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Cations and transport of galactose in kidney-cortex slices
SHORT COMMUNICATIONS
367
Cations and transport of galactose in kidney-cortex slices KRANE AND CRANE1 reported that galactose can accumulate against a concentration gradient when kidney-cortex slices are incubated aerobically in a sodium acetate medium. They also found that the up-hill transport was inhibited by dinitrocresol, phloridzin and by anaerobiosis. As it is known that the transport of some sugars, both up-hill and diffusion-type, in the intestine is linked with the active transport of sodium ~ and that the transport of glucose in the surviving guinea-pig intestine is not only sodium-depertdent but is also influenced by the presence of K ÷ (ref. 3) an attempt was made here to establish whether some similar relationship might not hold with respect to the transport of galactose in kidney cortex. The method used was similar to that of KRANE AND CRANE1; however, unlabelled galactose was used throughout. Kidney-cortex slices were prepared from rabbit kidney rapidly removed from the animal and kept iced during the cutting procedure. The slices were then maintained at room temperature in a medium of the following composition: NaC1, o.125 M; KC1, o.oi M; CHaCOONa , O.Oli M; CaC12, o.ooo74 M; potassium phosphate buffer (pH 7.3), 0.0074 M. The composition of this medium was altered with respect to the amount of Na + and K +, either by equimolecular replacement of K+ with Na +, or by equimolecular replacement of Na + with Li+, as indicated in the subsequent text. The slices were then transferred to a medium of the same composition placed in small Erlenmeyer flasks with a side arm, containing galactose also dissolved in the same medium, so as to give a 3.3 rmV/ concentration after mixing the contents. Incubation was then carried out on a Dubnoff shaker at 25 ° and galaetose tipped in from the side arm after a Io-min equilibration period, with oxygen or nitrogen in the gas phase; it was stopped after a period ranging from 2 9° min, counting from the moment of tipping in the galactose solution. The slices were then quickly blotted, weighed on a torsion balance and homogenized in a Potter-Elvehjem homogenizer with 2 ml 50o ZnSO 4 and 2 ml 0.3 N Ba(OH)e. After standing for at least 3 ° rain, the suspension was centrifuged at approx. IOOOx g and an aliquot of the supernatant used for the determination of reducing sugars by the method of SOMOGYI AND NELSON4,5. Radioactive galactose was not used, since it had been demonstrated that galactose enters the kidney cells as such and is not phosphorylated at a rate that might affect the results of the estimation. It was found several times that the initial pre-incubation value of the reducing-sugar content of the slices was of the order of 0.5 mg glucose equivalent/ml intracellular water. In such cases the experimental values obtained were not considered to be valid. It could be shown, in agreement with previous findings, that the up-hill transport of galactose depends on the presence of air in the gas phase and that it is inhibited by phloridzin, considered to be a specific inhibitor of sugar transport (e.g. ref. 6). The investigation was extended to the use of ouabain, described as a selective inhibitor of the "sodium pump" without affecting other metabolic processes 7. At a concentration of 5"1o-4 M, ouabain inhibited galactose transport beyond the diffusionequilibrium level, the steady-state value with ouabain being the same aerobically and anaerobically (Table I). This finding is in qualitative agreement with those made by CRANE2 in hamster intestine. The role of the Na + in the medium was tested by replacing Na + with Li + in the presence of 5o m M K +. Here again (cf. Table I) it could be seen that Na + is prerequisite Biochim. Biophys. Acta, 54 (I961) 367-369
368
SHORT COMMUNICATIONS TABLE I STEADY-STATE
CONCENTRATIONS
OF
GALACTOSE
IN
KIDNEY-CORTEN
SLICES
I n c u b a t i o n a t 25 °, in 5 ° h i m K +, w i t h o.6o m g g a l a c t o s e / m l . Gas phase
Oxygen Oxygen Oxygen Oxygen Nitrogen Nitrogen Nitrogen Nitrogen
Galactose(mg/ml
Medium
intracellular water)
No a d d i t i o n 5' IO ~ M p h l o r i d z i n 5" IO 4 M o u a b a i n Na + r e p l a c e d b y Li + No a d d i t i o n 5" lO-4 3 I p h l o r i d z i n 5" io 4 M o u a b a i n Na ~ r e p l a c e d b y Li +
2.47 o.28 o.55 o.61 o.6o o.3o o.54 o.63
for up-hill galactose transport to take place. It appears that the aerobic up-hilI transport of galactose is a process occurring concurrently with passive galactose diffusion into the cell and t h a t Na ÷ (as low as IO mM) is required for its full operation. On increasing the total Na + concentration in the medium to 16o m M (at the expense of K +) the steady-state level of galactose was found to be some IO ~!'olower than that at the optimal concentration of between IO and 136 mM. It was found that the concentration of K + in the medium is of importance for the steady-state level of galactose reached on incubation. In the absence of K + the final level attained is only slightly higher than would correspond to a diffusion equilibrium and when additional corrections (such as for the a m o u n t of glycogen split during incubation) are taken it m a y be concluded that actually no up-hill transport of galactose takes place in the absence of K + (Fig. i). The optimum concentration of K + was found to be approx. 5o mM, lower steady-state levels being obtained on increasing the K + concentration above this value. This decrease is apparently not due to the lower concentration of Na + as the latter was still sufficient for full performance in the experiments where Na + was partly replaced with K +. Moreover, there are indications t h a t at higher K + concentrations the energy metabolism of the cel[ 3.0
N2.o
1.O o
____Consen t r_a_Lj£n__of_ g_aL%cf.os_e_ i_n__tb _e__m_e_dju_m .....
g
2 0
40
80
120
[K÷](mm Fig. I. The effect of e x t e r n a l K + c o n c e n t r a t i o n on t h e i n t r a c e l l u l a r s t e a d y - s t a t e level of g a l a c t o s e a f t e r 6o-min i n c u b a t i o n a t 25 °. The v a r i o u s s y m b o l s refer to i n d i v i d u a l e x p e r i m e n t s .
Biochim. ~iophys. ,'tcla, 54 (19611 367-3()9
SHORT COMMUNICATIONS
369
m a y be affected s. Thus, both Na + and K + seem to be necessary for up-hill transport of galactose to take place in kidney-cortex slices. The mechanism of the effect of Na + m a y well be related to that suggested by CRANEz, in that up-hill galactose transport is inhibited both by phloridzin and ouabain (by the latter only aerobically) and that Na + is required for its operation; whether the same holds for K + cannot be decided at present. The ultimate source of energy for the process m a y well be identical with ATP, this being suggested by the ionic requirements and susceptibility to inhibitors being identical with those described for ATPase in the cell membrane (e.g. ref. 9). There is one point to be mentioned in this connection, viz. that of the localization of the ion effect. It is generally assumed that the sodium or the sodium-potassium pump of the tubular cell is located at the basal (peritubular) membrane 1°,11. For accumulation of galactose to occur in the cells it would be expected that the transporting mechanism be localized in the tubular membrane, being of the type described for the intestinal brush border. Thus the problem of the ion-linked transporting mechanism for galactose will require further study.
Laboratory for Cellular Metabolism, Institute of Biology, Czechoslovak Academy of Sciences, Prague (Czechoslovakia)
A. KLEINZELLER A. KOTYK
1 S. M. KRANE AND R. K. CRANE, J. Biol. Chem., 234 (I959) 211. 2 R. K. CRANE, in Symposium Membrane Transport and .~Ielabglism, Publishing H o u s e of the Czechoslovak A c a d e m y of Sciences, Prague, and Academic Press, Inc., New York, 1961, p. 447E. RII
Received May 26th, 1961 Biochim. Biophys. Acta, 54 (1961) 367-369
Metabolism of uridine diphosphate 91ucuronic acid by liver and kidney GINSBERG et al. z showed that kidney particulate preparations metabolized UDPglucuronic acid to D-glucuronic acid and glucuronic acid 1-phosphate. The present investigation was carried out to determine whether UDP-glucuronic acid was metabolized in a similar manner by liver. The results presented here show that while kidney particulate contained high levels of UDP-glucuronic acid pyrophosphatase and glucuronic acid 1-phosphate phosphatase, liver particulate possessed high pyrophosphatase activity but very little phosphatase activity. Accordingly, incubation of UDP-glucuronic acid with Biochim. Biophys. Acta, 54 (1961) 369-372
367
Cations and transport of galactose in kidney-cortex slices KRANE AND CRANE1 reported that galactose can accumulate against a concentration gradient when kidney-cortex slices are incubated aerobically in a sodium acetate medium. They also found that the up-hill transport was inhibited by dinitrocresol, phloridzin and by anaerobiosis. As it is known that the transport of some sugars, both up-hill and diffusion-type, in the intestine is linked with the active transport of sodium ~ and that the transport of glucose in the surviving guinea-pig intestine is not only sodium-depertdent but is also influenced by the presence of K ÷ (ref. 3) an attempt was made here to establish whether some similar relationship might not hold with respect to the transport of galactose in kidney cortex. The method used was similar to that of KRANE AND CRANE1; however, unlabelled galactose was used throughout. Kidney-cortex slices were prepared from rabbit kidney rapidly removed from the animal and kept iced during the cutting procedure. The slices were then maintained at room temperature in a medium of the following composition: NaC1, o.125 M; KC1, o.oi M; CHaCOONa , O.Oli M; CaC12, o.ooo74 M; potassium phosphate buffer (pH 7.3), 0.0074 M. The composition of this medium was altered with respect to the amount of Na + and K +, either by equimolecular replacement of K+ with Na +, or by equimolecular replacement of Na + with Li+, as indicated in the subsequent text. The slices were then transferred to a medium of the same composition placed in small Erlenmeyer flasks with a side arm, containing galactose also dissolved in the same medium, so as to give a 3.3 rmV/ concentration after mixing the contents. Incubation was then carried out on a Dubnoff shaker at 25 ° and galaetose tipped in from the side arm after a Io-min equilibration period, with oxygen or nitrogen in the gas phase; it was stopped after a period ranging from 2 9° min, counting from the moment of tipping in the galactose solution. The slices were then quickly blotted, weighed on a torsion balance and homogenized in a Potter-Elvehjem homogenizer with 2 ml 50o ZnSO 4 and 2 ml 0.3 N Ba(OH)e. After standing for at least 3 ° rain, the suspension was centrifuged at approx. IOOOx g and an aliquot of the supernatant used for the determination of reducing sugars by the method of SOMOGYI AND NELSON4,5. Radioactive galactose was not used, since it had been demonstrated that galactose enters the kidney cells as such and is not phosphorylated at a rate that might affect the results of the estimation. It was found several times that the initial pre-incubation value of the reducing-sugar content of the slices was of the order of 0.5 mg glucose equivalent/ml intracellular water. In such cases the experimental values obtained were not considered to be valid. It could be shown, in agreement with previous findings, that the up-hill transport of galactose depends on the presence of air in the gas phase and that it is inhibited by phloridzin, considered to be a specific inhibitor of sugar transport (e.g. ref. 6). The investigation was extended to the use of ouabain, described as a selective inhibitor of the "sodium pump" without affecting other metabolic processes 7. At a concentration of 5"1o-4 M, ouabain inhibited galactose transport beyond the diffusionequilibrium level, the steady-state value with ouabain being the same aerobically and anaerobically (Table I). This finding is in qualitative agreement with those made by CRANE2 in hamster intestine. The role of the Na + in the medium was tested by replacing Na + with Li + in the presence of 5o m M K +. Here again (cf. Table I) it could be seen that Na + is prerequisite Biochim. Biophys. Acta, 54 (I961) 367-369
368
SHORT COMMUNICATIONS TABLE I STEADY-STATE
CONCENTRATIONS
OF
GALACTOSE
IN
KIDNEY-CORTEN
SLICES
I n c u b a t i o n a t 25 °, in 5 ° h i m K +, w i t h o.6o m g g a l a c t o s e / m l . Gas phase
Oxygen Oxygen Oxygen Oxygen Nitrogen Nitrogen Nitrogen Nitrogen
Galactose(mg/ml
Medium
intracellular water)
No a d d i t i o n 5' IO ~ M p h l o r i d z i n 5" IO 4 M o u a b a i n Na + r e p l a c e d b y Li + No a d d i t i o n 5" lO-4 3 I p h l o r i d z i n 5" io 4 M o u a b a i n Na ~ r e p l a c e d b y Li +
2.47 o.28 o.55 o.61 o.6o o.3o o.54 o.63
for up-hill galactose transport to take place. It appears that the aerobic up-hilI transport of galactose is a process occurring concurrently with passive galactose diffusion into the cell and t h a t Na ÷ (as low as IO mM) is required for its full operation. On increasing the total Na + concentration in the medium to 16o m M (at the expense of K +) the steady-state level of galactose was found to be some IO ~!'olower than that at the optimal concentration of between IO and 136 mM. It was found that the concentration of K + in the medium is of importance for the steady-state level of galactose reached on incubation. In the absence of K + the final level attained is only slightly higher than would correspond to a diffusion equilibrium and when additional corrections (such as for the a m o u n t of glycogen split during incubation) are taken it m a y be concluded that actually no up-hill transport of galactose takes place in the absence of K + (Fig. i). The optimum concentration of K + was found to be approx. 5o mM, lower steady-state levels being obtained on increasing the K + concentration above this value. This decrease is apparently not due to the lower concentration of Na + as the latter was still sufficient for full performance in the experiments where Na + was partly replaced with K +. Moreover, there are indications t h a t at higher K + concentrations the energy metabolism of the cel[ 3.0
N2.o
1.O o
____Consen t r_a_Lj£n__of_ g_aL%cf.os_e_ i_n__tb _e__m_e_dju_m .....
g
2 0
40
80
120
[K÷](mm Fig. I. The effect of e x t e r n a l K + c o n c e n t r a t i o n on t h e i n t r a c e l l u l a r s t e a d y - s t a t e level of g a l a c t o s e a f t e r 6o-min i n c u b a t i o n a t 25 °. The v a r i o u s s y m b o l s refer to i n d i v i d u a l e x p e r i m e n t s .
Biochim. ~iophys. ,'tcla, 54 (19611 367-3()9
SHORT COMMUNICATIONS
369
m a y be affected s. Thus, both Na + and K + seem to be necessary for up-hill transport of galactose to take place in kidney-cortex slices. The mechanism of the effect of Na + m a y well be related to that suggested by CRANEz, in that up-hill galactose transport is inhibited both by phloridzin and ouabain (by the latter only aerobically) and that Na + is required for its operation; whether the same holds for K + cannot be decided at present. The ultimate source of energy for the process m a y well be identical with ATP, this being suggested by the ionic requirements and susceptibility to inhibitors being identical with those described for ATPase in the cell membrane (e.g. ref. 9). There is one point to be mentioned in this connection, viz. that of the localization of the ion effect. It is generally assumed that the sodium or the sodium-potassium pump of the tubular cell is located at the basal (peritubular) membrane 1°,11. For accumulation of galactose to occur in the cells it would be expected that the transporting mechanism be localized in the tubular membrane, being of the type described for the intestinal brush border. Thus the problem of the ion-linked transporting mechanism for galactose will require further study.
Laboratory for Cellular Metabolism, Institute of Biology, Czechoslovak Academy of Sciences, Prague (Czechoslovakia)
A. KLEINZELLER A. KOTYK
1 S. M. KRANE AND R. K. CRANE, J. Biol. Chem., 234 (I959) 211. 2 R. K. CRANE, in Symposium Membrane Transport and .~Ielabglism, Publishing H o u s e of the Czechoslovak A c a d e m y of Sciences, Prague, and Academic Press, Inc., New York, 1961, p. 447E. RII
Received May 26th, 1961 Biochim. Biophys. Acta, 54 (1961) 367-369
Metabolism of uridine diphosphate 91ucuronic acid by liver and kidney GINSBERG et al. z showed that kidney particulate preparations metabolized UDPglucuronic acid to D-glucuronic acid and glucuronic acid 1-phosphate. The present investigation was carried out to determine whether UDP-glucuronic acid was metabolized in a similar manner by liver. The results presented here show that while kidney particulate contained high levels of UDP-glucuronic acid pyrophosphatase and glucuronic acid 1-phosphate phosphatase, liver particulate possessed high pyrophosphatase activity but very little phosphatase activity. Accordingly, incubation of UDP-glucuronic acid with Biochim. Biophys. Acta, 54 (1961) 369-372