KREBS, Biochem. J., lO 7 (1968) 659G. M. FIMOGNARI, G. A. PORTER AND I. S. EDELMAN, Biochim. Biophys. Acta, 135 (1967) 89. G. ~V. G. SHARP AND A. LEAF, J. Gen. Physiol., 51 (1968) 27IS. M. Z. FALCHUK AND G. W. G. SHARP, Biochim. Biophys. Acta, 153 (1968) 706. N. BAUMAN AND C. J. HILL, Biochemistry, 7 (1968) 1322.
Received April I3th, 197o Biochim. Biophys. Acta, 215 (197 o) 216-219